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 priority from U.S. Provisional Application No. 63 / 089,978, filed on 9 October 2020, the entire contents of which are incorporated herein by reference.

[0002] Sequence listing reference This application includes a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. A copy of the ASCII file, created on 7 October 2021, is named UTFBP1250WO_ST25.txt and is 50.6 bytes in size.

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

[0004] 2. Explanation of related technologies Human metapneumovirus (hMPV) is a respiratory virus belonging to the Pneumoviridae family that had been transmitted in humans for at least half a century prior to its discovery in 2001 (van den Hoogen et al., 2001). Infection occurs almost universally by the age of five, and the burden of reinfection continues throughout life (van den Hoogen et al., 2001). However, infants (6-12 months), the elderly, and immunocompromised individuals have an increased risk of hospitalization due to more severe illnesses such as pneumonia and bronchitis (Deffrasnes et al., 2007). Despite the disease burden presented by hMPV, there are no approved vaccines or treatments for prevention or therapy. As a member of the Pneumoviridae family (recently elevated from a subfamily within Paramyxoviridae (paramyxoviruses)), hMPV is an enveloped negative-strand RNA virus. Viruses within this family encode three membrane proteins expressed on their surface. In the case of hMPV, these are small hydrophobic (SH), adherent (G), and fusion (F) proteins (Shafagati & Williams, 2018).

[0005] As a class I virus fusion glycoprotein, hMPV F is initially translated as a single polypeptide precursor (F0). Proteolytic cleavage converts F0 to disulfide-linked F2 and F1 subunits (Figure 1A). Three F2 / F1 heterodimers then associate to form a metastable prefusion trimer that constitutes the active protein. In cell cultures, this proteolytic activation can be achieved by adding trypsin, which cleaves the protein at single-nucleotide cleavage sites (van den Hoogen et al., 2001; Skiadopoulos et al., 2006; Schickli et al., 2005). During spontaneous infection, hMPV F0 is cleaved by trypsin-like extracellular serine proteases such as TMPRSS2, but the extent to which this occurs in producing cells compared to target cells is not clearly understood (Shirogane et al., 2008). The N-terminus of the mature F1 subunit contains a hydrophobic sequence called the fusion peptide, located within the internal cavity of the pre-fusion 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 expanding and opening, or "breathing" (Bangaru et al., 2019, Watanabe et al., 2019, Gilman et al., 2019). Recently, a human antibody targeting the trimer interface of hMPV F has been described, suggesting that pre-fusion hMPV F undergoes this transient opening in vivo (Huang et al., 2020). To facilitate membrane fusion, the metastable pre-fusion F protein undergoes a substantial conformational change to release the fusion peptide and extend into the host cell membrane. This unstable pre-hairpin intermediate folds over itself again to form a highly stable 6-helix bundle in the so-called post-fusion structure, consisting of trimers of the N-terminal and C-terminal heptad repeats (HRA and HRB, respectively) (Mas et al., 2016).Given its essential role in viral entry, vaccine candidates against hMPV generally include the F protein. In other words, potential vaccination strategies against hMPV involve targeting its fusion (F) glycoprotein, which is essential for viral infection. However, there remains a need for stabilized F proteins that can be used to identify drug candidates and stimulate an effective immune response against the F protein. [Overview of the project]

[0006] Accordingly, this specification provides a pre-fusion structural variant of thermally stable hMPV F. The introduction of a disulfide bond by introducing a pair of cystine mutations improved protein expression and thermal stability. Individual hydrophobic, electrostatic interaction, and charge reduction mutations are also beneficial. In addition, combining multiple beneficial mutations further improved desired protein properties.

[0007] In one embodiment, the Specified Information provides an engineered protein comprising a metapneumovirus (MPV) F protein extradomain having at least 90% identity with (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, wherein the engineered protein comprises at least one mutation in 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 The substitutions include those at the positions corresponding to 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 embodiments, the manipulated 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. Sequence ID 3 corresponds to the DW-1 variant sequence. Sequence ID 4 corresponds to the F protein of hMPV A1 NL / 1 / 00 strain (GenBank: AAK62968.2). Sequence ID 5 corresponds to the F protein of hMPV A2 NL / 00 / 17 strain (GenBank: ACJ70115.1).Sequence ID 6 corresponds to the F protein of the hMPV B1 NL / 1 / 99 strain (GenBank: AY525843.1). Sequence ID 7 corresponds to the F protein of the hMPV B2 TN / 99 / 419 strain (GenBank: AAS92882.1).

[0008] In some embodiments, the manipulated protein includes a proline substitution corresponding to A / D185P. In some embodiments, the manipulated protein includes a substitution from RQSR (any one residue 99-102 from SEQ ID NOs. 4-7; SEQ ID NO. 9) to RRRR (any one residue 99-102 from SEQ ID NOs. 1-3; SEQ ID NO. 10). In some embodiments, the manipulated protein includes a proline substitution corresponding to A / D185P and a substitution from RQSR (any one residue 99-102 from SEQ ID NOs. 4-7; SEQ ID NO. 9) to RRRR (any one residue 99-102 from SEQ ID NOs. 1-3; SEQ ID NO. 10). In some embodiments, the manipulated protein includes a substitution from any one residue 87-104 from SEQ ID NOs. 1-7 to GGGGSGGGGSR (SEQ ID NO. 8).

[0009] In some embodiments, the manipulated proteins are 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 The manipulated disulfide bond includes, among others, 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 embodiments, the manipulated protein includes manipulated 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. In some embodiments, the manipulated protein includes manipulated disulfide bonds containing pairs of cysteine ​​substitutions corresponding to A140C and A147C. In some embodiments, the manipulated protein includes manipulated disulfide bonds containing pairs of cysteine ​​substitutions corresponding to V104C and N457C. In some embodiments, the manipulated protein includes manipulated disulfide bonds containing pairs of cysteine ​​substitutions corresponding to L110C and N322C. In some embodiments, the manipulated protein contains a manipulated disulfide bond comprising a pair of cysteine ​​substitutions corresponding to T365C and V463C.

[0010] In some embodiments, the manipulated protein includes substitutions at positions corresponding to L219 and / or V231. In some embodiments, the manipulated protein includes substitutions corresponding to L219K and / or V231I. In some embodiments, the manipulated protein includes pairs of cysteine ​​substitutions corresponding to T127C and N153C. In some embodiments, the manipulated protein includes pairs of cysteine ​​substitutions corresponding to L110C and N322C. In some embodiments, the manipulated protein includes pairs of cysteine ​​substitutions corresponding to A140C and A147C. In some embodiments, the manipulated protein includes substitutions corresponding to G366S.

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

[0012] In some embodiments, the manipulated protein includes a manipulated disulfide bond containing a pair of cysteine ​​substitutions corresponding to A116C and A338C. In some embodiments, the manipulated protein includes a manipulated disulfide bond containing a pair of cysteine ​​substitutions corresponding to T365C and V463C. In some embodiments, the manipulated protein includes a manipulated disulfide bond containing a pair of cysteine ​​substitutions corresponding to T127C and N153C. In some embodiments, the manipulated protein includes a manipulated disulfide bond containing a pair of cysteine ​​substitutions corresponding to T365C and Q455C. In some embodiments, the manipulated protein includes at least one additional manipulated disulfide bond.

[0013] In some embodiments, the manipulated protein includes cavity-filling substitutions at positions 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 embodiments, the manipulated protein includes cavity-filling substitutions at positions corresponding to L105, V118, I137, S149, L158, L165, or Q426. In some embodiments, the manipulated protein includes a substitution corresponding to L105I or L105W. In some embodiments, the manipulated protein includes a substitution corresponding to L158W. In some embodiments, the manipulated protein includes a substitution corresponding to V118F or V118M. In some embodiments, the manipulated protein includes a substitution corresponding to Q426W. In some embodiments, the manipulated protein includes a substitution corresponding to L165F. In some embodiments, the manipulated protein includes a substitution corresponding to S149V or S149I. In some embodiments, the manipulated protein includes a substitution at the position corresponding to I137. In some embodiments, the manipulated protein includes a substitution corresponding to I137L.

[0014] In some embodiments, the manipulated proteins are G106W, A107F, T160M, L158W, I128F, A190M, V118F, V118M, Q426W, L165F, V191I, T160V, S149V, I137L, S149I, V169I, N46V, T49I, V / I122L, S192L, This includes cavity filling replacement selected from the group consisting of T317L, V162F, V162W, L105I, L105F, L105W, L134I, A117M, S347M, S347K, S347Q, V47M, G261M, I268M, S470Y, V231I, A374V, I217V, and / or S355F.

[0015] In some embodiments, the manipulated protein contains 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 embodiments, the manipulated protein includes substitutions at positions corresponding to S376, G366, and / or S194. In some embodiments, the manipulated protein includes substitutions corresponding to S376T, G366S, and / or S194Q.

[0017] In some embodiments, the manipulated protein includes a substitution at the position corresponding to K166. In some embodiments, the manipulated protein includes a substitution corresponding to K166E.

[0018] In some embodiments, the manipulated protein includes a substitution at the position corresponding to H435 that modifies pH sensitivity. In some embodiments, the manipulated protein includes a substitution corresponding to H435E, H435D, or H435N.

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

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

[0021] In some embodiments, the engineered protein comprises substitutions corresponding to T127C, N153C, A185P, T365C, V463C, L219K, and V231I. In some embodiments, 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 embodiments, the engineered protein comprises substitutions corresponding to T127C, N153C, T365C, V463C, L219K, and V231I. In some embodiments, 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 embodiments, the engineered protein comprises substitutions corresponding to L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, N368H, E453Q, and V463C. In some embodiments, the engineered protein comprises 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) to RRRR (SEQ ID NO: 10). In some embodiments, the engineered protein comprises substitutions corresponding to L110C, T127C, A140C, A147C, N153C, L219K, V231I, N322C, T365C, N368H, E453Q, and V463C. In some embodiments, 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 substitutions corresponding to GGGGSGGGGSR (SEQ ID NO: 8) from residues 87 - 104 of any one of SEQ ID NOs: 1 - 7.

[0024] In some embodiments, the engineered protein may include any combination of the substitutions disclosed in Table 1. In some embodiments, the engineered protein may include any combination of the substitutions disclosed in Table 1 in combination with the proline substitution corresponding to A / D185P. In some embodiments, the engineered protein may include any combination of the substitutions disclosed in Table 1 in combination with the substitution corresponding to the change from 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 embodiments, the engineered protein may include any combination of the substitutions disclosed in Table 1 in combination with the proline substitution corresponding to A / D185P and the substitution corresponding to the change from 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).

[0025] In some embodiments, the engineered protein includes 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 includes the trimerization domain of T4 fibritin.

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

[0029] In some embodiments, the manipulated protein contains 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, a trimer of an engineered metapneumovirus (MPV) F protein is provided herein, comprising at least one subunit described in any one of the embodiments of the engineered protein. In some embodiments, the trimer is more stable in its pre-fusion conformation compared to a trimer of a wild-type metapneumovirus (MPV) F subunit. In some embodiments, the trimer comprises at least one engineered disulfide bond between the subunits. In some embodiments, the at least one engineered disulfide bond between the subunits is formed by substitutions corresponding to S316C and D421C.

[0031] In one embodiment, as described herein, Pharmacologically acceptable carriers; and, (i) the manipulated protein described in any one of the embodiments of the manipulated protein, or (ii) the manipulated trimer described in any one of the embodiments of the manipulated trimer A pharmaceutical composition is provided which includes the above. In some embodiments, the pharmaceutical composition further comprises an adjuvant.

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

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

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

[0035] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that this detailed description and specific examples, while illustrating preferred embodiments of the present invention, are given only as illustrative examples, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

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

[0037] [Figure 1]Substitutions beneficial for hMPV F stabilization. (A) Schematic diagram of the hMPV F protein extradomain. Disulfide bonds and N-glycosylation sites are highlighted. Residue numbers indicating the start and end of F1 and F2 are shown below the bars. (B) Side view of the trimer hMPV F (PDB ID: 5WB0) extradomain in the pre-fusion structure. One protomer is shown as a ribbon diagram, and the other two are shown as white molecular surfaces. Each inset corresponds to the antigen site where the substitution is located. [Figure 2A] Characterization of single-substitution hMPV F variants. Relative expression levels of individual purified variants calculated by the area under the curve (AUC) of the peak fraction of the SEC. Variants are grouped by design. The horizontal dotted line shows the calculated AUC concentration of the base construct, normalized to 100% for comparison. [Figure 2B] Characterization of single-substituted hMPV F variants. Size exclusion chromatography (SEC) of purified F variants, grouped by design (proline, polarity, cavity packing, and disulfide). Vertical dotted lines indicate peak retention volumes for the hMPV F basic construct. [Figure 2C] Characterization of single-substituted hMPV F variants. Differential scanning fluorescence (DSF) analysis of the thermal stability of disulfide variants. Vertical dotted lines indicate the melting temperatures for the basic constructs. [Figure 2D] Characterization of single-substituted hMPV F variants. SDS-PAGE analysis of hMPV F base constructs and single-substituted F variants. Molecular weight reference is shown on the left in kDa units. [Figure 3A] Image of an electrophoretic gel showing the expression of F protein substitution variants (cavitary mutations). Molecular weight reference is shown in kDa units on the left. [Figure 3B] Image of an electrophoresis gel showing the expression of the F protein substitution variant (disulfide mutation). Molecular weight reference is shown in kDa units on the left. [Figure 3C] Image of an electrophoretic gel showing the expression of an F protein substitution variant (proline mutation). Molecular weight reference is shown in kDa units on the left. [Figure 3D] Image of an electrophoresis gel showing the expression of F protein substitution variants (electrostatic mutations). Molecular weight reference is shown on the left in kDa units. [Figure 4] Characterization of polysubstituted hMPV F variants. (A-C) SEC of purified polysubstituted hMPV F variants from 3 cycles of replicates. Vertical dotted lines indicate peak retention volumes for the hMPV F base construct. (D) DSF analysis of the thermal stability of polysubstituted F variants. Vertical dotted lines indicate melting temperatures for the base construct. (E) SEC traces of DS-CavEs2 purified from 1 L cultures of FreeStyle 293-F cells. (F) Binding of heat-treated or long-term stored DS-CavEs2 to MPE8 Fab, measured by biolayer interference. Vertical dotted lines indicate the end of the association event. Untreated DS-CavEs2 was included as a control. [Figure 5] Differential scanning fluorescence (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. Vertical dotted lines indicate characteristic peak retention volumes for the base construct (BaseCon). [Figure 7] (A) Modified hMPV F variant bound to prefusion-preferred antibody MPE8. (A) Side view of the atomic model of the hMPV F variant (DSx2) bound to MPE8 Fab, shown as a ribbon diagram. The constant region of MPE8 Fab is omitted for clarity. The side chains of the two disulfide substitutions in DSx2 are highlighted with bars. (B) Magnified view of the binding interface between the MPE8 light chain CDR and the antigen site II / V of the F protein. (C) Magnified view of the binding interface between the MPE8 heavy chain CDR and F. The main chain of CDR3 converges toward antigen site III, which is highlighted as a transparent surface. Key residues forming polar interactions are indicated with bars. [Figure 8]The structure of hMPV F DS-CavEs2 exhibits its pre-fusion three-dimensional structure. (A) Side view of the atomic model of the apo-hMPV F variant (DS-CavEs2) in the pre-fusion three-dimensional structure. This model (refractory brick-colored ribbon) is superimposed on the pre-fusion F structure (silver ribbon, PDB ID: 5WB0). The introduced substitutional side chains are highlighted with bars. Each inset corresponds to the antigen site where superposition is performed. (B) Representative 2D class-averaged image of DS-CavEs2 complexed with MPE8 Fab. [Figure 9] Structural comparison of hMPV F DSx2 and its associated PDB structure 5WB0. Overlay of a single protomer from PDBID:5WB0(hMPV F) and hMPV F DSx2. [Modes for carrying out the invention]

[0038] Detailed explanation The human metapneumovirus (hMPV) fusion (F) protein is essential for viral entry and is a key target for neutralizing antibody and vaccine development. While the pre-fusion structure is considered an optimal vaccine antigen, previously described pre-fusion F protein expression has been insufficient and poorly stabilized. In this invention, the structure of hMPV F was used to guide the design and characterize the engineered hMPV F protein. In some embodiments, the engineered hMPV F protein described in the embodiments is stabilized in the pre-membrane fusion structure. Such engineered proteins can be used to stimulate, for example, an immune response specific to the anti-hMPV F protein. In further embodiments, the engineered F protein can be used to detect F protein-binding antibodies in a sample. Therefore, the engineered proteins provided herein enable a more effective method for vaccination against hMPV and simultaneously enable a novel assay method for detecting anti-hMPV F protein antibodies, for example, in biological samples.

[0039] I. The nature of this disclosure The hMPV F protein, like other class I fusion proteins, is readily induced by host factors and transitions from a metastable pre-fusion state to a highly stable post-fusion state. Using pre-fusion stabilizing proteins such as DS-Cav1 as vaccineogens has been shown to induce higher neutralizing titers in animal models compared to using post-fusion proteins. This led to the idea of ​​using the pre-fusion structure of hMPV F (PDB ID: 5WB0) as a guide for strategically introducing mutations one at a time, specifically in regions that undergo substantial conformational changes during pre-to-post transition. Similar to recent successes in manipulating the SARS-CoV-2 spike, multiple proline substitutions increased protein expression while maintaining F in its pre-fusion conformation. The role of A107P in the fusion peptide is quite similar to that of the F817P substitution from HexaPro (Hsieh et al., 2020), likely by imposing rigidity on the fusion peptide and also by capping the helix with the pre-fusion structure. Interestingly, replacing Ala with Phe, an equivalent residue on RSV F, resulted in a slight decrease in protein expression compared to the basic 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. At antigen site V, there are three proline substitutions that lead to increased protein yield. Given that the side chains of these residues are partially exposed on the surface of the trimer, they are considered suboptimal for the vaccine antigen.

[0040] The implementation of interprotomer or intersubunit salt bridges is a more effective method than intraprotomer salt bridges and tends to efficiently maintain trimer viral proteins in a relatively compact three-dimensional structure. For example, the K588E mutation from gp41 electrostatically interacts with K62 or K492 from gp120, which is favorable for HIV-1 Env to remain in a closed three-dimensional structure before fusion (Rutten et al., 2018). Within this design, the L219K substitution from the F1 subunit may form a salt bridge with either E80 from the F2 subunit or D209 from the F1 subunit. The 73E substitution from the F2 subunit may also form a salt bridge with R198 from the neighboring F1 subunit. Both variants enhance expression and have longer retention times in SEC, which implicitly suggests a very similar role for salt bridge design in class I fusion proteins. On the other hand, intraprotomeral salt bridge designs such as variant N145E eliminated hMPV F expression (Table 1). Similarly, this type of salt bridge was not very effective in stabilizing the SARS-CoV-2 spike. Reducing repulsion caused by charge clusters at the protomer interface is another approach to prevent trimer opening. Charge clusters (E453 / D454) were found in the HRB region proximal to the base of the trimer. Therefore, replacing Glu453 with its equivalent Gln may reduce charge repulsion caused by negatively charged clusters. 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 made hMPV F a more compact trimer and retained the native quaternary structure, as evidenced by the SEC elution profile (Figure 2B).

[0041] The cavity-filling approach was also very effective in stabilizing loosely assembled viral proteins in their pre-fusion conformation. For example, the S190F substitution in the Cav1 variant successfully filled the cavity between sites V and II, and the V207L ​​substitution, with only a single CH2 addition, successfully filled the pocket between sites Φ. One of the most highly expressed variants, V231I, surprisingly, is located in domain IIIb (site II), a region that does not undergo conformational changes during the transition from pre-fusion to post-fusion. The other two substitutions that enhanced expression (S149I, I137L) are both located in domain IIIa (site V) and appear to aggregate relative to each other to stabilize the highly flexible α2 and β3.

[0042] Among the strategies used to stabilize hMPV F, the introduction of disulfide bonds likely yielded the highest success rate. The L110C / N322C substitution was designed to trap the fusion peptide within the central cavity, while the T365C / V463C substitution was designed to lock the HRB to a membrane-proximal region. By immobilizing the fusion peptide or HRB to a region that remains constant during the transition from pre-fusion to post-fusion, the F protein was retained in its pre-fusion conformation, and its thermal stability 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 different types of disulfide design strategies. These disulfide bonds are placed in regions undergoing conformational changes (e.g., domain IIIa (site V)), but they appear to stabilize the pre-fusion state by preventing the refolding of the HRA near the central helix. This is similar to the Q162C / L168C substitution used in the F protein of Wenza virus 3 (Stewart-Jones et al., 2018), suggesting that this type of disulfide design may be a common approach for stabilizing class I viral fusion proteins. They can prevent the refolding of the HRA, potentially forcing it to refold into the pre-fusion conformation. Similar disulfide designs that restrict the local flexibility of the secondary structure in regions undergoing conformational changes during the transition from pre-fusion to post-fusion have also been successfully applied to the SARS-CoV-2 spike. Interestingly, the V104C / N457C substitution inhibits furin protease digestion of F, resulting in a single species of F0 on SDS-PAGE. The furin cleavage site proximal to the fusion peptide may be embedded in the central cavity due to the disulfide design. This variant may be practical 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 for producing 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 this invention, by combining multiple beneficial modifications, we have obtained DS-CavEs2, one of the best constructs, including the design of disulfide bonds, cavity filling, and electrostatic stabilization. DS-CavEs2 exhibits 10-fold higher protein expression, enhanced thermal stability, and retains the pre-fusion epitope even after thermal stress and long-term storage at 4°C. Two disulfide substitutions at site V, a cavity-filling substitution at site II, and the introduction of another disulfide bond proximal to the fusion peptide did not disrupt the conformation of the distal half of the membrane of hMPV F, which contains the most potent neutralizing epitope in RSV F (Figure 8A) (Graham et al., 2017, Gilman et al., 2016). In contrast, the T365C / V463C substitution altered the relative position of the α10 helix. However, this proximal membrane region is unlikely to be immunogenic, and no neutralizing antibodies targeting this region have been found in RSV F.

[0044] Another version of DS-CavEs2 in which the furin site is replaced with a flexible glycine-serine (GS) linker or polyglycine linker generates a single-stranded 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 construct this construct by recombination, this design may be more cost-effective for the industrial production of vaccine antigens.

[0045] Notably, both pre-fusion-stabilized F constructs crystallized as monomers, even when complexed with MPE8, which recognizes epitopes 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 nsEM visualization of trimer F particles was possible (Figure 8B), these data suggest that the hMPV F trimer is in equilibrium with the dissociated monomer, even when fused to the trimer-forming motif. This is consistent with recent results demonstrating that some class I viral fusion proteins undergo trimer opening or "respiration," and native antibodies binding 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] Pre-fusion-stabilized class I virus fusion proteins are known to induce high neutralizing antibody titers in animals and humans, often an order of magnitude higher than those induced by the fused antigen (Crank et al., 2019; McLellan et al., 2013; Stewart-Jones et al., 2018). Previous studies showed little difference in immunogenicity between pre-fusion and fused hMPV F proteins (Battles et al., 2017), but these studies were conducted using a 10 μg antigen dose and fused F proteins that are now known to contain some amount of pre-fusion-like protein that has not yet adopted the fused three-dimensional structure. Pre-fusion-stabilized hMPV F antigen is expected to induce higher neutralizing antibody titers in mice than the fused F protein. These results are more consistent with other immunogenicity studies of pre-fusion stabilized viral proteins than with 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 and facilitate the isolation of potent, broadly reactive monoclonal antibodies that may be useful for passive prophylaxis in high-risk cohorts.

[0047] II. Definition It should be understood that the general description above and the detailed description below are illustrative and descriptive only and do not limit the claimed invention. In this application, the use of the singular includes the plural unless otherwise explicitly stated. In this application, the use of “or” means “and / or” unless otherwise explicitly stated. Furthermore, the use of the term “including,” and other forms such as “including” and “included,” is not limiting. Also, terms such as “element” or “component” include both elements and components containing one unit, and elements and components containing more than one subunit, unless otherwise explicitly stated. Also, the use of the term “portion” may include part or all of a moiety.

[0048] As used herein, the notation "a" or "an" may mean one or more. As used herein, as used in conjunction with the phrase "including" in a claim(s), the term "a" or "an" may mean one or more than one.

[0049] The use of the term “or” in the claims is used to mean “and / or” unless explicitly directed to mean only the options or the options are not mutually exclusive, even if this disclosure supports a definition that refers only to the options and “and / or” alone. As used herein, “another” may mean at least the second or later one.

[0050] As used herein, the term "approximately" when referring to measurable values ​​such as quantity and duration is intended to include a variation of up to ±10% from the stated value. Unless otherwise indicated, all numbers used herein and in the claims to represent properties such as the quantity of components and molecular weight, reaction conditions, etc., should be understood to be modified by the term "approximately" in all instances. Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations, which may vary depending on the desired properties to be obtained by the subject matter of the disclosed invention. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be interpreted at least in consideration of the reported number of significant figures and by applying common rounding techniques. Although the numerical ranges and parameters described for the broad scope of the invention are approximations, the numerical values ​​described in the specific examples are reported as accurately as possible. However, each numerical value inherently contains certain errors that inevitably arise from the standard deviation found in their respective test measurements.

[0051] When used herein, the term “essentially not present” with respect to a specified component is used herein to mean that none of the specified components are intentionally included in the composition and / or are present only as impurities or in trace amounts. Therefore, the total amount of a specified component resulting from any unintended impurities in the composition is well below 0.05%, preferably below 0.01%. Most preferably, a composition is one in which no amount of a specified component can 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. “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, such as antibodies naturally occurring in camelids that may contain only heavy chains, it may contain fewer chains. Antibodies may originate from a single source or they may be “chimeric,” meaning that different parts of an antibody may originate from two different antibodies, as further described below. Antigen-binding proteins, antibodies, or binding fragments are produced in hybridomas by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. Unless otherwise indicated, the term “antibody” includes antibodies containing two full-length heavy chains and two full-length light chains, as well as their derivatives, variants, fragments, and mutant proteins, examples of which are described below. Furthermore, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimes”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof, respectively. In some embodiments, this term also encompasses peptide bodies.

[0053] The structural unit of native antibodies is a tetramer. Each such tetramer typically consists of a pair of two identical polypeptide chains, each pair having one full-length "light" chain (about 25 kDa in certain embodiments) and one full-length "heavy" chain (about 50–70 kDa in certain embodiments). The amino-terminus of each chain typically contains a variable region of about 100–110 or more amino acids, which is typically responsible for antigen recognition. The carboxyl-terminus of each chain typically defines a constant region that can perform effector functions. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining antibody isotypes 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 the 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, the heavy chain also containing a "D" region of about 10 amino acids. See, for example, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (the whole is referenced for all purposes). The variable region of each light / heavy chain pair typically forms the antigen-binding site.

[0054] The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chain of an antibody, typically containing approximately 120–130 amino acids at the amino terminus in the heavy chain and approximately 100–110 amino acids at the amino terminus in the light chain. In certain embodiments, the variable regions of different antibodies can differ significantly in amino acid sequence, even between antibodies of the same species. The variable region of an antibody typically determines the specificity of a particular antibody to its target.

[0055] The variable regions typically exhibit the same general structure, with a relatively conserved framework region (FR) linked by three hypervariable regions (also called complementarity-determining regions or CDRs). The CDRs from each pair of chains are typically aligned by the framework region, which can enable binding to specific epitopes. From the N-terminus to the C-terminus, both the light-chain and heavy-chain variable regions typically contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain typically follow the definitions in 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, the antibody heavy chain binds to the antigen in the absence of the antibody light chain. In certain embodiments, the antibody light chain binds to the antigen in the absence of the antibody heavy chain. In certain embodiments, the antibody binding region binds to the antigen in the absence of the antibody light chain. In certain embodiments, the antibody binding region binds to the antigen in the absence of the antibody heavy chain. In certain embodiments, individual variable regions specifically bind to the antigen in the absence of other variable regions.

[0057] In certain embodiments, the definitive demarcation of the CDR and the identification of residues constituting the antibody binding site are accomplished by elucidating the structure of the antibody and / or the structure of the antibody-ligand complex. In certain embodiments, this may be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, the CDR region can be identified or roughly predicted using various analytical methods. Examples of such methods include, but are not limited to, the Kabat definition, Chothia definition, AbM definition, and contact definition.

[0058] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, for example, Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but takes into account the location of certain structural loop regions. See, for example, Chothia et al., J.Mol.Biol., 196:901-17 (1986), Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses a suite of integrated computer programs developed by the Oxford Molecular Group to model antibody structures. See, for example, Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989), “AbM TM See, “A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from its primary sequence using a combination of a knowledge database and ab initio methods, such as the method 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 the analysis of available complex crystal structures. See, for example, MacCallum et al., J.Mol.Biol., 5:732-45 (1996).

[0059] Conventionally, the CDR regions of the heavy chain are typically designated H1, H2, and H3, and are numbered sequentially from the amino terminus to the carboxyl terminus. The CDR regions of the light chain are typically designated L1, L2, and L3, and are numbered sequentially from the amino terminus to the carboxyl terminus.

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

[0061] The term "heavy chain" includes full-length heavy chains and fragments thereof that have a variable region sequence sufficient to confer binding specificity. A full-length heavy chain comprises a variable region domain VH and three constant region domains CH1, CH2, and CH3. The VH domain is located at the amino terminus of the polypeptide, and the CH domains are located at the carboxyl terminus, with CH3 being the closest to the carboxyl terminus of the polypeptide. Heavy chains 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 possessing 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 hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990) and Kostelny et al., J. Immunol., 148:1547-1553 (1992).

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

[0064] The "Fc" region contains two heavy chain fragments, each containing the CH1 and CH2 domains of the antibody. These two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0065] The "Fv region" includes variable regions from both the heavy and light chains, but lacks a steady-state region.

[0066] An antibody that "specifically binds to" or "is specific to" a particular polypeptide or epitope on a particular polypeptide is an antibody that binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on a particular polypeptide. For example, the hMPV F protein-specific antibody of the present invention is specific to the hMPV F protein. In some embodiments, the antibody that binds to the 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, e.g., 10⁻⁸ M to 10⁻¹³ M, e.g., 10⁻⁹ M to 10⁻¹³ M).

[0067] When used in the context of antigen-binding proteins (e.g., antibodies or their antigen-binding fragments) competing for the same epitope, the term “competing” means competition between antigen-binding proteins, as determined by the assay, where the antigen-binding protein being tested (e.g., antibody or its antigen-binding fragment) blocks or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., ligand, or reference antibody) to a common antigen (e.g., hMPV F or its fragment). Whether one antigen-binding protein competes with another antigen-binding protein can be determined using a number of competitive binding assays, such as solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive 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); and solid-phase direct labeling RIA using I-125 labeling (see, e.g., Morel et al.). This can be determined using solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct-labeled 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 cell carrying one 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 cell 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 (competing 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 an adjacent epitope 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, an excess of competing antigen-binding proteins will attempt to inhibit (e.g., reduce) the specific binding of the reference antigen-binding protein to the common antigen by at least 40–45%, 45–50%, 50–55%, 55–60%, 60–65%, 65–70%, 70–75%, or more than 75%. In some cases, binding is inhibited by at least 80–85%, 85–90%, 90–95%, 95–97%, or more than 97%.

[0068] As used herein, the term “epitope” refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. Epitopes can be either linear or structural. Linear epitopes are formed by a continuous sequence of amino acids from the antigen and interact with the antibody based on their primary structure. Structural epitopes, on the other hand, consist of discontinuous segments of the amino acid sequence of the antigen and interact with the antibody based on the 3D structure of the antigen. Generally, epitopes are approximately 5 or 6 amino acid long. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen.

[0069] The term "host cell" means a cell that has been transformed, or can be transformed, by a nucleic acid sequence to express the gene of interest. This term includes offspring of a parent cell, regardless of whether the offspring's morphology or genetic structure is identical to that of the original parent cell, as 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 sequence alignment and comparison. "Identity percentage" means the percentage of identical residues among amino acids or nucleotides in the molecules being compared, calculated based on the size of the smallest molecule among those being compared. For these calculations, gaps in alignment (if any) are preferably handled by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that may be used to determine the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, 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 the percentage of identity, the sequences being compared are typically aligned to yield the greatest possible match between them. An example of a computer program that can be used to determine the percentage of 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 two polypeptides or polynucleotides whose sequence identity percentage is to be determined. The sequences are aligned so that their respective corresponding amino acids or nucleotides best match (a "matched range" as determined by the algorithm). A gap opening penalty (calculated as 3 × mean diagonal element, where “mean diagonal element” is the average of the diagonal elements of the comparison matrix used, and “diagonal element” is the score or number assigned to each perfect match of amino acids by a particular comparison matrix) and a gap extension penalty (which is typically 1 / 10 × gap opening penalty), as well as comparison matrices such as PAM 250 or BLOSUM 62, are used in conjunction with the algorithm. In certain embodiments, standard comparison matrices (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.USA89:10915-10919 for the BLOSUM 62 comparison matrix) are also used by the algorithm.

[0072] Examples of parameters that can be used when determining the identity percentage of polypeptides 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 a match 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 these two full-length sequences. Therefore, the selected alignment method (GAP program) may be modified to result in alignment of at least 50 or more consecutive amino acids of the target polypeptide, if so desired.

[0074] As used herein, the term “covalent” refers to associations via intramolecular interactions, such as covalent, metallic, and / or ionic bonds, or associations via intermolecular interactions, such as hydrogen bonds or non-covalent bonds.

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

[0076] The terms "polynucleotide" or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. The nucleotides constituting a polynucleotide may be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide bond modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniladeates, and phosphoramidates.

[0077] As used herein, “vector” refers to a nucleic acid molecule that, when introduced into a host cell, thereby produces a transformed host cell. A vector may include nucleic acid sequences that enable replication within the host cell, such as origins of replication. A vector may also include 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 nucleic acids and / or proteins other than those native to the cell. A vector may optionally include substances that help achieve entry of nucleic acids into the cell, such as viral particles, liposomes, or protein coatings.

[0078] The terms “polypeptide” or “protein” mean a macromolecule having the amino acid sequence of a native protein, i.e., a protein produced by a naturally occurring, non-recombinant cell, or it includes molecules produced by genetically engineered or recombinant cells that have the amino acid sequence of a native protein, or molecules having one or more amino acid deletions, additions, and / or substitutions of the native sequence. The term also includes amino acid polymers, where one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. The terms “polypeptide” and “protein” explicitly encompass hMPV F protein-binding proteins, antibodies, or sequences having one or more amino acid deletions, additions, and / or substitutions of an antigen-binding protein. The term “polypeptide fragment” refers to a polypeptide having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions compared to a 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 to 500 amino acid long. For example, a fragment may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunologically functional fragments of an antibody containing a binding domain. In the case of hMPV F protein-binding antibodies, useful fragments include, but are not limited to, CDR regions, variable domains of the heavy and / or light chains, a portion of an antibody chain containing two CDRs, or simply its variable region, etc.

[0079] The 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 the pharmaceutically acceptable delivery of the fusion proteins disclosed herein. Generally, the properties of the carrier will depend on the specific mode of administration used. For example, parenteral formulations typically include an injection solution containing a pharmaceutically and physiologically acceptable fluid as a vehicle, such as water, saline, equilibrium salt solution, aqueous dextrose, or glycerol. In the case of solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical composition may contain small amounts of non-toxic auxiliary substances such as humectants or emulsifiers, preservatives, and pH buffers, for example, sodium acetate or sorbitan monolaurate.

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

[0081] As used herein, the terms “therapeutically effective dose” or “effective dosage” refer to the dosage or concentration of a drug that is effective in treating a disease or condition. For example, with respect to the use of a monoclonal antibody or its antigen-binding fragment disclosed herein for the treatment of a viral infection.

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

[0083] III. hMPV F protein Human metapneumovirus (hMPV) is an enveloped, minus-chain virus belonging to the family Pneumoviridae, discovered in 2001, but having circulated 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). Initially non-functional, it requires a proteolytic cleavage event to form F1 and F2 subunits, which are covalently linked by disulfide bonds. The new N-terminus of the F2 polypeptide contains a hydrophobic sequence that will be inserted into the host cell membrane during the process of eventual fusion of the virus and host cell membranes. At some point, either in transit or on the membrane surface, the F protein associates with itself to form a metastable trimer in its so-called pre-fusion structure. The hMPV fusion protein is cleaved extracellularly by trypsin-like proteases. An unknown triggering event occurs, and under its influence, the F protein undergoes a dramatic conformational change: after extending the fusion peptide into the host cell membrane, it folds over itself again, forming a 6-helix bundle in the so-called post-fusion structure. The energy difference between the extended intermediate and the post-fusion structure provides the energy necessary for membrane fusion.

[0084] Historically, the structure of paramyxovirus fusion proteins has been classified into domains that generally divide the initially elucidated pre-fusion protein into head, neck, and stem. The nomenclature of assigning three general domains (DI, DII, and DIII) to the pre-fusion structure of additional structures continues. The family pneumoviridae, which was formerly a subfamily of paramyxoviruses, also retains this convention. The domains of hMPV are vaguely classified into the same three domains, and two additional regions of heptad repeats are similarly defined. Heptad repeat A (HRA) is located at the N-terminus of F1 within DIIIa, and heptad repeat B is located at the C-terminus outside the defined domain region and prior to the protein's transmembrane domain. However, due to structural similarities, the protein regions can be more accurately described using the transition of antigen site nomenclature used for the fusion protein of respiratory syncytial virus (RSV), another member of the family pneumoviridae. DIIIa undergoes maximum structural reconfiguration when the fusion peptide is released from the internal cavity of the trimer, and as the fusion peptide is inserted into the target membrane, the HRA forms a 3-coil bundle. Finally, the HRB associates around the outside of the HRA bundle to form 6HB in the post-fusion structure.

[0085] Recently, stabilization of the pre-fusion structure of class I fusion proteins has yielded promising results as vaccine antigens in clinical trials, and both the RSV vaccine and the SARS-coronavirus-2 vaccine utilize 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 pre-fusion structures of these two proteins are highly similar (Battles et al., 2017; van den hoogen et al., 2002). Based on its elucidated pre-fusion structure, a few different strategies have been used to stabilize RSV F in its pre-fusion structure, including the introduction of proline, 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 more optimally filled by hydrophobic residue substitution, and the introduction of a disulfide bond was introduced into the fusion peptide region. In the case of PR-DM, a proline residue was introduced to prevent the loop region from being reconfigured into the extended alpha-helix seen in the post-fusion structure. In RSV F, the region of charge repulsion was also identified and reduced. Similarly, the elucidated pre-fusion structure enabled the stabilization of the coronavirus by introducing two proline substitutions (S-2P) into the hinge region. Within the fields of HIV (SOSIP) and influenza, there are examples where introducing stabilizing mutations has resulted in well-functioning pre-fusion reagents.

[0086] Recently, using knowledge gained from RSV F research, hMPV F was stabilized in its pre-fusion structure. First, the F2 / F1 cleavage site sequence "RQSR" was replaced with a polybasic "RRRR" sequence to enable efficient cleavage by furin-like proteases in producing cells. Next, mimicking the RSV F stabilization strategy (Krarup et al., 2015), proline was introduced into the helix-loop-helix region of F1 at the apex of the distal trimer. This manipulation strategy allowed the pre-trimeric crystal structure of hMPV F to be obtained, but protein expression was insufficient, suggesting that further manipulation is needed (Battles et al., 2017). Furthermore, previous serum depletion assays and mouse immunization experiments showed no significant difference in antigenicity between pre-fusion and post-fusion hMPV F (Battles et al., 2017). In contrast, pre-fusion RSV F induced a more robust neutralizing antibody response than post-fusion RSV F, and serum depletion experiments demonstrated that most of the RSV neutralizing activity in human serum was limited to the pre-fusion structure (Sastre et al., 2005; Magro et al., 2012). These data suggest that a more stable pre-fusion hMPV F construct is needed to investigate these inconsistent results.

[0087] For this purpose, a strategy for stabilizing the F protein by mutations in the coding sequence of the F protein is demonstrated herein. Using the published pre-fusion hMPV F structure, the introduction of additional amino acid substitutions was guided. It was found that several beneficial substitution combinations had an additive effect on the desired protein properties. Details of the mutations analyzed and provided herein are shown in Table 1 below. Mutant proteins were expressed as detailed in the examples, and the amount of protein-trimer complex produced was determined.

[0088] (Table 1) F protein substitutions and mutations TIFF2023544834000001.tif225158TIFF2023544834000002.tif206158TIFF2023544834000003.tif211163TIFF2023544834000004.tif100163

[0089] IV. Pharmaceutical preparations This disclosure provides a pharmaceutical composition comprising an engineered hMPV F protein. Such composition can be used to stimulate an immune response, such as as part of a vaccine formulation.

[0090] When nucleic acid molecules encoding the manipulated hMPV F protein are used in a pharmaceutical composition, the nucleic acid molecules may contain, or consist of, covalently linked deoxyribonucleotides and / or ribonucleotides, or analogs thereof. The nucleic acid molecules described herein generally contain phosphodiester bonds, but may also include nucleic acid analogs having at least one different bond, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite bond, as well as peptide nucleic acid backbone and bonds. Mixtures of native polynucleotides and analogs may be prepared, but alternatively, mixtures of different polynucleotide analogs, as well as mixtures of native polynucleotides and analogs, may be prepared. Nucleic acid molecules may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be induced before or after polymer organization. Non-nucleotide components may be interposed in the nucleotide sequence. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeling components. This term also includes both double-stranded and single-stranded molecules. Unless otherwise specified or requested, 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 consists of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, if the polynucleotide is RNA, uracil (U) instead of thymine. Thus, the term “nucleic acid sequence” is the alphabetical representation of a nucleic acid molecule. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses not only the explicitly indicated sequence but also its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions may be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue.

[0091] In some embodiments, the nucleic acids of this disclosure comprise one or more modified nucleosides containing a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides may possess desirable properties, such as enhanced nuclease stability or increased binding affinity to a target nucleic acid, compared to oligonucleotides comprising only nucleosides containing the native sugar moiety. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a sugar substitute. Such a sugar substitute may comprise one or more substitutions corresponding to the substitution of the substituted sugar moiety.

[0092] In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-crosslinked 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 may be allyl, amino, azide, thio, O-allyl, or O-C1~C 10 Alkyl, O--C1~C 10 Selected from substituted alkyl groups, 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 substituted or unsubstituted C1-C 10 It is 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 contains more than one non-crosslinked sugar substituent, e.g., TF-5'-methyl sugar moiety (for additional 5',2'-bis-substituted sugar moieties and nucleosides, see, for example, PCT international application WO2008 / 101157).

[0093] A nucleoside containing a 2'-substituted sugar moiety is referred to as a 2'-substituted nucleoside. In some embodiments, a 2'-substituted nucleoside includes a 2'-substituted group selected from halo, allyl, amino, azide, 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, alkalil, aralkyl, O-alkalil, 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 substituted or unsubstituted C1-C 10 These are alkyl groups. 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 groups.

[0094] In some embodiments, the 2'-substituted nucleoside comprises a 2'-substituted group 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 substituted or unsubstituted C1-C 10 It is alkyl.

[0095] In some embodiments, the 2'-substituted nucleoside includes a sugar moiety comprising 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, the 2'-substituted nucleoside comprises a sugar moiety containing a 2'-substituted substituent selected from F, O--CH3, and OCH2CH2OCH3.

[0097] In some embodiments, the nucleoside of the Disclosure comprises one or more unmodified nucleic acid bases. In certain embodiments, the nucleoside of the Disclosure comprises one or more modified nucleic acid bases.

[0098] In some embodiments, the modified nucleic acid base is selected from universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines include, as defined herein, 2-aminopropyl adenine, 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-propynyl CH3)uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6 -Includes azouracil, cytosine, and thymine, 5-uracil (pseudracil), 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, indiscriminate bases, size-expanding bases, and fluorinated bases. Further modified nucleic acid bases include tricyclic pyrimidines, such as phenoxazinecytidine ([5,4-b][1,4]benzoxazine-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiadin-2(3H)-one), G-clamps, such as substituted phenoxazinecytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazine-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indole-2-one), and pyridoindolecytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one).Modified nucleic acid bases may also include those in which a purine or pyrimidine base is replaced by another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Further examples of nucleic acid bases 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 teaching certain of the above-mentioned modified nucleic acid bases, as well as the preparation of other modified nucleic acid bases, 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, and 5,484,90. This includes 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 herein by reference in its entirety.

[0100] Furthermore, additional modifications may be carried out at other positions on the oligonucleotide, particularly at 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 oligonucleotide of the present disclosure involves chemically linking one or more additional non-ligand moieties or conjugates to the oligonucleotide to enhance the activity, cell distribution, or cell uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, e.g., cholesterol moieties, cholic acid, thioethers, e.g., hexyl-5-tritylthiol, thiocholesterol, aliphatic chains, e.g., dodecanediol or undecyl residues, phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chains, or adamantane acetate, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties. In some embodiments, the nucleic acid molecule encoding the chimeric hMPV / RSV F protein is a modified RNA, such as modified mRNA. Modified (m)RNA is a specific chemical modification intended to confer increased stability and lower immunogenicity to mRNA, thereby facilitating the expression of therapeutically important proteins. For example, N1-methyl-pseudridine (N1mΨ) is superior in translational ability to several other nucleoside modifications and their combinations. In some embodiments, the (m)RNA molecule used herein may have uracil replaced with pseudouracil, such as a 1-methyl-3'-pseudridine base. In some embodiments, some of the uracils are replaced, while in others, all of the uracils are replaced. The (m)RNA may include a 5' cap, a 5' UTR element, optionally a codon-optimized open reading frame, a 3' UTR element, and a poly(A) sequence and / or polyadenylation signal.

[0101] Nucleic acid molecules may be delivered as naked nucleic acid molecules, or in a delivery vehicle such as lipid nanoparticles, whether native or modified. The lipid nanoparticles may contain one or more nucleic acids present in a weight ratio of about 5:1 to about 1:100 relative to the lipid nanoparticles. In some embodiments, the nucleic acid to lipid nanoparticle weight ratio 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 within that range.

[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 ionizable groups such as alkylamines and one or more hydrophobic groups such as alkyl groups of C6 or higher.

[0103] In some embodiments of this disclosure, lipid nanoparticles are mixed with one or more steroids or steroid derivatives. In some embodiments, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, in some embodiments, the term “steroid” refers to a class of compounds having a 4-ring, 17-carbon cyclic structure, which may further include one or more substitutions including alkyl groups, alkoxy groups, hydroxyl groups, oxo groups, acyl groups, or double bonds between two or more carbon atoms.

[0104] In some embodiments of this disclosure, lipid nanoparticles are mixed with one or more PEGylated lipids (or PEG lipids). In some embodiments, this disclosure includes the use of any lipid to which a PEG group is attached. In some embodiments, the PEG lipid is a diglyceride, also containing 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 having a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG ceramide conjugates, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropane-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified diastearoyl phosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is determined by the molecular weight of the PEG component in 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 weights of the PEG modification range from 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 may be used in this disclosure are taught in U.S. Patent 5,820,873, WO2010 / 141069, or U.S. Patent 8,450,298, which are incorporated herein by reference.

[0105] In some embodiments of this disclosure, lipid nanoparticles are mixed with one or more phospholipids. In some embodiments, any lipid also contains phosphate groups. In some embodiments, the phospholipid is one or two long-chain C6-C 24 The structure contains 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 phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipids define lipids and lipid-like molecules that have both positive and negative charges.

[0106] In some aspects of this disclosure, lipid nanoparticles containing a compound comprising a lipophilic component and a cationic component are provided, wherein 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 pH greater than 8, 9, 10, 11, or 12, and are uncharged. 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 also contains C6-C 24 The compounds may further contain one or more lipid components, such as two or more fatty acids having alkyl or alkenyl carbon groups. These lipid groups may be linked via ester bonds or further added 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, a composition is provided that contains a compound containing a lipophilic component and a cationic component, where the cationic component is ionizable. In some embodiments, an ionizable cationic lipid refers to a lipid and lipid-like molecule having a nitrogen atom capable of acquiring a charge (pKa). These lipids may be known as cationic lipids in the literature. These molecules having an amino group typically have 2 to 6 hydrophobic chains, often C6 to C 24 alkyl or alkenyl such as alkyl or alkenyl groups, but may have at least 1 or more than 6 tails.

[0108] In some embodiments, the amount of lipid nanoparticles having a nucleic acid molecule encapsulated therein in the pharmaceutical composition is from about 0.1 w / w% to about 50 w / w%, about 0.25 w / w% to about 25 w / w%, about 0.5 w / w% to about 20 w / w%, about 1 w / w% to about 15 w / w%, about 2 w / w% to about 10 w / w%, about 2 w / w% to about 5 w / w%, or about 6 w / w% to about 10 w / w%. In some embodiments, the amount of lipid nanoparticles having a nucleic acid molecule encapsulated therein in the pharmaceutical composition is 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 therefrom.

[0109] In some embodiments, the disclosure includes one or more sugars incorporated into a pharmaceutical composition. In some embodiments, the sugars used herein are saccharides. These saccharides may be used to function as antifreeze agents to protect the pharmaceutical composition from destabilization during the drying process. These water-soluble excipients include carbohydrates or saccharides, such as disaccharides like sucrose, trehalose, or lactose; trisaccharides like fructose, glucose, or galactose that constitute raffinose; polysaccharides like 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, treitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, inositol, boremitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, or polyglycitol.

[0110] In some embodiments, the amount of sugar in the pharmaceutical composition is approximately 25 w / w% to approximately 98 w / w%, 40 w / w% to approximately 95 w / w%, 50 w / w% to approximately 90 w / w%, 50 w / w% to approximately 70 w / w%, or approximately 80 w / w% to approximately 90 w / w%. In some embodiments, the amount of sugar in the pharmaceutical composition is approximately 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%, or approximately 95 w / w%, or any range derivable within that range.

[0111] In some embodiments, the pharmaceutically acceptable polymer is a copolymer. The pharmaceutically acceptable polymer may further comprise one, two, three, four, five, or six subunits of distinct types of polymer subunits. These polymer subunits may comprise polyoxypropylene, polyoxyethylene, or similar subunits. In particular, the pharmaceutically acceptable polymer may comprise at least one hydrophobic subunit and at least one hydrophilic subunit. Specifically, 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, an expression cassette is used to express the engineered hMPV F protein, either for subsequent purification and delivery to cells / targets, or for direct use in a virus-based delivery approach. This specification provides an expression vector containing one or more nucleic acids encoding the engineered hMPV F protein.

[0113] Expression requires that appropriate signals are provided within the vector and that various regulatory elements, such as enhancers / promoters from both viral and mammalian sources, drive the expression of the engineered hMPV F protein in the cell. Throughout this application, the term “expression cassette” is intended to include any type of gene construct containing nucleic acid encoding a gene product, in which part or all of the coding sequence of the nucleic acid can be transcribed and translated, i.e., under the control of a promoter. “Promoter” refers to a DNA sequence recognized by the cell’s synthetic mechanism or introduced synthetic mechanism, which is 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 gene expression. “Expression vector” is intended to include an expression cassette contained within a gene construct that is replicable and therefore contains one or more of the origin of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.

[0114] The term promoter will be used herein to refer to a group of transcriptional regulatory modules clustered around an initiation site for RNA polymerase II. Most of the ideas about how promoters are organized come from analyses of several viral promoters, including analyses of HSV thymidine kinase (tk) and SV40 initial transcription units. These studies, enhanced by more recent efforts, show that promoters are composed of individual 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 the promoter for mammalian terminal deoxynucleotidyltransferase genes and the promoter for SV40 late genes, individual elements that overlap the start site themselves help to fix the start site.

[0116] Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the initiation site, although in recent years, some promoters have been shown to contain functional elements downstream of the initiation site as well. The spacing between promoter elements is often flexible, so that promoter function is maintained even if the elements are reversed or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased up to 50 bp, beyond which activity begins to decrease. Depending on the promoter, individual elements may function cooperatively or independently to activate transcription.

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

[0118] Enhancers are genetic elements located at distant locations on the same DNA molecule that increase transcription from a promoter. Enhancers are organized very similarly to promoters; that is, they consist of many individual elements, each of which binds to one or more transcription proteins. The fundamental distinction between enhancers and promoters is operational. The enhancer region as a whole must be capable of stimulating transcription from a distance, but this does not necessarily apply to the promoter region or its components. On the other hand, a promoter must have one or more elements at a specific site and orientation that direct the initiation of RNA synthesis, whereas enhancers lack these specificities. Promoters and enhancers often overlap, are contiguous, and frequently appear to have very similar modular structures.

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

[0120] Promoter and / or enhancer include, for example, immunoglobulin light chain, immunoglobulin heavy chain, T cell receptor, HLA DQa and / or DQbeta, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, β-actin, muscle creatine kinase (MCK), prealbumin (trans tyretin), elastase I, metallothionein (MTII), collagenase, albumin, α-fetoprotein, t-globin, β-globin, c-fos, c-HA-ras, insulin, neuronal contact molecule (NCAM), α1-antitrypsin (antitrypain), H2B (TH2B) histone, mouse and / or type I collagen, glucose regulatory proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN) I) These may include platelet-derived growth factor (PDGF), SV40, polyoma, retrovirus, papillomavirus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon leukemia virus.

[0121] When cDNA insertions are used, it is typically desirable to include a polyadenylation signal to achieve proper polyadenylation of the gene transcript. Any polyadenylation sequence, such as human growth hormone and the SV40 polyadenylation signal, may be used. Terminators are also intended as elements of the expression cassette. These elements can serve to enhance the message level and minimize read-through from the cassette to other sequences.

[0122] Several methods exist for introducing expression vectors into cells. In certain embodiments, the expression constructs include viruses or engineered constructs derived from viral genomes. Certain viruses are attractive candidates for exogenous gene transfer into mammalian cells due to their ability to enter cells via receptor-mediated endocytosis, integrate into the host cell genome, and stably and efficiently express viral genes. These have relatively low capacity for exogenous DNA sequences and a limited host spectrum. Furthermore, their oncogenic potential and cytopathic effects in tolerant cells raise safety concerns. Although they can only accommodate up to 8 kB of exogenous genetic material, they can be readily 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. An “adenovirus expression vector” is intended to contain a construct that (a) supports the packaging of the construct and (b) contains sufficient adenovirus sequences to express the modified hMPV F protein cloned therein. In this context, expression does not require the synthesis of the gene product.

[0124] The expression vector contains a genetically modified form of adenovirus. Knowledge of the adenovirus's genetic makeup—a 36kB linear double-stranded DNA virus—allows for the substitution of large fragments of adenovirus DNA with foreign sequences up to 7kB. In contrast to retroviruses, adenovirus infection of host cells does not result in chromosomal integration because adenovirus DNA can replicate episomal without potential genotoxicity. Furthermore, adenoviruses are structurally stable, and no genome rearrangements have been detected after large-scale amplification. Adenoviruses can infect virtually all epithelial cells, regardless of their cell cycle stage. To date, adenovirus infections are thought to be associated only with mild illnesses in humans, such as acute respiratory diseases.

[0125] Adenoviruses are particularly well-suited as gene transfer vectors due to their moderate genome size, ease of handling, high titer, broad target cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeat sequences (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 separated by the initiation 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) leads to the synthesis of these proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut-off. The late gene products, 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 16.8mu) is particularly efficient during the later stages of infection, and all mRNA produced from this promoter is preferred for translation because it possesses a 5'-tripertite leader (TPL) sequence. In one system, recombinant adenovirus is produced from homologous recombination between a shuttle vector and a proviral vector. Due to the possibility of recombination between the two proviral vectors, wild-type adenovirus can be produced from this process. Therefore, it is essential to isolate a single clone of the virus from individual plaques and examine its genomic structure.

[0126] The generation and propagation of current adenovirus vectors with replication defects depend on a specific helper cell line called 293, which is transformed from human embryonic kidney cells with an Ad5 DNA fragment and constitutively expresses the E1 protein. Since the E3 region is not essential to the adenovirus genome, current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, D3, or both regions. Adenoviruses can typically package approximately 105% of their wild-type genome, providing about 2kb of extra DNA. Combined with approximately 5.5kb of replaceable DNA in the E1 and E3 regions, the maximum capacity of current adenovirus vectors is less than 7.5kb, or about 15% of the vector's total length. Over 80% of the adenovirus genome remains in the vector backbone, becoming a source of vector-derived cytotoxicity. Furthermore, the replication defect in E1-deleted viruses is incomplete.

[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 tolerant to human adenovirus. Such cells include, for example, Vero cells or other mesenchymal or epithelial monkey fetal cells. As described above, the preferred helper cell line is 293.

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

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

[0130] In embodiments, in particular, the vector is an AAV vector. AAV is a small virus that infects humans and several other primate species. AAV is not currently known to cause disease. Its apparent lack of pathogenicity is further supported by the fact that the virus elicits a very mild immune response. In many cases, AAV vectors are integrated into the host cell genome, which may be important for certain applications, but can also have undesirable consequences. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without being integrated into the host cell genome, although in native viruses, some integration of the virus-carried gene into the host genome does actually occur. These characteristics make AAV a very attractive candidate for creating viral vectors for gene therapy and for creating isogeneic 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. This virus is a small (20 nm) replication-deficient virus that lacks an envelope.

[0131] Wild-type AAV has attracted considerable interest from gene therapy researchers due to several characteristics. Among the most prominent is the virus's apparent lack of pathogenicity. It can also infect non-dividing cells and possesses the ability to stably integrate into the host cell genome at a specific site on human chromosome 19 (designated AAVS1). This characteristic makes it somewhat more predictable than retroviruses, which sometimes pose a threat of random insertions and mutagenesis, potentially leading to cancer. While AAV genomes are most frequently integrated at the aforementioned site, random integration into the genome occurs at a very low frequency. However, the development of AAV as a gene therapy vector eliminated this integration capability by removing rep and cap from the vector's DNA. The desired gene, along with a promoter to drive gene transcription, is inserted between inverted terminal repeats (ITRs) that aid in concatemer formation in the nucleus, after the single-stranded vector DNA has been 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 host cell's lifetime. In dividing cells, AAV DNA is lost during cell division because episomal DNA is not replicated along with 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, which appears to be limited to the production of neutralizing antibodies, while they do not induce a clearly defined cytotoxic response. This characteristic, along with their ability to infect quiescent cells, demonstrates their superiority over adenoviruses as vectors for human gene therapy.

[0132] The AAV genome consists of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-stranded, with a length of approximately 4.7 kilobases. This genome contains inverted terminal repeat sequences (ITRs) at both ends of the DNA strand, as well as two open reading frames (ORFs), rep and cap. The former consists of four duplicated genes encoding the Rep protein, which is required throughout the AAV life cycle, while the latter contains duplicated nucleotide sequences of the capsid proteins VP1, VP2, and VP3, which interact together to form an icosahedral-symmetrical capsid.

[0133] Inverted end repeat (ITR) sequences each contain 145 base pairs. They are so named due to their symmetry, which has been shown to be necessary for the efficient multiplication of the AAV genome. The feature that gives these sequences this property is their ability to form hairpins, which contributes to so-called self-priming, enabling primase-independent synthesis of the second DNA strand. ITRs have also been shown to be necessary for both the integration of AAV DNA into the host cell genome (chromosome 19 in humans) and its rescue from there, as well as for the efficient capsid formation of AAV DNA combined with the generation of fully assembled deoxyribonuclease-resistant AAV particles.

[0134] Regarding gene therapy, the ITR appears to be the only sequence required in cis alongside the therapeutic gene, while the structural (cap) and packaging (rep) proteins can be delivered trans. This assumption has led to the establishment of many methods for the efficient production of recombinant AAV (rAAV) vectors containing reporter or therapeutic genes. However, it has also been reported that the ITR is not the only element required in cis for effective replication and capsid formation. A small group of researchers has identified a sequence called a cis-acting Rep-dependent element (CARE) located inside the coding sequence of the rep gene. CARE has been shown to enhance replication and capsid formation when present in cis.

[0135] In some embodiments, the Disclosure provides a pharmaceutical composition containing one or more salts. The salts may 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 composition may contain one or more phosphates, for example, to produce a phosphate buffer. The phosphate buffer may contain each of the phosphates to buffer the solution to a pH of 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, or any range within which it can be derived.

[0136] In certain embodiments, the disclosure includes one or more excipients incorporated into a pharmaceutical composition. “Excipient” means a pharmaceutically acceptable carrier, which is a relatively inert substance used to facilitate the administration or delivery of an API to a subject, or to facilitate the processing of an API into a drug formulation that can be pharmaceutically used for delivery to a site of action within a subject. Furthermore, these compounds may be used as diluents to obtain a dosage that can be easily measured or administered to a patient. Non-limiting examples of excipients include polymers, stabilizers, surfactants, surface modifiers, dissolution accelerators, buffers, capsule encapsulants, antioxidants, preservatives, nonionic wetting or clarifying agents, thickeners, and absorption enhancers.

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

[0138] The compositions may also contain small amounts of wetting agents or emulsifiers, or pH buffers, if desired. These compositions may take the form of liquids, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceuticals are described in "Remington's Pharmaceutical Sciences." Such compositions are intended to contain a prophylactic or therapeutically effective amount of antibody or fragments thereof, preferably in a purified form, together with a suitable amount of carrier that provides a form for appropriate administration to the patient. The formulation should be suitable for the mode of administration, which may be oral, intravenous, intra-arterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or mechanical ventilation.

[0139] The manipulated proteins described herein may be formulated for parenteral administration, for example, for injection via intradermal, intravenous, intramuscular, subcutaneous, intratumoral, or even intraperitoneal routes. Antibodies may be administered via local routes directly to the mucosa, for example, by nasal spray, inhalation, or nebulizer. Pharmaceutically acceptable salts include salts and salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0140] Generally, the components of the compositions of this disclosure are supplied either separately or mixed together in a unit dosage form, for example, as dry lyophilized powder or anhydrous concentrate in airtight containers such as ampoules or sachets indicating the amount of the active agent. If the composition is to be administered by inhalation, it may be dispensed in an inhalation bottle containing pharmaceutical-grade sterile water or saline solution. If the composition is to be administered by injection, ampoules of sterile water for injection or saline solution may be provided so that the components can be mixed before administration.

[0141] The compositions of this disclosure may be formulated in neutral or salt form. 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] Medication can be administered in a single-dose or multi-dose schedule. Multi-dose administration may be used in a primary immunization schedule and / or a booster immunization schedule. In a multi-dose schedule, various doses may be administered via the same or different routes. Multi-dose administration will typically be given at intervals of at least one week (e.g., approximately 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 16 weeks, etc.).

[0143] The compositions disclosed herein may be used to treat both children and adults. Therefore, human subjects may be under 1 year of age, 1 to 5 years of age, 5 to 16 years of age, 16 to 55 years of age, 55 to 65 years of age, or at least 65 years of age.

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

[0145] V. Immunodetection Methods Still in further embodiments, the present disclosure relates to an immunodetection method for conjugating, purifying, isolating, quantifying, and otherwise generally detecting hMPV F protein-binding antibodies. While such a method can be applied in a conventional sense, another application is in quality control and monitoring of vaccine stocks, where the antibody can be used to assess the quantity or integrity (i.e., long-term stability) of the antigen. Alternatively, this method may be used to screen various antibodies for a suitable / desired reactivity profile.

[0146] Some immunoassay methods include, to name a few, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradioquantification, fluorescence immunoassay, chemiluminescence assay, bioluminescence assay, and Western blotting. In particular, competitive assays for the detection and quantification of hMPV F protein-binding antibodies are also provided. Generally, immunoconjugation methods involve obtaining a sample suspected to contain hMPV F protein-binding antibodies and contacting the sample with the antigen according to this disclosure, and possibly under conditions effective in enabling 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 bound 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. By washing unwanted components from the column, the hMPV F protein-binding antibodies immunocomplexed with the immobilized antigen remain, and these are collected by removing the antigen from the column.

[0148] The immunoconjugation method also includes a method for detecting and quantifying the amount of hMPV F protein or related components in a sample, as well as the detection and quantification of any immune complexes formed during the conjugation process. Here, a sample suspected to contain hMPV F protein is obtained, the sample is contacted with an antibody that binds to hMPV F protein or its components, and then the amount of immune complexes formed under specific conditions is detected and quantified. In terms of antigen detection, the biological sample to be analyzed may be any sample suspected to contain 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 for enabling the formation of an immune complex (primary immune complex) and for a sufficient period of time generally involves simply adding the antigen composition to the sample and incubating the mixture for a period of time long enough for the antigen to form an immune complex (i.e., bind to) the hMPV F protein-binding antibody. After this time, the sample-antibody composition, such as tissue sections, ELISA plates, dot blots, or Western blots, is generally washed to remove any nonspecifically bound antibody species so that only the specifically bound antibodies within the primary immune complex are detectable.

[0150] In general, the detection of immune complex formation is well known in the art and can be achieved by applying a number of approaches. These methods generally rely on the detection of labels or markers, such as radioactive, fluorescent, biological, and enzyme 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. Naturally, as is known in the art, additional advantages may be found in the use of secondary antibodies and / or secondary binding ligands such as biotin / avidin ligand binding configurations.

[0151] The antibody used for detection may be linked to a detectable label, in which case the label is simply detected to determine the amount of primary immunocomplex in the composition. Alternatively, the first antibody to be bound within the primary immunocomplex may be detected using a second binding ligand having binding affinity to that antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is often an antibody itself and can therefore be called a "secondary" antibody. The primary immunocomplex is brought into contact with the labeled secondary binding ligand or antibody under conditions effective to enable the formation of the secondary immunocomplex and for a sufficient period of time for this purpose. The secondary immunocomplex is then generally washed to remove any nonspecifically bound labeled secondary antibody or ligand, and the remaining label in the secondary immunocomplex is then detected.

[0152] A further method involves detecting primary immune complexes using a two-step approach. As described above, a secondary immune complex is formed using a second binding ligand, such as an antibody having binding affinity to the antibody in question. After washing, the secondary immune complex is brought into contact with a third binding ligand or antibody having binding affinity to the second antibody under conditions effective for enabling the formation of another immune complex (tertiary immune complex) and for a sufficient period of time for this purpose. The third ligand or antibody is linked to a detectable label so that the thus formed tertiary immune complex can be detected. This system may allow for signal amplification if desired.

[0153] One immunoassay 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 biotin bound to complexed biotin. In this method, the sample to be tested is first incubated in a solution containing the antibody from the first step. 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 sequential solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with additional biotin sites added to the antibody / antigen complex at each step. The amplification step is repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the antibody from the second step against biotin. This antibody from the second step is labeled with an enzyme that can be used, for example, to detect the presence of the antibody / antigen complex by histoenzymology using a chromogenic substrate. Through appropriate amplification, a conjugate that can be viewed macroscopically can be produced.

[0154] Another known immunodetection method utilizes the immunoPCR (polymerase chain reaction) technique. While the PCR method is similar to the Cantor method up to incubation with biotinylated DNA, instead of using multiple streptavidin and biotinylated DNA incubations, the DNA / biotin / streptavidin / antibody complex is washed with a low-pH or high-salt buffer that releases the antibody. The resulting wash is then used to perform a PCR reaction with suitable primers and an appropriate control. At least theoretically, the enormous amplification capacity and specificity of PCR can be utilized to detect a single antigen molecule.

[0155] 1. ELISA Immunoassays, in their simplest and most direct sense, are binding assays. Certain preferred immunoassays include 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 apparent that detection is not limited to these techniques, and Western blotting, dot blotting, FACS analysis, etc., may also be used.

[0156] In one exemplary ELISA, the antibody of this disclosure is immobilized on a selected surface exhibiting protein affinity, such as a well of a polystyrene microtiter plate. A test composition suspected to contain the hMPV F protein is then added to the well. After binding and removal of nonspecifically bound immunocomplexes by washing, the bound antigen can be detected. Detection may also be achieved by adding another anti-hMPV F protein antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by adding a second anti-hMPV F protein antibody, followed by the addition of a third antibody having binding affinity to the second antibody, the third antibody being 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 the surface of a well and then contacted with the anti-hMPV F protein antibody of the present disclosure. After binding and removal of nonspecifically bound immune complexes by washing, the bound anti-hMPV F protein antibody is detected. If the first anti-hMPV F protein antibody is ligated to a detectable label, the immune complex may be detected directly. Again, the immune complex may be detected using a second antibody having binding affinity to the first anti-hMPV F protein antibody, wherein the second antibody is ligated to a detectable label.

[0158] Regardless of the format used, ELISAs share certain common characteristics, including coating, incubation, and binding, removal of nonspecifically bound species by washing, and detection of bound immune complexes. These are described below.

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

[0160] In ELISA, it is perhaps more common to use secondary or tertiary detection methods rather than direct procedures. Therefore, after binding proteins or antibodies to the wells, reducing background by coating with a non-reactive material, and removing unbound material by washing, the immobilized surface is brought into contact with the biological sample of test under conditions effective in enabling immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a labeled tertiary antibody or a secondary binding ligand or antibody combined with a third binding ligand.

[0161] "Conditions effective in enabling immune complex (antigen / antibody) formation" means 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 help reduce nonspecific background.

[0162] "Preferred" conditions also mean that the incubation is at a temperature sufficient to allow for effective bonding or lasts for such a period of time. The incubation step may typically be at a temperature of approximately 25°C to 27°C for about 1 to 2 to 4 hours, or at about 4°C overnight.

[0163] Following all incubation steps in the ELISA, the contact surface is washed to remove any uncomplexed material. Preferred washing procedures include washing with a solution such as PBS / Tween or borate buffer. The formation of specific immunocomplexes between the test sample and the initially bound material, and subsequent washing, can determine even the presence of trace amounts of immunocomplexes.

[0164] To provide a means of detection, the second or third antibody will have an associated label to enable detection. Preferably, this will be an enzyme that produces color development upon incubation with a suitable chromogenic substrate. Therefore, it would be desirable to contact or incubate, for example, the first and second immunocomplexes with urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase conjugated antibodies for a certain period of time and under conditions favorable for the development of further immunocomplex formation (e.g., incubation for 2 hours at room temperature 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 urea, bromocresol purple, 2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), or H2O2 (if the peroxidase is enzyme-labeled). Then, quantification is achieved by measuring the degree of the resulting color, for example, using a visible spectrum spectrophotometer.

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

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

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

[0169] To make proteins accessible for antibody detection, they are moved from the gel onto a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed at the top of the gel, and a stack of filter paper is placed on top of it. The entire stack is placed in a buffer, which moves upwards towards the filter paper by capillary action, carrying the proteins with it. Another method for moving proteins is called electroblotting, which uses an electric current to draw the proteins from the gel onto the PVDF or nitrocellulose membrane. The proteins move from 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 thin surface layer for detection (see below). Both types of membranes are selected because of their non-specific protein-binding properties (i.e., they bind equally well to all proteins). 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 withstand repeated probing as well. 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 indirectly using a labeled protein A or secondary labeled antibody that binds to the Fc region of the primary antibody, following an unlabeled primary antibody.

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

[0171] In short, frozen sections can be prepared by rehydrating 50 ng of frozen "pulverized" tissue in phosphate-buffered saline (PBS) in a small plastic capsule at room temperature; pelletizing the particles by centrifugation; re-turbidifying them in viscous embedding medium (OCT); inverting and / or again pelletizing the capsule by centrifugation; rapidly freezing in isopentane at -70°C; cutting off the plastic capsule and / or removing the frozen cylinder from the tissue; fixing the tissue cylinder on a cryostat microtome chuck; and / or cutting out 25 to 50 serial sections from the capsule. Alternatively, a frozen whole tissue sample may be used to cut out serial sections.

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

[0173] 4. Immunodetection kits Still in further embodiments, the disclosure relates to an immunodetection kit for use in conjunction with the immunodetection method described above. Since the hMPV F protein can be detected using an antibody, an antibody may be included in the kit. The immunodetection kit would therefore contain, in a suitable container, a first antibody that binds to the hMPV F protein, and optionally an immunodetection reagent. Alternatively, the hMPV F protein-binding antibody may be detected using an hMPV F protein antigen. In this case, the immunodetection kit would therefore contain, 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 reagent of the kit may take any of a variety of forms, including a detectable label associated with or bound to the antibody. A detectable label associated with or bound to a secondary ligand is also intended. An exemplary secondary ligand is a secondary antibody having binding affinity to the first antibody.

[0175] Further preferred immunodetection reagents for use in this kit include a two-component reagent comprising a secondary antibody having binding affinity to a first antibody, together with a third antibody having binding affinity to a second antibody, wherein the third antibody is linked to a detectable label. As described above, several exemplary labels are known in the art, and all such labels may be used in connection with this disclosure.

[0176] The kit may further include suitably fractionated compositions of the hMPV F protein, whether labeled or unlabeled, which can be used to create standard curves for detection assays. The kit may contain the antibody-labeled conjugate in complete conjugate form, in intermediate form, or as separate parts that are conjugated by the user of the kit. The components of the kit may be packaged in either an aqueous medium or in lyophilized form.

[0177] The kit's container means would generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody can be placed, or preferably preferably dispensed. The kits of this disclosure would also typically include means for containing the antibody, antigen, and any other reagent containers, tightly sealed for commercial sale. Such containers may include injection-molded or blow-molded plastic containers into which the desired vial is held.

[0178] 5. Flow cytometry and FACS The antibodies of this disclosure may also be used in flow cytometry or FACS. Flow cytometry is a laser or impedance-based technique used in many detection assays, including cell counting, cell sorting, biomarker detection, and protein engineering. This technique allows for simultaneous multi-parameter analysis of the physical and chemical properties of up to several thousand particles per second by suspending cells in a flowing fluid and passing them through an electrical detector. Flow cytometry is conventionally used in the diagnosis of disorders, but it has many other applications in basic research, clinical practice, and clinical trials.

[0179] Fluorescent cell sorting (FACS) is a specialized type of cytometry. It provides a method for sorting a heterogeneous mixture of living cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence properties of each cell. Generally, this technique involves drawing a cell suspension into the center of a narrow, rapidly flowing liquid stream. The stream is arranged so that the cells are far apart relative to their diameter. An oscillating mechanism causes the cell stream to split 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 charging ring is positioned just at the 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 droplet as it splits from the stream. The charged droplets then fall through an electrostatic deflection system, which deflects the droplets into containers based on their charge.

[0180] In certain embodiments, for use in flow cytometry or FACS, the antibody of the Disclosure is labeled with a fluorophore, then bound to cells of interest, which are analyzed by a flow cytometer or parsed by a FACS machine. [Examples]

[0181] The following embodiments are included to illustrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments are representative of techniques found by the inventors to function well in the implementation of the present invention and can therefore be considered to constitute a preferred mode for its implementation. However, those skilled in the art will understand that, in light of this disclosure, similar or comparable results can still be obtained with many modifications in the specific embodiments disclosed without departing from the spirit and scope of the present 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 their efficacy was confirmed by DNA sequencing. FreeStyle 293F cells (ThermoFisher) were co-transfected with polyethyleneimine (PEI) using plasmids encoding the F variant and furin in a 4:1 ratio. Three hours after transfection, kifunensin was added to a final concentration of 5 μM, and for large-scale transfection, 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 application to 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). For initial variant screening and characterization, monosubstituted and combined substituted hMPV F variants were purified from 40 mL cell cultures. 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 cotransfected into FreeStyle 293F cells in a 1:1 ratio via PEI. A stop codon was introduced before the hinge region of the heavy chain to generate the MPE8 antigen-binding fragment (Fab). To purify the MPE8 Fab, the filtered 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, flash-frozen in liquid nitrogen, and then stored at -80°C.

[0184] Differential scanning calorimetry. A purified hMPV variant at a final concentration of 1 μM was mixed with 5-fold SYPRO Orange protein gel stain (ThermoFisher) in a white opaque 96-well plate (VWR). The mixture was then measured using a Roche LightCycler 480 II by continuous fluorescence scanning (λex=465 nm, λem=580 nm) at a temperature gradient of 4.4 °C / min and a temperature range of 25 °C to 95 °C. The data were plotted as derivatives of the melting curve.

[0185] MPE8 binding analysis by biolayer interference. To examine the epitope integrity of hMPV F under various temperature stresses, aliquots of DS-CavEs2 were incubated in a thermocycler at 37°C, 50°C, or 70°C for 30 minutes, or left at 4°C for 2.5 months, and then tested for MPE8 binding by BLI using Octet RED96e (ForteBio). Briefly, an anti-human Fab-CH1 second-generation (FAB2G) biosensor (ForteBio) was used to capture an equal volume 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. The biosensor containing the captured MPE8 was then immersed in 50 nM heat-treated DS-CavEs2, and the association rate was measured. After a 600-second association step, a 600-second dissociation step was performed in wells containing only buffer. The binding curve was aligned to baseline, and the buffer was subtracted.

[0186] Negative staining EM. The fused hMPV F was heat-treated at 70°C for 10 minutes, and then applied to a CF-400-Cu grid (Electron Microscopy Sciences) that had been plasma-washed with a 4:1 ratio of O2 / H2 for 45 seconds in a Solarus 950 plasma washing system (Gatan). The grid was stained using methylamine tungstate (Nanoprobes). Pre-fused, stabilized hMPV F was incubated with 2x molar excess MPE8 Fab in 1x PBS at room temperature for 30 minutes. The hMPV-F:Fab complex was 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 onto the CF-400-Cu grid. The grid was imaged at a magnification of 92,000x (corresponding to a calibration pixel size of 1.63 Å / pixel) using a Talos F200C TEM microscope (Thermo Fisher Scientific) equipped with a Ceta 16M detector. CTF estimation and particle collection were performed using cisTEM (Grant et al., 2018). Subsequently, the particles were sent to cryoSPARC v2.15.0 for 2D classification (Punjani et al., 2017).

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

[0188] (Table 2) Statistics on crystallographic data collection and refinement The values ​​in parentheses in TIFF2023544834000005.tif216161 are for the outermost shell with the highest resolution.

[0189] Enzyme-linked immunosorbent assay. Panels of pre-fusion specific monoclonal antibodies (MFP10, Ac967, Ac1025, and MPE8) (Corti et al., 2013) or non-pre-fusion specific antibodies (MF11, MF14) (Battles et al., 2017) 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 post-fusion hMPV F were applied to antibody-coated wells at room temperature for 1 hour. Unbound F was removed by washing three times with 0.1% Tween-20 in PBS. Bound F was then detected by adding an anti-His-tagged mAb conjugated with horseradish peroxidase (HRP) (Bio-Rad), followed by three washes with 0.1% Tween-20 in PBS. Next, an HRP substrate (Sigma) was added for color development, and the optical density was read at 492 nm using an ELISA plate reader.

[0190] Example 1 - Structural-based design of pre-fusion stabilized hMPV F Similar to other class I viral fusion glycoproteins, the pre-fusion hMPV F protein exhibits a metastable state and readily converts to a stable post-fusion structure during induction. To stabilize the F protein in the pre-fusion state, the proline substitution A185P was introduced into the helix-loop-helix region at the apex of the trimer. This substitution allowed us to obtain the pre-fusion structure of hMPV, but the low expression level of this construct hindered its potential application as a vaccine candidate. Therefore, we also used the H368N substitution (Schowalter et al., 2009), which has been previously shown to increase protein expression levels, similar to the previously described pre-fusion-stabilized F protein BV-115 (Battles et al., 2017). From this basic construct, 97 variants were designed based on the pre-fusion (PDB ID: 5WB0) and post-fusion (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 employed included disulfide bonds to lock regions that substantially move during transition from pre-fusion to post-fusion, hydrophobic residues to fill internal cavities, polar residues to counteract internal charge imbalances, and proline substitutions favorable to the pre-fusion conformation while unfavorable to 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, and many of these variants exhibited higher thermal stability.

[0191] (Table 3) Exemplary F protein variants TIFF2023544834000006.tif223151TIFF2023544834000007.tif221152TIFF2023544834 000008.tif234161TIFF2023544834000009.tif232157TIFF2023544834000010.tif24161

[0192] Example 2 - Single substitution F variant The expression profiles of the 42 individual variants are summarized in Figure 2A, and the size exclusion chromatography (SEC) traces of the selected variants from each design category are shown in Figure 2B. Throughout this example, it should be noted that the values ​​of the protein yield multiplier change provided in parentheses relate to the original values ​​determined when each construct was designed. The values ​​given in Table 3 are from experiments in which all constructs were reexpressed 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 reexpression, all constructs were expressed better than in the original expression. The lower yield of the base construct in the original expression was the main reason for the higher yield multiplier values ​​at the original values.

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

[0194] Salt bridges were introduced 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 (Figure 2A, B), and both showed longer retention times in SEC. The introduction of a pair of substitutions L66D / K188R increased expression by 1.6-fold. E453Q showed a 1.9-fold increase in protein yield [2.8-fold increase], but also showed a significant rightward shift of the trimer peak in SEC, suggesting that the trimer may be in a relatively closed three-dimensional structure (Figure 2C). In addition, several 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] (Figure 2A, B). One other variant, S376T, also showed a 2.4-fold increase in protein yield compared to the base construct. Two other variants, S149I and G366S, showed 2.3-fold and 1.7-fold increases in protein expression [2.3-fold and 2.9-fold increases] compared to the base construct, and eluted as monodisperse peaks in SEC.

[0195] Pre-fusion stabilization disulfide crosslinking was tested to improve the protein expression level and thermal stability of hMPV F. Two examples, L110C / N322C and T365C / V463C, showed increases of 2.5 and 1.7 times [2.8 and 2.4 times] in protein yield and 5.6°C and 6.2°C in Tm, respectively, compared to the base construct (Figures 2A, B, C). Variant A116C / A338C also improved thermal stability compared to the base construct, but did not increase expression levels. On the other hand, A140C / A147C and T127C / N153C, both located in domain IIIa (site V) (i.e., α2, α3, β3, and β4 (Figure 1B)), significantly increased protein expression levels by 3.1 times and 2.8 times [6.0 times and 4.8 times], respectively (Figures 2A, B). T127C / N153C moderately improved thermal stability (Figure 2C). The V104C / N457C variant showed reduced furin cleavage compared to the base construct (Figure 2D), increased Tm by 4°C, and decreased protein expression levels (Figures 2B, C). Overall, about 20 of the 42 variants increased protein expression levels (Figure 2A), and of these, 6 variants showed increased thermal stability.

[0196] Example 3 - Polysubstituted F variant The single substitution combination was introduced into three different variants containing either two disulfide bonds, one disulfide bond and one cavity filler, or one disulfide bond and one salt bridge. In all three combination variants, the substitution T365C / V463C was included due to its significant improvement in thermal stability. All three variants (T127C / N153C / T365C / V463C, V231I / T365C / V463C, L219K / T365C / V463C) showed further increases of 1.2, 1.9, and 1.2 times in protein yield compared to their parent construct T365C / V463C, and the Tm of variants containing two disulfide bonds increased by 1.7°C compared to the parent T365C / V463C construct and 6.4°C compared to the base construct (Figures 4A, D). The T127C / N153C / T365C / V463C variant was named DSx2 and further introduced with either L219K or V231I. DSx2 / L219K and DSx2 / V231I showed further increases of 1.1-fold and 1.6-fold compared to DSx2 (Figures 4B, 6). Furthermore, variants containing all beneficial mutations (T127C / N153C / T365C / V463C / L219K / V231I) produced 16 mg of protein from 1 L of FreeStyle 293-F cells, showing the highest expression level among all constructs with a further 1.8-fold increase compared to DSx2 and a Tm of 60.7°C (Figures 4D, 5). This variant was newly named DS-CavEs, and by further introducing one disulfide bond design (L110C / N322C) into DS-CavEs, a pentasubstituted variant named MM-1 (T127C / N153C / T365C / V463C / L219K / V231I / L110C / N322C) was generated. Regarding MM-1, protein expression levels decreased by 25% compared to DS-CavEs, demonstrating a substantial improvement in Tm (67.6°C). Another variant, named MM-1H (T127C / N153C / T365C / V463C / L219K / V231I / / L110C / N322C / N386H), was created by reverting H368N back to the wild-type His368 compared to MM-1.Regarding MM-1H, protein expression was 25% lower compared to DS-CavEs, and it showed an increased Tm (65.2°C) (Figure 4C, D). This enhanced thermal stability due to the introduction of L110C / N322C may be very advantageous for vaccine antigens. Due to the benefits indicated 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, referred to as MM-4H, showed minimal differences in expression yield and a 1.0°C increase in Tm (Figure 4C, D).

[0197] Furthermore, the A140C / A147C and E453Q substitutions were introduced into MM-1H (which also corresponds to introducing E453Q into MM-4H), and the resulting molecule was named DS-CavEs2. Large-scale expression of DS-CavEs-2 produced 15.7 mg of pre-fusion stabilized F from 1 L of FreeStyle 293-F cells (Figure 4E). The antigenic surface of DS-CavEs2 is well-conserved. The affinity of DS-CavEs2 for the pre-fusion specific antibody MPE8 (Wen et al., 2017) remained unchanged after incubation at 50°C for 30 minutes and was equivalent to that of the basic construct (Figure 4F). From nsEM analysis, DS-CavEs2 appeared to be a well-folded pre-fusion trimer, with each protomer conjugated by MPE8 Fab. DS-CavEs2 with the additional substitution enhances expression by more than 10 times and exhibits the highest thermal stability (Tm 71.8°C) while retaining the correct pre-fusion structure (Figure 4D), suggesting its potential for vaccine development.

[0198] Example 4 - Crystal structure of the MPE8-bonded DSx2 construct To determine the effect of multiple substitutions on the three-dimensional structure of hMPV F in the combined mutant DSx2 construct, the crystal structure of the protein complexed with the pre-fusion specific antibody MPE8 was obtained. This protein complex was crystallized in space group P2 and diffracted by X-rays to a resolution of 2.2 Å. After model construction and refinement, this structure was found to have 19.68% and 23.5% R, respectively. work and R freeIt was initially found that these properties were improved to 21.3% and 24.2%, respectively, upon further structural refinement (Table 2). Compared to the previously determined hMPV F structure (PDBID:5WB0), DSx2 retained its pre-fusion conformation and had 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 a resolution of 2.5 Å from a crystal of space group P21 (Table 2). In the absence of MPE8, DS-CavEs2 retained its pre-fusion conformation, and the RMSD across the 428 Cα atoms shared with PDBID:5WB0 was 2.3 Å (Figure 8A). Clear electron densities were observed for all disulfide bond substitutions (Cys127 / Cys153, Cys140 / Cys147, Cys110 / Cys322, Cys365 / Cys463) and cavity-filling substitution (I231). Superposition of the distal half of the membrane of DS-CavEs2 (sites II, V, and φ) with the previous hMPV F structure (PDBID:5WB0) revealed substantial migration of antigen site IV toward the central triple axis (Figure 8A). The superposition of site IV from both structures revealed the presence of a rigid body bending at the center of two long β-chains (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 alter the local conformation. In contrast, the Cys365 / Cys463 substitution pulled the α10 helix away from the central triple axis, thus altering the downward orbit of the HRB (Figure 8A). Negative staining electron microscopy (nsEM) analysis was performed on MPE8 complexed with DS-CavEs2. After 2D class averaging, multiple classes indicated DS-CavEs2 as a well-folded pre-fusion trimer linked by two or three MPE8 Fabs, demonstrating that DS-CavEs2 can adopt a trimer conformation (Figure 8B).

[0200] Example 6 - Pre-fusion stabilized hMPV F variant as immunogen To investigate whether pre-fusion-stabilized hMPV F variants function as immunogens, BALB / c mice will be immunized at 3-week intervals with either pre-fusion (e.g., basic construct, DSx2, and DS-CavEs2) or post-fusion F antigens enhanced with CpG. Serum will be collected 10 days after the second immunization. Pre-fusion-stabilized F constructs are expected to elicit higher neutralizing antibody titers against hMPV A1 and / or hMPV B1 compared to post-fusion F antigens.

[0201] All methods disclosed and claimed herein can be carried out and performed without undue experimentation in light of this disclosure. Although the compositions and methods of the present invention are 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 steps or sequences 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 chemically and physiologically related active substances may be substituted for the active substances described herein, insofar as the same or similar results are achieved. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to fall within the spirit, scope, and concept of the invention as defined by the appended claims.

[0202] References The following references are expressly incorporated herein by reference to the extent that they provide exemplary procedural or other details that supplement the details set forth herein. TIFF2023544834000011.tif202160TIFF2023544834000012.tif238160TIFF2023544834000013.tif238160TIFF2023544834000014.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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