Prefusion stabilized HMPV F protein

Abstract

Provided herein are engineered hMPV F proteins. In some aspects, the engineered F proteins exhibit enhanced conformational stability and / or antigenicity. Also provided are methods for using the engineered F proteins as diagnostics, in screening platforms, and / or in vaccine compositions.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 089,978, filed October 9, 2020, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web, and is hereby incorporated by reference in its entirety. The ASCII copy, created on October 7, 2021, is named UTFBP1250WO_ST25.txt and is 50.6 bytes in size.

[0003] 1.Technical Field The present disclosure relates generally to the fields of medicine, virology, immunology, and protein engineering. More specifically, the present disclosure relates to an engineered human metapneumovirus (hMPV) F protein and its use in drug design and vaccine formulation. [Background technology]

[0004] 2. Description of Related Technology Human metapneumovirus (hMPV) is a respiratory virus of the family Pneumoviridae that has been circulating in humans for at least half a century prior to its discovery in 2001 (van den Hoogen et al., 2001). Infection occurs almost ubiquitously by age 5 years, and the burden of reinfection continues throughout life (van den Hoogen et al., 2001). However, infants (6-12 months), the elderly, and immunocompromised individuals are at increased risk of hospitalization due to more severe disease, including pneumonia and bronchitis (Deffrasnes et al., 2007). Despite the disease burden presented by hMPV, there are no approved vaccines or therapeutics for prevention or treatment. As a member of the family Pneumoviridae (recently promoted from a subfamily within Paramyxoviridae), hMPV is an enveloped, negative-stranded RNA virus. Viruses within this family encode three membrane proteins expressed on the surface. In the case of hMPV, these are the small hydrophobic (SH), attachment (G), and fusion (F) proteins (Shafagati & Williams, 2018).

[0005] As a class I viral fusion glycoprotein, hMPV F is initially translated as a single polypeptide precursor (F0). Proteolytic cleavage converts F0 into disulfide-linked F2 and F1 subunits (Figure 1A). Three F2 / F1 heterodimers then associate into a metastable prefusion trimer that constitutes the active form of the protein. In cell culture, this proteolytic activation can be accomplished by the addition of trypsin, which cleaves the protein at a monobasic cleavage site (van den Hoogen et al., 2001; Skiadopoulos et al., 2006; Schickli et al., 2005). During natural infection, hMPV F0 is cleaved by trypsin-like extracellular serine proteases such as TMPRSS2, but the extent to which this occurs in producer cells versus target cells is unclear (Shirogane et al., 2008). The N-terminus of the mature F1 subunit contains a hydrophobic sequence, termed the fusion peptide, that is located within the internal cavity of the prefusion F trimer (Battles et al., 2017). For other class I fusion proteins, such as human respiratory syncytial virus F (RSV F) and influenza hemagglutinin (HA), the trimer has been shown to be unstable and capable of transiently spreading open or "breathing" (Bangaru et al., 2019; Watanabe et al., 2019; Gilman et al., 2019). Recently, human antibodies targeting the trimer interface of hMPV F have been described, suggesting that prefusion hMPV F undergoes this transient opening in vivo (Huang et al., 2020). To facilitate membrane fusion, the metastable prefusion F protein undergoes substantial conformational changes to liberate the fusion peptide and extend into the host cell membrane. This unstable pre-hairpin intermediate refolds upon itself to form a highly stable six-helix bundle composed of trimers of N- and C-terminal heptad repeats (HRA and HRB, respectively) in the so-called postfusion conformation (Mas et al., 2016).Considering its essential role in viral entry, vaccine candidates against hMPV generally contain the F protein. In other words, a potential vaccination strategy against hMPV is by targeting its fusion (F) glycoprotein, which is essential for viral infection. However, there remains a need for a stabilized F protein that can be used to identify drug candidates and stimulate an effective immune response against the F protein. Summary of the Invention

[0006] Thus, provided herein are thermostable hMPV F prefusion conformation variants. Introduction of a pair of cystine mutations to introduce disulfide bonds improved protein expression and thermostability. Individual hydrophobic, electrostatic, and charge-reducing mutations are also beneficial. In addition, combinations of multiple beneficial mutations further improved desired protein properties.

[0007] In one embodiment, provided herein is an engineered protein comprising a metapneumovirus (MPV) F protein ectodomain having at least 90% identity to (i) amino acids 19-489 of any of SEQ ID NOs: 1, 2, and 4-7, or (ii) amino acids 19-484 of SEQ ID NO: 3, the engineered protein comprising at least one mutation relative to any one of SEQ ID NOs: 1-7, the at least one mutation being K166, N342, A / D185, K188, T49, V262, H435, E26, G439, ​​N46, L158, A161, L50, V162, E51, R163, V104, N457, L110, N322, A113, D336, A116, A338, A140, A147. , S291, S443, S293, S444, S355, V442, T365, V463, S22, G53, V169, E305, L302, V47, A159, T127, N153, G121, I / F258, G106, A107, T160, I128, A190, V118, Q426, L165, V191, S149, I137, V / I122, S192, T317 , L105, L134, A117, S347, G261, I268, S470, L473, S265, L460, F48, Q455, V231, A374, I217, S376, G366, S194, L219, A344, A86, T114, V148, D461, L66, L73, N145, Q195, E453, and / or H368. In some aspects, the engineered protein has at least 95% identity to (i) amino acids 19-489 of any of SEQ ID NOs: 1, 2, and 4-7, or (ii) amino acids 19-484 of SEQ ID NO: 3. SEQ ID NO: 1 corresponds to the BV-115 variant sequence. SEQ ID NO: 2 corresponds to the JSM-1147 variant sequence. SEQ ID NO: 3 corresponds to the DW-1 variant sequence. SEQ ID NO: 4 corresponds to the F protein of hMPV A1 NL / 1 / 00 strain (GenBank: AAK62968.2). SEQ ID NO: 5 corresponds to the F protein of hMPV A2 NL / 00 / 17 strain (GenBank: ACJ70115.1).SEQ ID NO: 6 corresponds to the F protein of the hMPV B1 NL / 1 / 99 strain (GenBank: AY525843.1). SEQ ID NO: 7 corresponds to the F protein of the hMPV B2 TN / 99 / 419 strain (GenBank: AAS92882.1).

[0008] In some aspects, the engineered protein comprises a proline substitution corresponding to A / D185P. In some aspects, the engineered protein comprises a substitution corresponding to RQSR (residues 99-102 of any one of SEQ ID NOS:4-7; SEQ ID NO:9) to RRRR (residues 99-102 of any one of SEQ ID NOS:1-3; SEQ ID NO:10). In some aspects, the engineered protein comprises a proline substitution corresponding to A / D185P and a substitution corresponding to RQSR (residues 99-102 of any one of SEQ ID NOS:4-7; SEQ ID NO:9) to RRRR (residues 99-102 of any one of SEQ ID NOS:1-3; SEQ ID NO:10). In some aspects, the engineered protein comprises a substitution corresponding to GGGGSGGGGSR (SEQ ID NO:8) to residues 87-104 of any one of SEQ ID NOS:1-7.

[0009] In some aspects, the engineered proteins include E26C and G439C, N46C and L158C, T49C and A161C, L50C and V162C, E51C and R163C, E51C and K166C, V104C and N457C, L110C and N322C, A113C and D336C, A116C and A338C, A140C and A147C, S291C and S443C, S293C and S443C, S293C and S444C , comprising engineered disulfide bonds including pairs of cysteine ​​substitutions corresponding to S355C and V442C, T365C and V463C, S22C and H435C, G53C and K166C, G53C and V169C, E305C and N457C, S291C and L302C, V47C and A159C, T127C and N153C, G121C and I / F258C, F48C and T160C, and / or T365C and Q455C. In some aspects, the engineered protein comprises an engineered disulfide bond comprising a pair of cysteine ​​substitutions corresponding to A116C and A338C, T365C and V463C, T127C and N153C, T365C and Q455C, V104C and N457C, L110C and N322C, or A140C and A147C. In some aspects, the engineered protein comprises an engineered disulfide bond comprising a pair of cysteine ​​substitutions corresponding to A140C and A147C. In some aspects, the engineered protein comprises an engineered disulfide bond comprising a pair of cysteine ​​substitutions corresponding to V104C and N457C. In some aspects, the engineered protein comprises an engineered disulfide bond comprising a pair of cysteine ​​substitutions corresponding to L110C and N322C. In some aspects, the engineered protein comprises an engineered disulfide bond comprising a pair of cysteine ​​substitutions corresponding to T365C and V463C.

[0010] In some aspects, the engineered protein comprises substitutions at positions corresponding to L219 and / or V231. In some aspects, the engineered protein comprises substitutions corresponding to L219K and / or V231I. In some aspects, the engineered protein comprises a pair of cysteine ​​substitutions corresponding to T127C and N153C. In some aspects, the engineered protein comprises a pair of cysteine ​​substitutions corresponding to L110C and N322C. In some aspects, the engineered protein comprises a pair of cysteine ​​substitutions corresponding to A140C and A147C. In some aspects, the engineered protein comprises a substitution corresponding to G366S.

[0011] In some aspects, the engineered protein comprises substitutions at positions corresponding to Q426, T49, L187, L473, and / or S347. In some aspects, the engineered protein comprises substitutions corresponding to Q426W, T49E, L187F, L473F, and / or S347Q.

[0012] In some aspects, the engineered protein comprises an engineered disulfide bond comprising a pair of cysteine ​​substitutions corresponding to A116C and A338C. In some aspects, the engineered protein comprises an engineered disulfide bond comprising a pair of cysteine ​​substitutions corresponding to T365C and V463C. In some aspects, the engineered protein comprises an engineered disulfide bond comprising a pair of cysteine ​​substitutions corresponding to T127C and N153C. In some aspects, the engineered protein comprises an engineered disulfide bond comprising a pair of cysteine ​​substitutions corresponding to T365C and Q455C. In some aspects, the engineered protein comprises at least one additional engineered disulfide bond.

[0013] In some aspects, the engineered protein comprises a cavity filling substitution at a position corresponding to G106, A107, T160, L158, I128, A190, V118, Q426, L165, V191, T160, S149, I137, S149, V169, N46, T49, V / I122, S192, T317, V162, L105, L134, A117, S347, V47, G261, I268, S470, V231, A374, I217, and / or S355. In some aspects, the engineered protein comprises a cavity filling substitution at a position corresponding to L105, V118, I137, S149, L158, L165, or Q426. In some embodiments, the engineered protein comprises a substitution corresponding to L105I or L105W. In some embodiments, the engineered protein comprises a substitution corresponding to L158W. In some embodiments, the engineered protein comprises a substitution corresponding to V118F or V118M. In some embodiments, the engineered protein comprises a substitution corresponding to Q426W. In some embodiments, the engineered protein comprises a substitution corresponding to L165F. In some embodiments, the engineered protein comprises a substitution corresponding to S149V or S149I. In some embodiments, the engineered protein comprises a substitution at a position corresponding to I137. In some embodiments, the engineered protein comprises a substitution corresponding to I137L.

[0014] In some embodiments, the engineered protein is selected from the group consisting of G106W, A107F, T160M, L158W, I128F, A190M, V118F, V118M, Q426W, L165F, V191I, T160V, S149V, I137L, S149I, V169I, N46V, T49I, V / I122L, S192L, and / or S355F.

[0015] In some aspects, the engineered protein comprises a proline substitution selected from the group consisting of A86P, A107P, A113P, T114P, V148P, S443P, D461P, L130P, L141P, K142P, E146P, L151P, N153P, V162P, A / D185P, D186P, L187P, K188P, N342P, and A344P.

[0016] In some aspects, the engineered protein comprises substitutions at positions corresponding to S376, G366, and / or S194. In some aspects, the engineered protein comprises substitutions corresponding to S376T, G366S, and / or S194Q.

[0017] In some aspects, the engineered protein comprises a substitution at a position corresponding to K166. In some aspects, the engineered protein comprises a substitution corresponding to K166E.

[0018] In some embodiments, the engineered protein comprises a substitution at a position corresponding to H435 that modulates pH sensitivity, hi some embodiments, the engineered protein comprises a substitution corresponding to H435E, H435D, or H435N.

[0019] In some aspects, the engineered protein comprises electrostatic interaction substitutions at positions corresponding to L66, L73, N145, Q195, E453, L66, K188, H368, D461, T49, and / or V262. In some aspects, the engineered protein comprises substitutions corresponding to L66N, L73E, N145E, Q195K, E453Q, L66D, K188R, H368R, D461E, T49E, and / or V262D.

[0020] In some aspects, the engineered protein includes substitutions corresponding to L110C, T127C, A140C, A147C, N153C, L219K, V231I, N322C, T365C, E453Q, and / or V463C.

[0021] In some aspects, the engineered protein comprises substitutions corresponding to T127C, N153C, A185P, T365C, V463C, L219K, and V231I. In some aspects, the engineered protein comprises substitutions corresponding to T127C, N153C, A185P, T365C, V463C, L219K, V231I, and RQSR (residues 99-102 of any one of SEQ ID NOs:1-7) to RRRR (SEQ ID NO:10). In some aspects, the engineered protein comprises substitutions corresponding to T127C, N153C, T365C, V463C, L219K, and V231I. In some aspects, the engineered protein comprises a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:14 or 16.

[0022] In some aspects, the engineered protein includes substitutions corresponding to L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, N368H, E453Q, and V463C. In some aspects, the engineered protein includes substitutions corresponding to L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, N368H, E453Q, V463C, and RQSR (residues 99-102 of any one of SEQ ID NOs:1-7) through RRRR (SEQ ID NO:10). In some aspects, the engineered protein comprises substitutions corresponding to L110C, T127C, A140C, A147C, N153C, L219K, V231I, N322C, T365C, N368H, E453Q, and V463C. In some aspects, the engineered protein comprises a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15 or 17.

[0023] In some embodiments, the engineered protein comprises a substitution from residues 87-104 of any one of SEQ ID NOs:1-7 corresponding to GGGGSGGGGSR (SEQ ID NO:8).

[0024] In some aspects, the engineered protein may include any set of substitutions disclosed in Table 1. In some aspects, the engineered protein may include any set of substitutions disclosed in Table 1 in combination with a proline substitution corresponding to A / D185P. In some aspects, the engineered protein may include any set of substitutions disclosed in Table 1 in combination with a substitution corresponding to RQSR (residues 99-102 of any one of SEQ ID NOS:4-7; SEQ ID NOS:9) to RRRR (residues 99-102 of any one of SEQ ID NOS:1-3; SEQ ID NOS:10). In some aspects, the engineered protein may include any set of substitutions disclosed in Table 1 in combination with a proline substitution corresponding to A / D185P and a substitution corresponding to RQSR (residues 99-102 of any one of SEQ ID NOS:4-7; SEQ ID NOS:9) to RRRR (residues 99-102 of any one of SEQ ID NOS:1-3; SEQ ID NOS:10).

[0025] In some aspects, the engineered protein comprises a combination of at least one engineered disulfide bond, at least one cavity filling substitution, and at least one proline substitution.

[0026] In some embodiments, the engineered protein has at least 95% identity to the amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the engineered hMPV F protein ectodomain has 95% identity to SEQ ID NO:3.

[0027] In some embodiments, the engineered protein is fused or conjugated to a trimerization domain. In some embodiments, the engineered protein is fused or conjugated to a trimerization domain. In some embodiments, the trimerization domain comprises the trimerization domain of T4 fibritin.

[0028] In some embodiments, the engineered protein is fused or conjugated to a transmembrane domain. In some embodiments, the engineered protein is fused to a transmembrane domain. In some embodiments, the transmembrane domain comprises the transmembrane domain of a metapneumovirus (MPV) F protein.

[0029] In some embodiments, the engineered protein comprises an N-terminal signal sequence. In some embodiments, the N-terminal signal sequence is MSWKVMIIISLLITPQHG (residues 1-18 of SEQ ID NO:6 or 7; SEQ ID NO:11). In some embodiments, the N-terminal signal sequence is MSWKVVIIFSLLITPQHG (residues 1-18 of any one of SEQ ID NOs:1-5).

[0030] In one embodiment, provided herein is an engineered metapneumovirus (MPV) F protein trimer comprising at least one subunit according to any one of the embodiments of the engineered protein. In some aspects, the trimer is stabilized in a pre-fusion conformation compared to a trimer of wild-type metapneumovirus (MPV) F subunits. In some aspects, the trimer comprises at least one engineered disulfide bond between the subunits. In some aspects, the at least one engineered disulfide bond between the subunits is formed by substitutions corresponding to S316C and D421C.

[0031] In one embodiment, the present specification provides a pharma- ceutically acceptable carrier; and (i) an engineered protein according to any one of the engineered protein embodiments; or (ii) an engineered trimer according to any one of the engineered trimer embodiments. In some aspects, the pharmaceutical composition further comprises an adjuvant.

[0032] In one embodiment, provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of the engineered protein according to any one of the embodiments of the engineered protein. In some aspects, the nucleic acid comprises a DNA expression vector. In some aspects, the nucleic acid comprises an mRNA.

[0033] In one embodiment, provided herein is a method for preventing a metapneumovirus (MPV) infection or a disease associated with a metapneumovirus infection in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition described in any one of the pharmaceutical composition embodiments or a nucleic acid molecule described in any one of the nucleic acid molecule embodiments.

[0034] In one embodiment, provided herein is a composition comprising an engineered protein according to any one of the present embodiments bound to an antibody.

[0035] [The present invention 1001] 1. An engineered protein comprising a metapneumovirus (MPV) F protein ectodomain, the engineered protein having at least 90% identity to (i) amino acids 19 to 489 of any of SEQ ID NOs: 1, 2, and 4 to 7, or (ii) amino acids 19 to 484 of SEQ ID NO: 3, wherein the engineered protein comprises at least one mutation with respect to any one of SEQ ID NOs: 1 to 7, the at least one mutation being selected from the group consisting of K166, N342, A / D185, K188, T49, V262, H435, E26, G439, ​​N46, L158, A161, L50, V162, E51, R163, V104, N457, L110, N322, A113, D336, A116, A338, A140, A147, S291, S443, S293. , S444, S355, V442, T365, V463, S22, G53, V169, E305, L302, V47, A159, T127, N153, G121, I / F258, G1 06, A107, T160, I128, A190, V118, Q426, L165, V191, S149, I137, V / I122, S192, T317, L105, L134, A11 7, S347, G261, I268, S470, L473, S265, L460, F48, Q455, V231, A374, I217, S376, G366, S194, L219, A344, A86, T114, V148, D461, L66, L73, N145, Q195, E453, and / or H368. [The present invention 1002] 1001. An engineered protein of the invention comprising a proline substitution corresponding to A / D185P. [The present invention 1003] 1001. An engineered protein of the present invention comprising a substitution corresponding to RQSR (residues 99-102 of any one of SEQ ID NOs:1-7) to RRRR (SEQ ID NO:10). [The present invention 1004] 1001. An engineered protein of the present invention comprising a proline substitution corresponding to A / D185P and a substitution corresponding to RQSR (residues 99-102 of any one of SEQ ID NOs: 1-7) to RRRR (SEQ ID NO: 10). [The present invention 1005] 1001. An engineered protein of the present invention comprising a substitution of residues 87-104 of any one of SEQ ID NOs:1-7 with GGGGSGGGGSR (SEQ ID NO:8). [The present invention 1006] E26C and G439C, N46C and L158C, T49C and A161C, L50C and V162C, E51C and R163C, E51C and K166C, V104C and N457C, L110C and N322C, A113C and D336C, A116C and A338C, A140C and A147C, S291C and S443C, S293C and S443C, S293C and S444C, S355C and Engineered disulfide bonds including pairs of cysteine ​​substitutions corresponding to V442C, T365C and V463C, S22C and H435C, G53C and K166C, G53C and V169C, E305C and N457C, S291C and L302C, V47C and A159C, T127C and N153C, G121C and I / F258C, F48C and T160C, and / or T365C and Q455C. The engineered protein of any of claims 1001 to 1005, comprising: [The present invention 1007] Engineered disulfide bonds containing pairs of cysteine ​​substitutions corresponding to A116C and A338C, T365C and V463C, T127C and N153C, T365C and Q455C, V104C and N457C, L110C and N322C, or A140C and A147C. The engineered protein of the present invention 1006, comprising: [The present invention 1008] An engineered disulfide bond containing a pair of cysteine ​​substitutions corresponding to A140C and A147C 1007. An engineered protein of the present invention comprising: [The present invention 1009] An engineered disulfide bond containing a pair of cysteine ​​substitutions corresponding to V104C and N457C 1007. An engineered protein of the present invention comprising: [The present invention 1010] An engineered disulfide bond containing a pair of cysteine ​​substitutions corresponding to L110C and N322C 1007. An engineered protein of the present invention comprising: [The present invention 1011] An engineered disulfide bond containing a pair of cysteine ​​substitutions corresponding to T365C and V463C 1007. An engineered protein of the present invention comprising: [The present invention 1012] The engineered protein of the invention 1011 further comprising a substitution at a position corresponding to L219 and / or V231. [The present invention 1013] The engineered protein of the invention 1011 further comprising substitutions corresponding to L219K and / or V231I. [The present invention 1014] The engineered protein of any of claims 1011 to 1013, further comprising a pair of cysteine ​​substitutions corresponding to T127C and N153C. [The present invention 1015] The engineered protein of any of claims 1011 to 1013, further comprising a pair of cysteine ​​substitutions corresponding to L110C and N322C. [The present invention 1016] The engineered protein of any of claims 1011 to 1015, further comprising a pair of cysteine ​​substitutions corresponding to A140C and A147C. [The present invention 1017] A further substitution corresponding to G366S, any of the engineered proteins of the present invention 1011 to 1016. [The present invention 1018] The engineered protein of the invention 1011 further comprising substitutions at positions corresponding to Q426, T49, L187, L473, and / or S347. [The present invention 1019] The engineered protein of the invention 1011 further comprising substitutions at positions corresponding to Q426, T49, L187, L473, and / or S347. [The present invention 1020] The engineered protein of the invention 1011, further comprising substitutions corresponding to Q426W, T49E, L187F, L473F, and / or S347Q. [The present invention 1021] An engineered disulfide bond containing a pair of cysteine ​​substitutions corresponding to A116C and A338C 1007. An engineered protein of the present invention comprising: [The present invention 1022] An engineered disulfide bond containing a pair of cysteine ​​substitutions corresponding to T365C and V463C 1007. An engineered protein of the present invention comprising: [The present invention 1023] An engineered disulfide bond containing a pair of cysteine ​​substitutions corresponding to T127C and N153C 1007. An engineered protein of the present invention comprising: [The present invention 1024] An engineered disulfide bond containing a pair of cysteine ​​substitutions corresponding to T365C and Q455C 1007. An engineered protein of the present invention comprising: [The present invention 1025] 1007. The engineered protein of the present invention, further comprising at least one additional engineered disulfide bond. [The present invention 1026] 1006. The engineered protein of any of claims 1001-1005, comprising a cavity-filling substitution at a position corresponding to G106, A107, T160, L158, I128, A190, V118, Q426, L165, V191, T160, S149, I137, S149, V169, N46, T49, V / I122, S192, T317, V162, L105, L134, A117, S347, V47, G261, I268, S470, V231, A374, I217, and / or S355. [The present invention 1027] 1026. An engineered protein of the invention comprising a cavity-filling substitution at a position corresponding to L105, V118, I137, S149, L158, L165, or Q426. [The present invention 1028] An engineered protein of the invention 1027 comprising a substitution corresponding to L105I or L105W. [The present invention 1029] An engineered protein of the invention 1027 comprising a substitution corresponding to L158W. [The present invention 1030] An engineered protein of the invention 1027 comprising a substitution corresponding to V118F or V118M. [The present invention 1031] An engineered protein of the invention 1027 which contains a substitution corresponding to Q426W. [The present invention 1032] An engineered protein of the invention 1027 comprising a substitution corresponding to L165F. [The present invention 1033] The engineered protein of the present invention 1027, comprising a substitution corresponding to S149V or S149I. [The present invention 1034] An engineered protein of the invention 1033 further comprising a substitution at the position corresponding to I137. [The present invention 1035] An engineered protein of the invention 1034 comprising a substitution corresponding to I137L. [The present invention 1036] G106W, A107F, T160M, L158W, I128F, A190M, V118F, V118M, Q426W, L165F, V191I, T16 0V, S149V, I137L, S149I, V169I, N46V, T49I, V / I122L, S192L, T317L, V162F, V162W, L 105I, L105F, L105W, L134I, A117M, S347M, S347K, S347Q, V47M, G261M, I268M, S470Y, V231I, A374V, I217V, and / or S355F. [The present invention 1037] 6. The engineered protein of any of claims 1001 to 1005, comprising a proline substitution selected from the group consisting of A86P, A107P, A113P, T114P, V148P, S443P, D461P, L130P, L141P, K142P, E146P, L151P, N153P, V162P, A / D185P, D186P, L187P, K188P, N342P, and A344P. [The present invention 1038] 6. The engineered protein of any of claims 1001 to 1005, comprising substitutions at positions corresponding to S376, G366, and / or S194. [The present invention 1039] An engineered protein of the invention 1038 comprising substitutions corresponding to S376T, G366S, and / or S194Q. [The present invention 1040] 6. The engineered protein of any of claims 1001 to 1005, comprising a substitution at the position corresponding to K166. [The present invention 1041] An engineered protein of the invention 1040 comprising a substitution corresponding to K166E. [The present invention 1042] 6. The engineered protein of any of claims 1001 to 1005, comprising a substitution at the position corresponding to H435 that modulates pH sensitivity. [The present invention 1043] An engineered protein of the invention 1042 comprising a substitution corresponding to H435E, H435D, or H435N. [The present invention 1044] 1006. The engineered protein of any of claims 1001 to 1005, comprising electrostatic interaction substitutions at positions corresponding to L66, L73, N145, Q195, E453, L66, K188, H368, D461, T49, and / or V262. [The present invention 1045] 1044. An engineered protein of the invention comprising substitutions corresponding to L66N, L73E, N145E, Q195K, E453Q, L66D, K188R, H368R, D461E, T49E, and / or V262D. [The present invention 1046] 6. The engineered protein of any of claims 1001 to 1005, comprising substitutions corresponding to L110C, T127C, A140C, A147C, N153C, L219K, V231I, N322C, T365C, E453Q, and / or V463C. [The present invention 1047] 6. The engineered protein of any of claims 1001 to 1005, comprising substitutions corresponding to T127C, N153C, A185P, T365C, V463C, L219K, and V231I. [The present invention 1048] 1005. The engineered protein of any of claims 1001 to 1005, comprising T127C, N153C, A185P, T365C, V463C, L219K, V231I, and a substitution corresponding to RQSR (residues 99 to 102 of any one of SEQ ID NOs: 1 to 7) to RRRR (SEQ ID NO: 10). [The present invention 1049] 6. The engineered protein of any of claims 1001 to 1005, comprising substitutions corresponding to T127C, N153C, T365C, V463C, L219K, and V231I. [The present invention 1050] 6. The engineered protein of any of claims 1001 to 1005, comprising substitutions corresponding to L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, N368H, E453Q, and V463C. [The present invention 1051] 1005. The engineered protein of any of claims 1001 to 1005, comprising L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, N368H, E453Q, V463C, and a substitution corresponding to RQSR (residues 99 to 102 of any one of SEQ ID NOs: 1 to 7) to RRRR (SEQ ID NO: 10). [The present invention 1052] 6. The engineered protein of any of claims 1001 to 1005, comprising substitutions corresponding to L110C, T127C, A140C, A147C, N153C, L219K, V231I, N322C, T365C, N368H, E453Q, and V463C. [The present invention 1053] The engineered protein of any of claims 1006 to 1052, comprising a substitution of residues 87 to 104 of any one of SEQ ID NOs: 1 to 7 corresponding to GGGGSGGGGSR (SEQ ID NO: 8). [The present invention 1054] The engineered protein of any of claims 1001 to 1053, comprising a combination of at least one engineered disulfide bond, at least one cavity-filling substitution, and at least one proline substitution. [The present invention 1055] The engineered protein of any of claims 1001 to 1054, comprising a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14 or 16. [The present invention 1056] The engineered protein of any one of claims 1001 to 1054, comprising a polypeptide sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15 or 17. [The present invention 1057] An engineered protein according to any one of claims 1001 to 1056, having at least 95% identity to the amino acid sequence of SEQ ID NO: 1 to 3. [The present invention 1058] The engineered protein of any of claims 1001 to 1056, comprising an engineered hMPV F protein ectodomain having 95% identity to SEQ ID NO:3. [The present invention 1059] The engineered protein of any of claims 1001 to 1058, wherein said protein is fused or conjugated to a trimerization domain. [The present invention 1060] The engineered protein of the present invention, wherein said protein is fused to a trimerization domain. [The present invention 1061] 1060. The engineered protein of the present invention, wherein said trimerization domain comprises the trimerization domain of T4 fibritin. [The present invention 1062] The engineered protein of any of claims 1001 to 1058, wherein said protein is fused or conjugated to a transmembrane domain. [The present invention 1063] 1062. The engineered protein of claim 1062, wherein said protein is fused to a transmembrane domain. [The present invention 1064] 1062. The engineered protein of the present invention, wherein said transmembrane domain comprises the transmembrane domain of a metapneumovirus (MPV) F protein. [The present invention 1065] The engineered protein of any of claims 1001 to 1062, comprising an N-terminal signal sequence. [The present invention 1066] 1065. The engineered protein of the present invention, wherein said N-terminal signal sequence is MSWKVMIIISLLITPQHG (SEQ ID NO:11). [The present invention 1067] A trimer of an engineered metapneumovirus (MPV) F protein comprising at least one subunit of any of claims 1001 to 1066. [The present invention 1068] The engineered trimer of the present invention 1067, wherein said trimer is stabilized in a pre-fusion conformation relative to a trimer of wild-type metapneumovirus (MPV) F subunit. [The present invention 1069] 1067. The engineered trimer of the present invention, wherein said trimer comprises at least one engineered disulfide bond between the subunits. [The present invention 1070] The engineered trimer of claim 1069, wherein at least one engineered disulfide bond between said subunits is selected from S316C and D421C. [The present invention 1071] A pharma- ceutically acceptable carrier, and (i) any one of the engineered proteins of the present invention 1001 to 1066, or (ii) any one of the engineered trimers of the present invention 1067 to 1070. 13. A pharmaceutical composition comprising: [The present invention 1072] The composition of the present invention 1071 further comprising an adjuvant. [The present invention 1073] A nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of any one of the engineered proteins of the present inventions 1001 to 1066. [The present invention 1074] The nucleic acid of claim 1073, wherein the nucleic acid comprises a DNA expression vector. [The present invention 1075] The nucleic acid of claim 1073, wherein the nucleic acid comprises mRNA. [The present invention 1076] A method for preventing metapneumovirus (MPV) infection or a disease associated with metapneumovirus infection in a subject, the method comprising administering to the subject an effective amount of any of the pharmaceutical compositions of the present inventions 1071 to 1072 or any of the nucleic acid molecules of the present inventions 1073 to 1075. [The present invention 1077] A pharmaceutical composition according to any one of claims 1071 to 1072 or a nucleic acid molecule according to any one of claims 1073 to 1075 for use in treating or preventing a metapneumovirus (MPV) infection or a disease associated with a metapneumovirus infection in a subject. [The present invention 1078] Use of the pharmaceutical composition of any of claims 1071 to 1072 or the nucleic acid molecule of any of claims 1073 to 1075 in the manufacture of a medicament for the treatment or prevention of a metapneumovirus (MPV) infection or a disease associated with a metapneumovirus infection. [The present invention 1079] A composition comprising an engineered protein of any of claims 1001 to 1066 or an engineered trimer of any of claims 1067 to 1070 bound to an antibody. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that this detailed description and the specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0036] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0037] [Figure 1] Beneficial substitutions for hMPV F stabilization. (A) Schematic of the hMPV F protein ectodomain. Disulfide bonds and N-glycosylation sites are highlighted. Residue numbers indicating the beginning and end of F1 and F2 are indicated below the bars. (B) Side view of the trimeric hMPV F (PDB ID: 5WB0) ectodomain in the prefusion conformation. One promoter is shown as a ribbon diagram and the other two as white molecular surfaces. Each inset corresponds to the antigenic site where the substitution is located. [Figure 2A] Characterization of single substitution hMPV F variants. Relative expression of purified individual variants calculated by area under the curve (AUC) of the SEC peak fraction. Variants are grouped by design. The horizontal dotted line indicates the calculated AUC concentration of the base construct, which has been normalized to 100% for comparison. [Figure 2B] Characterization of single substitution hMPV F variants. Size exclusion chromatography (SEC) of purified F variants grouped by design (proline, polar, cavity filling, and disulfide). The vertical dotted lines indicate the peak retention volumes for the hMPV F base construct. [Figure 2C]Characterization of single substitution hMPV F variants. Differential scanning fluorimetry (DSF) analysis of the thermal stability of disulfide variants. The vertical dotted line indicates the melting temperature for the base construct. [Figure 2D] Characterization of single substitution hMPV F variants. SDS-PAGE analysis of hMPV F base constructs and single substitution F variants. Molecular weight standards are indicated in kDa on the left. [Figure 3A] Image of an electrophoretic gel showing expression of F protein substitution variants (cavity mutations). Molecular weight standards are indicated in kDa on the left. [Figure 3B] Image of an electrophoretic gel showing expression of F protein substitution variants (disulfide mutations). Molecular weight standards are indicated in kDa on the left. [Figure 3C] Image of an electrophoretic gel showing expression of F protein substitution variants (proline mutations). Molecular weight standards are indicated in kDa on the left. [Figure 3D] Image of an electrophoretic gel showing expression of F protein substitution variants (electrostatic mutations). Molecular weight standards are indicated in kDa on the left. [Figure 4] Characterization of multi-substituted hMPV F variants. (A-C) SEC of purified multi-substituted hMPV F variants from three repeated cycles. The vertical dotted line indicates the peak retention volume for the hMPV F base construct. (D) DSF analysis of the thermostability of multi-substituted F variants. The vertical dotted line indicates the melting temperature for the base construct. (E) SEC trace of DS-CavEs2 purified from a 1 L culture of FreeStyle 293-F cells. (F) Binding of heat-treated or long-term stored DS-CavEs2 to MPE8 Fab as measured by Biolayer Interferometry. The vertical dotted line indicates the end of the association event. Untreated DS-CavEs2 was included as a control. [Diagram 5] Differential scanning fluorimetry (DSF) analysis of the thermal stability of F protein substitution variants. The vertical dotted line indicates the first melting temperature for JSM-1147. [Figure 6]Size-exclusion chromatography of purified F protein substitution variants. The vertical dotted line indicates the characteristic peak retention volume for the base construct (BaseCon). [Figure 7] Engineered hMPV F variant bound to prefusion-preferred antibody MPE8. (A) Side view of the atomic model of hMPV F variant (DSx2) bound to MPE8 Fab, shown as a ribbon diagram. The constant region of MPE8 Fab has been omitted for clarity. The side chains of the two disulfide substitutions in DSx2 are highlighted with sticks. (B) Close-up of the binding interface of MPE8 light chain CDRs and antigenic site II / V of the F protein. (C) Close-up of the binding interface of MPE8 heavy chain CDRs and F. The main chain of CDR3 is clustered against antigenic site III, which is highlighted as a transparent surface. Key residues that form polar interactions are shown with sticks. [Figure 8] The structure of hMPV F DS-CavEs2 in the prefusion conformation. (A) Side view of the atomic model of the apo hMPV F variant (DS-CavEs2) in the prefusion conformation. This model (firebrick ribbon) is superimposed with the structure of prefusion F (silver ribbon, PDB ID: 5WB0). The side chains of the introduced substitutions are highlighted with sticks. Each inset corresponds to an antigenic site where the superposition is being performed. (B) Representative 2D class average image of DS-CavEs2 complexed with MPE8 Fab. [Figure 9] Structural comparison of hMPV F DSx2 with the related PDB structure 5WB0. Superposition of single promoters from PDBID:5WB0 (hMPV F) and hMPV F DSx2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Detailed Description Human metapneumovirus (hMPV) fusion (F) protein is essential for virus entry and is an important target for neutralizing antibody and vaccine development. Although the pre-fusion conformation is considered to be the optimal vaccine antigen, previously described pre-fusion F proteins have been poorly expressed and poorly stabilized. In the present invention, the structure of hMPV F was used to guide the design and characterization of engineered hMPV F proteins. In some aspects, the engineered hMPV F proteins described in the embodiments are stabilized in the conformation that exists before membrane fusion. Such engineered proteins can be used, for example, to stimulate an immune response specific to anti-hMPV F protein. In a further aspect, the engineered F protein can be used to detect F protein-binding antibodies in a sample. Thus, the engineered proteins provided herein enable more effective methods for vaccination against hMPV while simultaneously enabling new assay methods for detecting anti-hMPV F protein antibodies, for example, in biological samples.

[0039] I. Aspects of the Disclosure The hMPV F protein, similar to other class I fusion proteins, is readily induced by host factors to transition from a metastable prefusion state to a highly stable postfusion state. The use of prefusion-stabilized proteins such as DS-Cav1 as vaccinogens has been found to elicit higher neutralization titers in animal models compared to using postfusion proteins. This inspired the use of the prefusion structure of hMPV F (PDB ID: 5WB0) as a guide to strategically introduce mutations one at a time, specifically to regions that undergo substantial conformational changes during the pre-to-post transition. Similar to recent successes for engineering the SARS-CoV-2 spike, multiple proline substitutions increased protein expression while retaining F in the prefusion conformation. The role of A107P in the fusion peptide is quite similar to that of the F817P substitution from HexaPro (Hsieh et al., 2020), presumably by imposing rigidity on the fusion peptide and also capping the helix in the prefusion conformation. Interestingly, replacing Ala with Phe, the equivalent residue on RSV F, slightly reduced protein expression compared to the base construct (Table 1). D461P in HRB may also perform the same function as A107P or A113P, which were the most effective proline substitutions in the fusion peptide. There are three proline substitutions in antigen site V that lead to increased protein yields. Considering that the side chains of these residues are partially exposed on the surface of the trimer, they are considered suboptimal for vaccine antigens.

[0040] The implementation of interprotomer or intersubunit salt bridges tends to efficiently hold trimeric viral proteins in a relatively compact conformation in a more effective manner than intraprotomer salt bridges. For example, the K588E mutation from gp41 electrostatically interacts with K62 or K492 from gp120, which favors HIV-1 Env to stay in a prefusion closed conformation (Rutten et al., 2018). Among our design, the L219K substitution from the F1 subunit could form a salt bridge with either E80 from the F2 subunit or D209 from the F1 subunit. The 73E substitution from the F2 subunit could also form a salt bridge with R198 from the neighboring F1 subunit. Both variants had enhanced expression and longer retention time in SEC, implying a similar role of salt bridge design for class I fusion proteins. On the other hand, intraprotomer salt bridge designs such as variant N145E abolished the expression of hMPV F (Table 1). Similarly, this type of salt bridge was less successful in stabilizing the SARS-CoV-2 spike. Reducing the repulsion caused by charge clusters at the protomer interface is another approach to block trimer opening. Charge clusters (E453 / D454) were found in the HRB region proximal to the base of the trimer. Thus, replacing Glu453 with the equivalent Gln may reduce the charge repulsion caused by the negatively charged cluster. Similar to the E487Q substitution from RSV F or the K588F substitution from Ebola GP (McLellan et al., 2013; Rutten et al., 2020), this variant rendered hMPV F a more compact trimer and retained the native quaternary structure, as evident by the SEC elution profile (Figure 2B).

[0041] Cavity-filling approaches were also highly effective in stabilizing loosely assembled viral proteins in the prefusion conformation. For example, the S190F substitution in the Cav1 variant successfully fills the cavity between sites V and II, and the V207L ​​substitution successfully fills the pocket between sites Φ with only a single CH2 addition. One of the highest expressing variants, V231I, is surprisingly located in domain IIIb (site II), a region that does not undergo conformational changes during the transition from prefusion to postfusion. The other two substitutions that enhanced expression (S149I, I137L), both located in domain IIIa (site V), appear to assemble against each other to stabilize the highly flexible α2 and β3.

[0042] Among the strategies used to stabilize hMPV F, the introduction of disulfide bonds probably yields the highest success rate. The L110C / N322C substitution was designed to trap the fusion peptide in the central cavity, and the T365C / V463C substitution was designed to lock the HRB to the membrane-proximal region. By anchoring the fusion peptide or HRB to a region that remains constant during the pre- to post-fusion transition, the F protein was retained in the pre-fusion conformation and its thermostability was significantly improved. This approach has been successfully used for several class I viral fusion proteins (McLellan et al., 2013; Stewart-Jones et al., 2018; Sanders et al., 2013). This design is reminiscent of the DS variant for RSV F and SOSIP for HIV-1 Env. In contrast, the T127C / N153C or A140C / A147C substitutions represent a different kind of disulfide design strategy. Although these disulfide bonds are placed in regions that undergo conformational changes (e.g., domain IIIa (site V)), they appear to stabilize the prefusion state by blocking refolding of HRA near the central helix. This is similar to the Q162C / L168C substitutions used in the F protein of uenza virus 3 (Stewart-Jones et al., 2018), suggesting that this kind of disulfide design could be a general approach to stabilize class I viral fusion proteins. They could block refolding of HRA, potentially forcing it to refold into a prefusion conformation. Similar disulfide designs that restrict the local flexibility of the secondary structure in regions that undergo conformational changes during the pre- to post-fusion transition have also been successfully applied to the SARS-CoV-2 spike. Interestingly, the V104C / N457C substitutions block furin protease digestion of F, resulting in a single species of F0 in SDS-PAGE. The furin cleavage site proximal to the fusion peptide may be buried in the central cavity due to disulfide design. This variant may be useful for protease-free production of vaccine antigens.

[0043] Combining multiple beneficial modifications that increase protein expression and stability has proven to be an effective strategy to produce optimized pre-fusion antigens (Joyce et al., 2016, Krarup et al., 2015, McLellan et al., 2013, Rutten et al., 2020, Rutten et al., 2018, Jiachen et al., 2021, Hsieh et al., 2020). In the present invention, combining multiple beneficial modifications resulted in one of the best constructs, DS-CavEs2, including disulfide bond, cavity filling, and electrostatic stabilization designs. DS-CavEs2 has 10-fold higher protein expression, enhanced thermostability, and retains pre-fusion epitopes after heat stress and long-term storage at 4°C. The introduction of two disulfide substitutions at site V, a cavity-filling substitution at site II, and another disulfide bond proximal to the fusion peptide did not disrupt the conformation of the membrane-distal half of hMPV F, which harbors the most potent neutralizing epitopes in RSV F (Fig. 8A) (Graham et al., 2017; Gilman et al., 2016). In contrast, the T365C / V463C substitutions changed the relative position of the α10 helix. However, this membrane-proximal region is unlikely to be immunogenic, and no neutralizing antibodies targeting this region in RSV F have been found.

[0044] Another version of DS-CavEs2, in which the furin site is replaced with a flexible glycine-serine (GS) or polyglycine linker, generates a single-chain form of the pre-fusion F trimer. In some embodiments, the linker has the sequence GGSGGS (SEQ ID NO: 12) or GGGGGG (SEQ ID NO: 13). Given that no furin protease is required to recombinantly produce this construct, this design may be more cost-effective for industrial production of vaccine antigens.

[0045] Notably, both prefusion stabilized F constructs crystallized as monomers, even when complexed with MPE8, which recognizes an epitope spanning adjacent protomers. Other groups have also crystallized monomeric hMPV F bound to antibodies that recognize different antigenic sites (Huang et al., 2020; Wen et al., 2012). Given that visualization of trimeric F particles by nsEM was possible (Figure 8B), these data suggest that hMPV F trimers are in equilibrium with dissociated monomers, even when fused to trimerization motifs. This is consistent with recent results demonstrating that several class I viral fusion proteins undergo trimer opening or "breathing," and native antibodies that bind to the trimer interface of influenza HA and hMPV F have been isolated (Bangaru et al., 2019; Watanabe et al., 2019; Gilman et al., 2019).

[0046] Prefusion-stabilized class I viral fusion proteins are known to elicit high neutralizing antibody titers in animals and humans, often an order of magnitude higher than those induced by postfusion antigens (Crank et al., 2019; McLellan et al., 2013; Stewart-Jones et al., 2018). Previous studies have shown little difference in the immunogenicity of prefusion and postfusion hMPV F proteins (Battles et al., 2017), but those studies were performed with a 10 μg antigen dose and with postfusion F proteins that are now known to be contaminated with some amount of prefusion-like protein that has not yet adopted a postfusion conformation. Prefusion-stabilized hMPV F antigens are expected to elicit higher neutralizing antibody titers in mice than postfusion F proteins. These results would be more consistent with other immunogenicity studies of prefusion stabilized viral proteins than previous hMPV F studies (Crank et al., 2019; van den Hoogen et al., 2002; Stewart-Jones et al., 2018). The stabilized proteins described herein should accelerate the development of hMPV F vaccine candidates as well as facilitate the isolation of potent, broadly reactive monoclonal antibodies that may be useful for passive prophylaxis of high-risk cohorts.

[0047] II. Definition It is to be understood that both the general description above and the detailed description below are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless expressly stated otherwise. In this application, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "comprising" and other forms such as "comprises" and "includes" is not limiting. Also, the use of terms such as "element" or "component" encompasses both elements and components that contain one unit and elements and components that contain more than one subunit, unless expressly stated otherwise. Also, the use of the term "portion" can include a portion or the entire portion of a moiety.

[0048] As used herein, the term "a" or "an" may mean one or more. As used herein, in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0049] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to only alternatives or the alternatives are not mutually exclusive, even if the disclosure supports a definition that refers only to alternatives as well as "and / or." As used herein, "another" may mean at least a second or more.

[0050] The term "about" as used herein when referring to measurable values, such as amounts, durations of time, and the like, is intended to encompass variations of up to ±10% from the stated value. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weights, reaction conditions, and the like used in the specification and claims should be understood as being modified in all occurrences by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the disclosed subject matter. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0051] As used herein, the term "essentially free" with respect to a specified component is used herein to mean that none of the specified components are intentionally incorporated into the composition and / or are present only as contaminants or in trace amounts. Thus, the total amount of the specified components resulting from any unintentional contaminants of the composition is well below 0.05%, preferably below 0.01%. Most preferred are compositions in which any amount of the specified components cannot be detected by standard analytical methods.

[0052] The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with an intact antibody for specific binding to a target antigen, including, for example, chimeric, humanized, fully human, and bispecific antibodies. An "antibody" is a species of antigen-binding protein. An intact antibody generally contains at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, such as antibodies naturally occurring in camelids that may contain only heavy chains. An antibody may be derived from only a single source, or may be "chimeric," that is, different portions of the antibody may be derived from two different antibodies, as described further below. Antigen-binding proteins, antibodies, or binding fragments are produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are described below. Additionally, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

[0053] The structural unit of a natural antibody comprises a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (in certain embodiments, about 25 kDa) and one full-length "heavy" chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically contains a variable region of about 100-110 or more amino acids that are typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that may be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant regions are typically linked by a "J" region of about 12 or more amino acids, of which the heavy chain also includes a "D" region of about 10 more amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)), which is incorporated by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair typically form the antigen-binding site.

[0054] The term "variable region" or "variable domain" refers to a portion of an antibody's light and / or heavy chains that typically comprises approximately 120-130 amino acids at the amino terminus in the heavy chain and about 100-110 amino terminal amino acids in the light chain. In certain embodiments, the variable regions of different antibodies differ significantly in amino acid sequence, even among antibodies of the same species. The variable regions of an antibody typically determine the specificity of a particular antibody for its target.

[0055] Variable regions typically exhibit the same general structure, with relatively conserved framework regions (FR) linked by three hypervariable regions (also called complementarity determining regions or CDRs). The CDRs from the two chains of each pair are typically aligned by the framework regions, which may allow binding to a specific epitope. From the N-terminus to the C-terminus, both the light chain variable region and the heavy chain variable region typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain typically follows the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk J. Mol. Biol., 196:901-917 (1987), or Chothia et al., Nature, 342:878-883 (1989).

[0056] In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

[0057] In certain embodiments, definitive demarcation of the CDRs and identification of the residues that constitute the antibody binding site are accomplished by solving the structure of the antibody and / or the structure of the antibody-ligand complex. In certain embodiments, this can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various analytical methods can be used to identify or approximately predict the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

[0058] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but it takes into account the location of certain structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses a comprehensive suite of computer programs produced by the Oxford Molecular Group to model antibody structures. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989), “AbM TM See, e.g., "A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of antibodies from the primary sequence using a combination of knowledge databases and ab initio methods such as those described in Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0059] By convention, the CDR regions of the heavy chain are typically referred to as H1, H2, and H3, numbered sequentially from the amino terminus to the carboxy terminus, and the CDR regions of the light chain are typically referred to as L1, L2, and L3, numbered sequentially from the amino terminus to the carboxy terminus.

[0060] The term "light chain" includes full-length light chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain contains a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa and lambda chains.

[0061] The term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain contains a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide and the CH domain is at the carboxyl-terminus, with CH3 being closest to the carboxy-terminus of the polypeptide. The heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0062] Bispecific or bifunctional antibodies are typically artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0063] The term "antigen" refers to a substance that can induce an adaptive immune response. Specifically, an antigen is a substance that serves as a target for the receptor of the adaptive immune response. Typically, an antigen is a molecule that binds to an antigen-specific receptor but cannot itself induce an immune response in the body. Antigens are usually proteins and polysaccharides, and less frequently lipids. As used herein, antigens also include immunogens and haptens.

[0064] The "Fc" region comprises two heavy chain fragments comprising the CH1 and CH2 domains of an antibody, the two heavy chain fragments being held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0065] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0066] An antibody that "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is an antibody that binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope of a polypeptide. For example, an hMPV F protein-specific antibody of the invention is specific for an hMPV F protein. In some embodiments, an antibody that binds to an hMPV F protein has a dissociation constant (Kd) of ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10-8 M or less, e.g., 10-8 M to 10-13 M, e.g., 10-9 M to 10-13 M).

[0067] The term "compete" when used in the context of antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof) competing for the same epitope refers to competition between the antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) being tested blocks or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand, or reference antibody) to a common antigen (e.g., hMPV F or a fragment thereof). Whether one antigen-binding protein competes with another antigen-binding protein can be determined by a number of types of competitive binding assays, e.g., solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIAs using I-125 labels (see, e.g., Morel et al., 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing any of these, an unlabeled test antigen-binding protein, and a labeled reference antigen-binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Usually, the test antigen-binding protein is present in excess.Antigen binding proteins identified by competitive assays (competitor antigen binding proteins) include antigen binding proteins that bind to the same epitope as the reference antigen binding protein, and antigen binding proteins that bind to adjacent epitopes that are sufficiently proximal to the epitope bound by the reference antigen binding protein to cause steric hindrance. Additional details regarding methods for determining competitive binding are provided in the Examples herein. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of the reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0068] The term "epitope" as used herein refers to a specific atom or group of amino acids on an antigen to which an antibody binds. An epitope can be either a linear epitope or a conformational epitope. A linear epitope is formed by a continuous amino acid sequence from an antigen and interacts with an antibody based on their primary structure. On the other hand, a conformational epitope is composed of a discontinuous section of the amino acid sequence of an antigen and interacts with an antibody based on the 3D structure of the antigen. In general, an epitope is approximately 5 or 6 amino acids long. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope in the antigen.

[0069] The term "host cell" refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of a parent cell, whether or not the morphology or genetic make-up of the progeny is identical to that of the original parent cell, so long as the gene of interest is present.

[0070] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percent of identical residues between the amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably handled by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press, Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press, Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press, von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press, Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press, and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0071] When calculating percent identity, the sequences being compared are typically aligned to maximize the match between sequences.An example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387, Genetics Computer Group, University of Wisconsin, Madison, Wis.).The computer algorithm GAP is used to align the two polypeptides or polynucleotides whose percent sequence identity is to be determined.The sequences are aligned to optimize their corresponding amino acids or nucleotides (the "matched range" as determined by the algorithm). A gap opening penalty (which is calculated as 3× the average diagonal element, where the "average diagonal element" is the average of the diagonal elements of the comparison matrix being used and a "diagonal element" is the score or number assigned to each perfect amino acid match according to a particular comparison matrix) and gap extension penalty (which is usually 1 / 10× the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62, are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix and Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919 for the BLOSUM 62 comparison matrix) are also used by the algorithm.

[0072] Examples of parameters that can be used in determining percent identity of polypeptide or nucleotide sequences using the GAP program can be found in Needleman et al., 1970, J. Mol. Biol. 48:443-453.

[0073] A particular alignment scheme for aligning two amino acid sequences may result in matching of only short regions of the two sequences, and this small aligned region may have very high sequence identity even if there is no significant relationship between the two full-length sequences. Thus, the selected alignment method (GAP program) may be adjusted to result in an alignment spanning at least 50 or other number of consecutive amino acids of the target polypeptide, if so desired.

[0074] As used herein, the term "linked" refers to an association through intramolecular interactions, such as covalent, metallic, and / or ionic bonds, or through intermolecular interactions, such as hydrogen bonds or non-covalent bonds.

[0075] The term "operably linked" refers to an arrangement of elements such that the components so described are configured to perform their normal functions. Thus, a given signal peptide operably linked to a polypeptide directs the secretion of the polypeptide from a cell. In the case of a promoter, a promoter operably linked to a coding sequence directs the expression of the coding sequence. Promoters or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression of the coding sequence. For example, untranslated but transcribed intervening sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0076] The term "polynucleotide" or "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleotides that make up a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates, and phosphoramidates.

[0077] As used herein, a "vector" refers to a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may contain a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector may also contain one or more therapeutic genes and / or selectable marker genes, as well as other genetic elements known in the art. A vector can transduce, transform, or infect a cell, thereby causing the cell to express a nucleic acid and / or protein other than the nucleic acid and / or protein native to the cell. A vector optionally includes materials that help achieve entry of the nucleic acid into the cell, such as a viral particle, a liposome, a protein coat, and the like.

[0078] The term "polypeptide" or "protein" refers to a polymer having the amino acid sequence of a native protein, i.e., a protein produced by a non-recombinant cell in the natural form, or it includes molecules produced by genetic engineering or recombinant cells that have the amino acid sequence of a native protein, or molecules that have one or more amino acid deletions, additions, and / or substitutions of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. The terms "polypeptide" and "protein" expressly encompass hMPV F protein binding proteins, antibodies, or sequences that have one or more amino acid deletions, additions, and / or substitutions of antigen binding proteins. The term "polypeptide fragment" refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to the full-length native protein. Such fragments may also contain modified amino acids compared to the native protein. In certain embodiments, the fragments are about 5-500 amino acids in length. For example, a fragment can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies that contain the binding domain. In the case of hMPV F protein binding antibodies, useful fragments include, but are not limited to, the CDR regions, the variable domains of the heavy and / or light chains, a portion of the antibody chain containing two CDRs or simply the variable region thereof, and the like.

[0079] Pharmaceutically acceptable carriers useful in the present invention are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975) describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations usually comprise injectable fluids that contain pharma- ceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like, as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0080] As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. A subject may be a patient, which refers to a human who visits a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0081] As used herein, the term "therapeutically effective amount" or "effective dosage" refers to a dosage or concentration of a drug effective to treat a disease or condition, e.g., with respect to the use of the monoclonal antibodies or antigen-binding fragments thereof disclosed herein to treat a viral infection.

[0082] As used herein, "treating" a condition or "treatment" includes preventing or alleviating the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or any combination thereof.

[0083] III. hMPV F protein Human metapneumovirus (hMPV) is an enveloped, negative-stranded virus of the family Pneumoviridae that was discovered in 2001, but had been distributing for at least half a century prior to its discovery. The hMPV fusion (F) protein is one of three surface glycoproteins encoded by the viral genome. As a class I fusion, hMPV F is initially translated as a single polypeptide precursor (F0). Being initially non-functional, a proteolytic cleavage event is required to form the F1 and F2 subunits that are covalently linked by disulfide bonds. The new N-terminus of the F2 polypeptide contains a hydrophobic sequence that will insert into the host cell membrane during a process that ultimately fuses the viral and host cell membranes. At some point, either during transit or at the membrane surface, the F protein associates with itself to form a metastable trimer in the so-called prefusion conformation. The hMPV fusion protein is cleaved extracellularly by a trypsin-like protease. An unknown triggering event affects the F protein to undergo a dramatic conformational change in which it extends the fusion peptide into the host cell membrane and then refolds back on itself to form a six-helix bundle in the so-called post-fusion conformation. The energy difference between the extended intermediate and the post-fusion conformation provides the energy required for membrane fusion.

[0084] Historically, the structures of paramyxovirus fusion proteins have been classified into domains that generally compartmentalize the initially elucidated prefusion proteins into head, neck, and stalk. The nomenclature assigning three general domains (DI, DII, and DIII) to the prefusion structures of additional structures continues. The family pneumoviridae also retains this convention, as they were formerly a subfamily of paramyxoviruses. The domains of hMPV are loosely classified into the same three domains, and two additional regions of heptad repeats are similarly defined. Heptad repeat A (HRA) is at the N-terminus of F1, located within DIIIa, and heptad repeat B is at the C-terminus outside the defined domain region and preceding the transmembrane domain of the protein. However, due to structural similarities, the shift in antigenic site nomenclature used for the fusion protein of respiratory syncytial virus (RSV), another member of the pneumoviridae family, can be used to more precisely describe the protein regions. DIIIa undergoes its greatest conformational rearrangement when the fusion peptide is released from the internal cavity of the trimer, with HRA forming a three-coil bundle as the fusion peptide is inserted into the target membrane, and finally HRB associates around the outside of the HRA bundle to form 6HB in the postfusion conformation.

[0085] Recently, stabilization of class I fusion proteins in their prefusion conformation has produced promising results as vaccine antigens in clinical trials, with both RSV and SARS-coronavirus-2 vaccines using this approach (Baden et al., 2020; Keech et al., 2020; Williams et al., 2020; Crank et al., 2019). RSV shares approximately 33% sequence identity with hMPV, and the two prefusion structures are highly similar (Battles et al., 2017; van den hoogen et al., 2002). Based on its elucidated prefusion structure, a few different strategies have been used to stabilize RSV F in its prefusion conformation, including the introduction of prolines, disulfide bonds, and cavity-filling substitutions (Joyce et al., 2016; Krarup et al., 2015; McLellan et al., 2013). In the case of DS-Cav1, the internal cavity was filled more optimally by hydrophobic residue substitutions and the introduction of disulfide bonds was introduced in the fusion peptide region. In the case of PR-DM, proline residues were introduced to prevent the loop region from rearranging into the extended alpha-helix seen in the post-fusion conformation. In RSV F, regions of charge repulsion were also identified and reduced. Similarly, the elucidated pre-fusion structure allowed the stabilization of coronaviruses by introducing two proline substitutions (S-2P) in the hinge region. There are also examples within the HIV (SOSIP) and influenza fields where engineering stabilizing mutations has led to well-behaved pre-fusion reagents.

[0086] Recently, hMPV F was stabilized in a prefusion conformation using knowledge gained from RSV F studies. First, the F2 / F1 cleavage site sequence "RQSR" was replaced with a polybasic "RRRR" sequence to allow efficient cleavage by furin-like proteases in producer cells. Then, mimicking the RSV F stabilization strategy (Krarup et al., 2015), proline was introduced into the helix-loop-helix region in F1 at the membrane-distal trimer apical end. This engineering strategy allowed the hMPV F trimer prefusion crystal structure to be obtained, but the protein was poorly expressed, suggesting that further engineering is required (Battles et al., 2017). Furthermore, previous serum depletion assays and mouse immunization experiments showed that there were no significant differences in antigenicity between prefusion and postfusion hMPV F (Battles et al., 2017). In contrast, prefusion RSV F induced a more robust neutralizing antibody response than postfusion RSV F, and serum depletion experiments demonstrated that most of the RSV neutralizing activity in human serum bound exclusively to the prefusion conformation (Sastre et al., 2005; Magro et al., 2012). These data suggested that more stable prefusion hMPV F constructs were required to explore these discordant results.

[0087] To this end, a strategy for stabilizing the F protein by mutation of the coding sequence of the F protein is demonstrated herein. The published pre-fusion hMPV F structure was used to guide the engineering of additional amino acid substitutions. Combinations of multiple beneficial substitutions were found to have additive effects on the desired protein properties. Details of the mutations analyzed and provided herein are shown in Table 1 below. The mutant proteins were expressed as detailed in the Examples and the amounts of protein and trimeric complexes produced were determined.

[0088] Table 1. F protein substitutions and mutations TIFF0007660671000001.tif225158TIFF0007660671000002.tif206158TIFF0007660671000003.tif211163TIFF0007660671000004.tif100163

[0089] IV. Pharmaceutical Preparations The present disclosure provides pharmaceutical compositions comprising engineered hMPV F proteins, which can be used to stimulate an immune response, such as as part of a vaccine formulation.

[0090] When a nucleic acid molecule encoding an engineered hMPV F protein is used in a pharmaceutical composition, the nucleic acid molecule may comprise or consist of deoxyribonucleotides and / or ribonucleotides, or their analogs, covalently linked together. The nucleic acid molecules described herein generally contain phosphodiester bonds, but may optionally include nucleic acid analogs, which may have at least one different bond, such as phosphoramidate, phosphorothioate, phosphorodithioate, or O-methyl phosphoramidite bonds, as well as peptide nucleic acid backbones and bonds. Mixtures of naturally occurring polynucleotides and analogs can be made, but alternatively, mixtures of different polynucleotide analogs, as well as mixtures of naturally occurring polynucleotides and analogs may be made. The nucleic acid molecule may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling component. The term also includes both double-stranded and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form. A nucleic acid molecule is composed of a specific sequence of the four nucleotide bases, namely adenine (A), cytosine (C), guanine (G), thymine (T), and, if the polynucleotide is RNA, uracil (U) in place of thymine. Thus, the term "nucleic acid sequence" is the alphabetical representation of a nucleic acid molecule. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses not only the sequence explicitly depicted, but also its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0091] In some embodiments, the nucleic acids of the present disclosure contain one or more modified nucleosides that contain modified sugar moieties. Such compounds that contain one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with target nucleic acids, compared to oligonucleotides that contain only nucleosides that contain natural sugar moieties. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogates may contain one or more substitutions that correspond to the substitutions of the substituted sugar moiety.

[0092] In some embodiments, the modified sugar moiety is a substituted sugar moiety that contains one or more non-bridging sugar substituents, including, but not limited to, substituents at the 2' and / or 5' positions. Examples of suitable sugar substituents at the 2' position include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the sugar substituent at the 2' position is allyl, amino, azido, thio, O-allyl, O-C1-C2-C3-amino, aryl ... 10 Alkyl, O--C1~C 10 substituted alkyl, OCF3, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn), and O--CH2--C(=O)--N(Rm)(Rn), where each Rm and Rn is independently H or a substituted or unsubstituted C1-C 10 and alkyl. Examples of sugar substituents at the 5' position include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In some embodiments, the substituted sugar comprises more than one non-bridging sugar substituent, such as a TF-5'-methyl sugar moiety (see, e.g., PCT International Application WO2008 / 101157 for additional 5',2'-bis substituted sugar moieties and nucleosides).

[0093] Nucleosides that include a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In some embodiments, a 2'-substituted nucleoside comprises a 2'-substituent selected from halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S, or N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn), or O--CH2--C(=O)--N(Rm)(Rn), where each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 These 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0094] In some embodiments, the 2'-substituted nucleoside comprises a 2'-substituent selected from F, NH2, N3, OCF3, O--CH3, O(CH2)3NH2, CH2-CH=CH2, O--CH2-CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O--(CH2)2--O--N(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O--CH2--C(=O)--N(Rm)(Rn), where each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 It is an alkyl.

[0095] In some embodiments, 2'-substituted nucleosides comprise a sugar moiety that includes a 2'-substituent selected from F, OCF3, O--CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2--O--N(CH3)2, --O(CH2)2O(CH2)2N(CH3)2, and O--CH2--C(=O)--N(H)CH3.

[0096] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety that includes a 2'-substituent selected from F, O--CH3, and OCH2CH2OCH3.

[0097] In some embodiments, the nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, the nucleosides of the present disclosure comprise one or more modified nucleobases.

[0098] In some embodiments, the modified nucleobase is selected from universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases, as defined herein. The 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines include 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-aminopropyluracil, 5-propynylcytosine, 5-hydroxymethylcytosine, 5-aminopropyluracil ... -azo-uracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).Modified nucleobases can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Additional nucleobases include those disclosed in U.S. Patent 3,687,808 and in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859.

[0099] Representative U.S. patents that teach the preparation of certain of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Patents 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,900, and 5,512,112. Nos. 8, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,681,941, 5,750,692, 5,763,588, 5,830,653, and 6,005,096, each of which is incorporated by reference in its entirety.

[0100] Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide. For example, one additional modification of the ligand-conjugated oligonucleotides of the present disclosure involves chemically linking one or more additional non-ligand moieties or conjugates to the oligonucleotide that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholic acid, thioethers, such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains, such as dodecanediol or undecyl residues, phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties. In some aspects, the nucleic acid molecule encoding the chimeric hMPV / RSV F protein is a modified RNA, such as a modified mRNA. Modified (m)RNA contemplates certain chemical modifications that confer increased stability and reduced immunogenicity to the mRNA, thereby facilitating the expression of therapeutically important proteins. For example, N1-methyl-pseudouridine (N1mΨ) is superior to some other nucleoside modifications and their combinations in terms of translational capacity. In some embodiments, the (m)RNA molecules used herein may have uracils replaced with pseudouracils, such as 1-methyl-3'-pseudouridylyl bases. In some embodiments, some of the uracils are replaced, while in other embodiments, all of the uracils are replaced. The (m)RNA may include a 5' cap, a 5' UTR element, an optional codon-optimized open reading frame, a 3' UTR element, and a poly(A) sequence and / or a polyadenylation signal.

[0101] The nucleic acid molecule, whether native or modified, may be delivered as a naked nucleic acid molecule or in a delivery vehicle such as a lipid nanoparticle. The lipid nanoparticle may include one or more nucleic acids present in a weight ratio of about 5:1 to about 1:100 relative to the lipid nanoparticle. In some embodiments, the weight ratio of nucleic acid to lipid nanoparticle is about 5:1, 2.5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, or any value derivable therein.

[0102] In some embodiments, the lipid nanoparticles used herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 lipids. These lipids may include triglycerides, phospholipids, steroids or sterols, PEGylated lipids, or groups having an ionizable group such as an alkylamine and one or more hydrophobic groups such as C6 or higher alkyl groups.

[0103] In some aspects of the present disclosure, lipid nanoparticles are mixed with one or more steroids or steroid derivatives.In some embodiments, steroids or steroid derivatives include any steroids or steroid derivatives.As used herein, in some embodiments, the term "steroid" refers to a class of compounds that have a 4-ring 17-carbon ring structure, which may further include one or more substitutions, including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or double bonds between two or more carbon atoms.

[0104] In some aspects of the disclosure, the lipid nanoparticles are mixed with one or more PEGylated lipids (or PEG lipids). In some embodiments, the disclosure includes using any lipid to which a PEG group is attached. In some embodiments, the PEG lipid is a diglyceride, which also includes a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C24 long chain alkyl or alkenyl groups, or C6-C24 fatty acid groups, attached to a linker group with a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamines and phosphatidic acids, PEG-ceramide conjugates, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified diastearoyl phosphatidylethanolamines or PEG-modified dimyristoyl-sn-glycerols. In some embodiments, the PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight is about 200 to about 500, about 400 to about 5,000, about 500 to about 3,000, or about 1,200 to about 3,000. The molecular weight of the PEG modification is about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. Some non-limiting examples of lipids that can be used in the present disclosure are taught by US Pat. No. 5,820,873, WO2010 / 141069, or US Pat. No. 8,450,298, which are incorporated herein by reference.

[0105] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more phospholipids. In some embodiments, any lipid that also contains a phosphate group. In some embodiments, the phospholipid is one or two long chain C6-C 24 It is a structure containing an alkyl or alkenyl group, glycerol or sphingosine, one or two phosphate groups, and optionally an organic small molecule. In some embodiments, the organic small molecule is an amino acid, a sugar, or an amino-substituted alkoxy group such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipids are defined as lipids and lipid-like molecules that have both positive and negative charges.

[0106] In some aspects of the present disclosure, lipid nanoparticles are provided that contain a compound containing a lipophilic component and a cationic component, where the cationic component is ionizable. In some embodiments, the ionizable cationic lipid contains one or more groups that are protonated at physiological pH, but can be deprotonated at pHs above 8, 9, 10, 11, or 12, and do not have a charge. The ionizable cationic group may contain one or more protonable amines that can form a cationic group at physiological pH. The ionizable cationic lipid compound may also be a C6-C 24 They may further include one or more lipid components, such as two or more fatty acids with alkyl or alkenyl carbon groups. These lipid groups may be linked via ester bonds or further attached to sulfur atoms via Michael addition reactions. In some embodiments, these compounds may be dendrimers, dendrons, polymers, or combinations thereof.

[0107] In some aspects of the present disclosure, compositions are provided that contain a compound containing a lipophilic component and a cationic component, where the cationic component is ionizable. In some embodiments, ionizable cationic lipids refer to lipids and lipid-like molecules that have a nitrogen atom that can acquire an electric charge (pKa). These lipids may be known in the literature as cationic lipids. These molecules with amino groups typically have 2-6 hydrophobic chains, often C6-C 24 The alkyl or alkenyl groups may have at least one or more than six tails, such as alkyl or alkenyl groups.

[0108] In some embodiments, the amount of lipid nanoparticles having encapsulated nucleic acid molecules in the pharmaceutical composition is from about 0.1 w / w% to about 50 w / w%, from about 0.25 w / w% to about 25 w / w%, from about 0.5 w / w% to about 20 w / w%, from about 1 w / w% to about 15 w / w%, from about 2 w / w% to about 10 w / w%, from about 2 w / w% to about 5 w / w%, or from about 6 w / w% to about 10 w / w%. In some embodiments, the amount of lipid nanoparticles with encapsulated nucleic acid molecules in the pharmaceutical composition is from about 0.1 w / w%, 0.25 w / w%, 0.5 w / w%, 1 w / w%, 2.5 w / w%, 5 w / w%, 7.5 w / w%, 10 w / w%, 15 w / w%, 20 w / w%, 25 w / w%, 30 w / w%, 35 w / w%, 40 w / w%, 45 w / w%, 50 w / w%, 55 w / w%, 60 w / w%, 65 w / w%, 70 w / w%, 75 w / w%, 80 w / w%, 85 w / w%, 90 w / w%, to about 95 w / w%, or any range derivable therein.

[0109] In some aspects, the present disclosure includes one or more sugars formulated in the pharmaceutical composition. In some embodiments, the sugars used herein are saccharides. These sugars may be used to act as cryoprotectants to protect the pharmaceutical composition from destabilization during the drying process. These water-soluble excipients include carbohydrates or sugars, such as disaccharides such as sucrose, trehalose, or lactose, trisaccharides such as fructose, glucose, galactose, which constitute raffinose, polysaccharides such as starch or cellulose, or sugar alcohols such as xylitol, sorbitol, or mannitol. In some embodiments, these excipients are solid at room temperature. Some non-limiting examples of sugar alcohols include erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotritol, maltotetraitol, or polyglycitol.

[0110] In some embodiments, the amount of sugar in the pharmaceutical composition is from about 25 w / w% to about 98 w / w%, 40 w / w% to about 95 w / w%, 50 w / w% to about 90 w / w%, 50 w / w% to about 70 w / w%, or about 80 w / w% to about 90 w / w%. In some embodiments, the amount of sugar in the pharmaceutical composition is from about 10 w / w%, 15 w / w%, 20 w / w%, 25 w / w%, 30 w / w%, 35 w / w%, 40 w / w%, 45 w / w%, 50 w / w%, 52.5 w / w%, 55 w / w%, 57.5 w / w%, 60 w / w%, 62.5 w / w%, 65 w / w%, 67.5 w / w%, 70 w / w%, 75 w / w%, 80 w / w%, 82.5 w / w%, 85 w / w%, 87.5 w / w%, 90 w / w% to about 95 w / w%, or any range derivable therein.

[0111] In some embodiments, the pharma- ceutically acceptable polymer is a copolymer. The pharma- ceutically acceptable polymer may further comprise 1, 2, 3, 4, 5, or 6 subunits of individual different types of polymer subunits. These polymer subunits may comprise polyoxypropylene, polyoxyethylene, or similar subunits. In particular, the pharma- ceutical acceptable polymer may comprise at least one hydrophobic subunit and at least one hydrophilic subunit. In particular, the copolymer may have a hydrophilic subunit on each side of the hydrophobic unit. The copolymer may have a hydrophilic subunit that is polyoxyethylene and a hydrophobic subunit that is polyoxypropylene.

[0112] In some embodiments, expression cassettes are used to express the engineered hMPV F protein, either for subsequent purification and delivery to a cell / subject, or for direct use in a viral-based delivery approach. Provided herein are expression vectors containing one or more nucleic acids encoding the engineered hMPV F protein.

[0113] Expression requires that appropriate signals are provided within the vector and include various regulatory elements such as enhancers / promoters from both viral and mammalian sources that drive expression of the engineered hMPV F protein in the cell. Throughout this application, the term "expression cassette" is intended to include any type of genetic construct containing a nucleic acid encoding a gene product, where part or all of the coding sequence of the nucleic acid can be transcribed and translated, i.e., under the control of a promoter. A "promoter" refers to a DNA sequence that is recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct position and orientation relative to the nucleic acid to control the initiation of RNA polymerase and expression of the gene. An "expression vector" is intended to include an expression cassette contained in a genetic construct that is capable of replication and thus includes one or more of an origin of replication, a transcription termination signal, a poly-A region, a selectable marker, and a multipurpose cloning site.

[0114] The term promoter will be used herein to refer to a group of transcriptional control modules clustered around an initiation site for RNA polymerase II. Most of the ideas about how promoters are organized come from the analysis of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies have been augmented by more recent efforts to show that promoters are composed of distinct functional modules, each of which consists of approximately 7-20 bp of DNA and contains one or more recognition sites for transcriptional activator or repressor proteins.

[0115] At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters that lack a TATA box, such as those for the mammalian terminal deoxynucleotidyl transferase gene and the SV40 late genes, individual elements that overlap the start site themselves help to anchor the start site.

[0116] Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but in recent years, some promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased to a spacing of 50 bp, after which activity begins to decrease. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0117] In certain embodiments, viral promoters such as human cytomegalovirus (CMV) immediate early gene promoter, SV40 early promoter, Rous sarcoma virus long terminal repeat, rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high levels of expression of the coding sequence of interest. The use of other viral or mammalian cell or bacterial phage promoters known in the art to achieve expression of the coding sequence of interest is also contemplated, so long as the expression level is sufficient for a given purpose. By using a promoter with well-known properties, the expression level and pattern of the protein of interest after transfection or transformation can be optimized. Furthermore, the selection of a promoter that is regulated in response to a specific physiological signal can allow for inducible expression of the gene product.

[0118] Enhancers are genetic elements that increase transcription from a promoter located at a distant location on the same DNA molecule. Enhancers are organized much like promoters; that is, they are composed of many individual elements, each of which binds one or more transcriptional proteins. The fundamental distinction between enhancers and promoters is an operational one. The enhancer region as a whole must be capable of stimulating transcription from a distance, but this is not necessarily true for the promoter region or its components. On the other hand, a promoter must have one or more elements at a specific site and in a specific orientation that direct the initiation of RNA synthesis, whereas enhancers lack these particularities. Promoters and enhancers are often overlapping and contiguous, and often appear to have a very similar modular organization.

[0119] Below is a list of promoters / enhancers and inducible promoters / enhancers that can be used in combination with a nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter / enhancer combination (according to the Eukaryotic Promoter Database EPDB) can also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional gene expression construct.

[0120] The promoter and / or enhancer may be, for example, an immunoglobulin light chain, an immunoglobulin heavy chain, a T-cell receptor, HLA DQ a and / or DQ β, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, β-actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, α-fetoprotein, t-globin, β-globin, c-fos, c-HA-ras, insulin, neuronal cell junction molecule (NCAM), α1-antitrypsin, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated protein (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet derived growth factor (PDGF), SV40, polyoma, retrovirus, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia virus.

[0121] When a cDNA insert is used, one will typically desire to include a polyadenylation signal to achieve proper polyadenylation of the gene transcript. Any polyadenylation sequence may be used, such as human growth hormone and SV40 polyadenylation signals. Terminators are also contemplated as elements of the expression cassette. These elements may function to enhance message levels and minimize read-through from the cassette to other sequences.

[0122] There are several ways in which expression vectors can be introduced into cells. In certain embodiments, the expression construct comprises a virus or an engineered construct derived from a viral genome. Certain viruses have become attractive candidates for the transfer of foreign genes into mammalian cells due to their ability to enter cells via receptor-mediated endocytosis and integrate into the host cell genome to stably and efficiently express viral genes. They have a relatively low capacity for foreign DNA sequences and a limited host spectrum. Furthermore, their carcinogenic potential and cytopathic effects in permissive cells raise safety concerns. They can only accommodate up to 8kB of foreign genetic material, but can be easily introduced into a variety of cell lines and experimental animals.

[0123] One method for in vivo delivery is the use of an adenovirus expression vector. By "adenovirus expression vector" we mean to include a construct that contains sufficient adenovirus sequences to (a) support packaging of the construct and (b) express an engineered hMPV F protein cloned therein. In this context, expression does not require that the gene product be synthesized.

[0124] Expression vectors include genetically engineered forms of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB linear double-stranded DNA virus, allows for the replacement of large pieces of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retroviruses, adenoviral infection of host cells does not result in chromosomal integration, since adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Adenoviruses are also structurally stable, and no genome rearrangements have been detected after extensive amplification. Adenoviruses can infect virtually all epithelial cells, regardless of their cell cycle stage. So far, adenoviral infection appears to be associated only with mild diseases, such as acute respiratory disease, in humans.

[0125] Adenoviruses are particularly suitable for use as gene transfer vectors due to their moderate genome size, ease of manipulation, high titer, wide target cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for regulating the transcription of the viral genome and a few cellular genes. Expression of the E2 region (E2A and E2B) results in the synthesis of these proteins for viral DNA replication. These proteins are involved in DNA replication, expression of the late genes, and shut-off of the host cell. The products of the late genes, including most of the viral capsid proteins, are expressed only after significant processing of a single primary transcript produced by the major late promoter (MLP). The MLP (located at 16.8 mu) is particularly efficient during the late stages of infection, and all mRNAs produced from this promoter carry the 5'-triplet leader (TPL) sequence, making it the preferred mRNA for translation. In one system, recombinant adenovirus is generated from homologous recombination between the shuttle vector and the proviral vector. Due to the possibility of recombination between the two proviral vectors, wild-type adenovirus can be generated from this process. It is therefore essential to isolate single clones of virus from individual plaques and examine their genomic structure.

[0126] The generation and propagation of current adenoviral vectors that are replication-deficient rely on a unique helper cell line, called 293, which is transformed from human embryonic kidney cells with Ad5 DNA fragments and constitutively expresses E1 protein. Because the E3 region is not essential to the adenoviral genome, current adenoviral vectors, with the help of 293 cells, carry foreign DNA in either the E1, D3, or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing an extra capacity of about 2 kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the E1 and E3 regions, the maximum capacity of current adenoviral vectors is less than 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenoviral genome remains in the vector backbone, which is a source of vector-derived cytotoxicity. Additionally, E1-deleted viruses are only partially replication-deficient.

[0127] Helper cell lines may be derived from human cells such as human fetal kidney cells, muscle cells, hematopoietic cells, or other mesenchymal or epithelial human fetal cells. Alternatively, helper cells may be derived from cells of other mammalian species that are permissive for human adenovirus. Such cells include, for example, Vero cells or other mesenchymal or epithelial monkey fetal cells. As mentioned above, the preferred helper cell line is 293.

[0128] The adenovirus of the present disclosure is replication-deficient or at least conditionally replication-deficient.The adenovirus may be of any of the 42 different known serotypes or subgroups A to F.Adenovirus type 5 of subgroup C is one exemplary starting material that can be used to obtain the conditionally replication-deficient adenovirus vector for use in the present invention.

[0129] Other viral vectors may also be used as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus, adeno-associated virus (AAV), and herpes virus may also be used. They offer several attractive features for various mammalian cells.

[0130] In embodiments, in particular embodiments, the vector is an AAV vector. AAV is a small virus that infects humans and some other primate species. AAV is not currently known to cause disease. The virus induces a very mild immune response, further supporting its apparent lack of pathogenicity. In many cases, AAV vectors integrate into the host cell genome, which may be important for certain applications, but may also have undesirable consequences. AAV-based gene therapy vectors can infect both dividing and quiescent cells and persist in an extrachromosomal state without integration into the host cell genome, whereas in native viruses, some integration of the virus-carried genes into the host genome does occur. These characteristics make AAV a very attractive candidate for generating viral vectors for gene therapy and for generating isogenic human disease models. Recent human clinical trials using AAV for gene therapy in the retina have shown promise. AAV belongs to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. The virus is a small (20 nm), non-enveloped, replication-defective virus.

[0131] Wild-type AAV has attracted considerable interest from gene therapy researchers due to several characteristics. Chief among these is the virus's apparent lack of pathogenicity. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) on human chromosome 19. This characteristic makes it somewhat more predictable than retroviruses, which present the threat of random insertion and mutagenesis that sometimes leads to the development of cancer. The AAV genome integrates most frequently at the sites mentioned, while random integration into the genome occurs at a very low frequency. However, the development of AAV as a gene therapy vector eliminated this integration ability by removing the rep and cap from the vector's DNA. The desired gene together with a promoter to drive transcription of the gene is inserted between inverted terminal repeats (ITRs), which aid in the formation of concatemers in the nucleus after the single-stranded vector DNA is converted to double-stranded DNA by the host cell DNA polymerase complex. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact throughout the life of the host cell. In dividing cells, AAV DNA disappears with cell division because episomal DNA is not replicated along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at a very low frequency. AAVs also exhibit very low immunogenicity that appears to be limited to the generation of neutralizing antibodies, while they do not induce a well-defined cytotoxic response. This feature, together with the ability to infect quiescent cells, indicates their superiority over adenoviruses as vectors for human gene therapy.

[0132] The AAV genome is made of either positive or negative stranded single-stranded deoxyribonucleic acid (ssDNA) that is approximately 4.7 kilobases long. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs), rep and cap. The former consists of four overlapping genes that code for the Rep protein required throughout the AAV life cycle, while the latter contains overlapping nucleotide sequences for the capsid proteins VP1, VP2, and VP3, which interact together to form the capsid with icosahedral symmetry.

[0133] The inverted terminal repeat (ITR) sequences contain 145 bases each. They are so named because of their symmetry, which has been shown to be required for efficient multiplication of the AAV genome. A feature of these sequences that confers this property is their ability to form hairpins, which contribute to the so-called self-priming that allows primase-independent synthesis of the second DNA strand. The ITRs have also been shown to be required for both integration and rescue of AAV DNA into and from the host cell genome (chromosome 19 in humans) and for efficient encapsidation of AAV DNA in combination with the generation of fully assembled deoxyribonuclease-resistant AAV particles.

[0134] For gene therapy, the ITRs appear to be the only sequences required in cis next to the therapeutic gene, while the structural (cap) and packaging (rep) proteins can be delivered in trans. With this assumption, many methods have been established for the efficient production of recombinant AAV (rAAV) vectors containing reporter or therapeutic genes. However, it has also been published that the ITRs are not the only elements required in cis for efficient replication and encapsidation. A few research groups have identified sequences called cis-acting Rep-dependent elements (CAREs) that are internal to the coding sequence of the rep gene. CAREs have been shown to enhance replication and encapsidation when present in cis.

[0135] In some aspects, the disclosure provides pharmaceutical compositions containing one or more salts. The salts can be inorganic potassium or sodium salts, such as potassium chloride, sodium chloride, dibasic potassium phosphate, monobasic potassium phosphate, dibasic sodium phosphate, or monobasic sodium phosphate. The pharmaceutical compositions can include one or more phosphate salts, such as to produce a phosphate buffer. The phosphate buffer can include each of the phosphate salts to buffer the solution to about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 pH, or any range derivable therein.

[0136] In some aspects, the present disclosure includes one or more excipients formulated in the pharmaceutical composition. "Excipient" refers to a pharma- ceutically acceptable carrier, a relatively inert substance used to facilitate administration or delivery of an API to a subject, or to facilitate processing of an API into a drug formulation that can be pharma- ceutically used for delivery to a site of action in a subject. Additionally, these compounds may be used as diluents to obtain dosages that can be easily measured or administered to a patient. Non-limiting examples of excipients include polymers, stabilizers, surfactants, surface modifiers, solubility enhancers, buffers, encapsulating agents, antioxidants, preservatives, non-ionic wetting or clarifying agents, thickeners, and absorption enhancers.

[0137] In specific embodiments, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency for use in animals, and more particularly in humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water, and preferably include an adjuvant. Water is a particular carrier when the pharmaceutical composition is administered by injection, such as intramuscular injection. Saline solutions and aqueous solutions of dextrose and glycerol may also be used as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0138] The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations may contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceuticals are described in "Remington's Pharmaceutical Sciences." Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which may be oral, intravenous, intraarterial, buccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.

[0139] The engineered proteins of the disclosure described herein can be formulated for parenteral administration, e.g., for injection via intradermal, intravenous, intramuscular, subcutaneous, intratumoral, or even intraperitoneal routes. Antibodies can alternatively be administered by topical routes directly to mucous membranes, e.g., by nasal drops, inhalation, or by nebulizer. Pharmaceutically acceptable salts include acid salts and salts formed with inorganic acids, e.g., hydrochloric or phosphoric acid, or organic acids, e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases, e.g., isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0140] In general, the components of the composition of the present disclosure are supplied either separately or mixed together in unit dosage form, for example as dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent.When the composition is to be administered by inhalation, it can be dispensed by an inhalation bottle containing sterile water or saline of pharmaceutical grade.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0141] The compositions of the present disclosure may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0142] Dosing can be according to a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or in a booster immunization schedule. In a multiple dose schedule, the various doses may be given by the same or different routes. Multiple doses will typically be administered at least one week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

[0143] The compositions disclosed herein may be used to treat both children and adults. Thus, a human subject may be less than 1 year old, 1-5 years old, 5-16 years old, 16-55 years old, 55-65 years old, or at least 65 years old.

[0144] Preferred routes of administration include, but are not limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, and intraocular injections. Particularly preferred routes of administration include intramuscular, intradermal, and subcutaneous injections.

[0145] V. Immunodetection Methods In still further embodiments, the present disclosure relates to immunodetection methods for binding, purifying, harvesting, quantifying, and otherwise generally detecting hMPV F protein binding antibodies. While such methods can be applied in the conventional sense, another application is in quality control and monitoring of vaccine stocks, where the antibodies can be used to assess the amount or integrity (i.e., long-term stability) of the antigen. Alternatively, the methods may be used to screen various antibodies for appropriate / desired reactivity profiles.

[0146] Some immunodetection methods include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), immunoradiometric assays, fluorescent immunoassays, chemiluminescence assays, bioluminescence assays, and Western blots, to name a few. In particular, competitive assays for the detection and quantification of hMPV F protein binding antibodies are also provided. In general, immunobinding methods include obtaining a sample suspected of containing hMPV F protein binding antibodies and contacting the sample with an antigen according to the present disclosure, optionally under conditions effective to allow the formation of an immune complex.

[0147] These methods include methods for detecting or purifying hMPV F protein binding antibodies or hMPV F protein from a sample. The antigen is preferably linked to a solid support, such as in the form of a column matrix, and a sample suspected of containing hMPV F protein binding antibodies is applied to the immobilized antigen. Unwanted components are washed from the column, leaving hMPV F protein binding antibodies immunocomplexed to the immobilized antigen, which are then collected by removing the antigen from the column.

[0148] The immunobinding method also includes a method for detecting and quantifying the amount of hMPV F protein or related components in a sample, as well as detecting and quantifying any immune complexes formed during the binding process. Here, a sample suspected of containing hMPV F protein would be obtained, the sample would be contacted with an antibody that binds to the hMPV F protein or components thereof, and then the amount of immune complexes formed under specific conditions would be detected and quantified. In terms of antigen detection, the biological sample analyzed can be any sample suspected of containing hMPV F protein, such as tissue sections or specimens, homogenized tissue extracts, biological fluids including blood and serum (e.g., nasal swabs), or secretions such as feces or urine.

[0149] Contacting a selected biological sample with an antigen under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) generally involves simply adding an antigen composition to the sample and incubating the mixture for a period of time long enough for the antigen to form immune complexes with (i.e., bind to) the hMPV F protein-binding antibodies. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, is generally washed to remove any non-specifically bound antibody species so that only the specifically bound antibodies in the primary immune complexes are detected.

[0150] In general, detection of immune complex formation is well known in the art and can be achieved by application of a number of approaches. These methods are generally based on the detection of labels or markers, such as any of the following: radioactive, fluorescent, biological, and enzymatic tags. Patents relating to the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Of course, additional advantages may be found with the use of secondary binding ligands, such as secondary antibodies and / or biotin / avidin ligand binding arrangements, as is known in the art.

[0151] The antibody used in detection may itself be linked to a detectable label, and then this label is simply detected, thereby determining the amount of primary immune complexes in the composition. Alternatively, the first antibody that becomes bound in the primary immune complex may be detected using a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is often itself an antibody, and therefore may be referred to as a "secondary" antibody. The primary immune complex is contacted with a labeled secondary binding ligand or antibody under conditions effective to allow the formation of secondary immune complexes and for a period of time sufficient for this purpose. The secondary immune complex is then generally washed to remove any non-specifically bound labeled secondary antibody or ligand, and the label remaining in the secondary immune complex is then detected.

[0152] Further methods include the detection of primary immune complexes by a two-step approach. As described above, a second binding ligand, such as an antibody, having binding affinity for the antibody is used to form secondary immune complexes. After washing, the secondary immune complexes are again contacted with a third binding ligand or antibody having binding affinity for the second antibody under conditions effective to allow the formation of immune complexes (tertiary immune complexes) and for a period of time sufficient therefor. The third ligand or antibody is linked to a detectable label so that the tertiary immune complexes thus formed can be detected. This system may allow for signal amplification if desired.

[0153] One immunodetection method uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and then a second antibody is used to detect the biotin bound to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first-step antibody. If the target antigen is present, a portion of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing a second-step antibody against biotin. This second-step antibody is labeled with an enzyme that can be used to detect the presence of the antibody / antigen complex, for example, by histoenzymology using a chromogenic substrate. By suitable amplification, macroscopically visible conjugates can be produced.

[0154] Another known immunodetection method utilizes the immuno-PCR (polymerase chain reaction) technique. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, but instead of using multiple streptavidin and biotinylated DNA incubations, the DNA / biotin / streptavidin / antibody complex is washed out with a low pH or high salt buffer, which releases the antibody. The resulting wash is then used to perform a PCR reaction using suitable primers along with appropriate controls. At least in theory, the enormous amplification power and specificity of PCR can be exploited to detect single antigen molecules.

[0155] 1. ELISA Immunoassays, in their simplest and most direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it is readily understood that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analysis, and the like may also be used.

[0156] In one exemplary ELISA, the antibodies of the present disclosure are immobilized on a selected surface that exhibits protein affinity, such as the wells of a polystyrene microtiter plate. A test composition suspected of containing hMPV F protein is then added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection may be achieved by the addition of another anti-hMPV F protein antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection may also be achieved by the addition of a second anti-hMPV F protein antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, of which the third antibody is linked to a detectable label.

[0157] In another exemplary ELISA, a sample suspected of containing hMPV F protein (e.g., potentially infected cells) is immobilized on a well surface and then contacted with an anti-hMPV F protein antibody of the present disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-hMPV F protein antibody is detected. If the first anti-hMPV F protein antibody is linked to a detectable label, the immune complex may be detected directly. Again, the immune complex may be detected using a second antibody that has binding affinity for the first anti-hMPV F protein antibody, where the second antibody is linked to a detectable label.

[0158] Regardless of the format used, ELISAs have certain features in common, such as coating, incubating, and binding, removal of non-specifically bound species by washing, and detection of bound immune complexes, which are described below.

[0159] When coating a plate with either antigen or antibody, the wells of the plate are generally incubated with a solution of the antigen or antibody, either overnight or for a specified period of time. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with respect to the test antiserum. These include solutions of bovine serum albumin (BSA), casein, or milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, thus reducing the background caused by nonspecific binding of the antiserum onto the surface.

[0160] In ELISA, it is perhaps more common to use secondary or tertiary detection means rather than a direct procedure. Thus, after binding of protein or antibody to the well, reducing background by coating with a non-reactive material, and removing unbound material by washing, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.

[0161] By "under conditions effective to allow immune complex (antigen / antibody) formation" is meant conditions that preferably include diluting the antigen and / or antibody with a solution such as BSA, bovine gamma globulin (BGG), or phosphate buffered saline (PBS) / Tween. These added agents also tend to assist in reducing nonspecific background.

[0162] "Suitable" conditions also mean that incubation is at a temperature or for a period of time sufficient to allow for effective binding. Incubation steps are typically at temperatures preferably between about 25°C and 27°C for about 1-2-4 hours, or may be overnight at about 4°C.

[0163] All incubation steps in the ELISA are followed by washing the contact surface to remove uncomplexed material. A preferred washing procedure involves washing with a solution such as PBS / Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the presence of even minute amounts of immune complexes can be determined.

[0164] To provide a means of detection, the second or third antibody will have a label associated therewith to allow detection. Preferably, this will be an enzyme that will generate color development upon incubation with an appropriate chromogenic substrate. Thus, for example, it may be desirable to contact or incubate the first and second immune complexes with urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase conjugated antibodies for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation at room temperature for 2 hours in a PBS-containing solution such as PBS-Tween).

[0165] After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate, such as, for example, urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), or H2O2 (if peroxidase is the enzyme label). Quantitation is then achieved by measuring the degree of color produced, for example, using a visible spectrum spectrophotometer.

[0166] 2. Western Blot Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by polypeptide length (denaturing conditions) or by the 3D structure of the protein (native / non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF) where they are probed (detected) using antibodies specific for the target protein.

[0167] Samples may be taken from whole tissues or from cell cultures. In most cases, solid tissues are first mechanically disrupted using a blender (for larger sample volumes), a homogenizer (for smaller volumes), or by sonication. Cells may also be disrupted by one of the mechanical methods mentioned above. Various combinations of detergents, salts, and buffers may be used to promote cell lysis as well as to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent digestion of the sample by its own enzymes. Tissue preparation is often performed at low temperatures to avoid protein denaturation.

[0168] Gel electrophoresis is used to separate the proteins of a sample. The separation of proteins can be by isoelectric point (pI), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful method for determining proteins. It is also possible to use two-dimensional (2D) gels, which spread the proteins from a single sample in two dimensions. Proteins are separated according to their isoelectric point (the pH at which their net charge is neutral) in the first dimension and according to their molecular weight in the second dimension.

[0169] To make the proteins accessible to antibody detection, they are transferred from within the gel onto a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed on top of the gel and a stack of filter papers is placed on top of it. The entire stack is placed in a buffer solution, which moves up the paper by capillary action, carrying the proteins with it. Another method for transferring proteins is called electroblotting, which uses an electric current to draw the proteins from the gel onto a PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the structure they had in the gel. As a result of this blotting process, the proteins are exposed on a surface layer for detection (see below). Both types of membranes are chosen because of their nonspecific protein binding properties (i.e., they bind all proteins equally well). Protein binding is based on hydrophobic interactions as well as charged interactions between the membrane and the protein. Nitrocellulose membranes are cheaper than PVDF but are much more fragile and do not hold up well to repeated probing. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be confirmed by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once transferred, the protein is detected using a labeled primary antibody, or an unlabeled primary antibody followed by indirect detection using labeled Protein A or a secondary labeled antibody that binds to the Fc region of the primary antibody.

[0170] 3. Immunohistochemistry The antibodies of the present disclosure may also be used in conjunction with both fresh frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). Methods for preparing tissue blocks from these particulate specimens have been used successfully in previous IHC studies of various prognostic factors and are well known to those of skill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0171] Briefly, frozen sections may be prepared by rehydrating 50 ng of frozen "ground" tissue in phosphate-buffered saline (PBS) at room temperature in small plastic capsules; pelleting the particles by centrifugation; resuspending them in viscous embedding medium (OCT); mixing by inversion and / or pelleting again by centrifugation; flash-freezing in isopentane at -70°C; cutting off the plastic capsule and / or removing the frozen cylinder from the tissue; fixing the tissue cylinder onto a cryostat microtome chuck; and / or cutting 25-50 serial sections from the capsule. Alternatively, frozen whole tissue samples may be used for cutting serial sections.

[0172] Permanent sections may be prepared by a similar method, which involves rehydrating 50 mg of sample in a plastic microcentrifuge tube; pelleting; resuspending in 10% formalin and fixing for 4 hours; washing / pelleting; resuspending in warm 2.5% agar; pelleting; chilling in ice water to harden the agar; removing the tissue / agar block from the tube; infiltrating and / or embedding the block in paraffin; and / or cutting up to 50 permanent serial sections. Again, whole tissue samples may be substituted.

[0173] 4. Immunodetection Kit In yet a further embodiment, the present disclosure relates to an immunodetection kit for use with the immunodetection method described above. The antibody may be included in the kit, since the antibody may be used to detect the hMPV F protein. The immunodetection kit would therefore include, in a suitable container means, a first antibody that binds to the hMPV F protein, and optionally an immunodetection reagent. Alternatively, the hMPV F protein antigen may be used to detect the hMPV F protein-bound antibody. In this case, the immunodetection kit would therefore include, in a suitable container, the hMPV F protein antigen.

[0174] In certain embodiments, the antibody or antigen may be pre-bound to a solid support, such as a column matrix and / or a well of a microtiter plate. The immunodetection reagents of the kit may take any one of a variety of forms, including a detectable label associated with or linked to the antibody. Detectable labels associated with or bound to a secondary binding ligand are also contemplated. An exemplary secondary ligand is a secondary antibody that has binding affinity for the first antibody.

[0175] Further suitable immunodetection reagents for use in the present kits include bicomponent reagents that include a second antibody having binding affinity for the first antibody together with a third antibody having binding affinity for the second antibody, where the third antibody is linked to a detectable label. As noted above, several exemplary labels are known in the art, and any such label may be used in connection with the present disclosure.

[0176] The kit may further comprise a suitably aliquoted composition of hMPV F protein, whether labeled or unlabeled, that may be used to generate a standard curve for the detection assay. The kit may contain the antibody-label conjugate either in fully conjugated form, in the form of an intermediate, or as a separate moiety that is conjugated by the user of the kit. The components of the kit may be packaged either in aqueous media or in lyophilized form.

[0177] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody may be placed, or preferably suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials may be held.

[0178] 5. Flow Cytometry and FACS The antibody of the present disclosure may also be used in flow cytometry or FACS. Flow cytometry is a laser or impedance-based technology used in many detection assays, including cell counting, cell sorting, biomarker detection, and protein engineering. This technology allows for simultaneous multiparameter analysis of the physical and chemical properties of up to thousands of particles per second by suspending cells in a flowing fluid and passing them through an electrical detection device. Flow cytometry is routinely used in the diagnosis of disorders, but has many other applications in basic research, clinical practice, and clinical trials.

[0179] Fluorescence activated cell sorting (FACS) is a specialized type of cytometry. It provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence properties of each cell. In general, the technique involves drawing a cell suspension into the center of a rapidly flowing narrow liquid stream. The flow is arranged so that the cells are separated by a large distance relative to the diameter of the cells. A vibration mechanism causes the stream of cells to break up into individual droplets. Just before the stream splits into droplets, it passes through a fluorescence measurement station where the fluorescence of each cell is measured. A charged ring is placed at the exact point where the stream splits into droplets. A charge is placed on the ring just before the fluorescence intensity is measured, and an opposite charge is captured on the droplets as they split from the stream. The charged droplets then fall through an electrostatic deflection system that deflects them into containers based on their charge.

[0180] In certain embodiments, for use in flow cytometry or FACS, the antibodies of the present disclosure are labeled with a fluorophore and then bound to cells of interest, which are analyzed with a flow cytometer or sorted by a FACS machine. EXAMPLES

[0181] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the examples that follow are representative of techniques discovered by the inventors to work well in the practice of the invention, and therefore may be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0182] Materials and Methods Protein expression and purification. All hMPV F variants were constructed in plasmids containing His and StrepTag II tags by Gibson assembly and validated by DNA sequencing. Plasmids encoding F variants and furin were used in a 4:1 ratio to co-transfect FreeStyle 293F cells (ThermoFisher) with polyethylenimine (PEI). Three hours after transfection, kifunensine was added to a final concentration of 5 μM, and for large-scale transfections, Pluronic F-68 was added to a final concentration of 0.1 v / v%. Six days after transfection, the filter-sterilized supernatant was applied to a StrepTactin column (IBA) for initial purification, followed by a Superose 6 10 / 300 or Superdex 200 10 / 300 size-exclusion column (SEC) (GE Healthcare) to obtain monodisperse fractions in SEC buffer (2 mM Tris pH 8.0, 200 mM NaCl, and 0.03% NaN3). Single- and combination-substituted hMPV F variants were purified from 40 mL cell cultures for initial variant screening and characterization. Large-scale expression of DS-CavEs2 was purified using a Superose 6 16 / 600 column.

[0183] Plasmids encoding the heavy and light chains of MPE8 were co-transfected with PEI in a 1:1 ratio into FreeStyle 293F cells. A stop codon was introduced before the hinge region of the heavy chain to generate the antigen-binding fragment (Fab) of MPE8. To purify MPE8 Fab, the filter-sterilized supernatant was first applied to a CaptureSelect™ IgG-CH1 affinity matrix (ThermoFisher) and then to a Superdex 200 column (GE Healthcare) to obtain monodisperse fractions in PBS buffer. All protein samples were concentrated to 5-10 mg / ml, then flash frozen in liquid nitrogen and then stored at -80°C.

[0184] Differential Scanning Calorimetry. Purified hMPV variants at a final concentration of 1 μM were mixed with 5x final concentration of SYPRO Orange protein gel stain (ThermoFisher) in a white opaque 96-well plate (VWR). The mixtures were then measured by continuous fluorescence scanning (λex=465 nm, λem=580 nm) using a Roche LightCycler 480 II with a temperature ramp rate of 4.4°C / min and a temperature range of 25°C to 95°C. Data were plotted as the derivative of the melting curve.

[0185] MPE8 binding analysis by biolayer interference. To examine the integrity of the epitope of hMPV F under various temperature stresses, aliquots of DS-CavEs2 were incubated at 37°C, 50°C, or 70°C for 30 min in a thermocycler or left at 4°C for 2.5 months and then tested for MPE8 binding by BLI using an Octet RED96e (ForteBio). Briefly, an anti-human Fab-CH1 second generation (FAB2G) biosensor (ForteBio) was used to capture an equal amount of MPE8 Fab at a concentration of 30 nM in a buffer consisting of 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.005 v / v% Tween 20, and 1 mg / ml BSA, and the biosensor with captured MPE8 was then immersed in 50 nM heat-treated DS-CavEs2 to measure the association rate. After a 600 s association step, a 600 s dissociation step was performed in wells containing buffer only. Binding curves were baseline aligned and buffer subtracted.

[0186] Negative stain EM. Post-fusion hMPV F was heat treated at 70°C for 10 min and then applied to CF-400-Cu grids (Electron Microscopy Sciences) that were plasma cleaned with 4:1 O2 / H2 for 45 s in a Solarus 950 plasma cleaner (Gatan). Grids were stained using methylamine tungstate (Nanoprobes). Pre-fusion stabilized hMPV F was incubated with a 2-fold molar excess of MPE8 Fab in 1x PBS for 30 min at room temperature. hMPV-F:Fab complexes were diluted to a concentration of 0.03 mg / mL in 2 mM Tris (pH 8.0), 200 mM NaCl, and 0.02% NaN3, and then deposited on CF-400-Cu grids. Grids were imaged at 92,000x magnification (corresponding to a calibrated pixel size of 1.63 Å / pixel) on a Talos F200C TEM microscope (Thermo Fisher Scientific) equipped with a Ceta 16M detector. CTF estimation and particle collection were performed in a cisTEM (Grant et al., 2018). Particles were then sent to cryoSPARC v2.15.0 for 2D classification (Punjani et al., 2017).

[0187] X-ray crystallography of prefusion stabilized F and complexed with MPE8. DS-CavEs2 crystals were generated by hanging drop vapor diffusion by mixing 500 nl of DS-CavEs2 (10 mg / ml) with 500 nl of reservoir solution containing 0.1 M MES (pH 6.0) and 12% (v / v) PEK 20k. Crystals were immersed in a reservoir supplemented with 20% glycerol and frozen in liquid nitrogen. Diffraction data were collected to 2.5 Å at SBC beamline 19ID (Advanced Photon Source, Argonne National Laboratory). Crystals of DSx2 in complex with MPE8 Fab were grown by sitting drop vapor diffusion by mixing 100 nl of the complex (5.4 mg / ml) with 50 nl of reservoir solution containing 10% (v / v) isopropanol, 0.1 M HEPES (pH 7.5), and 20% (w / v) PEG4000. Crystals were frozen directly in liquid nitrogen without the addition of cryoprotectant. Diffraction data for a single crystal diffracting to 2.2 Å were collected at SBC beamline 19ID (Advanced Photon Source, Argonne National Laboratory). Data were indexed and integrated with iMOSFLM (Battye et al., 2011) and then merged and scaled using Aimless (Evans & Murshudov, 2013). Molecular replacement was performed in Phaser (McCoy et al., 2007) and the model was then subjected to multiple rounds of model building and refinement in Coot (Emsley & Cowtan, 2004) and Phenix (Adams et al., 2002), respectively. Data collection and refinement statistics can be found in Table 2.

[0188] Table 2. Crystallographic data collection and refinement statistics TIFF0007660671000005.tif216161The values ​​in parentheses are for the outermost shell with the highest resolution.

[0189] Enzyme-linked immunosorbent assay. A panel of hMPV F prefusion-specific (MFP10, Ac967, Ac1025, and MPE8) (Corti et al., 2013) or non-prefusion-specific (MF11, MF14) (Battles et al., 2017) antibodies were individually immobilized on 96-well microtiter plates overnight at 4 °C. After a blocking step with 1% BSA in PBS, serial dilutions starting with 4 ng of heat-treated or untreated postfusion hMPV F were applied to the antibody-coated wells for 1 h at room temperature. Unbound F was removed by washing three times with 0.1% Tween-20 in PBS. Bound F was then detected by adding anti-His tag mAb conjugated with horseradish peroxidase (HRP) (Bio-Rad), followed by three washes with 0.1% Tween-20 in PBS. HRP substrate (Sigma) was then added for color development, and the optical density was read at 492 nm using an ELISA plate reader.

[0190] Example 1 - Structure-based design of prefusion stabilized hMPV F Similar to other class I viral fusion glycoproteins, the prefusion hMPV F protein exhibits a metastable state and easily converts to a stable postfusion conformation during induction. To stabilize the F protein in the prefusion state, a proline substitution A185P was introduced into the trimer apical helix-loop-helix region. Although this substitution allowed the prefusion structure of hMPV to be obtained, the low expression level of this construct hindered its potential application as a vaccine candidate. Therefore, the H368N substitution (Schowalter et al., 2009), previously shown to increase protein expression, similar to the previously described prefusion stabilized F protein BV-115 (Battles et al., 2017), was also used. From this basic construct, 97 variants were designed based on the prefusion (PDB ID: 5WB0) and postfusion (PDB ID: 5L1X) structures of hMPV F, and each variant was then expressed and characterized in terms of production yield, monodispersity, thermal stability, and antigenicity. Exemplary variants are shown in Table 3. Strategies used included disulfide bonds to lock regions that move substantially during pre- to post-fusion transition, hydrophobic residues to fill internal cavities, polar residues to counter internal charge imbalance, and proline substitutions that favor the pre-fusion conformation and disfavor F1 refolding. Regions that move more than 5 Å during transition are highlighted in blue in Figure 1A (Battles et al., 2017), and the best substitutions from each category are shown in Figure 1B. Overall, 36 variants with single substitutions increased protein expression, with many of these variants exhibiting higher thermostability.

[0191] Table 3: Exemplary F protein variants TIFF0007660671000006.tif223151TIFF0007660671000007.tif221152TIFF0007660671 000008.tif234161TIFF0007660671000009.tif232157TIFF0007660671000010.tif24161

[0192] Example 2 - Single Substitution F Variants The expression profiles of 42 individual variants are summarized in Figure 2A, and size exclusion chromatography (SEC) traces of selected variants from each design category are shown in Figure 2B. Note that throughout this example, the protein yield fold change values ​​provided in parentheses refer to the original values ​​determined when each of the constructs was designed. The values ​​given in Table 3 are from experiments in which all constructs were re-expressed and purified in parallel, and for each experiment the area under the curve in the SEC chromatogram is compared to the base construct control. In the re-expression, all constructs expressed better than in the original expression. The lower yield of the base construct in the original expression was the main factor behind the higher yield fold values ​​in the original values.

[0193] Nine variants with proline substitutions were designed, expressed, and characterized. Six of the nine variants enhanced protein expression (Fig. 2A,B). Two variants, A107P and A113P, both located within the fusion peptide, showed a 2.9-fold and 1.9-fold increase in protein yield compared to the base construct, respectively [5.1-fold and 3.3-fold increase] (Fig. 2B). Note that A107P showed a rightward shift in the SEC peak compared to the base construct, suggesting a more compact trimeric structure (Fig. 2B). The only substitution in domain I (site I), designed to cap the helix, A344P, showed a two-fold increase in protein yield but slightly decreased the Tm by about 0.6°C. We also attempted to engineer D461P to cap α10 of HRB, which resulted in a 1.8-fold increase in protein yield [a 2.2-fold increase] and a rightward shift in the SEC peak compared to the base construct, indicative of a more compact trimeric structure. Finally, T114P, E146P, and V148P all increased protein expression.

[0194] Salt bridges were engineered into the MPV F protein to neutralize the internal charge imbalance. L73E and L219K increased protein expression by 1.5-fold and 1.4-fold [2.6-fold and 1.4-fold], respectively (Fig. 2A,B), and both showed longer retention times in SEC. A pair of substitutions, L66D / K188R, was introduced, which increased expression by 1.6-fold. E453Q showed a 1.9-fold increase [2.8-fold increase] in protein yield, but also showed a significant rightward shift of the trimer peak in SEC, suggesting that the trimer may be in a relatively closed conformation (Fig. 2C). In addition, some cavity-filling variants showed beneficial effects on the stabilization of the F protein. For example, V231I in domain IIIb (site II) increased protein expression by 2.7-fold [3.6-fold] (Fig. 2A,B). One other variant, S376T, also showed a 2.4-fold increase in protein yield over the base construct. Two other variants, S149I and G366S, showed a 2.3-fold and 1.7-fold increase in protein expression [2.3-fold and 2.9-fold increase] over the base construct and eluted as monodisperse peaks on SEC.

[0195] Prefusion-stabilizing disulfide bridges were tested for improving protein expression and thermal stability of hMPV F. Two examples, L110C / N322C and T365C / V463C, showed a 2.5-fold and 1.7-fold increase in protein yield [2.8-fold and 2.4-fold increase] and a 5.6°C and 6.2°C increase in Tm, respectively, compared to the base construct (Fig. 2A,B,C). Variant A116C / A338C also improved thermal stability but did not increase expression compared to the base construct. Meanwhile, A140C / A147C and T127C / N153C, all located in domain IIIa (site V) (i.e., α2, α3, β3, and β4 (Fig. 1B)), significantly increased protein expression by 3.1-fold and 2.8-fold [6.0-fold and 4.8-fold], respectively (Fig. 2A,B). T127C / N153C moderately improved thermostability (Figure 2C). The V104C / N457C variant showed reduced furin cleavage (Figure 2D), a 4°C increase in Tm, and reduced protein expression levels compared to the base construct (Figure 2B,C). Overall, about 20 of the 42 variants increased protein expression (Figure 2A), and of these, 6 variants showed increased thermostability.

[0196] Example 3 - Multi-substituted F variants Combinations of single substitutions were engineered into three different variants containing either two disulfide bonds, one disulfide bond and one cavity-filling, or one disulfide bond and one salt bridge. In all three combination variants, the substitutions T365C / V463C were included due to their significant improvement in thermal stability. All three variants (T127C / N153C / T365C / V463C, V231I / T365C / V463C, L219K / T365C / V463C) showed an additional 1.2-fold, 1.9-fold, and 1.2-fold increase in protein yield over its parent construct T365C / V463C, and the Tm of the variant containing two disulfide bonds was further increased by 1.7°C compared to the parent T365C / V463C construct and 6.4°C compared to the base construct (Figure 4A,D). The T127C / N153C / T365C / V463C variant was named DSx2 and was further engineered into it with either L219K or V231I. DSx2 / L219K and DSx2 / V231I showed an additional 1.1-fold and 1.6-fold increase compared to DSx2 (Figures 4B, 6). Furthermore, the variant containing all beneficial mutations (T127C / N153C / T365C / V463C / L219K / V231I) showed the highest expression level of all constructs with an additional 1.8-fold increase compared to DSx2, producing 16 mg of protein from 1 L of FreeStyle 293-F cells, with a Tm of 60.7°C (Figures 4D, 5). This variant was newly named DS-CavEs, and one disulfide bond design (L110C / N322C) was further engineered into DS-CavEs to generate a penta-substituted variant (T127C / N153C / T365C / V463C / L219K / V231I / L110C / N322C) named MM-1. For MM-1, protein expression was reduced by 25% compared to DS-CavEs, and showed a substantial improvement in Tm (67.6°C). Another variant, designated MM-1H (T127C / N153C / T365C / V463C / L219K / V231I / / L110C / N322C / N386H), was generated by reverting H368N to wild-type His368 compared to MM-1.For MM-1H, protein expression was reduced by 25% compared to DS-CavEs and showed an increased Tm (65.2°C) (Figure 4C,D). This enhanced thermostability due to the introduction of L110C / N322C may be highly advantageous for vaccine antigens. Due to the benefit exhibited by the additional disulfide bond, the introduction of A140C / A147C was explored based on its favorable expression profile as a single substitution (Figure 2B). This construct, designated MM-4H, showed minimal difference in expression yield and an increase in Tm of 1.0°C (Figure 4C,D).

[0197] Furthermore, A140C / A147C and E453Q substitutions were introduced into MM-1H (which also corresponds to the introduction of E453Q into MM-4H) and named DS-CavEs2. Large-scale expression of DS-CavEs-2 yielded 15.7 mg of prefusion-stabilized F from 1 L of FreeStyle 293-F cells (Figure 4E). The antigenic surface of DS-CavEs2 is well preserved. The affinity of DS-CavEs2 for the prefusion-specific antibody MPE8 (Wen et al., 2017) did not change after incubation at 50°C for 30 min and is comparable to that of the base construct (Figure 4F). From nsEM analysis, DS-CavEs2 appears to be a well-folded prefusion trimer, with each protomer bound by MPE8 Fab. DS-CavEs2, which has additional substitutions, enhanced expression by more than 10-fold and showed the highest thermostability (Tm 71.8°C) in the correct pre-fusion conformation (Figure 4D), suggesting its potential for vaccine development.

[0198] Example 4 - Crystal structure of MPE8-bound DSx2 construct To determine the effect of multiple substitutions on the conformation of hMPV F for the combinatorial mutant DSx2 construct, a crystal structure of the protein complexed with the prefusion-specific antibody MPE8 was obtained. The protein complex crystallized in space group P2 and diffracted X-rays to 2.2 Å resolution. After model building and refinement, the structure had R work and R freeThese improved to 21.3% and 24.2%, respectively, upon further refinement of the structure (Table 2). Compared to the previously determined hMPV F structure (PDBID: 5WB0), DSx2 retained the prefusion conformation with an overall RMSD of 1.8 Å for 427 Cα residues (Figure 9).

[0199] Example 5 - Structure of MPE8-bound DS-CavEs2 The crystal structure of DS-CavEs2 was determined to 2.5 Å resolution from crystals in space group P21 (Table 2). In the absence of MPE8, DS-CavEs2 retained the prefusion conformation with an RMSD of 2.3 Å over 428 Cα atoms shared with PDBID:5WB0 (Figure 8A). Clear electron density was observed for all disulfide bond substitutions (Cys127 / Cys153, Cys140 / Cys147, Cys110 / Cys322, Cys365 / Cys463) and the cavity-filling substitution (I231). Superposition of the membrane-distal half of DS-CavEs2 (sites II, V, and φ) with the previous hMPV F structure (PDBID:5WB0) revealed a substantial shift of antigen site IV toward the central three-fold axis (Figure 8A). Superposition of site IV from both structures showed the presence of a rigid body bending at the center of two long β-strands (β1 and β22) connecting the upper and lower halves of the F protein (Figure 8A). Similar to the DSx2 structure, the two disulfide bond substitutions at site V did not change the local conformation. In contrast, the Cys365 / Cys463 substitutions pulled the α10 helix away from the central three-fold axis, thus changing the downward trajectory of the HRB (Figure 8A). Negative staining electron microscopy (nsEM) analysis was performed on MPE8 complexed to DS-CavEs2. After 2D class averaging, multiple classes showed DS-CavEs2 as a well-folded prefusion trimer bound by two or three MPE8 Fabs, demonstrating that DS-CavEs2 can adopt a trimeric conformation (Figure 8B).

[0200] Example 6 - Prefusion stabilized hMPV F variants as immunogens To investigate whether prefusion stabilized hMPV F variants function as immunogens, BALB / c mice are immunized with either CpG-augmented prefusion (e.g., base constructs, DSx2 and DS-CavEs2) or postfusion F antigens at 3-week intervals. Serum is collected 10 days after the second immunization. Prefusion stabilized F constructs are expected to elicit higher neutralizing antibody titers against hMPV A1 and / or hMPV B1 compared to postfusion F antigens.

[0201] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods and in the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results are achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0202] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are expressly incorporated herein by reference. TIFF0007660671000011.tif202160TIFF0007660671000012.tif238160TIFF0007660671000013.tif238160TIFF0007660671000014.tif48160

Claims

1. A nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence of an engineered protein comprising a metapneumovirus (MPV) F protein ectodomain having at least 90% identity to amino acids 19-489 of any one of SEQ ID NOs:4-7, wherein the engineered protein comprises an engineered disulfide bond comprising a pair of cysteine ​​substitutions corresponding to T127C and N153C, and / or L110C and N322C relative to any one of SEQ ID NOs:4-7, and wherein the nucleic acid molecule is an mRNA molecule.

2. The nucleic acid molecule of claim 1, wherein the engineered protein further comprises substitutions at positions corresponding to L219 and / or V231.

3. The nucleic acid molecule of claim 1, wherein the engineered protein further comprises substitutions corresponding to L219K and V231I.

4. The nucleic acid molecule of claim 1, wherein the engineered protein further comprises a pair of cysteine ​​substitutions corresponding to T365C and V463C.

5. The nucleic acid molecule of claim 1, wherein the engineered protein comprises a pair of cysteine ​​substitutions corresponding to T127C and N153C, a pair of cysteine ​​substitutions corresponding to T365C and V463C, and a substitution corresponding to L219K.

6. The nucleic acid molecule of claim 1, wherein the engineered protein comprises a pair of cysteine ​​substitutions corresponding to T127C and N153C, a pair of cysteine ​​substitutions corresponding to T365C and V463C, and a substitution corresponding to V231I.

7. The nucleic acid molecule of claim 1, wherein the engineered protein further comprises a proline substitution corresponding to A / D185P.

8. The nucleic acid molecule of claim 1, wherein the engineered protein further comprises a substitution of residues corresponding to positions 87-104 of any one of SEQ ID NOs: 4-7 with GGGGSGGGGGSR (SEQ ID NO: 8).

9. The nucleic acid molecule of claim 1, wherein the engineered protein further comprises electrostatic interaction substitutions at positions corresponding to L66, L73, N145, Q195, E453, K188, H368, D461, T49, and / or V262.

10. The nucleic acid molecule of claim 9, wherein the electrostatic interaction substitutions include substitutions corresponding to L66N, L73E, N145E, Q195K, E453Q, L66D, K188R, H368R, D461E, T49E, and / or V262D.

11. The nucleic acid molecule of claim 1, wherein the engineered protein comprises substitutions corresponding to T127C, N153C, T365C, V463C, L219K, and V231I.

12. The nucleic acid molecule of claim 1, wherein the engineered protein comprises substitutions corresponding to T127C, N153C, A185P, T365C, V463C, L219K, and V231I.

13. The nucleic acid molecule of claim 1, wherein the engineered protein comprises the following substitutions: T127C, N153C, A185P, T365C, V463C, L219K, V231I, and RQSR (residues corresponding to positions 99-102 of any one of SEQ ID NOs:4-7) to RRRR (SEQ ID NO:10).

14. The nucleic acid molecule of claim 1, wherein the engineered protein comprises substitutions corresponding to L110C, T127C, A140C, A147C, N153C, L219K, V231I, N322C, T365C, E453Q, and V463C.

15. The nucleic acid molecule of claim 1, wherein the engineered protein comprises substitutions corresponding to L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, E453Q, and V463C.

16. The nucleic acid molecule of claim 1, wherein the engineered protein comprises the following substitutions: L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, E453Q, V463C, and RQSR (residues corresponding to positions 99-102 of any one of SEQ ID NOs:4-7) to RRRR (SEQ ID NO:10).

17. The nucleic acid molecule of claim 1, wherein the engineered protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

14.

18. The nucleic acid molecule of claim 1, wherein the engineered protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

15.

19. The nucleic acid molecule of claim 1, wherein the engineered protein comprises a polypeptide sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

2.

20. The nucleic acid molecule of claim 1, wherein the engineered protein comprises an hMPV F protein ectodomain having at least 95% identity to amino acids 19-489 of SEQ ID NO:

4.

21. The nucleic acid molecule of claim 1, wherein the engineered protein is fused to a trimerization domain.

22. 22. The nucleic acid molecule of claim 21, wherein the trimerization domain comprises the trimerization domain of T4 fibritin.

23. The nucleic acid molecule of claim 1, wherein the engineered protein is fused to a transmembrane domain.

24. The nucleic acid molecule of claim 1, wherein the engineered protein comprises an N-terminal signal sequence.

25. A pharmaceutical composition comprising a pharma- ceutically acceptable carrier and the nucleic acid molecule of claim 1.

26. 26. The pharmaceutical composition of claim 25, further comprising an adjuvant.

27. A pharmaceutical composition for preventing metapneumovirus (MPV) infection or a disease associated with metapneumovirus infection in a subject, comprising the nucleic acid molecule of claim 1.

28. A nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of an engineered protein comprising a metapneumovirus (MPV) F protein ectodomain having the amino acid sequence of SEQ ID NO: 15, wherein the nucleic acid molecule is an mRNA molecule.

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