Respiratory syncytial virus F protein and nanostructures and their applications

Abstract

This document provides a recombinant polypeptide comprising an engineered extracellular domain of a respiratory syncytial virus (RSV) fusion (F) protein, wherein the extracellular domain comprises an engineered C-terminal α-helical region and / or amino acid substitutions, the amino acid substitutions stabilizing the F protein in its pre-fusion conformation. This disclosure also provides a two-component protein nanostructure comprising a first trimer component and a second pentamer component. This document provides a composition for use in vaccination, inducing an immune response, or treating or preventing RSV disease.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 583,097, filed September 15, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Merging of sequence lists by reference This application contains a sequence list electronically submitted in XML format, and the entire sequence list is hereby incorporated by reference. The XML copy was created on September 6, 2024, named 061291-517001WO.xml, and is 340 KB 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 typically include the viral membrane fusion protein as an antigen.

[0004] Structural information about viral membrane fusion proteins enables structure-based design of recombinant antigens for vaccines. See Graham et al., Annu Rev Med. 70:91-104 (2019). For example, WO 2014 / 160463 A1 describes stabilizing the pre-fusion conformation of respiratory syncytial virus (RSV) F protein by introducing amino acid substitutions for S155C, S290C, S190F, and V207L ​​(collectively, “DS-Cav1”) into the fusion (F) protein. Stabilization of the pre-fusion conformation of the RSV F protein improves the immune response to the protein. Since the RSV F protein is trimerized in its pre-fusion conformation, another approach is to fuse the C-terminus of the trimerized domain to an engineered extracellular domain of the RSV F protein. Another technique used in structure-based vaccine design is to display the engineered extracellular domain of the RSV F protein on protein nanostructures. WO 2018 / 187325 A1 describes a computationally designed two-component protein structure that self-assembles to display the variant DS-Cav1 RSV F protein or other antigens.

[0005] The need for viral membrane fusion proteins stabilized through designed amino acid substitution has not been met. This disclosure provides recombinant peptides and related compositions and methods to address this need for respiratory syncytial virus (RSV). Summary of the Invention

[0006] This invention generally relates to recombinant polypeptides comprising an extracellular domain of a viral membrane fusion (F) protein having (a) an engineered C-terminal α-helical segment; (b) amino acid substitutions of the F protein stably in its pre-fusion conformation; or a combination of (a) and (b). Advantageously, the disclosed modifications to the extracellular domain of the F protein improve thermal stability, conformational stability, antigenicity, and / or immunogenicity compared to a reference protein lacking these modifications. Further provided are trimeric protein complexes comprising such RSV F extracellular domain polypeptides and self-assembled protein nanostructures, as well as various other compositions, methods, and uses, including as vaccines. In another aspect, this disclosure provides a nanostructure comprising a trimeric component containing a helical forming segment as disclosed herein. In another aspect, this disclosure provides a helical forming segment as disclosed herein.

[0007] In one 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) a C-terminal helical forming segment relative to SEQ ID NO: 1 between about residue 500 and about residue 530, wherein the one or more amino acid substitutions are selected such that the segment forms a stable α-helical homotrimer.

[0008] 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 (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.

[0009] 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 (c) one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 56, 58, 154, 187, 296 or 298.

[0010] 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 (d) one, two, three or more amino acid substitutions relative to SEQ ID NO: 1 at positions 75, 216, 218 or 219.

[0011] 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 (e) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 92, 232, 235, 238, 249, 250, or 254. 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 (f) one, two, three, or more amino acid substitutions relative to SEQ ID NO: 1 at positions 67, 137, or 339.

[0012] 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 (g) a substitution of the furin protease cleavage site at about residue 100 to about residue 140 relative to SEQ ID NO: 1.

[0013] 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 any combination of (h) (a)-(g).

[0014] In some embodiments, the extracellular domain comprises (a) a 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 one or more amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

[0015] In some embodiments, the C-terminal helical forming segment contains about 10 to about 30 residues.

[0016] 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.

[0017] In some embodiments, the segment comprises (a) 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; (b) 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; (c) 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; (d) 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; (e) relative to SEQ ID NO: 1) The amino acid at position S509 is substituted, wherein S is replaced by A, I, L, M, V, F, W, Y, G, T, preferably A, I, L, M, V; (f) The amino acid at position D510 relative to SEQ ID NO: 1 is substituted, wherein D is replaced by any amino acid, preferably D, E, K, N, Q, R, S, T, Y; (g) The amino acid at position E511 relative to SEQ ID NO: 1 is substituted, wherein E is replaced by any amino acid; (h) The amino acid at position L512 relative to SEQ ID NO: 1 is substituted, wherein L is replaced by D, E, K, N, Q, R, S, T, Y, preferably A, I, L, M, V, F, W, Y, G, T; (i) Relative to SEQ ID NO: 1. The amino acid at position L513 is substituted, 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; (j) The amino acid at position H514 relative to SEQ ID NO: 1 is substituted, wherein H is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; (k) The amino acid at position N515 relative to SEQ ID NO: 1 is substituted, wherein N is replaced by any amino acid other than P, preferably A, I, L, M, V, F, W, Y, G; (l) The amino acid at position V516 relative to SEQ ID NO: 1 is substituted, wherein V is replaced by A, I, L, M, V, F, W, Y, G or T, S, K; (m) 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; (n) An amino acid substitution relative to SEQ ID NO: 1 at position T518, wherein T is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y.(o) a substitution of the amino acid at position G519 relative to SEQ ID NO: 1, wherein G is substituted with any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; and / or any combination of (p)(a)-(o).

[0018] In some embodiments, the segment comprises (a) an amino acid substitution relative to SEQ ID NO: 1 at position L503, wherein F is substituted with Q, V, K, R, N, L; (b) 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; (c) an amino acid substitution relative to SEQ ID NO: 1 at position F505, wherein F is substituted with I, V, N, T, L; (d) 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; (e) 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; (f) an amino acid substitution relative to SEQ ID NO: (g) Amino acid substitution at position K508 relative to SEQ ID NO: 1, wherein R is substituted by T, M, V, or R; (h) Amino acid substitution at position S509 relative to SEQ ID NO: 1, wherein S is substituted by T, I, K, Q, M, E, V, or S; (h) Amino acid substitution at position D510 relative to SEQ ID NO: 1, wherein D is substituted by S, K, N, D, or E; (i) Amino acid substitution at position E511 relative to SEQ ID NO: 1, wherein E is substituted by R, S, E, K, A, T, or L; (j) Amino acid substitution at position L512 relative to SEQ ID NO: 1, wherein L is substituted by V, N, T, or L; (k) Amino acid substitution at position L513 relative to SEQ ID NO: 1, wherein L is substituted by D, T, H, K, E, N, or R; (l) Amino acid substitution relative to SEQ ID NO: 1. An amino acid substitution at position H514, wherein H is replaced by A, N, E, S, V, K, T, or D; (m) An amino acid substitution at position N515 relative to SEQ ID NO: 1, wherein N is replaced by I, E, L, T, or Q; (n) An amino acid substitution at position V516 relative to SEQ ID NO: 1, wherein V is replaced by E, I, K, N, R, or Q; (o) An amino acid substitution at position N517 relative to SEQ ID NO: 1, wherein N is replaced by A, S, K, E, or R; (p) An amino acid substitution at position T518 relative to SEQ ID NO: 1, wherein T is replaced by K, S, Q, R, D, or E; (q) An amino acid substitution at position G519 relative to SEQ ID NO: 1, wherein G is replaced by V, L, or I; (r) An amino acid substitution at position I520 relative to SEQ ID NO: 1, wherein G is replaced by K, Q, E, N, or T.(s) Amino acid substitutions relative to SEQ ID NO: 1 at position P521, wherein G is substituted by H, D, E, K, R, N, Q; (t) Amino acid substitutions relative to SEQ ID NO: 1 at position E522, wherein G is substituted by L, R, I, V; (u) Amino acid substitutions relative to SEQ ID NO: 1 at position A523, wherein G is substituted by E, V, L, K, R, I; (v) Amino acid substitutions relative to SEQ ID NO: 1 at position P524, wherein G is substituted by A, K, T, E, R; (w) Amino acid substitutions relative to SEQ ID NO: 1 at position R525, wherein G is substituted by H, R, S, L, N, E, D; (x) Amino acid substitutions relative to SEQ ID NO: 1 at position D526, wherein G is substituted by I, L, V, R; (y) Amino acid substitutions relative to SEQ ID NO: 1) An amino acid substitution at position G527, wherein G is substituted by E, K, Q, or D; (z) An amino acid substitution at position Q528 relative to SEQ ID NO: 1, wherein G is substituted by D, K, S, R, or A; (aa) An amino acid substitution at position A529 relative to SEQ ID NO: 1, wherein G is substituted by T or L; (ab) An amino acid substitution at position Y530 relative to SEQ ID NO: 1, wherein G is substituted by L, E, or T; (ac) An amino acid substitution at position V531 relative to SEQ ID NO: 1, wherein G is substituted by A, R, or K; (ad) An amino acid substitution at position R532 relative to SEQ ID NO: 1, wherein G is substituted by V or A; and / or (ae) any combination of (a)-(ad).

[0019] In some embodiments, the segment comprises a polypeptide sequence listed in Table 2A or Table 2B, or a polypeptide sequence having one to five amino acid substitutions.

[0020] In some embodiments, the 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 having 1 to 5 amino acid substitutions.

[0021] 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.

[0022] In some embodiments, the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO:10).

[0023] 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.

[0024] 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.

[0025] In some embodiments, the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and D486A.

[0026] In some embodiments, the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and T249P.

[0027] In some embodiments, the polypeptide includes a heteropolymerized domain at the C-terminus of its extracellular domain.

[0028] In some implementations, the multimerizing domain is a trimerizing domain.

[0029] In some embodiments, the polymerized domain comprises 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% of the same polypeptide sequence as I53-50A (SEQ ID NO: 144) or I53-50A ΔCys (SEQ ID NO: 145).

[0030] In some embodiments, the extracellular domain comprises amino acid substitutions S155C, S290C, S190F, and V207L.

[0031] In some embodiments, the extracellular domain comprises a polypeptide sequence that is 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% identical to the following SEQ ID NO: 6, SEQ ID NO: 6. Optionally lacking the p27 peptide shown in bold, where “X” refers to the site involving the added C-terminal helical segment and can be any amino acid: QNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVCKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTFRVLDLKNYINNQLLPMLNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPL STYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCN TDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSXXXXXXXXXXXXXXXXX (SEQ ID NO: 6).

[0032] In some embodiments, the extracellular domain comprises a polypeptide sequence that is 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% identical to the following SEQ ID NO: 7, SEQ ID NO: 7 optionally lacking the p27 peptide shown in bold, where “X” refers to a site involving the added C-terminal helical segment and may be any amino acid: QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVS TYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCN VDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSXXXXXXXXXXXXXXXXX (SEQ ID NO:7).

[0033] In some embodiments, the extracellular domain comprises a polypeptide sequence that is 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% identical to the following SEQ ID NO: 8, SEQ ID NO: 8 optionally lacking the p27 peptide shown in bold: QNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVCKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTFRVLDLKNYINNQLLPMLNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIND MPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKY DCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSREIIRAINIVRKIASEK (SEQ ID NO: 8).

[0034] In some embodiments, the extracellular domain comprises a polypeptide sequence that is 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% identical to the following SEQ ID NO: 9, SEQ ID NO: 9 optionally lacking the p27 peptide shown in bold: QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLIND MPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKY DCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSREIIRAINIVRKIASEK (SEQ ID NO:9).

[0035] In some embodiments, the polypeptide comprises 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% of the sequence identical to any one or more of SEQ ID NO: 1-9, 76-296.

[0036] In another aspect, this disclosure provides a trimeric protein complex comprising the polypeptide of this disclosure.

[0037] 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 lacking modification (a)-(h).

[0038] In some implementations, stability is increased, as determined by storage at approximately 40°C, compared to the trimer protein complex lacking modification (a)-(h).

[0039] In some implementations, thermal stability is increased compared to a reference RSV F protein containing amino acid substitutions consisting essentially of S155C, S290C, S190F, and V207L ​​(DS-Cav1).

[0040] In another aspect, this disclosure provides a protein nanostructure comprising a trimer component, said trimer component comprising the polypeptide described herein.

[0041] In some embodiments, the nanostructure is a two-component nanostructure comprising a first trimer component and a second pentamer component.

[0042] In some embodiments, the first trimer component comprises engineered extracellular domains of a respiratory syncytial virus (RSV) fusion (F) polypeptide and an I53-50A polypeptide.

[0043] In some embodiments, the first trimer component comprises a fusion protein, which comprises, in N-terminus to C-terminus, an RSV fusion (F) polypeptide, an amino acid linker, and an I53-50A polypeptide.

[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, 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 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% of the same polypeptide sequence as 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 identical to SEQ ID NO: 20 or 71 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% identical polypeptide sequence.

[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, 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 a polymerized region with or without I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) 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% of the same polypeptide sequence; and / or a second pentameric component, wherein the pentameric component comprises 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% of the same polypeptide sequence as 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 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 region of I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) 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% of the same polypeptide sequence; and / or a second pentameric component, wherein the pentameric component comprises 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% of the same polypeptide sequence as SEQ ID NO: 20 or 71.

[0047] 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 region of I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) 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% of the same polypeptide sequence; and / or a second pentameric component, wherein the pentameric component comprises 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% of the same polypeptide sequence as SEQ ID NO: 20 or 71.

[0048] In some embodiments, the trimer component comprises 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% of the same polypeptide sequence as any of the sequences listed in Table 14 or without the underlined and / or bold / italic polypeptide sequence.

[0049] In some embodiments, the pentamer component comprises 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% of the same polypeptide sequence as any one or more of SEQ ID NO: 20, 44, 45, 52, 71, 73, 74.

[0050] In another aspect, this disclosure provides a pharmaceutical composition comprising a polypeptide, protein complex, or nanostructure of the present disclosure.

[0051] In another aspect, this disclosure provides a vaccine comprising a polypeptide, protein complex, or nanostructure of this disclosure.

[0052] In another aspect, this disclosure provides a method for vaccinating a subject, the method comprising administering the composition described herein to the subject.

[0053] 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.

[0054] In another aspect, this disclosure provides a method for treating or preventing RSV disease in a subject, the method comprising administering the composition described herein to the subject.

[0055] In another aspect, this disclosure provides compositions for use in vaccination, generating an immune response, or treating or preventing RSV disease.

[0056] In another aspect, this disclosure provides compositions, methods, or uses as described herein. In yet 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.

[0057] 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

[0058] 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 This shows a structural model of the RSV F protein (PDB 4MMU) in its pre-fusion conformation, with stabilization elements separating it into five distinct spaces. Spaces 1-4 are targeted by stabilization mutations. Space 5 refers to the C-terminus of the protein.

[0059] Figure 2 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).

[0060] Figure 3 Displays ddG scores, with representative designs highlighted.

[0061] Figure 4 Displays the hydrophobicity rating of the design. Mean (solid line), standard deviation (dashed line), and WT (dotted line).

[0062] Figure 5 This shows representative electron micrographs of the protein nanostructures described in this article.

[0063] Figure 6 The neutralizing titers against RSV / B (strain B18537) induced by various nanostructure immunogens based on RSV / B antigens are shown.

[0064] Figure 7 The display shows the neutralizing titers against RSV / A (Tracy strain) induced by various nanostructure immunogens based on RSV / A antigens.

[0065] Figure 8 This shows a structural comparison of the cryo-EM structures of the RSV F extracellular domain of RSV / A.023 and DS-Cav1 (PDB 7LUE) fused with foldon. The C-terminal α-helical segment added in RSV / A.023 is shown in dark gray and surrounded by a dashed box. The antibody structure was removed from the PDB 7LUE model before image generation.

[0066] Figure 9 This shows a structural comparison of the C-terminal regions of the cryo-EM structures of the RSV F extracellular domain of RSV / A.023 and DS-Cav1 (PDB 7LUE) fused with foldon. 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. Detailed Implementation

[0067] 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 may be practiced without departing from the spirit of the invention.

[0068] 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 below are described more fully by reference to the entire specification.

[0069] I. Definition Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well.

[0070] 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 measurements 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.

[0071] The term “at least” followed by a number is used in this document to indicate the starting point of a range that begins with said number (which can be a range with or without an upper limit, depending on the definition of the variable). For example, “at least 1” means 1 or greater than 1.

[0072] The term "at most" followed by a number is used herein to indicate the end of a range that ends with said number (which 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 with a lower limit of the first number and an upper limit of the second number. For example, 25 to 100 mm means a range with a lower limit of 25 mm and an upper limit of 100 mm.

[0073] 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 residue is 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 is 75.0% identical to 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 indicated by EMBOSS Needle (available at ebi.ac.uk / Tools / psa / emboss_needle / on the World Wide Web) (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. Appl. 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 (available at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln on the World Wide Web).

[0074] For example, sequence identity can be determined using standard methods commonly used to compare the similarity of two polypeptide or two polynucleotide sequences. Using a computer program (e.g., EMBOSS Needle or BLAST), two polypeptide or two polynucleotide sequences are aligned to achieve optimal matching of their respective residues (along the full length of one or both sequences, or along a predetermined portion of one or both sequences). The program provides default open penalties and preset vacancy penalties, as well as scoring matrices such as PAM 250 (the standard scoring matrix; see Dayhoff et al., Atlas of Protein Sequence and Structure, Vol. 5, Supplement 3 (1978)) that can be used in conjunction with the computer program.

[0075] 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. The smallest segment of a protein that forms an α-helix is ​​generally considered to be approximately 6-7 amino acids long. 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 therein, 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.

[0076] 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, the term "α-helical homotrimer" as used herein does not include the heptapeptide repeat region of gp41 or its recombinant variants.

[0077] As used herein, for example in the term "stable α-helical homotrimer," the term "stable" 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 a larger trimeric protein complex (e.g., a trimeric antigen). In some embodiments, such as those predicted by calculation or determined experimentally, stabilizing the α-helical homotrimer includes stabilizing the trimeric protein complex by 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 stabilizing α-helical homotrimer is a “specific” homotrimer.

[0078] As used in this article, "conservative amino acid substitution" means: hydrophobic amino acids (Ala, Gly, Met, Val, Ile, Leu, Thr) are replaced by other hydrophobic amino acids; hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains; amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains; amino acids with negatively charged side chains (Asp, Glu) are replaced by other amino acids with negatively charged side chains; and polar amino acids (Cys, Ser, Thr, Asn, Gly, Tyr) are replaced by other polar amino acids.

[0079] Throughout this specification and the appended claims, unless the context otherwise requires, the words “comprise” and variations thereof, 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.

[0080] II. Engineered extracellular domains This disclosure provides 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 region. This disclosure further provides a recombinant polypeptide comprising amino acid substitutions that stably conform to the pre-fusion conformation of the RSV F protein. In other embodiments, this disclosure provides a recombinant polypeptide comprising amino acid substitutions having an engineered C-terminal α-helical region that stably conforms to the pre-fusion conformation of the RSV F protein.

[0081] 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 cells (McLellan et al., Science. 2013; 342(6158):592-8). The pre-fusion F protein possesses important epitopes 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 1 Within 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.

[0082] Illustrative sequences are shown in Table 1. The design used native RSV / BF protein sequences (GenBank: WDV37446.1). (Predicted) transmembrane regions are residues 527-549 and are in bold / underlined. Signal peptides are in italics and underlined.

[0083] Table 1.

[0084] In some embodiments, the extracellular domain is 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% identical to SEQ ID NO: 1. In some embodiments, the extracellular domain is 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% identical to SEQ ID NO: 3. In some embodiments, the extracellular domain is 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% identical to SEQ ID NO: 4. In some embodiments, the extracellular domain is 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% identical to SEQ ID NO: 180. In some embodiments, the extracellular domain is 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% identical to SEQ ID NO: 181. In some embodiments, the extracellular domain is 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% identical to SEQ ID NO: 182.

[0085] 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 that bind to 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 (utilizing Rosetta Remodel), 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 was determined by plotting ddG (Rosetta remodel) as a function of linker length or ddG (RFdiffusion) normalized to linker length. Helical constraints were then modeled for 6–14 additional amino acids.

[0086] Illustrative sequences are shown in Table 2A. 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.

[0087] Table 2A. C-terminal α-helical region (Rosetta remodel)

[0088] In some implementations, modeling has shown that the following substitutions will stabilize the F protein portion in a helical conformation.

[0089] In some embodiments, the extracellular domain includes (a) a 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, said one or more amino acid substitutions being selected such that said segment forms a stable α-helical homotrimer.

[0090] Table 2B. C-terminal α-helical region of RSV (RF diffusion)

[0091] Table 2C. Possible substitutions (RF diffusion) at positions 503-532

[0092] 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.

[0093] The computational design described herein has detailed yield information regarding desired amino acid substitutions that, individually or in groups, 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 an amino acid substitution at one or more of 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.

[0094] In some embodiments, the segment comprises (a) 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; (b) 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; (c) 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; (d) 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; (e) relative to SEQ ID NO: 1) The amino acid at position S509 is substituted, wherein S is replaced by A, I, L, M, V, F, W, Y, G, T, preferably A, I, L, M, V; (f) The amino acid at position D510 relative to SEQ ID NO: 1 is substituted, wherein D is replaced by any amino acid, preferably D, E, K, N, Q, R, S, T, Y; (g) The amino acid at position E511 relative to SEQ ID NO: 1 is substituted, wherein E is replaced by any amino acid; (h) The amino acid at position L512 relative to SEQ ID NO: 1 is substituted, wherein L is replaced by D, E, K, N, Q, R, S, T, Y, preferably A, I, L, M, V, F, W, Y, G, T; (i) Relative to SEQ ID NO: 1. The amino acid at position L513 is substituted, 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; (j) The amino acid at position H514 relative to SEQ ID NO: 1 is substituted, wherein H is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; (k) The amino acid at position N515 relative to SEQ ID NO: 1 is substituted, wherein N is replaced by any amino acid other than P, preferably A, I, L, M, V, F, W, Y, G; (l) The amino acid at position V516 relative to SEQ ID NO: 1 is substituted, wherein V is replaced by A, I, L, M, V, F, W, Y, G or T, S, K; (m) 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; (n) An amino acid substitution relative to SEQ ID NO: 1 at position T518, wherein T is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y.(o) a substitution of the amino acid at position G519 relative to SEQ ID NO: 1, wherein G is substituted with any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; and / or any combination of (p)(a)-(o).

[0095] In some embodiments, the segment comprises (a) an amino acid substitution relative to SEQ ID NO: 1 at position L503, wherein F is substituted with Q, V, K, R, N, or L; (b) 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, or Y; (c) an amino acid substitution relative to SEQ ID NO: 1 at position F505, wherein F is substituted with I, V, N, T, or L; (d) 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, or S; (e) 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, or Q; and (f) an amino acid substitution relative to SEQ ID NO: (g) Amino acid substitution at position K508 relative to SEQ ID NO: 1, wherein R is substituted by T, M, V, or R; (h) Amino acid substitution at position D510 relative to SEQ ID NO: 1, wherein D is substituted by S, K, N, D, or E; (i) Amino acid substitution at position E511 relative to SEQ ID NO: 1, wherein E is substituted by R, S, E, K, A, T, or L; (j) Amino acid substitution at position L512 relative to SEQ ID NO: 1, wherein L is substituted by V, N, T, or L; (k) Amino acid substitution at position L513 relative to SEQ ID NO: 1, wherein L is substituted by D, T, H, K, E, N, or R; (l) Amino acid substitution relative to SEQ ID NO: 1. An amino acid substitution at position H514, wherein H is replaced by A, N, E, S, V, K, T, or D; (m) An amino acid substitution at position N515 relative to SEQ ID NO: 1, wherein N is replaced by I, E, L, T, or Q; (n) An amino acid substitution at position V516 relative to SEQ ID NO: 1, wherein V is replaced by E, I, K, N, R, or Q; (o) An amino acid substitution at position N517 relative to SEQ ID NO: 1, wherein N is replaced by A, S, K, E, or R; (p) An amino acid substitution at position T518 relative to SEQ ID NO: 1, wherein T is replaced by K, S, Q, R, D, or E; (q) An amino acid substitution at position G519 relative to SEQ ID NO: 1, wherein G is replaced by V, L, or I; (r) An amino acid substitution at position I520 relative to SEQ ID NO: 1, wherein G is replaced by K, Q, E, N, or T.(s) Amino acid substitutions relative to SEQ ID NO: 1 at position P521, wherein G is substituted with H, D, E, K, R, N, or Q; (t) Amino acid substitutions relative to SEQ ID NO: 1 at position E522, wherein G is substituted with L, R, I, or V; (u) Amino acid substitutions relative to SEQ ID NO: 1 at position A523, wherein G is substituted with E, V, L, K, R, or I; (v) Amino acid substitutions relative to SEQ ID NO: 1 at position P524, wherein G is substituted with A, K, T, E, or R; (w) Amino acid substitutions relative to SEQ ID NO: 1 at position R525, wherein G is substituted with H, R, S, L, N, E, or D; (x) Amino acid substitutions relative to SEQ ID NO: 1 at position D526, wherein G is substituted with I, L, V, or R; (y) Amino acid substitutions relative to SEQ ID NO: 1) An amino acid substitution at position G527, wherein G is substituted by E, K, Q, or D; (z) An amino acid substitution at position Q528 relative to SEQ ID NO: 1, wherein G is substituted by D, K, S, R, or A; (aa) An amino acid substitution at position A529 relative to SEQ ID NO: 1, wherein G is substituted by T or L; (ab) An amino acid substitution at position Y530 relative to SEQ ID NO: 1, wherein G is substituted by L, E, or T; (ac) An amino acid substitution at position V531 relative to SEQ ID NO: 1, wherein G is substituted by A, R, or K; (ad) An amino acid substitution at position R532 relative to SEQ ID NO: 1, wherein G is substituted by V or A; and / or (ae) any combination of (a)-(ad).

[0096] In some embodiments, the segment comprises a polypeptide sequence listed in Table 2A, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the segment comprises a polypeptide sequence listed in Table 2A, 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 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.

[0097] 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).

[0098] 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.

[0099] In another aspect, this disclosure provides an α-helical segment comprising a polypeptide sequence listed in Table 2A or Table 2B, 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.

[0100] 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 can also 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.

[0101] Table 3A. Stabilization Substitution

[0102] The implementation schemes for the alternative combinations are shown in Table 3B.

[0103] Table 3B.

[0104] In some implementations, the extracellular domain contains amino acid substitutions for S155C, S290C, S190F, and V207L.

[0105] 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.

[0106] 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.

[0107] 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 can have other effects on structure, such as creating hydrophobicity or charge-charge interactions within the structure (e.g., salt bridges). These mutations are listed in Table 4A.

[0108] Table 4A

[0109] 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.

[0110] The substitution combinations are shown in Table 4B.

[0111] Table 4B.

[0112] 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.

[0113] In some embodiments, RSV F protein mutants comprise disulfide bond mutations selected from the following groups: 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.

[0114] In some implementations, the RSV F protein mutant contains one or more cavity-filling mutations selected from the groups shown in Table 4C.

[0115] Table 4C. Disulfide bond mutations

[0116] 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.

[0117] In some implementations, the RSV F protein mutant contains at least one electrostatic mutation selected from the group shown in Table 4D.

[0118] Table 4D. Electrostatic Transitions

[0119] In some implementations, the RSV F protein mutant contains the mutant D486S.

[0120] The substitution combinations are shown in Table 4E.

[0121] Table 4E.

[0122] 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.

[0123] 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.

[0124] The native sequence of the RSV / BF protein (GenBank: WDV37446.1) is shown below, with the (predicted) transmembrane region underlined, and the C-terminal helix (residues 492-501) of the native sequence also underlined. The signal peptide is in bold / italic and underlined.

[0125] In some embodiments, the extracellular domain comprises a polypeptide sequence that is 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% identical to 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 may be any amino acid: QNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAI ASGIAVCKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTFRVLDLKNYINNQLLPMLNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKI MTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSXXXXXXXXXXXXXXXXX (SEQ ID NO: 6).

[0126] In some embodiments, the extracellular domain comprises a polypeptide sequence that is 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% identical to the following SEQ ID NO: 7, wherein SEQ ID NO: 7 optionally lacks the p27 peptide shown in bold, wherein “X” indicates a site involving the added C-terminal helical segment and may be any amino acid: QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAI ASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKK LMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSXXXXXXXXXXXXXXXXX (SEQ ID NO: 7).

[0127] In some embodiments, the extracellular domain comprises 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% of the same polypeptide sequence as the following SEQ ID NO: 8, wherein SEQ ID NO: 8 optionally lacks the p27 peptide shown in bold: QNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAI ASGIAVCKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTFRVLDLKNYINNQLLPMLNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKI MTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSREIIRAINIVRKIASEK (SEQ ID NO: 8).

[0128] In some embodiments, the extracellular domain comprises a polypeptide sequence that is 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% identical to the following SEQ ID NO: 9, wherein SEQ ID NO: 9 optionally lacks the p27 peptide shown in bold: QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAI ASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKK LMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSREIIRAINIVRKIASEK (SEQ ID NO: 9).

[0129] In some embodiments, the polypeptide comprises 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% identical to any one or more of SEQ ID NO: 1-9.

[0130] Illustrative sequences containing the extracellular domains and C-terminal α-helical regions of various RSV F proteins are shown in Table 4F. Signal peptides are underlined.

[0131] In some embodiments, the extracellular domain contains 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% of the sequence shown in Table 4F.

[0132] Table 4F.

[0133] 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.

[0134] furin cleavage site The RSV F protein is cleaved by the protease furin during expression. This article provides a construct that replaces the furin cleavage site with a glycine-serine linker. The sequence is provided in Table 5A. In some embodiments, the extracellular domain of the RSV F protein contains an uncleaved furin cleavage site.

[0135] Table 5A. Frin protease cleavage linkers

[0136] connector In some implementations, the recombinant peptide and the protein nanostructure can be genetically fused so that both exist within a single peptide, referred to as a "fusion protein." The bond between the peptide and the protein nanostructure allows the recombinant peptide to be displayed on the exterior of the self-assembled protein nanostructure.

[0137] 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 an antigenic fragment thereof 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 an antigenic fragment thereof to the protein nanostructure. In some embodiments, the encoded polypeptide may comprise a linker between regions. In some embodiments, the polypeptide is a fusion protein comprising a recombinant RSV polypeptide, a linker, and protein nanostructure component polypeptides. In some embodiments, the polypeptide is a fusion protein comprising, in N-terminal to C-terminal order, a recombinant RSV polypeptide, a linker, and protein nanostructure component polypeptides. The linker may be a polypeptide. A variety of 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 composed of glycine and serine residues). In some embodiments, 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 the Glys-Ser linker are presented in Table 5B.

[0138] Table 5B.

[0139] In some embodiments, the linker comprises 3 to 30 amino acid residues. In some embodiments, the linker comprises 4 to 24 amino acid residues. In some embodiments, the linker comprises 8 to 24 amino acid residues. In some embodiments, the linker comprises 10 to 24 amino acid residues. In some embodiments, the linker comprises 12 to 24 amino acid residues. In some embodiments, the linker comprises 16 to 24 amino acid residues. In some embodiments, the linker comprises 18 to 24 amino acid residues. In some embodiments, the linker comprises 20 to 24 amino acid residues. In some embodiments, the linker comprises 4 to 20 amino acid residues. In some embodiments, the linker comprises 8 to 20 amino acid residues. In some embodiments, the linker comprises 10 to 20 amino acid residues. In some embodiments, the linker comprises 12 to 20 amino acid residues. In some embodiments, the linker comprises 16 to 20 amino acid residues. In some embodiments, the linker comprises 8 to 18 amino acid residues. In some embodiments, the linker comprises 12 to 16 amino acid residues. In some embodiments, the linker comprises 3 amino acid residues. In some embodiments, the linker comprises 4 amino acid residues. In some embodiments, the linker comprises 5 amino acid residues. In some embodiments, the linker comprises 6 amino acid residues. In some embodiments, the linker comprises 7 amino acid residues. In some embodiments, the linker comprises 8 amino acid residues. In some embodiments, the linker comprises 8 amino acid residues. In some embodiments, the linker comprises 10 amino acid residues. In some embodiments, the linker comprises 11 amino acid residues. In some embodiments, the linker comprises 12 amino acid residues. In some embodiments, the linker comprises 13 amino acid residues. In some embodiments, the linker comprises 14 amino acid residues. In some embodiments, the linker comprises 15 amino acid residues. In some embodiments, the linker comprises 16 amino acid residues. In some embodiments, the linker comprises 17 amino acid residues. In some embodiments, the linker comprises 18 amino acid residues. In some embodiments, the linker comprises 19 amino acid residues. In some embodiments, the linker comprises 20 amino acid residues. In some embodiments, the linker comprises 21 amino acid residues. In some embodiments, the linker comprises 22 amino acid residues. In some embodiments, the linker comprises 23 amino acid residues. In some embodiments, the linker comprises 24 amino acid residues. In some embodiments, the linker comprises 25 amino acid residues. In some embodiments, the linker comprises 26 amino acid residues. In some embodiments, the linker comprises 27 amino acid residues. In some embodiments, the linker comprises 28 amino acid residues. In some embodiments, the linker comprises 29 amino acid residues. In some embodiments, the linker comprises 30 amino acid residues.

[0140] 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: 320).

[0141] Trimerization domain In some embodiments, the peptide may include a trimerizing domain, such as FoldOn or GCN4 trimer. In some embodiments, the linker sequence includes FoldOn, wherein the FoldOn sequence is GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 179).

[0142] In some embodiments, the polypeptide may include a trimerizing domain, wherein the trimerizing domain sequence is DKIEEILSKIYHIENEIARIKKLIGE (GEN) (SEQ ID NO: 270). In some embodiments, the polypeptide may include a trimerizing domain, wherein the trimerizing domain sequence is EKFHQIEKEFSEVEGRIQDLEK (HA) (SEQ ID NO: 271).

[0143] In some embodiments, the polypeptide may include a trimerizing domain, wherein the trimerizing domain sequence is EDKIEEILSKIYHIENEIARIKKLIGEA (coiled isoleucine zipper) (SEQ ID NO: 272).

[0144] In some embodiments, the polypeptide may include a trimerizing domain, wherein the trimerizing domain sequence is GSGYIPEAPRDGQAYVRKDGEWVLLSTFL (phage T4 fibrin) (SEQ ID NO: 273).

[0145] In some embodiments, the trimerizing sequence is RMKQIEDKIEEILSKIYHIENEIARIKKLIGEA (GCN4) (SEQ ID NO: 274). In some embodiments, the trimerizing domain is a GCN4 variant. In some embodiments, the GCN4 variant sequence is RMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGR (SEQ ID NO: 275), RMKQIEDKIEEILSKIYHIENEIARIKKLIGNRTGGR (SEQ ID NO: 276), RMKQIEDKIENITSKIYHIENEIARIKKLIGNRTGGR (SEQ ID NO: 277), RMKQIEDKIEEILSKIYNITNEIARIKKLIGNRTGGR (SEQ ID NO: 278), or RMKQIEDKIENITSKIYNITNEIARIKKLIGNRTGGR (SEQ ID NO: 279).

[0146] Illustrative sequences comprising the extracellular domains, C-terminal α-helical regions, and FoldOn of various RSV F proteins are shown in Table 5C. Signal peptides are italicized and underlined. The underlined FoldOn sequence may be replaced by any of the trimerizing domains described herein or any of the multimerizing domains described in Table 6 to produce embodiments comprising such other trimerizing domains.

[0147] In some embodiments, the trimeric protein complex comprises 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% of 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 bolded. 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.

[0148] Table 5C.

[0149] 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 2A or Table 2B, or a polypeptide sequence having 1 to 5 amino acid substitutions. In some embodiments, the polypeptide comprises a trimeric pathogen protein linked to the α-helical segment at the 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 having 1 to 5 amino acid substitutions. In some embodiments, the polypeptide comprises an antigen at the N-terminus of the segment.

[0150] 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.

[0151] The protein nanostructures of the present invention may 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 optionally a 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 component, wherein the assembly domain forms a trimer associated by 3-fold rotational symmetry, and / or the second component is a pentamer component, 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 may be arranged together such that members of each component are correlated with each other by symmetry operators. 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 US Patent Publication No. 2015 / 0356240 A1.

[0152] 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). This can increase the immunogenicity of the antigen, free from theoretical constraints. 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) may 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 may be used. Alternatively, various bioconjugated linkers may be used. In some embodiments of this disclosure, the antigen may contain other polypeptide sequences in addition to the RSV F protein.

[0153] 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)

[0154] 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.

[0155] 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.

[0156] 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 connect two proteins is applicable to the protein nanostructures disclosed in this invention. Specifically, chemistry used to generate 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 carriers are provided, for example, by US Patent Publication No. 2008 / 0145373 A1.

[0157] Protein nanostructures can employ various coupling techniques to attach antigens to their 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).

[0158] 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 carry a negative charge on at least one surface, and the core polypeptide is engineered to carry a positive charge on at least one surface, or both positive and negative charges. This can promote 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, attachment is achieved using computational design of protein-protein interfaces.

[0159] polypeptide sequence Patent Publication No. US 2015 / 0356240 A1 describes various methods for designing protein assemblies. As described in US Patent Publication No. US 2016 / 0122392 A1 and International Patent Publication No. WO 2014 / 124301 A1, isolated peptides of SEQ ID NO: 13-63 are designed to be capable of self-assembling in pairs to form protein nanostructures, such as icosahedral particles. The design involves designing suitable interface residues for each member of the peptide pair, which can be assembled to form the protein nanostructure. The protein nanostructures thus formed include symmetrically repeating, non-natural, non-covalent peptide-peptide interfaces that orient a first assembly domain and a second assembly domain to the protein nanostructure, such as a protein nanostructure having icosahedral symmetry. Therefore, 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 identified residues in Table 6 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 added to the N-terminus (e.g., to form a helical extension).

[0160] Table 6.

[0161] Table 6 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 6 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 that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in length to the polypeptides selected from the group consisting of SEQ ID NO: 13-46, and identical 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 that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in length, and identical at least at the interface positions identified at 20%, 25%, 33%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100%.

[0162] As with proteins in general, peptides are expected to tolerate some changes to their designed sequences 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, Phe, Thr, Trp) 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, Gln) 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.

[0163] 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): an isolated polypeptide comprising an amino acid sequence that is 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% identical in length to the amino acid sequence indicated by SEQ ID NO, and / or identical at at least one identified interface location: 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)).

[0164] 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).

[0165] 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.

[0166] 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 / 0137234 A1; and U.S. Patent Publication No. 2019 / 0155988 A2, each of which is incorporated herein by reference in its entirety.

[0167] 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 that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in length to the amino acid sequence indicated by SEQ ID NO, and / or identical at at least one identified interface site): 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).

[0168] Various protein nanostructures are known in the art and described in, for example, U.S. Patent Publications US2015 / 0356240 A1, US2016 / 0122392 A1, US2018 / 0030429 A1, US2019 / 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 (60 monomers) of the trimer 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 assemble, for example, within expression cells or when separated trimers (or monomers) are mixed under suitable conditions.

[0169] 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 that is 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% identical to that of SEQ ID NO: 107. In some embodiments, the assembly domain comprises amino acid substitutions at positions 1, 2, 3, and 4 compared to SEQ ID NO: 107. In some embodiments, the assembly domain comprises amino acid substitutions at positions 1, 2, 3, and 4 compared to SEQ ID NO: 107. In some embodiments, multiple amino acid substitutions are made by replacing nonpolar residues (e.g., A, L, I, M, V, F, or W) with polar residues. In some embodiments, some or all amino acid substitutions are made by replacing small nonpolar residues (e.g., A, L, I, M, or V) with polar residues. In some embodiments, the protein nanostructure comprises amino acid substitutions E33L or E33V; K61L or K61M; D187A or D187V; and / or R190A. In some embodiments, the protein nanostructure comprises amino acid substitutions E33L, K61M, D187V, and R190A. In some embodiments, the protein nanostructure comprises amino acid substitutions E33V, K61L, D187A, and R190A. In some embodiments, the assembly domain comprises amino acid substitutions (e.g., K129A) that inactivate the enzymatic activity of the assembly domain. In some embodiments, the assembly domain may contain other amino acid substitutions (e.g., MI3; E56M or E56K; P186I; E191A; and / or K194A). In some embodiments, the assembly domain contains amino acid substitutions by removing cysteine ​​residues. In some embodiments, the assembly domain contains C76A and / or C100A substitutions.

[0170] In one aspect, this disclosure provides a protein nanostructure comprising a recombinant polypeptide comprising 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 comprising one or more amino acid substitutions relative to SEQ ID NO: 1 between about residue 500 and about residue 530, the one or more 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) relative to SEQ ID NO: (a) Substitution of one, two, three or more amino acids at positions 75, 216, 218 or 219; (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 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).

[0171] 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 comprises a polypeptide sequence that is 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% identical to I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64).

[0172] In another aspect, this disclosure provides a trimeric protein complex comprising the polypeptide of this disclosure. 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).

[0173] In some implementations, the first trimer component comprises engineered extracellular domains of a respiratory syncytial virus (RSV) fusion (F) peptide and an I53-50A peptide.

[0174] In some embodiments, the first trimer component comprises a fusion protein, which comprises, in N-terminus to C-terminus, an RSV fusion (F) polypeptide, an amino acid linker, and an I53-50A polypeptide.

[0175] 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 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% of the same polypeptide sequence as 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 identical to SEQ ID NO: 20 or 71 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% identical polypeptide sequence.

[0176] 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 an amino acid 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 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% of the same polypeptide sequence as 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 relative to SEQ ID NO: 10; 20 or 71 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% identical polypeptide sequence 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, the polymerized domain comprising I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). NO:64) 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% of the same polypeptide sequence; and / or a second pentamer component, wherein the pentamer component comprises a polypeptide sequence 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% of the same polypeptide sequence as SEQ ID NO: 20 or 71.

[0177] 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 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, the polymerized domain comprising 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% of the same polypeptide sequence as 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 identical to SEQ ID NO: 20 or 71 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% identical polypeptide sequence.

[0178] 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 of I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) 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% of the same polypeptide sequence; and / or a second pentameric component, wherein the pentameric component comprises 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% of the same polypeptide sequence as SEQ ID NO: 20 or 71.

[0179] 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 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 I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) 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% of the same polypeptide sequence; and / or a second pentameric component, wherein the pentameric component comprises 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% of the same polypeptide sequence as SEQ ID NO: 20 or 71.

[0180] 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) 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% of the same polypeptide sequence; and / or a second pentameric component, wherein the pentameric component comprises 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% of the same polypeptide sequence as SEQ ID NO: 20 or 71.

[0181] 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 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, the polymerized domain comprising I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) 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% of the same polypeptide sequence; and / or a second pentameric component, wherein the pentameric component comprises 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% of the same polypeptide sequence as SEQ ID NO: 20 or 71.

[0182] 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 a polypeptide sequence that is 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% identical to any of the sequences listed in Table 14, and comprising a C-terminal helical forming segment comprising the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10); and a polymerized domain comprising a polymerized domain that is identical to I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 10). 64) 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% of the same polypeptide sequence; and / or a second pentameric component, wherein the pentameric component comprises 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% of the same polypeptide sequence as SEQ ID NO: 20 or 71.

[0183] In some embodiments, the trimer component comprises 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% of the same polypeptide sequence as any of the sequences listed in Table 14 or without the underlined and / or bold / italic polypeptide sequence.

[0184] In another aspect, this disclosure provides a protein nanostructure comprising a trimer component, said trimer component comprising the polypeptide described herein. In some embodiments, the nanostructure is a two-component nanostructure comprising a first trimer component and a second pentamer component. In some embodiments, the pentamer component comprises 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% of the polypeptide sequence identical to any one or more of SEQ ID NO: 20, 44, 45, 52, 71, 73, 74.

[0185] 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 that is 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% identical to any of the following sequences: MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKVELIGNENHGLYLADQYVKGIAKSRKS. (SEQ ID NO: 114) MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDAQN. (SEQ ID NO:115) NFHQDCEAGLNRTVNLKFHSSYVYLSMASYFNRDDVALSNFAKFFRERSEEKEHAEKLIEYQNQRGGRVFLQSVEKPERDDWANGLEALQTALKLQKSVNQALLDLHAVAADKSDPHMTDFLESPYLSESVETIKKLGDHITSLKKLWSSHPGMAEYLFNKHTLG. (SEQ ID NO: 116) QFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS. (SEQ ID NO: 117) SGESQVRQNFKPEMEEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDGS. (SEQ ID NO: 118) 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 VLPs, AP205 capsid protein VLPs, and bacteriophage VLPs (e.g., bacteriophages). Display can be achieved on these and other platforms 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. Protein nanostructures may be, for example, dioxetine synthase nanoparticles as described in, for example, Geng et al., PLoS Pathog. 17(9):e1009897 (2021). Protein nanostructures may be, for example, ferritin nanoparticles as described in, for example, Joyce et al., bioRxiv 2021.05.09.443331 and U.S. Patent Publication US 2019 / 0330279 A1.

[0186] IV. Polynucleotides In another aspect, this disclosure provides polynucleotides encoding the antigen, first component, and / or second component of this disclosure. The polynucleotide sequence may comprise RNA or DNA. As used herein, "polynucleotide" refers to those that have been removed from their normal surrounding polynucleotide sequence 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 epitope tags, output and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, it will be apparent to those skilled in the art which nucleic acid sequences will encode the proteins of this disclosure.

[0187] 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 vector. 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 vector, such as spermine, polyethyleneimine, chitosan, or polyurethane. In some embodiments, the delivery medium is a polymeric delivery system, such as a polyamide-amine (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.

[0188] 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 polycation 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 non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0189] In various embodiments, the average diameter of the lipid nanoparticles is 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, LNP is 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, for example, in 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 / 0308. No. 304, No. 2014 / 0200257, No. 2013 / 086373, No. 2013 / 0338210, No. 2013 / 0323269, No. 2013 / 0245107, No. 2013 / 0195920, No. 2013 / 0123338, No. 2013 / 0022649, No. 2013 / 0017223, No. 2012 / 0295832, No. 2012 / 0183581, No. 201 No. 2 / 0172411, No. 2012 / 0027803, No. 2012 / 0058188, No. 2011 / 0311583, No. 2011 / 0311582, No. 2011 / 0262527, No. 2011 / 0216622, No. 2011 / 0117125, No. 2011 / 0091525, No. 2011 / 0076335, No. 2011 / 0060032, No. 2010 / 013058 No. 8, No. 2007 / 0042031, No. 2006 / 0240093, No. 2006 / 0083780, No. 2006 / 0008910, No. 2005 / 0175682, No. 2005 / 017054, No. 2005 / 0118253, No. 2005 / 0064595, No. 2004 / 0142025, No. 2007 / 0042031, No. 1999 / 009076 and PCT Publication No. WO The contents of the patents described in Nos. 99 / 39741, 2017 / 004143, 2017 / 075531, 2015 / 199952, 2014 / 008334, 2013 / 086373, 2013 / 086322, 2013 / 016058, 2013 / 086373, 2011 / 141705, 2017 / 049245, 2010 / 144740, 2017 / 075531 and 2001 / 07548 are incorporated herein by reference.

[0190] Other exemplary lipids and LNPs and their manufacture 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.

[0191] VI. Pharmaceutical Composition This disclosure also provides pharmaceutical compositions. Such pharmaceutical compositions can be used to induce 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).

[0192] In some embodiments, the pharmaceutical composition may also include excipients and / or additives. Examples of such agents are surfactants, stabilizers, complexing agents, antioxidants, or preservatives, flavoring agents, vitamins, or other additives known in the art that prolong the shelf life of the finished pharmaceutical preparation. Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or its salts, such as disodium EDTA, citric acid, hypozinotriacetic acid, and their salts. In some embodiments, preservatives include, but are not limited to, those that protect the solution from contamination by pathogenic particles, 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.

[0193] 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 (e.g., Ringer's solution), alcohols, oils, gelatin, carbohydrates (e.g., lactose, amylose, or starch), fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and pigments. Such formulations may be sterilized and, if necessary, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, and / or aromatic substances, and analogs that do not adversely react with the compounds of this disclosure. Those skilled in the art will recognize that other pharmaceutical excipients may be used in this disclosure.

[0194] 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.

[0195] In another aspect, this disclosure provides a pharmaceutical composition comprising a polypeptide, protein complex, or nanostructure of the present disclosure.

[0196] VII. Vaccines In another aspect, this disclosure provides a vaccine comprising a polypeptide, protein complex, or nanostructure as disclosed herein.

[0197] In some implementations, the vaccine contains an adjuvant.

[0198] In some embodiments, the pharmaceutical compositions provided herein are administered as RSV vaccines, such as RSV / A vaccines and RSV / B vaccines or bivalent RSV A / B vaccines.

[0199] adjuvant Adjuvants or immunostimulants may 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.

[0200] Adjuvants may include, but are not limited to, natural or synthetic adjuvants. Adjuvants may be organic or inorganic.

[0201] The adjuvant 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 (with monolayer liposomes containing 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.

[0202] 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.

[0203] Other available adjuvants 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 Any adjuvants on page 13 of Volume 2012 (2012), Article ID 831486, the contents of which are incorporated herein by reference in their entirety.

[0204] 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 E112KmCT-E112K and / or matrix-S.

[0205] In some embodiments, the adjuvant comprises squalene. In some embodiments, the adjuvant comprises aluminum hydroxide. In some embodiments, the adjuvant comprises AS01. E .

[0206] VIII. Usage Instructions In another aspect, this disclosure provides methods of administering the compositions, pharmaceutical compositions, or vaccines described herein.

[0207] 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 compositions of the present disclosure for use in vaccination, generating an immune response, or treating or preventing RSV disease. In another aspect, this disclosure provides compositions, methods, or uses as described herein. In another aspect, this disclosure provides a method of preparing a composition, the method comprising culturing host cells modified to express one or more polypeptides as described herein.

[0208] In some embodiments, the method includes administering the vaccine described herein. In some embodiments, the subject is simultaneously immunized against respiratory syncytial virus (RSV) 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 via intranasal injection. In one aspect, this disclosure provides a pre-filled syringe containing the vaccine described herein. In one aspect, this disclosure provides a kit containing either the vaccine described herein or a pre-filled syringe described herein.

[0209] 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 70 μg to about 75 μg, about 100 μg to about 125 μg, about 100 μg to about 150 μg, about 125 μg to about 175 μg, about 200 μg to about 250 μg, about 225 μg to about 300 μg, or about 250 μg to about 350 μg of protein nanostructures.

[0210] 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 125 μg to about 150 μg, about 150 μg to about 175 μg, about 175 μg to about 200 μg, about 200 μg to about 250 μg, or about 250 μg to about 300 μg of protein nanostructures.

[0211] In some implementations, the subjects are at risk of RSV disease. In some implementations, the subjects are adults aged 60 years or older. In some implementations, the subjects are healthy adults aged 18-45 years. In some implementations, the subjects are pregnant women between the 32nd and 36th weeks of pregnancy. In some implementations, the subjects are pregnant women between the 30th and 38th weeks of pregnancy. In some implementations, the subjects are pregnant women between the 28th and 38th weeks of pregnancy.

[0212] Any aspect or implementation described herein may be combined with any other aspect or implementation disclosed herein.

[0213] Example The following examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

[0214] 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.

[0215] 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 thought to form a helical bundle in the case of the native protein. The structure of the pre-fusion F protein is typically 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 typically replaced by a four-residue linker (SAIG) and a trimerized FoldOn domain. The predicted transmembrane domain begins at residue 527. The sequence of the native RSV / BF protein (GenBank: WDV37446.1) is shown here, with the transmembrane domain 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 modified antigens for use in vaccines. Our method 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 new 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 trimerized domains, such as the I53-50A multimerized domain. 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 conformation (i.e., tryptophan exposed to solvents).

[0216] 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. Typically, 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, this sequence is used for remodeling: It can generate blueprints in which amino acid residues are configured to match native sequences. A The model starts with the native sequence but allows substitutions (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 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.

[0217] Alternatively, RFdiffusion can be used for remodeling. The relaxed structures used as input for remodeling are also used as input for RFdiffusion, 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 Remodel are also preserved for diffusion. Unlike Rosetta Remodel, all structural data for subsequent residues are ignored. This is a limitation of RFdiffusion, not a scientific constraint on the design problem. Weights within and between protomers are set to 1, with secondary guide decay and guide scaling set to 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.

[0218] The design was analyzed based on the following criteria: 1) ColabFold validated the design using Rossetta by predicting the same ordered terminal helices as the design model (assuming the ColabFold method provides 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 away from 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., (long-range state) – (trimeric state).

[0219] 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 Remodel, 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 become difficult when constructing larger segments due to the increased degrees of freedom. The ideal linker length is 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).

[0220] Computational modeling of RSV / B proteins (using Rosetta Remodel) generated artificial polypeptide sequences, predicting that each sequence would form a stable α-helix, as shown in Table 7. 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 mode. 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 mode. 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 mode. All backbone sampling of these elements in the centroid mode was performed simultaneously, and sequence design in the all-atom mode was also performed simultaneously. Manually refined designs are used to remove exposed hydrophobic residues or buried polar residues, with the same characteristics of preferential selection from the nearest residue in the WT sequence or rational selection in the case of suboptimal WT residues.

[0221] 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 redesigned C-terminus 1, the C-terminus is further laterally from the antigen's axis of symmetry. Figure 2 Therefore, this modification minimizes the 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 7), where the antigen also contains the DS-Cav1 mutation.

[0222] Table 7. Illustrative C-end spiral formation section

[0223] The native sequence includes the C-terminal α-helical segment ISQVNEKINQSLAFIRRSDE (SEQ ID NO: 317).

[0224] 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: 319) 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.

[0225] native ISQVNEKINQSLA F IR RS DELLHNVN (SEQ Id NO: 318) Remodeling ISQVNEKINQSRE I IR AI NI V RK I ASEK (SEQ ID NO: 319) 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.

[0226] The published structures of RSV proteins typically do not include residues from the C-terminus to approximately 500 residues. These residues are not present in the recombinant protein under study, or are not visible in the observed electron density. Nevertheless, modeling suggests that the following substitutions will stabilize this portion of the F protein in its helical conformation.

[0227] Table 8. Possible replacements at locations 505-516

[0228] 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.

[0229] Small-scale screening showed expression of three of the four selected designs. Table 9 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 binds to both the pre-fusion monomer and the trimer, while AM14 binds only to 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.

[0230] Table 9. Summary of antibody binding screening data for the designed RSV / BF protein

[0231] 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.

[0232] Computational modeling was used to identify amino acid substitutions that stabilize the RSV / BF protein in its pre-fusion conformation. These mutations are listed in Table 10.

[0233] Table 10. Stabilization Substitution

[0234] Based on molecular modeling, the expected synergistic substitution combinations include:

[0235] The RSV F protein is cleaved by the protease furin during expression. Constructs replacing the furin cleavage sites (residues 104-140) with native linkers were also tested. The linker sequences are provided in Table 11, and were tested between residues 103 and 141.

[0236] Table 11. Frin protease cleavage linkers

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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 the I53-50A fusion chaperone. Various designs were characterized by increased binding to AM14 (pre-fusion) or decreased binding to 4D7 (post-fusion). Figure 1 The selected scaled-up protein was subjected to a six-day thermal stress test.

[0241] 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 with C-terminal remodeling generally showed greater thermal stability under storage (i.e., a reduced rate of 4D7 binding reduction).

[0242] The constructs selected for thermal denaturation and storage tests are shown in Table 12. 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 were controls containing the DS-Cav1 substitution. The data in Table 12 show that the C-terminal α-helix region itself increases thermal stability by up to approximately 25 °C (compare constructs RSV / B.002 with RSV / B.195, and constructs 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.

[0243] Table 12

[0244] 1 Based on RSV / 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 13. Representative electron micrographs are shown in... Figure 5 (RSV / B.195, with DS-Cav)

[0245] Table 13

[0246] 1 Based on hRSV / B / Australia / VIC-RCH056 / 2019 strain 2Except 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 13 are shown in Table 14. SEQ ID NO: 1 is used as a reference sequence. In each case, the signal peptide at the N-terminus (underlined) or the tag at the C-terminus may have a known substitution or deletion. RSV F proteins are 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 in bold and italics and can be removed via furin-based cleavage during antigen production in cell cultures.

[0247] Table 14.

[0248] The relative expression and antibody binding for each design are shown in Table 15.

[0249] Table 15. Relative expression of BLI and antibody binding

[0250] The mutations of the designed constructs used in the experiments are shown in Table 16. 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 described above, 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 with a known substitute or omitted. “o” indicates the use of an amino acid substitution.

[0251] Table 16. Mutations of the constructs used in the experiment

[0252] 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 14 and 16, 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 12). 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, demonstrating 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 provides a more robust improvement in the thermal stability of both RSV / A and RSV / B antigens. Furthermore, as evaluated by DLS, both RSV / A.013 and RSV / A.023 could be assembled into nanostructures in vitro with the addition of I53-50B (Table 13).

[0253] 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, RSV / B neutralizing titers 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 μg or 0.1 μg, all of which were fortified with AddaVax™ adjuvant (… Figure 6 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 μg unadjuvanted or 0.01 μg with AddaVax adjuvant. Figure 7 However, mice immunized with 1 μg of unadjuvanted RSV / A.023 nanostructures did indeed have significantly higher RSV / A neutralizing titers than mice immunized with the same dose of unadjuvanted RSV / A.03.

[0254] The cryogenic EM structure of the F extracellular domain of RSV / A.023 at 3.35 Å was resolved. Both the F extracellular domain and the C-terminal helical region were resolved, while the I53-50AΔcys domain fused to the antigen remained unresolved due to the flexibility of the linkers between these domains and the antigen extracellular domain. The overall structure of the F extracellular domain (…) Figure 8 A) A publicly disclosed low-temperature EM structure similar to the soluble DS-Cav1 construct ( Figure 8 B, PDB 7LUE), indicating that the F extracellular domain of RSV / A.023 is appropriately formed for use in the vaccine. The C-terminal helical forming segment forms hydrophobic interactions between each subunit as intended by the design scheme. Figure 9 A), and confirmed the addition of this structural segment to the publicly designed common structure relative to the stabilized RSV F extracellular domain. Figure 9 B).

[0255] Materials and methods Small-scale transfection: Multiple designed RSV / B expression, antigenicity, and thermostability were screened via 96 deep-well transfection. Expi293 cells in logarithmic growth phase were counted and transfected at 2.5 x 10⁻⁶ cells / wells. 6 Seed cells / ml. Incubate cells overnight at 36°C with shaking (120 rpm). Count cells the next day and dilute to 3 x 10⁶ cells / ml at 0.6 ml per well. Transfect cells transiently as follows: Dilute 1000 μg of plasmid DNA 5x master mix to a final volume of 35 ml with OptiMEM™ and mix gently in individual 96-well plates. Dilute the transporter 5 transfection reagent 5x master mix to a final volume of 35 ml with OptiMEM™ and mix gently. Add the diluted transporter 5 to the diluted DNA, mix, incubate at room temperature for 10 min, then add 42 μl to each well while gently shaking the plate. Return cells to the incubator and incubate with shaking at 1050 rpm for 4 days.

[0256] Biolayer Interferometry: The concentrations of antibodies 16A8 (ATUM), AM14, 4D7, D25, and palizumab (Creative Biolabs) were normalized to 10 μg / mL in sufficient volume in BLI assay buffer (PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4) to load 80 μL into each well of a black 384-well microplate (Thermo Scientific, 460518). Briefly, on an Octet rh16 instrument, the protein G biosensor (Sartorius, 18-5082) was immersed in the assay buffer for 60 seconds to obtain a baseline. Next, the biosensor was immersed in each antibody for 60 seconds to immobilize the present but unsaturated antibodies, followed by an additional baseline step. The immobilized antibody was associated with 80 μL of RSV / B supernatant for 120 seconds, and then the biosensor was immersed back into the assay buffer for 120 seconds to observe any possible dissociation. 16A8 is a monoclonal antibody that recognizes I53-50A and is used to estimate relative expression levels. AM14, D25, 4D7, and palizumab are specific for RSV F protein.

[0257] Large-scale transfection: Subgroups of the transient expression construct were generated at a 1-liter scale based on data from 96-well screening. Expi293 cells in logarithmic growth phase were counted and seeded at 2.5 x 10⁶ cells / ml in 220 ml of each of four 1-L flasks (total volume 880 ml). Cells were incubated overnight at 36°C with shaking (120 rpm). Cells were counted the next day and diluted to 3 x 10⁶ cells / ml in 232.5 ml of each 1-L flask. 6 Cells / ml. Transfect cells transiently as follows: Dilute 1000 μg plasmid DNA to a final volume of 35 ml with OptiMEM™ and mix gently. Dilute 2.5 ml of transporter 5 transfection reagent to a final volume of 35 ml with OptiMEM™ and mix gently. Add the diluted transporter 5 to the diluted DNA, mix, incubate at room temperature for 10 minutes, then add 17.5 ml dropwise to each 1 L flask while gently vortexing. Return the cells to the incubator and agitate for 4 days. One day after transfection, adjust the temperature to 33°C to increase protein yield.

[0258] Immobilized metal affinity chromatography: Add 4 mL Ni per 1 liter of cell supernatant 2+IMAC resin (Indigo, CubeBiotech catalog number 75103) was equilibrated to IMAC wash buffer (20 mM Tris pH 8.0, 300 mM NaCl, 30 mM imidazole). Tris pH 8.0 was added at 50 mM / L and NaCl was added to a final volume of 300 mM / L. The cell supernatant was incubated overnight at 4°C with stirring. After overnight incubation, the cell supernatant was transferred to a gravity column and the flow fraction was collected. The resin was then washed with 40 mL of IMAC wash buffer and the flow fraction was collected. The column was sealed and 8 mL of IMAC elution buffer (20 mM Tris pH 8.0, 300 mM NaCl, 500 mM imidazole) was added to each column and incubated for 10 minutes. The column was not stopped and the elution fraction was collected. The elution incubation was repeated twice. An SDS-PAGE gel was prepared to confirm that the protein of interest was captured in the elution fraction.

[0259] Differential scanning fluorescence: Nano-DSF thermal homogenization was used to estimate the T onset and melting temperatures (Tm) of antigen samples using SYPRO orange protein gel staining agent (Invitrogen) on UNcle Nano-DSF (UNchained Laboratories). The antigen sample was normalized to approximately 1 mg / mL (or 0.3–0.45 mg / mL for low-expression constructs) by adding the antigen sample to PCR tubes and then adding buffer (20 mM Tris pH 7.4, 250 mM NaCl, 4% sucrose) to a final volume of 31.5 μL. SYPRO was diluted from 5000X to a 200X working stock solution by adding 4 μL of SYPRO to 96 μL of buffer. Then, 3.5 μL of the 200X stock solution was added to each PCR tube to bring SYPRO to 20X. Triplets of the SYPRO-containing antigen sample dilution were applied to quartz capillary cassettes (UNi, UNchained Laboratories) and placed in UNcle. Data were collected using a temperature homogenization method from 15°C to 95°C (samples were held at 15°C for 300 seconds prior to data collection), with data collected in 1°C increments. Improved T-initiation and T-m were observed for all constructs compared to RSV / A.03 and RSV / B.002.

[0260] Accelerated Storage: After incubating antigen samples at 4°C or 40°C for 7 days, the binding of RSV F-specific antibodies to the trimeric antigen I53-50AΔcys fusion protein was evaluated. Antibody concentrations were normalized to 10 μg / mL in BLI assay buffer (PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4) by an adequate volume to load 80 μL into each well of a black 384-well microplate (Thermo Scientific, 460518). Briefly, on an Octet rh16 instrument, the protein G biosensor (Sartorius, 18-5082) was immersed in the assay buffer for 60 seconds to obtain a baseline. Next, the biosensor was immersed in each antibody for 60 seconds to immobilize any present but unsaturated antibodies, followed by an additional baseline step. The immobilized antibody was associated with 80 μL of purified RSV antigen (concentration normalized to 10 μg / mL) and incubated at 4 °C and 40 °C for 7 days for 120 seconds each. The biosensor was then immersed back into the assay buffer for 120 seconds to observe any possible dissociation. The new design exhibited higher AM14 binding and lower 4D7 binding than the controls (RSV / A.03 and RSV / B.002), indicating fewer post-fusion features and a denser trimer. After 7 days at 40 °C, decreased D25 and AM14 binding and increased 4D7 binding were observed for RSV / A.03 and RSV / B.002, while for the other constructs tested, binding of all antibodies was unaffected up to 7 days at 40 °C.

[0261] Assembly: The molar concentration of RSV / B or RSV / A trimers fused to I53-50AΔcys and I53-50B (second component, using the sequence of I53-50B.4PosT1, SEQ ID NO:46) was determined using UV-Vis spectroscopy. Absorbance values ​​at 280 nm were collected and divided by the calculated molar extinction coefficient (ExPASy). An in vitro assembly reaction to generate nanostructures with RSV / B antigen was performed, with the following components added: RSV F trimers fused to I53-50AΔcys were added to a PCR tube with a 1.5× molar excess of I53-50B; assembly buffer (20 mM Tris pH 7.4, 250 mM NaCl, 4% sucrose) was added to the sample in the PCR tube; and finally, I53-50B was added to the reaction to achieve a final assembly concentration of 7.5 μM and a final volume of 47.5 μL. Prior to subsequent measurements using dynamic light scattering (DLS), the reactants were incubated at ambient temperature with gentle shaking for approximately 30 minutes. Under the tested conditions, all constructs demonstrated assembly capability. Prior to nsEM analysis or immunogenicity studies, the assembled nanostructures were further purified by size exclusion chromatography on a Superose 6Increase 10 / 300 GL column to 20 mM Tris pH 7.4, 250 mM NaCl, and 4% sucrose.

[0262] Dynamic light scattering (DLS): The hydrodynamic diameter (Dh) and polydispersity (Pd%) of RSV / B nanostructure assemblies on UNcle Nano-DSF (UNchained Laboratories) were measured using dynamic light scattering (DLS). The setup included an increase in viscosity due to 4% sucrose in the buffer, which was interpreted as Dh measurements by the UNcle client software. Triplets of RSV / B nanostructure assemblies were applied to quartz capillary boxes (UNi, UNchained Laboratories), and measurements were performed using autoattenuated laser light, with 10 acquisitions per sample for 5 seconds each. Data were collected at 22°C, and all tested constructs produced monodisperse nanostructures of the expected size.

[0263] Electron Microscopy: For negative staining electron microscopy (nsEM), RSV F protein nanostructures were diluted to 75 μg / mL with 20 mM Tris pH 8.0, 150 mM NaCl, and 5% glycerol before and after freezing. 3 μL of sample was applied to the carbon side of two glow discharge (Pelco EasiGLOW) thick carbon-copper 400-mesh grids (EMS, CF400-Cu-TH). The samples were incubated on the grids for approximately 1 minute and then blotted dry with Grade 1 filter paper (Whatman). Immediately, 3 μL of 0.75% UF staining agent was applied to the carbon side of the grids and incubated for approximately 1 minute. The staining agent was blotted dry with filter paper, and this process was repeated twice. The grids were allowed to air dry for 5 minutes and then imaged at 57K magnification (Gatan camera) on a Talos L120C electron microscope. The micrographs show the correct self-assembly of the monodisperse nanostructures.

[0264] Immunogenicity studies: Two immunogenicity studies were conducted in 6–8 week old female BALB / c mice to evaluate neutralizing antibody responses induced by RSV / A and RSV / B designs. To evaluate the design of RSV / A.03, RSV / A.013, and RSV / A.023 nanostructures based on RSV / A, mice were immunized with 0.01 μg, 1 μg, or 5 μg of the nanostructure protein. The 0.01 μg dose was adjuvanted with the oil-in-water emulsion AddaVax™, while the 1 μg and 5 μg doses were not adjuvanted. Mice were immunized on days 0 and 21, and then sacrificed on day 35. Serum collected on day 35 was used for neutralization assays using the RSV / A Tracy strain. Similarly, the nanostructures of RSV / B designs RSV / B.002, RSV / B.093, RSV / B.195, RSV / B.160, and RSV / B.171 were evaluated. Mice were immunized on days 0 and 21 with samples of nanostructures supplemented with AddaVax™ adjuvant at doses of 0.02 μg or 0.1 μg. Serum samples collected during terminal blood collection on day 35 were used for neutralization assays with RSV / B strain 18537. RSV / A and RSV / B neutralization assays were performed in Hep-2 cells. Serial dilutions of serum samples were prepared in 96-well plates. Equal volumes of virus were added to each dilution and incubated for 1.5 h, followed by the addition of Hep-2 cells. The plates were incubated for 6–8 days, then fixed and stained with 10% neutral formalin and 0.01% crystal violet. The neutralizing antibody titer was defined as the final dilution at which the viral cytopathic effect was reduced by 50%. One-way ANOVA was used to determine statistically significant differences between groups immunized with different designs at the same dose.

[0265] Cryo-electron microscopy: The IMAC-purified trimer RSV / A.023 sample was further purified on a Superdex 200 Increase10 / 300 GL column to 20 mM Tris pH 7.4, 250 mM NaCl, and further concentrated to 0.88 mg / mL, followed by mesh preparation. The concentrated sample was then cryo-filtered using a Quantifoil R 1.2 / 1.3 AU 300 porous mesh. Data collection was performed using a Glacios 200keV microscope equipped with a Falcon IV detector (0.91 Å / pixel). The C3 symmetry model of RSVA023 was reconstructed from a PDB 4MMU using COOT. The final atomic structure was refined in Phenix and validated using MolProbity and half-graph cross-validation. Structural analysis was performed using COOT, Chimera, and PyMol.

[0266] Example 4. Method for generating C-terminal diffusion The relaxed structures used as input to the Rosetta Remodel were also used as input to RFdiffusion, 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 the Rosetta Remodel were also preserved for diffusion. Unlike the Rosetta Remodel, all structural data for subsequent residues were ignored. This is a limitation of RFdiffusion, not a scientific constraint on the design problem. Weights within and between protomers were set to 1, with secondary guide decay and guide 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 remodeled 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 remodeled domain. The top 2% based on MPNN sample scores were selected for computational characterization.

[0267] abbreviation

[0268] By incorporating references The full disclosure of each of the patent documents or scientific documents mentioned in this article is incorporated herein by reference for all purposes.

[0269] equivalent The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Therefore, the foregoing embodiments should be considered illustrative rather than limiting of the invention as described herein. Consequently, the scope of the invention is indicated by the appended claims rather than the foregoing description, and all variations within the equivalent meaning and scope of the claims are intended to be included therein.

Claims

1. A recombinant polypeptide comprising 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 one or more 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).

2. The polypeptide of claim 1, 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 one or more amino acid substitutions being selected such that the segment forms a stable α-helical homotrimer.

3. The polypeptide of claim 2, wherein the C-terminal helical region comprises about 10 to about 30 residues.

4. The polypeptide of claim 2, 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.

5. The polypeptide of claim 2, wherein the segment comprises: (a) 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; (b) The amino acid at position I506 of SEQ ID NO: 1 is substituted relative to I, wherein I is substituted by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y or A, I, L, V; (c) 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; (d) 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; (e) The amino acid at position S509 of SEQ ID NO: 1 is substituted relative to S, wherein S is substituted by A, I, L, M, V, F, W, Y, G, T, preferably A, I, L, M, V; (f) The amino acid substitution at position D510 relative to SEQ ID NO: 1, wherein D is replaced by any amino acid, preferably D, E, K, N, Q, R, S, T, Y; (g) Amino acid substitution at position E511 relative to SEQ ID NO: 1, wherein E is replaced by any amino acid; (h) The amino acid at position L512 of SEQ ID NO: 1 is substituted with D, E, K, N, Q, R, S, T, Y, preferably A, I, L, M, V, F, W, Y, G, T; (i) The amino acid at position L513 of SEQ ID NO: 1 is substituted relative to L, wherein L is substituted 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; (j) The amino acid at position H514 of SEQ ID NO: 1 is substituted relative to H, wherein H is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; (k) The amino acid at position N515 relative to SEQ ID NO: 1 is substituted, wherein N is replaced by any amino acid other than P, preferably A, I, L, M, V, F, W, Y, G; (l) 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; (m) The amino acid substituted at position N517 relative to SEQ ID NO: 1, wherein N is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; (n) 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; (o) The amino acid at position G519 of SEQ ID NO: 1 is substituted relative to G, wherein G is replaced by any amino acid other than P, preferably D, E, K, N, Q, R, S, T, Y; and / or Any combination of (p), (a), and (o).

6. The polypeptide of claim 2, wherein the segment comprises: (a) The amino acid substitution at position L503 relative to SEQ ID NO: 1, wherein F is replaced by Q, V, K, R, N, L; (b) The amino acid at position A504 relative to SEQ ID NO: 1, wherein I is replaced by any amino acid other than P, preferably S, T, L, A, Q, K, E, Y; (c) The amino acid substitution at position F505 relative to SEQ ID NO: 1, wherein F is replaced by I, V, N, T, L; (d) 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; (e) The amino acid at position R507 of SEQ ID NO: 1 is substituted relative to R, wherein R is substituted by any amino acid other than P, preferably A, N, K, E, D, or Q; (f) The amino acid substitution at position K508 relative to SEQ ID NO: 1, wherein R is replaced by T, M, V, or R; (g) 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; (h) The amino acid substitution at position D510 relative to SEQ ID NO: 1, wherein D is replaced by S, K, N, D, or E; (i) 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; (j) The amino acid substitution at position L512 relative to SEQ ID NO: 1, wherein L is replaced by V, N, T, or L; (k) 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; (l) 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; (m) The amino acid substitution at position N515 relative to SEQ ID NO: 1, wherein N is replaced by I, E, L, T, or Q; (n) 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; o) The amino acid substitution at position N517 relative to SEQ ID NO: 1, wherein N is replaced by A, S, K, E, or R; (p) The amino acid substitution at position T518 relative to SEQ ID NO: 1, wherein T is replaced by K, S, Q, R, D, E; (q) The amino acid substitution at position G519 relative to SEQ ID NO: 1, wherein G is replaced by V, L, or I; (r) The amino acid substitution at position I520 relative to SEQ ID NO: 1, wherein G is replaced by K, Q, E, N, T; (s) 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; (t) The amino acid substitutions at position E522 relative to SEQ ID NO: 1, wherein G is replaced by L, R, I, V; (u) The amino acid substitution at position A523 relative to SEQ ID NO: 1, wherein G is replaced by E, V, L, K, R, I; (v) Amino acid substitutions relative to SEQ ID NO: 1 at position P524, wherein G is replaced by A, K, T, E, or R; (w) The amino acid substitutions relative to SEQ ID NO: 1 at position R525, wherein G is replaced by H, R, S, L, N, E, D; (x) The amino acid substitution at position D526 relative to SEQ ID NO: 1, wherein G is replaced by I, L, V, R; (y) The amino acid substitution at position G527 relative to SEQ ID NO: 1, wherein G is replaced by E, K, Q, or D; (z) The amino acid substitution at position Q528 relative to SEQ ID NO: 1, wherein G is replaced by D, K, S, R, or A; (aa) The amino acid substitution at position A529 relative to SEQ ID NO: 1, wherein G is replaced by T or L; (ab) Amino acid substitutions at position Y530 relative to SEQ ID NO: 1, wherein G is replaced by L, E, and T; (ac) Amino acid substitutions at position V531 relative to SEQ ID NO: 1, wherein G is replaced by A, R, or K; (ad) Amino acid substitutions relative to SEQ ID NO: 1 at position R532, wherein G is substituted by V or A; and / or Any combination of (ae) (a)-(ad).

7. The polypeptide of claim 2, wherein the segment comprises a polypeptide sequence listed in Table 2A or Table 2B, or a polypeptide sequence having 1 to 5 amino acid substitutions.

8. The polypeptide of claim 2, 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.

9. The polypeptide of claim 2, wherein the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10), or a polypeptide sequence having 1 to 5 amino acid substitutions.

10. The polypeptide of claim 2, wherein the segment comprises the polypeptide sequence NQSREIIRAINIVRKIASEK (SEQ ID NO: 10).

11. The polypeptide of any one of claims 1-10, 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.

12. The polypeptide of any one of claims 1-10, 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.

13. The polypeptide of claim 11, wherein the extracellular domain comprises amino acid substitutions of D489A, T400D, E487R, and K498A.

14. The polypeptide of claim 11, wherein the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and D486A.

15. The polypeptide of claim 11, wherein the extracellular domain comprises amino acid substitutions of F488W, D489A, T400D, E487R, K498A, and T249P.

16. The polypeptide of any one of claims 1-14, wherein the polypeptide comprises a heteropolymerized domain at the C-terminus of the extracellular domain.

17. The polypeptide of claim 16, wherein the polymerizing domain is a trimerizing domain.

18. The polypeptide of claim 16 or claim 17, wherein the polymerized domain comprises 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% of the same polypeptide sequence as I53-50A (SEQ ID NO: 19) or I53-50A ΔCys (SEQ ID NO: 64).

19. The polypeptide of any one of claims 1-18, wherein the extracellular domain comprises amino acid substitutions of S155C, S290C, S190F, and V207L.

20. The polypeptide of any one of claims 1-19, wherein the extracellular domain comprises a polypeptide sequence that is 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% identical to the following: QNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAI ASGIAVCKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTFRVLDLKNYINNQLLPMLNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKI MTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSXXXXXXXXXXXXXXXXX (SEQ ID NO: 6), optionally lacking the p27 peptide shown in bold.

21. The polypeptide of any one of claims 1-20, wherein the extracellular domain comprises a polypeptide sequence that is 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% identical to the following: QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAI ASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKK LMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSXXXXXXXXXXXXXXXXX (SEQ ID NO: 7), optionally lacking the p27 peptide shown in bold.

22. The polypeptide of any one of claims 1-20, wherein the extracellular domain comprises a polypeptide sequence that is 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% identical to the following: QNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAI ASGIAVCKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTFRVLDLKNYINNQLLPMLNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKI MTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSREIIRAINIVRKIASEK (SEQ ID NO: 8), optionally lacking the p27 peptide shown in bold.

23. The polypeptide of any one of claims 1-20, wherein the extracellular domain comprises a polypeptide sequence that is 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% identical to the following: QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAI ASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKK LMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSREIIRAINIVRKIASEK (SEQ ID NO: 9), optionally lacking the p27 peptide shown in bold.

24. The polypeptide of any one of claims 1-20, wherein the polypeptide comprises 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% identical to any one or more of SEQ ID NO: 1-9.

25. A trimeric protein complex comprising a polypeptide according to any one of claims 1 to 24.

26. The trimeric protein complex of claim 25, wherein 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 lacking modification (a)-(h).

27. The trimeric protein complex of claim 25, wherein the stability, as determined by storage at about 40°C, is increased compared to the trimeric protein complex lacking modification (a)-(h).

28. The trimeric protein complex of claim 26, 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).

29. A protein nanostructure comprising a trimer component, said trimer component comprising a polypeptide according to any one of claims 1 to 24.

30. The nanostructure of claim 29, wherein the nanostructure is a two-component nanostructure comprising a first trimer component and a second pentamer component.

31. The nanostructure of claim 30, wherein the first trimer component comprises engineered extracellular domains of a respiratory syncytial virus (RSV) fusion (F) polypeptide and an I53-50A polypeptide.

32. The nanostructure of any one of claims 29 to 31, wherein the first trimer 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.

33. The nanostructure according to any one of claims 29 to 32, 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 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% identical polypeptide sequences to 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 that is 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% identical to that of SEQ ID NO: 20 or 71.

34. The nanostructure according to any one of claims 29 to 32, 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 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% identical polypeptide sequences to 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 that is 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% identical to that of SEQ ID NO: 20 or 71.

35. The nanostructure according to any one of claims 29 to 32, 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 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% identical polypeptide sequences to 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 that is 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% identical to that of SEQ ID NO: 20 or 71.

36. The nanostructure of any one of claims 29 to 32, 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 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% identical polypeptide sequences to 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 that is 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% identical to that of SEQ ID NO: 20 or 71.

37. The nanostructure of any one of claims 29 to 36, wherein the trimer component comprises 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% the same polypeptide sequence as any one of the sequences listed in Table 14 or without the underlined and / or bold / italic polypeptide sequence.

38. The nanostructure of any one of claims 29 to 37, wherein the pentamer component comprises 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% of the same polypeptide sequence as any one or more of SEQ ID NO: 20, 44, 45, 52, 71, 73, 74.

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

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

41. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 24, a protein complex according to any one of claims 25 to 28, a nanostructure according to any one of claims 29 to 38, a polynucleotide according to claim 39, or a delivery medium according to claim 40.

42. A vaccine comprising a polypeptide according to any one of claims 1 to 24, a protein complex according to any one of claims 25 to 28, a nanostructure according to any one of claims 29 to 38, a polynucleotide according to claim 39, or a delivery medium according to claim 40.

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

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

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

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

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

48. A lipid nanoparticle (LNP) comprising the polynucleotide of claim 39.

49. A host cell comprising the polynucleotide of claim 39.

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

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