WO2019165019A1

WO2019165019A1 - Antibodies to human respiratory syncytial virus protein f pre-fusion conformation and methods of use therefor

Info

Publication number: WO2019165019A1
Application number: PCT/US2019/018871
Authority: WO
Inventors: Jr. James E. Crowe; Jarrod MOUSA
Original assignee: Vanderbilt University
Priority date: 2018-02-21
Filing date: 2019-02-21
Publication date: 2019-08-29

Abstract

The present disclosure is directed to antibodies binding to human respiratory syncytial virus F protein, including both neutralizing and non-neutralizing antibodies, and methods for use thereof.

Description

DESCRIPTION

ANTIBODIES TO HUMAN RESPIRATORY SYNCYTIAL VIRUS PROTEIN F PRE- FUSION CONFORMATION AND METHODS OF USE THEREFOR

PRIORITY CLAIM

This application claims benefit of priority to U.S. Provisional Application Serial No. 62/633,308, filed February 21, 2018, 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

Viral bronchiolitis consistently remains a burden among young children. Respiratory syncytial virus (RSV) is chief among these infections as the leading cause of viral bronchiolitis and viral pneumonia in infants and children (Hall et al, 2009 and Shefali-Patel et al, 2012). The related pneumovirus, human metapneumovirus (hMPV), contributes to this burden, with infection resulting in 5-10% of hospitalizations due to lower respiratory tract infections (Williams et al, 2004, Principi and Esposito, 2014 and Panda et al, 2014). hMPV was first identified in 2001, yet it is thought to have infected the human population for at least fifty years (van den Hoogen et al, 2001). As major human pathogens, RSV and hMPV are among the few infectious viruses with global impact for which there is no licensed vaccine. Palivizumab (Bates et al, 2014) (Synagis) has become the standard of care for prophylactic treatment against RSV, yet the availability and effectiveness of palivizumab for preventing disease is limited. Furthermore, a second generation antibody candidate, motavizumab, has been developed with higher affinity and more potent neutralization, but was not approved for prophylactic use (Wu, et al, 2007). Although palivizumab is accessible for prophylactic RSV treatment, such a treatment is not available for hMPV infection.

Recent attempts to develop an RSV vaccine have focused on the highly conserved fusion (F) protein, a type I F protein that has both pre- and post-fusion conformations (Lopez et al, 1998 and Dutch, 2010). Pneumovirus F proteins are synthesized as inactive precursors (Fo) that are cleaved during cellular processing into two disulfide-linked domains (Fi and F₂). Upon activation, the F proteins cause fusion of viral and cell membranes. The RSV F pre- fusion conformation is meta-stable, easily transitioning to the post-fusion conformation upon viral attachment to host cells. Recombinant RSV F protein readily converts to the post-fusion conformation (McLellan et al, 2011), and formalin inactivation of RSV was shown to result in the pre- to post-fusion rearrangement of the fusion protein (Killikelly et al, 2016). Stabilization of the RSV F protein in the pre-fusion conformation has proven successful, resulting in Ds-Cavl (disulfide-linked, cavity-filled) and SC-TM (single-chain-triple mutant) variants with enhanced stability in recombinant expression (McLellan et al, 2013 and Krarup et al, 2015). Both pre-fusion variants have been characterized structurally, as has post-fusion RSV F (McLellan et al, 2011).

Several major neutralizing epitopes exist on RSV F, based on functional and structural data (FIG. SI). Antigenic site II (Mousa et al, 2016) recognized by the humanized murine mAbs palivizumab and motavizumab, and antigenic site IV (McLellan et al, 2010) recognized by murine mAb 101F, are preserved in both the pre-fusion and post-fusion RSV F conformations. Pre-fusion-specific mAbs have been characterized that defined new antigenic sites including mAbs D25 (site 0) (McLellan etal, 2013), MPE8 (site ΙΠ) (Corti etal, 2013; Wen et al, 2017), and the recently discovered mAb hRSV90 (site Vffl) (Mousa et al, 2017). A quaternary epitope has been described by recognition via human mAb AM14 (Gilman et al, 2015). Several antibodies targeting the hMPV F protein have been isolated using murine hybridoma or phage display techniques, and the known hMPV F antigenic sites appear structurally similar to those on RSV F (Williams ei a/., 2007, Ulbrandteia/., 2008 and Ulbrandt et al, 2006). A monomelic hMPV F protein has been characterized structurally in complex with a neutralizing antibody DS7 (Wen et al, 2012), and in the post-fusion conformation (Mas etal, 2016).

RSV and hMPV F share -36% sequence homology, and human antibodies that cross- neutralize RSV and hMPV have been described (Corti et al, 2013, Wen et al, 2017, Mas et al, 2016, Schuster et al, 2014; Gilman et al, 2016). The human mAb MPE8 was found to neutralize four different pneumovirus es, including RSV, hMPV, bovine RSV, and pneumonia virus of mice (Corti et al, 2013), and a similar mAb 25P13 has been described (Wen et al, 2017). The human mAb 54G10 recognizes the site IV region of the RSV F protein, cross-reacts with hMPV F protein, and protects against viral infection in vivo (Schuster et al, 2014). Of the antigenic sites described thus far, site IV consists partially of a predominantly linear region demonstrated in the co-crystallization of the mouse-derived mAb 101F in complex with a 15- mer peptide (McLellan etal, 2010). MAb 101F also was shown to cross-react with both RSV and hMPV (Mas et al, 2016). The inventors recently reported several human antibodies generated from human B cells that primarily target antigenic sites II and VIII (Mousa et al, 2016 and Mousa et al, 2017). However, structural and functional data regarding human mAb binding to antigenic site IV is lacking. Recent work described the generation and characterization of over 300 human mAbs generated via B cell sorting, and those mAbs targeting antigenic site IV were recognized as a substantial percentage (-15-40%) in the human repertoire (Gilman et al. , 2016). Previous reports have described binding of the murine-derived mAb 101F (McLellan et al, 2010, Mas et al, 2016 and Wu et al, 2007), as well as several binding sites (TV, V, VI) near the site IV epitope (Lopez et al, 1998).

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 clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; 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 may be 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 clone-paired variable sequences as set forth in Table 1, 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 antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, 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 is 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 clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by clone- paired variable sequences as set forth in Table 1, 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 antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, 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 is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment.

The antibody or antibody fragment may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, such as a LALA, N297, GASD/ALIE, or is glycan modified to alter (eliminate or enhance) FcR interactions, such as enzymatic or chemical addition or removal of glycans, or genetic modification of a glycosylation pattern, or comprises an Fc portion mutated to alter FcRn interactions to increase in vivo half-life and in vivo protection, such as a YTE or LS mutation.

The antibody or antibody fragment may recognize an epitope on RSV F protein in antigenic site IV and may neutralize RSV and human metapneumovirus. The antibody or antibody fragment may be administered prior to infection, or after infection. Delivering may comprise 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 a provided a monoclonal antibody, wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, 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 antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, 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 is 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 RSV F protein in antigenic site IV, and/or may neutralize RSV and HMPV. The antibody or antibody fragment may further comprise a cell penetrating peptide and/or is an intrabody.

In still yet another embodiment, there is provided a hybridoma or engineered cell encoding an antibody or antibody fragment wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, 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 antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, 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 is 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 RSV F protein in antigenic site IV, and/or may neutralize RSV and HMPV. The antibody or antibody fragment may further comprise a cell penetrating peptide and/or is an intrabody.

A further embodiment comprises a vaccine formulation comprising one or more antibodies or antibody fragments characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, 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 antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, 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 is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. At least one of said antibodies may be a chimeric antibody, is bispecific antibody, and/or is an IgG. At least one of said antibodies or antibody fragments may recognize an epitope on RSV F protein in antigenic site IV, and/or may neutralize RSV and HMPV. A least one of said antibodies or antibody fragments may further comprise a cell penetrating peptide and/or is an intrabody. The antibody or antibody fragment may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, such as a LALA, N297, GASD/ALIE, or is glycan modified to alter (eliminate or enhance) FcR interactions, such as enzymatic or chemical addition or removal of glycans, or genetic modification of a glycosylation pattern, or comprises an Fc portion mutated to alter FcRn interactions to increase in vivo half-life and in vivo protection, such as a YTE or LS mutation.

An additional embodiment comprises a method of identifying an anti-human respiratory syncytial virus (hRSV) protein F site IV-specific neutralizing monoclonal antibody or polyclonal serum comprising (a) contacting a candidate antibody or serum with hRSV protein F in the presence of a known site IV-specific neutralizing antibody or antigen binding fragment thereof; (b) assessing binding of said candidate antibody or serum to hRSV protein F; and (c) identifying said candidate antibody or serum as a protein F site IV-specific neutralizing antibody when said known site IV-specific neutralizing antibody or antigen binding fragment thereof blocks binding of said candidate antibody or serum to hRSV protein F. The method may further comprise performing a control reaction where said candidate antibody or serum is contacted with hRSV protein F in the absence of a known site IV-specific neutralizing antibody or fragment thereof. Detection may comprise ELISA, RIA or Western blot. The known site IV-specific neutralizing antibody or fragment thereof may be encoded by clone-paired variable sequences as set forth in Table 1, 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 known site IV-specific neutralizing antibody or fragment thereof may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, 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. Other embodiments include (i) a monoclonal antibody or fragment thereof, wherein said antibody or fragment thereof recognizes an epitope on RSV F protein in antigenic site IV, such as where said antibody or antibody fragment is specific for an epitope on RSV F protein in antigenic site IV, and (ii) a monoclonal antibody or fragment thereof, wherein said antibody or antibody fragment recognizes an epitope on RSV F protein in antigenic site IV and neutralizes RSV and HMPV.

In still another embodiment, there is provided a method of identifying the presence of human respiratory syncytial virus (hRSV) protein F site IV protective antigen in a vaccine or virus preparation using human respiratory syncytial virus (hRSV) protein F site IV-specific monoclonal or polyclonal neutralizing antibodies comprising (a) contacting a candidate vaccine or virus composition with a known site IV-specific neutralizing antibody or antigen binding fragment thereof; (b) assessing binding of said candidate vaccine or virus composition to a known site IV-specific neutralizing antibody or antigen binding fragment; and (c) identifying said candidate vaccine or virus composition as containing the protein F site IV protective epitope when one or more known site IV-specific neutralizing antibodies bind to the candidate vaccine or virus composition.

Also provided is 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. The 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. The first antibody or antibody fragment 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. The first antibody or antibody fragment 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. The first antibody or antibody fragment may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2. The first antibody or antibody fragment may comprise light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2. The first 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 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. The second antibody or antibody fragment 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. The second antibody or antibody fragment 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. The second antibody or antibody fragment may comprise light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2. The second antibody or antibody fragment 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 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-D. Epitope binning and hMPV F cross-reactivity. (FIG. 1A) Epitope binning with the newly generated mAbs on post-fusion RSV A2 F protein revealed the mAbs competed for binding to antigenic site IV with the previously described mAbs 101F and S4G10. (FIG. IB) Epitope binning on pre-fusion RSV A2 F SC-TM suggested binding at antigenic site IV, as competition was not observed with site II mAb motavizumab nor site 0 mAb D2S. (FIG. 1C) Binding and neutralization curves indicate mAb 17E10 cross-reacts with hMPV F. The top graph displays ELISA binding data with post-fusion hMPV Al F protein. Only cross-reactive mAbs 17E10, 54G10, 101F, and MPE8 show binding to the protein, while the site IV mAbs 2N6, 3M3, and 6F18 show no binding. Each data point is the average of three independent experiments, each with four technical replicates. Error bars represent the standard deviation. The bottom graph show neutralization for the cross-reactive mAbs, with D2S used as a negative control. MAbs 17E10, MPE8, and 101F neutralize hMPV F while the D25 control shows no reduction in virus. Data points are the average of three technical replicates, and error bars indicate the standard deviation. (FIG. ID) Epitope binning using post-fusion hMPV F. MAbs 101F, 17E10, and S4G10 display a similar competition binding pattern to that observed with RSV F protein. The mAbs compete for a site unique from site III mAbs 2SP13 and MPE8, and DS7. For epitope binning, data indicate the percent binding of the second antibody in the presence of the first antibody, compared with the second antibody alone. Cells filled in black indicate full competition, in which <33% of the uncompeted signal was observed, intermediate competition (gray) if signal was between 33% and 66%, and noncompeting (white) if signal was >66%. Antigenic sites are highlighted at the top and side based on competition-binding with the control mAbs D25 (site 0), 131-2a (site I), palivizumab (PALI) or motavizumab (MOTA) (site Π), or 101F (site IV). FIGS. 2A-C. Characterization of antigenic site IV mutations. (FIG. 2A) Alanine- scanning mutagenesis binding values for the generated site IV mAbs, compared with palivizumab and mAb D2S controls. The mAb reactivity for each RSV F variant was calculated relative to that of wild-type RSV F. Error bars indicate the measurement range of two independent experiments. Labels in figure from top to bottom correspond to bars left to right. (FIG. 2B) Overlay of RSV F (SEQ ID NO: 52) and hMPV F (SEQ ID NO: 53) sequences and crystal structures (PDB IDs: 3RRR, 5L1X, overlaid at chain H for each structure) at antigenic site IV, with RSV F residues from the alanine-scanning mutagenesis shown. Conserved residues between RSV F and hMPV F are displayed in red font. In the crystal structure overlay, RSV F residues are shown in cyan and hMPV F residues are shown in blue. (FIG. 2C) ELIS A EC so values for recombinant post-fusion or pre-fusion (SC-TM) mutant proteins for the site IV mAbs or controls. Neutralization ICso values also are displayed for the RSV strain A2 F variant R429A. FIG.3. Binding curves determined by biolayer interferometry for mAbs targeting antigenic site IV. Streptavidin biosensors were incubated with biotinylated peptides, and then mAbs were tested for binding in real-time. Hie first dashed line indicates the end of the peptide loading step, and the second dashed line indicates the beginning of the mAb binding step. After binding, mAbs were allowed to dissociate in real-time. The data are representative chromatograms from one experiment Wild-type (WT) and mutant peptides consist of RSV F residues 422-436. The 30-mer A peptide includes RSV F residues 407-436, and the 30-mer B peptide includes RSV F residues 422-451.

FIG. 4. Multiple binding modes at antigenic site IV. Each site IV mAb was complexed with RSV A2 post-fusion F, and negative-strain electron microscopy images were generated using random-conical tilt analysis. MAbs 3M3 and 6F18 share a similar binding mode, while mAb 2N6 binds antigenic site IV at an angle allowing bypass of the Arg429 residue. MAb 17E10 binds to RSV F at an angle 42° from the plane of the other site IV mAbs. This unique binding pose likely mediates cross-reactivity with hMPV. 3-D reconstructions are displayed for each mAb-RSV F complex, and 2D class averages are displayed below the reconstructions.

FIG. 5. RSV 3M3 protects cotton rats from RSV replication. The inventors tested purified RSV 3M3 IgG in a prophylactic model to see if pretreatment with the mAB would protect against RSV infection in the lungs. Female cotton rats ~8 weeks old were treated prophylactically with antibody (day -1), using a 0.1 mg/kg antibody dose by the intraperitoneal route. RSV 3M3 was compared with similarly prepared (negative control) mAb to dengue virus (DENV 2D22 given at 1 mg/kg). Animals were inoculated with 10^s RSV A2 virus (day 0), then animals were sacrificed and lungs removed (day +4). Virus isolated through bronchoalveolar lavage was performed, then BAL fluids were tested for the presence of virus in a virus plaque assay using HEp-2 cell monolayer cultures, which were fixed and immunostained cells after 4 days. The RSV 3M3 mAb prevented virus replication in the lungs.

FIG. 6. MAbs RSV 3M3 exhibits excellent neutralizing activity against an RSV subgroup B strain. The inventors used the RSV field strain designated RSV B-l (Karron et al, 1997; Crowe et al, 19%), which was obtained from BEI Resources (BEI Resources catalog #NR-4052; Lot #BPR 348-00). Plaque reduction neutralization was performed in HEp-2 cell monolayer cultures, with ~ 80 plaques per well. The RSV 3M3 mAb exhibited superior activity compared to the recombinant forms of antibodies based on the sequences of reference antibodies MEDI8897 or palivizumab. The site VIII hRSV90 mAb (Mousa et al, 2017) was included as a positive control and also exhibited superior activity compared to the recombinant forms of antibodies based on the sequences of reference antibodies MEDI8897 or palivizumab. A negative control antibody (DENV 2D22 directed to dengue virus) did not neutralize at the threshold of 50% plaque reduction. SFIG. 1. Summary of known antigenic sites on the RSV F protein.

SFIG.2. ELISA binding curves for the newly generated site IV mAbs and controls to RSV F protein and construct variants. ECso values for these curves are displayed in Table 1. Zika NS1 protein was used as a negative control. Each data point is the average of three independent experiments, each with four technical replicates. Error bars represent the standard deviation.

SFIG.3. Neutralization curves for the newly generated site IV mAbs and controls.

ICso values are displayed in Table 1. An Ebola virus-specific mAb EBOV284 was included as a negative control. Data points indicate the average of three technical replicates. Error bars represent the standard deviation. SFIG. 4A-C. Critical residues for mAb 17E10 binding. (SFIG 4A) A shotgun mutagenesis mutation library for RSV F protein encompassing 368 mutations, where each amino acid was individually mutated to alanine, was constructed. Each well contained one mutant with a defined substitution. Reactivity results for a representative 384-well plate are shown. Eight positive (wild-type RSV F) and eight negative (mock-transfected) control wells were included on each plate. (SFIG. 4B) Human HEK-293T cells cells expressing the RSV F mutation library were tested for immunoreactivity with 17E10, which was measured using an Intellicyt high-throughput flow cytometer. Using algorithms described elsewhere (Davidson and Doranz, 2014), clones with reactivity of <30% relative to that of wild-type RSV F yet >70% reactivity for a different RSV F mAb were identified to be critical for 17E10 binding. (SFIG. 4C) Critical residues identified for 3M3, 6F18, and 17E10 are listed with the mean binding reactivies for each mAb as well as control antibodies palivizumab and D25. Reactivities are expressed as a percentage of the reactivity of the wild type with ranges (maximum minus minimum values) given in parentheses. Values shaded in gray are for critical residues. Data shown is the average of two replicates values.

SFIGS. 5A-B. ELISA binding curves for the site IV mAbs and controls to (SFIG. 5A) pre-fusion (SC-TM) or (SFIG. SB) post-fusion RSV A2 mutant proteins. Each data point is the average of three independent experiments, each with four technical replicates. Error bars indicate the standard deviation. ECso values are shown in FIG. 2C.

SFIG. 6. Plaque-reduction assay curves for the newly generated site IV mAbs and controls for neutralization of the RSV A2 R429A mutant virus. ICso values are displayed in FIG. 2C. Data points indicate the average of three technical replicates. Error bars represent the standard deviation.

SFIG. 7. ELISA binding curves of site IV mAbs to biotinylated site IV 15-mer peptides coated on streptavidin ELISA plates. Data points are the average of two technical replicates. Error bars indicate the range of the two measurements.

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.

To further define the molecular basis for human antibody site IV-mediated binding, as well as the cross-reactivity between RSV and hMPV at antigenic site IV, the inventor generated additional human antibodies against post-fusion RSV F. Herein, four of these mAbs that were specific to antigenic site IV are described, including the identification of several binding modes at antigenic site IV.

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 reinfected every five to seven years, with symptoms looking like a sinus infection or a cold (infections can also be asymptomatic).

Hie 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 RN A 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, andNA2. 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 Medlmmune), 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. Π. 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 pyrimi dines, 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. Antibodies of the Present Disclosure

Antibodies according to the present disclosure may be defined, in the first instance, by their 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 CDR's 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 epitope that lies within what the inventors now term "antigenic site VIII." This epitope is located between sites Π and 0, while also being close to the trimer-dependent mAb AM14 site and distant from antigenic site IV. Residues 16, 173, 174, 194, and 201 of RSV F all appear to have some involvement.

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.

Hie 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 SO 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. Hie 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 IgGi 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 x 1ο⁶ 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 (/. 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 stablizdng and coagulating agent. However, it is contemplated that dimers or mummers 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- l,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 Afunctional 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 arninooxy-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 display 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 etal, 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 (I VIG) 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 inj ection, /^'. 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). Hie 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, "chromium, ³⁶chlorine, "cobalt, "cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, 'hydrogen, iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, "selenium, "sulphur, technicium⁹⁹"¹ 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 technetium⁹⁹¹¹¹ 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 SNCh, 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 diethylenetriarninepentaacetic acid (DTP A) 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 diemylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriarninetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3a-6a-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 HI 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 DN A/biotin/strqjtavidin/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 nonspecific 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 ef a/., 1990).

Briefly, frozen-sections may be prepared by rebydrating 50 ng of frozen "pulverized" tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resus pending 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; res us pending 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.

Hie 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. Quality Control and Vaccine Testing

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 the 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

Ethics statement Participation of healthy human adult subjects was approved by the Vanderbilt University Institutional Review Board, and blood samples were obtained only after informed written consent.

Human hybridoma generation. Participation of healthy human adult subjects was approved by the Vanderbilt University Institutional Review Board, and blood samples were obtained only after informed consent. PBMCs were isolated from healthy human donor blood samples using Ficoll-Histopaque density gradient centrifugation. Approximately ten million PBMCs were mixed with 17 mL of transforming cell culture medium (Medium A, StemCell Technologies), 8 μg/mL of CpG (phosphorothioate-modified oligodeoxynucleotide ZOEZOEZZZZZOEEZOEZZZT (SEQ ID NO: 41), Invitrogen), 3 μg/mL of Chk2 inhibitor II (Sigma), 1 μg/mL of cyclosporine A (Sigma), and 4.5 mL of filtered supernatant from a culture of B95.8 cells (ATCC VR-1492) containing Epstein-Barr virus (EBV). After one week, transformed PBMCs were expanded into four 96-well culture plates using cell line expansion culture medium (Medium A containing 8 μβ/πιί CpG, 3 μ¾/ηιί Chk2 inhibitor II, and ten million irritated heterologous human PBMCs (Nashville Red Cross)). After one week, culture supematants were screened by ELISA against the post-fusion RSV A2 F protein. Cells from positive wells were fused with HMMA2.5 myeloma cells by electrofusion (Yu et al, 2008). Fused cells were plated in 384-well plates in growth medium containing 100 μΜ hypoxanthine, 0.4 μΜ aminopterin, 16 μΜ thymidine (HAT Media Supplement, Sigma), and 7 μg/mL ouabain (Sigma). Hybridomas were screened after two weeks for mAb production by ELISA, and cells from wells with reactive supematants were expanded to 96-well plates for one week before being screened again by ELISA, and then subjected to single-cell fluorescence-activated sorting. After cell sorting into 384-well plates containing Medium E (StemCell Technologies), hybridomas were screened by ELISA before expansion into both 48- well and 12- well plates. Enzyme-linked immunosorbent assay (ELISA) for binding to RSV F and hMPV

F proteins. For recombinant protein capture ELISA, 384-well plates were treated with 2 μg/mL of antigen for one hour at 37 °C or overnight at 4 °C. Following this, plates were blocked for one hour with 2% milk supplemented with 2% goat serum Primary mAbs and culture supernatants were applied to wells for one hour following three washes with PBS-T. Plates were washed with PBS-T four times before applying 25 μΣ, 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 five times with PBS-T, and 25 μΣ, of phosphatase substrate solution (1 mg/mL phosphatase substrate in 1 M Tris HC1 pH 9.6, 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. ELISA experiments using biotintylated peptides were conducted by coating pre-blocked streptavidin-coated plates (Fisher) with 10 μg/mL peptide for two hours. After three washes with PBS-T, plates were coated with primary mAbs for one hour. The remaining steps were conducted as described above.

Human mAb and Fab production and purification. Biologically cloned hybridoma cell lines were expanded in Medium E until approximately 80% confluent in 75-cm² flasks. For antibody production, cells from one 75-cm² cell culture flask were collected with a cell scraper and expanded to four 225-cm² cell culture flasks in serum-free medium (Hybridoma- SFM, GIBCO). After 30 days, supernatants were sterile filtered using 0.45 um pore size filter devices. For antibody purification, HiTrap MabSelectSure columns (GE Healthcare Life Sciences) were used to purify antibodies using the manufacturer's protocol. To obtain Fab fragments, papain digestion was performed following the manufacturer's protocol (Pierce Fab Preparation Kit, Thermo Scientific). Fab fragments were purified by removing IgG and Fc contaminants using a HiTrap MabSelectSure column (GE Healthcare Life Sciences).

Production and purification or recombinant RSV F, hMPV F, motavizumab, mAb 101F, mAb 54G10, mAb MPE8, and mAb D25. Plasmids encoding cDNAs for pre-fusion (SC-TM) or post-fusion RSV subgroup A strain A2, and subgroup B strain 18537 pre-fusion (DsCavl , a gift from Barney Graham) were expanded in K coli DH5a cells, and plasmids were purified using Qiagen Plasmid Maxiprep kits (Qiagen). Plasmids encoding cDNAs for the protein sequences of mAb 101F, mAb MPE8, and mAb D25, and motavizumab heavy and light chain sequences were cloned into vectors encoding human IgGl and lambda or kappa light chain constant regions, respectively. Vectors encoding the heavy and light chain sequences of 54G10 were a gift from Dennis Burton. MAb 131-2a protein was obtained from Sigma. Commercial preparations of palivizumab (Medimmune) were obtained from the pharmacy at Vanderbilt University Medical Center. For each liter of protein expression, 1.3 mg of plasmid DNA was mixed with 2 mg of polyethylenimine in Opti-MEM I cell culture medium (Fisher). After 10 min, the DNA mixture was added to HEK293F cells (ThermoFisher R79007) at 1 x 10⁶ cells/mL. The culture supernatant was harvested after 5 days, and the protein was purified by HiTrap Excel column (GE Healthcare Life Sciences) for RSV F protein variants or HiTrap MabSelectSure columns for mAbs. Assays for competition-binding and peptide binding. All studies were conducted on an Octet Red ForteOio biolayer interferometry system using anti-penta-HIS biosensor tips for competition and streptavidin-coated sensors for peptide binding. For competition, an initial baseline was obtained in kinetics buffer (ForteOio, diluted 1:10 in PBS), followed by antigen loading with 20 μg/mL of his-tagged RSV F protein being immobilized onto 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 mAb for 300 s. Following this, biosensors were immersed into wells containing 100 μg/mL of a second mAb for 300 s. The percent binding of the second mAb in the presence of the first mAb was determined by comparing the maximum 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 un-competed binding was considered intermediate competition, and < 33% was considered competing. To assess binding of mAbs to biotintylated peptides, streptavidin-coated sensors were immersed in kinetics buffer for 60 s, followed by immersion into biotintylated peptides at 5 μg/mL for 20 s. Following another 60 s baseline step, sensors were immersed in mAbs for 120 s for association. Dissociation from the peptide then was measured by immersing sensors in kinetics buffer for 120 s.

Antibody epitope mapping. Shotgun mutagenesis epitope mapping of anti-RSV F antibodies was performed using an alanine scanning mutagenesis library for RSV F protein (RSV-A2; NCBI ref # FJ614814), covering 368 surface-exposed residues identified from crystal structures of both the pre-fusion and post-fusion conformations of RSV F. An RSV F expression construct was mutated to change each residue to an alanine (and alanine residues to serine). The resulting 368 mutant RSV F expression constructs were sequence confirmed and arrayed into a 384- well plate (one mutation per well). Library screening was performed as described previously (Davidson and Doranz, 2014). The RSV F alanine scan library clones were transfected individually into human HEK-293T cells (ATCC CRL-3216) and allowed to express for 16 h before fixing cells in 4% (vol/vol) paraformaldehyde (Electron Microscopy Sciences) in PBS plus calcium and magnesium. Cells were incubated with mAbs, diluted in 10% (vol/vol) normal goat serum (NGS), for 1 h at room temperature, followed by a 30 min incubation with 3.75 ug/mL Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) in 10% NGS. Cells were washed twice with PBS without calcium or magnesium and resuspended in Cellstripper (Cellgro) plus 0.1% BSA (Sigma- Aldrich). Cellular fluorescence was detected using a high-throughput flow cytometer (Intellicyt). Before library screening, to ensure that the signals were within the linear range of detection, the optimal screening concentrations for each mAb were determined using an independent immunofluorescence titration curve against cells expressing wild-type RSV F. Antibody reactivity against each mutant protein clone was calculated relative to wild-type protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type protein transfected controls. Mutations within clones were identified as critical to the mAb epitope if they did not support reactivity of the test mAb, but supported reactivity of other antibodies. This counter-screen strategy facilitates the exclusion of RSV F protein mutants that are misfolded or have an expression defect The detailed algorithms used to interpret shotgun mutagenesis data are described elsewhere (Davidson and Doranz, 2014).

RSV and hMPV neutralization experiments. Plaque reduction assays for RSV were conducted by first serially diluting mAbs, and incubating mAbs with a suspension of infectious RSV A2 or Long strain viruses for 1 hr. Following this, confluent HEp-2 (ATCC CCL-23) cell monolayers cultures in 24-well plates, maintained in Opti-MEM I (Fisher) supplemented with 2% fetal bovine serum at 37 °C in a CO2 incubator, were inoculated with 50 μΣ, of the antibody: virus mixture for 1 hr. After the hour, cells were overlaid with 1 mL of 0.75% methylcellulose dissolved in Opti-MEM I + 2% fetal bovine serum Cells were incubated for four days, after which plaques were visualized by fixing cells with 10% neutral-buffered formalin and staining with crystal violet. For the hMPV neutralization experiments, serially diluted mAbs were incubated for 1 hour with hMPV Jpn03-1 B2 strain. Vera cell (ATCC CCL-81) monolayers, maintained as described above, were overlaid with the mAb/virus mixture for one hour. Cells were overlaid with 0.75% methylcellulose dissolved in Opti-MEM + 0.0005% toypsin-EDTA. After five days, the cells were fixed with 10% neutral buffered formalin. Cells were immunostained by incubating with 1:1000 dilution each of and -hMPV nucleoprotein antibody (Meridian Life Science C01851M) and anti-hMPV fusion protein antibody (Meridian Life Science C01852M) for one hour. After washing three times with water, the cells were incubated with 1:2000 of goat anti-mouse IgG (H+L) peroxidase labeled human serum absorbed antibody (SeraCare 074-1806) for one hour. After washing three times with water, the cells were overlaid with TrueBlue peroxidase substrate (SeraCare 54-78-00). Immunostained plaques were counted manually, and data from all neutralization experiments were analyzed with Prism software (GraphPad) to obtain ICso values. Assembly and rescue of recombinant RSV A2-mKate2-R429A. Site-directed mutagenesis was performed on a subclone of the A2 F gene to introduce the R429A mutation using primers:

429F: 5 '-GC ATCCAATAAAAATGCTGGAATC ATAAAGAC-3 ' (SEQ ID NO: 42) and 429R: 5 '-GTCTTT ATGATTCC AGC ATTTTT ATTGGATGC-3 ' (SEQ ID NO: 43)

(Integrated DNA Technologies, Coralville, IA). Once sequence confirmed, the R429A A2 F gene was ligated into the bacterial artificial chromosome (BAC) pSynk-A2-mKate2 using SacII/Sall restriction sites. pSynk-A2-mKate2 contains the antigenomic cDNA of RSV A2 with an mKate2 gene encoding the far-red monomelic Katushka-2 fluorescent reporter protein in the first position (Meng et al, 2015). The recombinant virus was rescued by co-transfecting the RSV antigenomic BAC and four codon-optimized helper plasmids expressing the RSV L, N, M2-1 and P proteins into BSRT7/5 cells as previously described (Hotard et al, 2012). Master and working stocks were subsequently propagated and harvested in HEp-2 (ATCC CCL-23) cells (Hotard et al, 2012, Rostad et al, 2016 and Stobart et al, 2016), and the virus was titrated by immunodetection plaque assay as described (Stokes et al, 2011).

EM sample preparation and data collection. The complex was generated by mixing excess of human Fab with RSV F protein and incubation at 37°C for 1 h followed by size- exclusion chromatography (S200, 16/300; GE Healthcare Life Sciences) in 50 mM Tris pH 7.5, 50 mM NaCl. The sample (5 ul at 5 μg/ml) was applied to a glow-discharged copper grid coated with continuous carbon (EMS 400 mesh) for 1 min washed and blotted. Freshly prepared uranyl formate (0.75%) was added for lmin blotted and air dried as described (Obi etal, 2004).

Data collection was done using FEI Tecnai F20 microscope operated at 200kV and equipped with Gatan Ultras can 4k x 4k CCD camera. The Random Comical Tilt (RCT) image pairs were acquired semi-automatically using SerialEM 3.6.3 (Mastronarde, 2005) at nominal magnification of 50,000X with pixel size of 2.18A and defocus of 1.2-1.8 um. The tilt pair was collected at -60° and 0°. To generate a 3D model of the RSV F-Fab complexes, the inventor used the RCT approach. Data processing was done using Scipion suit (scipion.cnbxsic.es/docs^in/view/TWiki/WebHome; 2017). First tilt-pairs from the micrographs were picked using xmipp3 - tilt pairs particle picking (De la Rosa-Trevin, 2013 and Sorzano et al, 2013) with box size of 160 x 160 pixels. The particles were extract and binned by 2 to generate a box size of 80 x 80 pixels (4.36 A/px). The images were normalized using xmipp3 - extract particle pairs (De la Rosa-Trevin, 2013; Sorzano et al, 2013). The untilted particles were band-pass filtered before aligning and classification using xmipp3 - cl2d (De la Rosa-Trevin, 2013 and Sorzano etal, 2013). From the 2D analysis, the inventor selected well-aligned classes with clear RSV-F protein-Fab complexes and performed 3D reconstruction using the corresponding tilted particles using xmipp3 - random conical tilt (De la Rosa-Trevin, 2013 and Sorzano et al, 2013). This initial model was then used for further 3D refinement using both the tilted images and 10% of the untilted particles in RELION - 3D auto-refine (Scheres, 2012). Volume display and figures were done in UCSF Chimera (Pettersen et al, 2004).

Example 2 - Results

Generation and epitope specificity of RSV F-specific human monoclonal antibodies. To understand binding modes at antigenic site IV by human antibodies, the inventor generated new RSV F-specific human mAbs using human hybridoma technology (Y u etal, 2008 and Smith and Crowe, 2015): designated mAbs 2N6, 3M3, 6F18, and 17E10. These mAbs were generated similar to those previously described to the RSV F protein (Mousa et al, 2016, Wen et al, 2017 and Mousa et al, 2017). Half maximal effective concentration (ECso) for each mAb was measured by enzyme-linked immunosorbent assay (ELISA) (Table A, SFIG. 2). Each mAb had comparable binding to F proteins representing viruses across the RSV antigenic subgroups, using RSV A strain A2 and RSV B strain 18537, as well as pre-fusion (single chain-triple mutant (SC-TM) and disulfide-cavity filling (DsCavl)) and post-fusion RSV F proteins. These findings suggested the epitope for the generated mAbs is present in both pre-fusion and post-fusion conformations. The mAbs were tested for neutralization of RSV A2 and RSV Long viruses (subgroup A), and RSV 18537 B and RSV WV/401R viruses (subgroup B) (Table A, SFIG. 3). Each mAb neutralized all strains with one especially potent mAb designated 3M3 having a half maximal inhibitory concentration (ICso) of 3 ng/mL for the RSV Long strain. The mAb sequences were analyzed by IMGT (Brochet etal, 2008), and each mAb is predicted to utilize a unique V gene sequence compared to each other (Table B). Furthermore, the heavy chain CDR3 length varies between 14-17 amino acids, except for mAb 17E10, which contains just 8 amino acids.

The generated mAbs were tested for epitope specificity using a biolayer interferometry- based competition-binding assay (Mousaef a/., 2016 and Mousaef a/., 2017). For this purpose, post-fusion RSV F or the pre-fusion-stabilized SC-TM variant (McLellan et al, 2013) were loaded onto sensor tips, and mAbs were competed for binding to each protein. In measuring competition on the post-fusion RSV F protein, the isolated mAbs share a unique antigenic site that did not overlap with either site II (recognized by palivizumab) or site I (recognized by murine mAb 131-2a) (FIG. 1A). Instead, the mAbs competed with each other, the previously described mouse-human chimeric mAb 101F (Wu et al, 2007), and the human mAb S4G10 (Schuster et al, 2014), both of which target antigenic site IV. When loading sensors with pre- fusion RSV F A2 SC-TM protein, the pre-fusion-specific mAb D25 (McLellan et al, 2013) was used to confirm the presence of the pre-fusion conformation, and to identify site 0 (FIG. IB). As expected, the site 0 mAb D2S did not compete with the site IV mAbs nor motavizumab at site II. Competition-binding patterns were consistent in assays using F protein in either pre- fusion or post-fusion conformation, further suggesting the mAbs target antigenic site IV, as this epitope is retained in both conformations of the F protein.

Multiple binding modes at antigenic site IV. The previously described site IV mAb 101F was reported to neutralize both RSV and hMPV (Mas et al, 2016). Similarly, mAb S4G10 neutralizes hMPV, as well as RSV at high concentrations, while also reducing hMPV and RSV titers in vivo (Schuster et al, 2014). As the newly generated mAbs competed for binding to F with both of these known RSV/hMPV cross-reactive mAbs, the inventor tested whether the newly isolated mAbs were cross-reactive to hMPV F by first testing binding in ELISA to the hMPV F protein. The mAb designated 17E10 bound to hMPV F, while the remaining three mAbs did not show detectable binding when tested at concentrations up to 20 μg/πlL (FIG. 1C). The cross-reactive mAb 17E10 neutralized hMPV infection in a neutralization assay at a level comparable to that of the site IV mAb S4G10, and the site ΠΙ mAb MPE8 (FIG. 1C). To confirm binding near site IV on hMPV F, competition-binding studies were performed using recombinant post-fusion hMPV F protein. Indeed, mAb 17E10 competed with mAbs 101 F and S4G10, but its pattern of competition differed from that of the site ΙΠ mAbs MPE8 and 25P13 (Wen et al, 2017), and the hMPV F-specific mAb DS7 Fab (Wen et al, 2012) (FIG. ID). These data suggest that there are several distinct epitopes within antigenic site IV, and particular interactions or binding poses can induce cross-reactivity with hMPV F.

To determine residues important for the generated mAbs, the inventor used an alanine- scanning mutagenesis F protein library expressed in HEK-293T cells coupled with flow cytometric detection of mAb binding or loss of binding. Each RSV F construct was transfected and allowed to express for 16 h before fixing cells with 4% (vol/vol) paraformaldehyde. The fixed cells were incubated with each mAbs, followed by incubation with a fluorescent secondary antibody, and cellular fluorescence was detected by flow cytometry. From the library, no mutant disrupted binding of mAb 2N6 significantly, suggesting no single residue alone is critical for binding. The R429 residue that has been previously identified as the principal contact residue for mAbs recognizing antigenic site IV was critical for binding of mAbs 3M3 and 6F18, yet this residue did not affect binding of mAbs 17E10 or 2N6 (FIG. 2A, SIG. 4). Instead, G430A and I432A mutants resulted in loss of mAb 17E10 binding to RSV F. All three mutations were shown previously to reduce binding of mAb 101F to cell surface- expressed proteins (Brochet et al. , 2008). Sequence alignment of the RSV and hMPV F proteins revealed a conserved amino acid motif of GIIK at antigenic site IV (FIG. 2B), while the corresponding R429 residue in hMPV F is replaced by a valine in the hMPV protein. Thus, the inventor hypothesized that amino acids within the conserved GIIK sequence were important for the cross-reactivity of mAb 17E10. The inventor further investigated this hypothesis using recombinantly expressed post- fusion RSV F mutant proteins postF-R429A, postF-G430A, or postF-I432A. Binding of each of the new site IV mAbs was tested by ELISA and, as expected, 3M3 failed to bind the postF- R429A mutant (FIG. 2C, FIG. S5). MAb 6F18 retained binding to the postF-R429A protein, different from the results where binding was lost to cell-surface expressed mutant protein (FIGS. 2A and 2C; SFIGS. 4-5). Another difference observed was the binding of 17E10 to the postF-G430A and postF-I432A mutants, which did not abrogate binding. These differences can be explained by the presence of the pre-fusion conformation in the cell surface-expressed system, while the recombinant protein is in the post-fusion conformation.

To account for these differences, the inventor generated site IV residue mutants in the pre-fusion RSV A2 F SC-TM construct, which were confirmed via binding of mAb D2S. In this case, binding matched data from the cell surface-expressed system, where binding for 6F18 was now lost at R429A, and 17E10 had reduced binding to the G430A mutant. Similarly, the R429A mutation abolished 101F binding only to F in the pre-fusion conformation but not to post-fusion F. The R429 residue was shown to bind deep into the antibody heavy /light chain interface of 101F in the reported crystal structure (McLellan et al, 2010), and to be important for binding of 101F to a site IV-derived peptide (Wu et al, 2007). The pre-fusion I432A mutant could not be tested as protein could not be obtained despite multiple expression attempts. The cross-reactive mAb 54G10 had reduced binding to the postF-R429A and postF-G430A mutants, and the preF-G430A mutant. Based on these data, it appears the mAbs S4G10, 6F18, 17E10, and 101F have additional contacts in post-fusion RSV F, which are absent in pre-fusion RSV F. To clarify the differential binding between cell-surface expressed F, pre-fusion F, and post-fusion F, the inventor generated a recombinant RSV R429A escape mutant virus and tested neutralization for each of the mAbs (FIG. 2C; SFIG 6). As expected, mAb 6F18, 101F, and 3M3 did not neutralize the virus, while neutralizing activity was retained for mAbs 17E10 and 2N6, consistent with the pre-fusion F R429A ELISA binding data. The inventor could not rescue mutants for G430A or I432A.

As antigenic site IV consists at least partially of a linear epitope, peptides have been used previously to characterize mAb binding (McLellan etal, 2010). Using a set of synthesized biotinylated 15-mer peptides consisting of residues 422-436, the inventor tested binding of 2N6, 3M3, 6F18, 17E10, 101F, and 54G10, using streptavidin-coupled biosensors and streptavidin- coated ELISA plates. Only mAbs 17E10 and 101F bound the wild-type 15-mer peptide by biosensor or ELISA (FIG. 3; SFIG. 7). Mutant peptides containing R429A, G430A, or I432A mutations abolished binding of both 17E10 and 101F. Although the R429 residue was found to be nonessential for mAb 17E10 binding, it is likely the mAb makes other contacts outside of the 15-mer region that are important for binding in full-length RSV F. Since the other RSV F-specific mAbs failed to bind to the peptides, peptides extending 15 residues in either N- terminal or C -terminal direction consisting of residues 407-436 (30-mer A) and 422-451 (30- mer B) were used to identify binding residues flanking the 15-mer region. For the 30-mer A peptide, binding was significantly enhanced for mAbs 17E10 and 101F, and binding was now observed for mAb 2N6 (FIG. 3). The 30-mer B peptide did not have this effect, suggesting additional contact residues for 17E10, 101F, and 2N6 extend in the N-terminal direction. As the 30-mer B did not affect binding, it is unlikely the increased binding to the 30-mer A peptide was due to increased peptide length. It is worth noting that only mAbs 17E10 and 101F bound to the 15-mer peptide, as this suggests that cross-reactive mAbs target this conserved area. However, it is clear that peptides to not properly recapitulate binding to recombinant proteins for antigenic site IV.

Electron microscopy determines the structural basis for cross-reactivity. To further probe the structural basis for the different binding motifs and the mAb cross-reactivity with hMPV F, the inventor generated complexes of the new mAbs 2N6, 3M3, 6F18, and 17E10 with post-fusion RSV F protein and generated 3-D reconstructions by negative-stain electron microscopy (FIG. 4). The EM reconstructions include an elongated post-fusion RSV F protein with two fragment-antigen binding domains bound to the viral protein. Binding poses for mAbs 3M3 and 6F18, both of which rely on R429 for binding, were very similar. The two mAbs formed T-shaped complexes with the wide-axis of the Fab molecule perpendicular to the long- axis of post-fusion RSV F. In the case of mAb 2N6, an alternative binding pose was observed, as the Fab is rotated approximately 90° from the orientation of Fabs 3M3 and 6F18. These data help explain the retention of binding of mAb 2N6 to all mutants tested, as the unique binding pose allows contact residues outside of the canonical antigenic site IV. Finally, the complex of Fab 17E10 with RSV F was quite different from that of the others, with a binding angle 42° shifted from the other mAbs. This binding angle of 17E10 may be indicative of cross-reactivity between RSV and MPV, while also allowing mAb 17E10 to bind and neutralize RSV strains, including the R429A mutant. A similar binding angle shifted from the 90° line was observed for the murine-derived mAb 101F to RSV F (Mas et al, 2016) (FIG. 4), and mAb cross- reactivity with hMPV F may be partially defined by this binding pose.

MAbs RSV 3M3 exhibited excellent neutralizing activity against a representative RSV subgroup B strain. RSV B-l was obtained from BEI Resources (BEI Resources catalog #NR-4052; Lot #BPR 348-00). The RSV 3M3 antibody exhibited superior activity compared to the recombinant forms of antibodies based on the sequences of reference antibodies MEDI8897 or palivizumab. A negative control antibody (DENV 2D22 directed to dengue virus) did not neutralize at the threshold of 50% plaque reduction. The site VIII hRS V90 mAb (Mousa et al, 2017) was included as a positive control and also exhibited superior activity compared to the recombinant forms of antibodies based on the sequences of reference antibodies MEDI8897 or palivizumab. Example 3 - Discussion

Here the inventor defines the complexity of antigenic site IV on the RSV F protein by identifying a panel of neutralizing human mAbs, each with diverse binding modes. The idea presented that mAbs targeting the same antigenic site can have highly diverse binding modes likely applies to all antibody-antigen interactions. Antigenic site IV is preserved in both precision and post-fusion RSV F. However, amino acids surrounding antigenic site IV change dramatically between the two conformations. Therefore, binding of mAbs at this site can be quite diverse depending on whether a mAb is tested for binding to pre-fusion or post-fusion F. A summary of the diverse binding characteristics of each mAb is shown in SI Table. Previous studies suggested antigenic site IV site adopts a mostly linear conformation, and the corresponding antibodies appeared to form a single class of RSV-specific neutralizing antibodies. The inventor finds, in contrast, that human antibodies to this site recognize epitopes in diverse manners, and the potency and breadth of the neutralizing activity of those antibodies is associated with the fine details of the epitope, the binding angle, and other features of the antibody-antigen interaction. To understand the binding modes at antigenic site IV, the inventor generated a panel of new antibodies and present four mAbs that target antigenic site IV mAbs, each with unique binding requirements. Of those new clones, mAb 3M3 is quite potent, and mAb 17E10 is cross-reactive with hMPV F. The inventor cannot define the original ontogeny of mAb 17E10 as the donors were adults likely who have been infected multiple times with RSV and hMPV, thus the antibody could have been generated originally to RSV F or hMPV F, and then further mutated during subsequent infections. In general, mAbs are generated and optimized based on binding, as the germinal center reaction does not select for neutralizing capacity during affinity maturation. MAb 17E10 does neutralize hMPV much better than RSV, yet this does not prove that it was generated in response to hMPV. Rather, it is most clear that 17E10 binds to hMPV F in an orientation that facilitates more efficient inhibition of the pre- to post-fusion transition than for RSV F. This finding is similar to the previously discovered mAb 54G10, for which the extent of neutralization of hMPV and RSV were quite different. Reported ICso values for mAb 54G10 were 60 ng/mL for hMPV B2 and 14,200 ng/mL for RSV A2 (Schuster etal, 2014), similar values to those the inventor observed in this report. The binding pose of 17E10, while facilitating cross-reactivity, appears to be less efficient at inhibiting the pre- to post-fusion transition for RSV F.

The amino acid R429 in RSV F has been the defining contact residue of site IV based on studies performed on the binding properties of antibodies isolated from previous phage display library experiments (Crowe etal, 1998) and the murine/humanized-murine mAb 101F (Wu etal, 2007). While the two mAbs 3M3 and 6F18 required R429 for binding, mAbs 17E10 and 2N6 bound to cell surface-expressed or recombinantly expressed protein independently of this residue. The inventor also found site IV-based cross-neutralization of RSV and hMPV is associated with recognition of the G430 and 1432 residues, which are shared in RSV and hMPV F proteins, unlike R429, which is present only in RSV F. Furthermore, the binding poses of mAbs 17E10 and 2N6 show unique features allowing for the bypass of R429. No single amino acid was determined critical for binding of mAb 2N6, suggesting a mode of binding unique from mAbs 3M3, 6F18, 17E10, 101F, or 54G10. Further investigation of this binding mode is warranted through by X-ray crystallography.

MAb 17E10 binds in a pose much different than the other mAbs, which could determine cross-reactivity with hMPV. This pose is similar to that of the cross-reactive murine-derived mAb 101F, suggesting the tilted binding pose relative to antigenic site IV is indicative of cross- reactivity with hMPV F. MAb 17E10 binds at an approximately 56° tilt and 101F at a 32° tilt, in contrast to the other mAbs that are not cross-reactive and bind close to 0° tilt. The tilt angle may indeed be a determinant of cross-reactivity with hMPV F. Binding of 101F to pre-fusion F is dependent on the R429 residue and the G430A residues, while 17E10 bypasses R429 and utilizes G430 and 1432. Therefore, the high tilt angle by 17E10 compared to 101F likely allows this residue switch interaction.

Site IV is a robust antigenic site for the RSV immune repertoire as evidenced by the activity of the potently neutralizing mAb 3M3, and it is one of two known antigenic sites on the RSV F protein that induces cross-reactive mAbs. Current thought in the RSV field is that the most potent neutralizing mAbs (D2S, hRSV90) are directed toward the pre-fusion F conformation, particularly toward site 0 and site VIII. However, here the inventor describes a very potent mAb 3M3 that is directed toward antigenic site IV, suggesting antigenic site IV may be useful for incorporation into next-generation vaccine formulations. A limitation of this study is the small number of mAbs generated. The inventor cannot predict the frequency of 3M3-like or 17E10-like mAbs in the human repertoire, although a previous study has shown site IV mAbs in general represent a substantial portion of the human repertoire to RSV F (Gilman et al, 2016). These studies identify a new potent human mAb that could be used in prophylactic or therapeutic applications, and the data reveal important structural features of the F protein that could be used in rational structure-based vaccine design. For instance, as a vaccine antigen, the linear peptide that has been used as the canonical representation of antigenic site IV likely would be insufficiently immunogenic as a human vaccine antigen, however, incorporating residues slightly outside of this region and masking R429 may result in the production of potent and cross-neutralizing pan-Pneumovirus antibodies. Further structural analysis of cross-reactive site IV mAbs could facilitate development of a vaccine that

protects against both RSV and hMPV.

TABLE 1 - NUCLEOTIDE SEQUENCES FOR ANTIBODY VARIABLE REGIONS

Clone Variable Sequence Region SEQ

ID NO: caagtgcagctacaacaatggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtcta 1 RSV 2N6 - tggtgggtccttcagtgaaacctactggagttggatccgccagtccccaaaggggctggagtggattggggag

Heavy atcagtcgtagtggaagcaccaactacaacccgtccctcaagagtcgagtcaccatatcagtagacacgtcca

agaaccatttctccctgaagctgagctctgtgacccccgcggacacggctgtgtattactgtgcggccccccgg agagtggatggctacaacctacttgactcctggggccagggaaccgtggtcaccgtctcctca

RSV 2N6 - cagtctgtgctgacgcagccgccctcagtgtctggggccccaggacagacggtcaccatctcctgcactgatac 2 Light cagatccaacatcgggtcaggttatgatgtacactggtaccagcaacttccaggaacagcccccaaactcctca

tctatggtaacaccaatcgaccctcaggggtctctgaccgattctctggctccaagtctggctcctcagcctccct ggccatagctggtctccaggctgaggatgaggctgattattactgccagtcctttgacaacagccggactgcctt ttatatcttcggatccgggaccagagtgaccgttctg

RSV 3M3 - caggtgcagttagttcagtggggtgctggactggttaagccttctgaaacactgtctctgacatgtgccgtgtat 3 Heavy ggcggaacctttagcggctacttctggaactggatcagacaacctcctggaaaaggactggagtggattggcg

agatcaatcacggcggcaccaccaactacaattctagcctgaagtctagagtgaccatcagcatggacatgag caagaaccagttctacctgaaggtgaagagcgtgacagccgccgatacagccgtgtactactgttctagagga gtggctgatagaattagctctagctggcactacgacctgtggggactgggcacactggttacagtgtctagc

RSV 3M3 - agctatgtcctgacccaacctccttctgtgtctgttgctcctggacaaacagctagactgacatgtggcgccaac 4 Light aatatcggcaacgacaggatccactggtatcagcagcaacctggacaagctcctgttctggtggtgtttgaaga

cgtgcacagacctagcggcattccagccagatttagcggcagcaatagcggcaatacagctacactgttcatc agcagagtggagccaggagatgaggccgattactactgtcaggtgtgggatagcgagaatgatcatcccgtgt ttggcacaggcacaagagtgaccgttctg

RSV 6F18 - caggtgcagctggtgcagtcaggcccaggactggtgaggccctcgcagaccctctcagtcacctgtgacatctc 5 Heavy cggggacagagtctccagtaatagtgctgtttggaactggatcaggcagtccccatcgagaggccttgagtgg

ctgggaaggacatactacaggtccaagtggtatgatgattacgcaggatctgtgaagagtcgaatgaccatga acgtagacacatccaggaaccgggtctccctgcagctaaattcagtgacttccgaggacacggctgtctattac tgtgcaaggtcccaggatgacagtagtggttatcacgaagatttttttgacttctggggccagggaaccctggtc accgtctcctca RSV 6F18 - gacattgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcaagtc 6 Light cggccagagtgttttcttcaacttcaacaacaagaactacttagcttggtaccagcagaaaccaggacagcctc ctaagctgctcatgtactgggcatctacccgggaatccggggtccctgaccgattcactggcagcgggtctggg acagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcagcaatactataga atgccgtacacttttggccaggggaccaagctggagaccaaa

RSV 17E10 gaggtccagctggtgcagagtggggggggcctggtcaaacccggagggagcctgcgcctgtcttgtgctgtga 7 - Heavy gtggctttacattttccgacgcatggatgtactgggtgcggcaggcaccaggcaagggactggagtgggtggg caggatcaagcggagagtggacggcaccacaaccgattatggcgcccctgtgaagggccggttcacaatctct agggacgatagccgctccgtgctgtctctgcagatggacagcctgaagacagaggataccggcatctactatt gttccgtcctgtattcactgcagcattgggggaggggcactctggtcaccgtctcatcc

RSV 17E10 caggctgtcgtgacccaggaaccctctctgaccgtgtcccccggcggaaccgtgactctgacttgcggctccac 8 - Light aactgaactggtgaccgacgatcactaccccttctggtttcagcagaggccaggacaggcacctagaaccctg atctatgacacagtgcacaggcactcttgggcaccagcacggttcagcggatccctgggaggaggcaaggcc gccctgacactgagcggagcacagccagaggatgaggccgtgtactattgcctgctgtcctacaataatatgct ggtctttggagggggaactaaactgactgtgctg

TABLE 2 - PROTEIN SEQUENCES FOR ANTIBODY VARIABLE REGIONS

Clone Variable Sequence SEQ

NO:

RSV 2N6 ^VQLQQWGAGLLKPSETTLSLTCAWGGSFSETYWSWIRQSPKGLEWIGEISRSGSTNYNPSLK g Heavy SRvTIWDTSKNHFSLKLSSVTPADTAVYYCAAPRRVDGYNLLDSWGQGTvVTVSS

RSV 2N6 -QSVLTQPPSVSGAPGQTvTISCTDTRSNIGSGYDVHWYQQLPGTAPKLLIYGNTNRPSGVSDRlO Light FSGSKSGSSASLAIAGLQAEDEADYYCQSFDNSRTAFYIFGSGTRVTVL

RSV 3M3 -QVQLVQWGAGLVKPSETLSLTCAVYGGTFSGYFWNWI RQPPGKG LEWIG El N HGGTTNYNS 11 Heavy SLKSRvTISMDMSKNQFYLKVKSVTAADTAWYCSRGVADRISSSWHYDLWGLGTLVTVSS

RSV 3M3 -SYVLTQPPSVSVAPGQJARLTCG AN N IG N D Rl H WYQQQPGQAPVLWFED VH RPSG I PARFS 12 Ught GSNSGNTATLFISRVEPGDEADYYCQVWDSENDHPVFGTGTRVTVL

RSV 6F18 ^VQLVQSGPGLVRPSQTI^VTCDISGDRVSSNSAV^A/NWIRQSPSRGLEWLGRTYYRSKWYD 13 Heavy DYAGSVKSRMTMNVDTSRNRVSLQLNSVTSEDTAVYYCARSQDDSSGYHEDFFDFWGQGT

LVTVSS

RSV 6F18 -DIVMTQSPDSLAVSLGERATINCKSGQSVFFNFNNKNYLAVVYQQKPGQPPKLLMYWASTRE 14 Ught SGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQYYRMPYTFGQGTKLETK

RSV 17E10EVQLV(¾GGGLVKPGGSLRLSCAVSGFTFSDAWMYVvVRQAPGKGLEVVVGRIKRRVDGTTT 15

- Heavy DYGAPVKGRFTISRDDSRSVLSLQMDSLKTEDTGIYYCSVLYSLQHWGRGTLVTVSS

RSV 17E10(^vVTQEPSLWSPGGTvTLTCGSTTELvTDDHYPFWFQQRPGQAPRTLIYDTVHRHSWAPA16

- Ught RFSGSLGGGKAALTLSGAQPEDEAVYYCLLSYNNMLVFGGGTKLTVL

* * * * * * * * * * * * * * * * * 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 clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; 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 clone-paired variable sequences as set forth in Table 1.

7. The method of claims 1-5, wherein said 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.

8. The method of claims 1-5, wherein said 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.

9. The method of claims 1-5, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

10. The method of claims 1-5, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2.

11. The method of claims 1 -5, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

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 clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

14. The method of claim 13, the antibody or antibody fragment is encoded by clone-paired light and heavy chain variable sequences as set forth in Table 1.

15. The method of claim 13-14, the antibody or antibody fragment is encoded by clone- paired light and heavy chain variable sequences having 95% identify to as set forth in Table 1.

16. The method of claim 13-14, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone- paired sequences from Table 1.

17. The method of claim 13, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

18. The method of claim 13, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2.

19. The method of claim 13, encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

20. The method of claims 13-19, wherein said antibody is a chimeric antibody, a bispecific antibody, and/or is an IgG, or wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment.

21. The method of claims 13-20, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, such as a LALA, N297, GASD/ALIE, or is glycan modified to alter (eliminate or enhance) FcR interactions, such as enzymatic or chemical addition or removal of glycans, or genetic modification of a glycosylation pattern, or comprises an Fc portion mutated to alter FcRn interactions to increase in vivo half-life and in vivo protection, such as a YTE or LS mutation.

22. The method of claims 13-20, wherein said antibody or antibody fragment recognizes an epitope on RSV F protein in antigenic site IV, and/or said antibody or antibody fragment neutralizes RSV and human metapneumo virus .

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, genetic delivery of an RNA or DNA segment or vector encoding the antibody or antibody fragment, such as a VEE replicon, by injection with a needle, electroporation device, or other physical methods of gene delivery.

26. A monoclonal antibody, wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

27. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences according to clone-paired sequences from Table 1.

28. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired sequences from Table 1.

29. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 1.

30. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

31. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

32. The monoclonal antibody of claims 26-31 , wherein said antibody is a chimeric antibody, a bispecific antibody, and/or is an IgG, or wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment.

33. The monoclonal antibody of claims 26-32, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, such as a LALA, N297, GASD/ALIE, or is glycan modified to alter (eliminate or enhance) FcR interactions, such as enzymatic or chemical addition or removal of glycans, or genetic modification of a glycosylation pattern, or comprises an Fc portion mutated to alter FcRn interactions to increase in vivo half-life and in vivo protection, such as a YTE or LS mutation.

34. The monoclonal antibody of claims 26-33, wherein said antibody or antibody fragment recognizes an epitope on RSV F protein in antigenic site IV, and/or neutralizes RSV and human metapneumovirus.

35. The monoclonal antibody of claim 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 clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

37. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences according to clone- paired sequences from Table 1.

38. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired variable sequences from Table 1.

39. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having 95% identity to clone-paired variable sequences from Table 1.

40. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

41. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired variable sequences from Table 2.

42. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

43. The hybridoma or engineered cell of claims 36-42, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, such as a LALA, N297, GASD/ALIE, or is glycan modified to alter (eliminate or enhance) FcR interactions, such as enzymatic or chemical addition or removal of glycans, or genetic modification of a glycosylation pattern, or comprises an Fc portion mutated to alter FcRn interactions to increase in vivo half-life and in vivo protection, such as a YTE or LS mutation.

44. 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, or wherein said antibody is a chimeric antibody, a bispecific antibody, and/or is an IgG.

45. The hybridoma or engineered cell of claims 26-33, wherein said antibody or antibody fragment recognizes an epitope on RSV F protein in antigenic site IV, and/or neutralizes RSV and 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 clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.

48. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments is encoded by light and heavy chain variable sequences according to clone- paired sequences from Table 1.

49. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired sequences from Table 1.

50. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments is encoded by light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 1..

51. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments comprises light and heavy chain variable sequences according to clone- paired sequences from Table 2.

52. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

53. The vaccine formulation of claims 47-52, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, such as a LALA, N297, GASD/ALIE, or is glycan modified to alter (eliminate or enhance) FcR interactions, such as enzymatic or chemical addition or removal of glycans, or genetic modification of a glycosylation pattern, or comprises an Fc portion mutated to alter FcRn interactions to increase in vivo half-life and in vivo protection, such as a YTE or LS mutation.

54. The vaccine formulation of claims 47-52, 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 wherein at least one of said antibodies 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 RSV F protein in antigenic site IV, and/or neutralizes RSV and 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 rV-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 a known site IV-specific neutralizing antibody or antigen binding fragment thereof;

(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 IV-specific neutralizing when said known site IV-specific neutralizing antibodies or antigen binding fragment thereof 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 a known site IV-specific neutralizing antibody or fragment thereof.

59. The method of claims 57-58, wherein detection comprises ELIS A, RIA or Western blot.

60. The method of claims 57-59, wherein the known site IV-specific neutralizing antibody or fragment thereof is encoded by clone-paired variable sequences as set forth in Table 1.

61. The method of claims 57-60, wherein the known site IV-specific neutralizing antibody or fragment thereof 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.

62. The method of claims 57-60, wherein the known site IV-specific neutralizing antibody or fragment thereof is encoded by light and heavy chain variable sequences having 95% identity to clone-paired sequences as set forth in Table 1.

63. The method of claims 57-60, wherein the known site IV-specific neutralizing antibody or fragment thereof comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

64. The method of claims 57-60, wherein the known site IV-specific neutralizing antibody or fragment thereof comprises light and heavy chain variable sequences having 70%, 80% or 90% identity to clone-paired sequences from Table 2.

65. The method of claims 57-60, wherein the known site IV-specific neutralizing antibody or fragment thereof comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

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.

67. A monoclonal antibody or fragment thereof, wherein said antibody or fragment thereof recognizes an epitope on RSV F protein in antigenic site IV.

68. The monoclonal antibody or fragment thereof of claim 67, wherein said antibody or antibody fragment neutralizes RSV and metapneumovirus.

69. A method of identifying the presence of human respiratory syncytial virus (hRSV) protein F site IV protective antigen in a vaccine or virus preparation using human respiratory syncytial virus (hRSV) protein F site IV-specific monoclonal or polyclonal neutralizing antibodies comprising:

(a) contacting a candidate vaccine or virus composition with a known site IV- specific neutralizing antibody or antigen binding fragment thereof; (b) assessing binding of said candidate vaccine or virus composition to a known site rV-specific neutralizing antibody or antigen binding fragment; and

(c) identifying said candidate vaccine or virus composition as containing the protein F site IV protective epitope when one or more known site IV-specific neutralizing antibodies bind to the candidate vaccine or virus composition.

70. 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.

71. The method of claim 70, wherein said sample comprises recombinantly produced antigen.

72. The method of claim 70, wherein said sample comprise a vaccine formulation or vaccine production batch.

73. The method of claims 70-72, wherein detection comprises ELISA, RIA, western blot, a biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometric staining.

74. The method of claims 70-73, wherein the first antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1.

75. The method of claims 70-73, 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.

76. The method of claims 70-73, 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.

77. The method of claims 70-73, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

78. The method of claims 70-73, 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.

79. The method of claims 70-73, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

80. The method of claims 70-79, wherein the first antibody fragment is a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment.

81. The method of claims 70-80, further comprising steps (a) and (b) a second time to determine the antigenic stability of the antigen over time.

82. The method of claims 70-81, 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.

83. The method of claim 82, wherein the second antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1.

84. The method of claim 82, 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.

85. The method of claim 82, 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.

86. The method of claims 82, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

87. The method of claim 82, 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.

88. The method of claim 82, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

89. The method of claim 82, wherein the second antibody fragment is a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment.

90. The method of claim 82, further comprising steps (c) and (d) a second time to determine the antigenic stability of the antigen over time.