Viral proteins and nanostructures and uses thereof

Abstract

This article provides recombinant peptides comprising engineered extracellular domains of viral proteins derived from enveloped viruses. It also provides two-component protein nanostructures and compositions for use in vaccination, generating immune responses, or treating or preventing viral infections.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 583,117, filed September 15, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation of sequence lists This application contains a sequence list submitted electronically in XML format, and the entire sequence list is hereby incorporated by reference. The XML copy was created on September 11, 2024, named 061291-518001WO.xml, and is 1,130KB in size. Background Technology

[0003] When an enveloped virus encounters a target cell, its viral membrane fusion protein undergoes a conformational change, driving the fusion of the viral envelope with the target cell membrane. This fusion process delivers the viral genome into the target cell. For many enveloped viruses, an adaptive immune response to the viral membrane fusion protein is a key source of protective immunity, partly because neutralizing antibodies can inhibit this fusion process. Therefore, vaccines for enveloped viruses often include the viral membrane fusion protein as an antigen.

[0004] The need for viral membrane fusion proteins stabilized through designed amino acid substitution has not been met. This disclosure provides recombinant peptides, along with related compositions and methods, to address this need for respiratory syncytial virus (RSV), hMPV, PIV3, PIV5, SARS-CoV-2, and Nipah virus. Summary of the Invention

[0005] In one aspect, this disclosure provides a recombinant polypeptide comprising an engineered extracellular domain of a trimeric pathogenic (e.g., viral) protein, wherein, relative to a native reference sequence of the pathogenic (e.g., viral) protein, the extracellular domain comprises a C-terminal helical forming segment comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In another aspect, this disclosure provides a nanostructure comprising a trimeric component comprising the helical forming segment as disclosed herein. In yet another aspect, this disclosure provides the helical forming segment as disclosed herein.

[0006] In some embodiments of the recombinant peptide, the C-terminal helical forming region has improved hydrophobic stacking compared to the native reference sequence. In some embodiments, the C-terminal helical forming region comprises about 7 to about 31 residues. In some embodiments, the amino acid substitutions comprise polar, charged, and / or hydrophobic amino acids. In some embodiments, the C-terminal helical forming region comprises a peptide sequence according to any of the following: LXXTIXXLLXIXXXLXXXL (SEQ ID NO: 566), LVXTXKXLXDLIXXLXXLLXKLXX (SEQ ID NO: 567), LNKVKKXVXXLXXXVXXLEKXLX (SEQ ID NO: 568), EKIXXAIKKAXKL (SEQ ID NO: 569), EXIXKAIKXLXXXXX (SEQ ID NO: 570), XKXXEXXXXVXXXXXXXXX (SEQ ID NO: 571), XXLKKAAXIXKKXLKXX (SEQ ID NO: 572).

[0007] In some implementations, the C-terminal helical forming region contains a polypeptide sequence according to any of the common sequences in Table 24.

[0008] In some embodiments, the segment comprises a polypeptide sequence according to any of the following: L X2X2T I X2X2L L X2I[V / I] X2X2L [I / L] X2X2L (SEQ ID NO: 573), LV [A / T] T X2K X2L X2D LI X2X2L [K / E]X2L L X2K L X2X2 (SEQ ID NO: 574) or LNKVKK X2V X2X2L X2X2X2V X2X2L EK X2L X2 (SEQ ID NO: 575), wherein X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

[0009] In some embodiments, the segment comprises a polypeptide sequence according to any of the following: EKI X2X2A IKK AX2K L (SEQ ID NO: 576), E X2I X2K AIK X2L [L / X2] X2X2[X1 / X2] X2 (SEQ ID NO: 577). 、X2K [X1 / T] [L / E]E [T / A] X1X2[I / X2] V X2X2[X1 / X2] [X1 / X2] X2X2X1X2X2 (SEQ ID NO: 578) or X2X2L KKAA X2I X1K K X1L K X2X2 (SEQ ID NO: 579), wherein X1 is a nonpolar residue selected from A, I, L, and M, and X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K, and H, preferably a wild-type amino acid. In some embodiments, the segment comprises a polypeptide sequence listed in Table 25A or Table 25B. In some embodiments, the native reference sequence of the viral protein is any one of SEQ ID NO: 1, 104, 327, 382, ​​459, 499.

[0010] In some embodiments, the recombinant polypeptide comprises an engineered extracellular domain of an hMPV fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 104 between about residues 470 and about residues 500, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 104, the residues creating hydrophobic contacts between segments in the α-helical homotrimer. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 104 between about residues 470 and about residues 490, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 7 to about 21 residues.

[0011] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 104 at position Q471, wherein Q is substituted by any one of A, D, E, I, Q, R, S, T; (2) an amino acid substitution relative to SEQ ID NO: 104 at position A472, wherein A is substituted by any one of A, D, E, I, K, R, S, T, Y; (3) an amino acid substitution relative to SEQ ID NO: 104 at position L473, wherein L is substituted by any one of A, I, L, M, Q, S, T, W; (4) an amino acid substitution relative to SEQ ID NO: 104 at position V474, wherein V is substituted by any one of A, D, E, I, K, L, N, Q, S, T; and (5) an amino acid substitution relative to SEQ ID NO: 104 The amino acid at position D475 is substituted, wherein D is substituted by any one of A, D, E, H, K, N, Q, R, S, T; (6) The amino acid at position Q476 of SEQ ID NO: 104 is substituted, wherein Q is substituted by any one of A, D, E, H, I, K, L, M, N, Q, T, V; (7) The amino acid at position S477 of SEQ ID NO: 104 is substituted, wherein S is substituted by any one of A, E, I, K, L, M, N, Q, R, S, T, V; (8) The amino acid at position N478 of SEQ ID NO: 104 is substituted, wherein N is substituted by any one of A, D, E, K, N, Q, R, S, T; (9) The amino acid at position N478 of SEQ ID NO: 104 is substituted, wherein N is substituted by any one of A, D, E, K, N, Q, R, S, T; (9) The amino acid at position N478 of SEQ ID NO: 104 is substituted, wherein D is substituted by any one of A, D, E, K, N, Q, R, S, T; 104 The amino acid at position R479 is substituted, wherein R is substituted by any one of A, D, E, F, I, K, L, M, N, Q, R, S, T, W, Y, (10) relative to SEQ ID NO: 104 The amino acid at position I480 is substituted, wherein I is substituted by any one of A, I, L, M, R, S, T, V, (11) relative to SEQ ID NO: 104 The amino acid at position L481 is substituted, wherein L is substituted by any one of D, E, I, K, L, M, N, Q, R, S, T, (12) relative to SEQ ID NO: 104 The amino acid at position S482 is substituted, wherein S is substituted by any one of A, D, E, K, Q, R, S, T, (13) relative to SEQ ID NO: 104 substituted with an amino acid at position S483, wherein S is substituted by any one of A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, Y, (14) relative to SEQ ID NO: 104 substituted with an amino acid at position A484, wherein A is substituted by any one of A, D, E, I, K, L, M, R, S, T, V, Y, (15) relative to SEQ ID NO: 104 substituted with an amino acid at position E485,(16) where E is replaced by any one of D, E, G, K, L, Q, R, S, T, (17) where the amino acid at position K486 relative to SEQ ID NO: 104 is replaced by any one of A, E, I, K, L, Q, R, S, T, (18) where the amino acid at position N488 relative to SEQ ID NO: 104 is replaced by any one of E, I, K, L, N, Q, R, S, V, (19) where the amino acid at position T489 relative to SEQ ID NO: 104 is replaced by any one of A, D, E, K, S, and (20) any combination of (1)-(19). In some embodiments, the segment comprises a polypeptide sequence listed in Table 6B, or a polypeptide sequence having one to five amino acid substitutions.

[0012] In some embodiments, the extracellular domain includes a C-terminal helical forming segment relative to SEQ ID NO: 104, between about residues 470 and about residues 500, comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 16 to about 30 residues.

[0013] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 104 at position A472, wherein A is substituted by any one of T, N, K, R, E, S; (2) an amino acid substitution relative to SEQ ID NO: 104 at position L473, wherein L is substituted by any one of T, I, V; (3) an amino acid substitution relative to SEQ ID NO: 104 at position V474, wherein V is substituted by any one of E, Q, L, D; (4) an amino acid substitution relative to SEQ ID NO: 104 at position D475, wherein D is substituted by any one of E, D, K; (5) an amino acid substitution relative to SEQ ID NO: 104 at position Q476, wherein Q is substituted by any one of Q, R, D, A, T, S, K; (6) an amino acid substitution relative to SEQ ID NO: 104 at position S477, wherein S is substituted by any one of I, V, L; and (7) an amino acid substitution relative to SEQ ID NO: 104 The amino acid at position N478 is substituted, wherein N is substituted by any one of K, E, N, S; (8) The amino acid at position R479 relative to SEQ ID NO: 104 is substituted, wherein R is substituted by any one of T, D, E, S, Y, A; (9) The amino acid at position I480 relative to SEQ ID NO: 104 is substituted, wherein I is substituted by any one of L, N; (10) The amino acid at position L481 relative to SEQ ID NO: 104 is substituted, wherein L is substituted by any one of T, D, E, K, Q, S, N; (11) The amino acid at position S482 relative to SEQ ID NO: 104 is substituted, wherein S is substituted by any one of E, D, S, T, Q, K; (12) The amino acid at position S483 relative to SEQ ID NO: 104 is substituted, wherein S is substituted by any one of R, K, L, E, A; (13) The amino acid at position S483 relative to SEQ ID NO: 104 is substituted, wherein S is substituted by any one of R, K, L, E, A; (14) The amino acid at position A484 of SEQ ID NO: 104 is substituted, wherein A is substituted by any of V, I, or M; (15) The amino acid at position E485 of SEQ ID NO: 104 is substituted, wherein E is substituted by any of E, A, K, H, Q, S, or N; (16) The amino acid at position K486 of SEQ ID NO: 104 is substituted, wherein K is substituted by any of S, E, K, R, V, D, or H; (17) The amino acid at position G487 of SEQ ID NO: 104 is substituted, wherein G is substituted by any of I or L; (18) The amino acid at position N488 of SEQ ID NO: 104 is substituted, wherein N is substituted by any of E, K, or R; (19) The amino acid at position T489 of SEQ ID NO: 104 is substituted.Where T is replaced by any one of K, E, S, R, Q, (19) the amino acid at position S490 of SEQ ID NO: 104 is replaced by any one of E, V, T, R, L, (20) the amino acid at position G491 of SEQ ID NO: 104 is replaced by any one of GL, I, V, (21) the amino acid at position R492 of SEQ ID NO: 104 is replaced by any one of E, Q, S, A, D, (22) the amino acid at position E493 of SEQ ID NO: 104 is replaced by any one of A, E, N, L, K, Q, S, (23) the amino acid at position N494 of SEQ ID NO: 104 is replaced by any one of I, L, (24) the amino acid at position N494 of SEQ ID NO: 104 is replaced by any one of SEQ ID NO: 104. An amino acid substitution at position L495, wherein L is substituted by any of K, L, T, V, I; (25) An amino acid substitution at position Y496 relative to SEQ ID NO: 104, wherein Y is substituted by any of K, E, R, Q; (26) An amino acid substitution at position F497 relative to SEQ ID NO: 104, wherein F is substituted by any of D, R, E, Q; (27) An amino acid substitution at position Q498 relative to SEQ ID NO: 104, wherein Q is substituted by any of V, L; and / or (28) Any combination of (1)-(27). In some embodiments, the segment comprises a polypeptide sequence listed in Table 6D, or a polypeptide sequence having 1 to 5 amino acid substitutions.

[0014] In some embodiments, the extracellular domain further comprises one, two, three or more amino acid substitutions relative to SEQ ID NO: 104 at positions 63, 97, 98, 99, 100, 101, 102, 140, 147, 153, 185, 188, 219, 231, 294, 365, 368, 450, 463 or 470.

[0015] In some embodiments, the recombinant polypeptide comprises an engineered extracellular domain of a PIV3 fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 327 between about residues 460 and about residues 490, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 327, the residues creating hydrophobic contacts between segments in the α-helical homotrimer. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 104 between about residues 460 and about residues 480, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 20 to about 28 residues.

[0016] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 327 at position L460, wherein L is substituted by any one of L, M, and V; (2) an amino acid substitution relative to SEQ ID NO: 327 at position N461, wherein N is substituted by N; (3) an amino acid substitution relative to SEQ ID NO: 327 at position K462, wherein K is substituted by any one of K and R; (4) an amino acid substitution relative to SEQ ID NO: 327 at position V463, wherein V is substituted by any one of L, V, and T; (5) an amino acid substitution relative to SEQ ID NO: 327 at position K464, wherein K is substituted by any one of A, K, and Q; (6) an amino acid substitution relative to SEQ ID NO: 327 at position S465, wherein S is substituted by any one of K and S; (7) an amino acid substitution relative to SEQ ID NO: 327 at position D466, wherein D is substituted by any one of E and K; and (8) an amino acid substitution relative to SEQ ID NO: 327 at position D466, wherein D is substituted by any one of E and K. (9) The amino acid substitution at position L467 of SEQ ID NO: 327, wherein L is substituted by any one of V, L, or T; (10) The amino acid substitution at position E468 of SEQ ID NO: 327, wherein E is substituted by any one of K, D, or E; (11) The amino acid substitution at position S470 of SEQ ID NO: 327, wherein S is substituted by any one of I, L, M, Y, F, or W; (12) The amino acid substitution at position K471 of SEQ ID NO: 327, wherein K is substituted by any one of L, W, A, or I; (13) The amino acid substitution at position E472 of SEQ ID NO: 327, wherein E is substituted by any one of K or E; (14) The amino acid substitution at position E468 of SEQ ID NO: 327, wherein E is substituted by any one of K or E; 327 The amino acid at position W473 is substituted, wherein W is substituted by any of E, I, K, Q, (15) The amino acid at position Y474 relative to SEQ ID NO: 327 is substituted, wherein Y is substituted by any of L, M, T, V, E, (16) The amino acid at position R475 relative to SEQ ID NO: 327 is substituted, wherein R is substituted by any of S, K, R, A, (17) The amino acid at position R476 relative to SEQ ID NO: 327 is substituted, wherein R is substituted by any of K, E, S, N, (18) The amino acid at position S477 relative to SEQ ID NO: 327 is substituted, wherein S is substituted by any of K, D, E, and (19) Any combination of (1)-(18).

[0017] In some embodiments, the segment comprises a polypeptide sequence listed in Table 7B, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment relative to SEQ ID NO: 327, between about residues 465 and about residues 490, comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 14 to about 30 residues.

[0018] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 327 at position S465, wherein S is substituted by any one of E, K, D, S, N, Q, T, R, A; (2) an amino acid substitution relative to SEQ ID NO: 327 at position D466, wherein D is substituted by any one of D, R, K, E, M, Q, A, S, N; (3) an amino acid substitution relative to SEQ ID NO: 327 at position L467, wherein L is substituted by any one of I, V, L; (4) an amino acid substitution relative to SEQ ID NO: 327 at position E468, wherein E is substituted by any one of E, K, S, D, R, H, T, N, A; (5) an amino acid substitution relative to SEQ ID NO: 327 at position E469, wherein E is substituted by any one of K, S, E, N, T, Q, H, D, Y; and (6) an amino acid substitution relative to SEQ ID NO: 327 substituted amino acid at position S470, wherein S is substituted by any one of L, D, V, I, A, N, T; (7) substituted amino acid at position K471 relative to SEQ ID NO: 327, wherein K is substituted by any one of E, T, L, K, N, I, R, Q, S; (8) substituted amino acid at position E472 relative to SEQ ID NO: 327, wherein E is substituted by any one of E, K, Q, S, H, R, T; (9) substituted amino acid at position W473 relative to SEQ ID NO: 327, wherein W is substituted by any one of R, Q, K, E, T, S, I, N; (10) substituted amino acid at position Y474 relative to SEQ ID NO: 327, wherein Y is substituted by any one of V, L, I, Q, T; (11) substituted amino acid at position Y474 relative to SEQ ID NO: 327. 327 substituted amino acid at position R475, wherein R is substituted by any one of H, K, D, T, E, S, R, N, Q, A, (12) relative to SEQ ID NO: 327 substituted amino acid at position R476, wherein R is substituted by any one of A, T, H, E, D, K, R, Q, S, (13) relative to SEQ ID NO: 327 substituted amino acid at position S477, wherein S is substituted by any one of I, L, V, (14) relative to SEQ ID NO: 327 substituted amino acid at position N478, wherein N is substituted by any one of E, L, K, I, R, S, Q, (15) relative to SEQ ID NO: 327 substituted amino acid at position Q479, wherein Q is substituted by any one of K, H, E, N, Q, R, T, A, S, (16) relative to SEQ ID NO: 327 is an amino acid substitution at position K480, wherein K is replaced by any one of K, R, E, T, S, L, A, I, or V.(17) An amino acid substituted relative to SEQ ID NO: 327 at position L481, wherein L is substituted by any one of L, V, or I; (18) An amino acid substituted relative to SEQ ID NO: 327 at position D482, wherein D is substituted by any one of K, A, E, S, H, T, N, D, or R; (19) An amino acid substituted relative to SEQ ID NO: 327 at position S483, wherein S is substituted by any one of Q, T, E, A, S, N, D, K, or L; (20) An amino acid substituted relative to SEQ ID NO: 327 at position I484, wherein I is substituted by any one of I, L, A, or V; (21) An amino acid substituted relative to SEQ ID NO: 327 at position G485, wherein G is substituted by any one of L, K, R, E, or I; (22) An amino acid substituted relative to SEQ ID NO: 327 is an amino acid substitution at position S486, wherein S is substituted by any of T, A, E, R, H, D, S, and / or any combination of (23) (1)-(22). In some embodiments, the segment comprises a polypeptide sequence listed in Table 7D, or a polypeptide sequence having 1 to 5 amino acid substitutions.

[0019] In some embodiments, the recombinant polypeptide comprises an engineered extracellular domain of a PIV5 fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 382 between about residues 460 and about residues 490, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 382, ​​the residues creating hydrophobic contacts between segments in the α-helical homotrimer. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 104 between about residues 460 and about residues 480, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 6 to about 26 residues.

[0020] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 382 at position A463, wherein A is substituted by any one of L, T, V, and A; (2) an amino acid substitution relative to SEQ ID NO: 382 at position L464, wherein L is substituted by any one of K, I, Q, A, W, and E; (3) an amino acid substitution relative to SEQ ID NO: 382 at position Q465, wherein Q is substituted by any one of K, Q, T, E, S, and R; (4) an amino acid substitution relative to SEQ ID NO: 382 at position H466, wherein H is substituted by any one of K, A, E, L, I, W, R, Q, T, D, and Y; (5) an amino acid substitution relative to SEQ ID NO: 382 at position L467, wherein L is substituted by any one of V, I, L, M, FA, T, C, and H; and (6) an amino acid substitution relative to SEQ ID NO: 382 substituted amino acid at position A468, wherein A is substituted by any one of D, T, K, L, E, R, I, N, S; (7) substituted amino acid at position Q469 relative to SEQ ID NO: 382, ​​wherein Q is substituted by any one of E, K, S, T, A, R, Q, D; (8) substituted amino acid at position S470 relative to SEQ ID NO: 382, ​​wherein S is substituted by any one of A, K, L, I, T, S, V, H, Y, E, W, FR, Q, M; (9) substituted amino acid at position D471 relative to SEQ ID NO: 382, ​​wherein D is substituted by any one of T, E, V, L, S, I, A, K, Y, W; (10) substituted amino acid at position T472 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of K, E, R, S, T, A, D, L; (11) substituted amino acid at position T472 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of K, E, R, S, T, A, D, L; (11) substituted amino acid at position T472 relative to SEQ ID NO: 382, ​​wherein Q is substituted by any one of E, K, S, T, A, D, L; (12) The amino acid at position Y473 of SEQ ID NO: 382 is substituted, wherein Y is substituted by any one of T, K, S, R, Q, D, E, I, H, M; (13) The amino acid at position L474 of SEQ ID NO: 382 is substituted, wherein L is substituted by any one of T, S, L, A, D, W, Q, I, Y, V, K, E; (14) The amino acid at position S475 of SEQ ID NO: 382 is substituted, wherein S is substituted by any one of T, E, I, K, S, Q, A, L, R, D; (15) The amino acid at position I477 of SEQ ID NO: 382 is substituted, wherein I is substituted by any one of K, Q, R, D, T, E, I, Y, S, L.(16) An amino acid substituted at position T478 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of E, K, S, D, W, L, Q, I, T; (17) An amino acid substituted at position S479 relative to SEQ ID NO: 382, ​​wherein S is substituted by any one of R, K, Q, S, A, D, E; (18) An amino acid substituted at position A480 relative to SEQ ID NO: 382, ​​wherein A is substituted by any one of S, K; (19) An amino acid substituted at position T481 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of E, D, S, K, M, N, A, T; (20) An amino acid substituted at position T482 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of R, S, Q, L, K; (21) An amino acid substituted at position T482 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of R, S, Q, L, K; (22) The amino acid substitution at position T483 of SEQ ID NO: 382, ​​wherein T is substituted by any of K, A, or S; (23) The amino acid substitution at position S484 of SEQ ID NO: 382, ​​wherein S is substituted by any of S, E, D, or Y; (24) The amino acid substitution at position V485 of SEQ ID NO: 382, ​​wherein V is substituted by any of Q or T; (25) The amino acid substitution at position L486 of SEQ ID NO: 382, ​​wherein L is substituted by K; (26) The amino acid substitution at position S487 of SEQ ID NO: 382, ​​wherein S is substituted by any of S or K; (27) The amino acid substitution at position I488 of SEQ ID NO: 382, ​​wherein I is substituted by any of S or K; and / or (28) Any combination of (1)-(26).

[0021] In some embodiments, the segment comprises a polypeptide sequence listed in Table 8B, or a polypeptide sequence having one to five amino acid substitutions.

[0022] In some embodiments, the polypeptide comprises an engineered extracellular domain of the SARS-CoV2 spike (S) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 459 between about residues 1140 and about residues 1170, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 459, the residues creating hydrophobic contacts between segments in the α-helical homotrimer. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 459 between about residues 1140 and about residues 1170, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 10 to about 25 residues.

[0023] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 459 at position D1147, wherein D is substituted by any one of E, D, and K; (2) an amino acid substitution relative to SEQ ID NO: 459 at position S1148, wherein S is substituted by any one of T, S, and K; (3) an amino acid substitution relative to SEQ ID NO: 459 at position F1149, wherein F is substituted by A; (4) an amino acid substitution relative to SEQ ID NO: 459 at position K1150, wherein K is substituted by any one of I, A, L, and M; (5) an amino acid substitution relative to SEQ ID NO: 459 at position E1151, wherein E is substituted by any one of K, S, D, R, and E; (6) an amino acid substitution relative to SEQ ID NO: 459 at position E1152, wherein E is substituted by any one of I, Y, K, T, R, and E; and (7) an amino acid substitution relative to SEQ ID NO: 459 substituted amino acid at position L1153, wherein L is substituted by any one of T or A; (8) substituted amino acid at position D1154 relative to SEQ ID NO: 459, wherein D is substituted by any one of L, I, E, T, M, V; (9) substituted amino acid at position K1155 relative to SEQ ID NO: 459, wherein K is substituted by any one of E, K, T, R; (10) substituted amino acid at position Y1156 relative to SEQ ID NO: 459, wherein Y is substituted by any one of I, V, K, R; (11) substituted amino acid at position F1157 relative to SEQ ID NO: 459, wherein F is substituted by any one of V, A, I, Y, T, S; (12) substituted amino acid at position K1158 relative to SEQ ID NO: 459, wherein K is substituted by any one of L, R, S, K, D, W, N, I; (13) substituted amino acid at position K1158 relative to SEQ ID NO: 459 is substituted with an amino acid at position N1159, wherein N is substituted with any one of K, T, Q, I, R, E; (14) is substituted with an amino acid at position H1160 relative to SEQ ID NO: 459, wherein H is substituted with any one of I, L, R, E, K, S; (15) is substituted with an amino acid at position T1161 relative to SEQ ID NO: 459, wherein T is substituted with any one of L, N, I, A, S, W, Y; (16) is substituted with an amino acid at position S1162 relative to SEQ ID NO: 459, wherein S is substituted with any one of K, S, T, R; (17) is substituted with an amino acid at position P1163 relative to SEQ ID NO: 459, wherein P is substituted with any one of E, D, R, K, I, A.(18) An amino acid substitution relative to SEQ ID NO: 459 at position D1164, wherein D is substituted by any one of W, S, M, D, T, I, N; (19) An amino acid substitution relative to SEQ ID NO: 459 at position V1165, wherein V is substituted by any one of E, A, K, L; (20) An amino acid substitution relative to SEQ ID NO: 459 at position D1166, wherein D is substituted by any one of K, S; (21) An amino acid substitution relative to SEQ ID NO: 459 at position L1167, wherein L is substituted by any one of R, K; (22) An amino acid substitution relative to SEQ ID NO: 459 at position G1168, wherein G is substituted by any one of K, S; (23) An amino acid substitution relative to SEQ ID NO: 459 at position D1169, wherein D is substituted by S; (24) An amino acid substitution relative to SEQ ID NO: 459. Amino acid substitution at position I1170, where I is substituted by S, and / or any combination of (25)(1)-(24).

[0024] In some embodiments, the segment comprises a polypeptide sequence listed in Table 9B, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment relative to SEQ ID NO: 459, between about residues 1145 and about residues 1175, comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 12 to about 22 residues.

[0025] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 459 at position D1147, wherein D is substituted by any one of Q, T, E, S, N, D, K; (2) an amino acid substitution relative to SEQ ID NO: 459 at position S1148, wherein S is substituted by any one of T, K, N, R, S, A, E; (3) an amino acid substitution relative to SEQ ID NO: 459 at position F1149, wherein F is substituted by any one of L, T, I, V; (4) an amino acid substitution relative to SEQ ID NO: 459 at position K1150, wherein K is substituted by any one of K, Q, R, H, S, E; (5) an amino acid substitution relative to SEQ ID NO: 459 at position E1151, wherein E is substituted by any one of E, N, A, S, K, T, D; and (6) an amino acid substitution relative to SEQ ID NO: 459 substituted amino acid at position E1152, wherein E is substituted by any one of E, T, V, R, K, N; (7) substituted amino acid at position L1153 relative to SEQ ID NO: 459, wherein L is substituted by any one of S, V, T, A; (8) substituted amino acid at position D1154 relative to SEQ ID NO: 459, wherein D is substituted by any one of T, L, E, I, V; (9) substituted amino acid at position K1155 relative to SEQ ID NO: 459, wherein K is substituted by any one of H, E, S, T, Q; (10) substituted amino acid at position Y1156 relative to SEQ ID NO: 459, wherein Y is substituted by any one of L, E, I, T, A, S, R; (11) substituted amino acid at position F1157 relative to SEQ ID NO: 459, wherein F is substituted by any one of T, V, I, A, S, M; (12) substituted amino acid at position F1157 relative to SEQ ID NO: 459. (13) The amino acid at position K1158 of SEQ ID NO: 459 is substituted, wherein K is substituted by any one of K, E, N, R, T, A, Q, I; (14) The amino acid at position H1160 of SEQ ID NO: 459 is substituted, wherein H is substituted by any one of L, M, A, E, T, Y, I, S; (15) The amino acid at position T1161 of SEQ ID NO: 459 is substituted, wherein T is substituted by any one of L, I; (16) The amino acid at position S1162 of SEQ ID NO: 459 is substituted, wherein S is substituted by any one of S, R, K, N, E, Q; (17) The amino acid at position P1163 of SEQ ID NO: 459 is substituted.Wherein P is substituted by any of E, S, T, R, (18) the amino acid at position D1164 of SEQ ID NO:459 is substituted, wherein D is substituted by any of T, M, A, (19) the amino acid at position V1165 of SEQ ID NO:459 is substituted, wherein V is substituted by any of A, L, and / or (20) any combination of (1)-(19).

[0026] In some embodiments, the segment comprises a polypeptide sequence listed in Table 9D, or a polypeptide sequence having one to five amino acid substitutions.

[0027] In some embodiments, the recombinant polypeptide comprises an engineered extracellular domain of the Nipah fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 499 between about residues 460 and about residues 490, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 499, the residues creating hydrophobic contacts between segments in the α-helical homotrimer. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 499 between about residues 460 and about residues 490, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 16 to about 33 residues.

[0028] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 499 at position M463, wherein M is substituted by any one of I, A, T, L, M; (2) an amino acid substitution relative to SEQ ID NO: 499 at position N464, wherein N is substituted by N; (3) an amino acid substitution relative to SEQ ID NO: 499 at position Q465, wherein Q is substituted by any one of E, L, K, T, S, I, D; (4) an amino acid substitution relative to SEQ ID NO: 499 at position S466, wherein S is substituted by S; (5) an amino acid substitution relative to SEQ ID NO: 499 at position L467, wherein L is substituted by any one of M, L, I, V, T; (6) an amino acid substitution relative to SEQ ID NO: 499 at position Q468, wherein Q is substituted by any one of E, K, A, T, S, D, R, I, Q; and (7) an amino acid substitution relative to SEQ ID NO: 499 substituted with an amino acid at position Q469, wherein Q is substituted by any one of R, S, K, T, E, Q; (8) substituted with an amino acid at position S470 relative to SEQ ID NO: 499, wherein S is substituted by any one of T, L, I, V, A; (9) substituted with an amino acid at position K471 relative to SEQ ID NO: 499, wherein K is substituted by any one of K, A, W, E, L, I; (10) substituted with an amino acid at position D472 relative to SEQ ID NO: 499, wherein D is substituted by any one of K, T, R, Q, E; (11) substituted with an amino acid at position Y473 relative to SEQ ID NO: 499, wherein Y is substituted by any one of W, D, K, Y, I, M, E, T; (12) substituted with an amino acid at position I474 relative to SEQ ID NO: 499, wherein I is substituted by any one of I, V, M, L, A; (13) substituted with an amino acid at position I474 relative to SEQ ID NO: 499; (14) The amino acid at position K475 of SEQ ID NO: 499 is substituted, wherein K is substituted by any one of T, K, M, R, E, L, A, S; (15) The amino acid at position E476 of SEQ ID NO: 499 is substituted, wherein E is substituted by any one of K, S, A, E, T, D; (16) The amino acid at position A477 of SEQ ID NO: 499 is substituted, wherein A is substituted by any one of L, I, V, FT, A, M, W, K, Y; (17) The amino acid at position Q478 of SEQ ID NO: 499 is substituted, wherein Q is substituted by any one of I, K, A, L, E, D, S, Y; (18) The amino acid at position R479 of SEQ ID NO: 499 is substituted, wherein R is substituted by any one of A, S, K, R, T, L, E.(18) An amino acid substituted relative to SEQ ID NO: 499 at position L480, wherein L is substituted by any one of K, E, R, Y, T, Q; (19) An amino acid substituted relative to SEQ ID NO: 499 at position L481, wherein L is substituted by any one of W, I, V, L, E, S, Q, A, T; (20) An amino acid substituted relative to SEQ ID NO: 499 at position D482, wherein D is substituted by any one of K, Q, E, W, T, S, A; (21) An amino acid substituted relative to SEQ ID NO: 499 at position T483, wherein T is substituted by any one of S, T, K, R, Q; (22) An amino acid substituted relative to SEQ ID NO: 499 at position V484, wherein V is substituted by any one of R, E, I, S, Y, L, K, D; (23) An amino acid substituted relative to SEQ ID NO: 499 substituted with an amino acid at position N485, wherein N is substituted with any of I, R, K, W, E, T; (24) substituted with an amino acid at position P486 relative to SEQ ID NO: 499, wherein P is substituted with any of A, T, R, K, Q; (25) substituted with an amino acid at position S487 relative to SEQ ID NO: 499, wherein S is substituted with any of K, R, T, S; (26) substituted with an amino acid at position L488 relative to SEQ ID NO: 499, wherein L is substituted with any of E, V, L, K; (27) substituted with an amino acid at position I489 relative to SEQ ID NO: 499, wherein I is substituted with any of E, Q, L, K, R; and / or (28) any combination of (1)-(27).

[0029] In some embodiments, the segment comprises a polypeptide sequence listed in Table 10B, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment relative to SEQ ID NO: 499, between about residues 460 and about residues 490, comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 12 to about 26 residues.

[0030] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 499 at position M463, wherein M is substituted by any one of L, I, V; (2) an amino acid substitution relative to SEQ ID NO: 499 at position N464, wherein N is substituted by N; (3) an amino acid substitution relative to SEQ ID NO: 499 at position Q465, wherein Q is substituted by any one of Q, T, N, D, E, S, K, R, A; (4) an amino acid substitution relative to SEQ ID NO: 499 at position S466, wherein S is substituted by S; (5) an amino acid substitution relative to SEQ ID NO: 499 at position L467, wherein L is substituted by any one of I, V, L, A; (6) an amino acid substitution relative to SEQ ID NO: 499 at position Q468, wherein Q is substituted by any one of S, K, T, D, E, R, Q; and (7) an amino acid substitution relative to SEQ ID NO: 499 substituted with an amino acid at position Q469, wherein Q is substituted by any one of S, Q, N, E, A, D, K, T, R; (8) substituted with an amino acid at position S470 relative to SEQ ID NO: 499, wherein S is substituted by any one of L, A, I, V, N; (9) substituted with an amino acid at position K471 relative to SEQ ID NO: 499, wherein K is substituted by any one of E, Q, S, R, K, A, T, D, L, N; (10) substituted with an amino acid at position D472 relative to SEQ ID NO: 499, wherein D is substituted by any one of K, T, E, Q, D, N, S, A; (11) substituted with an amino acid at position Y473 relative to SEQ ID NO: 499, wherein Y is substituted by any one of A, S, R, T, V, E, I, K, L, D, Q; (12) substituted with an amino acid at position Y473 relative to SEQ ID NO: 499. (13) The amino acid at position I474 of SEQ ID NO: 499 is substituted, wherein I is substituted by any one of L, I, V; (14) The amino acid at position K475 of SEQ ID NO: 499 is substituted, wherein K is substituted by any one of K, H, D, T, A, S, R, Q, E, N; (15) The amino acid at position E476 of SEQ ID NO: 499 is substituted, wherein E is substituted by any one of K, R, S, E, A, T, H, D; (16) The amino acid at position A477 of SEQ ID NO: 499 is substituted, wherein A is substituted by any one of A, L, I, V; (17) The amino acid at position R479 of SEQ ID NO: 499 is substituted.(18) Relative to the amino acid at position L480 of SEQ ID NO: 499, wherein L is replaced by any one of D, L, E, K, T, R, V, I, Q; (19) Relative to the amino acid at position L481 of SEQ ID NO: 499, wherein L is replaced by any one of L, V, I; (20) Relative to the amino acid at position D482 of SEQ ID NO: 499, wherein D is replaced by any one of E, K, N, D, L, Q, H; (21) Relative to the amino acid at position T483 of SEQ ID NO: 499, wherein T is replaced by any one of E, K, S, Q, A, T; (22) Relative to the amino acid at position V484 of SEQ ID NO: 499, wherein V is replaced by any one of V, L, I; (23) Relative to SEQ ID NO: 499 substituted with an amino acid at position N485, wherein N is substituted with any one of R, K, L, V, E, Q, I; (24) substituted with an amino acid at position P486 relative to SEQ ID NO: 499, wherein P is substituted with any one of R, E, A, S, L; (25) substituted with an amino acid at position S487 relative to SEQ ID NO: 499, wherein S is substituted with any one of Q, R, T, S, L; (26) substituted with an amino acid at position L488 relative to SEQ ID NO: 499, wherein L is substituted with any one of L; and / or (27) any combination of (1)-(26).

[0031] In some embodiments, the segment comprises a polypeptide sequence listed in Table 10D, or a polypeptide sequence having one to five amino acid substitutions.

[0032] In some embodiments, the recombinant polypeptide comprises an engineered extracellular domain of a respiratory syncytial virus (RSV) fusion (F) protein, wherein the extracellular domain comprises (a) a C-terminal helical forming segment relative to SEQ ID NO: 1 between about residues 500 and about residues 530, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer; (b) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 140, 399, 400, 485, 486, 487, 488, 489, 494, or 498; (c) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 56, 58, 154, 187, 296, or 298; (d) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 75, 216, 218, or 219; and (e) relative to SEQ ID NO: 1. (a) Substitution of one, two, three or more amino acids at positions 92, 232, 235, 238, 249, 250 or 254; (f) Substitution of one, two, three or more amino acids at positions 67, 137 or 339 relative to SEQ ID NO: 1; (g) Substitution of the furin cleavage site at about residue 100 to about residue 140 relative to SEQ ID NO: 1 with a non-cleavable linker; or (h) Any combination of (a)-(g).

[0033] In some embodiments, the extracellular domain includes a C-terminal helical forming segment relative to SEQ ID NO: 1, between about residues 500 and about residues 530, comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 10 to about 30 residues.

[0034] In some embodiments, the segment comprises substitutions of two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 1, which create hydrophobic contacts between segments in the α-helical isotrimester.

[0035] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 1 at position F505, wherein F is substituted with A, I, L, M, V, G, T; (2) an amino acid substitution relative to SEQ ID NO: 1 at position I506, wherein I is substituted with any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y or A, I, L, V; (3) an amino acid substitution relative to SEQ ID NO: 1 at position R507, wherein R is substituted with any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y or A, I, L, V; (4) an amino acid substitution relative to SEQ ID NO: 1 at position K508, wherein R is substituted with K, Q, R, preferably A, V, T, I; (5) relative to SEQ ID NO: 1. An amino acid substituted at position S509, wherein S is substituted by A, I, L, M, V, F, W, Y, G, T, preferably A, I, L, M, V; (6) An amino acid substituted at position D510 relative to SEQ ID NO: 1, wherein D is substituted by any amino acid, preferably D, E, K, N, Q, R, S, T, Y; (7) An amino acid substituted at position E511 relative to SEQ ID NO: 1, wherein E is substituted by any amino acid; (8) An amino acid substituted at position L512 relative to SEQ ID NO: 1, wherein L is substituted by D, E, K, N, Q, R, S, T, Y, preferably A, I, L, M, V, F, W, Y, G, T; (9) An amino acid substituted at position L512 relative to SEQ ID NO: 1, wherein L is substituted by D, E, K, N, Q, R, S, T, Y, preferably A, I, L, M, V, F, W, Y, G, T; 1. An amino acid substituted at position L513, wherein L is replaced by any amino acid, preferably A, I, L, M, V, F, W, Y, G, more preferably D, E, K, N, Q, R, S, T, Y; (10) An amino acid substituted at position H514 relative to SEQ ID NO: 1, wherein H is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; (11) An amino acid substituted at position N515 relative to SEQ ID NO: 1, wherein N is replaced by any amino acid other than P, preferably A, I, L, M, V, F, W, Y, G; (12) An amino acid substituted at position V516 relative to SEQ ID NO: 1, wherein V is replaced by A, I, L, M, V, F, W, Y, G or T, S, K; (13) An amino acid substituted at position V516 relative to SEQ ID NO: 1. An amino acid substitution at position N517, wherein N is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; (14) An amino acid substitution at position T518 relative to SEQ ID NO: 1, wherein T is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y.(15) An amino acid substitution relative to SEQ ID NO: 1 at position G519, wherein G is substituted with any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; and / or (16) any combination of (1)-(15).

[0036] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 1 at position L503, wherein F is substituted with Q, V, K, R, N, L; (2) an amino acid substitution relative to SEQ ID NO: 1 at position A504, wherein I is substituted with any amino acid other than P, preferably S, T, L, A, Q, K, E, Y; (3) an amino acid substitution relative to SEQ ID NO: 1 at position F505, wherein F is substituted with I, V, N, T, L; (4) an amino acid substitution relative to SEQ ID NO: 1 at position I506, wherein I is substituted with any amino acid other than P, preferably Q, N, K, R, V, S; (5) an amino acid substitution relative to SEQ ID NO: 1 at position R507, wherein R is substituted with any amino acid other than P, preferably A, N, K, E, D, Q; and (6) an amino acid substitution relative to SEQ ID NO: 1. The amino acid at position K508 is substituted, wherein R is substituted by T, M, V, R; (7) The amino acid at position S509 relative to SEQ ID NO: 1 is substituted, wherein S is substituted by T, I, K, Q, M, E, V, S; (8) The amino acid at position D510 relative to SEQ ID NO: 1 is substituted, wherein D is substituted by S, K, N, D, E; (9) The amino acid at position E511 relative to SEQ ID NO: 1 is substituted, wherein E is substituted by R, S, E, K, A, T, L; (10) The amino acid at position L512 relative to SEQ ID NO: 1 is substituted, wherein L is substituted by V, N, T, L; (11) The amino acid at position L513 relative to SEQ ID NO: 1 is substituted, wherein L is substituted by D, T, H, K, E, N, R; (12) The amino acid at position L513 relative to SEQ ID NO: 1 is substituted, wherein L is substituted by D, T, H, K, E, N, R; (13) The amino acid at position L513 relative to SEQ ID NO: 1 is substituted, wherein L is substituted by D, T, H, K, E, N, R; (14) The amino acid at position L512 relative to SEQ ID NO: 1 is substituted, wherein R is substituted by T, M, V, R; (15) The amino acid at position S509 relative to SEQ ID NO: 1 is substituted, wherein S is substituted by T, I, K, Q, M, E, V, R; (15) The amino acid at position D509 relative to SEQ ID NO: 1 is substituted, wherein S is substituted by T, I, K, Q, M, E, V, R; (15) The amino acid at position L512 relative to SEQ ID NO: 1 is substituted, wherein R is substituted by T, 1. The amino acid at position H514 is substituted, wherein H is substituted by A, N, E, S, V, K, T, D; (13) The amino acid at position N515 relative to SEQ ID NO: 1 is substituted, wherein N is substituted by I, E, L, T, Q; (14) The amino acid at position V516 relative to SEQ ID NO: 1 is substituted, wherein V is substituted by E, I, K, N, R, Q; (15) The amino acid at position N517 relative to SEQ ID NO: 1 is substituted, wherein N is substituted by A, S, K, E, R; (16) The amino acid at position T518 relative to SEQ ID NO: 1 is substituted, wherein T is substituted by K, S, Q, R, D, E; (17) The amino acid at position G519 relative to SEQ ID NO: 1 is substituted, wherein G is substituted by V, L, I; (18) The amino acid at position I520 relative to SEQ ID NO: 1 is substituted, wherein G is substituted by K, Q, E, N, T.(19) Amino acid substitutions relative to SEQ ID NO: 1 at position P521, wherein G is substituted by H, D, E, K, R, N, Q; (20) Amino acid substitutions relative to SEQ ID NO: 1 at position E522, wherein G is substituted by L, R, I, V; (21) Amino acid substitutions relative to SEQ ID NO: 1 at position A523, wherein G is substituted by E, V, L, K, RI; (22) Amino acid substitutions relative to SEQ ID NO: 1 at position P524, wherein G is substituted by A, K, T, E, R; (23) Amino acid substitutions relative to SEQ ID NO: 1 at position R525, wherein G is substituted by H, R, S, L, N, E, D; (24) Amino acid substitutions relative to SEQ ID NO: 1 at position D526, wherein G is substituted by I, L, V, R; (25) Amino acid substitutions relative to SEQ ID NO: (26) Amino acid substitution at position G527, wherein G is substituted by E, K, Q, or D; (27) Amino acid substitution at position Q528 relative to SEQ ID NO: 1, wherein G is substituted by D, K, S, R, or A; (28) Amino acid substitution at position A529 relative to SEQ ID NO: 1, wherein G is substituted by T or L; (29) Amino acid substitution at position Y530 relative to SEQ ID NO: 1, wherein G is substituted by L, E, or T; (20) Amino acid substitution at position V531 relative to SEQ ID NO: 1, wherein G is substituted by A, R, or K; (21) Amino acid substitution at position R532 relative to SEQ ID NO: 1, wherein G is substituted by V or A; and / or (32) Any combination of (1)-(30).

[0037] In some embodiments, the segment comprises a polypeptide sequence listed in Table 2B or Table 2C, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), NQSALWLEAAKYVKQAREKS (SEQ ID NO: 11), NQSAKNAEAAKIAEETKRKD (SEQ ID NO: 12), or NQSRETAKAVSAVK (SEQ ID NO: 75), or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10).

[0038] In some embodiments, the extracellular domain comprises (b) one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 140, 399, 400, 485, 486, 487, 488, 489, 494 or 498. In some embodiments, the extracellular domain comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 1: E487R + K498A, E487R + K498E, E487K + K498E, D486A + E487R + K498A, D486Q + E487R + K498A, D486E + E487A + D489A + T400D, D486A + E487M + K498A, E487Q, D486S, F488W + D489A + T400D + E487R + K498A, F140W + D489A + T400D + E487R + K498A, Q494I + S485I + K399A + 487R + 498A, Q494M + S485I + K399A; D486A + 487M + 498A, Q494L + S485A + K399V + D486A + 487M + 498A, Q494M + S485A + K399V + D486A + 487M + 498A, Q494A + S485F + K399V + D486A + 487M + 498Y, D489A + T400D + E487R + K498A, or D489A + T400D. In some embodiments, the extracellular domain contains amino acid substitutions for D489A, T400D, E487R, and K498A. In some embodiments, the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and D486A. In some embodiments, the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and T249P.

[0039] In some embodiments, the polypeptide includes a heteropolymerization domain at the C-terminus of its extracellular domain. In some embodiments, the polymerization domain is a trimerization domain. In some embodiments, the polymerization domain includes a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64). In some embodiments, the extracellular domain includes amino acid substitutions for S155C, S290C, S190F, and V207L.

[0040] In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 6, SEQ ID NO: 6 optionally lacking the p27 peptide shown in bold, and wherein “X” refers to a site involving the added C-terminal helical segment and can be any amino acid: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 7, wherein SEQ ID NO: 7 optionally lacks the p27 peptide shown in bold, wherein “X” refers to a site involving the added C-terminal helical segment and may be any amino acid: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 8, wherein SEQ ID NO: 8 optionally lacks the p27 peptide shown in bold: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 9, wherein SEQ ID NO: 9 optionally lacks the p27 peptide shown in bold: In some embodiments, the polypeptide comprises a sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 1-9.

[0041] In another aspect, this disclosure provides a trimeric protein complex comprising the polypeptide disclosed herein. In some embodiments, the thermal stability, as determined by nanoDSF, is increased by at least 10°C, at least 15°C, at least 20°C, about 10°C to about 30°C, about 10°C to about 20°C, or about 20°C to about 30°C, compared to a trimeric protein complex lacking modification (a)-(h). In some embodiments, the stability, as determined by storage at about 40°C, is increased compared to a trimeric protein complex lacking modification (a)-(h). In some embodiments, the thermal stability is increased compared to a reference RSV F protein comprising amino acid substitutions consisting substantially of S155C, S290C, S190F, and V207L ​​(DS-Cav1). In some embodiments, the thermal stability, as measured by nanoDSF, is increased by at least 10°C, at least 15°C, at least 20°C, about 10°C to about 30°C, about 10°C to about 20°C, or about 20°C to about 30°C compared to the trimeric protein complex lacking modification (a)-(g).

[0042] In some implementations, increased stability was observed by storage at approximately 40°C compared to the trimer protein complex lacking modification (a)-(g).

[0043] In another aspect, this disclosure provides a protein nanostructure comprising a trimeric component, said trimeric component comprising the polypeptide disclosed herein. In some embodiments, the nanostructure is a two-component nanostructure comprising a first trimeric component and a second pentameric component. In some embodiments, the first trimeric component comprises engineered extracellular domains of a respiratory syncytial virus (RSV) fusion (F) polypeptide and an I53-50A polypeptide. In some embodiments, the first trimeric component comprises a fusion protein comprising, in N-terminus to C-terminus, an RSV fusion (F) polypeptide, an amino acid linker, and an I53-50A polypeptide. In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 1 and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and a polymerized domain, the polymerized domain comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or a second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71 have polypeptide sequences with at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0044] In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, D489A, T400D, E487R, and K498A relative to SEQ ID NO: 1 and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and a polymerized domain comprising I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) A polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71 having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71.

[0045] In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and T249P relative to SEQ ID NO: 1 and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and a polymerized domain comprising a polymerized region with or without I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) A polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71 having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71.

[0046] In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and D486A relative to SEQ ID NO: 1 and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and a polymerized domain comprising a polymerized region with or without I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) A polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71 having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71.

[0047] In some embodiments, the trimer component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the sequences listed in Table 19. In some embodiments, the pentamer component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 20, 44, 45, 52, 71, 73, 74.

[0048] In another aspect, this disclosure provides a pharmaceutical composition comprising the peptides, protein complexes, or nanostructures disclosed herein. In another aspect, this disclosure provides a vaccine comprising the peptides, protein complexes, or nanostructures disclosed herein. In another aspect, this disclosure provides a trimeric protein complex comprising the peptides disclosed herein. In another aspect, this disclosure provides a protein nanostructure comprising a trimeric component comprising the peptides disclosed herein. In some embodiments, the nanostructure is a two-component nanostructure comprising a first trimeric component and a second pentameric component. In some embodiments, the first trimeric component comprises engineered extracellular domains of a respiratory syncytial virus (RSV) fusion (F) peptide and an I53-50A peptide. In some embodiments, the first trimeric component comprises engineered extracellular domains of a human metapneumovirus (hMPV) fusion (F) peptide and an I53-50A peptide. In some embodiments, the first trimeric component comprises engineered extracellular domains of an hMPV / A fusion (F) peptide and an I53-50A peptide. In some embodiments, the first trimer component comprises engineered extracellular domains of an hMPV / B fusion (F) peptide and an I53-50A peptide. In some embodiments, the first trimer component comprises engineered extracellular domains of a human parainfluenza virus type 3 (PIV3) fusion (F) peptide and an I53-50A peptide. In some embodiments, the first trimer component comprises engineered extracellular domains of a human parainfluenza virus type 5 (PIV3) fusion (F) peptide and an I53-50A peptide. In some embodiments, the first trimer component comprises engineered extracellular domains of a SARS-CoV-2 spike (S) peptide and an I53-50A peptide. In some embodiments, the first trimer component comprises engineered extracellular domains of a Nipah virus fusion (F) peptide and an I53-50A peptide. In some embodiments, the first trimer component comprises a fusion protein comprising, in N-terminus to C-terminus, an engineered fusion (F) peptide, an amino acid linker, and an I53-50A peptide. In some embodiments, the first trimer component comprises a fusion protein comprising, in N-terminal to C-terminal order, an engineered spike (S) polypeptide, an amino acid linker, and an I53-50A polypeptide. In some embodiments, the trimer component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the sequences listed in Table 19, or without the underlined and / or bold / italic polypeptide sequence.In some embodiments, the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 20, 44, 45, 52, 71, 73, 74.

[0049] In another aspect, this disclosure provides a method for vaccinating a subject, the method comprising administering to the subject the composition disclosed herein. In another aspect, this disclosure provides a method for generating an immune response in a subject, the method comprising administering to the subject the composition disclosed herein. In another aspect, this disclosure provides a method for treating or preventing a viral infection in a subject, the method comprising administering to the subject the composition disclosed herein. In another aspect, this disclosure provides compositions for vaccination, generating an immune response, or treating or preventing any viral infection disease disclosed herein. In another aspect, this disclosure provides compositions, methods, or uses as described herein. In another aspect, this disclosure provides a method for preparing a composition, the method comprising culturing host cells modified to express one or more polypeptides as described herein.

[0050] In another aspect, this disclosure provides a recombinant polypeptide for displaying molecules (such as antigens), the recombinant polypeptide comprising an α-helical segment and a polymerizing domain, wherein the α-helical segment comprises one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the α-helical segment has improved hydrophobic stacking. In some embodiments, the α-helical segment comprises about 7 to about 31 residues. In some embodiments, the amino acid substitutions comprise polar, charged, and / or hydrophobic amino acids.

[0051] In some embodiments, the α-helical segment comprises a polypeptide sequence according to any of the following: LXXTIXXLLXIXXXLXXXL (SEQ ID NO: 566), LVXTXKXLXDLIXXLXXLLXKLXX (SEQ ID NO: 567), LNKVKKXVXXLXXXVXXLEKXLX (SEQ ID NO: 568), EKIXXAIKKAXKL (SEQ ID NO: 569), EXIXKAIKXLXXXXX (SEQ ID NO: 570), XKXXEXXXXVXXXXXXXXX (SEQ ID NO: 571), XXLKKAAXIXKKXLKXX (SEQ ID NO: 572).

[0052] In some implementations, the α-helical segment comprises a polypeptide sequence according to any of the common sequences in Table 24.

[0053] In some embodiments, the α-helical segment comprises a polypeptide sequence according to the following: a) L X2X2T I X2X2L LX2I [V / I] X2X2L [I / L] X2X2L (SEQ ID NO: 573), b) LV [A / T] T X2K X2L X2D L IX2X2L [K / E] X2L L X2K L X2X2 (SEQ ID NO: 574), or c) LNKVKK X2V X2X2L X2X2X2VX2X2L EK X2L X2 (SEQ ID NO: 575), wherein X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

[0054] In some embodiments, the α-helical segment comprises a polypeptide sequence according to the following: a) EKI X2X2A IK KA X2K L (SEQ ID NO: 576), b) E X2I X2K AIK X2L [L / X2] X2X2[X1 / X2] X2 (SEQ ID NO: 577), and c) X2K [X1 / T] [L / E]E [T / A] X1X2[I / X2] V X2X2[X1 / X2] [X1 / X2] X2X2X1X2X2 (SEQ ID NO: 578), or d) X2X2L KKAA X2I X1K K X1L K X2X2 (SEQ ID NO: 576). 579), wherein X1 is a nonpolar residue selected from A, I, L and M, and X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

[0055] In some embodiments, the α-helix segment comprises a polypeptide sequence listed in Table 25A or Table 25B, or a polypeptide sequence thereof having 1 to 5 amino acid substitutions. In some embodiments, the α-helix segment comprises a polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), NQSALWLEAAKYVKQAREKS (SEQ ID NO: 11), NQSAKNAEAAKIAEETKRKD (SEQ ID NO: 12), or NQSRETAKAVSAVK (SEQ ID NO: 75), or a polypeptide sequence thereof having 1 to 5 amino acid substitutions.

[0056] In some embodiments, the polymerizing domain is I53-50A or a variant thereof. In some embodiments, the polymerizing domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64). In some embodiments, the polypeptide comprises an α-helix segment fused to the N-terminus of the polymerizing domain via a peptide bond or polypeptide linker. In some embodiments, the polypeptide comprises an antigenic polypeptide at the N-terminus of the α-helix segment.

[0057] In another aspect, this disclosure provides a polypeptide comprising an α-helical segment comprising a polypeptide sequence listed in Table 25A or Table 25B, or a polypeptide sequence having 1 to 5 amino acid substitutions. In another aspect, this disclosure provides a protein nanostructure comprising a trimeric component comprising the polypeptide described herein. In some embodiments, the nanostructure is a bicomponent nanostructure comprising a first trimeric component and a second pentameric component. In some embodiments, the nanostructure is a bicomponent nanostructure comprising a second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71.

[0058] In another aspect, this disclosure provides a pharmaceutical composition comprising the polypeptide or nanostructure described herein. In another aspect, this disclosure provides a vaccine comprising the polypeptide or nanostructure described herein. In another aspect, this disclosure provides a method of vaccinating a subject, the method comprising administering the composition described herein to the subject. In another aspect, this disclosure provides a method of inducing an immune response in a subject or treating or preventing a viral infection, the method comprising administering the polypeptide or nanostructure described herein to the subject.

[0059] In another aspect, this disclosure provides a method for preparing the polypeptides or nanostructures described herein, the method comprising culturing host cells modified to express one or more polypeptides as described herein.

[0060] In another aspect, this disclosure provides a recombinant polypeptide comprising an engineered extracellular domain of a respiratory syncytial virus (RSV) fusion (F) protein, wherein the extracellular domain comprises: (1) one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 140, 399, 400, 485, 486, 487, 488, 489, 494 or 498; (2) one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 56, 58, 154, 187, 296 or 298; (3) one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 75, 216, 218 or 219; (4) one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 92, 232, 235, 238, 249, 250 or 254; and (5) substitutions relative to SEQ ID NO: 1. One, two, three or more amino acids are substituted at position 67, 137 or 339; (6) the substitution of the furin cleavage site at about residue 100 to about residue 140 relative to SEQ ID NO: 1 is replaced by a non-cleavable linker; or (7) any combination of (1)-(6).

[0061] In some embodiments, the extracellular domain comprises one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 140, 399, 400, 485, 486, 487, 488, 489, 494 or 498.

[0062] In some embodiments, the extracellular domain comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 1: E487R + K498A, E487R + K498E, E487K + K498E, D486A + E487R + K498A, D486Q + E487R + K498A, D486E + E487A + D489A + T400D, D486A + E487M + K498A, E487Q, D486S, F488W + D489A + T400D + E487R + K498A, F140W + D489A + T400D + E487R + K498A, Q494I + S485I + K399A + 487R + 498A, Q494M + S485I + K399A; D486A + 487M + 498A, Q494L + S485A + K399V + D486A + 487M + 498A, Q494M + S485A + K399V + D486A + 487M + 498A, Q494A + S485F + K399V + D486A + 487M + 498Y, D489A + T400D + E487R + K498A or D489A + T400D.

[0063] In some embodiments, the extracellular domain comprises amino acid substitutions of D489A, T400D, E487R, and K498A. In some embodiments, the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and D486A. In some embodiments, the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and T249P.

[0064] In some embodiments, the polypeptide comprises a heteropolymerization domain. In some embodiments, the polymerization domain is a trimerization domain. In some embodiments, the polymerization domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64). In some embodiments, the extracellular domain comprises amino acid substitutions S155C, S290C, S190F, and V207L.

[0065] In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 6, SEQ ID NO: 6 optionally lacking the p27 peptide shown in bold, and wherein “X” refers to a site involving the added C-terminal helical segment and can be any amino acid: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 7, wherein SEQ ID NO: 7 optionally lacks the p27 peptide shown in bold, wherein “X” refers to a site involving the added C-terminal helical segment and may be any amino acid: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 8, wherein SEQ ID NO: 8 optionally lacks the p27 peptide shown in bold: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 9, wherein SEQ ID NO: 9 optionally lacks the p27 peptide shown in bold: In some embodiments, the polypeptide comprises a sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 1-9.

[0066] In another aspect, this disclosure provides a trimeric protein complex comprising the polypeptide shown herein.

[0067] In some embodiments, the thermal stability, as determined by nanoDSF, is increased by at least 10°C, at least 15°C, at least 20°C, about 10°C to about 30°C, about 10°C to about 20°C, or about 20°C to about 30°C, compared to the trimeric protein complex without modification (1)-(7).

[0068] In some embodiments, stability is increased by storage at approximately 40°C compared to the trimer protein complex lacking modifications (1)-(7). In some embodiments, thermal stability is increased compared to a reference RSV F protein comprising amino acid substitutions consisting essentially of S155C, S290C, S190F, and V207L ​​(DS-Cav1).

[0069] In another aspect, this disclosure provides a protein nanostructure comprising a trimeric component, said trimeric component comprising the polypeptide described herein. In some embodiments, the nanostructure is a two-component nanostructure comprising a first trimeric component and a second pentameric component. In some embodiments, the first trimeric component comprises engineered extracellular domains of a respiratory syncytial virus (RSV) fusion (F) polypeptide and an I53-50A polypeptide. In some embodiments, the first trimeric component comprises a fusion protein comprising, in N-terminus to C-terminus, an RSV fusion (F) polypeptide, an amino acid linker, and an I53-50A polypeptide.

[0070] In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 1; and a polymerized domain, the polymerized domain comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or a second pentameric component, wherein the pentameric component comprises an amino acid substitution at positions S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 1; and / or a second pentameric component, wherein the pentameric component comprises an amino acid substitution at positions S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 1; and / or a second pentameric component, wherein the second ... 20 or 71 have polypeptide sequences with at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0071] In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, D489A, T400D, E487R, and K498A relative to SEQ ID NO: 1; and a polymerized domain, the polymerized domain comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or a second pentameric component, wherein the pentameric component comprises an amino acid substitution at positions S155C, S290C, S190F, V207L, D489A, T400D, E487R, and K498A relative to SEQ ID NO: 1; and / or a second pentameric component, wherein the pentameric component comprises an amino acid substitution at positions S155C, S290C, S190F, V207L, D489A, T400D, E487R, and K498A relative to SEQ ID NO: 1; and / or a second pentameric component, wherein the second ... 20 or 71 have polypeptide sequences with at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0072] In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and T249P relative to SEQ ID NO: 1; and a polymerized domain, the polymerized domain comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or a second pentameric component, wherein the pentameric component comprises an amino acid substitution at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and T249P relative to SEQ ID NO: 1; ... second pentameric component comprises an amino acid substitution at positions S155C, S290C, S190F, V207L, F488W, D489A, T489A 20 or 71 have polypeptide sequences with at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0073] In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and D486A relative to SEQ ID NO: 1; and a polymerized domain, the polymerized domain comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or a second pentameric component, wherein the pentameric component comprises an amino acid substitution at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and D486A relative to SEQ ID NO: 1; ... second pentameric component comprises an amino acid substitution at positions S155C, S290C, S190F, V207L, F488W, D489A, T489A 20 or 71 have polypeptide sequences with at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0074] In some embodiments, the trimer component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the sequences listed in Table 19 or without the underlined and / or bold / italic polypeptide sequence.

[0075] In some embodiments, the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 20, 44, 45, 52, 71, 73, 74.

[0076] In another aspect, this disclosure provides a pharmaceutical composition comprising the polypeptides, protein complexes, or nanostructures described herein. In another aspect, this disclosure provides a vaccine composition comprising the polypeptides, protein complexes, or nanostructures described herein. In another aspect, this disclosure provides a method of vaccinating a subject, the method comprising administering the composition described herein to the subject. In another aspect, this disclosure provides a method of generating an immune response in a subject, the method comprising administering the composition described herein to the subject. In another aspect, this disclosure provides a method of treating or preventing RSV disease in a subject, the method comprising administering the composition described herein to the subject. In another aspect, this disclosure provides a composition described herein for use in vaccination, generating an immune response, or treating or preventing RSV disease. In another aspect, this disclosure provides a method of preparing a composition described herein, the method comprising culturing host cells modified to express one or more polypeptides as described herein. In another aspect, this disclosure provides compositions, methods, or uses as described herein.

[0077] Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein. Other aspects, embodiments, and advantages of the invention will become apparent from the following detailed description. Attached Figure Description

[0078] These and other features, aspects, and advantages of the invention will be better understood with reference to the following description and accompanying drawings, in which: Figure 1 A structural model of the RSV F protein (PDB 4MMU) in its pre-fusion conformation is shown, with stabilizing elements separating it into five distinct spaces. Spaces 1-4 are targeted by stabilizing mutations. Space 5 refers to the C-terminus of the protein.

[0079] Figure 2 This image shows close-up views of the C-terminal structure of the RSV F protein, determined by X-ray crystallography of pre-fusion RSV F (PDB 4MMU) before and after remodeling. The remodeled residues (residues 503-509) are outlined with coarser black highlights (left), and additional structures added by remodeling are shown in black (right).

[0080] Figure 3 The ddG score is displayed, with representative designs highlighted.

[0081] Figure 4 The hydrophobicity score of the design is displayed. Mean (solid line), standard deviation (dashed line), and WT (dotted line).

[0082] Figure 5 Representative electron micrographs of protein nanostructures as described in this article are shown.

[0083] Figure 6 A shows the structural models of the PIV5 F protein before (left) and after (right) C-terminal remodeling. Predicted omitted or unstructured regions (left, not shown) are represented by α-helical structures (right, dark black).

[0084] Figure 6 B shows the structural models of the PIV3 F protein before (left) and after (right) C-terminal remodeling.

[0085] Figure 6 C shows the structural models of the Nipah F protein before (left) and after (right) C-terminal remodeling.

[0086] Figure 6 D shows the structural models of the hMPV F protein before (left) and after (right) C-terminal remodeling.

[0087] Figure 6 E shows the structural models of the SARS-CoV-2 S protein before (left) and after (right) C-terminal remodeling.

[0088] Figure 7 The predicted ddG values ​​for the Paramyxoviridae family vary with remodeling length. Top left: PIV5, bottom: PIV3, top right: Nipah. Dotted lines represent averages, and solid lines represent WT sequences. Note that WT sequences only include structured residues present in the PDB.

[0089] Figure 8 This shows a representative remodeling design using RF diffusion from HMPV. The origin region is black, and the background from the input PDB is white.

[0090] Figure 9 The predicted ddG values ​​for the Pneumoviridae and Coronaviridae families vary with remodeling length. Top: HMPV, Bottom: SARS-CoV-2. Dotted lines represent mean values, and solid lines represent WT sequences. Note that WT sequences only include structured residues present in the PDB.

[0091] Figure 10 The predicted hydrophobicity of the Paramyxoviridae family varies with the position of the remodeled sequence. Top left: PIV5, bottom: PIV3, top right: Nipah. Dotted lines represent averages, and solid lines represent WT sequences. Note that WT sequences only include structured residues present in the PDB.

[0092] Figure 11The predicted hydrophobicity of the Pneumoviridae and Coronaviridae families varies with the position of the remodeled sequence. Top: HMPV, Bottom: SARS-CoV-2. Dotted lines represent mean values, and solid lines represent WT sequences. It should be noted that WT sequences only include structured residues present in the PDB.

[0093] Figure 12 Principal component analysis of distances in the first group (parallel) remodeled sequences is shown.

[0094] Figure 13 Principal component analysis of distances in the second group (non-parallel) remodeled sequences is shown.

[0095] Figure 14 A-14C shows the location-specific probabilities for Group 1 (parallel). The probability represents the likelihood of remodeling the length. Figure 14 A shows the location-specific probability of cluster _p2. Figure 14 B shows the location-specific probability of cluster _p1. Figure 14 C represents the location-specific probability of cluster _p0.

[0096] Figure 15 A-15D shows the location-specific probabilities for group 2 (non-parallel). The probability represents the likelihood of remodeling the length. Figure 15 A shows the location-specific probability of cluster _o0. Figure 15 B shows the location-specific probability of cluster _o1. Figure 15 C represents the location-specific probability of cluster _o3. Figure 15 D represents the location-specific probability of cluster _o2.

[0097] Figure 16A-16G The location weights for each cluster are displayed. Figure 16A The position weights of cluster _p0 are displayed. Figure 16B The position weights of cluster _p1 are displayed. Figure 16C The position weights of cluster _p2 are displayed. Figure 16D The position weights of cluster _o0 are displayed. Figure 16E The position weights of cluster _o1 are displayed. Figure 16F The position weights of cluster _o2 are displayed. Figure 16G The position weights of cluster _o3 are displayed.

[0098] Figure 17 The neutralizing titers against RSV / B (strain B18537) induced by various nanostructured immunogens based on RSV / B antigens were demonstrated.

[0099] Figure 18The neutralizing titers against RSV / A (Tracy strain) induced by various nanostructure immunogens based on RSV / A antigens were demonstrated.

[0100] Figure 19 The cryo-EM structures of the RSV F extracellular domain of DS-Cav1 (PDB 7LUE) fused with a foldon (A) are shown for comparison. The C-terminal α-helical segment added in RSV / A.023 is shown in dark gray and surrounded by a dashed box. Antibody structures were removed from the PDB 7LUE model before image generation.

[0101] Figure 20 The cryo-EM structures of the RSV F extracellular domain of RSV / A.023 and DS-Cav1 (PDB 7LUE) fused with the fold are shown as a structural comparison of the C-terminal regions. The C-terminal α-helix segment added in RSV / A.023 is shown in dark gray and surrounded by a dashed box. Antibody structures were removed from the PDB 7LUE model before image generation.

[0102] Figure 21 The maximum binding of PIV3 F to monoclonal antibody 3×1, remodeled by biolayer interferometry, is shown (top) and the maximum binding normalized to the maximum binding of antibody 16A8 specific to anticomponent A.

[0103] Figure 22 The maximum binding of PIV3 F to monoclonal antibody PIA174, remodeled by biolayer interferometry, is shown (top) and the maximum binding normalized to the maximum binding of antibody 16A8 specific to anticomponent A.

[0104] Figure 23 The maximum binding to monoclonal antibody 16A8 was demonstrated via biolayer interference.

[0105] Figure 24 The maximum binding of PIV3 F with a universal C-terminal remodeling sequence to monoclonal antibody 16A8 was demonstrated by biolayer interferometry.

[0106] Figure 25 The maximum binding of PIV3 F with a universal C-terminal remodeling sequence to monoclonal antibody 3×1 via biolayer interferometry is shown (top), as well as the normalized maximum binding of the maximum binding to anti-component A specific antibody 16A8.

[0107] Figure 26 The maximum binding of PIV3 F with a universal C-terminal remodeling sequence to monoclonal antibody PIA174 via biolayer interferometry is shown (top), as well as the normalized maximum binding to anticomponent A specific antibody 16A8. Detailed Implementation

[0108] Before describing embodiments of this disclosure, it should be understood that such embodiments are provided by way of example only, and various alternatives to the embodiments of this disclosure described herein may be employed in the practice of this invention. Many variations, modifications, and substitutions will occur to those skilled in the art, and can be practiced without departing from the spirit of the invention.

[0109] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Various scientific dictionaries including those containing the terms included herein are well-known and available to those skilled in the art. While any methods and materials similar to or equivalent to those described herein may be used to practice or test this disclosure, some preferred methods and materials are described. Therefore, the terms defined immediately thereafter are described more fully by reference to the entire specification.

[0110] I. Definition Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well.

[0111] As used herein, the term "about" means a range of values ​​that include a specified value and that would be reasonably thought by one of ordinary skill in the art to be similar to the specified value. For example, using a measurement generally acceptable in the art, about means within the standard deviation. For example, about means a range extending to + / - 10%, + / - 5%, + / - 3%, or + / - 1% of the specified value.

[0112] The term "at least" followed by a number in this document is used to indicate the starting point of a range from which that number begins (the range can be an upper limit or an unlimited limit, depending on the defined variable). For example, "at least 1" means 1 or greater than 1.

[0113] The term "at most" followed by a number is used herein to indicate the end of a range that ends with that number (the range may be a range with a lower limit of 1 or 0 or an undefined lower limit, depending on the defined variable). For example, "at most 4" means 4 or less, and "at most 40%" means 40% or less. When a range is given in this specification as "(first number) to (second number)" or "(first number) - (second number)", this means a range where the lower limit is the first number and the upper limit is the second number. For example, 25 to 100 mm means a range where the lower limit is 25 mm and the upper limit is 100 mm.

[0114] In the case of two or more nucleic acid or peptide sequences, the term "identity" or "identity percentage" refers to two or more sequences or subsequences that are identical or have a specified percentage of the same amino acid residues or nucleotides when compared and aligned to obtain maximum correspondence. Sequence alignment methods used for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions in both sequences where the same nucleotide or amino acid residues are present. The sequence identity percentage is determined by dividing the number of matches in the alignment by the length of the reference sequence and then multiplying the resulting value by 100. For example, when aligned with a reference sequence having 1554 amino acids, a peptide sequence with 1166 matches has 75.0% identity with the test sequence (1166 ÷ 1554 × 100 = 75.0). When used herein, vacancies in the alignment do not reduce the sequence identity percentage. Unless otherwise stated, the best sequence alignment for comparison is performed by global alignment as described by Needleman and Wunsch, Mol. Biol. 48:443 (1970), as described by EMBOSSNeedle (on the World Wide Web ebi.ac.uk / Tools / psa / emboss_needle / ) (Madeira et al.) Nucleic Acids Res. 50(W1):W276-W279 (2022)) was implemented. Other alignment methods may be used, including but not limited to those described in the following literature: Devereux et al., Nucleic Acids Res. 12:387-95 (1984); Atschul et al. J. Mo. Biol. 215:403-10 (1990) (BLAST); Carrillo and Lipman Siam J. Applied Math.48(5) (1988); Computational Molecular Biology (edited by Lesk, AM, 1989); Biocomputing Informatics and Genome Projects, (edited by Smith, DW, 1993); Computer Analysis of Sequence Data, Part I, (edited by Griffin and Griffin, 1994); Sequence Analysis in Molecular Biology (von Heinje, 2012); Sequence Analysis Primer (edited by Gribskov and Devereux, J., 1993). Sequence identity was calculated using an implementation of the Needleman-Wunsch algorithm provided by the National Library of Medicine (on blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln on the World Wide Web).

[0115] For example, sequence identity can be determined using standard methods commonly used to compare the similarity of two polypeptide or two polynucleotide sequences. Using computer programs (such as the EMBOSS Needle or BLAST), two polypeptide or two polynucleotide sequences are aligned to achieve optimal matching of their individual residues (along the full length of one or both sequences, or along predetermined portions of one or both sequences). These programs provide default open and default empty penalties, as well as scoring matrices that can be used in conjunction with the computer program, such as PAM 250 (the standard scoring matrix; see Dayhoff et al., Atlas of Protein Sequence and Structure, Vol. 5, Supplement 3 (1978)).

[0116] As used herein, the term "helix-forming segment" refers to the portion of a protein or polypeptide that forms or is expected to form an α-helix. An α-helix is ​​a protein secondary structural element stabilized by hydrogen bonds between a carbonyl oxygen and an amino group every three residues at the helical turn. It is generally believed that the smallest segment of a protein forming an α-helix is ​​approximately 6-7 amino acids. Therefore, in some embodiments, the helical forming segment comprises about 5 to about 30 amino acid residues, about 7 to about 14 amino acid residues, about 7 to about 21 amino acid residues, about 7 to about 28 amino acid residues, about 7 to about 35 amino acid residues, about 7 to about 42 amino acid residues, or about 7 to about 49 amino acid residues; or any value therebetween, such as, but not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or more amino acids. In some embodiments, the helical forming segment forms a parallel triple helix bundle.

[0117] As used herein, the term "α-helical homotrimer" refers to a bundle of triple helices having parallel orientations. The term does not include hexagonal bundles, such as those assembled from three antiparallel double helical bundles; that is, as used herein, the term "α-helical homotrimer" does not include the heptapeptide repeat region of gp41 or its recombinant variants.

[0118] As used herein, the term "stable" in phrases such as "stable α-helical homotrimer" means that a protein structure (e.g., homotrimer) persists under suitable conditions. Stable protein structures can be detected by biophysical or biochemical methods known in the art—including, but not limited to, size exclusion chromatography, dynamic light scattering, electron microscopy, analytical ultracentrifugation, X-ray crystallography, nuclear magnetic resonance spectroscopy, circular dichroism, thermal denaturation, or interaction measurements. "Stable" α-helical homotrimers can be distinguished from unstable homotrimers, in part by structural analysis (e.g., by X-ray crystallography, NMR, or EM) or by measuring the effects of the α-helical homotrimer (e.g., by binding studies (BLI, SPR) or biophysical studies (thermal denaturation)). In some embodiments, stable α-helical homotrimers may be stable at room temperature and / or at high temperatures (e.g., 40°C). α-Helical homotrimers can form homotrimers independently or as part of larger trimeric protein complexes (such as trimeric antigens). In some embodiments, such as those predicted by calculation or determined experimentally, a stable α-helical homotrimer stabilizes the trimeric protein complex with a ΔΔG of at least -10, at least -20, at least -30, at least -40, at least -50, or at least -60. In some embodiments, the stable α-helical homotrimer is a “specific” homotrimer.

[0119] As used in this article, "conservative amino acid substitution" means that: hydrophobic amino acids (Ala, Gly, Met, Val, Ile, Leu, Phe, Thr, Trp) are substituted by other hydrophobic amino acids; hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are substituted by other hydrophobic amino acids with large side chains; amino acids with positively charged side chains (Arg, His, Lys) are substituted by other amino acids with positively charged side chains; amino acids with negatively charged side chains (Asp, Glu) are substituted by other amino acids with negatively charged side chains; and polar amino acids (Cys, Ser, Thr, Asn, Gly, Tyr) are substituted by other polar amino acids.

[0120] Throughout this specification and in the following claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising”, as well as “has” or “having” and “includes” or “including”, shall be understood to imply inclusion of the stated elements or steps or groups of elements or steps, but not to exclude any other elements or steps or groups of elements or steps. “consisting essentially of” or “consistses essentiallyly” indicates the exclusion of elements or steps that substantially affect the essential and novel features of the claimed invention.

[0121] II. Engineered extracellular domains This disclosure provides engineered extracellular domains of trimeric viral proteins, including but not limited to those from the Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Filoviridae, Herpesviridae, Orthomyxoviridae, Coronaviridae, Retroviridae, and Arenaviridae families. Table 1 shows the programmable viral fusion proteins. In some embodiments, the trimeric viral protein is an enveloped viral fusion protein.

[0122] Table 1.

[0123] In one aspect, this disclosure provides a recombinant polypeptide comprising an engineered extracellular domain of a trimeric viral protein, wherein, relative to the native reference sequence of the viral protein, the extracellular domain comprises a C-terminal helical forming segment comprising one or more amino acid substitutions selected such that the segment forms an α-helical homotrimer.

[0124] In some embodiments, the C-terminal helical forming region has improved hydrophobic stacking compared to the native reference sequence. In some embodiments, the C-terminal helical forming region comprises about 7 to about 31 residues. In some embodiments, the amino acid substitutions comprise polar, charged, and / or hydrophobic amino acids. In some embodiments, the C-terminal helical forming region comprises a polypeptide sequence according to any of the following: LXXTIXXLLXIXXXLXXXL (SEQ ID NO: 566), LVXTXKXLXDLIXXLXXLLXKLXX (SEQ ID NO: 567), LNKVKKXVXXLXXXVXXLEKXLX (SEQ ID NO: 568), EKIXXAIKKAXKL (SEQ ID NO: 569), EXIXKAIKXLXXXXX (SEQ ID NO: 570), XKXXEXXXXVXXXXXXXXX (SEQ ID NO: 571), XXLKKAAXIXKKXLKXX (SEQ ID NO: 572).

[0125] In some implementations, the C-terminal helical forming region contains a polypeptide sequence according to any of the common sequences in Table 24.

[0126] In some embodiments, the segment comprises a polypeptide sequence according to any of the following: L X2X2T I X2X2L L X2I[V / I] X2X2L [I / L] X2X2L (SEQ ID NO: 573), LV [A / T] T X2K X2L X2D LI X2X2L [K / E]X2L L X2K L X2X2 (SEQ ID NO: 574) or LNKVKK X2V X2X2L X2X2X2V X2X2L EK X2L X2 (SEQ ID NO: 575), wherein X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

[0127] In some embodiments, the segment comprises a polypeptide sequence according to any of the following: EKI X2X2A IKK AX2K L (SEQ ID NO: 576), E X2I X2K AIK X2L [L / X2] X2X2[X1 / X2] X2 (SEQ ID NO: 577). 、X2K [X1 / T] [L / E]E [T / A] X1X2[I / X2] V X2X2[X1 / X2] [X1 / X2] X2X2X1X2X2 (SEQ ID NO: 578) or X2X2L KKAA X2I X1K K X1L K X2X2 (SEQ ID NO: 579), wherein X1 is a nonpolar residue selected from A, I, L, and M, and X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K, and H, preferably a wild-type amino acid. In some embodiments, the segment comprises a polypeptide sequence listed in Table 25A or Table 25B. In some embodiments, the native reference sequence of the viral protein is any one of SEQ ID NO: 1, 104, 327, 382, ​​459, 499.

[0128] Respiratory syncytial virus (RSV) F protein The respiratory syncytial virus (RSV) F protein is the major conserved surface antigen of RSV, and antibodies against it are associated with protection against the disease. As demonstrated by the clinical efficacy of palizumab, a monoclonal antibody that binds to the F antigen and induces viral neutralization, the RSV F protein is a validated protective target against RSV infection (Johnson et al., J Infect Dis. 1997 Nov; 176(5): 1215-24). The RSV F protein is known to undergo significant structural changes from its pre-fusion to post-fusion form, catalyzing viral fusion with the host membrane to allow viral entry into the cell (McLellan et al., Science. 2013; 342(6158): 592-8). The pre-fusion F protein possesses important antigenic determinants that are lost during the transition to the post-fusion F protein (Melero et al., Vaccine. 2017; 35(3): 461-468). Antibody depletion studies of human serum absorbed with RSV F protein in any conformation have demonstrated that most neutralizing responses against RSV F protein target the pre-fusion structure (Krarup et al., Nat Commun. 2015;6:8143). These studies have also demonstrated the potential of antibodies binding to the post-fusion F protein to interfere with neutralization (Ngwuta et al., Sci Transl Med. 2015;7(309):309ra162). Generally, high levels of antibodies against RSV F protein are associated with protection against severe disease. However, generating high-titer neutralizing antibodies against RSV F protein remains challenging due to the specific biochemical properties of RSV F protein and the unpredictability of vaccine responses to RSV F. A structural model of the pre-fusion conformation of RSV F protein is shown in [Figure / Image / Insert Model ... Figure 1Within the structure, the stabilizing element is divided into five distinct spaces. Stabilization mutations target spaces 1-4. Space 5 refers to the C-terminus of the protein.

[0129] Illustrative sequences are shown in Table 2A. The design used native RSV / BF protein sequences (GenBank: WDV37446.1). The (predicted) transmembrane region is residues 527-549 and is in bold / underlined. The signal peptide is in italics and underlined. The proximal region surrounding the p27 peptide is in bold.

[0130] Table 2A.

[0131] In some implementations, RSV refers to RSV / A. In some implementations, RSV refers to RSV / B.

[0132] In some embodiments, the extracellular domain has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 1. In some embodiments, the extracellular domain has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3. In some embodiments, the extracellular domain has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 4. In some embodiments, the extracellular domain has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 5.

[0133] In another aspect, this disclosure provides a recombinant polypeptide comprising an engineered extracellular domain of a respiratory syncytial virus (RSV) fusion (F) protein, wherein the extracellular domain comprises: (a) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 140, 399, 400, 485, 486, 487, 488, 489, 494, or 498; (b) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 56, 58, 154, 187, 296, or 298; (c) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 75, 216, 218, or 219; (d) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 92, 232, 235, 238, 249, 250, or 254; and (e) relative to SEQ ID NO: 1. One, two, three or more amino acid substitutions at positions 67, 137 or 339; (f) substitutions relative to SEQ ID NO: 1 at about residue 100 to about residue 140 with non-cleavable linkers replacing the furin cleavage site; or (g) any combination of (a)-(f).

[0134] C-end spiral formation section In at least some cases, the C-terminal ends of the extracellular domains of many viral fusion proteins are known or predicted to be helical bundles mediated by helical transmembrane domains. The inventors have observed that in RSV F proteins, the C-terminal helical regions of the extracellular domain exhibit suboptimal hydrophobic stacking. Artificial polypeptide sequences were generated using computational modeling (with Rosetta remodeling), predicting that each sequence would form a stable α-helix. In the illustrative, non-limiting examples provided below, the helical backbone was first optimized with side chains represented as centroids, and then the side chains were designed in an all-atom model. The optimal linker length could be determined by plotting ddG (Rosetta remodeling) varying with linker length or ddG (RF diffusion) normalized to linker length. Helical constraints were then used to model 6–14 additional amino acids.

[0135] Illustrative sequences are shown in Table 2B. Residues 500-502 of the native RSV F protein are included as NQS (bold underline) and are conserved along with the native sequence in these embodiments, while many other amino acid residues are modified.

[0136] Table 2B. C-terminal α-spiral section (Rosetta remodeling)

[0137] In some implementations, modeling has shown that the following substitutions will stabilize the F protein portion in a helical conformation.

[0138] Illustrative sequences generated by RF diffusion are shown in Table 2C. Residues 500-502 of the native RSV F protein are included as NQS (bold underline) and are conserved along with the native sequence in these embodiments, while many other amino acid residues are modified. Table 2. C-terminal α-helix region of C.RSV (RF diffusion)

[0139] Table 2D. Possible substitutions (RF diffusion) at positions 503-532

[0140] In some embodiments, the C-terminal helical forming segment comprises about 10 to about 30 residues. In some embodiments, the segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 1, without being bound by theory, said residues may create hydrophobic contacts between the segment and the α-helix in the trimer.

[0141] The computational design described herein has detailed yield information regarding desired amino acid substitutions that, individually or in groups, can stabilize the extracellular domain of the RSV F protein. Illustrative, non-limiting amino acid substitutions that may be used are described below. In some embodiments, the C-terminal helical forming segment (“segment”) contains amino acid substitutions at positions 505-519 according to reference SEQ ID NO: 1. Those skilled in the art will readily understand that alignment with the reference sequence of this segment depends on preserving the helical structure of the segment, and therefore insertions and deletions in the alignment are not permitted when generating the sequence alignment for this segment. The inclusion of the starting amino acid (e.g., F in F505) herein is for clarity only; it should be understood that the modifications provided herein can be used with other RSV strains where the starting amino acid differs from the amino acid in the RSV / B reference strain sequence SEQ ID NO: 1.

[0142] In some embodiments, the segment comprises a polypeptide sequence listed in Table 2B, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the segment comprises a polypeptide sequence listed in Table 2B, or a polypeptide sequence having 1, 2, 3, 4, 5, or more amino acid substitutions. In some embodiments, the segment comprises a polypeptide sequence listed in Table 2C, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the segment comprises a polypeptide sequence listed in Table 2C, or a polypeptide sequence having 1, 2, 3, 4, 5, or more amino acid substitutions.

[0143] In some embodiments, the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), NQSALWLEAAKYVKQAREKS (SEQ ID NO: 11), NQSAKNAEAAKIAEETKRKD (SEQ ID NO: 12), or NQSRETAKAVSAVK (SEQ ID NO: 75), or a polypeptide sequence thereof having 1 to 5 amino acid substitutions. In some embodiments, the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), or a polypeptide sequence thereof having 1 to 5 amino acid substitutions. In some embodiments, the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10).

[0144] In some embodiments, the C-terminal helical forming region comprises about 5 to about 30 residues. In some embodiments, the C-terminal helical forming region comprises about 5 to about 25 residues. In some embodiments, the C-terminal helical forming region comprises about 5 to about 20 residues. In some embodiments, the C-terminal helical forming region comprises about 5 to about 15 residues. In some embodiments, the C-terminal helical forming region comprises about 5 to about 10 residues. In some embodiments, the C-terminal helical forming region comprises about 10 to about 30 residues. In some embodiments, the C-terminal helical forming region comprises about 10 to about 25 residues. In some embodiments, the C-terminal helical forming region comprises about 10 to about 20 residues. In some embodiments, the C-terminal helical forming region comprises about 10 to about 15 residues. In some embodiments, the C-terminal helical forming region comprises about 15 to about 30 residues. In some embodiments, the C-terminal helical forming region comprises about 15 to about 25 residues. In some embodiments, the C-terminal helical forming region comprises about 15 to about 20 residues.

[0145] In another aspect, this disclosure provides an α-helical segment comprising a polypeptide sequence listed in Table 25A or Table 25B, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the polypeptide comprises a trimeric pathogen protein linked to an N-terminus or C-terminus of the α-helical segment. In some embodiments, the C-terminal helical forming segment comprises at least 5 residues. In some embodiments, the C-terminal helical forming segment comprises at least 10 residues. In some embodiments, the C-terminal helical forming segment comprises at least 15 residues. In some embodiments, the C-terminal helical forming segment comprises at least 20 residues. In some embodiments, the C-terminal helical forming segment comprises at least 25 residues.

[0146] Stabilization replacement Computational modeling was used to identify amino acid substitutions that stabilize RSV / BF proteins in their pre-fusion conformation. Without being bound by theory, the following amino acid substitutions are described herein as “stabilizing substitutions” because they are predicted to stabilize RSV / BF proteins by increasing shape complementarity within the tertiary structure of the pre-fusion conformation. Amino acid substitutions may have other effects on structure, such as creating hydrophobicity or charge-charge interactions (e.g., salt bridges) within the structure. These mutations are listed in Table 3A.

[0147] Table 3A. Stabilization Substitution

[0148] The implementation schemes for the alternative combinations are shown in Table 3B.

[0149] Table 3B.

[0150] In some implementations, the extracellular domain contains amino acid substitutions for S155C, S290C, S190F, and V207L.

[0151] In some embodiments, the extracellular domain comprises one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 140, 399, 400, 485, 486, 487, 488, 489, 494 or 498.

[0152] In some embodiments, the extracellular domain comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 1: E487R + K498A; E487R + K498E; E487K + K498E; D486A + E487R + K498A; D486Q + E487R + K498A; D486E + E487A + D489A + T400D; D486A + E487M + K498A; E487Q; D486S; F488W + D489A + T400D + E487R + K498A; F140W + D489A + T400D + E487R + K498A; Q494I + S485I + K399A + 487R + 498A; Q494M + S485I + K399A; D486A + 487M + 498A; Q494L + S485A + K399V + D486A + 487M + 498A; Q494M + S485A + K399V + D486A + 487M + 498A; Q494A + S485F + K399V + D486A + 487M + 498Y; D489A + T400D + E487R + K498A; or D489A + T400D. In some embodiments, the extracellular domain contains amino acid substitutions for F488W, D489A, T400D, E487R, K498A, and D486A. In some implementations, the extracellular domain contains amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and T249P.

[0153] Additional substitutions in the stable pre-fusion conformation of the F protein Without being bound by theory, the following amino acid substitutions are predicted to stabilize RSV F proteins. Amino acid substitutions may have other structural effects, such as creating hydrophobicity or charge-charge interactions within the structure (e.g., salt bridges). These mutations are listed in Table 4A.

[0154] Table 4A

[0155] In some embodiments, the extracellular domain comprises one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 54, 55, 58, 66, 67, 88, 92, 98, 101, 103, 106, 140, 142, 144, 148, 149, 154, 155, 188, 190, 207, 215, 232, 235, 238, 249, 254, 279, 290, 296, 298, 361, 371, 399, 400, 428, 458, 485, 486, 487, 488, 489, 494, 495, or 498. In some embodiments, the extracellular domain comprises substitutions relative to SEQ ID NO: 1. 1. T54H, S55C, T58M, K66E, N67I, T67I, T67V, N88C, E92C, E92D, Q98C, Q101P, T103C, R106C, F140W, L142C, V144C, I148C, A149C, V154I, S155C, L188C, S190I, S215P, E232A, R235Y, S238C, T249P, N2 One, two, three or more amino acid substitutions at 54C, Q279C, V296A, V296I, A298L, Q361C, N371C, K399A, T400D, N428C, Y458C, S485I, D486A, D486S, D486N, E487M, E487Q, E487R, F488W, D489A, D489S, Q494M, V495Y or K498A.

[0156] The substitution combinations are shown in Table 4B.

[0157] Table 4B.

[0158] In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at S155C, S290C, S190F, and V207L. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at S55C, L188C, L142C, N371C, T54H, and V296I. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at S55C, L188C, and D486S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at S55C, L188C, T54H, and S190I. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at T103C, I148C, S190I, and D486S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at T103C, I148C, T54H, S190I, V296I, and D486S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at S55C, L188C, T54H, and D486S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at S55C, L188C, T54H, S190I, and D486S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at S155C, S290C, S190I, and D486S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at S55C, L188C, L142C, N371C, T54H, V296I, D486S, E487Q, and D498S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at S155C, S290C, T54H, S190I, and V296I.

[0159] In some embodiments, the RSV F protein mutant comprises disulfide bond mutations selected from the group consisting of: 55C and 188C; 155C and 290C; 103C and 148C; and 142C and 371C, such as S55C and L188C, S155C and S290C, T103C and I148C, or L142C and N371C. Examples of such mutation pairs include: 508C and 509C; 515C and 516C; 522C and 523C, such as K508C and S509C, N515C and V516C, or T522C and T523C.

[0160] In some implementations, the RSV F protein mutant contains one or more cavity-filling mutations selected from the groups shown in Table 4C.

[0161] Table 4C. Disulfide bond mutations

[0162] In some implementations, the RSV F protein mutant contains at least one cavity-filling mutation selected from the group consisting of T54H, S190I, and V296I.

[0163] In some implementations, the RSV F protein mutant contains at least one electrostatic mutation selected from the group shown in Table 4D.

[0164] Table 4D. Electrostatic Transitions

[0165] In some implementations, the RSV F protein mutant contains the mutant D486S.

[0166] The substitution combinations are shown in Table 4E.

[0167] Table 4E.

[0168] In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at T103C, I148C, S190I, and D486S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at T54H, S55C, L188C, and D486S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at T54H, T103C, I148C, S190I, V296I, and D486S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at T54H, S55C, L142C, L188C, V296I, and N371C. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at S55C, L188C, and D486S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at T54H, S55C, L188C, and S190I. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at S55C, L188C, S190I, and D486S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at T54H, S55C, L142C, L188C, V296I, N371C, D486S, E487Q, and D489S. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at T54H, S155C, S190I, S290C, and V296I. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at N67I and S215P. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at N67I, S215P, and E487Q. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at V56C and V164C. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at I57C and S190C. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at T58C and V164C. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at N165C and V296C. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at K168C and V296C. In some embodiments, the extracellular domain comprises amino acid substitutions relative to SEQ ID NO: 1 at M396C and F483C.

[0169] The combination of C-terminal spiral formation segment and stabilization substitution In some embodiments, this disclosure provides a recombinant polypeptide comprising an amino acid substitution having an engineered C-terminal α-helical segment, the amino acid substitution stabilizing the RSV F protein in its pre-fusion conformation.

[0170] The native sequence of the RSV / BF protein (GenBank: WDV37446.1) is shown below, where the (predicted) transmembrane region is in italics, and the C-terminal helix of the native sequence (residues 492-501) is also in bold / underlined. The signal peptide is in italics / underlined / underlined.

[0171] In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 6, SEQ ID NO: 6 optionally lacking the p27 peptide shown in bold, and wherein “X” refers to a site involving the added C-terminal helical segment and can be any amino acid: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 7, wherein SEQ ID NO: 7 optionally lacks the p27 peptide shown in bold, wherein “X” refers to a site involving the added C-terminal helical segment and may be any amino acid: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 8, wherein SEQ ID NO: 8 optionally lacks the p27 peptide shown in bold: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 9, wherein SEQ ID NO: 9 optionally lacks the p27 peptide shown in bold: In some embodiments, the polypeptide comprises a sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 1-9.

[0172] Illustrative sequences containing the extracellular domains and C-terminal α-helical units of various RSV F proteins are shown in Table 4F. Signal peptides are underlined. The proximal region surrounding the p27 peptide is in bold. In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence shown in Table 4F.

[0173] Table 4F.

[0174] In some embodiments, the extracellular domain includes any of the stabilizing mutations of the RSV F protein disclosed in U.S. Patent Nos. 9,950,058, 8,563,002, 11,261,239, 11,629,181, and 11,655,284, each of which is hereby incorporated in its entirety by reference.

[0175] furin cleavage site The RSVF protein is cleaved by the protease furin during expression. This article provides a construct in which the furin cleavage site is replaced by a glycine-serine linker. The sequence is provided in Table 5A. In some embodiments, the extracellular domain of the RSVF protein contains an uncleaved furin cleavage site.

[0176] Table 5A. Frin protease cleavage linkers

[0177] connector In some implementations, recombinant peptides and protein nanostructures can be genetically fused, so that they both exist within a single peptide, termed "fusion proteins." The bonding between the peptide and the protein nanostructure allows the recombinant peptide to be displayed on the exterior of the self-assembled protein nanostructure.

[0178] Various polypeptide sequences can be used to link proteins or their antigenic fragments to protein nanostructures. In some cases, the linker comprises a polypeptide sequence that may be included in the encoding polynucleotide sequence. Any suitable linker polypeptide can be used. In some embodiments, the linker imposes a rigid relative orientation of an antigenic protein (e.g., an extracellular domain from an RSV fusion protein) or its antigenic fragment onto the protein nanostructure. In some embodiments, the linker flexibly links an antigenic protein (e.g., an extracellular domain from an RSV fusion protein) or its antigenic fragment to the protein nanostructure. In some embodiments, the encoded polypeptide may include a linker between regions. In some embodiments, the polypeptide is a fusion protein comprising a recombinant RSV polypeptide, a linker, and a protein nanostructure component polypeptide. In some embodiments, the polypeptide is a fusion protein comprising, in N-terminal to C-terminal order, a recombinant RSV polypeptide, a linker, and a protein nanostructure component polypeptide. The linker can be a polypeptide. Various polypeptide sequences can be used and are well known in the art. In some embodiments, the linker may comprise a Gly-Ser linker of any suitable length (i.e., a linker consisting of glycine and serine residues). In some implementations, the length of the Gly-Ser linker can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues. Non-limiting examples of Glys-Ser linkers are presented in Table 5B.

[0179] Table 5B.

[0180] In some embodiments, the linker contains 3 to 30 amino acid residues. In some embodiments, the linker contains 4 to 24 amino acid residues. In some embodiments, the linker contains 8 to 24 amino acid residues. In some embodiments, the linker contains 10 to 24 amino acid residues. In some embodiments, the linker contains 12 to 24 amino acid residues. In some embodiments, the linker contains 16 to 24 amino acid residues. In some embodiments, the linker contains 18 to 24 amino acid residues. In some embodiments, the linker contains 20 to 24 amino acid residues. In some embodiments, the linker contains 4 to 20 amino acid residues. In some embodiments, the linker contains 8 to 20 amino acid residues. In some embodiments, the linker contains 10 to 20 amino acid residues. In some embodiments, the linker contains 12 to 20 amino acid residues. In some embodiments, the linker contains 16 to 20 amino acid residues. In some embodiments, the linker contains 8 to 18 amino acid residues. In some embodiments, the linker contains 12 to 16 amino acid residues. In some embodiments, the linker contains 3 amino acid residues. In some embodiments, the linker contains 4 amino acid residues. In some embodiments, the linker contains 5 amino acid residues. In some embodiments, the linker contains 6 amino acid residues. In some embodiments, the linker contains 7 amino acid residues. In some embodiments, the linker contains 8 amino acid residues. In some embodiments, the linker contains 8 amino acid residues. In some embodiments, the linker contains 10 amino acid residues. In some embodiments, the linker contains 11 amino acid residues. In some embodiments, the linker contains 12 amino acid residues. In some embodiments, the linker contains 13 amino acid residues. In some embodiments, the linker contains 14 amino acid residues. In some embodiments, the linker contains 15 amino acid residues. In some embodiments, the linker contains 16 amino acid residues. In some embodiments, the linker contains 17 amino acid residues. In some embodiments, the linker contains 18 amino acid residues. In some embodiments, the linker contains 19 amino acid residues. In some embodiments, the linker contains 20 amino acid residues. In some embodiments, the linker contains 21 amino acid residues. In some embodiments, the linker contains 22 amino acid residues. In some embodiments, the linker contains 23 amino acid residues. In some embodiments, the linker contains 24 amino acid residues. In some embodiments, the linker contains 25 amino acid residues. In some embodiments, the linker contains 26 amino acid residues. In some embodiments, the linker contains 27 amino acid residues. In some embodiments, the linker contains 28 amino acid residues. In some embodiments, the linker contains 29 amino acid residues.In some implementations, the linker contains 30 amino acid residues.

[0181] In some embodiments, the encoded polypeptide may include a linker between regions. In some embodiments, the polypeptide is a fusion protein comprising a recombinant RSV polypeptide, a linker, an N-terminal extension linker, and a protein nanostructure component polypeptide. In some embodiments, the polypeptide is a fusion protein comprising, in N-terminal to C-terminal order, a recombinant RSV polypeptide, a linker, an N-terminal extension linker, and a protein nanostructure component polypeptide. In some embodiments, the N-terminal extension linker is an I53-50A helical extension. In some embodiments, the polypeptide sequence of the N-terminal extension linker is EKAAKAEEAARK (SEQ ID NO: 665).

[0182] Trimerization domain In some embodiments, the peptide may include a trimerizing domain, such as a fold or a GCN4 trimer. In some embodiments, the linker sequence includes a fold, wherein the fold sequence is GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 1235).

[0183] In some embodiments, the polypeptide may include a trimerizing domain, wherein the trimerizing domain sequence is DKIEEILSKIYHIENEIARIKKLIGE (SEQ ID NO: 666) (GEN). In some embodiments, the polypeptide may include a trimerizing domain, wherein the trimerizing domain sequence is EKFHQIEKEFSEVEGRIQDLEK (SEQ ID NO: 667) (HA).

[0184] In some embodiments, the peptide may include a trimerizing domain, wherein the trimerizing domain sequence is EDKIEEILSKIYHIENEIARIKKLIGEA (Seq ID NO: 668) (a coiled helical isoleucine zipper).

[0185] In some embodiments, the peptide may include a trimerizing domain, wherein the trimerizing domain sequence is GSGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 669) (phage T4 fibrin).

[0186] In some embodiments, the trimerizing sequence is RMKQIEDKIEEILSKIYHIENEIARIKKLIGEA (SEQ ID NO: 670) (GCN4). In some embodiments, the trimerizing domain is a GCN4 variant. In some embodiments, the GCN4 variant sequence is RMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGR (SEQ ID NO: 671), RMKQIEDKIEEILSKIYHIENEIARIKKLIGNRTGGR (SEQ ID NO: 672), RMKQIEDKIENITSKIYHIENEIARIKKLIGNRTGGR (SEQ ID NO: 673), RMKQIEDKIEEILSKIYNITNEIARIKKLIGNRTGGR (SEQ ID NO: 674), or RMKQIEDKIENITSKIYNITNEIARIKKLIGNRTGGR (SEQ ID NO: 675).

[0187] Illustrative sequences containing various RSV F protein extracellular domains, C-terminal α-helical regions, and folds are shown in Table 5C. Signal peptides are italicized and underlined. The underlined fold sequence may be replaced by any of the trimerizing domains described herein or any of the multimerizing domains described in Table 11 to produce embodiments containing such other trimerizing domains.

[0188] In some embodiments, the trimeric protein complex comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence shown in Table 5C. In some embodiments, the trimeric protein complex can be used as a trimeric component of a protein nanostructure. The proximal region surrounding the p27 peptide is bold. In some embodiments, the p27 peptide can be removed from the extracellular domain of the RSV F protein via furin-based cleavage during antigen generation in cell cultures. Folded sequences are bold / underlined.

[0189] Table 5C.

[0190] In some embodiments, the recombinant polypeptide comprises an engineered extracellular domain of a respiratory syncytial virus (RSV) fusion (F) protein, wherein the extracellular domain comprises (a) a C-terminal helical forming segment relative to SEQ ID NO: 1 between about residues 500 and about residues 530, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer; (b) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 140, 399, 400, 485, 486, 487, 488, 489, 494, or 498; (c) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 56, 58, 154, 187, 296, or 298; (d) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 75, 216, 218, or 219; and (e) relative to SEQ ID NO: 1. (a) Substitution of one, two, three or more amino acids at positions 92, 232, 235, 238, 249, 250 or 254; (f) Substitution of one, two, three or more amino acids at positions 67, 137 or 339 relative to SEQ ID NO: 1; (g) Substitution of the furin cleavage site at about residue 100 to about residue 140 relative to SEQ ID NO: 1 with a non-cleavable linker; or (h) Any combination of (a)-(g).

[0191] In some embodiments, the extracellular domain includes a C-terminal helical forming segment relative to SEQ ID NO: 1, between about residues 500 and about residues 530, comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 10 to about 30 residues.

[0192] In some embodiments, the C-terminal helical forming segment includes substitutions of two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 1, which create hydrophobic contacts between segments in the α-helix and trimer.

[0193] In some embodiments, the C-terminal helical forming segment comprises (1) an amino acid substitution relative to SEQ ID NO: 1 at position F505, wherein F is substituted with A, I, L, M, V, G, T; (2) an amino acid substitution relative to SEQ ID NO: 1 at position I506, wherein I is substituted with any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y or A, I, L, V; (3) an amino acid substitution relative to SEQ ID NO: 1 at position R507, wherein R is substituted with any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y or A, I, L, V; (4) an amino acid substitution relative to SEQ ID NO: 1 at position K508, wherein R is substituted with K, Q, R, preferably A, V, T, I; (5) an amino acid substitution relative to SEQ ID NO: 1. An amino acid substituted at position S509, wherein S is substituted by A, I, L, M, V, F, W, Y, G, T, preferably A, I, L, M, V; (6) An amino acid substituted at position D510 relative to SEQ ID NO: 1, wherein D is substituted by any amino acid, preferably D, E, K, N, Q, R, S, T, Y; (7) An amino acid substituted at position E511 relative to SEQ ID NO: 1, wherein E is substituted by any amino acid; (8) An amino acid substituted at position L512 relative to SEQ ID NO: 1, wherein L is substituted by D, E, K, N, Q, R, S, T, Y, preferably A, I, L, M, V, F, W, Y, G, T; (9) An amino acid substituted at position L512 relative to SEQ ID NO: 1, wherein L is substituted by D, E, K, N, Q, R, S, T, Y, preferably A, I, L, M, V, F, W, Y, G, T; 1. An amino acid substituted at position L513, wherein L is replaced by any amino acid, preferably A, I, L, M, V, F, W, Y, G, more preferably D, E, K, N, Q, R, S, T, Y; (10) An amino acid substituted at position H514 relative to SEQ ID NO: 1, wherein H is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; (11) An amino acid substituted at position N515 relative to SEQ ID NO: 1, wherein N is replaced by any amino acid other than P, preferably A, I, L, M, V, F, W, Y, G; (12) An amino acid substituted at position V516 relative to SEQ ID NO: 1, wherein V is replaced by A, I, L, M, V, F, W, Y, G or T, S, K; (13) An amino acid substituted at position V516 relative to SEQ ID NO: 1. An amino acid substitution at position N517, wherein N is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; (14) An amino acid substitution at position T518 relative to SEQ ID NO: 1, wherein T is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y.(15) An amino acid substitution relative to SEQ ID NO: 1 at position G519, wherein G is substituted with any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; and / or (16) any combination of (1)-(15).

[0194] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 1 at position L503, wherein F is substituted with Q, V, K, R, N, L; (2) an amino acid substitution relative to SEQ ID NO: 1 at position A504, wherein I is substituted with any amino acid other than P, preferably S, T, L, A, Q, K, E, Y; (3) an amino acid substitution relative to SEQ ID NO: 1 at position F505, wherein F is substituted with I, V, N, T, L; (4) an amino acid substitution relative to SEQ ID NO: 1 at position I506, wherein I is substituted with any amino acid other than P, preferably Q, N, K, R, V, S; (5) an amino acid substitution relative to SEQ ID NO: 1 at position R507, wherein R is substituted with any amino acid other than P, preferably A, N, K, E, D, Q; and (6) an amino acid substitution relative to SEQ ID NO: 1. The amino acid at position K508 is substituted, wherein R is substituted by T, M, V, R; (7) The amino acid at position S509 relative to SEQ ID NO: 1 is substituted, wherein S is substituted by T, I, K, Q, M, E, V, S; (8) The amino acid at position D510 relative to SEQ ID NO: 1 is substituted, wherein D is substituted by S, K, N, D, E; (9) The amino acid at position E511 relative to SEQ ID NO: 1 is substituted, wherein E is substituted by R, S, E, K, A, T, L; (10) The amino acid at position L512 relative to SEQ ID NO: 1 is substituted, wherein L is substituted by V, N, T, L; (11) The amino acid at position L513 relative to SEQ ID NO: 1 is substituted, wherein L is substituted by D, T, H, K, E, N, R; (12) The amino acid at position L513 relative to SEQ ID NO: 1 is substituted, wherein L is substituted by D, T, H, K, E, N, R; (13) The amino acid at position L513 relative to SEQ ID NO: 1 is substituted, wherein L is substituted by D, T, H, K, E, N, R; (14) The amino acid at position L512 relative to SEQ ID NO: 1 is substituted, wherein R is substituted by T, M, V, R; (15) The amino acid at position S509 relative to SEQ ID NO: 1 is substituted, wherein S is substituted by T, I, K, Q, M, E, V, R; (15) The amino acid at position D509 relative to SEQ ID NO: 1 is substituted, wherein S is substituted by T, I, K, Q, M, E, V, R; (15) The amino acid at position L512 relative to SEQ ID NO: 1 is substituted, wherein R is substituted by T, 1. The amino acid at position H514 is substituted, wherein H is substituted by A, N, E, S, V, K, T, D; (13) The amino acid at position N515 relative to SEQ ID NO: 1 is substituted, wherein N is substituted by I, E, L, T, Q; (14) The amino acid at position V516 relative to SEQ ID NO: 1 is substituted, wherein V is substituted by E, I, K, N, R, Q; (15) The amino acid at position N517 relative to SEQ ID NO: 1 is substituted, wherein N is substituted by A, S, K, E, R; (16) The amino acid at position T518 relative to SEQ ID NO: 1 is substituted, wherein T is substituted by K, S, Q, R, D, E; (17) The amino acid at position G519 relative to SEQ ID NO: 1 is substituted, wherein G is substituted by V, L, I; (18) The amino acid at position I520 relative to SEQ ID NO: 1 is substituted, wherein G is substituted by K, Q, E, N, T.(19) Amino acid substitutions relative to SEQ ID NO: 1 at position P521, wherein G is substituted by H, D, E, K, R, N, Q; (20) Amino acid substitutions relative to SEQ ID NO: 1 at position E522, wherein G is substituted by L, R, I, V; (21) Amino acid substitutions relative to SEQ ID NO: 1 at position A523, wherein G is substituted by E, V, L, K, RI; (22) Amino acid substitutions relative to SEQ ID NO: 1 at position P524, wherein G is substituted by A, K, T, E, R; (23) Amino acid substitutions relative to SEQ ID NO: 1 at position R525, wherein G is substituted by H, R, S, L, N, E, D; (24) Amino acid substitutions relative to SEQ ID NO: 1 at position D526, wherein G is substituted by I, L, V, R; (25) Amino acid substitutions relative to SEQ ID NO: (26) Amino acid substitution at position G527, wherein G is substituted by E, K, Q, or D; (27) Amino acid substitution at position Q528 relative to SEQ ID NO: 1, wherein G is substituted by D, K, S, R, or A; (28) Amino acid substitution at position A529 relative to SEQ ID NO: 1, wherein G is substituted by T or L; (29) Amino acid substitution at position Y530 relative to SEQ ID NO: 1, wherein G is substituted by L, E, or T; (20) Amino acid substitution at position V531 relative to SEQ ID NO: 1, wherein G is substituted by A, R, or K; (21) Amino acid substitution at position R532 relative to SEQ ID NO: 1, wherein G is substituted by V or A; and / or (32) Any combination of (1)-(30).

[0195] In some embodiments, the segment comprises a polypeptide sequence listed in Table 2B or Table 2C, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), NQSALWLEAAKYVKQAREKS (SEQ ID NO: 11), NQSAKNAEAAKIAEETKRKD (SEQ ID NO: 12), or NQSRETAKAVSAVK (SEQ ID NO: 75), or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10).

[0196] In some embodiments, the extracellular domain comprises one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 140, 399, 400, 485, 486, 487, 488, 489, 494 or 498. In some embodiments, the extracellular domain comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 1: E487R + K498A, E487R + K498E, E487K + K498E, D486A + E487R + K498A, D486Q + E487R + K498A, D486E + E487A + D489A + T400D, D486A + E487M + K498A, E487Q, D486S, F488W + D489A + T400D + E487R + K498A, F140W + D489A + T400D + E487R + K498A, Q494I + S485I + K399A + 487R + 498A, Q494M + S485I + K399A; D486A + 487M + 498A, Q494L + S485A + K399V + D486A + 487M + 498A, Q494M + S485A + K399V + D486A + 487M + 498A, Q494A + S485F + K399V + D486A + 487M + 498Y, D489A + T400D + E487R + K498A, or D489A + T400D. In some embodiments, the extracellular domain contains amino acid substitutions for D489A, T400D, E487R, and K498A.

[0197] In some embodiments, the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and D486A. In some embodiments, the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and T249P.

[0198] In some embodiments, the polypeptide includes a heteropolymerization domain at the C-terminus of its extracellular domain. In some embodiments, the polymerization domain is a trimerization domain. In some embodiments, the polymerization domain includes a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64). In some embodiments, the extracellular domain includes amino acid substitutions for S155C, S290C, S190F, and V207L.

[0199] In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 6, SEQ ID NO: 6 optionally lacking the p27 peptide shown in bold, and wherein “X” refers to a site involving the added C-terminal helical segment and can be any amino acid: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 7, wherein SEQ ID NO: 7 optionally lacks the p27 peptide shown in bold, wherein “X” refers to a site involving the added C-terminal helical segment and may be any amino acid: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 8, wherein SEQ ID NO: 8 optionally lacks the p27 peptide shown in bold: In some embodiments, the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following SEQ ID NO: 9, wherein SEQ ID NO: 9 optionally lacks the p27 peptide shown in bold: In some embodiments, the polypeptide comprises a sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 1-9.

[0200] In another aspect, this disclosure provides a trimeric protein complex comprising the polypeptide disclosed herein. In some embodiments, the thermal stability, as determined by nanoDSF, is increased by at least 10°C, at least 15°C, at least 20°C, about 10°C to about 30°C, about 10°C to about 20°C, or about 20°C to about 30°C, compared to a trimeric protein complex lacking modification (a)-(h). In some embodiments, the stability, as determined by storage at about 40°C, is increased compared to a trimeric protein complex lacking modification (a)-(h). In some embodiments, the thermal stability is increased compared to a reference RSV F protein comprising amino acid substitutions consisting substantially of S155C, S290C, S190F, and V207L ​​(DS-Cav1).

[0201] In another aspect, this disclosure provides a protein nanostructure comprising a trimeric component, said trimeric component comprising the polypeptide disclosed herein. In some embodiments, the nanostructure is a two-component nanostructure comprising a first trimeric component and a second pentameric component. In some embodiments, the first trimeric component comprises engineered extracellular domains of a respiratory syncytial virus (RSV) fusion (F) polypeptide and an I53-50A polypeptide. In some embodiments, the first trimeric component comprises a fusion protein comprising, in N-terminus to C-terminus, an RSV fusion (F) polypeptide, an amino acid linker, and an I53-50A polypeptide. In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 1 and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and a polymerized domain, the polymerized domain comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or a second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71 have polypeptide sequences with at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0202] In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, D489A, T400D, E487R, and K498A relative to SEQ ID NO: 1 and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and a polymerized domain comprising I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) A polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71 having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71.

[0203] In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and T249P relative to SEQ ID NO: 1 and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and a polymerized domain comprising a polymerized region with or without I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) A polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71 having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71.

[0204] In some embodiments, the nanostructure is a two-component nanostructure comprising: a first trimeric component, wherein the first trimeric component comprises an engineered extracellular domain of an RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and D486A relative to SEQ ID NO: 1 and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and a polymerized domain comprising a polymerized region with or without I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) A polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71 having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide sequence of SEQ ID NO: 20 or 71.

[0205] In some embodiments, the trimer component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the sequences listed in Table 19. In some embodiments, the pentamer component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 20, 44, 45, 52, 71, 73, 74.

[0206] In another aspect, this disclosure provides a recombinant polypeptide comprising an α-helical segment and a polymerizing domain, wherein the segment comprises a polypeptide sequence listed in Table 25A or Table 25B, or a polypeptide sequence thereof having 1 to 5 amino acid substitutions. In some embodiments, the polypeptide comprises a trimeric pathogen protein linked to the α-helical segment at an N-terminus or C-terminus. In some embodiments, the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), NQSALWLEAAKYVKQAREKS (SEQ ID NO: 11), NQSAKNAEAAKIAEETKRKD (SEQ ID NO: 12), or NQSRETAKAVSAVK (SEQ ID NO: 75), or a polypeptide sequence thereof having 1 to 5 amino acid substitutions. In some embodiments, the polypeptide comprises an antigen at the N-terminus of the segment.

[0207] Human metapneumovirus (hMPV) hMPV is a negative-sense single-stranded RNA virus that causes upper and lower respiratory tract infections. hMPV shares substantial homology with respiratory syncytial virus (RSV) in its surface glycoprotein. The F protein, present as a trimer, is a type I glycoprotein.

[0208] Illustrative sequences are shown in Table 6A. The design was performed using the native hMPV F protein sequence. Signal peptides are italicized and underlined. Table 6A

[0209] In some embodiments, the extracellular domain of the hMPV F protein has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 179. In some embodiments, the extracellular domain of the hMPV F protein has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 180. In some embodiments, the extracellular domain of the hMPV F protein has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 181.

[0210] C-end spiral formation section The illustrative sequence of the C-terminal α-helical region is shown in Table 6B (Rosetta remodeling). Residues 468-470 of the native hMPV F protein are included as ENS (bold underline) and are conserved along with the native sequence in these embodiments, while many other amino acid residues are modified.

[0211] Table 6B. C-terminal α-spiral segment of hMPV (Rosetta remodeling)

[0212] In some embodiments, the C-terminal helical forming region comprises 5 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 5 to 20 residues. In some embodiments, the C-terminal helical forming region comprises 5 to 15 residues. In some embodiments, the C-terminal helical forming region comprises 5 to 10 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 20 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 15 residues.

[0213] In some implementations, modeling has shown that the following substitutions will stabilize the F protein moiety in a helical conformation. Illustrative sequences of possible substitutions are shown in Table 6C.

[0214] Table 6C. Possible replacements at positions 471-489 (Rosetta remodeling)

[0215] An illustrative sequence of the C-terminal α-helical region is shown in Table 6D (RF diffusion). Residues 469-471 of the native hMPV F protein are included as NSQ (bold underline) and are conserved along with the native sequence in these embodiments, while many other amino acid residues are modified.

[0216] Table 6. C-terminal α-helical region (RF diffusion) of hMPV.

[0217] In some embodiments, the C-terminal helical forming region contains 15 to about 35 residues. In some embodiments, the C-terminal helical forming region contains 15 to about 30 residues. In some embodiments, the C-terminal helical forming region contains 15 to about 25 residues. In some embodiments, the C-terminal helical forming region contains 15 to about 20 residues. In some embodiments, the C-terminal helical forming region contains 20 to about 25 residues. In some embodiments, the C-terminal helical forming region contains 25 to about 30 residues.

[0218] In some implementations, modeling has shown that the following substitutions will stabilize the F protein moiety in a helical conformation. Illustrative sequences of possible substitutions are shown in Table 6E.

[0219] Table 6E. Possible substitutions (RF diffusion) at positions 472-498

[0220] In some embodiments, the recombinant polypeptide comprises an engineered extracellular domain of an hMPV fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 104 between about residues 470 and about residues 500, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 104, the residues creating hydrophobic contacts between segments in the α-helical homotrimer. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 104 between about residues 470 and about residues 490, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 7 to about 21 residues.

[0221] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 104 at position Q471, wherein Q is substituted by any one of A, D, E, I, Q, R, S, T; (2) an amino acid substitution relative to SEQ ID NO: 104 at position A472, wherein A is substituted by any one of A, D, E, I, K, R, S, T, Y; (3) an amino acid substitution relative to SEQ ID NO: 104 at position L473, wherein L is substituted by any one of A, I, L, M, Q, S, T, W; (4) an amino acid substitution relative to SEQ ID NO: 104 at position V474, wherein V is substituted by any one of A, D, E, I, K, L, N, Q, S, T; and (5) an amino acid substitution relative to SEQ ID NO: 104 The amino acid at position D475 is substituted, wherein D is substituted by any one of A, D, E, H, K, N, Q, R, S, T; (6) The amino acid at position Q476 of SEQ ID NO: 104 is substituted, wherein Q is substituted by any one of A, D, E, H, I, K, L, M, N, Q, T, V; (7) The amino acid at position S477 of SEQ ID NO: 104 is substituted, wherein S is substituted by any one of A, E, I, K, L, M, N, Q, R, S, T, V; (8) The amino acid at position N478 of SEQ ID NO: 104 is substituted, wherein N is substituted by any one of A, D, E, K, N, Q, R, S, T; (9) The amino acid at position N478 of SEQ ID NO: 104 is substituted, wherein N is substituted by any one of A, D, E, K, N, Q, R, S, T; (9) The amino acid at position N478 of SEQ ID NO: 104 is substituted, wherein D is substituted by any one of A, D, E, K, N, Q, R, S, T; 104 The amino acid at position R479 is substituted, wherein R is substituted by any one of A, D, E, F, I, K, L, M, N, Q, R, S, T, W, Y, (10) relative to SEQ ID NO: 104 The amino acid at position I480 is substituted, wherein I is substituted by any one of A, I, L, M, R, S, T, V, (11) relative to SEQ ID NO: 104 The amino acid at position L481 is substituted, wherein L is substituted by any one of D, E, I, K, L, M, N, Q, R, S, T, (12) relative to SEQ ID NO: 104 The amino acid at position S482 is substituted, wherein S is substituted by any one of A, D, E, K, Q, R, S, T, (13) relative to SEQ ID NO: 104 substituted with an amino acid at position S483, wherein S is substituted by any one of A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, Y, (14) relative to SEQ ID NO: 104 substituted with an amino acid at position A484, wherein A is substituted by any one of A, D, E, I, K, L, M, R, S, T, V, Y, (15) relative to SEQ ID NO: 104 substituted with an amino acid at position E485,(16) where E is replaced by any one of D, E, G, K, L, Q, R, S, T, (17) where the amino acid at position K486 relative to SEQ ID NO: 104 is replaced by any one of A, E, I, K, L, Q, R, S, T, (18) where the amino acid at position N488 relative to SEQ ID NO: 104 is replaced by any one of E, I, K, L, N, Q, R, S, V, (19) where the amino acid at position T489 relative to SEQ ID NO: 104 is replaced by any one of A, D, E, K, S, and (20) any combination of (1)-(19). In some embodiments, the segment comprises a polypeptide sequence listed in Table 6B, or a polypeptide sequence having one to five amino acid substitutions.

[0222] In some embodiments, the extracellular domain includes a C-terminal helical forming segment relative to SEQ ID NO: 104, between about residues 470 and about residues 500, comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 16 to about 30 residues.

[0223] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 104 at position A472, wherein A is substituted by any one of T, N, K, R, E, S; (2) an amino acid substitution relative to SEQ ID NO: 104 at position L473, wherein L is substituted by any one of T, I, V; (3) an amino acid substitution relative to SEQ ID NO: 104 at position V474, wherein V is substituted by any one of E, Q, L, D; (4) an amino acid substitution relative to SEQ ID NO: 104 at position D475, wherein D is substituted by any one of E, D, K; (5) an amino acid substitution relative to SEQ ID NO: 104 at position Q476, wherein Q is substituted by any one of Q, R, D, A, T, S, K; (6) an amino acid substitution relative to SEQ ID NO: 104 at position S477, wherein S is substituted by any one of I, V, L; and (7) an amino acid substitution relative to SEQ ID NO: 104 The amino acid at position N478 is substituted, wherein N is substituted by any one of K, E, N, S; (8) The amino acid at position R479 relative to SEQ ID NO: 104 is substituted, wherein R is substituted by any one of T, D, E, S, Y, A; (9) The amino acid at position I480 relative to SEQ ID NO: 104 is substituted, wherein I is substituted by any one of L, N; (10) The amino acid at position L481 relative to SEQ ID NO: 104 is substituted, wherein L is substituted by any one of T, D, E, K, Q, S, N; (11) The amino acid at position S482 relative to SEQ ID NO: 104 is substituted, wherein S is substituted by any one of E, D, S, T, Q, K; (12) The amino acid at position S483 relative to SEQ ID NO: 104 is substituted, wherein S is substituted by any one of R, K, L, E, A; (13) The amino acid at position S483 relative to SEQ ID NO: 104 is substituted, wherein S is substituted by any one of R, K, L, E, A; (14) The amino acid at position A484 of SEQ ID NO: 104 is substituted, wherein A is substituted by any of V, I, or M; (15) The amino acid at position E485 of SEQ ID NO: 104 is substituted, wherein E is substituted by any of E, A, K, H, Q, S, or N; (16) The amino acid at position K486 of SEQ ID NO: 104 is substituted, wherein K is substituted by any of S, E, K, R, V, D, or H; (17) The amino acid at position G487 of SEQ ID NO: 104 is substituted, wherein G is substituted by any of I or L; (18) The amino acid at position N488 of SEQ ID NO: 104 is substituted, wherein N is substituted by any of E, K, or R; (19) The amino acid at position T489 of SEQ ID NO: 104 is substituted.Where T is replaced by any one of K, E, S, R, Q, (19) the amino acid at position S490 of SEQ ID NO: 104 is replaced by any one of E, V, T, R, L, (20) the amino acid at position G491 of SEQ ID NO: 104 is replaced by any one of GL, I, V, (21) the amino acid at position R492 of SEQ ID NO: 104 is replaced by any one of E, Q, S, A, D, (22) the amino acid at position E493 of SEQ ID NO: 104 is replaced by any one of A, E, N, L, K, Q, S, (23) the amino acid at position N494 of SEQ ID NO: 104 is replaced by any one of I, L, (24) the amino acid at position N494 of SEQ ID NO: 104 is replaced by any one of SEQ ID NO: 104. An amino acid substitution at position L495, wherein L is substituted by any of K, L, T, V, I; (25) An amino acid substitution at position Y496 relative to SEQ ID NO: 104, wherein Y is substituted by any of K, E, R, Q; (26) An amino acid substitution at position F497 relative to SEQ ID NO: 104, wherein F is substituted by any of D, R, E, Q; (27) An amino acid substitution at position Q498 relative to SEQ ID NO: 104, wherein Q is substituted by any of V, L; and / or (28) Any combination of (1)-(27). In some embodiments, the segment comprises a polypeptide sequence listed in Table 6D, or a polypeptide sequence having 1 to 5 amino acid substitutions.

[0224] In some embodiments, the extracellular domain further comprises one, two, three or more amino acid substitutions relative to SEQ ID NO: 104 at positions 63, 97, 98, 99, 100, 101, 102, 140, 147, 153, 185, 188, 219, 231, 294, 365, 368, 450, 463 or 470.

[0225] Human parainfluenza virus types 3 (PIV3) and 5 (PIV5) PIV is a negative-sense single-stranded RNA virus that causes a variety of respiratory diseases. It is a major cause of acute respiratory infections prevalent in infants and early childhood. The PIV F protein promotes viral fusion and cell entry.

[0226] The illustrative sequence of the native PIV3 F protein is shown in Table 7A.

[0227] Table 7A

[0228] In some implementations, the extracellular domain has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 327.

[0229] C-end spiral formation section The illustrative sequence of the C-terminal α-helical region is shown in Table 7B (Rosetta remodeling). Residues 456-459 of the native PIV3 F protein are included as ISIE (bold underline) and are conserved along with the native sequence in these embodiments, while many other amino acid residues are modified.

[0230] Table 7B. C-terminal α-helical region of PIV3 (Rosetta remodeling)

[0231] In some embodiments, the C-terminal helical forming region contains 20 to about 30 residues. In some embodiments, the C-terminal helical forming region contains 20 to about 25 residues. In some embodiments, the C-terminal helical forming region contains 25 to about 30 residues.

[0232] In some implementations, modeling has shown that the following substitutions will stabilize the F protein moiety in a helical conformation. Illustrative sequences of possible substitutions are shown in Table 7C.

[0233] Table 7C. Possible replacements at positions 460-477 (Rosetta remodeling)

[0234] An illustrative sequence of the C-terminal α-helical region is shown in Table 7D (RF diffusion). Residues 456-464 of the native MPV F protein are included as ISIELNKAK (bold underline) (or, ISIELNKVK) and are conserved along with the native sequence in these embodiments, while many other amino acid residues are modified.

[0235] Table 7D. C-terminal α-helical region of PIV3 (RF diffusion)

[0236] In some embodiments, the C-terminal helical forming region comprises 10 to 30 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 20 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 15 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 30 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 20 residues. In some embodiments, the C-terminal helical forming region comprises 20 to 30 residues. In some embodiments, the C-terminal helical forming region comprises 20 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 25 to 30 residues.

[0237] In some implementations, modeling has shown that the following substitutions will stabilize the F protein moiety in a helical conformation. Illustrative sequences of possible substitutions are shown in Table 7E.

[0238] Table 7E. Possible substitutions (RF diffusion) at positions 465-486

[0239] In some embodiments, the recombinant polypeptide comprises an engineered extracellular domain of a PIV3 fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 327 between about residues 460 and about residues 490, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 327, the residues creating hydrophobic contacts between segments in the α-helical homotrimer. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 104 between about residues 460 and about residues 480, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 20 to about 28 residues.

[0240] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 327 at position L460, wherein L is substituted by any one of L, M, and V; (2) an amino acid substitution relative to SEQ ID NO: 327 at position N461, wherein N is substituted by N; (3) an amino acid substitution relative to SEQ ID NO: 327 at position K462, wherein K is substituted by any one of K and R; (4) an amino acid substitution relative to SEQ ID NO: 327 at position V463, wherein V is substituted by any one of L, V, and T; (5) an amino acid substitution relative to SEQ ID NO: 327 at position K464, wherein K is substituted by any one of A, K, and Q; (6) an amino acid substitution relative to SEQ ID NO: 327 at position S465, wherein S is substituted by any one of K and S; (7) an amino acid substitution relative to SEQ ID NO: 327 at position D466, wherein D is substituted by any one of E and K; and (8) an amino acid substitution relative to SEQ ID NO: 327 at position D466, wherein D is substituted by any one of E and K. (9) The amino acid substitution at position L467 of SEQ ID NO: 327, wherein L is substituted by any one of V, L, or T; (10) The amino acid substitution at position E468 of SEQ ID NO: 327, wherein E is substituted by any one of K, D, or E; (11) The amino acid substitution at position S470 of SEQ ID NO: 327, wherein S is substituted by any one of I, L, M, Y, F, or W; (12) The amino acid substitution at position K471 of SEQ ID NO: 327, wherein K is substituted by any one of L, W, A, or I; (13) The amino acid substitution at position E472 of SEQ ID NO: 327, wherein E is substituted by any one of K or E; (14) The amino acid substitution at position E468 of SEQ ID NO: 327, wherein E is substituted by any one of K or E; 327 The amino acid at position W473 is substituted, wherein W is substituted by any of E, I, K, Q, (15) The amino acid at position Y474 relative to SEQ ID NO: 327 is substituted, wherein Y is substituted by any of L, M, T, V, E, (16) The amino acid at position R475 relative to SEQ ID NO: 327 is substituted, wherein R is substituted by any of S, K, R, A, (17) The amino acid at position R476 relative to SEQ ID NO: 327 is substituted, wherein R is substituted by any of K, E, S, N, (18) The amino acid at position S477 relative to SEQ ID NO: 327 is substituted, wherein S is substituted by any of K, D, E, and (19) Any combination of (1)-(18).

[0241] In some embodiments, the segment comprises a polypeptide sequence listed in Table 7B, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment relative to SEQ ID NO: 327, between about residues 465 and about residues 490, comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 14 to about 30 residues.

[0242] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 327 at position S465, wherein S is substituted by any one of E, K, D, S, N, Q, T, R, A; (2) an amino acid substitution relative to SEQ ID NO: 327 at position D466, wherein D is substituted by any one of D, R, K, E, M, Q, A, S, N; (3) an amino acid substitution relative to SEQ ID NO: 327 at position L467, wherein L is substituted by any one of I, V, L; (4) an amino acid substitution relative to SEQ ID NO: 327 at position E468, wherein E is substituted by any one of E, K, S, D, R, H, T, N, A; (5) an amino acid substitution relative to SEQ ID NO: 327 at position E469, wherein E is substituted by any one of K, S, E, N, T, Q, H, D, Y; and (6) an amino acid substitution relative to SEQ ID NO: 327 substituted amino acid at position S470, wherein S is substituted by any one of L, D, V, I, A, N, T; (7) substituted amino acid at position K471 relative to SEQ ID NO: 327, wherein K is substituted by any one of E, T, L, K, N, I, R, Q, S; (8) substituted amino acid at position E472 relative to SEQ ID NO: 327, wherein E is substituted by any one of E, K, Q, S, H, R, T; (9) substituted amino acid at position W473 relative to SEQ ID NO: 327, wherein W is substituted by any one of R, Q, K, E, T, S, I, N; (10) substituted amino acid at position Y474 relative to SEQ ID NO: 327, wherein Y is substituted by any one of V, L, I, Q, T; (11) substituted amino acid at position Y474 relative to SEQ ID NO: 327. 327 substituted amino acid at position R475, wherein R is substituted by any one of H, K, D, T, E, S, R, N, Q, A, (12) relative to SEQ ID NO: 327 substituted amino acid at position R476, wherein R is substituted by any one of A, T, H, E, D, K, R, Q, S, (13) relative to SEQ ID NO: 327 substituted amino acid at position S477, wherein S is substituted by any one of I, L, V, (14) relative to SEQ ID NO: 327 substituted amino acid at position N478, wherein N is substituted by any one of E, L, K, I, R, S, Q, (15) relative to SEQ ID NO: 327 substituted amino acid at position Q479, wherein Q is substituted by any one of K, H, E, N, Q, R, T, A, S, (16) relative to SEQ ID NO: 327 is an amino acid substitution at position K480, wherein K is replaced by any one of K, R, E, T, S, L, A, I, or V.(17) An amino acid substituted relative to SEQ ID NO: 327 at position L481, wherein L is substituted by any one of L, V, or I; (18) An amino acid substituted relative to SEQ ID NO: 327 at position D482, wherein D is substituted by any one of K, A, E, S, H, T, N, D, or R; (19) An amino acid substituted relative to SEQ ID NO: 327 at position S483, wherein S is substituted by any one of Q, T, E, A, S, N, D, K, or L; (20) An amino acid substituted relative to SEQ ID NO: 327 at position I484, wherein I is substituted by any one of I, L, A, or V; (21) An amino acid substituted relative to SEQ ID NO: 327 at position G485, wherein G is substituted by any one of L, K, R, E, or I; (22) An amino acid substituted relative to SEQ ID NO: 327 is an amino acid substitution at position S486, wherein S is substituted by any of T, A, E, R, H, D, S, and / or any combination of (23) (1)-(22). In some embodiments, the segment comprises a polypeptide sequence listed in Table 7D, or a polypeptide sequence having 1 to 5 amino acid substitutions.

[0243] The illustrative sequence of the native PIV5 F protein is shown in Table 8A.

[0244] Table 8A

[0245] In some implementations, the extracellular domain of the PIV5 protein has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 382.

[0246] C-end spiral formation section The illustrative sequence of the C-terminal α-helical region is shown in Table 8B (Rosetta remodeling). Residues 459-462 of the native PIV5 F protein are included as SLSD (bold underline) and are conserved along with the native sequence in these embodiments, while many other amino acid residues are modified.

[0247] Table 8B. C-terminal α-helical region of PIV5 (Rosetta remodeling)

[0248] In some embodiments, the C-terminal helical forming region comprises 5 to 30 residues. In some embodiments, the C-terminal helical forming region comprises 5 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 5 to 20 residues. In some embodiments, the C-terminal helical forming region comprises 5 to 15 residues. In some embodiments, the C-terminal helical forming region comprises 5 to 10 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 30 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 20 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 15 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 30 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 20 residues.

[0249] In some implementations, modeling has shown that the following substitutions will stabilize the F protein moiety in a helical conformation. Illustrative sequences of possible substitutions are shown in Table 8C.

[0250] Table 8C. Possible replacements at positions 463-488 (Rosetta remodeling)

[0251] In some embodiments, the recombinant polypeptide comprises an engineered extracellular domain of a PIV5 fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 382 between about residues 460 and about residues 490, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 382, ​​the residues creating hydrophobic contacts between segments in the α-helical homotrimer. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 104 between about residues 460 and about residues 480, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 6 to about 26 residues.

[0252] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 382 at position A463, wherein A is substituted by any one of L, T, V, and A; (2) an amino acid substitution relative to SEQ ID NO: 382 at position L464, wherein L is substituted by any one of K, I, Q, A, W, and E; (3) an amino acid substitution relative to SEQ ID NO: 382 at position Q465, wherein Q is substituted by any one of K, Q, T, E, S, and R; (4) an amino acid substitution relative to SEQ ID NO: 382 at position H466, wherein H is substituted by any one of K, A, E, L, I, W, R, Q, T, D, and Y; (5) an amino acid substitution relative to SEQ ID NO: 382 at position L467, wherein L is substituted by any one of V, I, L, M, FA, T, C, and H; and (6) an amino acid substitution relative to SEQ ID NO: 382 substituted amino acid at position A468, wherein A is substituted by any one of D, T, K, L, E, R, I, N, S; (7) substituted amino acid at position Q469 relative to SEQ ID NO: 382, ​​wherein Q is substituted by any one of E, K, S, T, A, R, Q, D; (8) substituted amino acid at position S470 relative to SEQ ID NO: 382, ​​wherein S is substituted by any one of A, K, L, I, T, S, V, H, Y, E, W, FR, Q, M; (9) substituted amino acid at position D471 relative to SEQ ID NO: 382, ​​wherein D is substituted by any one of T, E, V, L, S, I, A, K, Y, W; (10) substituted amino acid at position T472 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of K, E, R, S, T, A, D, L; (11) substituted amino acid at position T472 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of K, E, R, S, T, A, D, L; (11) substituted amino acid at position T472 relative to SEQ ID NO: 382, ​​wherein Q is substituted by any one of E, K, S, T, A, D, L; (12) The amino acid at position Y473 of SEQ ID NO: 382 is substituted, wherein Y is substituted by any one of T, K, S, R, Q, D, E, I, H, M; (13) The amino acid at position L474 of SEQ ID NO: 382 is substituted, wherein L is substituted by any one of T, S, L, A, D, W, Q, I, Y, V, K, E; (14) The amino acid at position S475 of SEQ ID NO: 382 is substituted, wherein S is substituted by any one of T, E, I, K, S, Q, A, L, R, D; (15) The amino acid at position I477 of SEQ ID NO: 382 is substituted, wherein I is substituted by any one of K, Q, R, D, T, E, I, Y, S, L.(16) An amino acid substituted at position T478 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of E, K, S, D, W, L, Q, I, T; (17) An amino acid substituted at position S479 relative to SEQ ID NO: 382, ​​wherein S is substituted by any one of R, K, Q, S, A, D, E; (18) An amino acid substituted at position A480 relative to SEQ ID NO: 382, ​​wherein A is substituted by any one of S, K; (19) An amino acid substituted at position T481 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of E, D, S, K, M, N, A, T; (20) An amino acid substituted at position T482 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of R, S, Q, L, K; (21) An amino acid substituted at position T482 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of R, S, Q, L, K; (22) The amino acid substitution at position T483 of SEQ ID NO: 382, ​​wherein T is substituted by any of K, A, or S; (23) The amino acid substitution at position S484 of SEQ ID NO: 382, ​​wherein S is substituted by any of S, E, D, or Y; (24) The amino acid substitution at position V485 of SEQ ID NO: 382, ​​wherein V is substituted by any of Q or T; (25) The amino acid substitution at position L486 of SEQ ID NO: 382, ​​wherein L is substituted by K; (26) The amino acid substitution at position S487 of SEQ ID NO: 382, ​​wherein S is substituted by any of S or K; (27) The amino acid substitution at position I488 of SEQ ID NO: 382, ​​wherein I is substituted by any of S or K; and / or (28) Any combination of (1)-(26).

[0253] In some embodiments, the segment comprises a polypeptide sequence listed in Table 8B, or a polypeptide sequence having one to five amino acid substitutions.

[0254] SARS-CoV-2 SARS-CoV-2 is a single-sense positive-sense RNA virus that can cause severe respiratory illness in humans. The SARS-CoV-2 viral spike (S) protein, a trimeric class I fusion glycoprotein, binds to angiotensin-converting enzyme 2 (ACE2), the entry receptor utilized by SARS-CoV-2. The coronavirus spike (S) protein is a major surface protein and a target of neutralizing antibodies in infected subjects or patients. Therefore, it should be considered a potential protective antigen in vaccine design.

[0255] Table 9A

[0256] In some implementations, the extracellular domain of the SARS-CoV-2 spike (S) protein has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 459.

[0257] C-end spiral formation section The illustrative sequence of the C-terminal α-helical region is shown in Table 9B (Rosetta remodeling). Residues 1147-1170 of the native SARS-CoV-2 S protein are included as LQPEL (bold underline) and are conserved along with the native sequence in these embodiments, while many other amino acid residues are modified.

[0258] Table 9B. C-terminal α-spiral region of SARS (Rosetta remodeling)

[0259] In some embodiments, the C-terminal helical forming region contains 10 to about 25 residues. In some embodiments, the C-terminal helical forming region contains 10 to about 20 residues. In some embodiments, the C-terminal helical forming region contains 10 to about 15 residues. In some embodiments, the C-terminal helical forming region contains 15 to about 25 residues. In some embodiments, the C-terminal helical forming region contains 15 to about 20 residues.

[0260] In some implementations, modeling has shown that the following substitutions will stabilize the F protein moiety in a helical conformation. Illustrative sequences of possible substitutions are shown in Table 9C. The numbers in this table reflect individual amino acid substitutions relative to the reference sequences described above.

[0261] Table 9C. Possible replacements at positions 1147-1170 (Rosetta remodeling)

[0262] The illustrative sequence of the C-terminal α-helical region is shown in Table 9D (RF diffusion). Residues 1147-1165 of the native SARS-CoV-2 spike (S) protein are included as LQPEL (bold underline) and are conserved along with the native sequence in these embodiments, while many other amino acid residues are modified.

[0263] Table 9D. C-terminal α-spiral region of SARS (RF spread)

[0264] In some embodiments, the C-terminal helical forming region contains 10 to about 25 residues. In some embodiments, the C-terminal helical forming region contains 10 to about 20 residues. In some embodiments, the C-terminal helical forming region contains 15 to about 25 residues.

[0265] In some implementations, modeling has shown that the following substitutions will stabilize the F protein moiety in a helical conformation. Illustrative sequences of possible substitutions are shown in Table 9E.

[0266] Table 9E. Possible substitutions (RF diffusion) at locations 1147-1165

[0267] In some embodiments, the engineered extracellular domain of the SARS-CoV2 spike (S) protein (wherein the extracellular domain comprises a C-terminal helical forming segment relative to SEQ ID NO: 459 between about residues 1140 and about residues 1170) contains one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 459, the residues creating hydrophobic contacts between segments in the α-helical homotrimer. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment relative to SEQ ID NO: 459 between about residues 1140 and about residues 1170, comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 10 to about 25 residues.

[0268] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 459 at position D1147, wherein D is substituted by any one of E, D, and K; (2) an amino acid substitution relative to SEQ ID NO: 459 at position S1148, wherein S is substituted by any one of T, S, and K; (3) an amino acid substitution relative to SEQ ID NO: 459 at position F1149, wherein F is substituted by A; (4) an amino acid substitution relative to SEQ ID NO: 459 at position K1150, wherein K is substituted by any one of I, A, L, and M; (5) an amino acid substitution relative to SEQ ID NO: 459 at position E1151, wherein E is substituted by any one of K, S, D, R, and E; (6) an amino acid substitution relative to SEQ ID NO: 459 at position E1152, wherein E is substituted by any one of I, Y, K, T, R, and E; and (7) an amino acid substitution relative to SEQ ID NO: 459 substituted amino acid at position L1153, wherein L is substituted by any one of T or A; (8) substituted amino acid at position D1154 relative to SEQ ID NO: 459, wherein D is substituted by any one of L, I, E, T, M, V; (9) substituted amino acid at position K1155 relative to SEQ ID NO: 459, wherein K is substituted by any one of E, K, T, R; (10) substituted amino acid at position Y1156 relative to SEQ ID NO: 459, wherein Y is substituted by any one of I, V, K, R; (11) substituted amino acid at position F1157 relative to SEQ ID NO: 459, wherein F is substituted by any one of V, A, I, Y, T, S; (12) substituted amino acid at position K1158 relative to SEQ ID NO: 459, wherein K is substituted by any one of L, R, S, K, D, W, N, I; (13) substituted amino acid at position K1158 relative to SEQ ID NO: 459 is substituted with an amino acid at position N1159, wherein N is substituted with any one of K, T, Q, I, R, E; (14) is substituted with an amino acid at position H1160 relative to SEQ ID NO: 459, wherein H is substituted with any one of I, L, R, E, K, S; (15) is substituted with an amino acid at position T1161 relative to SEQ ID NO: 459, wherein T is substituted with any one of L, N, I, A, S, W, Y; (16) is substituted with an amino acid at position S1162 relative to SEQ ID NO: 459, wherein S is substituted with any one of K, S, T, R; (17) is substituted with an amino acid at position P1163 relative to SEQ ID NO: 459, wherein P is substituted with any one of E, D, R, K, I, A.(18) An amino acid substitution relative to SEQ ID NO: 459 at position D1164, wherein D is substituted by any one of W, S, M, D, T, I, N; (19) An amino acid substitution relative to SEQ ID NO: 459 at position V1165, wherein V is substituted by any one of E, A, K, L; (20) An amino acid substitution relative to SEQ ID NO: 459 at position D1166, wherein D is substituted by any one of K, S; (21) An amino acid substitution relative to SEQ ID NO: 459 at position L1167, wherein L is substituted by any one of R, K; (22) An amino acid substitution relative to SEQ ID NO: 459 at position G1168, wherein G is substituted by any one of K, S; (23) An amino acid substitution relative to SEQ ID NO: 459 at position D1169, wherein D is substituted by S; (24) An amino acid substitution relative to SEQ ID NO: 459. Amino acid substitution at position I1170, where I is substituted by S, and / or any combination of (25)(1)-(24).

[0269] In some embodiments, the segment comprises a polypeptide sequence listed in Table 9B, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment relative to SEQ ID NO: 459, between about residues 1145 and about residues 1175, comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 12 to about 22 residues.

[0270] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 459 at position D1147, wherein D is substituted by any one of Q, T, E, S, N, D, K; (2) an amino acid substitution relative to SEQ ID NO: 459 at position S1148, wherein S is substituted by any one of T, K, N, R, S, A, E; (3) an amino acid substitution relative to SEQ ID NO: 459 at position F1149, wherein F is substituted by any one of L, T, I, V; (4) an amino acid substitution relative to SEQ ID NO: 459 at position K1150, wherein K is substituted by any one of K, Q, R, H, S, E; (5) an amino acid substitution relative to SEQ ID NO: 459 at position E1151, wherein E is substituted by any one of E, N, A, S, K, T, D; and (6) an amino acid substitution relative to SEQ ID NO: 459 substituted amino acid at position E1152, wherein E is substituted by any one of E, T, V, R, K, N; (7) substituted amino acid at position L1153 relative to SEQ ID NO: 459, wherein L is substituted by any one of S, V, T, A; (8) substituted amino acid at position D1154 relative to SEQ ID NO: 459, wherein D is substituted by any one of T, L, E, I, V; (9) substituted amino acid at position K1155 relative to SEQ ID NO: 459, wherein K is substituted by any one of H, E, S, T, Q; (10) substituted amino acid at position Y1156 relative to SEQ ID NO: 459, wherein Y is substituted by any one of L, E, I, T, A, S, R; (11) substituted amino acid at position F1157 relative to SEQ ID NO: 459, wherein F is substituted by any one of T, V, I, A, S, M; (12) substituted amino acid at position F1157 relative to SEQ ID NO: 459. (13) The amino acid at position K1158 of SEQ ID NO: 459 is substituted, wherein K is substituted by any one of K, E, N, R, T, A, Q, I; (14) The amino acid at position H1160 of SEQ ID NO: 459 is substituted, wherein H is substituted by any one of L, M, A, E, T, Y, I, S; (15) The amino acid at position T1161 of SEQ ID NO: 459 is substituted, wherein T is substituted by any one of L, I; (16) The amino acid at position S1162 of SEQ ID NO: 459 is substituted, wherein S is substituted by any one of S, R, K, N, E, Q; (17) The amino acid at position P1163 of SEQ ID NO: 459 is substituted.Wherein P is substituted by any of E, S, T, R, (18) the amino acid at position D1164 of SEQ ID NO:459 is substituted, wherein D is substituted by any of T, M, A, (19) the amino acid at position V1165 of SEQ ID NO:459 is substituted, wherein V is substituted by any of A, L, and / or (20) any combination of (1)-(19).

[0271] In some embodiments, the segment comprises a polypeptide sequence listed in Table 9D, or a polypeptide sequence having one to five amino acid substitutions.

[0272] Nipah virus Nipah virus is a highly pathogenic virus that has caused sporadic outbreaks of serious neurological and respiratory diseases.

[0273] Table 10A

[0274] In some embodiments, the extracellular domain of the Nipah F protein has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 499.

[0275] C-end spiral formation section The illustrative sequence of the C-terminal α-helical region is shown in Table 10B (Rosetta remodeling). Residues 460-462 of the native Nipah F protein are included as the ISS (bold underline) and are conserved along with the native sequence in these embodiments, while many other amino acid residues are modified.

[0276] Table 10B. Nipah's C-terminal α-helical segment (Rosetta remodeling)

[0277] In some embodiments, the C-terminal helical forming region comprises 15 to 35 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 30 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 20 residues. In some embodiments, the C-terminal helical forming region comprises 20 to 35 residues. In some embodiments, the C-terminal helical forming region comprises 20 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 25 to 35 residues. In some embodiments, the C-terminal helical forming region comprises 25 to 30 residues.

[0278] In some implementations, modeling has shown that the following substitutions will stabilize the F protein moiety in a helical conformation. Illustrative sequences of possible substitutions are shown in Table 10C.

[0279] Table 10C. Possible replacements at positions 463-489 (Rosetta remodeling)

[0280] The illustrative sequence of the C-terminal α-helical region is shown in Table 10D (RF diffusion). Residues 460-462 of the native Nipah F protein are included as the ISS (bold underline) and are conserved along with the native sequence in these embodiments, while many other amino acid residues are modified.

[0281] Table 10D. Nipah's C-terminal α-helical region (RF diffusion)

[0282] In some embodiments, the C-terminal helical forming region comprises 10 to 30 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 20 residues. In some embodiments, the C-terminal helical forming region comprises 10 to 15 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 30 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 25 residues. In some embodiments, the C-terminal helical forming region comprises 15 to 20 residues. In some embodiments, the C-terminal helical forming region comprises 20 to 30 residues. In some embodiments, the C-terminal helical forming region comprises 20 to 25 residues.

[0283] In some implementations, modeling has shown that the following substitutions will stabilize the F protein moiety in a helical conformation. Illustrative sequences of possible substitutions are shown in Table 10E.

[0284] Table 10E. Possible substitutions (RF diffusion) at positions 463-489

[0285] In some embodiments, the recombinant polypeptide comprises an engineered extracellular domain of the Nipah fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 499 between about residues 460 and about residues 490, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 499, the residues creating hydrophobic contacts between segments in the α-helical homotrimer. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO: 499 between about residues 460 and about residues 490, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 16 to about 33 residues.

[0286] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 499 at position M463, wherein M is substituted by any one of I, A, T, L, M; (2) an amino acid substitution relative to SEQ ID NO: 499 at position N464, wherein N is substituted by N; (3) an amino acid substitution relative to SEQ ID NO: 499 at position Q465, wherein Q is substituted by any one of E, L, K, T, S, I, D; (4) an amino acid substitution relative to SEQ ID NO: 499 at position S466, wherein S is substituted by S; (5) an amino acid substitution relative to SEQ ID NO: 499 at position L467, wherein L is substituted by any one of M, L, I, V, T; (6) an amino acid substitution relative to SEQ ID NO: 499 at position Q468, wherein Q is substituted by any one of E, K, A, T, S, D, R, I, Q; and (7) an amino acid substitution relative to SEQ ID NO: 499 substituted with an amino acid at position Q469, wherein Q is substituted by any one of R, S, K, T, E, Q; (8) substituted with an amino acid at position S470 relative to SEQ ID NO: 499, wherein S is substituted by any one of T, L, I, V, A; (9) substituted with an amino acid at position K471 relative to SEQ ID NO: 499, wherein K is substituted by any one of K, A, W, E, L, I; (10) substituted with an amino acid at position D472 relative to SEQ ID NO: 499, wherein D is substituted by any one of K, T, R, Q, E; (11) substituted with an amino acid at position Y473 relative to SEQ ID NO: 499, wherein Y is substituted by any one of W, D, K, Y, I, M, E, T; (12) substituted with an amino acid at position I474 relative to SEQ ID NO: 499, wherein I is substituted by any one of I, V, M, L, A; (13) substituted with an amino acid at position I474 relative to SEQ ID NO: 499; (14) The amino acid at position K475 of SEQ ID NO: 499 is substituted, wherein K is substituted by any one of T, K, M, R, E, L, A, S; (15) The amino acid at position E476 of SEQ ID NO: 499 is substituted, wherein E is substituted by any one of K, S, A, E, T, D; (16) The amino acid at position A477 of SEQ ID NO: 499 is substituted, wherein A is substituted by any one of L, I, V, FT, A, M, W, K, Y; (17) The amino acid at position Q478 of SEQ ID NO: 499 is substituted, wherein Q is substituted by any one of I, K, A, L, E, D, S, Y; (18) The amino acid at position R479 of SEQ ID NO: 499 is substituted, wherein R is substituted by any one of A, S, K, R, T, L, E.(18) An amino acid substituted relative to SEQ ID NO: 499 at position L480, wherein L is substituted by any one of K, E, R, Y, T, Q; (19) An amino acid substituted relative to SEQ ID NO: 499 at position L481, wherein L is substituted by any one of W, I, V, L, E, S, Q, A, T; (20) An amino acid substituted relative to SEQ ID NO: 499 at position D482, wherein D is substituted by any one of K, Q, E, W, T, S, A; (21) An amino acid substituted relative to SEQ ID NO: 499 at position T483, wherein T is substituted by any one of S, T, K, R, Q; (22) An amino acid substituted relative to SEQ ID NO: 499 at position V484, wherein V is substituted by any one of R, E, I, S, Y, L, K, D; (23) An amino acid substituted relative to SEQ ID NO: 499 substituted with an amino acid at position N485, wherein N is substituted with any of I, R, K, W, E, T; (24) substituted with an amino acid at position P486 relative to SEQ ID NO: 499, wherein P is substituted with any of A, T, R, K, Q; (25) substituted with an amino acid at position S487 relative to SEQ ID NO: 499, wherein S is substituted with any of K, R, T, S; (26) substituted with an amino acid at position L488 relative to SEQ ID NO: 499, wherein L is substituted with any of E, V, L, K; (27) substituted with an amino acid at position I489 relative to SEQ ID NO: 499, wherein I is substituted with any of E, Q, L, K, R; and / or (28) any combination of (1)-(27).

[0287] In some embodiments, the segment comprises a polypeptide sequence listed in Table 10B, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the extracellular domain comprises a C-terminal helical forming segment relative to SEQ ID NO: 499, between about residues 460 and about residues 490, comprising one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the C-terminal helical forming segment comprises about 12 to about 26 residues.

[0288] In some embodiments, the segment comprises (1) an amino acid substitution relative to SEQ ID NO: 499 at position M463, wherein M is substituted by any one of L, I, V; (2) an amino acid substitution relative to SEQ ID NO: 499 at position N464, wherein N is substituted by N; (3) an amino acid substitution relative to SEQ ID NO: 499 at position Q465, wherein Q is substituted by any one of Q, T, N, D, E, S, K, R, A; (4) an amino acid substitution relative to SEQ ID NO: 499 at position S466, wherein S is substituted by S; (5) an amino acid substitution relative to SEQ ID NO: 499 at position L467, wherein L is substituted by any one of I, V, L, A; (6) an amino acid substitution relative to SEQ ID NO: 499 at position Q468, wherein Q is substituted by any one of S, K, T, D, E, R, Q; and (7) an amino acid substitution relative to SEQ ID NO: 499 substituted with an amino acid at position Q469, wherein Q is substituted by any one of S, Q, N, E, A, D, K, T, R; (8) substituted with an amino acid at position S470 relative to SEQ ID NO: 499, wherein S is substituted by any one of L, A, I, V, N; (9) substituted with an amino acid at position K471 relative to SEQ ID NO: 499, wherein K is substituted by any one of E, Q, S, R, K, A, T, D, L, N; (10) substituted with an amino acid at position D472 relative to SEQ ID NO: 499, wherein D is substituted by any one of K, T, E, Q, D, N, S, A; (11) substituted with an amino acid at position Y473 relative to SEQ ID NO: 499, wherein Y is substituted by any one of A, S, R, T, V, E, I, K, L, D, Q; (12) substituted with an amino acid at position Y473 relative to SEQ ID NO: 499. (13) The amino acid at position I474 of SEQ ID NO: 499 is substituted, wherein I is substituted by any one of L, I, V; (14) The amino acid at position K475 of SEQ ID NO: 499 is substituted, wherein K is substituted by any one of K, H, D, T, A, S, R, Q, E, N; (15) The amino acid at position E476 of SEQ ID NO: 499 is substituted, wherein E is substituted by any one of K, R, S, E, A, T, H, D; (16) The amino acid at position A477 of SEQ ID NO: 499 is substituted, wherein A is substituted by any one of A, L, I, V; (17) The amino acid at position R479 of SEQ ID NO: 499 is substituted.(18) Relative to the amino acid at position L480 of SEQ ID NO: 499, wherein L is replaced by any one of D, L, E, K, T, R, V, I, Q; (19) Relative to the amino acid at position L481 of SEQ ID NO: 499, wherein L is replaced by any one of L, V, I; (20) Relative to the amino acid at position D482 of SEQ ID NO: 499, wherein D is replaced by any one of E, K, N, D, L, Q, H; (21) Relative to the amino acid at position T483 of SEQ ID NO: 499, wherein T is replaced by any one of E, K, S, Q, A, T; (22) Relative to the amino acid at position V484 of SEQ ID NO: 499, wherein V is replaced by any one of V, L, I; (23) Relative to SEQ ID NO: 499 substituted with an amino acid at position N485, wherein N is substituted with any one of R, K, L, V, E, Q, I; (24) substituted with an amino acid at position P486 relative to SEQ ID NO: 499, wherein P is substituted with any one of R, E, A, S, L; (25) substituted with an amino acid at position S487 relative to SEQ ID NO: 499, wherein S is substituted with any one of Q, R, T, S, L; (26) substituted with an amino acid at position L488 relative to SEQ ID NO: 499, wherein L is substituted with any one of L; and / or (27) any combination of (1)-(26).

[0289] In some embodiments, the segment comprises a polypeptide sequence listed in Table 10D, or a polypeptide sequence having one to five amino acid substitutions.

[0290] III. Protein Nanostructures This disclosure further provides protein nanostructures comprising any of the engineered extracellular domains described herein. For example, this disclosure provides protein nanostructures comprising a trimeric component and a pentameric component, the trimeric component comprising a recombinant polypeptide comprising an extracellular domain of a respiratory syncytial virus (RSV) viral membrane fusion (F) protein having an engineered C-terminal α-helical segment of the F protein stably in its pre-fusion conformation.

[0291] Compositions are further provided in which any of the α-helical segments described herein is used as a fusion with a trimeric protein complex or with a trimeric component of a nanostructure to stabilize the complex or component. For example, the α-helical segments described herein can be used without any antigen (e.g., an extracellular domain) or with an antigen or other molecule that is attached to the complex or nanostructure by other means (such as bioconjugation chemistry). In some embodiments, the α-helical segments described herein are used as a fusion protein with a monomeric antigen, including but not limited to the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein.

[0292] The protein nanostructures of the present invention can comprise multimeric protein assemblies suitable for displaying molecules such as antigens (e.g., engineered extracellular domains). In some embodiments described herein, the protein nanostructure comprises at least a first component displaying an engineered extracellular domain and an optional second component. The engineered extracellular domain may comprise one or more amino acid substitutions, C-terminal helical formation segments, or combinations thereof. The first component may comprise or consist of three copies of a fusion protein. In some embodiments, the fusion protein comprises an assembly domain having a protein sequence designed by computational methods to assemble into the nanostructure. In some embodiments, the first component is a trimer wherein the assembly domain forms a trimer associated by 3-fold rotational symmetry, and / or the second component is a pentamer wherein the assembly domain forms a pentamer associated by 5-fold rotational symmetry. In some embodiments, a combination of the two components forms an "icosahedral particle" with I53 symmetry. These components can be arranged together such that members of each component are associated with each other by a symmetry operator. A general computational method for designing self-assembled protein materials, involving the symmetry docking of protein building blocks in a target symmetric architecture, is disclosed in patent publication number US 2015 / 0356240 A1.

[0293] The term "core" in this document is used to describe the central portion of a protein nanostructure. For clarity, the term "core" as used herein does not include the molecule displayed by the nanostructure. The core can be used to assemble multiple copies of the displayed molecule, such as an antigen (e.g., an engineered extracellular domain). Without being bound by theory, this can increase the immunogenicity of the antigen. This disclosure contemplates nanostructures in which the core is non-covalently associated with the displayed antigen; covalently linked to the displayed antigen (e.g., by chemical conjugation); or, in a preferred embodiment, linked to the displayed antigen via a polypeptide linker in a fusion protein. In some embodiments, the fusion protein comprises a first polypeptide containing an antigen (e.g., an extracellular domain) and a first assembly domain. In some embodiments, the antigen (e.g., the extracellular domain) is non-covalently or covalently linked to the assembly domain. For example, the antigen (e.g., the extracellular domain) can be fused to a first component and configured to bind a portion of the first component or a chemical tag on the first component. For example, a streptavidin-Biotin (or neutral avidin-Biotin) linker can be used. Alternatively, various bioconjugated linkers can be used. In some embodiments of this disclosure, the antigen may contain other polypeptide sequences in addition to the RSV F protein.

[0294] In some embodiments, three copies of the antigenic (e.g., extracellular domain) peptide are displayed on a 3x axis. Therefore, the protein nanostructure is capable of displaying 60 monomeric antigenic (e.g., extracellular domain) peptides. In some embodiments, the protein nanostructure is adapted to display up to 12, 24, or 60 monomers. In some embodiments, the component may comprise peptides linked to multiple engineered extracellular domains, such that the protein nanostructure displays different extracellular domains on the same nanostructure. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more different extracellular domains are displayed. Non-limiting illustrative protein nanostructures are provided by Bale et al. Science 353:389-94 (2016); Heinze et al. J. Phys. Chem B. 120:5945-5952 (2016); King et al. Nature 510:103-108 (2014); and King et al. Science 336:1171-71 (2012)

[0295] Attachment mode The protein nanostructures disclosed herein represent antigen proteins in various ways, including as gene fusions, or by other means disclosed herein. As used herein, “linked to” or “attached to” means any means known in the art for associating two polypeptides. Association can be direct or indirect, reversible or irreversible, weak or strong, covalent or non-covalent, and selective or non-selective.

[0296] In some implementations, attachment is achieved by genetically engineering to produce N-terminal or C-terminal fusions of potential antigenic peptides that form protein nanostructures.

[0297] In some embodiments, attachment is achieved through post-translational covalent attachment of one or more antigen proteins. In some embodiments, chemical crosslinking is used to non-specifically attach antigens to protein nanostructures. In some embodiments, chemical crosslinking is used to specifically attach antigen proteins to protein nanostructures (e.g., to a first polypeptide or a second polypeptide). Various specific and non-specific crosslinking chemistry methods, such as click chemistry and other methods, are known in the art. Generally, any crosslinking chemical / bioconjugate used to link two proteins can be applied to the protein nanostructures disclosed in this invention. In particular, chemistry used for generating immunoconjugates or antibody-drug conjugates can be used. In some embodiments, cleavable or non-cleavable linkers are used to generate protein nanostructures. Processes and methods for conjugating antigens to a carrier are provided, for example, by patent publication number US 2008 / 0145373 A1.

[0298] Protein nanostructures can employ various coupling techniques to attach antigens to the core, including but not limited to the SpyCatcher system described in Escolano et al. Nature 570:468-473 (2019), He et al. Sci Adv. 7(12):eabf1591 (2021), and Tan et al. Nat. Commun. 12(1):542 (2021).

[0299] In some embodiments, attachment is achieved through non-covalent attachment between the component and the extracellular domain. In some embodiments, the extracellular domain is engineered to have a negative charge on at least one surface, and the core polypeptide is engineered to have a positive charge on at least one surface, or both positive and negative charges. This can facilitate intermolecular association between the extracellular domain and the component core polypeptide via electrostatic forces. In some embodiments, shape complementarity is employed to attach the extracellular domain to the component core. Shape complementarity can be pre-existing or rationally designed. In some embodiments, computational design of protein-protein interfaces is used to achieve attachment.

[0300] In another aspect, this disclosure provides a trimeric protein complex comprising the polypeptide disclosed herein. In yet another aspect, this disclosure provides a protein nanostructure comprising a trimeric component comprising the polypeptide disclosed herein.

[0301] In some embodiments, the nanostructure is a two-component nanostructure comprising a first trimeric component and a second pentameric component. In some embodiments, the first trimeric component comprises engineered extracellular domains of a respiratory syncytial virus (RSV) fusion (F) peptide and an I53-50A peptide. In some embodiments, the first trimeric component comprises engineered extracellular domains of a human metapneumovirus (hMPV) fusion (F) peptide and an I53-50A peptide. In some embodiments, the first trimeric component comprises engineered extracellular domains of a human parainfluenza virus type 3 (PIV3) fusion (F) peptide and an I53-50A peptide. In some embodiments, the first trimeric component comprises engineered extracellular domains of a human parainfluenza virus type 5 (PIV3) fusion (F) peptide and an I53-50A peptide. In some embodiments, the first trimeric component comprises engineered extracellular domains of a SARS-CoV-2 spike (S) peptide and an I53-50A peptide. In some embodiments, the first trimer component comprises engineered extracellular domains of a Nipah virus fusion (F) polypeptide and an I53-50A polypeptide. In some embodiments, the first trimer component comprises a fusion protein comprising, in N-terminal to C-terminal order, an engineered fusion (F) polypeptide, an amino acid linker, and an I53-50A polypeptide. In some embodiments, the first trimer component comprises a fusion protein comprising, in N-terminal to C-terminal order, an engineered spike (S) polypeptide, an amino acid linker, and an I53-50A polypeptide. In some embodiments, the trimer component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the sequences listed in Table 19 or without the underlined and / or bold / italic polypeptide sequence. In some embodiments, the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 20, 44, 45, 52, 71, 73, 74.

[0302] polypeptide sequence Patent publication US 2015 / 0356240 A1 describes various methods for designing protein assemblies. As described in US Patent Publication US 2016 / 0122392 A1 and International Patent Publication WO 2014 / 124301 A1, the isolated peptides of SEQ ID NO:13-63 are designed to be able to self-assemble in pairs to form protein nanostructures, such as icosahedral particles. This design involves designing suitable interface residues for each member of the peptide pair, which can be assembled to form the protein nanostructure. The resulting protein nanostructures include symmetrically repeating, non-natural, non-covalent peptide-peptide interfaces that orient a first assembly domain and a second assembly domain toward the protein nanostructure, such as a protein nanostructure having icosahedral symmetry. Thus, in one embodiment, the first and second assembly domains of the components are selected from the group consisting of SEQ ID NO:13-63. In each case, N-terminal methionine residues present in the full-length protein are included but can be removed to prepare fusions not included in the sequence. The residues identified in Table 11 are numbered starting with the N-terminal methionine (not shown). In various embodiments, one or more additional residues are deleted from the N-terminus, and / or additional residues are added to the N-terminus (e.g., to form a helical extension).

[0303] Table 11.

[0304] Table 11 provides the amino acid sequences of the first and second assembly domains of embodiments of this disclosure. In each case, the sequence pairs together form an I53 polymer with icosahedral symmetry. The right column in Table 11 identifies the number of residues identified as present at the interface of the resulting assembled protein nanostructure in each illustrative polypeptide (i.e., "identified interface residues"). As can be seen, the number of interface residues of the illustrative polypeptides of SEQ ID NO:13-46 is in the range of 4-13. In various embodiments, the first and second assembly domains comprise amino acid sequences of polypeptides selected from the group consisting of SEQ ID NO:13-46, having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity in length, and having the same amino acid sequence at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 identified interface sites (depending on the number of interface residues of the given polypeptide). SEQ ID NO: 47-63 represents other amino acid sequences of the first and second assembly domains according to embodiments of the present disclosure. In other embodiments, the first and / or second assembly domains comprise amino acid sequences of polypeptides selected from the group consisting of SEQ ID NO: 13-63, having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity in length, and identical amino acid sequences at at least 20%, 25%, 33%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% identifiable interface positions.

[0305] As with proteins in general, peptides are expected to tolerate some changes to the designed sequence without disrupting subsequent assembly into protein nanostructures, especially when such changes involve conserved amino acid substitutions. As used herein, “conserved amino acid substitution” means: hydrophobic amino acids (Ala, Gly, Met, Val, Ile, Leu, Thr) are substituted with other hydrophobic amino acids; hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are substituted with other hydrophobic amino acids with large side chains; polar amino acids (Asp, Glu, Lys, Arg, Ser, Thr, Asn, Gly Tyr) are substituted with other polar amino acids; amino acids with positively charged side chains (Arg, His, Lys) are substituted with other amino acids with positively charged side chains; and amino acids with negatively charged side chains (Asp, Glu) are substituted with other amino acids with negatively charged side chains.

[0306] In various embodiments of the protein nanostructures of the present invention, the first assembly domain and the second assembly domain (or vice versa) comprise a polypeptide or a modified form thereof having an amino acid sequence selected from the following pairs (i.e., permissible modifications as disclosed for the polypeptides of the present invention: isolated polypeptides comprising an amino acid sequence indicated by SEQ ID NO having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identity in length, and / or the same amino acid sequence at at least one identified interface position): SEQ ID NO:13 and SEQ ID NO:14 (I53-34A and I53-34B); SEQ ID NO:15 and SEQ ID NO:16 (I53-40A and I53-40B); SEQ ID NO:15 and SEQ ID NO:36 (I53-40A and I53-40B.1); SEQ ID NO:35 and SEQ ID NO:16 (I53-40A.1 and I53-40B); SEQ ID NO:47 and SEQ ID NO:48 (types I53-40A and I53-40B); SEQ ID NO:17 and SEQ ID NO:18 (I53-47A and I53-47B); SEQ ID NO:17 and SEQ ID NO:39 (I53-47A and I53-47B.1); SEQ ID NO:17 and SEQ ID NO:40 (I53-47A and I53-47B.1NegT2); SEQ ID NO:37 and SEQ ID NO:18 (I53-47A.1 and I53-47B); SEQ ID NO:37 and SEQ ID NO:39 (I53-47A.1 and I53-47B.1); SEQ ID NO:37 and SEQ ID NO:40 (I53-47A.1 and I53-47B.1NegT2); SEQ ID NO:38 and SEQ ID NO:18 (I53-47A.1NegT2 and I53-47B); SEQ ID NO:38 and SEQ ID NO:39 (I53-47A.1NegT2 and I53-47B.1); SEQ ID NO:38 and SEQ ID NO:40 (I53-47A.1NegT2 and I53-47B.1NegT2); SEQ ID NO:49 and SEQ ID NO:50 (types I53-47A and I53-47B); SEQ ID NO:19 and SEQ ID NO:20 (I53-50A and I53-50B); SEQ ID NO:19 and SEQ ID NO:44 (I53-50A and I53-50B.1); SEQ ID NO:19 and SEQ ID NO:45 (I53-50A and I53-50B.1NegT2); SEQ ID NO:19 and SEQ ID NO:46 (I53-50A and I53-50B.4PosT1); SEQ ID NO:41 and SEQ ID NO:20 (I53-50A.1 and I53-50B); SEQ ID NO:41 and SEQ ID NO:44 (I53-50A.1 and I53-50B.1); SEQ ID NO:41 and SEQ ID NO:45 (I53-50A.1 and I53-50B.1NegT2); SEQ ID NO:41 and SEQ ID NO:46 (I53-50A.1 and I53-50B.4PosT1); SEQ ID NO:42 and SEQ ID NO:20 (I53-50A.1NegT2 and I53-50B); SEQ ID NO:42 and SEQ ID NO:44 (I53-50A.1NegT2 and I53-50B.1); SEQ ID NO:42 and SEQ ID NO:45 (I53-50A.1NegT2 and I53-50B.1NegT2); SEQ ID NO:42 and SEQ ID NO:46 (I53-50A.1NegT2 and I53-50B.4PosT1); SEQ ID NO:43 and SEQ ID NO:20 (I53-50A.1PosT1 and I53-50B); SEQ ID NO:43 and SEQ ID NO:44 (I53-50A.1PosT1 and I53-50B.1); SEQ ID NO:43 and SEQ ID NO:45 (I53-50A.1PosT1 and I53-50B.1NegT2); SEQ ID NO:43 and SEQ ID NO:46 (I53-50A.1PosT1 and I53-50B.4PosT1); SEQ ID NO:51 and SEQ ID NO:52 (types I53-50A and I53-50B); SEQ ID NO:21 and SEQ ID NO:22 (I53-51A and I53-51B); SEQ ID NO:23 and SEQ ID NO:24 (I52-03A and I52-03B); SEQ ID NO:25 and SEQ ID NO:26 (I52-32A and I52-32B); SEQ ID NO:27 and SEQ ID NO:28 (I52-33A and I52-33B) SEQ ID NO:29 and SEQ ID NO:30 (I32-06A and I32-06B); SEQ ID NO:31 and SEQ ID NO:32 (I32-19A and I32-19B); SEQ ID NO:33 and SEQ ID NO:34 (I32-28A and I32-28B); SEQ ID NO:35 and SEQ ID NO:36 (I53-40A.1 and I53-40B.1); SEQ ID NO:53 and SEQ ID NO:54 (T32-28A and T32-28B); SEQ ID NO:55 and SEQ ID NO:56 (T33-09A and T33-09B); SEQ ID NO:57 and SEQ ID NO:58 (T33-15A and T33-15B); SEQ ID NO:59 and SEQ ID NO:60 (T33-21A and T33-21B); SEQ ID NO:61 and SEQ ID NO:62 (T33-28A and T32-28B); and SEQ ID NO:63 and SEQ ID NO:56 (T33-31A and T33-09B (also known as T33-31B)).

[0307] In some embodiments, the assembly domains are I53_dn5B (trimer, optionally linked to the antigen) and I53_dn5A or I53_dn5A.1 or I53_dn5A.2 (pentamer). The I53_dn5 nanostructures are described in US 2022 / 0072120 A1, the contents of which are incorporated herein by reference. Variants of I53_dn5 may include one or more amino acid substitutions, such as C94A, C119A, W18G, K84R, M88P, E91D, L117I, or L120D (collectively, “I53_dn5A.1”; Ueda et al. eLife 9:e57659(2020)) or A25E, M88A, C119T, L120E, A127E, L131T, I132K, E133A, or deletions at positions 135-137 (“I53_dn5A.2”; Wang et al. bioRxiv 2022.08.04.502842).

[0308] In some embodiments, the extracellular domain is expressed as a fusion protein having a first assembly domain. In some embodiments, the first assembly domain and the extracellular domain are joined by a linker sequence.

[0309] Non-limiting examples of designed protein complexes that can be used in the protein nanostructures of this disclosure include those disclosed in U.S. Patent No. 9,630,994; International Patent Publication No. WO2018187325A1; U.S. Patent Publication No. 2018 / 0137234A1; and U.S. Patent Publication No. 2019 / 0155988 A2, each of which is incorporated herein by reference in its entirety.

[0310] In various embodiments of the protein nanostructures disclosed herein, the assembly domain is a polypeptide or a modified form thereof having an amino acid sequence selected from the following pairs (i.e., permissible modifications as disclosed for the polypeptides of the present invention: isolated polypeptides comprising an amino acid sequence indicated by SEQ ID NO having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity in length, and / or the same amino acid sequence at at least one identified interface location): SEQ ID NO: 65 and SEQ ID NO: 66 (T33_dn2A and T33_dn2B); SEQ ID NO: 67 and SEQ ID NO: 68 (T33_dn5A and T33_dn5B); SEQ ID NO: 69 and SEQ ID NO: 70 (T33_dn10A and T33_dn10B); or SEQ ID NO: 71 and SEQ ID NO: 72 (I53_dn5A and I53_dn5B).

[0311] Various protein nanostructures are known in the art and described in, for example, U.S. Patent Publications US2015 / 0356240 A1, US 2016 / 0122392 A1, US 2018 / 0030429 A1, US 2019 / 0341124 A1, and US2022 / 0072120 A1, the contents of which are incorporated herein by reference. In some embodiments, the protein nanostructure comprises a variant of KDPG aldolase (Protein Database Code 1WA3) engineered to self-assemble into a protein nanostructure as an assembly domain. In its native form, 1WA3 non-covalently assembles to form a trimer via a first interface (trimeric interface). When 20 copies of the trimer (60 monomers) are computationally docked to form a single-component icosahedral protein nanostructure, groups of five monomers of 1WA3 contact each other via a second interface (pentamer interface). By introducing amino acid substitutions, pentamer interfaces can be stabilized, allowing protein nanostructures to spontaneously self-assemble, for example, within expression cells or when separated trimers (or monomers) are mixed under suitable conditions.

[0312] In some embodiments, the pentamer interface comprises 1, 2, 3, 4 or more interface residues, such as residues at positions 33, 61, 187 and 190 according to SEQ ID NO: 107. In some embodiments, the assembly domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 107. In some embodiments, the assembly domain comprises amino acid substitutions at positions 1, 2, 3, and 4 of SEQ ID NO: 107 at positions 33, 61, 187 and 190. In some embodiments, the multiple amino acid substitutions are substitutions of polar residues for nonpolar residues (e.g., A, L, I, M, V, F or W). In some embodiments, some or all of the amino acid substitutions are substitutions of polar residues for small nonpolar residues (e.g., A, L, I, M or V). In some embodiments, the protein nanostructure comprises amino acid substitutions of E33L or E33V; K61L or K61M; D187A or D187V; and / or R190A. In some embodiments, the protein nanostructure comprises amino acid substitutions of E33L, K61M, D187V, and R190A. In some embodiments, the protein nanostructure comprises amino acid substitutions of E33V, K61L, D187A, and R190A. In some embodiments, the assembly domain comprises amino acid substitutions that inactivate the enzymatic activity of the assembly domain (e.g., K129A). In embodiments, the assembly domain may comprise other amino acid substitutions (e.g., MI3; E56M or E56K; P186I; E191A; and / or K194A). In some embodiments, the assembly domain comprises amino acid substitutions with cysteine ​​residues removed. In some embodiments, the assembly domain comprises C76A and / or C100A substitutions.

[0313] In some embodiments, the polypeptide comprises a heteropolymerization domain. In some embodiments, the polymerization domain is a trimerization domain. In some embodiments, the polymerization domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64). In some embodiments, the extracellular domain comprises amino acid substitutions S155C, S290C, S190F, and V207L.

[0314] In some embodiments, the trimer component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the sequences listed in Table 19 or without the underlined and / or bold / italic polypeptide sequence.

[0315] Ferritin-based nanostructures In some embodiments, the assembly domain is a ferritin polypeptide. In some embodiments, the assembly domain of the ferritin nanostructure comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the following sequences: In some implementations, the C-terminal helical forming segment links the antigen to any nanoparticles known in the art—including but not limited to HPV particles (using SpyCatcher) or ferritin.

[0316] Other nanostructures or nanoparticles In some embodiments, the extracellular domains described herein are displayed on any nanostructures or nanoparticles known in the art. Illustrative nanostructures and nanoparticles include, but are not limited to, human papillomavirus (HPV) virus-like particles (VLPs), Chikungunya virus VLPs, AP205 capsid protein VLPs, and bacteriophage VLPs (e.g., bacteriophages). Display can be achieved by generating fusion proteins of the extracellular domains with system-associated proteins, by bioconjugation chemistry (e.g., SpyCatcher), or by other means known in the art on these and other platforms. Protein nanostructures can be, for example, dioxetine synthase nanoparticles as described in, for example, Geng et al., PLoS Pathog. 17(9):e1009897 (2021). Protein nanostructures can be, for example, ferritin nanoparticles as described in, for example, Joyce et al., bioRxiv 2021.05.09.443331 and U.S. Patent Publication No. US 2019 / 0330279 A1.

[0317] In another aspect, this disclosure provides a recombinant polypeptide for displaying molecules (such as antigens), the recombinant polypeptide comprising an α-helical segment and a polymerizing domain, wherein the α-helical segment comprises one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer. In some embodiments, the α-helical segment has improved hydrophobic stacking. In some embodiments, the α-helical segment comprises about 7 to about 31 residues. In some embodiments, the amino acid substitutions comprise polar, charged, and / or hydrophobic amino acids.

[0318] In some embodiments, the α-helical segment comprises a polypeptide sequence according to any of the following: LXXTIXXLLXIXXXLXXXL (SEQ ID NO: 566), LVXTXKXLXDLIXXLXXLLXKLXX (SEQ ID NO: 567), LNKVKKXVXXLXXXVXXLEKXLX (SEQ ID NO: 568), EKIXXAIKKAXKL (SEQ ID NO: 569), EXIXKAIKXLXXXXX (SEQ ID NO: 570), XKXXEXXXXVXXXXXXXXX (SEQ ID NO: 571), XXLKKAAXIXKKXLKXX (SEQ ID NO: 572).

[0319] In some implementations, the α-helical segment comprises a polypeptide sequence according to any of the common sequences in Table 24.

[0320] In some embodiments, the α-helical segment comprises a polypeptide sequence according to the following: a) L X2X2T I X2X2L LX2I [V / I] X2X2L [I / L] X2X2L (SEQ ID NO: 573), b) LV [A / T] T X2K X2L X2D L IX2X2L [K / E] X2L L X2K L X2X2 (SEQ ID NO: 574), or c) LNKVKK X2V X2X2L X2X2X2VX2X2L EK X2L X2 (SEQ ID NO: 575), wherein X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

[0321] In some embodiments, the α-helical segment comprises a polypeptide sequence according to the following: a) EKI X2X2A IK KA X2K L (SEQ ID NO: 576), b) E X2I X2K AIK X2L [L / X2] X2X2[X1 / X2] X2 (SEQ ID NO: 577), and c) X2K [X1 / T] [L / E]E [T / A] X1X2[I / X2] V X2X2[X1 / X2] [X1 / X2] X2X2X1X2X2 (SEQ ID NO: 578), or d) X2X2L KKAA X2I X1K K X1L K X2X2 (SEQ ID NO: 576). 579), wherein X1 is a nonpolar residue selected from A, I, L and M, and X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

[0322] In some embodiments, the α-helix segment comprises a polypeptide sequence listed in Table 25A or Table 25B, or a polypeptide sequence thereof having 1 to 5 amino acid substitutions. In some embodiments, the α-helix segment comprises a polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), NQSALWLEAAKYVKQAREKS (SEQ ID NO: 11), NQSAKNAEAAKIAEETKRKD (SEQ ID NO: 12), or NQSRETAKAVSAVK (SEQ ID NO: 75), or a polypeptide sequence thereof having 1 to 5 amino acid substitutions.

[0323] In some embodiments, the polymerizing domain is I53-50A or a variant thereof. In some embodiments, the polymerizing domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64). In some embodiments, the polypeptide comprises an α-helix segment fused to the N-terminus of the polymerizing domain via a peptide bond or polypeptide linker. In some embodiments, the polypeptide comprises an antigenic polypeptide at the N-terminus of the α-helix segment.

[0324] In another aspect, this disclosure provides a polypeptide comprising an α-helical segment comprising a polypeptide sequence listed in Table 25A or Table 25B, or a polypeptide sequence having 1 to 5 amino acid substitutions. In another aspect, this disclosure provides a protein nanostructure comprising a trimeric component comprising the polypeptide described herein. In some embodiments, the nanostructure is a bicomponent nanostructure comprising a first trimeric component and a second pentameric component. In some embodiments, the nanostructure is a bicomponent nanostructure comprising a second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71.

[0325] IV. Polynucleotides In another aspect, this disclosure provides polynucleotides encoding any of the polypeptides, complexes, components, nanostructures, or other compositions of this disclosure. The polynucleotide sequences may comprise RNA or DNA. As used herein, “polynucleotide” refers to those that have been removed from their normal surrounding polynucleotide sequences in the genome or cDNA sequence. Such polynucleotide sequences may include additional sequences that can be used to facilitate the expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding antigenic determinant tags, output and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, those skilled in the art will appreciate which nucleic acid sequences will encode the proteins of this disclosure.

[0326] V. Delivery medium In some embodiments, a polynucleotide (e.g., mRNA) encoding a protein nanostructure is formulated in a delivery medium, said protein nanostructure comprising a component of a viral protein monomer containing a trimeric viral antigen. In some embodiments, the delivery medium is a non-viral carrier. In some embodiments, the delivery medium is a lipid nanoparticle (LNP). In some embodiments, the delivery medium is a liposome. In some embodiments, the delivery medium is a polymeric non-viral carrier, such as spermine, polyethyleneimine, chitosan, or polyurethane. In some embodiments, the delivery medium is a polymeric delivery system, such as polyamidoamine (PAA), poly-β-amino ester (PBAE), or polyethyleneimine (PEI). In some embodiments, the delivery medium is ferritin nanoparticles. In some embodiments, the delivery medium is an encapsulating protein.

[0327] In some embodiments, a polynucleotide (e.g., mRNA) encoding a protein nanostructure is formulated in nanoparticles, said protein nanostructure comprising a component of a viral protein monomer containing a trimeric viral antigen. In some embodiments, the nanoparticles are lipid nanoparticles (LNPs). In some embodiments, the polynucleotide is formulated in a lipid-polycationic complex, referred to as a cationic LNP. As a non-limiting example, the polycationic complex may include cationic peptides or polypeptides, such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, the polynucleotide is formulated in an LNP, said LNP comprising a noncationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0328] In various embodiments, the lipid nanoparticles have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, LNPs are substantially non-toxic. In some implementations, when present in LNPs, the polynucleotides are resistant to degradation by nucleases in aqueous solutions.Lipids containing polynucleotides and LNPs, and methods for their preparation, are described in, for example, U.S. Patent Nos. 8,569,256, 5,965,542 and U.S. Patent Publications Nos. 2021 / 0323914, 2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, and 2014. / 0308304, 2014 / 0200257, 2013 / 086373, 2013 / 0338210, 2013 / 0323269, 2013 / 0245107, 2013 / 0195920, 2013 / 0123338, 2013 / 0022649, 2013 / 0017223, 2012 / 0295832, 2012 / 0183581, 2 012 / 0172411, 2012 / 0027803, 2012 / 0058188, 2011 / 0311583, 2011 / 0311582, 2011 / 0262527, 2011 / 0216622, 2011 / 0117125, 2011 / 0091525, 2011 / 0076335, 2011 / 0060032, 2010 / 01305 88, 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, 1999 / 009076 and PCT publication number WO The contents of the patents mentioned in WO 2017 / 004143, WO 2017 / 075531, WO 2015 / 199952, WO 2014 / 008334, WO 2013 / 086373, WO 2013 / 086322, WO 2013 / 016058, WO2013 / 086373, WO2011 / 141705, WO 2017 / 049245, WO 2010 / 144740, WO / 2017 / 075531 and WO2001 / 07548 are incorporated herein by reference.

[0329] Other exemplary lipids and LNPs and their fabrication are known in the art—for example, in U.S. Patent Publication No. US2012 / 0276209; Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, MolTher nucleic Acids, 1: e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, e139; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which is incorporated herein by reference. Lipids and their manufacture can be found, for example, in U.S. Patent Publications 2015 / 0376115 and 2016 / 0376224, the contents of which are incorporated herein by reference.

[0330] VI. Pharmaceutical Composition This disclosure also provides pharmaceutical compositions. Such pharmaceutical compositions can be used to generate an immune response against an infectious disease in a subject. The pharmaceutical compositions of this disclosure may include pharmaceutically acceptable carriers. A detailed discussion of such carriers is available in [the following text is missing from the original extract]. Remington: The Science and Practice of Pharmacy Obtained from Chapter 30 of (23rd edition, 2021).

[0331] In some embodiments, the pharmaceutical composition may also contain excipients and / or additives. Examples of such agents are surfactants, stabilizers, complexing agents, antioxidants, or preservatives that prolong the shelf life of the finished pharmaceutical formulation, flavoring agents, vitamins, or other additives known in the art. Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or its salts, such as disodium salt, citric acid, hypozinotriacetic acid, and their salts. In some embodiments, preservatives include, but are not limited to, those that protect the solution from pathogenic particle contamination, including benzalkonium chloride or benzoic acid, or benzoates, such as sodium benzoate. Antioxidants include, but are not limited to, vitamins, provitamins, ascorbic acid, vitamin E, their salts, or esters.

[0332] Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline solutions, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose, amylose, or starch), fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and pigments, etc. Such formulations can be sterilized and, if necessary, can be mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, and / or aromatic substances, as well as analogs that do not react adversely with the compounds of this disclosure. Those skilled in the art will recognize that other pharmaceutical excipients may be used in this disclosure.

[0333] In some implementations, one or more tonic agents may be added to provide the desired ionic strength. Tonic agents as used herein include those that show no or only negligible pharmacological activity after administration. Inorganic and organic tonic modifiers may be used.

[0334] In another aspect, this disclosure provides a pharmaceutical composition comprising the polypeptide, protein complex, or nanostructure disclosed herein.

[0335] VII. Vaccines In another aspect, this disclosure provides a vaccine comprising a polypeptide, protein complex, or nanostructure as disclosed herein. In yet another aspect, this disclosure provides a vaccine comprising a polypeptide or nanostructure described herein.

[0336] In some implementations, the vaccine contains an adjuvant.

[0337] In some embodiments, the pharmaceutical compositions provided herein are administered as RSV vaccines, such as RSV / A and RSV / B vaccines or bivalent RSV A / B vaccines. In some embodiments, the pharmaceutical compositions provided herein are administered as hMPV vaccines. In some embodiments, the pharmaceutical compositions provided herein are administered as hMPV / A vaccines. In some embodiments, the pharmaceutical compositions provided herein are administered as hMPV / B vaccines. In some embodiments, the pharmaceutical compositions provided herein are administered as bivalent hMPV / A and hMPV / B vaccines. In some embodiments, the pharmaceutical compositions provided herein are administered as bivalent hMPV / A and RSV vaccines. In some embodiments, the pharmaceutical compositions provided herein are administered as PIV3 vaccines. In some embodiments, the pharmaceutical compositions provided herein are administered as PIV5 vaccines. In some embodiments, the pharmaceutical compositions provided herein are administered as SARS-CoV-2 vaccines. In some embodiments, the pharmaceutical compositions provided herein are administered as Nipah vaccines. In some embodiments, the pharmaceutical compositions provided herein are administered as bivalent RSV / hMPV vaccines.

[0338] adjuvant Adjuvants or immunostimulants can also be administered together with or in combination with lipid nanoparticle compositions. The advantages of adjuvants include, but are not limited to, enhancing the immunogenicity of antigens, altering the nature of the immune response, reducing the amount of antigen required for successful immunization, reducing the frequency of required booster immunizations, and improving immune responses in older adults and immunocompromised vaccine recipients. These agents can be administered co-administered via any route, such as intramuscular, subcutaneous, intravenous, or intradermal injection.

[0339] Adjuvants can be, but are not limited to, natural or synthetic adjuvants. Adjuvants can be organic or inorganic.

[0340] Adjuvants may be selected from any of the following categories: (1) mineral salts, such as aluminum hydroxide and aluminum phosphate or calcium phosphate gels; (2) emulsions, including oil emulsions and surfactant-based formulations, such as microfluidic cleaner-stabilized oil-in-water emulsions, purified saponins, oil-in-water emulsions, and stabilized water-in-oil emulsions; (3) particulate adjuvants, such as virions (monolithic liposomes combined with influenza hemagglutinin), structured complexes of saponins and lipids, and poly(lactic-co-glycolic acid) (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; (6) inert mediators, such as gold particles; (7) microbial adjuvants; (8) tonicotinic compounds; (9) carbohydrates; or combinations thereof.

[0341] Adjuvants for nucleic acid vaccines (DNA) have been disclosed, for example, in Kobiyama et al., Vaccines, 2013, 1(3), 278-292, the contents of which are incorporated herein by reference in their entirety. Any of the adjuvants disclosed by Kobiyama et al. may be used in vaccines as described herein.

[0342] Other adjuvants that can be used include those listed in web-based vaccine adjuvant databases, such as violinet.org / vaxjo / , and described by Sayers et al., for example. J. Biomedicine and Biotechnology Volume 2012 (2012), Article ID 831486, page 13, any adjuvants, the content of the data is incorporated herein by reference in its entirety.

[0343] Specific adjuvants may include cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, potassium aluminum sulfate adjuvant, aluminum gel, ISCOM(s)™, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, CpG DNA vaccine adjuvant, cholera toxin, cholera toxin B subunit, liposomes, saponin vaccine adjuvant, DDA adjuvant, squalene-based adjuvant, Etx B subunit adjuvant, IL-12 vaccine adjuvant, LTK63 vaccine mutant adjuvant, TiterMax Gold adjuvant, Ribi vaccine adjuvant, Montanide ISA 720 adjuvant, Corynebacterium-derived P40 vaccine adjuvant, MPL™ adjuvant, AS04, AS02, AS01. ELipopolysaccharide vaccine adjuvant, cell wall acyl dipeptide adjuvant, CRL1005, inactivated Corynebacterium parvum vaccine adjuvant, Montanide ISA 51, Bordetella pertussis component vaccine adjuvant, cationic liposome vaccine adjuvant, amantadine dipeptide vaccine adjuvant, Arlacel A, VSA-3 adjuvant, aluminum vaccine adjuvant, Polygen vaccine adjuvant, ADJUMER™, seaweed dextran, Bay R1005, Theramide®, stearoyl tyrosine, Spol, Algammulin, AVRIDINE®, calcium phosphate gel, CTA1-DD gene fusion protein, DOC / alum complex, gamma inulin, Gerbu adjuvant, GM-CSF, GMDP, recombinant hIFN-γ / interferon-g, interleukin-1β, interleukin-2, interleukin-7, Sclavo peptide, Rehydragel LV, Rehydragel HPA, Loxoribine, MF59, MTP-PE liposomes, Murametide, Murapamitine, D-Morapamitine, NAGO, nonionic surfactant vesicles, PMMA, protein cochleates, QS-21, SPT (antigen preparation), nanoemulsion vaccine adjuvants, AS03, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, LTR192G vaccine adjuvant, Escherichia coli heat-labile toxin, LT, amorphous aluminum hydroxyphosphate sulfate adjuvant, calcium phosphate vaccine adjuvant, Montanide incomplete Seppic adjuvant. Adjuvant, Imiquimod, Resiquimod, AF03, flagellin, poly(I:C), ISCMATRIX®, Abisco-100 vaccine adjuvant, albumin-heparin microparticle vaccine adjuvant, AS-2 vaccine adjuvant, B7-2 vaccine adjuvant, DHEA vaccine adjuvant, antibody against co-stimulatory molecules containing immunoliposomes, SAF-1, Sendai proteoliposome, Sendai-containing lipid matrix, threonyl muramyl dipeptide (TMDP), Ty particle vaccine adjuvant, Bupivacaine vaccine adjuvant, DL-PGL (poly(DL-lactide-co-glycolide)) vaccine adjuvant, IL-15 vaccine adjuvant, LTK72 vaccine adjuvant, MPL-SE vaccine adjuvant, non-toxic mutant of cholera toxin E112K (mCT-E112K) and / or matrix-S.

[0344] In some embodiments, the adjuvant comprises squalene. In some embodiments, the adjuvant comprises aluminum hydroxide. In some embodiments, the adjuvant comprises AS01. E .

[0345] VIII. Usage Instructions In another aspect, this disclosure provides methods of administering the compositions, pharmaceutical compositions or vaccines described herein.

[0346] In another aspect, this disclosure provides a method for vaccinating a subject, the method comprising administering the composition described herein to the subject. In another aspect, this disclosure provides a method for generating an immune response in a subject, the method comprising administering the composition described herein to the subject. In another aspect, this disclosure provides a method for treating or preventing a disease in a subject, including but not limited to RSV, hMPV, PIV3, PIV5, Nipah, and / or SARS-CoV-2, the method comprising administering the composition described herein to the subject. In another aspect, this disclosure provides compositions of the present disclosure for use in vaccination, generating an immune response, or treating or preventing a disease, including but not limited to RSV, hMPV, PIV3, PIV5, Nipah, and / or SARS-CoV-2. In another aspect, this disclosure provides compositions, methods, or uses as described herein. In another aspect, this disclosure provides a method for treating or preventing a coronavirus disease in a subject, the method comprising administering the composition described herein to the subject. In another aspect, this disclosure provides compositions of the present disclosure for use in vaccination, generating an immune response, or treating or preventing a coronavirus disease. In another aspect, this disclosure provides compositions, methods, or uses as described herein.

[0347] In another aspect, this disclosure provides a method for vaccinating a subject, the method comprising administering to the subject a composition described herein. In another aspect, this disclosure provides a method for generating an immune response in a subject or for treating or preventing viral infection, the method comprising administering to the subject a polypeptide or nanostructure described herein. In another aspect, this disclosure provides a method for preparing a polypeptide or nanostructure described herein, the method comprising culturing host cells modified to express one or more polypeptides as described herein.

[0348] In some embodiments, the method includes administering the vaccine described herein. In some embodiments, the subject is immunized against RSV, hMPV, PIV3, PIV5, Nipah, and / or SARS-CoV-2 S infection. In some embodiments, the subject is immunized against coronavirus infection. In some embodiments, the vaccine is administered via subcutaneous injection. In some embodiments, the vaccine is administered via intramuscular injection. In some embodiments, the vaccine is administered via intradermal injection. In some embodiments, the vaccine is administered intranasally. In one aspect, this disclosure provides a pre-filled syringe comprising the vaccine described herein. In one aspect, this disclosure provides a kit comprising the vaccine described herein or the pre-filled syringe described herein. In one aspect, this disclosure provides a kit comprising the vaccine described herein or the lyophilized vaccine described herein.

[0349] In some embodiments, the unit dose of the pharmaceutical composition comprises about 0.5 μg to about 1 μg, about 20 μg to about 25 μg, about 25 μg to about 50 μg, about 50 μg to about 70 μg, about 70 μg to about 75 μg, about 75 μg to about 100 μg, about 100 μg to about 125 μg, about 100 μg to about 150 μg, about 125 μg to about 150 μg, about 125 μg to about 175 μg, about 150 μg to about 175 μg, about 175 μg to about 200 μg, about 200 μg to about 250 μg, about 225 μg to about 300 μg, about 250 μg to about 300 μg, or about 250 μg to about 350 μg of protein nanostructures.

[0350] In some embodiments, the subject is at risk of disease, including but not limited to RSV, hMPV, PIV3, PIV5, Nipah, and / or SARS-CoV-2. In some embodiments, the subject is at risk of hMPV disease. In some embodiments, the subject is at risk of PIV3 disease. In some embodiments, the subject is at risk of PIV5 disease. In some embodiments, the subject is at risk of coronavirus disease. In some embodiments, the subject is an adult aged 60 years or older. In some embodiments, the subject is a healthy adult aged 18-45 years. In some embodiments, the subject is a pregnant woman in her 32nd to 36th week of pregnancy. In some embodiments, the subject is a pregnant woman in her 30th to 38th week of pregnancy. In some embodiments, the subject is a pregnant woman in her 28th to 38th week of pregnancy.

[0351] In another aspect, this disclosure provides a method for vaccinating a subject, the method comprising administering to the subject the composition disclosed herein. In another aspect, this disclosure provides a method for generating an immune response in a subject, the method comprising administering to the subject the composition disclosed herein. In another aspect, this disclosure provides a method for treating or preventing a viral infection in a subject, the method comprising administering to the subject the composition disclosed herein. In another aspect, this disclosure provides a composition for use in vaccination, generating an immune response, or treating or preventing any viral infectious disease disclosed herein. In another aspect, this disclosure provides compositions, methods, or uses as described herein. In another aspect, this disclosure provides a method for preparing a composition, the method comprising culturing host cells modified to express one or more polypeptides as described herein.

[0352] Any aspect or implementation described herein may be combined with any other aspect or implementation disclosed herein. Example The following examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

[0353] Example 1. Remodeling the C-terminus of the RSV F protein This embodiment describes remodeling the C-terminus of the RSV F protein to generate a stable helical forming segment.

[0354] Like other type I viral membrane fusion proteins, the RSV F protein forms a trimer with two main conformations (pre-fusion and post-fusion). The C-terminus of the extracellular domain adjacent to the transmembrane domain is believed to form a helical bundle in the case of the native protein. The pre-fusion F protein structure is generally modeled with the C-terminus as an α-helix, where the structured density ends at approximately residues 510 or 512 (e.g., PDB 5C6B and 5UDD, respectively). The native sequence after residue 513 is often replaced by a four-residue linker (SAIG) and a trimeric domain. The predicted transmembrane domain begins at residue 527. The sequence of the native RSV / BF protein (GenBank: WDV37446.1) is shown here, where the transmembrane domain is in bold / underlined: We hypothesize that poor structural resolution at the C-terminus of the extracellular domain reflects the incomplete hydrophobic stacking of helical bundles in the native protein when expressed in a recombinant manner. We have developed a circuit to remodel the C-terminus of the extracellular domain to generate improved antigens for use in vaccines. Our approach remodels the segments (corresponding to approximately residues 500 and 530 relative to the native sequence) into structurally more stable helical bundles by substituting residues (e.g., to generate novel non-covalent interactions, prevent residue conflicts, or modify the polypeptide backbone) and retaining or enhancing polar exposed surfaces, thereby reducing the self-association free energy of the protopolymer (e.g., predicted ddG and measured thermal denaturation temperature). The remodeling circuit involves manually selecting sequences predicted to form structures capable of acting as transposons to link the C-terminus of the extracellular domain to trimerizing domains, such as the I53-50A multimerizing domain. The manual selection is based on a combination of polypeptide sequence diversity and computational metrics, including geometric design space, hydrophobic core package, terminal availability, and the absence of obvious errors in the conformation (i.e., tryptophan exposed to solvents).

[0355] Structural models from protein databases (PDBs) are prepared for design by symmetrization, heteroatom removal, renumbering, relaxation, and labeling of glycosylation sites. These models are written into a Rosetta blueprint file to create a gradient between the native structure and the remodeled domain. Generally, the blueprint includes a two-residue native sequence remodeled using helical constraints; followed by one or more existing helical residues that facilitate C-terminal hydrophobic contacts, allowing for design; and then an α-helical segment of approximately 1–10 amino acids in length, determined empirically by the designer. For example, to remodel this sequence: Blueprints can be generated where amino acid residues are configured to match the native sequence. A The model starts with the native sequence but allows substitution (A), and is newly modeled as any amino acid (X) (top line), while the 3D structure of the polypeptide is set to match the native structure (.) or constrained to a helix (H): Use this blueprint or a similar blueprint to generate a design using Rosetta remodeling. Filter the remodeling results directly from the design model via ddG calculations and manually revert to remove any introduced glycosylation sites, exposed hydrophobic residues, or buried polar residues. Relax the resulting model and then calculate ddG again.

[0356] Alternatively, RF diffusion can be used for remodeling. The relaxed structures used as input for remodeling are also used as input for RF diffusion, except that only the C-terminal helix is ​​used as input for diffusion. This approach significantly reduces computation time. Positions with WT sequence identity from Rosetta remodeling are also preserved for diffusion. Unlike Rosetta remodeling, all structural data for subsequent residues are ignored. This is a limitation of RF diffusion, not a scientific constraint on the design problem. Weights within and between protomers are set to 1, with secondary guidance decay and guidance scaling of 2. The C-terminal length varies between 12 and 31 residues depending on the source virus, and 15 remodeling helices are generated for each length. Sequences are generated using protein MPNN, with a sampling temperature of 0.4°C and negative biases for C (-10) and F, G, P, W, and Y (-1). 100 sequences are generated for each remodeling domain. The top 2% based on MPNN sample scores are selected for computational characterization.

[0357] The design was analyzed based on the following criteria: 1) ColabFold validated the design using Rossetta by predicting ordered terminal helices consistent with the design model (assuming the ColabFold method can provide reliable results for a specific fusion protein); 2) ddG was reduced by 5–15 Rossetta energy units (REU); and 3) the design featured a well-packed hydrophobic core with no foreign elements (i.e., no hydrophobic stacking of helical segments between protomers). To calculate ddG, two models were generated, one in which all protomers were correctly contacted as trimers, and the other in which protomers moved distally to each other. Side chain repacking in both models was minimized, and then the two models were scored. ddG is the difference in scores, e.g. (distal state) – (trimeric state).

[0358] Figure 2 The structural model shown compares a representative experimental model (left) of the RSV F protein provided by the PDB 4MMU with a predicted structure (right) of a representative design. The optimal C-terminal length for remodeling was determined by plotting the average ddG against the length of the C-terminal helix. Figure 3 As shown in the diagram. When using Rosetta remodeling, the average ddG decreases until an optimal length is reached, at which point ddG tends to remain the same or increase again. This is likely because remodeling can be difficult when constructing larger segments due to the increased degrees of freedom. The ideal connector lengths are those close to the minimum ddG. In this case, the optimal C-terminal helix is ​​determined to terminate at approximately position 519. Empirically, it has been observed that ddG is minimized when the helical segment extends beyond the original position 513 by approximately 6 residues (i.e., to position 519).

[0359] Computational modeling of RSV / B proteins (remodeled using Rosetta) was used to generate artificial polypeptide sequences, predicting that each sequence would form a stable α-helix, as shown in Table 12. Residues 500-502 of the native RSV F protein were included as NQS. Residues Q501 and S502 were remodeled using helical constraints while preserving native sequence identity. This optimized the helical backbone of these residues, where side chains were represented as centroids, and then the side chains were reassembled in an all-atom model. Residues 503-509 were remodeled using both helical and no-sequence constraints. The helical backbone was first optimized using side chains represented as centroids, and the side chains were designed in an all-atom model. Therefore, there was some bias towards the native sequence. Six to 14 additional amino acids were added using helical constraints. During backbone sampling, the side chains were represented as valine centroids, and then the sequence was sampled in an all-atom model. All backbone sampling of these elements in the centroid model was performed simultaneously, and sequence design in the all-atom model was also performed simultaneously. The design is manually refined to remove exposed hydrophobic residues or buried polar residues that have the same identity as those selected preferentially from the nearest residue in the WT sequence or rationally selected in the case of the second-best WT residues.

[0360] The I53-50A molecule is highly suitable for fusion with many trimeric antigen genes and is characterized by a symmetrical N-terminus spaced approximately 5 nm apart. Due to the remodeling of the C-terminus 1, the C-terminus is laterally further from the axis of symmetry of the antigen. Figure 2 Therefore, this modification may minimize strain on gene fusions with I53-50A compared to antigen fragments that are typically studied and terminate at residue 513. Four sequences were selected for experimental testing as gene fusions with the I53-50A variant (I53-50Aδcys) (Table 12), in which the antigen also contains the DS-Cav1 mutation.

[0361] Table 12. Illustrative C-end spiral formation section

[0362] The native sequence includes the C-terminal α-helical segment ISQVNEKINQSLAFIRRSDE (SEQ ID NO: 713).

[0363] In the context, the C-terminal α-helix of the modified construct is ISQVNEKINQSRE I IR AI NI V RK I ASEK (SEQ ID NO: 714) is only nine residues longer than the known helical portion of the original structure and two residues shorter than the predicted helical segment. Contact residues are bolded and underlined.

[0364] native ISQVNEKINQSLA F IR RS DELLHNVN (SEQ ID NO: 715) Remodeling ISQVNEKINQSRE I IR AI NI V RK I ASEK (SEQ ID NO: 714) Although the WT sequence has a three-residue hydrophobic segment leading to the designed helix and a five-residue polar segment in the middle, which facilitates suboptimal stacking, the remodeled sequence is characterized by an alternating pattern of hydrophobic and polar segments, which does not have hydrophobic segments longer than two consecutive residues and does not have polar segments longer than three consecutive residues. Figure 4 The remodeled spiral has at least two hydrophobic sections at positions 508 and / or 509 and 511 and / or 512 and optimally four hydrophobic sections at positions 505 and / or 506, 508 and / or 509, 511 and / or 512 and 515 and / or 516.

[0365] The published structures of RSV proteins generally do not include residues from the C-terminus to approximately 500 residues. These residues are not present in the recombinant protein under study, or they are not visible in the observed electron density. Nevertheless, modeling suggests that the following substitutions will stabilize the F protein portion in a helical conformation.

[0366] Table 13. Possible replacements at locations 505-516

[0367] In some embodiments, polar amino acids refer to D, E, K, N, Q, R, S, T, and Y. In some embodiments, polar amino acids include charged amino acid residues. In some embodiments, charged amino acids refer to E, D, R, K, and H. In some embodiments, hydrophobic amino acids refer to A, I, L, M, V, F, Y, and W.

[0368] Small-scale screening showed expression of three of the four selected designs. Table 14 shows the binding of antibodies D25, AM14, and 4D7 to RSV / BF proteins fused to I53-50A to form a trimeric protein complex (but not assembled with I53-50B). Both D25 and AM14 are specific for the pre-fusion state; however, D25 can bind both the pre-fusion monomer and the trimer, while AM14 can only bind the blocked pre-fusion trimer. 4D7 is specific for the post-fusion state. C-terminal 1 expression is good and shows the highest binding to AM14.

[0369] Table 14. Summary of antibody binding screening data for the designed RSV / BF protein

[0370] Example 2. Design of stabilization substitution for RSV F protein This example describes a stabilization mutant group used to stabilize the pre-fusion state of the RSV F protein. Based on the structure of RSV F in its pre-fusion conformation compared to its post-fusion conformation (not shown), Figure 1 The stabilizing mutations at the interfaces between protopolymers are designed to reduce the energy of the pre-fusion state or increase the energy of the post-fusion state.

[0371] Computational modeling was used to identify amino acid substitutions that stabilize RSV / BF proteins in their pre-fusion conformation. These mutations are listed in Table 15.

[0372] Table 15. Stabilization Substitution

[0373] Based on molecular modeling, the expected synergistic substitution combinations include:

[0374] The RSV F protein is cleaved by the protease furin during expression. Constructs were also tested by replacing the cleavage sites (residues 104-140) of furin with native linkers. The linker sequences are provided in Table 16, and they were tested between residues 103 and 141.

[0375] Table 16. Frin protease cleavage linkers

[0376] Example 3. Experimental evaluation of RSV F protein This embodiment demonstrates that the C-terminal helical forming region described in Example 1 increases the thermal stability of the recombinant peptide by up to about 20-25°C or more, and increases storage stability under accelerated degradation conditions (stored at 40°C). Further improvements were observed when the C-terminal helical forming region was combined with the stabilizing mutation described in Example 2. The recombinant peptide retains its ability to self-assemble to form a two-component I53-50 type nanostructure.

[0377] Small-scale HEK293 expression was used to test recombinant peptides comprising the extracellular domain of the RSV / BF protein fused to I53-50AΔcys (B18537 strain with the DS-Cav1 mutation). Supernatants were screened for relative expression using a monoclonal antibody (16A8) specifically binding to I53-50A via biolayer interferometry (BLI). BLI was used to measure binding to known RSV F protein antibodies D25 (specific to the pre-fusion state), AM14 (specific to the pre-fusion state of the blocked trimer), and 4D7 (specific to the post-fusion state). Measurements were normalized to binding by palizumab (conformity-independent). Increased AM14 was observed in several designs characterized by mutations in space 1, C-terminal remodeling, or both.

[0378] The scaled-up protein formulations used for the selected designs were incubated at 4°C or 40°C for six days. Designs showing smaller losses in D25 or AM14 binding and smaller increases in 4D7 binding at 40°C were identified compared to the DS-Cav1 mutation alone. The C-terminal 1 design, including the remodeled C-terminus (Example 1), showed almost no reduction in AM14 binding and no increase in 4D7 binding.

[0379] Mutants were selected for combination analysis. Extracellular domain sequences from contemporary RSV / B strains (hRSV / B / Australia / VIC-RCH056 / 2019) were used in these experiments. Antibody binding was normalized to 16A8 mAb, which is specific for I53-50A fusion conjugates. Various designs were characterized by increased binding to AM14 (before fusion) or decreased binding to 4D7 (after fusion). Figure 1 The selected scaled-up protein was subjected to a six-day thermal stress test.

[0380] Fourteen designs were selected for further analysis after scale-up and purification. Antigen measurements confirmed increased AM14 binding in all test designs relative to the DS-Cav1 mutation alone. Constructs incorporating C-terminal remodeling generally showed greater thermal stability at storage (i.e., a reduced rate of 4D7 binding reduction).

[0381] The constructs selected for thermal denaturation and storage tests are shown in Table 17. All tested RSV / B constructs were based on the sequence of the hRSV / B / Australia / VIC-RCH056 / 2019 strain fused to I53-50AΔcys, including the DS-Cav1 mutation. All proteins were tested as soluble trimeric fusions (before assembly with I53-50B to form nanostructures). RSV / A.03 (based on strain A2) and RSV / B.002 are controls containing the DS-Cav1 substitution. The data in Table 17 show that the C-terminal α-helix region itself can increase thermal stability by up to approximately 25 °C (compare construct RSV / B.002 with RSV / B.195, and construct RSV / B.093 with RSV / B.189). Furthermore, all constructs with the C-terminal α-helix region maintained their pre-fusion conformation after seven days of storage at 40 °C. A construct RSV / B.093 without a C-terminal α-helical segment was also in a stable pre-fusion state at 40°C, but its melting temperature was lower than that of the construct containing the C-terminal remodeling.

[0382] Table 17

[0383] 1 Based on hRSV / B / Australia / VIC-RCH056 / 2019 strain 2 NQSREIIRAINIVRKIASEK (SEQ ID NO: 10) 3 Based on strain A2 4 Except for DS-Cav1 (S155C, S290C, S190F and V207L) The selected construct was incubated with the second component I53-50B to form a nanostructure. Dynamic light scattering (DLS) and negative staining electron microscopy (nsEM) confirmed the assembly of the nanostructure. The results are shown in Table 18. Representative electron micrographs are shown in... Figure 5 (RSV / B.195, with DS-Cav)

[0384] Table 18

[0385] 1 Based on hRSV / B / Australia / VIC-RCH056 / 2019 strain 2 Except for DS-Cav1 (S155C, S290C, S190F and V207L) 3 NQSREIIRAINIVRKIASEK (SEQ ID NO: 10) 4 Based on strain A2 The sequences of the designed constructs used in Table 18 are shown in Table 19. SEQ ID NO: 1 is used as a reference sequence. In each case, the signal peptide at the N-terminus shown underlined or the tag at the C-terminus may have a known substitution or deletion. The RSV F protein is known to cleave at two furin cleavage sites, resulting in the loss of a peptide sequence known as “p27”. (Rezende et al.) Front. Microbiol. , Vol. 14 (2023). As used herein, the term "peptide" includes peptides lacking the p27 peptide due to this cleavage reaction. The proximal region surrounding the p27 peptide is italicized and can be removed via furin-based cleavage during antigen production in cell cultures.

[0386] Table 19

[0387] The relative expression and antibody binding for each design are shown in Table 20.

[0388] Table 20. Relative expression of BLI and antibody binding

[0389] The mutations of the designed constructs used in the experiments are shown in Table 21. All sequences are characterized by the fusion of the extracellular domain of RSV F (with the DS-Cav1 mutation) with the I53-50AΔcys (SEQ ID NO: 64) gene via a glycine- and serine-based flexible linker. Designs containing a C-terminal α-helix segment place this segment at the C-terminus of the extracellular domain as previously described, and prior to the flexible linker. SEQ ID NO: 1 is used as a reference sequence. In each case, the signal peptide at the N-terminus or the tag at the C-terminus can be replaced or deleted with known substitutes. “o” indicates the use of an amino acid substitution.

[0390] Table 21. Mutations of the constructs used in the experiment

[0391] 1 500-NQSREIIRAINIVRKIASEK-519 To test whether these stabilization modifications could be generalized beyond RSV / B-based antigens, two novel designs were also evaluated in the case of RSV / A antigen sequences (RSV / A.013 and RSV / A.023). Both designs contained the DS-Cav1 mutation and gene fusion to I53-50AΔcys, with RSV / A.013 adding a C-terminal α-helix (equivalent to the RSV / B.195 design) and RSV / A.023 adding both a C-terminal α-helix and the D489A, T400D, E487R, and K498A mutations (equivalent to the RSV / B.171 design). The sequences and mutations in these designs are further detailed in Tables 19 and 21, respectively. Compared to the RSV / A.03 design excluding the C-terminal α-helix segment or the D489A, T400D, E487R, and K498A mutations, both thermal and storage stability at 40 °C were significantly increased (Table 17). RSV / A.013 and RSV / A.023 showed melt temperatures increased by 4.5 °C and 19.0 °C, respectively, compared to RSV / A.03. This demonstrates that the C-terminal α-helix segment can be used alone to improve the thermal stability of both RSV / A and RSV / B antigens, and that the combination of the C-terminal α-helix segment with further stabilizing mutations can more rigorously improve the thermal stability of both RSV / A and RSV / B antigens. Furthermore, as assessed by DLS, both RSV / A.013 and RSV / A.023 could be assembled into nanostructures in vitro with the addition of I53-50B (Table 18).

[0392] To evaluate the immunogenicity of different designs based on RSV / B or RSV / A, two in vivo studies were conducted in BALB / c mice. In one study, the RSV / B neutralizing titer induced by immunization with assembled nanostructures based on RSV / B.002, RSV / B.093, RSV / B.195, RSV / B.160, or RSV / B.171 at doses of 0.02 mg or 0.1 mg, all of which were fortified with AddaVax adjuvant (… Figure 17 No statistically significant differences were observed between any designs at any dose. Similarly, no statistically significant differences were observed between mice immunized with assembled nanostructures based on RSV / A.03, RSV / A.013, or RSV / A.023 at doses of 5 mg without adjuvant or 0.01 mg with AddaVax adjuvant. Figure 18 However, mice immunized with 1 mg of unadjuvanted RSV / A.023 nanostructures did have significantly higher RSV / A neutralizing titers than mice immunized with the same dose of unadjuvanted RSV / A.03.

[0393] Example 4. Method for generating C-terminal diffusion The relaxed structures used as input for Rosetta remodeling were also used as input for RF diffusion, except that only the C-terminal helix and neighboring residues were used as diffusion input. This significantly reduced computation time. Positions with WT sequence identity from Rosetta remodeling were also retained for diffusion. Unlike Rosetta remodeling, all structural data for subsequent residues were ignored. This is a constraint on RF diffusion, not a scientific constraint on the design problem. Weights within and between protomers were set to 1, with secondary guidance decay and guidance scaling of 2. Non-standard weights Base_epoch8_ckpt.pt were applied and C3 symmetry was performed. The C-terminal length varied between 12 and 31 residues depending on the source virus, and 15 remodeling helices were generated for each length. Sequences were generated using protein MPNN, with a sampling temperature of 0.4°C and negative biases for C (-10) and F, G, P, W, and Y (-1). 100 sequences were generated for each remodeling domain. The top 2% based on MPNN sample scores were selected for computational characterization.

[0394] A unique set of all-α-helical bundles is generated for each input structure. For most inputs, Rosetta remodeling (remodeling) and RF diffusion (diffusion) are used, with the exception of PIV5, where remodeling yields a large number of unique results. The number and quality of the output structures are highly variable, depending on the input structure. For example, C-terminal residues in most structures exhibit low data quality, likely due to local flexibility. This, combined with consistent evidence of a lack of effort to refine this region, may result in suboptimal bond angles and bond lengths. Furthermore, many fusion proteins are slightly asymmetric. Symmetry may have introduced strain. Overall, these effects can influence the quality and number of outputs through the ddG filter, as well as the results produced by diffusion. For that reason, both remodeling and diffusion are used when remodeling alone is insufficient to produce sufficiently high-quality outputs.

[0395] Based on the geometry of the input structure, the C-terminal structural domain of the remodeled structure is generally divided into two categories. When the input structural domain already consists of a relatively compact spiral structure (e.g., ...), ... Figure 6 A-6D), the remodeling domain continues the helical bundle, in which a straight or slightly twisted helical bundle with a remodeling length of 10 to 24 residues is optimal. Figure 7 The input domain consists of converging α-helices, and the remodeled domain features helical intersections with well-packed hydrophobic cores. Figure 6 E). RF diffusion can also generate output, where the helices converge into a tight helical bundle ( Figure 8 The optimal remodeling length for these constructs is greater than 10 residues. Figure 9 In some cases, all remodeling lengths produce significantly better scores than WT sequences. Figure 9 In this case, the design is selected based on its score relative to the average of that remodeling length.

[0396] All selected remodeling sequences produced helical bundles with repeating patterns of both hydrophobic and hydrophilic residues. In most cases, WT sequences exhibited similar patterns, differing only in that one of the repeats was significantly less hydrophobic than the remodeling sequence. For example, remodeling position 8 in PIV5 is serine, and in remodeling designs it is typically leucine, isoleucine, valine, or alanine. Figure 10 Designs with more distant C-terminal helices tend to produce patterns of polar and hydrophobic residues compared to designs with WT offsets. Figure 11 ).

[0397] PIV5The input structure of PIV5 is 4GIP (Ref. 4). PIV5 has a reserved glycan at position 457. The B factor increases significantly from residues 460 to 464, and therefore, for that reason, repacking at 459 and 460 is allowed, and de novo sequences are generated for subsequent residues. 76 remodeling sequences are generated, ranging in length from six (6) to 26 residues. The design generally improves hydrophobic stacking, especially at positions 470 and 471. Some short remodeling sequences have excellent predictive ddG, but according to the ddG plot, the optimal length is about 12-14 residues ( Figure 7 ).

[0398] PIV3 The input for PIV3 is 8DG8 (Ref. 5). There is no glycan in the C-terminal helical bundle of PIV3. The quality of the cryogenic EM plot deteriorates gradually along the length of the C-terminal helix, and there are no side chains resolved after residue 469. There is some suboptimal stacking at position 460, and therefore this position is allowed for design when using Rosetta remodeling. Since residue 461 is natively contacted with the rest of the extracellular domain, its identity is preserved, allowing for de novo design of subsequent positions. Residues after position 468 are removed. RF diffusion does not allow for simultaneous extension and partial diffusion, so the diffusion model starts at residue 465. Ten (10) sequences are generated by Rosetta remodeling and 44 sequences by diffusion. The optimal length is 14–16 residues (…). Figure 7 Therefore, a remodeling length of 14 or greater is chosen for RF diffusion.

[0399] Nipah The input for Nipah is 7UP9 (Ref. 6). Nipah contains a glycan at residue 464, which is preserved in all designs. Because Nipah has a low-entropy methionine residue at residue 463 and no significant contact with the rest of the extracellular domain, both remodeling and diffusion are allowed to design a de novo sequence starting at residue 463. This requires manual reversion of residues 464 and 466 to preserve the glycan. The optimal sequence length is approximately 10 residues. Figure 7 Therefore, it is used as the minimum remodeling length for RF diffusion. Fifty-three (53) sequences are selected.

[0400] HMPV The input PDB for HMPV is 5WB0 (Ref. 7). The C-terminal resolution of HMPV is much lower than that of RSV. For that reason, only positions 471 and 472 of the input structure are included in the sequence design; all residues after 470 are allowed to be designed de novo. The optimal remodeling length is 10 residues. Figure 9Furthermore, the minimum remodeling length for RF diffusion was set to 10. Interestingly, the RF diffusion circuitry struggled to produce remodeling ends that were well-predicted for HMPV. This is likely due to the interaction between the identity of the case provided for diffusion and ColabFold, rather than an inherent characteristic of the HMPV-F protein. Like RSV-F, the HMPV-F remodeling design tended to have a well-stacked hydrophobic core in three or four layers, starting at position 473.

[0401] RSV A small set of C-terminal sequences was generated using RF diffusion. Longer remodeling sequences up to 31 residues in length were fully predicted. The RSV design was based on a 4MMU (Reference 8).

[0402] SARS-CoV-2 Based on a combination of reasonable quality data and a well-constructed model in the relevant region, we selected 7LAB as the input structure (refer to 9). The design was selected based on the average score relative to that length. Figure 9 De novo sequence design begins at residue 1147. Optimal remodeling length >10 residues, but some shorter designs with a remodeling length of six residues form very tightly packed helical bundles. For RF diffusion, a minimum length of 10 is chosen. Although the arrangement of polar and hydrophobic residues is largely the same for both design and WT sequences (…),… Figure 11 However, the hydrophobic residues tend to be smaller, especially at positions 1149 and 1153. This allows for a more compact packing, thus allowing residues 1150 or 1154 to also be hydrophobic.

[0403] Experimental validation of C-terminal remodeling designs in PIV3: 53 C-terminal remodeling designs described in Tables 7B and 7D were fused to the I53-50AΔcys gene with a 12-residue Gly-Ser linker and expressed at a small scale in HEK293 cells. These designs were compared to a control (PIV3F.C) using GCN4 instead of C-terminal remodeling, except for many designs (PIV3F.55-95, e.g., containing SEQ ID NO: 716 to 756) that added novel stabilizing mutations to the F extracellular domain relative to PIV3F.C. Figure 21 ) and PIA174 ( Figure 22 To determine the pre-fusion conformation, the pre-fusion specific monoclonal antibody was bound to the CompA-specific monoclonal antibody 16A8 and normalized to account for differences in expression levels. Figure 23Forty other non-C-terminal remodeling designs attempting to stabilize the pre-fusion conformation were also included in the analysis. While only 8 out of 40 non-C-terminal remodeling designs were in a strongly pre-fusion state, 36 out of 53 C-terminal remodeling designs were in a strongly pre-fusion state, and most exhibited some 3×1 and PIA174 binding. Surprisingly, for many C-terminal remodeling designs, the 3×1 and PIA174 binding signals were higher relative to PIV3F.C, demonstrating that this design technique can provide superior antigenicity and / or expression levels compared to gene fusion with GCN4, and is commonly used in the field. Furthermore, this design strategy had a significantly higher success rate than designs testing stabilization mutations instead of C-terminal remodeling strategies.

[0404] The pre-fusion conformation of the PIV3 fusion protein can be stabilized by adding a trimerizing domain, such as GCN4, between the antigen and CompA, in addition to the antigen and CompA (PIV3F.C in Tables 22 and 23; containing SEQ ID NO: 327). To better understand the effects of C-terminal remodeling, we expressed and purified three C-terminal remodeling constructs in HEK293 or CHO cells. These three constructs (PIV3F.28, PIV3F.40, and PIV3F.44, containing SEQ ID NO: 355, 367, and 371, respectively) were selected based on higher levels of binding signals to 3×1 and PIA174 after small-scale expression. Purification yield was determined by UV-Vis, percentage of high molecular weight (HMW) material was determined by size exclusion ultra-high performance liquid chromatography (UPLC), and pre-fusion conformation was determined by antibody binding using BLI (Table 22). Thermodynamic properties were determined using nanoDSF with either the exogenous dye SYPRO or intrinsic tryptophan fluorescence and static light scattering to determine the aggregation initiation temperature (T). agg C-terminal remodeling design moderately reduced HMW content and improved yield and pre-fusion antibody binding. Unlike RSV, the change in thermal stability metrics was minimal. However, WT PIV3 F protein exhibits higher intrinsic thermal stability than RSV F.

[0405] Table 22: Characterization of PIV3 F constructs remodeled at WT and C ends

[0406] *The first value comes from the HEK expression, and the second value comes from the CHO expression. **Normalization of 16A8 binding via BLI's PIA174 and 3×1 binding** To further differentiate the C-terminal remodeling design from the WT antigen, the three selected designs were stored under stress at 25°C or 45°C for 30 or 14 days, respectively. Stability was measured by size exclusion ultra-high performance liquid chromatography (SU-UPLC). The main peak area corresponding to PIV3 F and the earlier elution peak corresponding to high molecular weight substances (HMWS) were integrated, and the percentage change relative to the sample stored at -80°C was calculated. The designed constructs were more robust to stress storage, as evidenced by a 36.1% loss of the main peak area and a corresponding increase in HMWS in the WT constructs after 30 days of storage at 25°C, compared to only a 2-8% loss / increase in the C-terminal remodeling constructs (Table 23).

[0407] Table 23: Stress storage stability of PIV3 F constructs remodeled at WT and C ends

[0408] Example 5. Common Sequence Analysis The structure is analyzed by measuring the helical end moments of two of the three protopolymers in the input trimer structure. These moments are measured by determining a vector between the N-terminal α-carbon and the α-carbon near the C-terminus, which is an integer number of helical turns following the first selected α-carbon. The dot product of the helical moments is a measure of helical orthogonality.

[0409] Common sequences are identified by first clustering the input structure based on the C-terminal geometry. The dot product of C-terminal moments is generally clustered into two groups with average values ​​of 0.92 + / - 0.03 and 0.77 + / - 0.6, referred to as "parallel" and "non-parallel," respectively. The former includes Paramyxoviridae and Coronaviridae, while the latter consists of Pneumoviridae. Sequences derived from parallel and non-parallel helices are compared separately. The comparison is based on structural alignment. For PIV5, the WT sequence LAAV ends the alignment, which interferes with clustering. Therefore, MPNN is used to generate sequences to replace LAAV. Similarly, retained glycosylation sites also interfere with clustering. Glycosylation residues are randomly replaced with Q, N, D, S, or T to match those positions in the alignment ( Figure 16A-16G Noise was introduced at position 1). The alignment s...

Claims

1. A recombinant polypeptide comprising an engineered extracellular domain of a triviral protein, wherein the extracellular domain comprises: C-terminal helical forming segment, the C-terminal helical forming segment comprising one or more amino acid substitutions relative to the native reference sequence of the viral protein, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

2. The recombinant polypeptide of claim 1, wherein the C-terminal helical forming region has improved hydrophobic stacking compared to the native reference sequence.

3. The recombinant polypeptide of claim 1 or claim 2, wherein the C-terminal helical forming region comprises about 7 to about 31 residues.

4. The recombinant polypeptide according to any one of claims 1 to 3, wherein the amino acid substitution comprises polar, charged and / or hydrophobic amino acids.

5. The recombinant polypeptide of any one of claims 1 to 4, wherein the C-terminal helical region comprises a polypeptide sequence according to any one of the following: LXXTIXXLLXIXXXLXXXL (SEQ ID NO: 566) LVXTXKXLXDLIXXLXXLLXKLXX (SEQ ID NO: 567) LNKVKKXVXXLXXXVXXLEKXLX (SEQ ID NO: 568) EKIXXAIKKAXKL (SEQ ID NO: 569) EXIXKAIKXLXXXXX (SEQ ID NO: 570) XKXXEXXXXVXXXXXXXXX (SEQ ID NO: 571) XXLKKAAXIXKKXLKXX (SEQ ID NO: 572).

6. The recombinant polypeptide of any one of claims 1 to 5, wherein the C-terminal helical forming region comprises a polypeptide sequence according to any one of the common sequences in Table 24.

7. The recombinant polypeptide of any one of claims 1 to 6, wherein the segment comprises a polypeptide sequence according to the following: a) L X2X2T I X2X2L L X2I [V / I] X2X2L [I / L] X2X2L (SEQ ID NO: 573); b) LV [A / T] T X2K X2L X2D LI X2X2L [K / E] X2L L X2K L X2X2 (SEQ ID NO:574); or c) LNKVKK X2V X2X2L X2X2X2V X2X2L EK X2L X2 (SEQ ID NO: 575), wherein X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

8. The recombinant polypeptide of any one of claims 1 to 6, wherein the segment comprises a polypeptide sequence according to the following: a) EKI X2X2A IKKA X2K L (SEQ ID NO: 576); b) E X2I X2K AIK X2L [L / X2] X2X2[X1 / X2] X2 (SEQ ID NO: 577); and c) X2K [X1 / T] [L / E] E [T / A] X1X2[I / X2] V X2X2[X1 / X2] [X1 / X2] d) X2X2L KKAA X2I X1K K X1L K X2X2 (SEQ ID NO: 579), wherein X1 is a nonpolar residue selected from A, I, L and M, and X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

9. The recombinant polypeptide of any one of claims 1 to 8, wherein the segment comprises a polypeptide sequence listed in Table 25A or Table 25B.

10. The recombinant polypeptide of any one of claims 1 to 9, wherein the native reference sequence of the viral protein is any one of SEQ ID NO: 1, 104, 327, 382, ​​459, 499.

11. The recombinant polypeptide of any one of claims 1 to 10, wherein the recombinant polypeptide comprises an engineered extracellular domain of an hMPV fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions between about residues 470 and about residues 500 relative to SEQ ID NO: 104, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

12. The polypeptide of claim 11, wherein the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 104, the residues creating hydrophobic contacts between the segments in the α-helical isotrimester.

13. The polypeptide of claim 11 or claim 12, wherein the extracellular domain comprises the C-terminal helical forming segment containing one or more amino acid substitutions between about residues 470 and about residues 490 relative to SEQ ID NO:104, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

14. The polypeptide of any one of claims 11 to 13, wherein the C-terminal helical region comprises about 7 to about 21 residues.

15. The polypeptide of any one of claims 11 to 14, wherein the segment comprises: (1) The amino acid substitution at position Q471 relative to SEQ ID NO: 104, wherein Q is substituted by any one of A, D, E, I, Q, R, S, T; (2) The amino acid substitution at position A472 relative to SEQ ID NO: 104, wherein A is substituted by any one of A, D, E, I, K, R, S, T, Y; (3) The amino acid at position L473 relative to SEQ ID NO: 104 is replaced by any one of A, I, L, M, Q, S, T, W; (4) The amino acid at position V474 relative to SEQ ID NO: 104, wherein V is replaced by any one of A, D, E, I, K, L, N, Q, S, T; (5) The amino acid substitution at position D475 relative to SEQ ID NO: 104, wherein D is substituted by any one of A, D, E, H, K, N, Q, R, S, T; (6) The amino acid at position Q476 relative to SEQ ID NO: 104, wherein Q is replaced by any one of A, D, E, H, I, K, L, M, N, Q, T, V; (7) The amino acid substituted at position S477 relative to SEQ ID NO: 104, wherein S is substituted by any one of A, E, I, K, L, M, N, Q, R, S, T, V; (8) The amino acid substitution at position N478 relative to SEQ ID NO: 104, wherein N is substituted by any one of A, D, E, K, N, Q, R, S, T; (9) The amino acid at position R479 relative to SEQ ID NO: 104, wherein R is replaced by any one of A, D, E, F, I, K, L, M, N, Q, R, S, T, W, Y; (10) The amino acid substituted at position I480 relative to SEQ ID NO: 104, wherein I is substituted by any one of A, I, L, M, R, S, T, V; (11) The amino acid at position L481 relative to SEQ ID NO: 104, wherein L is replaced by any one of D, E, I, K, L, M, N, Q, R, S, T; (12) The amino acid substitution at position S482 relative to SEQ ID NO: 104, wherein S is substituted by any one of A, D, E, K, Q, R, S, T; (13) The amino acid substituted at position S483 relative to SEQ ID NO: 104, wherein S is substituted by any one of A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, Y; (14) The amino acid at position A484 relative to SEQ ID NO: 104, wherein A is replaced by any one of A, D, E, I, K, L, M, R, S, T, V, Y; (15) The amino acid substitution at position E485 relative to SEQ ID NO: 104, wherein E is substituted by any one of D, E, G, K, L, Q, R, S, T; (16) The amino acid substitution at position K486 relative to SEQ ID NO: 104, wherein K is substituted by any one of A, E, I, K, L, Q, R, S, T; (17) The amino acid substitution at position G487 relative to SEQ ID NO: 104, wherein G is substituted by any one of A, E, I, K, L, R, S, T, V; (18) The amino acid substitution at position N488 relative to SEQ ID NO: 104, wherein N is substituted by any one of E, I, K, L, N, Q, R, S; (19) An amino acid substitution relative to SEQ ID NO: 104 at position T489, wherein T is substituted by any one of A, D, E, K, S; and / or (20) Any combination of (1)-(19).

16. The polypeptide of any one of claims 11 to 15, wherein the segment comprises a polypeptide sequence listed in Table 6B, or a polypeptide sequence having one to five amino acid substitutions.

17. The polypeptide of any one of claims 11 to 16, wherein the extracellular domain comprises the C-terminal helical forming segment containing one or more amino acid substitutions between about residues 470 and about residues 500 relative to SEQ ID NO:104, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

18. The polypeptide of any one of claims 11 to 17, wherein the C-terminal helical region comprises about 16 to about 30 residues.

19. The polypeptide of any one of claims 11 to 18, wherein the segment comprises: (1) The amino acid substitution at position A472 relative to SEQ ID NO: 104, wherein A is substituted by any one of T, N, K, R, E, S; (2) The amino acid substitution at position L473 relative to SEQ ID NO: 104, wherein L is substituted by any one of T, I, or V; (3) The amino acid substitution at position V474 relative to SEQ ID NO: 104, wherein V is substituted by any one of E, Q, L, or D; (4) The amino acid substitution at position D475 relative to SEQ ID NO: 104, wherein D is substituted by any one of E, D, or K; (5) The amino acid substitution at position Q476 relative to SEQ ID NO: 104, wherein Q is substituted by any one of Q, R, D, A, T, S, K; (6) The amino acid substitution at position S477 relative to SEQ ID NO: 104, wherein S is substituted by any one of I, V, L; (7) The amino acid substitution at position N478 relative to SEQ ID NO: 104, wherein N is substituted by any one of K, E, N, or S; (8) The amino acid substitution at position R479 relative to SEQ ID NO: 104, wherein R is substituted by any one of T, D, E, S, Y, A; (9) The amino acid substitution at position I480 relative to SEQ ID NO: 104, wherein I is substituted by either L or N; (10) The amino acid substitution at position L481 relative to SEQ ID NO: 104, wherein L is substituted by any one of T, D, E, K, Q, S, N; (11) The amino acid substitution at position S482 relative to SEQ ID NO: 104, wherein S is substituted by any one of E, D, S, T, Q, K; (12) The amino acid substitution at position S483 relative to SEQ ID NO: 104, wherein S is substituted by any one of R, K, L, E, A; (13) The amino acid substitution at position A484 relative to SEQ ID NO: 104, wherein A is substituted by any one of V, I, or M; (14) The amino acid substituted at position E485 relative to SEQ ID NO: 104, wherein E is substituted by any one of E, A, K, H, Q, S, N; (15) The amino acid substitution at position K486 relative to SEQ ID NO: 104, wherein K is substituted by any one of S, E, K, R, V, D, H; (16) The amino acid substitution at position G487 relative to SEQ ID NO: 104, wherein G is substituted by either I or L; (17) The amino acid substitution at position N488 relative to SEQ ID NO: 104, wherein N is substituted by any one of E, K, or R; (18) The amino acid substitution at position T489 relative to SEQ ID NO: 104, wherein T is substituted by any one of K, E, S, R, Q; (19) The amino acid substitution at position S490 relative to SEQ ID NO: 104, wherein S is substituted by any one of E, V, T, R, L; (20) The amino acid substitution at position G491 relative to SEQ ID NO: 104, wherein G is substituted by any one of GL, I, V; (21) The amino acid substitution at position R492 relative to SEQ ID NO: 104, wherein R is substituted by any one of E, Q, S, A, D; (22) The amino acid substitution at position E493 relative to SEQ ID NO: 104, wherein E is substituted by any one of A, E, N, L, K, Q, S; (23) The amino acid substitution at position N494 relative to SEQ ID NO: 104, wherein N is substituted by either I or L; (24) The amino acid substitution at position L495 relative to SEQ ID NO: 104, wherein L is substituted by any one of K, L, T, V, I; (25) The amino acid substitution at position Y496 relative to SEQ ID NO: 104, wherein Y is substituted by any one of K, E, R, Q; (26) The amino acid substitution at position F497 relative to SEQ ID NO: 104, wherein F is substituted by any one of D, R, E, Q; (27) An amino acid substitution at position Q498 relative to SEQ ID NO: 104, wherein Q is substituted by either V or L; and / or (28) Any combination of (1)-(27).

20. The polypeptide of any one of claims 11 to 19, wherein the segment comprises a polypeptide sequence listed in Table 6D, or a polypeptide sequence having one to five amino acid substitutions.

21. The polypeptide of any one of claims 11 to 20, wherein the extracellular domain further comprises: The substitution of one, two, three or more amino acids at positions 63, 97, 98, 99, 100, 101, 102, 140, 147, 153, 185, 188, 219, 231, 294, 365, 368, 450, 463 or 470 relative to SEQ ID NO:

104.

22. The recombinant polypeptide of any one of claims 1 to 10, wherein the recombinant polypeptide comprises an engineered extracellular domain of a PIV3 fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions between about residues 460 and about residues 490 relative to SEQ ID NO: 327, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

23. The polypeptide of claim 22, wherein the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 327, the residues creating hydrophobic contacts between the segments in the α-helical isotrimester.

24. The polypeptide of claim 22 or claim 23, wherein the extracellular domain comprises the C-terminal helical forming segment containing one or more amino acid substitutions between about residues 460 and about residues 480 relative to SEQ ID NO:104, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

25. The polypeptide of any one of claims 22 to 24, wherein the C-terminal helical region comprises about 20 to about 28 residues.

26. The polypeptide of any one of claims 22 to 25, wherein the segment comprises: (1) An amino acid substitution relative to SEQ ID NO: 327 at position L460, wherein L is substituted by any one of L, M, or V; (2) The amino acid substitution at position N461 relative to SEQ ID NO: 327, wherein N is replaced by N; (3) The amino acid substitution at position K462 relative to SEQ ID NO: 327, wherein K is substituted by either K or R; (4) The amino acid substitution at position V463 relative to SEQ ID NO: 327, wherein V is substituted by any one of L, V, or T; (5) The amino acid substitution at position K464 relative to SEQ ID NO: 327, wherein K is substituted by any one of A, K, or Q; (6) An amino acid substitution at position S465 relative to SEQ ID NO: 327, wherein S is substituted by either K or S; (7) An amino acid substitution at position D466 relative to SEQ ID NO: 327, wherein D is substituted by either E or K; (8) The amino acid substitution at position L467 relative to SEQ ID NO: 327, wherein L is replaced by any one of V, L, or T; (9) The amino acid substitution at position E468 relative to SEQ ID NO: 327, wherein E is substituted by any one of K, D, and E; (10) The amino acid substitution at position E469 relative to SEQ ID NO: 327, wherein E is substituted by any one of T, Q, K, and E; (11) The amino acid substitution at position S470 relative to SEQ ID NO: 327, wherein S is substituted by any one of I, L, M, Y, F, W; (12) The amino acid substitution at position K471 relative to SEQ ID NO: 327, wherein K is substituted by any one of L, W, A, I; (13) The amino acid substitution at position E472 relative to SEQ ID NO: 327, wherein E is substituted by either K or E; (14) The amino acid substitution at position W473 relative to SEQ ID NO: 327, wherein W is substituted by any one of E, I, K, or Q; (15) The amino acid substitution at position Y474 relative to SEQ ID NO: 327, wherein Y is substituted by any one of L, M, T, V, E; (16) The amino acid substitution at position R475 relative to SEQ ID NO: 327, wherein R is substituted by any one of S, K, R, or A; (17) An amino acid substitution at position R476 relative to SEQ ID NO: 327, wherein R is substituted by any one of K, E, S, N; (18) An amino acid substitution at position S477 relative to SEQ ID NO: 327, wherein S is substituted by any one of K, D, or E; and / or (19) Any combination of (1)-(18).

27. The polypeptide of any one of claims 22 to 26, wherein the segment comprises a polypeptide sequence listed in Table 7B, or a polypeptide sequence having one to five amino acid substitutions.

28. The polypeptide of any one of claims 22 to 27, wherein the extracellular domain comprises the C-terminal helical forming segment containing one or more amino acid substitutions between about residues 465 and about residues 490 relative to SEQ ID NO:327, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

29. The polypeptide of any one of claims 22 to 28, wherein the C-terminal helical region comprises about 14 to about 30 residues.

30. The polypeptide of any one of claims 22 to 29, wherein the segment comprises: (1) An amino acid substitution relative to SEQ ID NO: 327 at position S465, wherein S is substituted by any one of E, K, D, S, N, Q, T, R, A; (2) The amino acid substitution at position D466 relative to SEQ ID NO: 327, wherein D is substituted by any one of D, R, K, E, M, Q, A, S, N; (3) The amino acid substitution at position L467 relative to SEQ ID NO: 327, wherein L is replaced by any one of I, V, and L; (4) The amino acid at position E468 relative to SEQ ID NO: 327, wherein E is replaced by any one of E, K, S, D, R, H, T, N, A; (5) The amino acid substitution at position E469 relative to SEQ ID NO: 327, wherein E is substituted by any one of K, S, E, N, T, Q, H, D, Y; (6) The amino acid substitution at position S470 relative to SEQ ID NO: 327, wherein S is substituted by any one of L, D, V, I, A, N, T; (7) The amino acid substitution at position K471 relative to SEQ ID NO: 327, wherein K is substituted by any one of E, T, L, K, N, I, R, Q, S; (8) The amino acid substitution at position E472 relative to SEQ ID NO: 327, wherein E is substituted by any one of E, K, Q, S, H, R, T; (9) The amino acid substitution at position W473 relative to SEQ ID NO: 327, wherein W is substituted by any one of R, Q, K, E, T, S, I, N; (10) The amino acid substitution at position Y474 relative to SEQ ID NO: 327, wherein Y is substituted by any one of V, L, I, Q, T; (11) The amino acid substituted at position R475 relative to SEQ ID NO: 327, wherein R is substituted by any one of H, K, D, T, E, S, R, N, Q, A; (12) The amino acid substitution at position R476 relative to SEQ ID NO: 327, wherein R is substituted by any one of A, T, H, E, D, K, R, Q, S; (13) The amino acid substitution at position S477 relative to SEQ ID NO: 327, wherein S is substituted by any one of I, L, V; (14) The amino acid substituted at position N478 relative to SEQ ID NO: 327, wherein N is substituted by any one of E, L, K, I, R, S, Q; (15) The amino acid substitution at position Q479 relative to SEQ ID NO: 327, wherein Q is substituted by any one of K, H, E, N, Q, R, T, A, S; (16) The amino acid substitution at position K480 relative to SEQ ID NO: 327, wherein K is substituted by any one of K, R, E, T, S, L, A, I, V; (17) The amino acid substitution at position L481 relative to SEQ ID NO: 327, wherein L is substituted by any one of L, V, or I; (18) The amino acid substitution at position D482 relative to SEQ ID NO: 327, wherein D is substituted by any one of K, A, E, S, H, T, N, D, R; (19) The amino acid substitution at position S483 relative to SEQ ID NO: 327, wherein S is substituted by any one of Q, T, E, A, S, N, D, K, L; (20) The amino acid substitution at position I484 relative to SEQ ID NO: 327, wherein I is substituted by any one of I, L, A, V; (21) The amino acid substitution at position G485 relative to SEQ ID NO: 327, wherein G is substituted by any one of L, K, R, E, I; (22) An amino acid substitution relative to SEQ ID NO: 327 at position S486, wherein S is substituted by any one of T, A, E, R, H, D, S; and / or (23) Any combination of (1)-(22).

31. The polypeptide of any one of claims 22 to 30, wherein the segment comprises a polypeptide sequence listed in Table 7D, or a polypeptide sequence having 1 to 5 amino acid substitutions.

32. The recombinant polypeptide of any one of claims 1 to 10, wherein the recombinant polypeptide comprises an engineered extracellular domain of a PIV5 fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions between about residues 460 and about residues 490 relative to SEQ ID NO: 382, ​​the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

33. The polypeptide of claim 32, wherein the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 382, ​​the residues creating hydrophobic contacts between the segments in the α-helical isotrimester.

34. The polypeptide of claims 32 to 33, wherein the extracellular domain comprises the C-terminal helical forming segment containing one or more amino acid substitutions between about residues 460 and about residues 480 relative to SEQ ID NO: 382, ​​the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

35. The polypeptide of any one of claims 32 to 34, wherein the C-terminal helical region comprises about 6 to about 26 residues.

36. The polypeptide of any one of claims 32 to 35, wherein the segment comprises: (1) The amino acid substitution at position A463 relative to SEQ ID NO: 382, ​​wherein A is substituted by any one of L, T, V, and A; (2) The amino acid substitution at position L464 relative to SEQ ID NO: 382, ​​wherein L is substituted by any one of K, I, Q, A, W, E; (3) The amino acid substitution at position Q465 relative to SEQ ID NO: 382, ​​wherein Q is substituted by any one of K, Q, T, E, S, R; (4) The amino acid at position H466 relative to SEQ ID NO: 382, ​​wherein H is replaced by any one of K, A, E, L, I, W, R, Q, T, D, Y; (5) The amino acid substitution at position L467 relative to SEQ ID NO: 382, ​​wherein L is replaced by any one of V, I, L, M, FA, T, C, H; (6) The amino acid substitution at position A468 relative to SEQ ID NO: 382, ​​wherein A is substituted by any one of D, T, K, L, E, R, I, N, S; (7) The amino acid substitution at position Q469 relative to SEQ ID NO: 382, ​​wherein Q is substituted by any one of E, K, S, T, A, R, Q, D; (8) The amino acid substituted at position S470 relative to SEQ ID NO: 382, ​​wherein S is substituted by any one of A, K, L, I, T, S, V, H, Y, E, W, FR, Q, M; (9) The amino acid substitution at position D471 relative to SEQ ID NO: 382, ​​wherein D is substituted by any one of T, E, V, L, S, I, A, K, Y, W; (10) The amino acid substitution at position T472 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of K, E, R, S, T, A, D, L; (11) The amino acid at position Y473 relative to SEQ ID NO: 382, ​​wherein Y is replaced by any one of T, K, S, R, Q, D, E, I, H, M; (12) The amino acid substituted at position L474 relative to SEQ ID NO: 382, ​​wherein L is substituted by any one of T, S, L, A, D, W, Q, I, Y, V, K, E; (13) The amino acid substituted at position S475 relative to SEQ ID NO: 382, ​​wherein S is substituted by any one of T, E, I, K, S, Q, A, L, R, D; (14) The amino acid substitution at position A476 relative to SEQ ID NO: 382, ​​wherein A is substituted by any one of R, K, A, S, E, I, T, D, Q; (15) The amino acid at position I477 relative to SEQ ID NO: 382, ​​wherein I is replaced by any one of K, Q, R, D, T, E, I, Y, S, L; (16) The amino acid substitution at position T478 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of E, K, S, D, W, L, Q, I, T; (17) The amino acid substitution at position S479 relative to SEQ ID NO: 382, ​​wherein S is substituted by any one of R, K, Q, S, A, D, E; (18) An amino acid substitution at position A480 relative to SEQ ID NO: 382, ​​wherein A is substituted by either S or K; (19) The amino acid substitution at position T481 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of E, D, S, K, M, N, A, T; (20) The amino acid substitution at position T482 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of R, S, Q, L, K; (21) An amino acid substitution at position T483 relative to SEQ ID NO: 382, ​​wherein T is substituted by any one of K, A, or S; (22) The amino acid substitution at position S484 relative to SEQ ID NO: 382, ​​wherein S is substituted by any one of S, E, D, Y; (23) An amino acid substitution at position V485 relative to SEQ ID NO: 382, ​​wherein V is substituted by either Q or T; (24) The amino acid substitution at position L486 relative to SEQ ID NO: 382, ​​wherein L is replaced by K; (25) An amino acid substitution at position S487 relative to SEQ ID NO: 382, ​​wherein S is substituted by either S or K; (26) An amino acid substitution at position I488 relative to SEQ ID NO: 382, ​​wherein I is substituted by either S or K; and / or (27) Any combination of (1)-(26).

37. The polypeptide of any one of claims 32 to 36, wherein the segment comprises a polypeptide sequence listed in Table 8B, or a polypeptide sequence having one to five amino acid substitutions.

38. The recombinant polypeptide of any one of claims 1 to 10, wherein the recombinant polypeptide comprises an engineered extracellular domain of a SARS-CoV2 spike (S) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions between about residues 1140 and about residues 1170 relative to SEQ ID NO: 459, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

39. The polypeptide of claim 38, wherein the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 459, the residues creating hydrophobic contacts between the segments in the α-helical isotrimester.

40. The polypeptide of claim 38 or claim 39, wherein the extracellular domain comprises the C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO:459 between about residues 1140 and about residues 1170, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

41. The polypeptide of any one of claims 38 to 40, wherein the C-terminal helical region comprises about 10 to about 25 residues.

42. The polypeptide of any one of claims 38 to 41, wherein the segment comprises: (1) The amino acid substitution at position D1147 relative to SEQ ID NO: 459, wherein D is substituted by any one of E, D, or K; (2) The amino acid substitution at position S1148 relative to SEQ ID NO: 459, wherein S is substituted by any one of T, S, or K; (3) The amino acid substitution at position F1149 relative to SEQ ID NO: 459, wherein F is replaced by A; (4) The amino acid substitution at position K1150 relative to SEQ ID NO: 459, wherein K is substituted by any one of I, A, L, M; (5) The amino acid substitution at position E1151 relative to SEQ ID NO: 459, wherein E is substituted by any one of K, S, D, R, and E; (6) The amino acid substitution at position E1152 relative to SEQ ID NO: 459, wherein E is substituted by any one of I, Y, K, T, R, and E; (7) An amino acid substitution at position L1153 relative to SEQ ID NO: 459, wherein L is substituted by either T or A; (8) The amino acid substitution at position D1154 relative to SEQ ID NO: 459, wherein D is substituted by any one of L, I, E, T, M, V; (9) The amino acid substitution at position K1155 relative to SEQ ID NO: 459, wherein K is substituted by any one of E, K, T, or R; (10) The amino acid substitution at position Y1156 relative to SEQ ID NO: 459, wherein Y is substituted by any one of I, V, K, R; (11) The amino acid substitution at position F1157 relative to SEQ ID NO: 459, wherein F is substituted by any one of V, A, I, Y, T, S; (12) The amino acid substituted at position K1158 relative to SEQ ID NO: 459, wherein K is substituted by any one of L, R, S, K, D, W, N, I; (13) The amino acid substitution at position N1159 relative to SEQ ID NO: 459, wherein N is substituted by any one of K, T, Q, I, R, E; (14) The amino acid substitution at position H1160 relative to SEQ ID NO: 459, wherein H is substituted by any one of I, L, R, E, K, S; (15) The amino acid substitution at position T1161 relative to SEQ ID NO: 459, wherein T is substituted by any one of L, N, I, A, S, W, Y; (16) The amino acid substitution at position S1162 relative to SEQ ID NO: 459, wherein S is substituted by any one of K, S, T, R; (17) The amino acid substitution at position P1163 relative to SEQ ID NO: 459, wherein P is substituted by any one of E, D, R, K, I, A; (18) The amino acid substitution at position D1164 relative to SEQ ID NO: 459, wherein D is substituted by any one of W, S, M, D, T, I, N; (19) The amino acid substitution at position V1165 relative to SEQ ID NO: 459, wherein V is substituted by any one of E, A, K, L; (20) The amino acid substitution at position D1166 relative to SEQ ID NO: 459, wherein D is substituted by either K or S; (21) The amino acid substitution at position L1167 relative to SEQ ID NO: 459, wherein L is substituted by either R or K; (22) The amino acid substitution at position G1168 relative to SEQ ID NO: 459, wherein G is substituted by either K or S; (23) The amino acid substitution at position D1169 relative to SEQ ID NO: 459, wherein D is replaced by S; (24) An amino acid substitution at position I1170 relative to SEQ ID NO: 459, wherein I is substituted by S; and / or (25) Any combination of (1)-(24).

43. The polypeptide of any one of claims 38 to 42, wherein the segment comprises a polypeptide sequence listed in Table 9B, or a polypeptide sequence having one to five amino acid substitutions.

44. The polypeptide of any one of claims 38 to 43, wherein the extracellular domain comprises the C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO:459 between about residues 1145 and about residues 1175, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

45. The polypeptide of any one of claims 38 to 44, wherein the C-terminal helical region comprises about 12 to about 22 residues.

46. ​​The polypeptide of any one of claims 38 to 45, wherein the segment comprises: (1) The amino acid substitution at position D1147 relative to SEQ ID NO: 459, wherein D is substituted by any one of Q, T, E, S, N, D, K; (2) The amino acid substitution at position S1148 relative to SEQ ID NO: 459, wherein S is substituted by any one of T, K, N, R, S, A, E; (3) The amino acid substitution at position F1149 relative to SEQ ID NO: 459, wherein F is replaced by any one of L, T, I, V; (4) The amino acid substitution at position K1150 relative to SEQ ID NO: 459, wherein K is substituted by any one of K, Q, R, H, S, E; (5) The amino acid substitution at position E1151 relative to SEQ ID NO: 459, wherein E is substituted by any one of E, N, A, S, K, T, or D; (6) The amino acid substitution at position E1152 relative to SEQ ID NO: 459, wherein E is substituted by any one of E, T, V, R, K, N; (7) The amino acid substitution at position L1153 relative to SEQ ID NO: 459, wherein L is substituted by any one of S, V, T, or A; (8) The amino acid substitution at position D1154 relative to SEQ ID NO: 459, wherein D is substituted by any one of T, L, E, I, V; (9) The amino acid substitution at position K1155 relative to SEQ ID NO: 459, wherein K is substituted by any one of H, E, S, T, Q; (10) The amino acid substitution at position Y1156 relative to SEQ ID NO: 459, wherein Y is substituted by any one of L, E, I, T, A, S, R; (11) The amino acid substitution at position F1157 relative to SEQ ID NO: 459, wherein F is substituted by any one of T, V, I, A, S, M; (12) The amino acid substitution at position K1158 relative to SEQ ID NO: 459, wherein K is substituted by any one of K, E, N, R, T, A, Q, I; (13) The amino acid substitution at position N1159 relative to SEQ ID NO: 459, wherein N is substituted by any one of T, E, A, K, Q; (14) The amino acid substituted at position H1160 relative to SEQ ID NO: 459, wherein H is substituted by any one of L, M, A, E, T, Y, I, S; (15) An amino acid substitution at position T1161 relative to SEQ ID NO: 459, wherein T is substituted by either L or I; (16) The amino acid substitution at position S1162 relative to SEQ ID NO: 459, wherein S is substituted by any one of S, R, K, N, E, Q; (17) The amino acid substitution at position P1163 relative to SEQ ID NO: 459, wherein P is substituted by any one of E, S, T, R; (18) The amino acid substitution at position D1164 relative to SEQ ID NO: 459, wherein D is substituted by any one of T, M, or A; (19) An amino acid substitution at position V1165 relative to SEQ ID NO: 459, wherein V is substituted by either A or L; and / or (20) Any combination of (1)-(19).

47. The polypeptide of any one of claims 38 to 46, wherein the segment comprises a polypeptide sequence listed in Table 9D, or a polypeptide sequence having one to five amino acid substitutions.

48. The recombinant polypeptide of any one of claims 1 to 10, the recombinant polypeptide comprising an engineered extracellular domain of a Nipah fusion (F) protein, wherein the extracellular domain comprises a C-terminal helical forming segment containing one or more amino acid substitutions between about residues 460 and about residues 490 relative to SEQ ID NO: 499, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

49. The polypeptide of claim 48, wherein the C-terminal helical forming segment comprises substitutions at two or more, three or more, or four or more residues relative to the reference sequence SEQ ID NO: 499, the residues creating hydrophobic contacts between the segments in the α-helical isotrimester.

50. The polypeptide of claim 48 or claim 49, wherein the extracellular domain comprises the C-terminal helical forming segment containing one or more amino acid substitutions relative to SEQ ID NO:NopahX between about residue 460 and about residue 490, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

51. The polypeptide of any one of claims 48 to 50, wherein the C-terminal helical region comprises about 16 to about 33 residues.

52. The polypeptide of any one of claims 48 to 51, wherein the segment comprises: (1) The amino acid substitution at position M463 relative to SEQ ID NO: 499, wherein M is substituted by any one of I, A, T, L, and M; (2) The amino acid substitution at position N464 relative to SEQ ID NO: 499, wherein N is replaced by N; (3) The amino acid substitution at position Q465 relative to SEQ ID NO: 499, wherein Q is substituted by any one of E, L, K, T, S, I, D; (4) The amino acid substitution at position S466 relative to SEQ ID NO: 499, wherein S is replaced by S; (5) The amino acid substitution at position L467 relative to SEQ ID NO: 499, wherein L is replaced by any one of M, L, I, V, T; (6) The amino acid substitution at position Q468 relative to SEQ ID NO: 499, wherein Q is substituted by any one of E, K, A, T, S, D, R, I, Q; (7) The amino acid substitution at position Q469 relative to SEQ ID NO: 499, wherein Q is substituted by any one of R, S, K, T, E, and Q; (8) The amino acid substitution at position S470 relative to SEQ ID NO: 499, wherein S is substituted by any one of T, L, I, V, A; (9) The amino acid substitution at position K471 relative to SEQ ID NO: 499, wherein K is substituted by any one of K, A, W, E, L, I; (10) The amino acid substitution at position D472 relative to SEQ ID NO: 499, wherein D is substituted by any one of K, T, R, Q, E; (11) The amino acid substitution at position Y473 relative to SEQ ID NO: 499, wherein Y is substituted by any one of W, D, K, Y, I, M, E, T; (12) The amino acid substitution at position I474 relative to SEQ ID NO: 499, wherein I is substituted by any one of I, V, M, L, A; (13) The amino acid substitution at position K475 relative to SEQ ID NO: 499, wherein K is substituted by any one of T, K, M, R, E, L, A, S; (14) The amino acid substitution at position E476 relative to SEQ ID NO: 499, wherein E is substituted by any one of K, S, A, E, T, or D; (15) The amino acid substitution at position A477 relative to SEQ ID NO: 499, wherein A is substituted by any one of L, I, V, FT, A, M, W, K, Y; (16) The amino acid substitution at position Q478 relative to SEQ ID NO: 499, wherein Q is substituted by any one of I, K, A, L, E, D, S, Y; (17) The amino acid substitution at position R479 relative to SEQ ID NO: 499, wherein R is substituted by any one of A, S, K, R, T, L, E; (18) The amino acid substitution at position L480 relative to SEQ ID NO: 499, wherein L is substituted by any one of K, E, R, Y, T, Q; (19) The amino acid substitution at position L481 relative to SEQ ID NO: 499, wherein L is substituted by any one of W, I, V, L, E, S, Q, A, T; (20) The amino acid substitution at position D482 relative to SEQ ID NO: 499, wherein D is substituted by any one of K, Q, E, W, T, S, A; (21) The amino acid substitution at position T483 relative to SEQ ID NO: 499, wherein T is substituted by any one of S, T, K, R, Q; (22) The amino acid substitution at position V484 relative to SEQ ID NO: 499, wherein V is substituted by any one of R, E, I, S, Y, L, K, D; (23) The amino acid substitution at position N485 relative to SEQ ID NO: 499, wherein N is substituted by any one of I, R, K, W, E, T; (24) The amino acid substitution at position P486 relative to SEQ ID NO: 499, wherein P is substituted by any one of A, T, R, K, Q; (25) The amino acid substitution at position S487 relative to SEQ ID NO: 499, wherein S is substituted by any one of K, R, T, and S; (26) The amino acid substitution at position L488 relative to SEQ ID NO: 499, wherein L is replaced by any one of E, V, L, K; (27) An amino acid substitution relative to SEQ ID NO: 499 at position I489, wherein I is substituted by any one of E, Q, L, K, R; and / or (28) Any combination of (1)-(27).

53. The polypeptide of any one of claims 48 to 52, wherein the segment comprises a polypeptide sequence listed in Table 10B, or a polypeptide sequence having one to five amino acid substitutions.

54. The polypeptide of any one of claims 48 to 53, wherein the extracellular domain comprises the C-terminal helical forming segment containing one or more amino acid substitutions between about residues 460 and about residues 490 relative to SEQ ID NO:499, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

55. The polypeptide of any one of claims 48 to 54, wherein the C-terminal helical region comprises about 12 to about 26 residues.

56. The polypeptide of any one of claims 48 to 55, wherein the segment comprises: (1) The amino acid substitution at position M463 relative to SEQ ID NO: 499, wherein M is substituted by any one of L, I, or V; (2) The amino acid substitution at position N464 relative to SEQ ID NO: 499, wherein N is replaced by N; (3) The amino acid at position Q465 relative to SEQ ID NO: 499, wherein Q is replaced by any one of Q, T, N, D, E, S, K, R, A; (4) The amino acid substitution at position S466 relative to SEQ ID NO: 499, wherein S is replaced by S; (5) The amino acid substitution at position L467 relative to SEQ ID NO: 499, wherein L is replaced by any one of I, V, L, or A; (6) The amino acid substitution at position Q468 relative to SEQ ID NO: 499, wherein Q is substituted by any one of S, K, T, D, E, R, and Q; (7) The amino acid substitution at position Q469 relative to SEQ ID NO: 499, wherein Q is substituted by any one of S, Q, N, E, A, D, K, T, R; (8) The amino acid substitution at position S470 relative to SEQ ID NO: 499, wherein S is substituted by any one of L, A, I, V, N; (9) The amino acid at position K471 relative to SEQ ID NO: 499, wherein K is replaced by any one of E, Q, S, R, K, A, T, D, L, N; (10) The amino acid substitution at position D472 relative to SEQ ID NO: 499, wherein D is substituted by any one of K, T, E, Q, D, N, S, A; (11) The amino acid at position Y473 relative to SEQ ID NO: 499, wherein Y is replaced by any one of A, S, R, T, V, E, I, K, L, D, Q; (12) The amino acid substitution at position I474 relative to SEQ ID NO: 499, wherein I is substituted by any one of L, I, and V; (13) The amino acid at position K475 relative to SEQ ID NO: 499, wherein K is replaced by any one of K, H, D, T, A, S, R, Q, E, N; (14) The amino acid substitution at position E476 relative to SEQ ID NO: 499, wherein E is substituted by any one of K, R, S, E, A, T, H, D; (15) The amino acid substitution at position A477 relative to SEQ ID NO: 499, wherein A is substituted by any one of A, L, I, V; (16) The amino acid substitution at position Q478 relative to SEQ ID NO: 499, wherein Q is substituted by any one of E, L, T, R, K, Q, S, I; (17) The amino acid substitution at position R479 relative to SEQ ID NO: 499, wherein R is substituted by any one of K, N, E, Q, S, T, H, R, A; (18) The amino acid substitution at position L480 relative to SEQ ID NO: 499, wherein L is replaced by any one of D, L, E, K, T, R, V, I, Q; (19) The amino acid substitution at position L481 relative to SEQ ID NO: 499, wherein L is replaced by any one of L, V, or I; (20) The amino acid substitution at position D482 relative to SEQ ID NO: 499, wherein D is substituted by any one of E, K, N, D, L, Q, H; (21) The amino acid substitution at position T483 relative to SEQ ID NO: 499, wherein T is substituted by any one of E, K, S, Q, A, T; (22) The amino acid substitution at position V484 relative to SEQ ID NO: 499, wherein V is substituted by any one of V, L, I; (23) The amino acid substitution at position N485 relative to SEQ ID NO: 499, wherein N is substituted by any one of R, K, L, V, E, Q, I; (24) The amino acid substitution at position P486 relative to SEQ ID NO: 499, wherein P is substituted by any one of R, E, A, S, L; (25) The amino acid substitution at position S487 relative to SEQ ID NO: 499, wherein S is substituted by any one of Q, R, T, S, L; (26) An amino acid substitution at position L488 relative to SEQ ID NO: 499, wherein L is substituted by any of L; and / or (27) Any combination of (1)-(26).

57. The polypeptide of any one of claims 48 to 56, wherein the segment comprises a polypeptide sequence listed in Table 10D, or a polypeptide sequence having one to five amino acid substitutions.

58. The recombinant polypeptide of any one of claims 1 to 10, wherein the recombinant polypeptide comprises an engineered extracellular domain of a respiratory syncytial virus (RSV) fusion (F) protein, wherein the extracellular domain comprises: (a) A C-terminal helical forming segment containing one or more amino acid substitutions between about residue 500 and about residue 530 relative to SEQ ID NO: 1, wherein the amino acid substitutions are selected such that the segment forms a stable α-helical homotrimer; (b) Substitution of one, two, three or more amino acids at positions 140, 399, 400, 485, 486, 487, 488, 489, 494 or 498 relative to SEQ ID NO: 1; (c) Substitution of one, two, three or more amino acids at positions 56, 58, 154, 187, 296 or 298 relative to SEQ ID NO: 1; (d) Substitution of one, two, three or more amino acids at positions 75, 216, 218 or 219 relative to SEQ ID NO: 1; (e) Substitution of one, two, three or more amino acids at positions 92, 232, 235, 238, 249, 250 or 254 relative to SEQ ID NO: 1; (f) Substitution of one, two, three or more amino acids at position 67, 137 or 339 relative to SEQ ID NO: 1; (g) Substitution of the furin cleavage site at about residue 100 to about residue 140 relative to SEQ ID NO: 1 with a non-cleavable linker; or Any combination of (h), (a), and (g).

59. The polypeptide of claim 58, wherein the extracellular domain comprises (a) the C-terminal helical forming segment relative to SEQ ID NO: 1 between about residue 500 and about residue 530, comprising one or more amino acid substitutions, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

60. The polypeptide of claim 59, wherein the C-terminal helical region comprises about 10 to about 30 residues.

61. The polypeptide of claim 59, wherein the segment comprises substitutions of the reference sequence SEQ ID NO: 1 at two or more, three or more, or four or more residues, the residues creating hydrophobic contacts between the segments in the α-helical isotrimester.

62. The polypeptide of claim 59, wherein the segment comprises: (1) The amino acid substitution at position F505 relative to SEQ ID NO: 1, wherein F is replaced by A, I, L, M, V, G, T; (2) The amino acid at position I506 of SEQ ID NO: 1 is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y or A, I, L, V; (3) The amino acid at position R507 of SEQ ID NO: 1 is substituted relative to R, wherein R is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y or A, I, L, V; (4) The amino acid substitution at position K508 relative to SEQ ID NO: 1, wherein R is substituted by K, Q, R, preferably A, V, T, I; (5) The amino acid at position S509 of SEQ ID NO: 1 is replaced by A, I, L, M, V, F, W, Y, G, T, preferably A, I, L, M, V; (6) The amino acid substituted at position D510 relative to SEQ ID NO: 1, wherein D is substituted by any amino acid, preferably D, E, K, N, Q, R, S, T, Y; (7) The amino acid substitution at position E511 relative to SEQ ID NO: 1, wherein E is substituted by any amino acid; (8) The amino acid at position L512 of SEQ ID NO: 1 is replaced by D, E, K, N, Q, R, S, T, Y, preferably A, I, L, M, V, F, W, Y, G, T; (9) The amino acid at position L513 of SEQ ID NO: 1 is replaced by any amino acid, preferably A, I, L, M, V, F, W, Y, G, more preferably D, E, K, N, Q, R, S, T, Y; (10) The amino acid at position H514 relative to SEQ ID NO: 1, wherein H is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; (11) The amino acid substituted at position N515 relative to SEQ ID NO: 1, wherein N is replaced by any amino acid other than P, preferably A, I, L, M, V, F, W, Y, G; (12) The amino acid substitution at position V516 relative to SEQ ID NO: 1, wherein V is substituted by A, I, L, M, V, F, W, Y, G or T, S, K; (13) The amino acid at position N517 of SEQ ID NO: 1 is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; (14) The amino acid substitution at position T518 relative to SEQ ID NO: 1, wherein T is substituted by any of the following except P, preferably D, E, K, N, Q, R, S, T, Y; (15) An amino acid substitution relative to SEQ ID NO: 1 at position G519, wherein G is substituted with any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; and / or (16) Any combination of (1)-(15).

63. The polypeptide of claim 59, wherein the segment comprises: (1) The amino acid substitution at position L503 relative to SEQ ID NO: 1, wherein F is replaced by Q, V, K, R, N, L; (2) The amino acid at position A504 of SEQ ID NO: 1 is replaced by any amino acid other than P, preferably S, T, L, A, Q, K, E, Y; (3) The amino acid substitution at position F505 relative to SEQ ID NO: 1, wherein F is replaced by I, V, N, T, L; (4) The amino acid at position I506 of SEQ ID NO: 1 is replaced by any amino acid other than P, preferably Q, N, K, R, V, or S; (5) The amino acid at position R507 of SEQ ID NO: 1 is replaced by any amino acid other than P, preferably A, N, K, E, D, or Q; (6) The amino acid substitution at position K508 relative to SEQ ID NO: 1, wherein R is replaced by T, M, V, or R; (7) The amino acid substitution at position S509 relative to SEQ ID NO: 1, wherein S is replaced by T, I, K, Q, M, E, V, S; (8) The amino acid substitution at position D510 relative to SEQ ID NO: 1, wherein D is replaced by S, K, N, D, or E; (9) The amino acid substitution at position E511 relative to SEQ ID NO: 1, wherein E is replaced by R, S, E, K, A, T, L; (10) The amino acid substitution at position L512 relative to SEQ ID NO: 1, wherein L is replaced by V, N, T, or L; (11) The amino acid substitution at position L513 relative to SEQ ID NO: 1, wherein L is replaced by D, T, H, K, E, N, R; (12) The amino acid substitution at position H514 relative to SEQ ID NO: 1, wherein H is replaced by A, N, E, S, V, K, T, or D; (13) The amino acid substitution at position N515 relative to SEQ ID NO: 1, wherein N is replaced by I, E, L, T, Q; (14) The amino acid substitution at position V516 relative to SEQ ID NO: 1, wherein V is replaced by E, I, K, N, R, or Q; (15) The amino acid substitution at position N517 relative to SEQ ID NO: 1, wherein N is replaced by A, S, K, E, or R; (16) The amino acid substitution at position T518 relative to SEQ ID NO: 1, wherein T is replaced by K, S, Q, R, D, E; (17) The amino acid substitution at position G519 relative to SEQ ID NO: 1, wherein G is replaced by V, L, or I; (18) The amino acid substitution at position I520 relative to SEQ ID NO: 1, wherein G is replaced by K, Q, E, N, T; (19) The amino acid substitutions relative to SEQ ID NO: 1 at position P521, wherein G is replaced by H, D, E, K, R, N, or Q; (20) The amino acid substitution at position E522 relative to SEQ ID NO: 1, wherein G is replaced by L, R, I, V; (21) The amino acid substitution at position A523 relative to SEQ ID NO: 1, wherein G is replaced by E, V, L, K, RI; (22) The amino acid substitution at position P524 relative to SEQ ID NO: 1, wherein G is replaced by A, K, T, E, or R; (23) The amino acid substitution at position R525 relative to SEQ ID NO: 1, wherein G is replaced by H, R, S, L, N, E, D; (24) The amino acid substitution at position D526 relative to SEQ ID NO: 1, wherein G is replaced by I, L, V, R; (25) The amino acid substitution at position G527 relative to SEQ ID NO: 1, wherein G is replaced by E, K, Q, or D; (26) The amino acid substitution at position Q528 relative to SEQ ID NO: 1, wherein E is replaced by D, K, S, R, or A; (27) The amino acid substitution at position A529 relative to SEQ ID NO: 1, wherein G is replaced by T or L; (28) The amino acid substitution at position Y530 relative to SEQ ID NO: 1, wherein G is replaced by L, E, and T; (29) The amino acid substitution at position V531 relative to SEQ ID NO: 1, wherein G is replaced by A, R, or K; (30) An amino acid substitution relative to SEQ ID NO: 1 at position R532, wherein G is substituted by V or A; and / or (31) Any combination of (1)-(30).

64. The polypeptide of claim 59, wherein the segment comprises a polypeptide sequence listed in Table 2B or Table 2C, or a polypeptide sequence having one to five amino acid substitutions.

65. The polypeptide of claim 59, wherein the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), NQSALWLEAAKYVKQAREKS (SEQ ID NO: 11), NQSAKNAEAAKIAEETKRKD (SEQ ID NO: 12) or NQSRETAKAVSAVK (SEQ ID NO: 75), or a polypeptide sequence having 1 to 5 amino acid substitutions.

66. The polypeptide of claim 59, wherein the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), or a polypeptide sequence having 1 to 5 amino acid substitutions.

67. The polypeptide of claim 59, wherein the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10).

68. The polypeptide of any one of claims 58 to 67, wherein the extracellular domain comprises (b) one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 140, 399, 400, 485, 486, 487, 488, 489, 494 or 498.

69. The polypeptide of any one of claims 58 to 67, wherein the extracellular domain comprises one or more of the following amino acid substitutions relative to SEQ ID NO:1: E487R + K498A; E487R + K498E; E487K + K498E; D486A + E487R + K498A; D486Q + E487R + K498A; D486E + E487A + D489A + T400D; D486A + E487M + K498A; E487Q; D486S; F488W + D489A + T400D + E487R + K498A; F140W + D489A + T400D + E487R + K498A; Q494I + S485I + K399A + 487R + 498A; Q494M + S485I + K399A; D486A + 487M + 498A; Q494L + S485A + K399V + D486A + 487M + 498A; Q494M + S485A + K399V + D486A + 487M + 498A; Q494A + S485F + K399V + D486A + 487M + 498Y; D489A + T400D + E487R + K498A; or D489A + T400D.

70. The polypeptide of claim 68, wherein the extracellular domain comprises amino acid substitutions of D489A, T400D, E487R, and K498A.

71. The polypeptide of claim 68, wherein the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and D486A.

72. The polypeptide of claim 68, wherein the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and T249P.

73. The polypeptide of any one of claims 58 to 72, wherein the polypeptide comprises a heteropolymerized domain at the C-terminus of the extracellular domain.

74. The polypeptide of claim 73, wherein the polymerizing domain is a trimerizing domain.

75. The polypeptide of claim 73 or claim 74, wherein the polymerized domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64).

76. The polypeptide of any one of claims 58 to 75, wherein the extracellular domain comprises amino acid substitutions of S155C, S290C, S190F, and V207L.

77. The polypeptide of any one of claims 58 to 76, wherein the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following: QNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAI ASGIAVCKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTFRVLDLKNYINNQLLPMLNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKI MTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSXXXXXXXXXXXXXXXXX (SEQ ID NO: 6), optionally lacking the p27 peptide shown in bold.

78. The polypeptide of any one of claims 58 to 77, wherein the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following: QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAI ASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKK LMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSXXXXXXXXXXXXXXXXX (SEQ ID NO: 7), optionally lacking the p27 peptide shown in bold.

79. The polypeptide of any one of claims 58 to 77, wherein the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following: QNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAI ASGIAVCKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTFRVLDLKNYINNQLLPMLNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKI MTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSREIIRAINIVRKIASEK (SEQ ID NO: 8), optionally lacking the p27 peptide shown in bold.

80. The polypeptide of any one of claims 58 to 77, wherein the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following: QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAI ASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKK LMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSREIIRAINIVRKIASEK (SEQ ID NO: 9), optionally lacking the p27 peptide shown in bold.

81. The polypeptide of any one of claims 58 to 77, wherein the polypeptide comprises a sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 1-9.

82. A trimeric protein complex comprising a polypeptide according to any one of claims 1 to 81.

83. The trimeric protein complex of claim 81, wherein the thermal stability, as measured by nanoDSF, is increased by at least 10°C, at least 15°C, at least 20°C, about 10°C to about 30°C, about 10°C to about 20°C, or about 20°C to about 30°C, compared to the trimeric protein complex lacking modification (a)-(h).

84. The trimeric protein complex of claim 82, wherein the stability measured by storage at about 40°C is increased compared to the trimeric protein complex lacking modification (a)-(h).

85. The trimeric protein complex of claim 83, wherein the increased thermal stability is compared to a reference RSV F protein comprising amino acid substitutions consisting substantially of S155C, S290C, S190F and V207L ​​(DS-Cav1).

86. A protein nanostructure comprising a trimer component, said trimer component comprising a polypeptide according to any one of claims 1 to 81.

87. The nanostructure of claim 86, wherein the nanostructure is a bicomponent nanostructure comprising the first trimeric component and the second pentameric component.

88. The nanostructure of claim 87, wherein the first trimeric component comprises engineered extracellular domains of a respiratory syncytial virus (RSV) fusion (F) polypeptide and an I53-50A polypeptide.

89. The nanostructure of any one of claims 86 to 88, wherein the first trimeric component comprises a fusion protein, the fusion protein comprising, in order from N-terminus to C-terminus, the RSV fusion (F) polypeptide, an amino acid linker, and the I53-50A polypeptide.

90. The nanostructure of any one of claims 86 to 89, wherein the nanostructure is a two-component nanostructure, the two-component nanostructure comprising: The first trimer component, wherein the first trimer component comprises an engineered extracellular domain of the RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 1 and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and Polymerized domains, said polymerized domains comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or The second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71.

91. The nanostructure of any one of claims 86 to 89, wherein the nanostructure is a two-component nanostructure, the two-component nanostructure comprising: The first trimer component, wherein the first trimer component comprises an engineered extracellular domain of the RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, D489A, T400D, E487R, and K498A relative to SEQ ID NO: 1 and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and Polymerized domains, said polymerized domains comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or The second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71.

92. The nanostructure of any one of claims 86 to 89, wherein the nanostructure is a two-component nanostructure, the two-component nanostructure comprising: The first trimer component, wherein the first trimer component comprises an engineered extracellular domain of the RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and T249P relative to SEQ ID NO: 1, and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and Polymerized domains, said polymerized domains comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or The second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71.

93. The nanostructure of any one of claims 86 to 89, wherein the nanostructure is a two-component nanostructure, the two-component nanostructure comprising: The first trimer component, wherein the first trimer component comprises an engineered extracellular domain of the RSV F polypeptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and D486A relative to SEQ ID NO: 1, and a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and Polymerized domains, said polymerized domains comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or The second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71.

94. The nanostructure of any one of claims 86 to 93, wherein the trimer component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the sequences listed in Table 19 or without an underlined and / or bold / italic polypeptide sequence.

95. The nanostructure of any one of claims 86 to 94, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 20, 44, 45, 52, 71, 73, 74.

96. A pharmaceutical composition comprising a polypeptide according to any one of claims 58 to 81, a protein complex according to any one of claims 82 to 85, or a nanostructure according to any one of claims 86 to 95.

97. The nanostructure of claim 86, wherein the nanostructure is a two-component nanostructure.

98. The nanostructure of claim 86, wherein the nanostructure is a single-component nanostructure.

99. The nanostructure of claim 97 or claim 98, wherein the nanostructure is a spherical nanostructure and / or a naturally occurring nanostructure, optionally a virus-like particle (VLP), optionally a human papillomavirus VLP.

100. The nanostructure of claim 97 or claim 99, wherein the nanostructure is a bicomponent nanostructure comprising the first trimeric component and the second pentameric component.

101. The nanostructure as claimed in any of the preceding claims, wherein the first trimeric component comprises engineered extracellular domains of a respiratory syncytial virus (RSV) fusion (F) polypeptide and an I53-50A polypeptide.

102. The nanostructure as claimed in any of the preceding claims, wherein the first trimeric component comprises engineered extracellular domains of a human metapneumovirus (hMPV) fusion (F) polypeptide and an I53-50A polypeptide.

103. The nanostructure as claimed in any of the preceding claims, wherein the first trimeric component comprises engineered extracellular domains of a human parainfluenza virus type 3 (PIV3) fusion (F) polypeptide and an I53-50A polypeptide.

104. The nanostructure as claimed in any of the preceding claims, wherein the first trimeric component comprises engineered extracellular domains of a human parainfluenza virus type 5 (PIV3) fusion (F) polypeptide and an I53-50A polypeptide.

105. The nanostructure as claimed in any of the preceding claims, wherein the first trimeric component comprises engineered extracellular domains of the SARS-CoV-2 spike (S) polypeptide and the I53-50A polypeptide.

106. The nanostructure as claimed in any of the preceding claims, wherein the first trimeric component comprises engineered extracellular domains of a Nipah virus fusion (F) polypeptide and an I53-50A polypeptide.

107. The nanostructure as claimed in any of the preceding claims, wherein the first trimeric component comprises a fusion protein, the fusion protein comprising, in N-terminus to C-terminus, the engineered fusion (F) polypeptide, an amino acid linker, and the I53-50A polypeptide.

108. The nanostructure as claimed in any of the preceding claims, wherein the first trimeric component comprises a fusion protein, the fusion protein comprising, in N-terminus to C-terminus, the engineered spike (S) polypeptide, an amino acid linker, and the I53-50A polypeptide.

109. The nanostructure as claimed in any of the preceding claims, wherein the trimeric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the sequences listed in Table 19 or without the underlined and / or bold / italic polypeptide sequence.

110. The nanostructure as claimed in any of the preceding claims, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 20, 44, 45, 52, 71, 73, 74.

111. A polynucleotide encoding a polypeptide, protein complex, or nanostructure as described in any of the preceding claims.

112. A delivery medium comprising a polynucleotide encoding a polypeptide, protein complex, or nanostructure as described in any of the preceding claims, wherein optionally, the delivery medium is a lipid nanoparticle (LNP).

113. A pharmaceutical composition or vaccine comprising a polypeptide, protein complex or nanostructure, polynucleotide or delivery medium according to any of the preceding claims.

114. A method of vaccinating a subject and / or generating an immune response in the subject, the method comprising administering to the subject a composition according to any of the preceding claims.

115. A method of treating or preventing a viral infection in a subject, the method comprising administering to the subject the composition according to any of the preceding claims.

116. The composition according to any of the preceding claims, wherein the composition is used for vaccination, generating an immune response, or treating or preventing any viral infection.

117. A method for preparing a composition according to any of the preceding claims, the method comprising culturing host cells modified to express one or more polypeptides as described herein.

118. A composition, method, or use as described herein.

119. A recombinant polypeptide for displaying a molecule such as an antigen, the recombinant polypeptide comprising an α-helical segment and a polymerizing domain, wherein the α-helical segment comprises one or more amino acid substitutions selected such that the segment forms a stable α-helical homotrimer.

120. The polypeptide of claim 119, wherein the α-helix segment has improved hydrophobic stacking compared to a reference α-helix segment without the amino acid substitutions.

121. The polypeptide of claim 119 or claim 120, wherein the α-helical segment comprises about 7 to about 31 residues.

122. The polypeptide of any one of claims 119 to 121, wherein the amino acid substitution comprises polar, charged and / or hydrophobic amino acids.

123. The polypeptide of any one of claims 119 to 122, wherein the α-helix segment comprises a polypeptide sequence according to any one of the following: LXXTIXXLLXIXXXLXXXL (SEQ ID NO: 566) LVXTXKXLXDLIXXLXXLLXKLXX (SEQ ID NO: 567) LNKVKKXVXXLXXXVXXLEKXLX (SEQ ID NO: 568) EKIXXAIKKAXKL (SEQ ID NO: 569) EXIXKAIKXLXXXXX (SEQ ID NO: 570) XKXXEXXXXVXXXXXXXXX (SEQ ID NO: 571) XXLKKAAXIXKKXLKXX (SEQ ID NO: 572).

124. The polypeptide of any one of claims 119 to 123, wherein the α-helical segment comprises a polypeptide sequence according to any one of the common sequences in Table 24.

125. The polypeptide of any one of claims 119 to 124, wherein the α-helix segment comprises a polypeptide sequence according to the following: a) L X2X2T I X2X2L L X2I [V / I] X2X2L [I / L] X2X2L (SEQ ID NO: 573); b) LV [A / T] T X2K X2L X2D LI X2X2L [K / E] X2L L X2K L X2X2 (SEQ ID NO:574); or c) LNKVKK X2V X2X2L X2X2X2V X2X2L EK X2L X2 (SEQ ID NO: 575), wherein X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

126. The polypeptide of any one of claims 119 to 124, wherein the α-helix segment comprises a polypeptide sequence according to the following: a) EKI X2X2A IKKA X2K L (SEQ ID NO: 576); b) E X2I X2K AIK X2L [L / X2] X2X2[X1 / X2] X2 (SEQ ID NO: 577); and c) X2K [X1 / T] [L / E] E [T / A] X1X2[I / X2] V X2X2[X1 / X2] [X1 / X2] d) X2X2L KKAA X2I X1K K X1L K X2X2 (SEQ ID NO: 579), wherein X1 is a nonpolar residue selected from A, I, L and M, and X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

127. The polypeptide of any one of claims 119 to 126, wherein the α-helical segment comprises a polypeptide sequence listed in Table 25A or Table 25B, or a polypeptide sequence having 1 to 5 amino acid substitutions.

128. The polypeptide of any one of claims 119 to 127, wherein the α-helical segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), NQSALWLEAAKYVKQAREKS (SEQ ID NO: 11), NQSAKNAEAAKIAEETKRKD (SEQ ID NO: 12), or NQSRETAKAVSAVK (SEQ ID NO: 75), or a polypeptide sequence having 1 to 5 amino acid substitutions.

129. The polypeptide of any one of claims 119 to 128, wherein the polymerizing domain is I53-50A or a variant thereof.

130. The polypeptide of any one of claims 119 to 129, wherein the polymerized domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64).

131. The polypeptide of any one of claims 119 to 130, wherein the polypeptide comprises an N-terminal fusion of the α-helical segment with the polymerized domain via a peptide bond or a peptide linker.

132. The polypeptide of any one of claims 119 to 131, wherein the polypeptide comprises an antigenic polypeptide at the N-terminus of the α-helical segment.

133. A polypeptide comprising an α-helical segment, wherein the α-helical segment comprises one or more amino acid substitutions, the amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

134. The polypeptide of claim 133, wherein the α-helix segment has improved hydrophobic stacking compared to a reference α-helix segment without the amino acid substitutions.

135. The polypeptide of claim 133 or claim 134, wherein the α-helical segment comprises about 7 to about 31 residues.

136. The polypeptide of any one of claims 133 to 135, wherein the amino acid substitution comprises polar, charged and / or hydrophobic amino acids.

137. The polypeptide of any one of claims 133 to 136, wherein the α-helix segment comprises a polypeptide sequence according to any one of the following: LXXTIXXLLXIXXXLXXXL (SEQ ID NO: 566) LVXTXKXLXDLIXXLXXLLXKLXX (SEQ ID NO: 567) LNKVKKXVXXLXXXVXXLEKXLX (SEQ ID NO: 568) EKIXXAIKKAXKL (SEQ ID NO: 569) EXIXKAIKXLXXXXX (SEQ ID NO: 570) XKXXEXXXXVXXXXXXXXX (SEQ ID NO: 571) XXLKKAAXIXKKXLKXX (SEQ ID NO: 572).

138. The polypeptide of any one of claims 133 to 137, wherein the α-helical segment comprises a polypeptide sequence according to any one of the common sequences in Table 24.

139. The polypeptide of any one of claims 133 to 138, wherein the α-helix segment comprises a polypeptide sequence according to the following: a) L X2X2T I X2X2L L X2I [V / I] X2X2L [I / L] X2X2L (SEQ ID NO: 573); b) LV [A / T] T X2K X2L X2D LI X2X2L [K / E] X2L L X2K L X2X2 (SEQ ID NO:574); or c) LNKVKK X2V X2X2L X2X2X2V X2X2L EK X2L X2 (SEQ ID NO: 575), wherein X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

140. The polypeptide of any one of claims 133 to 139, wherein the α-helix segment comprises a polypeptide sequence according to the following: a) EKI X2X2A IKKA X2K L (SEQ ID NO: 576); b) E X2I X2K AIK X2L [L / X2] X2X2[X1 / X2] X2 (SEQ ID NO: 577); and c) X2K [X1 / T] [L / E] E [T / A] X1X2[I / X2] V X2X2[X1 / X2] [X1 / X2] d) X2X2L KKAA X2I X1K K X1L K X2X2 (SEQ ID NO: 579), wherein X1 is a nonpolar residue selected from A, I, L and M, and X2 is a polar and charged residue selected from S, T, N, Q, E, D, R, K and H, preferably a wild-type amino acid.

141. The polypeptide of any one of claims 133 to 140, wherein the α-helical segment comprises a polypeptide sequence listed in Table 25A or Table 25B, or a polypeptide sequence having 1 to 5 amino acid substitutions.

142. The polypeptide of any one of claims 133 to 141, wherein the α-helical segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), NQSALWLEAAKYVKQAREKS (SEQ ID NO: 11), NQSAKNAEAAKIAEETKRKD (SEQ ID NO: 12) or NQSRETAKAVSAVK (SEQ ID NO: 75), or a polypeptide sequence having 1 to 5 amino acid substitutions.

143. A protein nanostructure comprising a trimer component, said trimer component comprising a polypeptide according to any one of claims 119 to 142.

144. The nanostructure of claim 143, wherein the nanostructure is a bicomponent nanostructure comprising the first trimeric component and the second pentameric component.

145. The nanostructure of claim 143, wherein the nanostructure is a bicomponent nanostructure comprising a second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71.

146. A pharmaceutical composition comprising a polypeptide according to any one of claims 119 to 142 or a nanostructure according to any one of claims 143 to 145.

147. A vaccine comprising a polypeptide according to any one of claims 119 to 142 or a nanostructure according to any one of claims 143 to 145.

148. A method of vaccinating a subject, the method comprising administering to the subject a polypeptide according to any one of claims 119 to 142 or a nanostructure according to any one of claims 143 to 145.

149. A method for generating an immune response or treating or preventing viral infection in a subject, the method comprising administering to the subject a polypeptide according to any one of claims 119 to 142, a nanostructure according to any one of claims 143 to 145, a pharmaceutical composition according to claim 146, or a vaccine according to claim 147.

150. A method for preparing a polypeptide according to any one of claims 119 to 142, a nanostructure according to any one of claims 143 to 145, a pharmaceutical composition according to claim 146, or a vaccine according to claim 147, the method comprising culturing host cells modified to express one or more polypeptides as described herein.

151. A recombinant polypeptide comprising an engineered extracellular domain of a respiratory syncytial virus (RSV) fusion (F) protein, wherein the extracellular domain comprises: (a) Substitution of one, two, three or more amino acids at positions 140, 399, 400, 485, 486, 487, 488, 489, 494 or 498 relative to SEQ ID NO: 1; (b) Substitution of one, two, three or more amino acids at positions 56, 58, 154, 187, 296 or 298 relative to SEQ ID NO: 1; (c) Substitution of one, two, three or more amino acids at positions 75, 216, 218 or 219 relative to SEQ ID NO: 1; (d) Substitution of one, two, three or more amino acids at positions 92, 232, 235, 238, 249, 250 or 254 relative to SEQ ID NO: 1; (e) Substitution of one, two, three or more amino acids at position 67, 137 or 339 relative to SEQ ID NO: 1; (f) Substitution of the furin cleavage site at about residue 100 to about residue 140 relative to SEQ ID NO: 1 with a non-cleavable linker; or (g) Any combination of (a)-(f).

152. The polypeptide of claim 152, wherein the extracellular domain comprises (a) one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 140, 399, 400, 485, 486, 487, 488, 489, 494 or 498.

153. The polypeptide of claim 151 or claim 152, wherein the extracellular domain comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 1: E487R + K498A; E487R + K498E; E487K + K498E; D486A + E487R + K498A; D486Q + E487R + K498A; D486E + E487A + D489A + T400D; D486A + E487M + K498A; E487Q; D486S; F488W + D489A + T400D + E487R + K498A; F140W + D489A + T400D + E487R + K498A; Q494I + S485I + K399A + 487R + 498A; Q494M + S485I + K399A; D486A + 487M + 498A; Q494L + S485A + K399V + D486A + 487M + 498A; Q494M + S485A + K399V + D486A + 487M + 498A; Q494A + S485F + K399V + D486A + 487M + 498Y; D489A + T400D + E487R + K498A; or D489A + T400D.

154. The polypeptide of claim 152, wherein the extracellular domain comprises amino acid substitutions of D489A, T400D, E487R, and K498A.

155. The polypeptide of claim 152, wherein the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and D486A.

156. The polypeptide of claim 152, wherein the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and T249P.

157. The polypeptide of any one of claims 151 to 156, wherein the polypeptide comprises a heteropolymerized domain.

158. The polypeptide of claim 157, wherein the polymerizing domain is a trimerizing domain.

159. The polypeptide of claim 157 or claim 158, wherein the polymerized domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64).

160. The polypeptide of any one of claims 151 to 159, wherein the extracellular domain comprises amino acid substitutions S155C, S290C, S190F, and V207L.

161. The polypeptide of any one of claims 151 to 160, wherein the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following: QNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAI ASGIAVCKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTFRVLDLKNYINNQLLPMLNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKI MTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSXXXXXXXXXXXXXXXXX (SEQ ID NO: 6), optionally lacking the p27 peptide shown in bold.

162. The polypeptide of any one of claims 151 to 161, wherein the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following: QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAI ASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKK LMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSXXXXXXXXXXXXXXXXX (SEQ ID NO: 7), optionally lacking the p27 peptide shown in bold.

163. The polypeptide of any one of claims 151 to 161, wherein the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following: QNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAI ASGIAVCKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTFRVLDLKNYINNQLLPMLNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKI MTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSREIIRAINIVRKIASEK (SEQ ID NO: 8), optionally lacking the p27 peptide shown in bold.

164. The polypeptide of any one of claims 151 to 161, wherein the extracellular domain comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following: QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAI ASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKK LMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSREIIRAINIVRKIASEK (SEQ ID NO: 9), optionally lacking the p27 peptide shown in bold.

165. The polypeptide of any one of claims 151 to 161, wherein the polypeptide comprises a sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 1-9.

166. A trimeric protein complex comprising a polypeptide according to any one of claims 151 to 165.

167. The trimeric protein complex of claim 166, wherein the thermal stability, as measured by nanoDSF, is increased by at least 10°C, at least 15°C, at least 20°C, about 10°C to about 30°C, about 10°C to about 20°C, or about 20°C to about 30°C, compared to the trimeric protein complex lacking modification (a)-(g).

168. The trimeric protein complex of claim 166, wherein the stability measured by storage at about 40°C is increased compared to the trimeric protein complex lacking modification (a)-(g).

169. The trimeric protein complex of claim 167, wherein the increased thermal stability is compared to a reference RSV F protein comprising amino acid substitutions consisting substantially of S155C, S290C, S190F and V207L ​​(DS-Cav1).

170. A protein nanostructure comprising a trimer component, said trimer component comprising a polypeptide according to any one of claims 151 to 165.

171. The nanostructure of claim 170, wherein the nanostructure is a bicomponent nanostructure comprising the first trimeric component and the second pentameric component.

172. The nanostructure of claim 171, wherein the first trimeric component comprises engineered extracellular domains of a respiratory syncytial virus (RSV) fusion (F) polypeptide and an I53-50A polypeptide.

173. The nanostructure of any one of claims 170 to 172, wherein the first trimeric component comprises a fusion protein, the fusion protein comprising, in order from N-terminus to C-terminus, the RSV fusion (F) polypeptide, an amino acid linker, and the I53-50A polypeptide.

174. The nanostructure of any one of claims 170 to 173, wherein the nanostructure is a two-component nanostructure, the two-component nanostructure comprising: The first trimer component, wherein the first trimer component comprises an engineered extracellular domain of the RSV F peptide, the extracellular domain comprising amino acid substitutions at positions S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 1; and Polymerized domains, said polymerized domains comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or The second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71.

175. The nanostructure of any one of claims 170 to 173, wherein the nanostructure is a two-component nanostructure, the two-component nanostructure comprising: The first trimer component, wherein the first trimer component comprises an engineered extracellular domain of the RSV F peptide, the extracellular domain comprising amino acid substitutions relative to SEQ ID NO: 1 at positions S155C, S290C, S190F, V207L, D489A, T400D, E487R, and K498A; and Polymerized domains, said polymerized domains comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or The second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71.

176. The nanostructure of any one of claims 170 to 173, wherein the nanostructure is a two-component nanostructure, the two-component nanostructure comprising: The first trimer component, wherein the first trimer component comprises an engineered extracellular domain of the RSV F peptide, the extracellular domain comprising amino acid substitutions relative to SEQ ID NO: 1 at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and T249P; and Polymerized domains, said polymerized domains comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or The second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71.

177. The nanostructure of any one of claims 170 to 173, wherein the nanostructure is a two-component nanostructure, the two-component nanostructure comprising: The first trimer component, wherein the first trimer component comprises an engineered extracellular domain of the RSV F peptide, the extracellular domain comprising amino acid substitutions relative to SEQ ID NO: 1 at positions S155C, S290C, S190F, V207L, F488W, D489A, T400D, E487R, K498A, and D486A; and Polymerized domains, said polymerized domains comprising a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64); and / or The second pentameric component, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20 or 71.

178. The nanostructure of any one of claims 170 to 177, wherein the trimer component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the sequences listed in Table 19 or without an underlined and / or bold / italic polypeptide sequence.

179. The nanostructure of any one of claims 170 to 178, wherein the pentameric component comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one or more of SEQ ID NO: 20, 44, 45, 52, 71, 73, 74.

180. A pharmaceutical composition comprising a polypeptide according to any one of claims 151 to 165, a protein complex according to any one of claims 166 to 169, or a nanostructure according to any one of claims 170 to 179.

181. A vaccine comprising a polypeptide according to any one of claims 151 to 165, a protein complex according to any one of claims 166 to 169, or a nanostructure according to any one of claims 170 to 179.

182. A method of vaccinating a subject, the method comprising administering to the subject the composition according to any of the preceding claims.

183. A method for generating an immune response in a subject, the method comprising administering to the subject a composition according to any of the preceding claims.

184. A method of treating or preventing RSV disease in a subject, the method comprising administering to the subject the composition according to any of the preceding claims.

185. The composition according to any of the preceding claims, wherein the composition is used for vaccination, generating an immune response, or treating or preventing RSV disease.

186. A method for preparing a composition according to any of the preceding claims, the method comprising culturing host cells modified to express one or more polypeptides as described herein.

187. A polynucleotide encoding a composition according to any of the preceding claims.

188. A delivery medium comprising the polynucleotide of claim 187, wherein optionally, the delivery medium is a lipid nanoparticle (LNP).

189. A lipid nanoparticle (LNP) comprising the polynucleotide as described in claim 187.

190. A host cell comprising the polynucleotide as described in claim 187.

191. A composition, method, or use as described herein.

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