WO2018140242A1

WO2018140242A1 - Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus

Info

Publication number: WO2018140242A1
Application number: PCT/US2018/013318
Authority: WO
Inventors: James E. Crowe, Jr.; Jarrod MOUSA
Original assignee: Vanderbilt University
Current assignee: Vanderbilt University
Priority date: 2017-01-27
Filing date: 2018-01-11
Publication date: 2018-08-02
Anticipated expiration: 2019-07-27

Abstract

The present disclosure is directed to antibodies binding to prefusion and postfusion forms of both human respiratory syncytial virus and human metapneumovirus F proteins, including neutralizing antibodies, and methods for use thereof.

Description

DESCRIPTION STRUCTURAL BASIS FOR ANTIBODY CROSS-NEUTRALIZATION OF RESPIRATORY SYNCYTIAL VIRUS AND HUMAN METAPNEUMOVIRUS

This application claims benefit of priority to U.S. Provisional Application Serial No. 62/451,323, filed January 27, 2017, the entire contents of which are hereby incorporated by reference. BACKGROUND

1. Field of the Disclosure

The present disclosure relates generally to the fields of medicine, infectious disease, and immunology. More particular, the disclosure relates to human antibodies binding to respiratory syncytial virus (RSV). 2. Background

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two closely related viruses that cause bronchiolitis and pneumonia in infants and the elderly (Collins & Crowe, 2013), with a significant health burden (Hall et al., 2013; Falsey et al., 2005; Hall, 2012; Edwards et al., 2013; Williams et al., 2004). There are no licensed vaccines or small molecule antiviral treatments specific to these two viruses currently. While a humanized murine monoclonal antibody (palivizumab) is approved to treat infants at high risk for RSV infection (Fenton et al., 2004; The IMapct-RSV Study Group, 1998), other treatments, as well as vaccines, for both viruses are still in development. Recent modeling of cross-immunity between RSV, HMPV and human parainfluenzaviruses 1-3 accounts for their epidemiological patterns of periodic outbreaks (Bhattacharyya et al., 2015), and suggests that a deeper understanding of host immunity to these viruses may lead to enhanced strategies for their control. Antibodies that cross-reactively neutralize both RSV and HMPV have been identified (Corti et al., 2013; Schuster et al., 2015) despite significant sequence differences in their respective F proteins (~36% identity). Identifying more of these types of antibodies will provide further opportunity for therapeutic intervention. SUMMARY

Thus, in accordance with the present disclosure, there is provided a method of detecting a human respiratory syncytial virus infection in a subject comprising (a) contacting a sample from said subject with an antibody or antibody fragment having heavy chain CDR1 GFTFSSYR (SEQ ID NO: 5), heavy chain CDR2 ITASSSYI (SEQ ID NO: 6), heavy chain CDR3 ARDENTGISHYWFDP (SEQ ID NO: 7), light chain CDR1 GSNLGADYG (SEQ ID NO: 8), light chain CDR2 GDR (SEQ ID NO: 9) and light chain CDR3 QSYDRSLNWV (SEQ ID NO: 10); and (b) detecting human respiratory syncytial virus in said sample by binding of said antibody or antibody fragment to a human respiratory syncytial virus antigen in said sample. The sample maybe a body fluid, such as blood, sputum, tears, saliva, mucous or serum, urine, exudate, transudate, tissue scrapings or feces. Detection may comprise ELISA, RIA or Western blot. The method may further comprise performing steps (a) and (b) a second time and determining a change in human respiratory syncytial virus antigen levels as compared to the first assay.

The antibody or antibody fragment may be encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, or by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. The antibody or antibody fragment may comprise heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, may comprise heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, or may comprise heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’)₂ fragment, or Fv fragment.

In another embodiment, there is provided a method of treating a subject infected with human respiratory syncytial virus, or reducing the likelihood of infection of a subject at risk of contracting human respiratory syncytial virus, comprising delivering to said subject an antibody or antibody fragment having heavy chain CDR1 GFTFSSYR (SEQ ID NO: 5), heavy chain CDR2 ITASSSYI (SEQ ID NO: 6), heavy chain CDR3 ARDENTGISHYWFDP (SEQ ID NO: 7), light chain CDR1 GSNLGADYG (SEQ ID NO: 8), light chain CDR2 GDR (SEQ ID NO: 9) and light chain CDR3 QSYDRSLNWV (SEQ ID NO: 10). The antibody or antibody fragment may be encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, or by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. The antibody or antibody fragment may comprise heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, may comprise heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, or may comprise heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment, a chimeric antibody and/or is an IgG.

The antibody or antibody fragment may recognize an epitope on pre-fusion and post- fusion RSV F protein in antigenic sites II (alternatively called site A) and IV, and optionally binds to human metapneumovirus F protein. The antibody or antibody fragment may neutralize RSV and human metapneumovirus. The antibody or antibody fragment may be administered prior to infection or administered after infection. Delivering comprises antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

In yet another embodiment, there is provided a monoclonal antibody or antibody fragment comprises heavy chain CDR1 GFTFSSYR (SEQ ID NO: 5), heavy chain CDR2 ITASSSYI (SEQ ID NO: 6), heavy chain CDR3 ARDENTGISHYWFDP (SEQ ID NO: 7), light chain CDR1 GSNLGADYG (SEQ ID NO: 8), light chain CDR2 GDR (SEQ ID NO: 9) and light chain CDR3 QSYDRSLNWV (SEQ ID NO: 10). The antibody or antibody fragment may be encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, or by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. The antibody or antibody fragment may comprise heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, may comprise heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, or may comprise heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment, or is a chimeric antibody, a bispecific antibody, and/or is an IgG. The antibody or antibody fragment may recognize an epitope on pre-fusion and post-fusion RSV F protein in antigenic sites II (alternatively called A) and IV, and optionally binds to human metapneumovirus F protein, and/or neutralizes RSV and/or human metapneumovirus. The antibody or antibody fragment may further comprise a cell penetrating peptide and/or is an intrabody.

In a further embodiment, there is provided a hybridoma or engineered cell encoding an antibody or antibody fragment wherein the antibody or antibody fragment is characterized by heavy chain CDR1 GFTFSSYR (SEQ ID NO: 5), heavy chain CDR2 ITASSSYI (SEQ ID NO: 6), heavy chain CDR3 ARDENTGISHYWFDP (SEQ ID NO: 7), light chain CDR1 GSNLGADYG (SEQ ID NO: 8), light chain CDR2 GDR (SEQ ID NO: 9) and light chain CDR3 QSYDRSLNWV (SEQ ID NO: 10). The antibody or antibody fragment may be encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, or by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. The antibody or antibody fragment may comprise heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, may comprise heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, or may comprise heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment. The antibody may be a chimeric antibody, a bispecific antibody, and/or is an IgG. The antibody or antibody fragment may recognize an epitope on pre-fusion and post- fusion RSV F protein in antigenic sites II (alternatively called A) and IV, and optionally binds to human metapneumovirus F protein, and/or neutralizes RSV and/or human metapneumovirus. The antibody or antibody fragment further comprises a cell penetrating peptide and/or is an intrabody.

In yet a further embodiment, there is provided a vaccine formulation comprising one or more antibodies or antibody fragments characterized by heavy chain CDR1 GFTFSSYR (SEQ ID NO: 5), heavy chain CDR2 ITASSSYI (SEQ ID NO: 6), heavy chain CDR3 ARDENTGISHYWFDP (SEQ ID NO: 7), light chain CDR1 GSNLGADYG (SEQ ID NO: 8), light chain CDR2 GDR (SEQ ID NO: 9) and light chain CDR3 QSYDRSLNWV (SEQ ID NO: 10). The antibody or antibody fragment may be encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, or by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. The antibody or antibody fragment may comprise heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, may comprise heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, or may comprise heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. At least one of said antibody fragments may be a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment, or is a chimeric antibody, is bispecific antibody, and/or is an IgG. The antibody or antibody fragment may recognize an epitope on pre-fusion and post-fusion RSV F protein in antigenic sites II (alternatively called A) and IV, and optionally binds to human metapneumovirus F protein, and/or neutralizes RSV and/or human metapneumovirus. At least one of said antibodies or antibody fragments may further comprise a cell penetrating peptide and/or is an intrabody.

An additional embodiment comprises a method of identifying anti-human respiratory syncytial virus (hRSV) protein F site A/IV-specific monoclonal antibody or polyclonal neutralizing antibodies comprising (a) contacting a candidate monoclonal antibody or polyclonal serum with hRSV protein F in the presence of the antibody or antibody fragment as described above; (b)assessing binding of said candidate monoclonal antibody or polyclonal serum to hRSV protein F; and (c) identifying said candidate monoclonal antibody or polyclonal serum as protein F site II (or A)/IV-specific neutralizing when said antibody or antibody fragment as described above blocks binding of said candidate monoclonal antibody or polyclonal serum to hRSV protein F.

The method may further comprise performing a control reaction where said candidate monoclonal antibody is contacted with hRSV protein F in the absence of the antibody or antibody fragment as described above. Detection may comprise ELISA, RIA or Western blot. The antibody or antibody fragment may be encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2, or by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. The antibody or antibody fragment may comprise heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, may comprise heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4, or may comprise heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. The antibody fragment may be a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab’)₂ fragment, or Fv fragment, or is a chimeric antibody, a bispecific antibody, and/or is an IgG.

Yet a further embodiment comprises a monoclonal antibody or fragment thereof, or hybridoma or engineered cell comprising the same, wherein said antibody or fragment thereof recognizes an epitope on pre-fusion and post-fusion RSV F protein in antigenic site II (or A)/IV, and also recognized an epitope on hMPV F protein.

Still yet another embodiment comprises a method of determining the antigenic integrity of an antigen comprising (a) contacting a sample comprising said antigen with a first antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (b) determining antigenic integrity of said antigen by detectable binding of said antibody or antibody fragment to said antigen. The sample may comprise recombinantly produced antigen. The sample may comprise a vaccine formulation or vaccine production batch. Detection may comprise ELISA, RIA, Western blot, a biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometric staining. The first antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1. The first antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. The antibody fragment may be a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment. The method may further comprising performing steps (a) and (b) a second time to determine the antigenic stability of the antigen over time. The method may further comprise (c) contacting a sample comprising said antigen with an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (d) determining antigenic integrity of said antigen by detectable binding of said antibody or antibody fragment to said antigen. The second antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1. The second antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. The second antibody fragment may be a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment. The method may further comprise performing steps (c) and (d) a second time to determine the antigenic stability of the antigen over time. The use of the word“a” or“an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.” The word“about” means plus or minus 5% of the stated number.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

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

FIGS. 1A-B. Overall structure of the RSV F:MPE8 complex. (FIGS. 1A-B) Structure of the complex of MPE8 scFv with prefusion RSV F. RSV F subunits are shown in surface format. Two subunits are colored light grey. The third subunit is colored by domain (DI: cyan; DII: green; DIII: magenta: heptad repeat B (HRB): blue). The MPE8 scFvs are shown in cartoon format with VH colored salmon and VL colored yellow. The view in FIG.1B is rotated 90˚ from FIG.1A and oriented down the 3-fold axis of the F trimer. MPE8 binds an epitope in the midsection of the RSV F ectodomain at the intersection of DI, DII and DIII domains from two subunits of the F trimer.

FIGS.2A-D. The MPE8 epitope and paratope. (FIG.2A) The MPE8 epitope is formed by residues from two adjacent F subunits. MPE8 is shown in cartoon format, with VH in pink and VL in yellow. The RSV F surface is shown, with interacting residues colored cyan and magenta in the two subunits as indicated. (FIG.2B) HCDR3 extends into a deep pocket at the F intersubunit interface, making contacts with both chains that are dependent on the prefusion F conformation. The HCDR3 extends underneath the Site A helix-turn-helix motif recognized by palivizumab/motavizumab. (FIG. 2C) MPE8 CDR loops form an extensive interface with RSV F, with HCDR1, HCDR2, LCDR1 and LCDR3 contacting one subunit. LCDR2 contacts a second subunit of F, while HCDR3 interacts with both. LCDR1 interacts with beta strands in HRA that refold to helices in the postfusion conformation. (FIG. 2D) HCDR3 folds over the surface of the VH and VL interface, covering residues of LCDR2 implicated in cross-reactive recognition of RSV and HMPV F.

FIGS. 3A-D. Structural basis for MPE8 cross-reactivity. (FIG. 3A) HCDR1, HCDR2 and HCDR3 interact with a contiguous patch of conserved residues within the MPE8 epitope on F. The RSV F epitope is colored by conservation (conserved: light orange; not conserved between RSV/HMPV: medium blue; not conserved between HMPV A/B strains: dark blue) (FIGS.3B) Location of predicted somatic mutations in mature MPE8. Somatically mutated residues are shown as spheres. Residue labels with yellow background indicate residues forming contacts with RSV F. The label for LCDR2 residue D50 is highlighted with a red box. MPE8 VH and VL are shown in cartoon format, colored pink and yellow, respectively. The view in panel (FIG.3C) is rotated 90˚ about the horizontal from FIG.3B. (FIG.3D) VL D50 within LCDR2 does not directly contact RSV F. D50 makes a hydrogen bond to the sidechain of HCDR3 residue T98, which could impact HCDR3 conformation and HMPV cross-reactivity.

FIGS. 4A-E. 25P13 is a unique RSV and HMPV cross-reactive neutralizing antibody that recognizes both the prefusion and postfusion forms of RSV F protein. (FIG.4A) 25P13 binds both RSV and HMPV F proteins and neutralizes both viruses. (FIG.4B) Epitope binning using the OctetRed system with anti-penta-HIS biosensors. Sensors were coated with RSV A2 fusion protein (DS-Cav1, prefusion conformation). Horizontal mAbs were loaded first, followed by the vertical mAbs. MAbs were judged to compete for the same site if maximum binding of the competing mAb was reduced to <33% of its un-competed binding (black boxes with white numbers). MAbs were considered non-competing if maximum binding of the competing mAb was >66% of its un-competed binding (white boxes with red numbers). Gray boxes with black numbers indicate an intermediate phenotype (between 33 and 66% of un-competed binding). (FIG. 4C) Epitope binning using OctetRed system with anti-penta-HIS biosensors. Sensors were coated with RSV A2 fusion protein (postfusion conformation, fusion peptide removed). Horizontal mAbs were loaded first, followed by the vertical mAbs. MAbs were judged to compete for the same site if maximum binding of the competing mAb was reduced to <33% of its un-competed binding (black boxes with white numbers). MAbs were considered non-competing if maximum binding of the competing mAb was >66% of its un-competed binding (white boxes with red numbers). Gray boxes with black numbers indicate an intermediate phenotype (between 33 and 66% of un-competed binding). (FIG. 4D) 25P13 uses the same VH and VL segments as MPE8 and shows homology in the HCDR3 and LCDR3 sequences (MPE8: HCDR1 = SEQ ID NO:11; HCDR2 = SEQ ID NO:12; HCDR3 = SEQ ID NO:13; LCDR1 = SEQ ID NO:15; LCDR2 = SEQ ID NO:16; LCDR3 = SEQ ID NO:17; 25P13: HCDR1 = SEQ ID NO:5; HCDR2 = SEQ ID NO:14; HCDR3 = SEQ ID NO:7; LCDR1 = SEQ ID NO:18; LCDR2 = SEQ ID NO:19; LCDR3 = SEQ ID NO:10). Many conserved amino acids of the 25P13 LCDR3 correspond to the F-contacting residues in MPE8 (indicated above the sequences), suggesting a similar mode of F engagement. (FIG.4E) Antibody ELISA assays with RSV F mutants that disrupt MPE8 binding (Corti et al., 2013) and lie within the MPE8 epitope (D310A and G307R). FIG. 5. Sequences of the MPE8 VH (SEQ ID NO: 20) and VL (SEQ ID NO: 21) domains. The Chothia numbering scheme for antibodies (used here) is shown above the sequences in single letter amino acid format. The IMGT sequence numbering used previously (Corti et al., 2013) is shown below the sequences.

FIG. 6. Schematics of MPE8 scFv constructs. The full mature VH and VL sequences are referred to as VHsm and VLsm, respectively. The inferred unmutated common ancestor sequences are referred to a VHgl and VLgl. The N94S mutation removes an N-linked glycosylation site and was previously shown not to affect MPE8 neutralization and is used here as the mature MPE8 sequence (Corti et al., 2013). Other mutations of the mature sequence are shown above the diagrams, with residues labeled in the Chothia numbering scheme (FIG.5). (GT(GGSGG)3GAS = SEQ ID NO:23) FIGS. 7A-B. Purification of the MPE8:RSV F complexes. (FIG. 7A) Gel filtration profiles of the purified RSV F DS-CAV1 and DS-CAV1:MPE8 complex. (FIG.7B) SDS-PAGE analysis of purified F:MPE8 complexes and a dissolved crystal.

FIGS.8A-D. Superposition of the postfusion RSV F onto the DS-CAV1:MPE8 complex. (FIG. 8A) The DSCAV1:MPE8 complex structure is shown with a single subunit of the F trimer represented in cartoon format in grey. The MPE8 scFv is shown as a semitransparent surface with VL in yellow and VH in pink. Residues that contact MPE8 in the two RSV F HRA β-strands are shown as blue spheres. (FIG. 8B) Superposition of a single subunit of the postfusion RSV F trimer (Swanson et al., 2011; McLellan et al., 2011) onto the DSCAV1:MPE8 complex. The postfusion RSV F is shown in cartoon format in grey with the translocated HRA β-strand residues shown as blue spheres. (FIG. 8CC) The DS-CAV1:MPE8 trimer structure is shown in cartoon format with two subunits of the F trimer colored grey and one colored blue. The F DII domain makes contacts to MPE8 VL in the prefusion conformation. (FIG. 8D) The postfusion RSV F trimer structure is shown in cartoon format with two subunits of the F trimer colored grey and one colored blue. In the postfusion conformation, the F DII domain makes significant clashes with the MPE8 VL, contributing to the prefusion specificity.

FIGS 9A-B. Mutations in F that disrupt MPE8 binding map to the interface. (FIG.9A) Surface representation of F showing the location of residues within the MPE8 epitope that disrupt MPE8 binding (Corti et al., 2013). The epitope is colored light purple, with magenta indicating the positions of residues R49, T50, L305, G307, I309 and D310 that selectively reduce MPE8 binding to RSV F. (FIG.9B) Close-up view of the F residues implicated in MPE8 binding, showing the positions of MPE8 HCDR2, HCDR3 and LCDR1. The F residues are shown in stick representation and colored magenta. The turn and C-terminal helix of the site A epitope are labeled.

FIG. 10. Sequence alignment of MPE8 and 25P13 VH and VL domains. Residue conservation is indicated below the sequences using CLUSTALX notation (“*” for conserved;“:” for strongly conservative mutations,“.” for weakly conservative mutations). CDR loops are indicated with a bar above the sequences and labeled. Conserved residues in the CDR loops and in contact with F are colored yellow. Non- conserved residues are colored blue. F contact residues are indicated with an“*” above the sequences. (VH-MPE8 = SEQ ID NO: 20; VL-MPE8 = SEQ ID NO: 21; VH- 25P13 = SEQ ID NO: 3; VL-25P13 = SEQ ID NO: 22)

FIGS. 11A-D. The MPE8 epitope is distinct from other structurally characterized anti-RSV F neutralizing antibodies. RSV F is shown in surface representation with antibodies shown in cartoon format, colored from N- (blue) to C- (red) termini for each chain. (FIG.11A) MPE8:RSV F complex determined here. (FIG. 11B) D25:F complex (3). (FIG.11C) Low resolution motavizumab:F complex (Gilman et al., 2015). (FIG.11D) Low resolution AM14:F complex (Gilman et al., 2015).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Respiratory syncytial virus (RSV) remains a major human pathogen, infecting the majority of infants before age two and causing reinfection throughout life. Despite decades of RSV research, there is no licensed RSV vaccine. Most candidate vaccines studied to date have incorporated the RSV fusion (F) surface glycoprotein, since the sequence of F is highly conserved among strains of RSV.

Here, the inventors examine the structural basis for cross-reactive antibody neutralization of the RSV and HMPV fusion (F) protein by two related, independently isolated antibodies, MPE8 that was previously reported and a new antibody designated 25P13. They solved the structure of the MPE8 antibody bound to RSV F and identified a related antibody (25P13) from an independent blood donor. The 25P13 antibody not only recognizes both RSV and HMPV, but also this antibody recognizes both the prefusion and post-fusion forms of the F proteins; MPE8 recognizes only the prefusion form of F. These results indicate that both antibodies use germline residues to interact with a conserved surface on F that could guide the emergence of cross-reactivity. The induction of similar cross-reactive neutralizing antibodies using structural vaccinology approaches, could enhance intrinsic cross-immunity to paramyxovirus associated respiratory diseases and the control of recurring outbreaks.

These and other aspects of the disclosure are described in detail below. I. Respiratory Syncytial Virus

Human respiratory syncytial virus (RSV) is a syncytial virus that causes respiratory tract infections. It is a major cause of lower respiratory tract infections and hospital visits during infancy and childhood. A prophylactic medication, palivizumab, can be employed to prevent human RSV in preterm (under 35 weeks gestation) infants, infants with certain congenital heart defects (CHD) or bronchopulmonary dysplasia (BPD), and infants with congenital malformations of the airway. Treatment is limited to supportive care (e.g., C-PAP), including oxygen therapy.

Human RSV is a negative-sense, single-stranded RNA virus of the family Pneumoviridae. Its name comes from the fact that F proteins on the surface of the virus cause the cell membranes on nearby cells to merge, forming syncytia. It was first isolated in 1956 from a chimpanzee, and called Chimpanzee Coryza Agent (CCA). Also in 1956, a new type of cytopathogenic myxovirus was isolated from a group of human infants with infantile croup. In temperate climates there is an annual epidemic during the winter months. In tropical climates, infection is most common during the rainy season. In the United States, 60% of infants are infected during their first RSV season, and nearly all children will have been infected with the virus by 2–3 years of age. Of those infected with RSV, 2–3% will develop bronchiolitis, necessitating hospitalization. Natural infection with HRSV induces protective immunity which wanes over time—possibly more so than other respiratory viral infections— and thus people can be infected multiple times. Sometimes an infant can become symptomatically infected more than once, even within a single HRSV season. Severe HRSV infections have increasingly been found among elderly patients. Young adults can be re- infected every five to seven years, with symptoms looking like a sinus infection or a cold (infections can also be asymptomatic).

The incubation time (from infection until symptoms arrive) is 4–5 days. For adults, HRSV produces mainly mild symptoms, often indistinguishable from common colds and minor illnesses. The Centers for Disease Control consider HRSV to be the“most common cause of bronchiolitis (inflammation of the small airways in the lung) and pneumonia in children under 1 year of age in the United States.” For some children, RSV can cause bronchiolitis, leading to severe respiratory illness requiring hospitalization and, rarely, causing death. This is more likely to occur in patients that are immunocompromised or infants born prematurely. Other HRSV symptoms common among infants include listlessness, poor or diminished appetite, and a possible fever.

Recurrent wheezing and asthma are more common among individuals who suffered severe HRSV infection during the first few months of life than among controls; whether HRSV infection sets up a process that leads to recurrent wheezing or whether those already predisposed to asthma are more likely to become severely ill with HRSV has yet to be determined.

Symptoms of pneumonia in immuno-compromised patients such as in transplant patients and especially bone marrow transplant patients should be evaluated to rule out HRSV infection. This can be done by means of polymerase chain reaction (PCR) testing for HRSV nucleic acids in peripheral blood samples if all other infectious processes have been ruled out or if it is highly suspicious for RSV such as a recent exposure to a known source of HRSV infection.

Complications include bronchiolitis or pneumonia, asthma, recurring infections, and acute otitis media. Transmission. The incubation period is 2–8 days, but is usually 4–6 days. RSV spreads easily by direct contact, and can remain viable for a half an hour or more on hands or for up to 5 hours on countertops. Childcare facilities allow for rapid child-to-child transmission in a short period of time. RSV can last 2–8 days, but symptoms may persist for up to three weeks.

The human RSV is virtually the same as chimpanzee coryza virus and can be transmitted from apes to humans, although transmission from humans to apes is more common. The virus has also been recovered from cattle, goats and sheep, but these are not regarded as major vectors of transmission and there is no animal reservoir of the virus.

Virology. Human RSV is a medium-sized (120-200 nm) enveloped virus that contains a lipoprotein coat and a linear negative-sense RNA genome (must be converted to an anti-sense genome prior to translation). The former contains virally encoded F, G, and SH lipoproteins. The F and G lipoproteins are the only two that target the cell membrane, and are highly conserved among RSV isolates. HRSV is divided into two antigenic subgroups, A and B, on the basis of the reactivity of the virus with monoclonal antibodies against the attachment (G) and fusion (F) glycoproteins. Subtype B is characterized as the asymptomatic strains of the virus that the majority of the population experiences. The more severe clinical illnesses involve subtype A strains, which tend to predominate in most outbreaks.

The genome is ~15,000 nucleotides in length and is composed of a single strand of RNA with negative polarity. It has 10 genes encoding 11 proteins. To date, 10 HRSV-A genotypes have been designated, GA1 to GA7, SAA1, NA1, and NA2. The HRSV-B genotypes include GB1 to GB4, SAB1 to SAB3, and BA1 to BA6. The genome of HRSV was completely sequenced in 1997.

Diagnosis. Human respiratory syncytial virus may be suspected based on the time of year of the infection; prevalence usually coincides with the winter flu season. Tests include (a) chest X-rays to check for typical bilateral perihilar fullness of bronchiolitis induced by the virus, (b) skin monitoring to check for hypoxemia, a lower than usual level of oxygen in the bloodstream, (c) blood tests to check white cell counts or to look for the presence of viruses, bacteria or other organisms, and (d) lab testing of respiratory secretions.

Several different types of laboratory tests are commercially available for diagnosis of RSV infection. Rapid diagnostic assays performed on respiratory specimens are available commercially. Most clinical laboratories currently utilize antigen detection tests. Compared with culture, the sensitivity of antigen detection tests generally ranges from 80% to 90%. Antigen detection tests and culture are generally reliable in young children but less useful in older children and adults. Sensitivity of virus isolation from respiratory secretions in cell culture varies among laboratories. RT-PCR assays are now commercially available. The sensitivity of these assays is equal to or exceeds the sensitivity of virus isolation and antigen detections methods. Highly sensitive RT-PCR assays should be considered when testing adults, because they may have low viral loads in their respiratory specimens.

Serologic tests are less frequently used for diagnosis. Although useful for research, a diagnosis using a collection of paired acute and convalescent sera to demonstrate a significant rise in antibody titer to HRSV cannot be made in time to guide care of the patient. On top of that, the antibody level does not always correlate with the acuteness or activity level of the infection.

RSV infection can be confirmed using tests for antigens or antibodies, or viral RNA by reverse transcription PCR. Quantification of viral load can be determined by various assay tests.

Prevention. As the virus is ubiquitous in all parts of the world, avoidance of infection is not possible. However, palivizumab (brand name Synagis manufactured by MedImmune), a moderately effective prophylactic drug, is available for infants at high risk. Palivizumab is a monoclonal antibody directed against RSV surface fusion protein. It is given by monthly injections, which are begun just prior to the RSV season and are usually continued for five months. HRSV prophylaxis is indicated for infants that are premature or have either cardiac or lung disease, but the cost of prevention limits use in many parts of the world.

Vaccine Research. A vaccine trial in 1960s using a formalin-inactivated vaccine (FI- RSV) increased disease severity in children who had been vaccinated. There is much active investigation into the development of a new vaccine, but at present no vaccine exists. Some of the most promising candidates are based on temperature sensitive mutants which have targeted genetic mutations to reduce virulence.

Scientists are attempting to develop a recombinant human respiratory syncytial virus vaccine that is suitable for intranasal instillation. Tests for determining the safety and level of resistance that can be achieved by the vaccine are being conducted in the chimpanzee, which is the only known animal that develops a respiratory illness similar to humans.

The development of a commercial human RSV vaccine has remained elusive. Recent breakthroughs have sparked continued interest in this highly sought after vaccine as the annual medical burden relating to human RSV has remained high, equal to Influenza and Pneumococcus.

Treatment. To date, treatment has been limited to supportive measures. Adrenaline, bronchodilators, steroids, antibiotics, and ribavirin confer“no real benefit.” Studies of nebulized hypertonic saline have shown that the use of nebulized 3% HS is a safe, inexpensive, and effective treatment for infants hospitalized with moderately severe viral bronchiolitis where respiratory syncytial virus (RSV) accounts for the majority of viral bronchiolitis cases. One study noted a 26% reduction in length of stay: 2.6 ± 1.9 days, compared with 3.5 ± 2.9 days in the normal-saline treated group (p=0.05). Supportive care includes fluids and oxygen until the illness runs its course. Salbutamol may be used in an attempt to relieve any bronchospasm if present. Increased airflow, humidified and delivered via nasal cannula, may be supplied in order to reduce the effort required for respiration. II. Monoclonal Antibodies and Production Thereof

A. General Methods

It will be understood that monoclonal antibodies binding to human respiratory syncytial virus will have several applications. These include the production of diagnostic kits for use in detecting and diagnosing human respiratory syncytial virus infection, as well as for treating the same. In these contexts, one may link such antibodies to diagnostic or therapeutic agents, use them as capture agents or competitors in competitive assays, or use them individually without additional agents being attached thereto. The antibodies may be mutated or modified, as discussed further below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent 4,196,265).

The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step for both these methods is immunization of an appropriate host or identification of subjects who are immune due to prior natural infection. As is well known in the art, a given composition for immunization may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine. As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non- specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants and aluminum hydroxide adjuvant.

In the case of human antibodies against natural pathogens, a suitable approach is to identify subjects that have been exposed to the pathogens, such as those who have been diagnosed as having contracted the disease, or those who have been vaccinated to generate protective immunity against the pathogen. Circulating anti-pathogen antibodies can be detected, and antibody producing B cells from the antibody-positive subject may then be obtained.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.

Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens or lymph nodes, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized or human or human/mouse chimeric cells. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp.65-66, 1986; Campbell, pp.75-83, 1984).

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods also is appropriate (Goding, pp. 71-74, 1986). Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10^-6 to 1 x 10- ⁸. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, infused cells (particularly the infused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. Ouabain is added if the B cell source is an Epstein Barr virus (EBV) transformed human B cell line, in order to eliminate EBV transformed lines that have not fused to the myeloma.

The preferred selection medium is HAT or HAT with ouabain. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a line of EBV-transformed B cells, as here, ouabain may also be used for drug selection of hybrids as EBV-transformed B cells are susceptible to drug killing, whereas the myeloma partner used is chosen to be ouabain resistant.

Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like. The selected hybridomas are then serially diluted or single-cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. When human hybridomas are used in this way, it is optimal to inject immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. Alternatively, human hybridoma cells lines can be used in vitro to produce immunoglobulins in cell supernatant. The cell lines can be adapted for growth in serum-free medium to optimize the ability to recover human monoclonal immunoglobulins of high purity.

MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the disclosure can be obtained from the purified monoclonal antibodies by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

It also is contemplated that a molecular cloning approach may be used to generate monoclonals. For this, RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 10⁴ times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.

Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Patent 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Patent 4,816,567 which describes recombinant immunoglobulin preparations; and U.S. Patent 4,867,973 which describes antibody-therapeutic agent conjugates. B. Antibody of the Present Disclosure

The antibody according to the present disclosure (exemplified by 25P13) may be defined, in the first instance, by binding specificity. Those of skill in the art, by assessing the binding specificity/affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims. In one aspect, there are provided monoclonal antibodies having clone-paired CDRs from the heavy and light chains as illustrated in Tables 3 and 4, respectively. Such antibodies may be produced by the clones discussed below in the Examples section using methods described herein.

In particular, the antibodies of the present disclosure, in one aspect, relate to the identification, through their binding specificity, of a previously unrecognized complex epitope that comprises elements of the previously designated antigenic site A (also called site II) and elements of site IV.

In a second aspect, the antibodies may be defined by their variable sequence, which include additional“framework” regions. These are provided in Tables 1 and 2 that encode or represent full variable regions. Furthermore, the antibodies sequences may vary from these sequences, optionally using methods discussed in greater detail below. For example, nucleic acid sequences may vary from those set out above in that (a) the variable regions may be segregated away from the constant domains of the light and heavy chains, (b) the nucleic acids may vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acids may vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids may vary from those set out above by virtue of the ability to hybridize under high stringency conditions, as exemplified by low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C, (e) the amino acids may vary from those set out above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or (f) the amino acids may vary from those set out above by permitting conservative substitutions (discussed below). Each of the foregoing applies to the nucleic acid sequences set forth as Table 1 and the amino acid sequences of Table 2. C. Engineering of Antibody Sequences

In various embodiments, one may choose to engineer sequences of the identified antibodies for a variety of reasons, such as improved expression, improved cross-reactivity or diminished off-target binding. The following is a general discussion of relevant techniques for antibody engineering. Hybridomas may be cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns.

Recombinant full length IgG antibodies were generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 Freestyle cells or CHO cells, and antibodies were collected an purified from the 293 or CHO cell supernatant.

The rapid availability of antibody produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to reduce the duration of process development programs. Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools, expressing a model antibody, in a disposable bioreactor: in a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g/L was achieved within 9 weeks of transfection.

Antibody molecules will comprise fragments (such as F(ab’), F(ab’)2) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form“chimeric” binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.

In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g., a chimeric, or CDR-grafted antibody). Alternatively, one may wish to make modifications, such as introducing conservative changes into an antibody molecule. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 ± 1), glutamate (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (- 3.4), phenylalanine (-2.5), and tyrosine (-2.3).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG1 can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency. Modifications in the Fc region can be introduced to extend the in vivo half-life of the antibody, or to alter Fc mediated fucntions such as complement activation, antibody dependent cellular cytotoxicity (ADCC), and FcR mediated phagocytosis. Other types of modifications include residue modification designed to reduce oxidation, aggregation, deamidation, and immunogenicity in humans. Other changes can lead to an increase in manufacturability or yield, or reduced tissue cross-reactivity in humans.

Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document. D. Single Chain Antibodies

A Single Chain Variable Fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.

Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alaine, serine and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approx. 5 × 10⁶ different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers. The recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.

In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge).

Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stablizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created. To link two different compounds in a step-wise manner, hetero- bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.

An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).

It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.

Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is“sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.

The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3'-dithiopropionate. The N-hydroxy- succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

U.S. Patent 4,680,338, describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.

U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques. E. Intrabodies

In a particular embodiment, the antibody is a recombinant antibody that is suitable for action inside of a cell– such antibodies are known as“intrabodies.” These antibodies may interfere with target function by a variety of mechanism, such as by altering intracellular protein trafficking, interfering with enzymatic function, and blocking protein-protein or protein-DNA interactions. In many ways, their structures mimic or parallel those of single chain and single domain antibodies, discussed above. Indeed, single-transcript/single-chain is an important feature that permits intracellular expression in a target cell, and also makes protein transit across cell membranes more feasible. However, additional features are required.

The two major issues impacting the implementation of intrabody therapeutic are delivery, including cell/tissue targeting, and stability. With respect to delivery, a variety of approaches have been employed, such as tissue-directed delivery, use of cell-type specific promoters, viral-based delivery and use of cell-permeability/membrane translocating peptides. With respect to the stability, the approach is generally to either screen by brute force, including methods that involve phage diplay and may include sequence maturation or development of consensus sequences, or more directed modifications such as insertion stabilizing sequences (e.g., Fc regions, chaperone protein sequences, leucine zippers) and disulfide replacement/modification.

An additional feature that intrabodies may require is a signal for intracellular targeting. Vectors that can target intrabodies (or other proteins) to subcellular regions such as the cytoplasm, nucleus, mitochondria and ER have been designed and are commercially available (Invitrogen Corp.; Persic et al., 1997). F. Purification

In certain embodiments, the antibodies of the present disclosure may be purified. The term“purified,” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it may naturally occur. Where the term“substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.

Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.

In purifying an antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

Commonly, complete antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody. Alternatively, antigens may be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter or bead. The antibodies is bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc.).

Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary. III. Active/Passive Immunization and Treatment/Prevention of Human respiratory syncytial virus Infection

A. Formulation and Administration

The present disclosure provides pharmaceutical compositions comprising anti-human respiratory syncytial virus antibodies and antigens for generating the same. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, or a peptide immunogen, and a pharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

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

Active vaccines are also envisioned where antibodies like those disclosed are produced in vivo in a subject at risk of Human respiratory syncytial virus infection. Such vaccines can be formulated for parenteral administration, e.g., formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Administration by intradermal and intramuscular routes are contemplated. The vaccine could alternatively be administered by a topical route directly to the mucosa, for example by nasal drops, inhalation, or by nebulizer. Pharmaceutically acceptable salts, include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Passive transfer of antibodies, known as artificially acquired passive immunity, generally will involve the use of intravenous or intramuscular injections. The forms of antibody can be human or animal blood plasma or serum, as pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, as high-titer human IVIG or IG from immunized or from donors recovering from disease, and as monoclonal antibodies (MAb). Such immunity generally lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non-human origin. However, passive immunity provides immediate protection. The antibodies will be formulated in a carrier suitable for injection, i.e., sterile and syringeable.

Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. IV. Antibody Conjugates

Antibodies of the present disclosure may be linked to at least one agent to from an antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or polynucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non- limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.

Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging." Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.

In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen, iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^99m and/or yttrium⁹⁰.¹²⁵I is often being preferred for use in certain embodiments, and technicium^99m and/or indium¹¹¹ are also often preferred due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present disclosure may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the disclosure may be labeled with technetium^99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).

Among the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.

Another type of antibody conjugates contemplated in the present disclosure are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in 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.

Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten- based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.

Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and may be used as antibody binding agents.

Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3 ^-6 ^-diphenylglycouril-3 attached to the antibody (U.S. Patents 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Patent 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p- hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Patent 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O’Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation. V. Immunodetection Methods

In still further embodiments, the present disclosure concerns immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting Human respiratory syncytial virus and its associated antigens. While such methods can be applied in a traditional sense, another use will be in quality control and monitoring of vaccine and other virus stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e., long term stability) of H1 antigens in viruses. Alternatively, the methods may be used to screen various antibodies for appropriate/desired reactivity profiles.

Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation of human respiratory syncytial virus antibodies directed to specific viral epitopes in samples also is provided. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, the immunobinding methods include obtaining a sample suspected of containing Human respiratory syncytial virus, and contacting the sample with a first antibody in accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes.

These methods include methods for purifying human respiratory syncytial virus or related antigens from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the Human respiratory syncytial virus or antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the human respiratory syncytial virus antigen immunocomplexed to the immobilized antibody, which is then collected by removing the organism or antigen from the column.

The immunobinding methods also include methods for detecting and quantifying the amount of human respiratory syncytial virus or related components in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing human respiratory syncytial virus or its antigens, and contact the sample with an antibody that binds Human respiratory syncytial virus or components thereof, followed by detecting and quantifying the amount of immune complexes formed under the specific conditions. In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing human respiratory syncytial virus or Human respiratory syncytial virus antigen, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid, including blood and serum, or a secretion, such as feces or urine.

Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to Human respiratory syncytial virus or antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art.

The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a“secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

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

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

Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule. A. ELISAs

Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.

In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the Human respiratory syncytial virus or Human respiratory syncytial virus antigen is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-Human respiratory syncytial virus antibody that is linked to a detectable label. This type of ELISA is a simple“sandwich ELISA.” Detection may also be achieved by the addition of a second anti-Human respiratory syncytial virus antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing the Human respiratory syncytial virus or Human respiratory syncytial virus antigen are immobilized onto the well surface and then contacted with the anti-Human respiratory syncytial virus antibodies of the disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-Human respiratory syncytial virus antibodies are detected. Where the initial anti-Human respiratory syncytial virus antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-Human respiratory syncytial virus antibody, with the second antibody being linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.

In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand. “Under conditions effective to allow immune complex (antigen/antibody) formation” means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.

The“suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so. Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

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

After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6- sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.

In another embodiment, the present disclosure contemplates the use of competitive formats. This is particularly useful in the detection of Human respiratory syncytial virus antibodies in sample. In competition based assays, an unknown amount of analyte or antibody is determined by its ability to displace a known amount of labeled antibody or analyte. Thus, the quantifiable loss of a signal is an indication of the amount of unknown antibody or analyte in a sample.

Here, the inventors propose the use of labeled Human respiratory syncytial virus monoclonal antibodies to determine the amount of Human respiratory syncytial virus antibodies in a sample. The basic format would include contacting a known amount of Human respiratory syncytial virus monoclonal antibody (linked to a detectable label) with Human respiratory syncytial virus antigen or particle. The Human respiratory syncytial virus antigen or organism is preferably attached to a support. After binding of the labeled monoclonal antibody to the support, the sample is added and incubated under conditions permitting any unlabeled antibody in the sample to compete with, and hence displace, the labeled monoclonal antibody. By measuring either the lost label or the label remaining (and subtracting that from the original amount of bound label), one can determine how much non-labeled antibody is bound to the support, and thus how much antibody was present in the sample. B. Western Blot

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

Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.

The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non- specific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies. C. Immunohistochemistry

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

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples may be used for serial section cuttings.

Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted. D. Immunodetection Kits

In still further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies may be used to detect Human respiratory syncytial virus or Human respiratory syncytial virus antigens, the antibodies may be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to Human respiratory syncytial virus or Human respiratory syncytial virus antigen, and optionally an immunodetection reagent.

In certain embodiments, the Human respiratory syncytial virus antibody may be pre- bound to a solid support, such as a column matrix and/or well of a microtitre plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kits include the two- component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.

The kits may further comprise a suitably aliquoted composition of the Human respiratory syncytial virus or Human respiratory syncytial virus antigens, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. VI. Antigen Stability Assessment/Vaccine Quality Control

The present disclosure also contemplates the use of antibodies and antibody fragments as described herein for use in assessing the antigenic integrity of a viral antigen in a sample. Biological medicinal products like vaccines differ from chemical drugs in that they cannot normally be characterized molecularly; antibodies are large molecules of significant complexity, and have the capacity to vary widely from preparation to preparation. They are also administered to healthy individuals, including children at the start of their lives, and thus a strong emphasis must be placed on their quality to ensure, to the greatest extent possible, that they are efficacious in preventing or treating life-threatening disease, without themselves causing harm.

The increasing globalization in the production and distribution of vaccines has opened new possibilities to better manage public health concerns, but has also raised questions about the equivalence and interchangeability of vaccines procured across a variety of sources. International standardization of starting materials, of production and quality control testing, and the setting of high expectations for regulatory oversight on the way these products are manufactured and used, have thus been the cornerstone for continued success. But it remains a field in constant change, and continuous technical advances in the field offer a promise of developing potent new weapons against our oldest public health threats, as well as new ones - malaria, pandemic influenza, and HIV, to name a few - but also put a great pressure on manufacturers, regulatory authorities, and the wider medical community to ensure that products continue to meet the highest standards of quality attainable.

Thus, one may obtain an antigen or vaccine from any source or at any point during a manufacturing process. The quality control processes may therefore begin with preparing a sample for an immunoassay that identifies binding of an antibody or fragment disclosed herein to a viral antigen. Such immunoassays are disclosed elsewhere in this document, and any of these may be used to assess the structural/antigenic integrity of the antigen. Standards for finding the sample to contain acceptable amounts of antigenically intact antigen may be established by regulatory agencies.

Another important embodiment where antigen integrity is assessed is in determining shelf-life and storage stability. Most medicines, including vaccines, can deteriorate over time. Therefore, it is critical to determine whether, over time, the degree to which an antigen, such as in a vaccine, degrades or destabilizes such that is it no longer antigenic and/or capable of generating an immune response when administered to a subject. Again, standards for finding the sample to contain acceptable amounts of antigenically intact antigen may be established by regulatory agencies.

In certain embodiments, viral antigens may contain more than one protective epitope. In these cases, it may prove useful to employ assays that look at the binding of more than one antibody, such as 2, 3, 4, 5 or even more antibodies. These antibodies bind to closely related epitopes, such that they are adjacent or even overlap each other. On the other hand, they may represent distinct epitopes from disparate parts of the antigen. By examining the integrity of multiple epitopes, a more complete picture of the antigen’s overall integrity, and hence ability to generate a protective immune response, may be determined. VII. Examples

The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 - Materials and Methods

Expression and purification of proteins. The gene encoding the stabilized prefusion DS-CAV1 RSV F trimer (McLellan et al., 2013a) was synthesized (GeneWiz ) and cloned into the PTT5 expression vector (National Research Council (NRC), Canada) with a T4-fibritin trimerization domain in-frame with the heptad repeat of the C-terminal HRB, and with Thrombin cleavage site. The wt MPE8 single chain Fv (scFv) gene was synthesized by Life Technologies as a GeneArt® Strings™ DNA Fragment, was cloned into the pCEP4 expression vector (Invitrogen) and contains a C-terminal TEV cleavage site and His6 tag. The RSV F and scFv expression plasmids were prepared using the Plasmid Mega® Kit (Qiagen) and transfected into suspension 293-6E cells (NRC) at a density of 1.8 to 2.0 million cells/mL using 25-kDa linear polyethylenimine (Polysciences), (PEI) following 293-6E cell protocols. Supernatants were harvested five days post-transfection by centrifugation (20 min at 8,000 x g at room temperature), filtered through 0.45 μm filters and dialyzed against 200 mM NaCl, 50 mM Na2HPO4 pH 7.4. The RSV F protein was purified by Co²⁺ affinity chromatography (TALON Resin, BD Biosciences) and size exclusion chromatography using a Superdex-200 column equilibrated in 25 mM sodium phosphate, pH 7.4, and 100 mM NaCl, 100 mM imidazole. The purified protein was concentrated with Amicon Ultra centrifugal filters with a 10 kD molecular weight cut-off (Millipore). RSV F mutants L305R, G307R and D310A were prepared from the DS-CAV1 RSV F construct (Biozilla– Mutagenesis). Additional MPE8 variants were synthesized and cloned into the PTT5 expression vector corresponding to hGL- lGL, hGL-lSM, hSM-lSM D50G and hGL-lGL G56D. The MPE8 mutant proteins were expressed similarly to wild-type MPE8 in 293-6E cells, with supernatants harvested five days post-transfection. The MPE8 proteins were purified by Co²⁺ affinity chromatography and size exclusion chromatography and concentrated as with wild-type MPE8.

RSV F DS-CAV1 mutants L305R, G307R, and D310A were expressed in expiCHO cells following the manufacturer’s protocol, harvested after six days, and purified by HiTrap TALON crude columns (GE Healthcare). cDNAs encoding the published variable gene sequences encoding the mAbs motavizumab, 101F, and D25 were synthesized (Genscript), and heavy and light chain sequences were cloned into vectors encoding human IgG1 and lambda or kappa light chain constant regions, respectively. Plasmids encoding the heavy and light chains of mAb 54G10 were a gift from Dr. Dennis Burton (Scripps Research Institute). Mab 131-2a protein was obtained from Sigma (MAB8599). Commercial preparations of palivizumab (Medimmune) were obtained from the pharmacy at Vanderbilt University Medical Center. Recombinant mAbs were expressed in HEK293F cells for 5 days following the manufacturer’s protocol and purified by HiTrap MabSelectSure columns (GE Healthcare).

Crystallization and structure determination of the MPE8:RSV F complex. Complexes of MPE8 scFvs with the DS-CAV1 RSV F variant were prepared by co-transfection of 2936E cells, followed by purification using Co²⁺ affinity chromatography and gel filtration chromatography. Complexes of MPE8 with the DS-CAV1 RSV F were observed using size exclusion chromatography with a Superdex-200 column (FIGS.7A-B) and verified by SDS– PAGE. Complexes were dialyzed into 25 mM Tris-HCl and 125 mM NaCl pH 7.0 and concentrated to 6-8 mg/mL. Crystals were grown from hanging drops with a well solution containing 0.1M Potassium Nitrate, 0.1M Citrate Phosphate pH 4.2, 1% Tacsimate pH 7.0, 14% PEG 6000. Crystals appeared after 3–10 days.

Native crystals were transferred to a cryoprotectant solution of 0.1M Potassium Nitrate, 0.1M Citrate Phosphate pH 4.2, 1% Tacsimate pH 7.0, 14% PEG 6000 and 15% glycerol, followed by flash cooling in liquid nitrogen. Data were collected at LS-CAT 21-ID-F beamline at the Advanced Photon Source (APS) - Argonne National Laboratory. Crystals belong to space group P3121 and exhibited significant diffraction anisotropy. The native data were initially processed to 3.1 Å with XDS and then submitted to the Diffraction Anisotropy Server, which truncated the data to 3.1 Å along the c* axis and 3.6 Å along the a*/b* axes (Table S1).

Molecular replacement searches were conducted with the program PHASER (Storoni et al., 2004) using the CCP4 suite of programs (Collaborative Computational Project, 1994). The PCSK9 Fab model (3H42) and prefusion RSV F model (4JHW) yielded clear molecular replacement solutions. Initial refinement provided an Rfree of 39.91% and R work of 41.14%. Automated model building to improve early models was performed using Phenix AutoBuild (Zwart et al., 2008). The complex structure was refined using Phenix Refine (Adams et al., 2002) followed by manual rebuilding with the program COOT (Emsley et al., 2004). The F model starts with residue 25 and ends with residue 509 (of 562 in the ectodomain construct). The Fv VH model starts with residue E1 and ends with residue S113, the Fv VL model starts with residue V3 and ends with residue L127 with one additional glycine derived from the construct. The final refinement statistics, native data, and phasing statistics are summarized in Table S1. Figures were generated with the program Pymol (world-wide-web at pymol.org).

Enzyme linked immunosorbent assay (ELISA) for binding to RSV F protein. 384- well plates were coated with 2 µg/mL of antigen overnight at 4 °C. Plates were blocked for with 2% milk supplemented with 2% goat serum for one hour, followed by three washes with PBS-T. Primary mAbs or B cell culture supernatants were applied to wells for two hours. Plates were washed with PBS-T four times before applying secondary antibody (goat anti-human IgG Fc, Meridian Life Science) at a dilution of 1:4,000 in blocking solution. After a one-hour incubation, the plates were washed six times with PBS-T, and phosphatase substrate solution (1 mg/mL phosphatase substrate in 1M Tris aminomethane, Sigma) was added to each well. The plates were incubated at room temperature before reading the optical density at 405 nm on a Biotek plate reader.

Isolation of the 25P13 antibody. 25P13 was isolated from the PBMCs of a Nashville Red Cross donor. PBMCs were isolated from human donor blood samples using Ficoll- Histopaque density gradient centrifugation. PBMCs were transformed with Epstein-Barr virus as described previously (Smith et al., 2012). Cells were screened by ELISA for binding to post- fusion RSV F, and positive wells were fused with HMMA2.5 myeloma cells using the previously published protocol (Smith et al., 2012. The 25P13 hybridoma was biologically cloned by single-cell fluorescence-activated sorting. The 25P13 hybridoma was expanded step- wise into 48-well and 12-well plates followed by 75-cm² flask in Media E (StemCell Technologies). Antibody production was accomplished by expanding the hybridoma to four 225-cm² cell culture flasks in serum-free medium (Hybridoma-SFM, GIBCO). After 21 days, supernatants were sterile filtered using 0.45 μm pore size filter devices. For antibody purification, HiTrap MabSelectSure columns (GE Healthcare Life Sciences) were used to purify antibodies using the manufacturer’s protocol.

RSV and HMPV neutralization experiments. 25P13 isolated from hybridoma supernatants were incubated 1:1 with a suspension of infectious RSV strain A2 for 1 hr. Following this, confluent HEp-2 cells, maintained in Opti-MEM I+GlutaMAX (Fisher) supplemented with 2% fetal bovine serum at 37 °C in a CO₂ incubator, in 24-well plates were inoculated with 50 µL of the antibody:virus mixture for one hr. After one hour, cells were overlaid with 1 mL of 0.75% methylcellulose dissolved in Opti-MEM I + GlutaMAX. Cells were incubated for four days after which the plaques were visualized by fixing cells with 10% neutral-buffered formalin and staining with crystal violet.

Neutralization experiments with hMPV used strain TN/96-12, which was obtained originally from a nasopharyngeal-wash specimen from a young child with upper respiratory tract illness in 1996 in Nashville, TN. Virus was grown in Vero cells with Opti-MEM I+ GlutaMAX medium (Fisher) 37 °C in a CO2 incubator. The medium for cell propagation was supplemented with 2% fetal bovine serum. Confluent Vero cell monolayer cultures in 24-well plates were washed once with Opti-MEM I+GlutaMAX to remove serum, and then incubated with HMPV:mAb mixtures for one hour. Following this, the cells were overlaid with 0.75% methylcellulose in Opti-MEM I+GlutaMAX containing 5 µg/mL trypsin. Cells were incubated for four days before being fixed with neutral buffered formalin. Plaques were visualized by immunoperoxidase staining where anti-HMPV F guinea pig serum (or a mouse mAb targeting the HMPV nucleoprotein (Meridian Life Science) was used at 1:1,000 dilution in milk to overlay the cells for one hour, followed by incubation with 1:1,000 diluted goat anti-guinea pig IgG or anti-mouse IgG coupled to horseradish peroxidase (Meridian Life Science) for one hour. TrueBlue peroxidase substrate (Kirkegaard and Perry) then was added to visualize plaques. Plaques were counted and compared to the virus control. Data were analyzed with Prism software (GraphPad) to obtain IC50 values.

Competition binding. Competition binding was conducted on an OctetRed system (ForteBio) using anti-penta-HIS biosensors (ForteBio) and kinetics buffer (ForteBio). After obtaining an initial baseline in kinetics buffer, 10 µg/mL of his-tagged RSV F postfusion or prefusion (DSCAV1) protein was immobilized onto anti-penta-HIS biosensor tips for 120 s. The baseline signal was measured again for 60 s before biosensor tips were immersed into wells containing 100 µg/mL primary antibody for 300 s. Following this, biosensors were immersed into wells containing 100 µg/mL of a second mAb for 300 s. Percent binding of a second mAbs in the presence of the first mAb was determined by comparing the maximal signal of the second mAb after the first mAb was added to the maximum signal of the second mAb alone. MAbs were considered non-competing if maximum binding of the second mAb was≥ 66% of its un-competed binding. A level between 33%–66% of its uncompeted binding was considered intermediate competition, and≤ 33% was considered competing.

Example 2 - Results

The RSV/HMPV F protein is a class I viral fusion protein that refolds to drive membrane fusion and virus entry (Jardetzky & Lamb, 2014; Wen et al., 2012; McLellan et al., 2013a; McLellan et al., 2011; Swanson et al., 2011; Yin et al., 2006; Mas et al., 2016), and it is the major target of the neutralizing antibody response. MPE8 was isolated from human B cells based on its ability to cross-neutralize a panel of RSV and HMPV strains (Corti et al., 2013). The inventors determined the crystal structure of an MPE8 variant in complex with a stabilized prefusion RSV F trimer (DS-CAV1) (Joyce et al., 2016; Boyington et al., 2016; Stewart-Jones et al., 2015; McLellan et al., 2013b). They generated single chain Fv (scFv) constructs for MPE8 and predicted unmutated common ancestor (UCA) variants that retain RSV F binding and neutralization (Corti et al., 2013) (FIGS. 5-6). Complexes of MPE8 with DS-CAV1 (McLellan et al., 2013b) were prepared by co-transfection of 293-6E cells, and purified using affinity and gel filtration chromatography (FIGS.7A-B). Optimized crystals of DS-CAV1 with the MPE8 scFv variant consisting of the predicted UCA heavy chain variable domain (VHgl) and the fully mature light chain variable domain (VLsm) were obtained and the structure was solved by molecular replacement (FIGS.1A-B, Methods and Table S1).

MPE8 binds an epitope near the midsection of the RSV F ectodomain (FIGS. 1A and 1B, consistent with previous mapping studies (Corti et al., 2013). The interface buries ~1,100 Å² of surface on each of the proteins (2,200 Å² total). The three scFvs are positioned with VH and VL domains aligned nearly perpendicular to the long axis of the F trimer and parallel to the predicted plane of the viral membrane. When viewed along the three-fold axis of the trimer, the MPE8 V domains radiate horizontally outwards from three apices of the F subunits, engaging the widest section of the F head domain (FIGS. 1B). MPE8 binds preferentially to prefusion F. Its conformational specificity has three apparent structural determinants. First, MPE8 engages RSV F residues located spanning two neighboring subunits of the trimer (FIGS. 1A-B), defining an intersubunit epitope present only in the prefusion conformation. Second, MPE8 contacts two β-strands of the prefusion heptad repeat A (HRA), which refold into a long helix and move away from the MPE8 interface in the postfusion form (McLellan et al., 2013a; Yin et al., 2006) (FIGS. 8A-B). Third, docking of MPE8 onto the post-fusion F structure indicates that steric clashes between F DII and the MPE8 VL domain could interfere with binding (FIGS.8C-D).

Most of the MPE8 epitope lies within 1 subunit of the F trimer, involving residues in DI and DIII, with a smaller contact area in DII of the neighboring subunit (FIG.2A). The DIII contacts overlap the helix-turn-helix motif of the palivizumab/motavizumab site A epitope (FIGS.2A-B). The VH domain lies distal to the F trimer interface, primarily contacting the F surface below and to one edge of site A (FIGS.2A-C). The HCDR3 loop extends underneath the site A motif in DIII to insert its tip into a pocket formed at the intersubunit interface (FIGS. 2B-C), with residues Y100 and N100A forming contacts dependent on the prefusion F conformation. HCDR3 residues (100B-100G) interact with the turn residues within site A (FIG. 2B). HCDR2 also forms interactions with the second helix of site A and with residues in DI, while HCDR1 contacts F below site A in DI (FIG.2C). RSV F mutations that have been shown previously to affect MPE8 binding also map to the structural interface (FIGS. 9A-B). The mutation of D310A would disrupt interactions with HCDR2 and many of these previously identified mutations line the groove along which HCDR3 extends (L305R, G307R, R49D and T50A).

The VL domain is positioned near the trimer interface with LCDR2 contacting DII of the adjacent subunit (FIG.2C). LCDR2 interactions across the trimer, together with HCDR3, would contribute to preferential binding to prefusion F. LCDR1 and LCDR3 interact with the first helix of site A (FIGS. 2A-D). LCDR1 also contacts two beta strands of the DIII HRA motif (F residues 178, 180 and 184-187), present only in the prefusion conformation (FIGS. 8A-D). The MPE8 paratope is a relatively flat surface comprised of the 6 CDR loops, with the heavy chain HCDR3 wrapping across the front of the VH domains and covering LCDR2 (FIG. 2D). HCDR1, HCDR2 and HCDR3 form an L-shaped surface that defines the majority of the MPE8 binding surface, with HCDR3 inserting into the F intersubunit groove.

The MPE8 epitope is only partially conserved in HMPV (FIG. 3A and Table S2). HCDR1 and HCDR2 anchor the antibody to the largest contiguous surface of conserved residues (FIG.3A). HCDR3 extends along a narrower conserved segment to contact additional, more fragmented patches of surface residue conservation. The light chain, particularly LCDR1, also contacts smaller, more isolated islands of conserved residues (FIG. 3A). MPE8 crossreactivity appears to be achieved by the formation of a large interface with a mosaic patchwork of conservation rather than by a narrow focus on a single conserved site. The predicted MPE8 UCA selectively neutralizes RSV, but not HMPV (Corti et al., 2013), indicating that cross-reactivity emerges as a result of somatic hypermutation that broadens its reactivity to HMPV. Fourteen residues in MPE8, 9 in VH and 5 in VL, are mutated in mature MPE8. VL residues play a major role in HMPV cross-reactivity (Corti et al., 2013). In the MPE8 structure, three of the five VL residues (D50, N52 and R93) are located in or near the RSV F interface (FIGS.3B-C). For VH, only three of the nine somatic mutations are within the RSV interface (residues S52A, Y58 and N100A in VHgl; FIGS.3B-C). VL D50 is a major determinant of cross-reactivity to HMPV F (Corti et al., 2013). Mutation of D50 to glycine, representing a reversion to the UCA, ablates HMPV F binding. D50 is located behind HCDR3 and does not directly contact F, but forms a hydrogen bond to HCDR3 residue T98 (FIG.3D). D50 may therefore indirectly stabilize an HCDR3 conformation that can bind both RSV and HMPV F, similar to somatic mutations observed in other antibodies. Mutation of VL R93S in LCDR3 reduces MPE8 binding to HMPV B strains (Corti et al., 2013). R93 lies above antigenic site A and forms a hydrogen bond with the main chain of RSV F residue 263 (FIGS. 3B-C). To a lesser extent, somatically mutated residues in VH have also been shown to influence MPE8 cross-reactivity (Corti et al., 2013). The structure indicates that these effects are likely due to VH residues Y58 and N100A in HCDR2 and HCDR3, respectively. Y58, the predicted germline residue, forms contacts to RSV F D310, an F residue important for MPE8 binding. N100A is at the tip of HCDR3 and is mutated to serine in the mature MPE8 sequence (FIGS.2B-C).

To further explore the diversity of cross-reactive antibodies to RSV and HMPV, the inventors identified a novel antibody (25P13) from a blood donor that also binds and neutralizes both RSV and HMPV F (FIG.4A).25P13 binding is stronger to a chimeric HMPV F protein with a grafted RSV F site A (RPM-1) (Wen et al., 2016) than it is to wild-type HMPV F, providing a preliminary mapping of its epitope (FIG.4A). Cross-competition assays with a panel of RSV specific antibodies show that 25P13 competes with MPE8, as well as motavizumab and palivizumab (FIG. 4B), while partially competing with 101F and 54G10 antibodies. The similar cross-competition pattern for MPE8 and 25P13 indicates that both antibodies overlap antigenic sites site A and IV. However, in contrast to MPE8, 25P13 binds more readily to post-fusion RSV F (FIG.4C).

The inventors sequenced the 25P13 heavy and light chain variable regions (FIG. 4D, FIG.10).25P13 uses the same VH gene segment (3-21*01) as MPE8 and a closely related or identical VL segment (FIG. 4D). Sequences of the HCDR and LCDR loops show significant homology. In particular, residues in MPE8 HCDR1 and HCDR2 are >80% conserved, indicating that 25P13 could engage the same conserved surface patch of residues on F similar to MPE8 (FIG.3A). Indeed, 25P13 and MPE8 both lose binding to RSV F DS-CAV1 with the D310A mutation (FIG. 4E and FIGS. 9A-B). LCDR3 is also well conserved (7 out of 10 residues), including two key residues (Y107 and R93) that are buried in the MPE8 complex with RSV F. HCDR3 exhibits the largest differences (~50% identity) and is shorter by two residues, indicating that the 25P13 HCDR3 would not insert as deeply into the intersubunit crevice in F and may not engage two subunits of F as observed for MPE8 (FIGS. 2A-D). A G307R mutation was previously shown to disrupt MPE8 binding (Corti et al., 2013). This mutation significantly reduces but does not eliminate 25P13 binding, consistent with interaction differences of its shorter HCDR3 loop (FIG.4E and FIGS.9A-B). Together, these data indicate that related cross-reactive neutralizing antibodies can be readily identified, which utilize germline HCDR1 and HCDR2 sequences to anchor interactions with F through a conserved surface determinant present in RSV and HMPV F proteins.

The MPE8 and 25P13 epitopes are distinct from other structurally characterized anti-F antibodies (FIGS.11A-D). D25 (McLellan et al., 2013a) and AM14 (Gilman et al., 2015) are two neutralizing antibodies specific for the prefusion F conformation, while motavizumab can bind both pre and post-fusion RSV F. D25 binds an epitope in the top of DIII that does not have any overlap with MPE8 (McLellan et al., 2013a). Low-resolution structures of AM14 and motavizumab with RSV F have been determined (Gilman et al., 2015) and binding of both of these antibodies would be sterically blocked by MPE8. The epitopes of AM14 and motavizumab partially overlap that of MPE8. However, only MPE8 engages the larger patch of conserved residues lying below the site A epitope that provides its nascent cross-reactivity (FIG. 3A). The newly isolated 25P13 antibody disclosed here is the only antibody that recognizes both HMPV and RSV F and also recognizes both prefusion and postfusion F of both viruses, thus it is the first member of a new class of RSV human antibodies developed.

Broadly neutralizing antibodies to HIV Env and to influenza virus HA have been shown in several cases to involve a focusing of the antibody interaction on highly conserved elements of an indispensible functional site (Lee & Wilson, 2015; Doria-Rose & Joyce, 2015; Schmidt et al., 2015; Zhou et al., 2015). MPE8 and 25P13 appear to achieve their cross-reactivity through an anchoring interaction mediated by germline HCDR1 & HCDR2 loops with a conserved F surface within DI, while fine tuning interactions across a patchwork of conserved and non-conserved epitope residues. However, the mode of binding of 25P13 can be distinguished from that of MPE8 because it recognizes both prefusion and postfusion F. Specific recognition of the prefusion F conformation through this cross-reactive site is not required for neutralization, as shown by the inventors’ observations with 25P13. Other potentially cross-reactive antibodies to RSV and HMPV, which bear the same VH domain as 25P13, likely are present as nascent variants within the antibody repertoire, providing an attractive target for structure-based vaccine design.

TABLE 1– NUCLEOTIDE SEQUENCES FOR ANTIBODY VARIABLE REGIONS Clone Variable Sequence Region SEQ

ID NO: 25P13 caggtgcagctggtggagtcgggggggggcgtggtcaagccgggggggtccctgagagtctcgtgtgttgtctct 1 heavy ggattcaccttcagttcctacagaatgcactgggtccgccaggcgccagggaagggactggagtgggtctcctcg

attactgctagtagttcttacataaactacgcagagtcagtgaagggccgattcaccatctccagagacaacgcc aagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggctgtttattactgtgcgagagacga gaatactggaatctcccactattggttcgacccctggggccagggaaccctggtcaccgtctcctcag

25P13 caggctgtggtgactcagccgccctcagtgtctggggccccagggcagagggtcatcatctcctgcactgggagc 2 light ggttccaacctcggggcagattatggtgtacactggtaccagcaacttccaggcacagcccccaaacttctcatc

tatggtgataggaatcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctgg ccatcactgggctccaggccgaggatgaggctgattattactgccagtcctatgacaggagcctgaattgggtgt tggcggagnaccaagctgaccntcnta

TABLE 2– PROTEIN SEQUENCES FOR ANTIBODY VARIABLE REGIONS Clone Variable Sequence SEQ ID

NO: 25P13 3 heavy QVQLVESGGGVVKPGGSLRVSCVVSGFTFSSYRMHWVRQAPGKGL

EWVSSITASSSYINYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAV YYCARDENTGISHYWFDPWGQGTLVTVSS 25P13 QAVVTQPPSVSGAPGQRVIISCTGSGSNLGADYGVHWYQQLPGTAP 4 light KLLIYGDRNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSY

DRSLNWVLAENQADNN

TABLE 3– CDR HEAVY CHAIN SEQUENCES Antibody CDRH1 CDRH2 CDRH3

(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO: ) 25P13

GFTFSSYR ITASSSYI ARDENTGISHYWFDP (5) (6) (7) TABLE 4– CDR LIGHT CHAIN SEQUENCES Antibody CDRL1 CDRL2 CDRL3

(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO: ) 2_5P13

GSNLGADYG GDR QSYDRSLNWV

(8) (9) (10)

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

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Claims

WHAT IS CLAIMED IS: 1. A method of detecting a human respiratory syncytial virus infection in a subject comprising: (a) contacting a sample from said subject with an antibody or antibody fragment having heavy chain CDR1 GFTFSSYR (SEQ ID NO: 5), heavy chain CDR2 ITASSSYI (SEQ ID NO: 6), heavy chain CDR3 ARDENTGISHYWFDP (SEQ ID NO: 7), light chain CDR1 GSNLGADYG (SEQ ID NO: 8), light chain CDR2 GDR (SEQ ID NO: 9) and light chain CDR3 QSYDRSLNWV (SEQ ID NO: 10); and

(b) detecting human respiratory syncytial virus in said sample by binding of said antibody or antibody fragment to a human respiratory syncytial virus antigen in said sample. 2. The method of claim 1, wherein said sample is a body fluid. 3. The method of claims 1-2, wherein said sample is blood, sputum, tears, saliva,

mucous or serum, urine, exudate, transudate, tissue scrapings or feces. 4. The method of claims 1-3, wherein detection comprises ELISA, RIA or Western blot. 5. The method of claims 1-4, further comprising performing steps (a) and (b) a second time and determining a change in human respiratory syncytial virus antigen levels as compared to the first assay. 6. The method of claims 1-5, wherein the antibody or antibody fragment is encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 7. The method of claims 1-5, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 8. The method of claims 1-5, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 9. The method of claims 1-5, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 10. The method of claims 1-5, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 11. The method of claims 1-5, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 12. The method of claims 1-11, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment. 13. A method of treating a subject infected with human respiratory syncytial virus, or reducing the likelihood of infection of a subject at risk of contracting human respiratory syncytial virus, comprising delivering to said subject an antibody or antibody fragment having heavy chain CDR1 GFTFSSYR (SEQ ID NO: 5), heavy chain CDR2 ITASSSYI (SEQ ID NO: 6), heavy chain CDR3 ARDENTGISHYWFDP (SEQ ID NO: 7), light chain CDR1 GSNLGADYG (SEQ ID NO: 8), light chain CDR2 GDR (SEQ ID NO: 9) and light chain CDR3 QSYDRSLNWV (SEQ ID NO: 10). 14. The method of claims 11-13, wherein the antibody or antibody fragment is encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 15. The method of claims 11-13, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 16. The method of claims 11-13, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 17. The method of claims 11-13, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 18. The method of claims 11-13, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 19. The method of claims 11-13, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 20. The method of claims 13-19, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment, a chimeric antibody and/or is an IgG. 21. The method of claims 13-20, wherein said antibody or antibody fragment recognizes an epitope on pre-fusion and post-fusion RSV F protein in antigenic sites II (alternatively called site A) and IV, and optionally binds to human metapneumovirus F protein. 22. The method of claim 21, wherein said antibody or antibody fragment neutralizes RSV and human metapneumovirus. 23. The method of claim 13-22, wherein said antibody or antibody fragment is administered prior to infection. 24. The method of claim 13-22, wherein said antibody or antibody fragment is administered after infection. 25. The method of claim 13-24, wherein delivering comprises antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment. 26. A monoclonal antibody or antibody fragment comprises heavy chain CDR1 GFTFSSYR (SEQ ID NO: 5), heavy chain CDR2 ITASSSYI (SEQ ID NO: 6), heavy chain CDR3 ARDENTGISHYWFDP (SEQ ID NO: 7), light chain CDR1 GSNLGADYG (SEQ ID NO: 8), light chain CDR2 GDR (SEQ ID NO: 9) and light chain CDR3 QSYDRSLNWV (SEQ ID NO: 10). 27. The monoclonal antibody or antibody fragment of claim 26, wherein the antibody or antibody fragment is encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 28. The monoclonal antibody or antibody fragment of claim 26, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 29. The monoclonal antibody or antibody fragment of claim 26, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 30. The monoclonal antibody or antibody fragment of claim 26, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 31. The monoclonal antibody or antibody fragment of claim 26, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 32. The monoclonal antibody or antibody fragment of claim 26, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 33. The monoclonal antibody of claims 26-32, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment, or is a chimeric antibody, a bispecific antibody, and/or is an IgG. 34. The monoclonal antibody of claims 26-33, wherein said antibody or antibody fragment recognizes an epitope on pre-fusion and post-fusion RSV F protein in antigenic sites II (alternatively called A) and IV, and optionally binds to human metapneumovirus F protein, and/or neutralizes RSV and/or human metapneumovirus. 35. The monoclonal antibody of claims 26-34, wherein said antibody or antibody fragment further comprises a cell penetrating peptide and/or is an intrabody. 36. A hybridoma or engineered cell encoding an antibody or antibody fragment wherein the antibody or antibody fragment is characterized by heavy chain CDR1 GFTFSSYR (SEQ ID NO: 5), heavy chain CDR2 ITASSSYI (SEQ ID NO: 6), heavy chain CDR3 ARDENTGISHYWFDP (SEQ ID NO: 7), light chain CDR1 GSNLGADYG (SEQ ID NO: 8), light chain CDR2 GDR (SEQ ID NO: 9) and light chain CDR3 QSYDRSLNWV (SEQ ID NO: 10). 37. The hybridoma or engineered cell of claim 36, wherein the antibody or antibody fragment is encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 38. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 39. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 40. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 41. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 42. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 43. The hybridoma or engineered cell of claims 36-42, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’)₂ fragment, or Fv fragment. 44. The hybridoma or engineered cell of claim 36-42, wherein said antibody is a chimeric antibody, a bispecific antibody, and/or is an IgG. 45. The hybridoma or engineered cell of claims 36-43, wherein said antibody or antibody fragment recognizes an epitope on pre-fusion and post-fusion RSV F protein in antigenic sites II (alternatively called A) and IV, and optionally binds to human metapneumovirus F protein, and/or neutralizes RSV and/or human metapneumovirus. 46. The hybridoma or engineered cell of claim 36-45, wherein said antibody or antibody fragment further comprises a cell penetrating peptide and/or is an intrabody. 47. A vaccine formulation comprising one or more antibodies or antibody fragments characterized by heavy chain CDR1 GFTFSSYR (SEQ ID NO: 5), heavy chain CDR2 ITASSSYI (SEQ ID NO: 6), heavy chain CDR3 ARDENTGISHYWFDP (SEQ ID NO: 7), light chain CDR1 GSNLGADYG (SEQ ID NO: 8), light chain CDR2 GDR (SEQ ID NO: 9) and light chain CDR3 QSYDRSLNWV (SEQ ID NO: 10). 48. The vaccine formulation of claim 47, wherein the antibody or antibody fragment is encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 49. The vaccine formulation of claim 47, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 50. The vaccine formulation of claim 47, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 51. The vaccine formulation of claim 47, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 52. The vaccine formulation of claim 47, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 53. The vaccine formulation of claim 47, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 54. The vaccine formulation of claims 47-53, wherein at least one of said antibody fragments is a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment, or is a chimeric antibody, is bispecific antibody, and/or is an IgG. 55. The vaccine formulation of claims 47-54, wherein said antibody or antibody fragment recognizes an epitope on pre-fusion and post-fusion RSV F protein in antigenic sites II (alternatively called A) and IV, and optionally binds to human metapneumovirus F protein, and/or neutralizes RSV and/or human metapneumovirus. 56. The vaccine formulation of claims 47-55, wherein at least one of said antibodies or antibody fragments further comprises a cell penetrating peptide and/or is an intrabody. 57. A method of identifying anti-human respiratory syncytial virus (hRSV) protein F site A/IV-specific monoclonal antibody or polyclonal neutralizing antibodies comprising: (a) contacting a candidate monoclonal antibody or polyclonal serum with hRSV protein F in the presence of the antibody or antibody fragment of claim 26; (b) assessing binding of said candidate monoclonal antibody or polyclonal serum to hRSV protein F; and

(c) identifying said candidate monoclonal antibody or polyclonal serum as protein F site II (or A)/IV-specific neutralizing when said antibody or antibody fragment of claim 26 blocks binding of said candidate monoclonal antibody or polyclonal serum to hRSV protein F. 58. The method of claim 57, further comprising performing a control reaction where said candidate monoclonal antibody is contacted with hRSV protein F in the absence of the antibody or antibody fragment of claim 26. 59. The method of claims 57-58, wherein detection comprises ELISA, RIA or Western blot. 60. The method of claims 57-59, wherein the antibody or antibody fragment is encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 61. The method of claims 57-59, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 62. The method of claims 57-59, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 1 and 2. 63. The method of claims 57-59, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 64. The method of claims 57-59, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 65. The method of claims 57-59, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 3 and 4. 66. The method of claims 57-65, wherein the antibody fragment is a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment, or is a chimeric antibody, a bispecific antibody, and/or is an IgG. 67. A monoclonal antibody or fragment thereof, or hybridoma or engineered cell comprising the same, wherein said antibody or fragment thereof recognizes an epitope on pre-fusion and post-fusion RSV F protein in antigenic site II (or A)/IV, and also recognized an epitope on hMPV F protein. 68. A method of determining the antigenic integrity of an antigen comprising:

(a) contacting a sample comprising said antigen with a first antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and

(b) determining antigenic integrity of said antigen by detectable binding of said antibody or antibody fragment to said antigen. 69. The method of claim 68, wherein said sample comprises recombinantly produced antigen. 70. The method of claim 68, wherein said sample comprise a vaccine formulation or vaccine production batch. 71. The method of claims 68-70, wherein detection comprises ELISA, RIA, western blot, a biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometric staining. 72. The method of claims 68-71, wherein the first antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1. 73. The method of claims 68-71 wherein said first antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1. 74. The method of claims 68-71, wherein said first antibody or antibody fragment is encoded by light and heavy chain variable sequences having 95% identity to clone- paired sequences as set forth in Table 1. 75. The method of claims 68-71, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2. 76. The method of claims 68-71, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2. 77. The method of claims 68-71, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. 78. The method of claims 68-77, wherein the first antibody fragment is a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab’)₂ fragment, or Fv fragment. 79. The method of claims 68-78, further comprising performing steps (a) and (b) a second time to determine the antigenic stability of the antigen over time. 80. The method of claims 68-79, further comprising: (c) contacting a sample comprising said antigen with an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and

(d) determining antigenic integrity of said antigen by detectable binding of said antibody or antibody fragment to said antigen. 81. The method of claim 80, wherein the second antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1. 82. The method of claim 80, wherein said second antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1. 83. The method of claim 80, wherein said second antibody or antibody fragment is encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1. 84. The method of claims 80, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2. 85. The method of claim 80, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80% or 90% identity to clone- paired sequences from Table 2. 86. The method of claim 80, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. 87. The method of claim 80, wherein the second antibody fragment is a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab’)₂ fragment, or Fv fragment. 88. The method of claim 80, further comprising performing steps (c) and (d) a second time to determine the antigenic stability of the antigen over time.