WO2019210144A1

WO2019210144A1 - Broadly neutralizing antibodies against hepatitis c virus

Info

Publication number: WO2019210144A1
Application number: PCT/US2019/029315
Authority: WO
Inventors: James E. Crowe, Jr.; Andrew I. FLYAK; Justin R. BAILEY
Original assignee: Vanderbilt University; The Johns Hopkins University
Priority date: 2018-04-27
Filing date: 2019-04-26
Publication date: 2019-10-31

Abstract

The present disclosure is directed to antibodies or antibody fragment binding to and neutralizing Hepatitis C virus and methods for use thereof.

Description

DESCRIPTION

BROADLY NEUTRALIZING ANTIBODIES AGAINST HEPATITIS C VIRUS

This application claims benefit of priority to U.S. Provisional Application Serial No. 62/663,853, filed April 27, 2018, the entire contents of which are hereby incorporated by reference. BACKGROUND

This invention was made with government support under K08 AI102761, U19 AI088791, and 1P30AI094189 and contracts HHSN272200900055C and HHSN272201400058C from the National Institutes of Health. The government has certain rights in the invention. 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 Hepatitis C virus, and methods of use therefor. 2. Background

Hepatitis C virus (HCV) infects ~185 million people worldwide and is a major cause of liver failure and hepatocellular carcinoma (1). With the recent development of potent, oral, interferon-free therapies, treatment of HCV infection has improved significantly. However, HCV eradication is unlikely to be achieved with treatment alone. Identification of those with HCV infection is challenging. Therapies are too costly for countries with the highest incidence. Reinfection can occur following treatment, and transmission of drug-resistant HCV is possible (Cox, 2015). The rate of acute HCV infection increased in most US states between 2010 and 2014, following an ongoing epidemic in opioid/heroin use (Zibbell et al., 2015; Conrad et al., 2015; Suryaprasad et al., 2014). This rising epidemic of acute HCV infection in the United States gives new urgency to prophylactic vaccine development efforts.

Broadly-neutralizing human monoclonal antibodies (bNAbs) capable of neutralizing diverse HCV strains have been isolated from HCV-infected individuals, proving that antibodies can target relatively conserved regions of the two HCV envelope glycoproteins (E1 and E2), despite the enormous genetic diversity of HCV (Hadlock et al., 2000; Keck et al., 2008; 2013; Giang et al., 2012; Krey et al., 2013; Johansson et al., 2007; Merat et al., 2016; Keck et al., 2011; 2012; Long et al. 2012; Law et al., 2008). Infusion of bNAbs is protective against infection in animal models of HCV (Law et al., 2008; Morin et al., 2012), , and a recent study also showed that bNAbs could abrogate established HCV infection in a humanized transgenic mouse model (de Jon et al., 2014). Given the efficacy of these bNAbs in blocking HCV infection, the molecular and genetic features of bNAbs and their epitopes may serve as a useful guide for rational HCV vaccine design.

Studies of the evolution of HIV-specific bNAbs have enabled an entire field of germline-targeted vaccine designs, and stabilization of envelope antigens (Burton et al., 2015; Bhiman et al., 2015; Bonsignori et al., 2016). However, studies of the natural evolution of HIV bNAbs still may not be the optimal system for fully understanding the fundamental principles of breadth and potency for bNAbs, because HIV-infected individuals do not clear their infections. In contrast, approximately 30% of individuals who become infected with HCV spontaneously clear the infection (Thomas et al., 2009), even though the viral diversity in HCV-infected individuals is comparable to or exceeds that of the diversity of HIV isolates in HIV-infected subjects (Martell et al., 1992; Simmonds et al., 1993; Pybus et al., 2001; Ray and Thomas; 2010). Spontaneous clearance of HCV has been associated with effective innate and T cell responses, but the inventors and others also have shown that spontaneous clearance is associated with early appearance of broadly-neutralizing antibodies against HCV in serum. Monoclonal antibodies (mAbs) from individuals with broadly neutralizing serum and clearance of HCV have not been isolated to date, so it is not known whether these mAbs have unique features relative to the mAbs previously isolated from individuals with persistent HCV infection. It is of interest to define the molecular basis for recognition and neutralization of an entire quasispecies of an antigenically diverse virus like HCV, with subsequent immune- mediated clearance.

SUMMARY

Thus, in accordance with the present disclosure, a method of detecting a Hepatitis C 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 Hepatitis C virus in said sample by binding of said antibody or antibody fragment to a Hepatitis C virus antigen in said sample. The sample may be a body fluid, such as blood, sputum, tears, saliva, mucous or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissues, urine, exudate, transudate, tissue scrapings or feces. Detection may comprise ELISA, RIA, lateral flow assay or Western blot. The method may further comprise performing steps (a) and (b) a second time and determining a change in Hepatitis C 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 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 Hepatitis C virus or reducing the likelihood of infection of a subject at risk of contracting Hepatitis C 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 a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab ^)2 fragment, or Fv fragment.

The antibody may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody may be a chimeric antibody or a bispecific antibody.

The antibody or antibody fragment may be administered prior to infection or after infection. The subject may be a pregnant female, a sexually active female, or a female undergoing fertility treatments. 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 still another embodiment, there is 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 a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab ^)₂ fragment, or Fv fragment.

The monoclonal antibody may be a chimeric antibody or is bispecific antibody. The antibody may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody or antibody fragment ma further comprise a cell penetrating peptide and/or may be an intrabody.

In an even further embodiment, there is provided a hybridoma or engineered cell encoding an antibody or antibody fragment wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. The hybridoma or engineered cell may be encoded 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 hybridoma or engineered cell may encoded 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 hybridoma or engineered cell may encode a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab ^)2 fragment, or Fv fragment.

The hybridoma or engineered cell may encode a chimeric antibody or a bispecific antibody. The hybridoma or engineered cell may encode an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The hybridoma or engineered cell may encode an antibody or antibody fragment further comprising a cell penetrating peptide and/or is an intrabody.

In a further embodiment, there is provided 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 a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab ^)2 fragment, or Fv fragment.

The at least one antibody may be a chimeric antibody or is bispecific antibody. The an least one antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The at least one antibody or antibody fragments may further comprise a cell penetrating peptide and/or is an intrabody.

A still further embodiment, there is provided a vaccine formulation comprising one or more expression vectors encoding a first antibody or antibody fragment as described above. The expression vector(s) may be Sindbis virus or VEE vector(s). The vaccine formulation may be formulated for delivery by needle injection, jet injection, or electroporation. The vaccine formulation may further comprise one or more expression vectors encoding for a second antibody or antibody fragment, such as a distinct antibody or antibody fragment as described above.

In an additional embodiment, there is provide a method of protecting the health of a placenta and/or fetus of a pregnant a subject infected with or at risk of infection with Hepatitis C 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 a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab ^)2 fragment, or Fv fragment.

The antibody may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody may be a chimeric antibody or a bispecific antibody. The antibody or antibody fragment may be administered prior to infection or after infection. The subject may be a pregnant female, a sexually active female, or a female undergoing fertility treatments. 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. The method may increase the size of the placenta as compared to an untreated control. The method may reduce viral load and/or pathology of the fetus as compared to an untreated control.

In still an additional a method of determining the antigenic integrity, correct conformation and/or correct sequence of a Hepatitis C virus 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, correct conformation and/or correct sequence of said antigen by detectable binding of said first antibody or antibody fragment to said antigen. The sample may comprise recombinantly produced antigen. The sample may comprise a vaccine formulation or vaccine production batch. Detection may comprise ELISA, RIA, western blot, a biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometric staining.

The first antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, may be encoded by light and heavy chain variable sequences having 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or may be encoded by light and heavy chain variable sequences having 95% identity to clone- paired sequences as set forth in Table 1. The first antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, 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 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 performing steps (a) and (b) a second time to determine the antigenic stability of the antigen over time.

The method may further comprise (c) contacting a sample comprising said antigen with a second 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 second antibody or antibody fragment to said antigen. The method may further comprise performing steps (c) and (d) a second time to determine the antigenic stability of the antigen over time.

The second 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 second 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 second antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab ^)2 fragment, or Fv fragment.

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 disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

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

FIGS 1A-B. Identification of subjects who spontaneously cleared HCV and possess broadly-neutralizing antibodies in plasma. (FIG. 1A) HCV viral loads of two subjects who spontaneously cleared HCV infection sampled periodically from the time of initial infection through viral clearance. Dashed line indicates limit of detection (LOD) of the viral load assay, which is 50 International Units (IU)/mL. Values below the LOD are set at 25 IU/mL and marked with gray circles. (-) or (+) indicate plasma samples that were HCV antibody-negative or positive by commercial antibody binding assay (EIA). White arrow indicates plasma samples tested for neutralizing breadth against the panel of 19 genotype 1 HCVpp. Stipled arrow indicates time points from which anti-HCV mAbs were isolated. Black arrow indicates plasma samples from which the viral quasispecies was sequenced by single genome amplification (SGA). (FIG.1B) Neutralizing breadth of plasma from the two subjects tested against a diverse panel of genotype 1a or 1b HCV pseudoparticles (HCVpp). Values shown are percent neutralization achieved by a 1:100 dilution of plasma, tested in duplicate. *For comparison, the median neutralization of each HCVpp by 42 subjects with persistent HCV infection, matched with subjects who cleared for duration of infection (Control plasma), is also shown.

FIG. 2. Intra-genotypic neutralizing breadth of anti-HCV monoclonal antibodies isolated from two subjects who spontaneously cleared HCV infection. Neutralizing breadth of mAbs against a diverse panel of genotype 1a or 1b HCV pseudoparticles (HCVpp). Neutralization patterns for the 5 most broadly-neutralizing mAbs are shown here; data for the remaining 10 mAbs are shown in FIG. 9. MAbs marked with blue were isolated from Subject 117 and mAbs marked with green were isolated from Subject 110. Values shown are percent neutralization achieved by 50 µg/mL of mAb. Values are means of two independent experiments, each performed in duplicate. For reference, previously described bNAbs AR4A and AR3C were tested in parallel against the same HCVpp panel. FIG. 3. Cross-genotypic neutralizing breadth of anti-HCV monoclonal antibodies. Neutralizing breadth of mAbs against a panel of genotype 1-6 replication competent hepatitis C viruses (HCVcc). The four mAbs with greatest neutralizing breadth in Figure 2 were tested. The name of each HCVcc strain is indicated with the viral subtype in parenthesis. Values shown are the means of two independent experiments, each performed in triplicate, and error bars represent standard deviations between experiments. The half maximal inhibitory concentration (IC50) of each mAb/HCVcc combination is shown. Curves with neutralization exceeding 50% at only the highest mAb concentration (50 µg/mL) were assigned an IC50 of 50 µg/mL.

FIGS. 4A-D. Epitope mapping of anti-HCV bNAbs. (FIG.4A) Critical binding residues for bNAbs, based on relative binding to strain H77 E1E2 or alanine scanning mutants spanning the full H77 E1E2 sequence. Binding residues are marked with green spheres superimposed on the H77 E2 core structure (31). For reference, contact residues for mAb AR3C, identified by Kong, et al., are indicated with blue spheres. Additional mAbs are shown in FIG.11. In the table, critical binding or contact residues shared by at least two mAbs are highlighted in red, and those shared by all four mAbs in purple. (FIGS. 4B-C) Competition-binding between mAbs. Six most broadly neutralizing mAbs from s117 (blue) and s110 (green) are shown, with additional mAbs shown in FIGS. 12A-B. Relative binding of 2 µg/mL of the biotinylated mAbs to strain 1a53 E1E2 in the presence or absence blocking mAbs at a concentration of 20 µg/mL. Combinations resulting in relative binding <0.7 or <0.35 are marked in yellow or red, respectively. (FIG. 4B) Competition-binding between novel mAbs and each other. Values represent the average of two independent experiments performed in duplicate. (FIG. 4C) Competition-binding between novel mAbs and reference bNAbs. Values represent the average of replicates from one experiment, except AR3C, which was tested in duplicate in two independent experiments. (FIG.4D) Clustering of the 6 most broadly-neutralizing mAbs (blue or green) with reference bNAbs (red) based upon neutralization profiling. For each mAb, neutralization of each of 19 HCVpp was measured, generating a neutralization profile, and pairwise spearman correlations were measured between these neutralization profiles. Circles at each intersection were scaled by the magnitude of the correlation between the indicated mAbs. Hierarchical clustering analysis using these pairwise correlations is depicted as a tree. Numbers at tree nodes are approximately unbiased (AU) test values (49), indicating strength of support for a particular cluster. FIGS. 5A-B. Role of somatic mutations in neutralization and binding of heterologous E1E2 proteins. (FIG. 5A) Neutralization of a heterologous genotype 1 HCVpp panel by HEPC3, HEPC3 with reversion of all somatic mutations in the light chain variable region to the germline encoded amino acids (L-RUA), HEPC3 with reversion of all somatic mutations in the heavy chain variable region (H-RUA), or HEPC3 with reversion of all somatic mutations in both light and heavy chain variable regions (H,L-RUA) at 50 µg/mL concentration of mAb, measured in duplicate. (FIG. 5B) Binding of serial dilutions of the indicated mAbs to genotype 1 E1E2 proteins, measured by ELISA. The individual reversion or combination of reversions introduced into HEPC3 are indicated on the vertical axis. The heatmap was generated using log10(Area Under the Curve) of binding of each mAb/E1E2 dilution series, which were measured in duplicate. Asterisks indicate significant differences between binding of each mAb to all E1E2 variants relative to binding of HEPC3 to the same E1E2 variants, measured by one-way ANOVA with adjustment for multiple comparisons (* p<0.05, ** p<0.005, **** p<.0001).

FIGS. 6A-B. HCV strain-specific effects of bNAb somatic mutations. (FIG. 6A) Binding of serial dilutions of HEPC3 or the indicated HEPC3 mAb variants to 4 different genotype 1 E1E2 protein variants, measured by ELISA. Values are means of duplicate wells, and error bars indicate standard deviations. (FIG.6B) Kinetic binding analysis of HEPC3 and HEPC3 mAb variants and soluble J6 strain (genotype 2a) E2 protein. Dissociation constants (KD) for each mAb are shown. Error bars represent the standard errors, which were calculated using a global fit mode that includes several analyte concentrations. Single amino acid reversions in HEPC3 are grouped by their location in HCDR1, 2, 3, or framework regions (Frm).

FIG.7. Longitudinal evolution of autologous E1E2 genes. Maximum-likelihood phylogenetic tree of E1E2 nucleotide sequences amplified by single genome amplification (SGA) from Subject 117 plasma at 7 longitudinal time points throughout the course of infection. Sequences are color-coded by date of sampling. Transmitted/founder (T/F) sequences inferred by phylogeny and date of sampling, and variants cloned for protein expression are indicated. Outgroup is composed of genotype 1a sequences from the heterologous E1E2 panel. Bootstrap values greater than 80 are indicated.

FIG. 8. Role of somatic mutations in binding of autologous E1E2 proteins. Binding of serial dilutions of HEPC3, HEPC3 with all heavy chain somatic mutations reverted to the germline-encoded amino acid (HEPC3 H-RUA), or HEPC3 with all somatic mutations reverted to the germline-encoded amino acid (HEPC3 H,L-RUA) to 21 unique autologous E1E2 proteins. Proteins are color-coded by date of sampling. Values are means of duplicate wells, and error bars indicate standard deviations. Median binding of an isotype control antibody to all E1E2 variants is shown as a control for nonspecific binding.

FIG.9. Neutralizing breadth of the 10 mAbs not shown in Figure 2 against a diverse panel of genotype 1a or 1b HCV pseudoparticles (HCVpp). Neutralization patterns for the 5 most broadly-neutralizing mAbs are shown in FIG. 2; data for the remaining 10 mAbs are shown here. MAbs marked with blue were isolated from Subject 117 and mAbs marked with green were isolated from Subject 110. Values shown are percent neutralization achieved by 50 µg/mL of mAb. Values are means of two replicate tests.

FIG.10. Binding of mAbs to native and denatured E1E2. Binding of 2 µg/mL of each mAb to native E1E2 (clone 1a53) or to the same E1E2 protein after boiling of E1E2 in detergent. Reference mAbs AR3C (conformational epitope) and HC33.8 (linear epitope) are included as controls. Values are the means of two replicate tests, and error bars indicate standard deviations.

FIG.11. Binding epitopes of 3 mAbs not shown in Figure 4. Binding residues were identified by measuring relative binding of mAbs to strain H77 E1E2 or alanine scanning mutants spanning the full H77 E1E2 sequence. Critical binding residues are marked with green spheres superimposed on the H77 E2 core structure (31). MAbs not shown here or in FIGS.4A-D did not have adequate affinity for strain H77 E1E2 to be mapped by this method.

FIGS.12A-B. Competition-binding between mAbs. Names of mAbs isolated from Subject 117 or Subject 110 are marked in blue or green, respectively. Binding of 2 µg/mL of the mAbs on the Y-axis (“Biotinylated mAbs”) to strain 1a53 E1E2 was measured in the presence or absence of the mAbs on the X-axis (“Blocking mAbs”) at a concentration of 20 µg/mL. Values shown are binding of the biotinylated mAb in the presence of blocking mAb, relative to binding in the absence of blocking mAb. Combinations resulting in relative binding <0.7 or <0.3 are marked in yellow or red, respectively. (FIG. 12A) Competition-binding between novel mAbs and each other. (FIG. 12B) Competition-binding between novel mAbs and a panel of previously published anti-HCV bNAbs. FIG. 13. Examples of correlations between neutralization profiles used for hierarchical clustering of mAbs in FIGS.4A-D. Each point indicates neutralization of an individual HCVpp by one antibody on the x-axis and a second antibody on the y- axis. Neutralization values are fraction unaffected (Fu) at 10 µg/mL of each antibody. Fu=infection in the presence of mAb/infection in the presence of nonspecific IgG. Spearman correlations (r) and p values are indicated for each mAb pair. HEPC3, HEPC74, and HEPC43 show strong, statistically significant correlations with each other and with AR3C, but no correlation with HC33.4.

FIGS.14A-D. Octet association/dissociation curves with HEPC3 mAb variants and J6 (genotype 2a) soluble E2. KD values generated from these curves are summarized in FIGS.6A-B.

FIG. 15. Ratio of nonsynonymous/synonymous mutations across all longitudinal E1E2 variants sequenced from subject 117. Analysis was performed with 20 codon windows and 1 codon steps. Hypervariable region 1 (HVR1) is shaded in gray, and the region spanning HEPC3 binding residues is shaded in blue.

FIG. 16. Highlighter plot indicating positions of amino acid differences in longitudinal E1E2 variants isolated from subject 117. Autologous variant transmitted/founder #3 (T/F#3) is used as the reference sequence, and sequences are arranged by their date of isolation. The locations of E1, E2, HVR1, and the region spanning HEPC3 binding residues are indicated.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS As discussed above, while effective treatment for HCV is available, these treatments are very expensive, and subject to potential drug resistance. To explore the possibility that effective immunotherapies for HCV are possible, the inventors prospectively followed a cohort of subjects from a time point prior to infection through the time of their spontaneous clearance of HCV. There they report an isolation of a panel of bNAbs from two of these subjects who spontaneously cleared HCV infection. They characterized the neutralizing breadth of these bNAbs, mapped the targeted epitopes, identified a germline heavy chain variable gene segment that was used by multiple bNAbs, and identified somatic mutations in one bNAb that were critical for breadth of recognition of heterologous envelope variants. They also defined the longitudinal evolution of the virus in the donor of this bNAb, allowing them to show that the bNAb unmutated ancestor could bind envelope proteins of early autologous transmitted/founder (T/F) viruses, and the mature bNAb could bind variants circulating immediately prior to viral clearance. Defining these determinants of bNAb breadth and the critical antigenic determinants favoring bNAb induction provides new insight into molecular mechanisms of immune-mediated clearance of HCV infection, and informs rational vaccine design. These and other aspects of the disclosure are described in detail below. I. Hepatitis C Virus

Hepatitis C virus (HCV) is a small (55–65 nm in size), enveloped, positive-sense single- stranded RNA virus of the family Flaviviridae. Hepatitis C virus is the cause of hepatitis C and some cancers such as liver cancer (hepatocellular carcinoma, abbreviated HCC) and lymphomas in humans. The hepatitis C virus belongs to the genus Hepacivirus, a member of the family Flaviviridae. Until recently it was considered to be the only member of this genus. However, a member of this genus has been discovered in dogs: canine hepacivirus. There is also at least one virus in this genus that infects horses. Several additional viruses in the genus have been described in bats and rodents.

The hepatitis C virus particle consists of a core of genetic material (RNA), surrounded by an icosahedral protective shell of protein, and further encased in a lipid (fatty) envelope of cellular origin. Two viral envelope glycoproteins, E1 and E2, are embedded in the lipid envelope.

Hepatitis C virus has a positive sense single-stranded RNA genome. The genome consists of a single open reading frame that is 9600 nucleotide bases long. This single open reading frame is translated to produce a single protein product, which is then further processed to produce smaller active proteins. This is why on publicly available databases, such as the European Bioinformatic Institute, the viral proteome only consists of 2 proteins.

At the 5' and 3' ends of the RNA, there are untranslated regions (UTRs) that are not translated into proteins but are important to translation and replication of the viral RNA. The 5' UTR has a ribosome binding site or internal ribosome entry site (IRES) that initiates the translation of a very long protein containing about 3,000 amino acids. The core domain of the HCV IRES contains a four-way helical junction that is integrated within a predicted pseudoknot. The conformation of this core domain constrains the open reading frame's orientation for positioning on the 40S ribosomal subunit. The large pre-protein is later cleaved by cellular and viral proteases into the 10 smaller proteins that allow viral replication within the host cell, or assemble into the mature viral particles. Structural proteins made by the hepatitis C virus include Core protein, E1 and E2; nonstructural proteins include NS2, NS3, NS4A, NS4B, NS5A, and NS5B.

The proteins of this virus are arranged along the genome in the following order: N terminal-core-envelope (E1)–E2–p7-nonstructural protein 2 (NS2)–NS3–NS4A–NS4B– NS5A–NS5B–C terminal. The mature nonstructural proteins (NS2 to NS5B) generation relies on the activity of viral proteinases. The NS2/NS3 junction is cleaved by a metal dependent autocatalytic proteinase encoded within NS2 and the N-terminus of NS3. The remaining cleavages downstream from this site are catalysed by a serine proteinase also contained within the N-terminal region of NS3.

The core protein has 191 amino acids and can be divided into three domains on the basis of hydrophobicity: domain 1 (residues 1–117) contains mainly basic residues with two short hydrophobic regions; domain 2 (residues 118–174) is less basic and more hydrophobic and its C-terminus is at the end of p21; domain 3 (residues 175–191) is highly hydrophobic and acts as a signal sequence for E1 envelope protein.

Both envelope proteins (E1 and E2) are highly glycosylated and important in cell entry. E1 serves as the fusogenic subunit and E2 acts as the receptor binding protein. E1 has 4–5 N- linked glycans and E2 has 11 N-glycosylation sites.

The p7 protein is dispensable for viral genome replication but plays a critical role in virus morphogenesis. This protein is a 63 amino acid membrane spanning protein which locates itself in the endoplasmic reticulum. Cleavage of p7 is mediated by the endoplasmic reticulum's signal peptidases. Two transmembrane domains of p7 are connected by a cytoplasmic loop and are oriented towards the endoplasmic reticulum's lumen. NS2 protein is a 21–23 kilodalton (kDa) transmembrane protein with protease activity. NS3 is 67 kDa protein whose N-terminal has serine protease activity and whose C- terminal has NTPase/helicase activity. It is located within the endoplasmic reticulum and forms a heterodimeric complex with NS4A—a 54 amino acid membrane protein that acts as a cofactor of the proteinase.

NS4B is a small (27 kDa) hydrophobic integral membrane protein with 4 transmembrane domains. It is located within the endoplasmic reticulum and plays an important role for recruitment of other viral proteins. It induces morphological changes to the endoplasmic reticulum forming a structure termed the membranous web.

NS5A is a hydrophilic phosphoprotein which plays an important role in viral replication, modulation of cell signaling pathways and the interferon response. It is known to bind to endoplasmic reticulum anchored human VAP proteins.

The NS5B protein (65 kDa) is the viral RNA dependent RNA polymerase. NS5B has the key function of replicating the HCV’s viral RNA by using the viral positive RNA strand as its template and catalyzes the polymerization of ribonucleoside triphosphates (rNTP) during RNA replication. Several crystal structures of NS5B polymerase in several crystalline forms have been determined based on the same consensus sequence BK (HCV-BK, genotype 1). The structure can be represented by a right-hand shape with fingers, palm, and thumb. The encircled active site, unique to NS5B, is contained within the palm structure of the protein. Recent studies on NS5B protein genotype 1b strain J4’s (HC-J4) structure indicate a presence of an active site where possible control of nucleotide binding occurs and initiation of de-novo RNA synthesis. De-novo adds necessary primers for initiation of RNA replication. Current research attempts to bind structures to this active site to alter its functionality in order to prevent further viral RNA replication.

An eleventh protein has also been described. This protein is encoded by a +1 frameshift in the capsid gene. It appears to be antigenic, but its function is unknown.

Replication of HCV involves several steps. The virus replicates mainly in the hepatocytes of the liver, where it is estimated that daily each infected cell produces approximately fifty virions (virus particles) with a calculated total of one trillion virions generated. The virus may also replicate in peripheral blood mononuclear cells, potentially accounting for the high levels of immunological disorders found in chronically infected HCV patients. HCV has a wide variety of genotypes and mutates rapidly due to a high error rate on the part of the virus' RNA-dependent RNA polymerase. The mutation rate produces so many variants of the virus it is considered a quasispecies rather than a conventional virus species. Entry into host cells occur through complex interactions between virions and cell-surface molecules CD81, LDL receptor, SR-BI, DC-SIGN, Claudin-1, and Occludin.

Once inside the hepatocyte, HCV takes over portions of the intracellular machinery to replicate. The HCV genome is translated to produce a single protein of around 3011 amino acids. The polyprotein is then proteolytically processed by viral and cellular proteases to produce three structural (virion-associated) and seven nonstructural (NS) proteins. Alternatively, a frameshift may occur in the Core region to produce an Alternate Reading Frame Protein (ARFP). HCV encodes two proteases, the NS2 cysteine autoprotease and the NS3-4A serine protease. The NS proteins then recruit the viral genome into an RNA replication complex, which is associated with rearranged cytoplasmic membranes. RNA replication takes places via the viral RNA-dependent RNA polymerase NS5B, which produces a negative strand RNA intermediate. The negative strand RNA then serves as a template for the production of new positive strand viral genomes. Nascent genomes can then be translated, further replicated or packaged within new virus particles. New virus particles are thought to bud into the secretory pathway and are released at the cell surface.

The virus replicates on intracellular lipid membranes. The endoplasmic reticulum in particular are deformed into uniquely shaped membrane structures termed‘membranous webs.’ These structures can be induced by sole expression of the viral protein NS4B. The core protein associates with lipid droplets and utilises microtubules and dyneins to alter their location to a perinuclear distribution. Release from the hepatocyte may involve the very low-density lipoprotein secretory pathway.

Based on genetic differences between HCV isolates, the hepatitis C virus species is classified into seven genotypes (1–7) with several subtypes within each genotype (represented by lower-cased letters). Subtypes are further broken down into quasispecies based on their genetic diversity. Genotypes differ by 30–35% of the nucleotide sites over the complete genome. The difference in genomic composition of subtypes of a genotype is usually 20–25%. Subtypes 1a and 1b are found worldwide and cause 60% of all cases.

Hepatitis C virus is predominantly a blood-borne virus, with very low risk of sexual or vertical transmission. Because of this mode of spread the key groups at risk are injecting drug users (IDUs), recipients of blood products and sometimes patients on haemodialysis. Common setting for transmission of HCV is also intra-hospital (nosocomial) transmission, when practices of hygiene and sterilization are not correctly followed in the clinic. A number of cultural or ritual practices have been proposed as a potential historical mode of spread for hepatitis C virus, including circumcision, genital mutilation, ritual scarification, traditional tattooing and acupuncture. It has also been argued that given the extremely prolonged periods of persistence of HCV in humans, even very low and undetectable rates of mechanical transmission via biting insects may be sufficient to maintain endemic infection in the tropic, where people receive large number of insect bites.

Identification of the origin of this virus has been difficult but genotypes 1 and 4 appear to share a common origin. A Bayesian analysis suggests that the major genotypes diverged about 300–400 years ago from the ancestor virus. The minor genotypes diverged about 200 years ago from their major genotypes. All of the extant genotypes appear to have evolved from genotype 1 subtype 1b.

A study of genotype 6 strains suggests an earlier date of evolution: ~1,100 to 1,350 years before the present (95% credible region, 600 to >2,500 years ago). The estimated rate of mutation was 1.8 × 10^-4 (95% credible region This genotype may be

the ancestor of the other genotypes.

A study of European, USA and Japanese isolates suggested that the date of origin of genotype 1b was ~1925. The estimated dates of origin of types 2a and 3a were 1917 and 1943 respectively. The time of divergence of types 1a and 1b was estimated to be 200–300 years.

A study of genotype 1a and 1b estimated the dates of origin to be 1914–1930 (95% credible interval: 1802–1957) for type 1a and 1911–1944 (95% credible interval: 1806–1965) for type 1b. Both types 1a and 1b underwent massive expansions in their effective population size between 1940 and 1960. The expansion of HCV subtype 1b preceded that of subtype 1a by at least 16 years (95% credible interval: 15–17 years). Both types appear to have spread from the developed world to the developing world.

The genotype 2 strains from Africa can be divided into four clades that correlate with their country of origin: (1) Cameroon and Central African Republic (2) Benin, Ghana and Burkina Faso (3) Gambia, Guinea, Guinea-Bissau and Senegal (4) Madagascar. There is also strong evidence now for the dissemination of hepatitis C virus genotype 2 from West Africa to the Caribbean by the Trans-Atlantic slave trade.

Genotype 3 is thought to have its origin in South East Asia.

These dates from these various countries suggests that this virus may have evolved in South East Asia and was spread to West Africa by traders from Western Europe. It was later introduced into Japan once that country's self-imposed isolation was lifted. Once introduced to a country its spread has been influenced by many local factors including blood transfusions, vaccination programmes, intravenous drug use and treatment regimes. Given the reduction in the rate of spread once screening for Hepatitis C in blood products was implemented in the 1990s it would seem that at least in recent times blood transfusion has been an important method of spreading for this virus. Additional work is required to determine the dates of evolution of the various genotypes and the timing of their spread across the globe. II. Monoclonal Antibodies and Production Thereof

An "isolated antibody" is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In particular embodiments, the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most particularly more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 basic heterotetramer units along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable region (V_H) followed by three constant domains (C_H) for each of the alpha and gamma chains and four CH domains for mu and isotypes. Each L chain has at the N-terminus, a variable region (V_L) followed by a constant domain (C_L) at its other end. The V_L is aligned with the V_H and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable regions. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.

The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on the amino acid sequences of their constant domains (CL). Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha, delta, epsilon, gamma and mu, respectively. They gamma and alpha classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term "variable" refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable regions of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD).

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31- 35 (H1), 50-65 (H2) and 95-102 (H3) in the V_H when numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and/or those residues from a "hypervariable loop" (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (H1), 52-56 (H2) and 95-101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and/or those residues from a "hypervariable loop"/CDR (e.g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the V_L, and 27-38 (H1), 56-65 (H2) and 105-120 (H3) in the V_H when numbered in accordance with the IMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res.27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res.28:219-221 (2000)). Optionally the antibody has symmetrical insertions at one or more of the following points 28, 36 (L1), 63, 74- 75 (L2) and 123 (L3) in the V_L, and 28, 36 (H1), 63, 74-75 (H2) and 123 (H3) in the V.sub.H when numbered in accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol. 309:657-670 (2001)).

By "germline nucleic acid residue" is meant the nucleic acid residue that naturally occurs in a germline gene encoding a constant or variable region. "Germline gene" is the DNA found in a germ cell (i.e., a cell destined to become an egg or in the sperm). A "germline mutation" refers to a heritable change in a particular DNA that has occurred in a germ cell or the zygote at the single-cell stage, and when transmitted to offspring, such a mutation is incorporated in every cell of the body. A germline mutation is in contrast to a somatic mutation which is acquired in a single body cell. In some cases, nucleotides in a germline DNA sequence encoding for a variable region are mutated (i.e., a somatic mutation) and replaced with a different nucleotide.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No.4,816,567) after single cell sorting of an antigen specific B cell, an antigen specific plasmablast responding to an infection or immunization, or capture of linked heavy and light chains from single cells in a bulk sorted antigen specific collection. The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

A. General Methods

It will be understood that monoclonal antibodies binding to Hepatitis C virus will have several applications. These include the production of diagnostic kits for use in detecting and diagnosing Hepatitis C 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 or vaccination with a licensed or experimental vaccine. 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 in animals 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 and in humans include alum, CpG, MFP59 and combinations of immunostimulatory molecules (“Adjuvant Systems”, such as AS01 or AS03). Additional experimental forms of inoculation to induce Hepatitis C virus -specific B cells is possible, including nanoparticle vaccines, or gene-encoded antigens delivered as DNA or RNA genes in a physical delivery system (such as lipid nanoparticle or on a gold biolistic bead), and delivered with needle, gene gun, transcutaneous electroporation device. The antigen gene also can be carried as encoded by a replication competent or defective viral vector such as adenovirus, adeno-associated virus, poxvirus, herpesvirus, or alphavirus replicon, or alternatively a virus like particle.

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 or to test the safety or efficacy of an experimental vaccine. Circulating anti-pathogen antibodies can be detected, and antibody encoding or 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, lymph nodes, tonsils or adenoids, bone marrow aspirates or biopsies, tissue biopsies from mucosal organs like lung or GI tract, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal or immune human 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). HMMA2.5 cells or MFP-2 cells are particularly useful examples of such cells. 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. In some cases, transformation of human B cells with Epstein Barr virus (EBV) as an initial step increases the size of the B cells, enhancing fusion with the relatively large-sized myeloma cells. Transformation efficiency by EBV is enhanced by using CpG and a Chk2 inhibitor drug in the transforming medium. Alternatively, human B cells can be activated by co-culture with transfected cell lines expressing CD40 Ligand (CD154) in medium containing additional soluble factors, such as IL-21 and human B cell Activating Factor (BAFF), a Type II member of the TNF superfamily. 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) and there are processes for better efficiency (Yu et al., 2008). Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10^-6 to 1 x 10^-8, but with optimized procedures one can achieve fusion efficiencies close to 1 in 200 (Yu et al., 2008). However, relatively low efficiency of fusion 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 medium. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the medium is supplemented with hypoxanthine. Ouabain is added if the B cell source is an 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 monoclonal antibodies. Single B cells labelled with the antigen of interest can be sorted physically using paramagnetic bead selection or flow cytometric sorting, then RNA can be isolated from the single cells and antibody genes amplified by RT-PCR. Alternatively, antigen- specific bulk sorted populations of cells can be segregated into microvesicles and the matched heavy and light chain variable genes recovered from single cells using physical linkage of heavy and light chain amplicons, or common barcoding of heavy and light chain genes from a vesicle. Matched heavy and light chain genes form single cells also can be obtained from populations of antigen specific B cells by treating cells with cell-penetrating nanoparticles bearing RT-PCR primers and barcodes for marking transcripts with one barcode per cell. The antibody variable genes also can be isolated by RNA extraction of a 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. For example, the epitope to which a given antibody bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids located within the antigen molecule (e.g., a linear epitope in a domain). Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within the antigen molecule (e.g., a conformational epitope).

Various techniques known to persons of ordinary skill in the art can be used to determine whether an antibody“interacts with one or more amino acids” within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays, such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Cross-blocking can be measured in various binding assays such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis, high-resolution electron microscopy techniques using single particle reconstruction, cryoEM, or tomography, crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem.73: 256A-265A. When the antibody neutralizes Hepatitis C virus, antibody escape mutant variant organisms can be isolated by propagating Hepatitis C virus in vitro or in animal models in the presence of high concentrations of the antibody. Sequence analysis of the Hepatitis C virus gene encoding the antigen targeted by the antibody reveals the mutation(s) conferring antibody escape, indicating residues in the epitope or that affect the structure of the epitope allosterically.

The term“epitope” refers to a site on an antigen to which B and/or T cells respond. B- cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see US 2004/0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the antibodies of the disclosure into groups of antibodies binding different epitopes.

The present disclosure includes antibodies that may bind to the same epitope, or a portion of the epitope. Likewise, the present disclosure also includes antibodies that compete for binding to a target or a fragment thereof with any of the specific exemplary antibodies described herein. One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference, the reference antibody is allowed to bind to target under saturating conditions. Next, the ability of a test antibody to bind to the target molecule is assessed. If the test antibody is able to bind to the target molecule following saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody is not able to bind to the target molecule following saturation binding with the reference antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference antibody.

To determine if an antibody competes for binding with a reference anti-Hepatitis C virus antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to the Hepatitis C virus antigen under saturating conditions followed by assessment of binding of the test antibody to the Hepatitis C virus antigen. In a second orientation, the test antibody is allowed to bind to the Hepatitis C virus antigen under saturating conditions followed by assessment of binding of the reference antibody to the Hepatitis C virus antigen. If, in both orientations, only the first (saturating) antibody is capable of binding to the Hepatitis C virus antigen, then it is concluded that the test antibody and the reference antibody compete for binding to the Hepatitis C virus antigen. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. Structural studies with EM or crystallography also can demonstrate whether or not two antibodies that compete for binding recognize the same epitope.

In another aspect, there are provided monoclonal antibodies having clone-paired CDRs from the heavy and light chains as illustrated in Tables 3 and 4, respectively. Such antibodies may be produced by the clones discussed below in the Examples section using methods described herein.

In another 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.

When comparing polynucleotide and polypeptide sequences, two sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins-- Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol.5, Suppl.3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol.183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11- 17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection. One particular example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res.25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The rearranged nature of an antibody sequence and the variable length of each gene requires multiple rounds of BLAST searches for a single antibody sequence. Also, manual assembly of different genes is difficult and error-prone. The sequence analysis tool IgBLAST (world-wide-web at ncbi.nlm.nih.gov/igblast/) identifies matches to the germline V, D and J genes, details at rearrangement junctions, the delineation of Ig V domain framework regions and complementarity determining regions. IgBLAST can analyze nucleotide or protein sequences and can process sequences in batches and allows searches against the germline gene databases and other sequence databases simultaneously to minimize the chance of missing possibly the best matching germline V gene.

In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparison of both strands.

For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. In one approach, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

Yet another way of defining an antibody is as a“derivative” of any of the below- described antibodies and their antigen-binding fragments. The term“derivative” refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen but which comprises, one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to a“parental” (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or non- naturally occurring amino acid residues. The term“derivative” encompasses, for example, as variants having altered CH1, hinge, CH2, CH3 or CH4 regions, so as to form, for example antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics. The term“derivative” additionally encompasses non-amino acid modifications, for example, amino acids that may be glycosylated (e.g., have altered mannose, 2-N- acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5- glycolneuraminic acid, etc. content), acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytic cleavage, linked to a cellular ligand or other protein, etc. In some embodiments, the altered carbohydrate modifications modulate one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In a specific embodiment, the altered carbohydrate modifications enhance antibody mediated effector function relative to the antibody lacking the carbohydrate modification. Carbohydrate modifications that lead to altered antibody mediated effector function are well known in the art (for example, see Shields, R. L. et al. (2002) J. Biol. Chem.277(30): 26733-26740; Davies J. et al. (2001) Biotechnology & Bioengineering 74(4): 288-294). Methods of altering carbohydrate contents are known to those skilled in the art, see, e.g., Wallick, S. C. et al. (1988) J. Exp. Med.168(3): 1099-1109; Tao, M. H. et al. (1989) J. Immunol. 143(8): 2595-2601; Routledge, E. G. et al. (1995) Transplantation 60(8):847-53; Elliott, S. et al. (2003) Nature Biotechnol.21:414-21; Shields, R. L. et al. (2002) J. Biol. Chem.277(30): 26733-26740).

A derivative antibody or antibody fragment can be generated with an engineered sequence or glycosylation state to confer preferred levels of activity in antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody- dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) functions as measured by bead-based or cell-based assays or in vivo studies in animal models.

A derivative antibody or antibody fragment may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In one embodiment, an antibody derivative will possess a similar or identical function as the parental antibody. In another embodiment, an antibody derivative will exhibit an altered activity relative to the parental antibody. For example, a derivative antibody (or fragment thereof) can bind to its epitope more tightly or be more resistant to proteolysis than the parental antibody. 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. 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. The following is a general discussion of relevant goals 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 can be generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 (e.g., Freestyle) cells or CHO cells, and antibodies can be collected and purified from the 293 or CHO cell supernatant. Other appropriate host cells systems include bacteria, such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco, with or without engineering for human-like glycans), algae, or in a variety of non-human transgenic contexts, such as mice, rats, goats or cows.

Expression of nucleic acids encoding antibodies, both for the purpose of subsequent antibody purification, and for immunization of a host, is also contemplated. Antibody coding sequences can be RNA, such as native RNA or modified RNA. Modified RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNAs, thereby facilitating expression of therapeutically important proteins. For instance, N1-methyl-pseudouridine (N1mY) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. In addition to turning off the immune/eIF2a phosphorylation-dependent inhibition of translation, incorporated N1mY nucleotides dramatically alter the dynamics of the translation process by increasing ribosome pausing and density on the mRNA. Increased ribosome loading of modified mRNAs renders them more permissive for initiation by favoring either ribosome recycling on the same mRNA or de novo ribosome recruitment. Such modifications could be used to enhance antibody expression in vivo following inoculation with RNA. The RNA, whether native or modified, may be delivered as naked RNA or in a delivery vehicle, such as a lipid nanoparticle.

Alternatively, DNA encoding the antibody may be employed for the same purposes. The DNA is included in an expression cassette comprising a promoter active in the host cell for which it is designed. The expression cassette is advantageously included in a replicable vector, such as a conventional plasmid or minivector. Vectors include viral vectors, such as poxviruses, adenoviruses, herpesviruses, adeno-associated viruses, and lentiviruses are contemplated. Replicons encoding antibody genes such as alphavirus replicons based on VEE virus or Sindbis virus are also contemplated. Delivery of such vectors can be performed by needle through intramuscular, subcutaneous, or intradermal routes, or by transcutaneous electroporation when in vivo expression is desired.

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

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

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

It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101 (incorporated herein by reference) states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 ± 1), glutamate (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (- 3.4), phenylalanine (-2.5), and tyrosine (-2.3). It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

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

The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG₁ can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.

Alternatively or additionally, it may be useful to combine amino acid modifications with one or more further amino acid modifications that alter C1q binding and/or the complement dependent cytotoxicity (CDC) function of the Fc region of an IL-23p19 binding molecule. The binding polypeptide of particular interest may be one that binds to C1q and displays complement dependent cytotoxicity. Polypeptides with pre-existing C1q binding activity, optionally further having the ability to mediate CDC may be modified such that one or both of these activities are enhanced. Amino acid modifications that alter C1q and/or modify its complement dependent cytotoxicity function are described, for example, in WO/0042072, which is hereby incorporated by reference.

One can design an Fc region of an antibody with altered effector function, e.g., by modifying C1q binding and/or FcgR binding and thereby changing CDC activity and/or ADCC activity.“Effector functions” are responsible for activating or diminishing a biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.). For example, one can generate a variant Fc region of an antibody with improved C1q binding and improved FcgRIII binding (e.g., having both improved ADCC activity and improved CDC activity). Alternatively, if it is desired that effector function be reduced or ablated, a variant Fc region can be engineered with reduced CDC activity and/or reduced ADCC activity. In other embodiments, only one of these activities may be increased, and, optionally, also the other activity reduced (e.g., to generate an Fc region variant with improved ADCC activity, but reduced CDC activity and vice versa).

FcRn binding. Fc mutations can also be introduced and engineered to alter their interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties. A collection of human Fc variants with improved binding to the FcRn have been described. High resolution mapping of the binding site on human IgG1 for FcgRI, FcgRII, FcgRIII, and FcRn and design of IgG1 variants with improved binding to the FcgR (Shields et al., 2001, J. Biol. Chem.276:6591-6604). A number of methods are known that can result in increased half- life (Kuo and Aveson, (2011)), including amino acid modifications may be generated through techniques including alanine scanning mutagenesis, random mutagenesis and screening to assess the binding to the neonatal Fc receptor (FcRn) and/or the in vivo behavior. Computational strategies followed by mutagenesis may also be used to select one of amino acid mutations to mutate.

The present disclosure therefore provides a variant of an antigen binding protein with optimized binding to FcRn. In a particular embodiment, the said variant of an antigen binding protein comprises at least one amino acid modification in the Fc region of said antigen binding protein, wherein said modification is selected from the group consisting of 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447 of the Fc region as compared to said parent polypeptide, wherein the numbering of the amino acids in the Fc region is that of the EU index in Kabat. In a further aspect of the disclosure the modifications are M252Y/S254T/T256E.

Additionally, various publications describe methods for obtaining physiologically active molecules whose half-lives are modified, see for example Kontermann (2009) either by introducing an FcRn-binding polypeptide into the molecules or by fusing the molecules with antibodies whose FcRn-binding affinities are preserved but affinities for other Fc receptors have been greatly reduced or fusing with FcRn binding domains of antibodies.

Derivatized antibodies may be used to alter the half-lives (e.g., serum half-lives) of parental antibodies in a mammal, particularly a human. Such alterations may result in a half- life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half- lives of the antibodies of the present disclosure or fragments thereof in a mammal, preferably a human, results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus reduces the frequency of the administration of said antibodies or antibody fragments and/or reduces the concentration of said antibodies or antibody fragments to be administered. Antibodies or fragments thereof having increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies or fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor.

Beltramello et al. (2010) previously reported the modification of neutralizing mAbs, due to their tendency to enhance dengue virus infection, by generating in which leucine residues at positions 1.3 and 1.2 of CH2 domain (according to the IMGT unique numbering for C-domain) were substituted with alanine residues. This modification, also known as“LALA” mutation, abolishes antibody binding to FcgRI, FcgRII and FcgRIIIa, as described by Hessell et al. (2007). The variant and unmodified recombinant mAbs were compared for their capacity to neutralize and enhance infection by the four dengue virus serotypes. LALA variants retained the same neutralizing activity as unmodified mAbs, but were completely devoid of enhancing activity. LALA mutations of this nature are therefore contemplated in the context of the presently disclosed antibodies.

Altered Glycosylation. A particular embodiment of the present disclosure is an isolated monoclonal antibody, or antigen binding fragment thereof, containing a substantially homogeneous glycan without sialic acid, galactose, or fucose. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, both of which may be attached to heavy chain or light chain constant regions respectively. The aforementioned substantially homogeneous glycan may be covalently attached to the heavy chain constant region. Another embodiment of the present disclosure comprises a mAb with a novel Fc glycosylation pattern. The isolated monoclonal antibody, or antigen binding fragment thereof, is present in a substantially homogenous composition represented by the GNGN or G1/G2 glycoform. Fc glycosylation plays a significant role in anti-viral and anti-cancer properties of therapeutic mAbs. The disclosure is in line with a recent study that shows increased anti- lentivirus cell-mediated viral inhibition of a fucose free anti-HIV mAb in vitro. This embodiment of the present disclosure with homogenous glycans lacking a core fucose, showed increased protection against specific viruses by a factor greater than two-fold. Elimination of core fucose dramatically improves the ADCC activity of mAbs mediated by natural killer (NK) cells but appears to have the opposite effect on the ADCC activity of polymorphonuclear cells (PMNs).

The isolated monoclonal antibody, or antigen binding fragment thereof, comprising a substantially homogenous composition represented by the GNGN or G1/G2 glycoform exhibits increased binding affinity for Fc gamma RI and Fc gamma RIII compared to the same antibody without the substantially homogeneous GNGN glycoform and with G0, G1F, G2F, GNF, GNGNF or GNGNFX containing glycoforms. In one embodiment of the present disclosure, the antibody dissociates from Fc gamma RI with a Kd of 1 x 10^-8 M or less and from Fc gamma RIII with a Kd of 1 x 10^-7 M or less.

Glycosylation of an Fc region is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. O- linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used. The recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequences are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. Thus, the presence of either of these peptide sequences in a polypeptide creates a potential glycosylation site.

The glycosylation pattern may be altered, for example, by deleting one or more glycosylation site(s) found in the polypeptide, and/or adding one or more glycosylation site(s) that are not present in the polypeptide. Addition of glycosylation sites to the Fc region of an antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). An exemplary glycosylation variant has an amino acid substitution of residue Asn 297 of the heavy chain. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original polypeptide (for O-linked glycosylation sites). Additionally, a change of Asn 297 to Ala can remove one of the glycosylation sites.

In certain embodiments, the antibody is expressed in cells that express beta (1,4)-N- acetylglucosaminyltransferase III (GnT III), such that GnT III adds GlcNAc to the IL-23p19 antibody. Methods for producing antibodies in such a fashion are provided in WO/9954342, WO/03011878, patent publication 20030003097A1, and Umana et al., Nature Biotechnology, 17:176-180, February 1999. Cell lines can be altered to enhance or reduce or eliminate certain post-translational modifications. such as glycosylation, using genome editing technology such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). For example, CRISPR technology can be used to eliminate genes encoding glycosylating enzymes in 293 or CHO cells used to express recombinant monoclonal antibodies.

Elimination of monoclonal antibody protein sequence liabilities. It is possible to engineer the antibody variable gene sequences obtained from human B cells to enhance their manufacturability and safety. Potential protein sequence liabilities can be identified by searching for sequence motifs associated with sites containing:

Such motifs can be eliminated by altering the synthetic gene for the cDNA encoding recombinant antibodies.

Protein engineering efforts in the field of development of therapeutic antibodies clearly reveal that certain sequences or residues are associated with solubility differences (Fernandez- Escamilla et al., Nature Biotech., 22 (10), 1302-1306, 2004; Chennamsetty et al., PNAS, 106 (29), 11937-11942, 2009; Voynov et al., Biocon. Chem., 21 (2), 385-392, 2010) Evidence from solubility-altering mutations in the literature indicate that some hydrophilic residues such as aspartic acid, glutamic acid, and serine contribute significantly more favorably to protein solubility than other hydrophilic residues, such as asparagine, glutamine, threonine, lysine, and arginine.

Stability. Antibodies can be engineered for enhanced biophysical properties. One can use elevated temperature to unfold antibodies to determine relative stability, using average apparent melting temperatures. Differential Scanning Calorimetry (DSC) measures the heat capacity, C_p, of a molecule (the heat required to warm it, per degree) as a function of temperature. One can use DSC to study the thermal stability of antibodies. DSC data for mAbs is particularly interesting because it sometimes resolves the unfolding of individual domains within the mAb structure, producing up to three peaks in the thermogram (from unfolding of the Fab, C_H2, and C_H3 domains). Typically unfolding of the Fab domain produces the strongest peak. The DSC profiles and relative stability of the Fc portion show characteristic differences for the human IgG₁, IgG₂, IgG₃, and IgG₄ subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun.355, 751-757, 2007). One also can determine average apparent melting temperature using circular dichroism (CD), performed with a CD spectrometer. Far-UV CD spectra will be measured for antibodies in the range of 200 to 260 nm at increments of 0.5 nm. The final spectra can be determined as averages of 20 accumulations. Residue ellipticity values can be calculated after background subtraction. Thermal unfolding of antibodies (0.1 mg/mL) can be monitored at 235 nm from 25-95 °C and a heating rate of 1 °C/min. One can use dynamic light scattering (DLS) to assess for propensity for aggregation. DLS is used to characterize size of various particles including proteins. If the system is not disperse in size, the mean effective diameter of the particles can be determined. This measurement depends on the size of the particle core, the size of surface structures, and particle concentration. Since DLS essentially measures fluctuations in scattered light intensity due to particles, the diffusion coefficient of the particles can be determined. DLS software in commercial DLA instruments displays the particle population at different diameters. Stability studies can be done conveniently using DLS. DLS measurements of a sample can show whether the particles aggregate over time or with temperature variation by determining whether the hydrodynamic radius of the particle increases. If particles aggregate, one can see a larger population of particles with a larger radius. Stability depending on temperature can be analyzed by controlling the temperature in situ. Capillary electrophoresis (CE) techniques include proven methodologies for determining features of antibody stability. One can use an iCE approach to resolve antibody protein charge variants due to deamidation, C-terminal lysines, sialylation, oxidation, glycosylation, and any other change to the protein that can result in a change in pI of the protein. Each of the expressed antibody proteins can be evaluated by high throughput, free solution isoelectric focusing (IEF) in a capillary column (cIEF), using a Protein Simple Maurice instrument. Whole-column UV absorption detection can be performed every 30 seconds for real time monitoring of molecules focusing at the isoelectric points (pIs). This approach combines the high resolution of traditional gel IEF with the advantages of quantitation and automation found in column-based separations while eliminating the need for a mobilization step. The technique yields reproducible, quantitative analysis of identity, purity, and heterogeneity profiles for the expressed antibodies. The results identify charge heterogeneity and molecular sizing on the antibodies, with both absorbance and native fluorescence detection modes and with sensitivity of detection down to 0.7 µg/mL.

Solubility. One can determine the intrinsic solubility score of antibody sequences. The intrinsic solubility scores can be calculated using CamSol Intrinsic (Sormanni et al., J Mol Biol 427, 478-490). The amino acid sequences for residues 95-102 (Kabat numbering) in HCDR3 of each antibody fragment such as a scFv can be evaluated via the online program to calculate the solubility scores. One also can determine solubility using laboratory techniques. Various techniques exist, including addition of lyophilized protein to a solution until the solution becomes saturated and the solubility limit is reached, or concentration by ultrafiltration in a microconcentrator with a suitable molecular weight cut-off. The most straightforward method is induction of amorphous precipitation, which measures protein solubility using a method involving protein precipitation using ammonium sulfate (Trevino et al., J Mol Biol, 366: 449- 460, 2007). Ammonium sulfate precipitation gives quick and accurate information on relative solubility values. Ammonium sulfate precipitation produces precipitated solutions with well- defined aqueous and solid phases and requires relatively small amounts of protein. Solubility measurements performed using induction of amorphous precipitation by ammonium sulfate also can be done easily at different pH values. Protein solubility is highly pH dependent, and pH is considered the most important extrinsic factor that affects solubility.

Autoreactivity. Generally, it is thought that autoreactive clones should be eliminated during ontogeny by negative selection, however it has become clear that many human naturally occurring antibodies with autoreactive properties persist in adult mature repertoires, and the autoreactivity may enhance the antiviral function of many antibodies to pathogens. It has been noted that HCDR3 loops in antibodies during early B cell development are often rich in positive charge and exhibit autoreactive patterns (Wardemann et al., Science 301, 1374-1377, 2003). One can test a given antibody for autoreactivity by assessing the level of binding to human origin cells in microscopy (using adherent HeLa or HEp-2 epithelial cells) and flow cytometric cell surface staining (using suspension Jurkat T cells and 293S human embryonic kidney cells). Autoreactivity also can be surveyed using assessment of binding to tissues in tissue arrays.

Preferred residues (“Human Likeness”). B cell repertoire deep sequencing of human B cells from blood donors is being performed on a wide scale in many recent studies. Sequence information about a significant portion of the human antibody repertoire facilitates statistical assessment of antibody sequence features common in healthy humans. With knowledge about the antibody sequence features in a human recombined antibody variable gene reference database, the position specific degree of“Human Likeness” (HL) of an antibody sequence can be estimated. HL has been shown to be useful for the development of antibodies in clinical use, like therapeutic antibodies or antibodies as vaccines. The goal is to increase the human likeness of antibodies to reduce potential adverse effects and anti-antibody immune responses that will lead to significantly decreased efficacy of the antibody drug or can induce serious health implications. One can assess antibody characteristics of the combined antibody repertoire of three healthy human blood donors of about 400 million sequences in total and created a novel “relative Human Likeness” (rHL) score that focuses on the hypervariable region of the antibody. The rHL score allows one to easily distinguish between human (positive score) and non-human sequences (negative score). Antibodies can be engineered to eliminate residues that are not common in human repertoires. 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 or B cell. 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 alanine, serine and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single- chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approx. 5 × 10⁶ different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers.

The recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.

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

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

An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent). It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.

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

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

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

U.S. Patent 4,680,338 describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug. U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques. E. Multispecific Antibodies

In certain embodiments, antibodies of the present disclosure are bispecific or multispecific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a single antigen. Other such antibodies may combine a first antigen binding site with a binding site for a second antigen. Alternatively, an anti-pathogen arm may be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcgR), such as FcgRI (CD64), FcgRII (CD32) and Fc gamma RIII (CD16), so as to focus and localize cellular defense mechanisms to the infected cell. Bispecific antibodies may also be used to localize cytotoxic agents to infected cells. These antibodies possess a pathogen-binding arm and an arm that binds the cytotoxic agent (e.g., saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab ^).sub.2 bispecific antibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti- Fc gamma RIII antibody and U.S. Pat. No.5,837,234 discloses a bispecific anti-ErbB2/anti-Fc gamma RI antibody. A bispecific anti-ErbB2/Fc alpha antibody is shown in WO98/02463. U.S. Pat. No.5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

According to a different approach, antibody variable regions with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, C_H2, and C_H3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant effect on the yield of the desired chain combination.

In a particular embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Techniques exist that facilitate the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a humanized bispecific antibody F(ab')₂ molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described (Merchant et al., Nat. Biotechnol.16, 677–681 (1998). doi:10.1038/nbt0798-677pmid:9661204). For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol., 148(5):1547-1553, 1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a V_H connected to a V_L by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and V_L domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

In a particular embodiment, a bispecific or multispecific antibody may be formed as a DOCK-AND-LOCK™ (DNL™) complex (see, e.g., U.S. Pat. Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examples section of each of which is incorporated herein by reference.) Generally, the technique takes advantage of the specific and high-affinity binding interactions that occur between a dimerization and docking domain (DDD) sequence of the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequence derived from any of a variety of AKAP proteins (Baillie et al., FEBS Letters.2005; 579: 3264; Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). The DDD and AD peptides may be attached to any protein, peptide or other molecule. Because the DDD sequences spontaneously dimerize and bind to the AD sequence, the technique allows the formation of complexes between any selected molecules that may be attached to DDD or AD sequences.

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147: 60, 1991; Xu et al., Science, 358(6359):85-90, 2017). A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present disclosure can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable regions. For instance, the polypeptide chain(s) may comprise VD1-(X1)_n-VD2-(X2)_n-Fc, wherein VD1 is a first variable region, VD2 is a second variable region, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH- CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable region polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable region polypeptides. The light chain variable region polypeptides contemplated here comprise a light chain variable region and, optionally, further comprise a C_L domain.

Charge modifications are particularly useful in the context of a multispecific antibody, where amino acid substitutions in Fab molecules result in reducing the mispairing of light chains with non-matching heavy chains (Bence-Jones-type side products), which can occur in the production of Fab-based bi-/multispecific antigen binding molecules with a VH/VL exchange in one (or more, in case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see also PCT publication no. WO 2015/150447, particularly the examples therein, incorporated herein by reference in its entirety).

Accordingly, in particular embodiments, an antibody comprised in the therapeutic agent comprises

(a) a first Fab molecule which specifically binds to a first antigen

(b) a second Fab molecule which specifically binds to a second antigen, and wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other,

wherein the first antigen is an activating T cell antigen and the second antigen is a target cell antigen, or the first antigen is a target cell antigen and the second antigen is an activating T cell antigen; and

wherein

i) in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index); or ii) in the constant domain CL of the second Fab molecule under b) the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CH1 of the second Fab molecule under b) the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index).

The antibody may not comprise both modifications mentioned under i) and ii). The constant domains CL and CH1 of the second Fab molecule are not replaced by each other (i.e., remain unexchanged).

In another embodiment of the antibody, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

In a particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

In a more particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

In an even more particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index). F. Chimeric Antigen Receptors

Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs)) are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell, with transfer of their coding sequence facilitated by retroviral vectors. In this way, a large number of target-specific T cells can be generated for adoptive cell transfer. Phase I clinical studies of this approach show efficacy.

The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta transmembrane and endodomain. Such molecules result in the transmission of a zeta signal in response to recognition by the scFv of its target. An example of such a construct is 14g2a-Zeta, which is a fusion of a scFv derived from hybridoma 14g2a (which recognizes disialoganglioside GD2). When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they recognize and kill target cells that express GD2 (e.g., neuroblastoma cells). To target malignant B cells, investigators have redirected the specificity of T cells using a chimeric immunoreceptor specific for the B-lineage molecule, CD19.

The variable portions of an immunoglobulin heavy and light chain are fused by a flexible linker to form a scFv. This scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression (this is cleaved). A flexible spacer allows to the scFv to orient in different directions to enable antigen binding. The transmembrane domain is a typical hydrophobic alpha helix usually derived from the original molecule of the signaling endodomain which protrudes into the cell and transmits the desired signal.

Type I proteins are in fact two protein domains linked by a transmembrane alpha helix in between. The cell membrane lipid bilayer, through which the transmembrane domain passes, acts to isolate the inside portion (endodomain) from the external portion (ectodomain). It is not so surprising that attaching an ectodomain from one protein to an endodomain of another protein results in a molecule that combines the recognition of the former to the signal of the latter.

Ectodomain. A signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and anchored in the cell membrane. Any eukaryotic signal peptide sequence usually works fine. Generally, the signal peptide natively attached to the amino-terminal most component is used (e.g., in a scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used

The antigen recognition domain is usually an scFv. There are however many alternatives. An antigen recognition domain from native T-cell receptor (TCR) alpha and beta single chains have been described, as have simple ectodomains (e.g., CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor). In fact almost anything that binds a given target with high affinity can be used as an antigen recognition region.

A spacer region links the antigen binding domain to the transmembrane domain. It should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition. The simplest form is the hinge region from IgG1. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. For most scFv based constructs, the IgG1 hinge suffices. However, the best spacer often has to be determined empirically.

Transmembrane domain. The transmembrane domain is a hydrophobic alpha helix that spans the membrane. Generally, the transmembrane domain from the most membrane proximal component of the endodomain is used. Interestingly, using the CD3-zeta transmembrane domain may result in incorporation of the artificial TCR into the native TCR a factor that is dependent on the presence of the native CD3-zeta transmembrane charged aspartic acid residue. Different transmembrane domains result in different receptor stability. The CD28 transmembrane domain results in a brightly expressed, stable receptor. Endodomain. This is the "business-end" of the receptor. After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used endodomain component is CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed.

"First-generation" CARs typically had the intracellular domain from the CD3 x- chain, which is the primary transmitter of signals from endogenous TCRs. "Second-generation" CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells. More recent, "third-generation" CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further augment potency. G. ADCs

Antibody Drug Conjugates or ADCs are a new class of highly potent biopharmaceutical drugs designed as a targeted therapy for the treatment of people with infectious disease. ADCs are complex molecules composed of an antibody (a whole mAb or an antibody fragment such as a single-chain variable fragment, or scFv) linked, via a stable chemical linker with labile bonds, to a biological active cytotoxic/anti-viral payload or drug. Antibody Drug Conjugates are examples of bioconjugates and immunoconjugates.

By combining the unique targeting capabilities of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, antibody-drug conjugates allow sensitive discrimination between healthy and diseased tissue. This means that, in contrast to traditional systemic approaches, antibody-drug conjugates target and attack the infected cell so that healthy cells are less severely affected.

In the development ADC-based anti-tumor therapies, an anticancer drug (e.g., a cell toxin or cytotoxin) is coupled to an antibody that specifically targets a certain cell marker (e.g., a protein that, ideally, is only to be found in or on infected cells). Antibodies track these proteins down in the body and attach themselves to the surface of cancer cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs or internalizes the antibody together with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released and kills the cell or impairs viral replication. Due to this targeting, ideally the drug has lower side effects and gives a wider therapeutic window than other agents. A stable link between the antibody and cytotoxic/anti-viral agent is a crucial aspect of an ADC. Linkers are based on chemical motifs including disulfides, hydrazones or peptides (cleavable), or thioethers (noncleavable) and control the distribution and delivery of the cytotoxic agent to the target cell. Cleavable and noncleavable types of linkers have been proven to be safe in preclinical and clinical trials. Brentuximab vedotin includes an enzyme-sensitive cleavable linker that delivers the potent and highly toxic antimicrotubule agent Monomethyl auristatin E or MMAE, a synthetic antineoplastic agent, to human specific CD30-positive malignant cells. Because of its high toxicity MMAE, which inhibits cell division by blocking the polymerization of tubulin, cannot be used as a single-agent chemotherapeutic drug. However, the combination of MMAE linked to an anti-CD30 monoclonal antibody (cAC10, a cell membrane protein of the tumor necrosis factor or TNF receptor) proved to be stable in extracellular fluid, cleavable by cathepsin and safe for therapy. Trastuzumab emtansine, the other approved ADC, is a combination of the microtubule-formation inhibitor mertansine (DM- 1), a derivative of the Maytansine, and antibody trastuzumab (Herceptin®/Genentech/Roche) attached by a stable, non-cleavable linker.

The availability of better and more stable linkers has changed the function of the chemical bond. The type of linker, cleavable or noncleavable, lends specific properties to the cytotoxic (anti-cancer) drug. For example, a non-cleavable linker keeps the drug within the cell. As a result, the entire antibody, linker and cytotoxic agent enter the targeted cancer cell where the antibody is degraded to the level of an amino acid. The resulting complex– amino acid, linker and cytotoxic agent– now becomes the active drug. In contrast, cleavable linkers are catalyzed by enzymes in the host cell where it releases the cytotoxic agent.

Another type of cleavable linker, currently in development, adds an extra molecule between the cytotoxic/anti-viral drug and the cleavage site. This linker technology allows researchers to create ADCs with more flexibility without worrying about changing cleavage kinetics. Researchers are also developing a new method of peptide cleavage based on Edman degradation, a method of sequencing amino acids in a peptide. Future direction in the development of ADCs also include the development of site-specific conjugation (TDCs) to further improve stability and therapeutic index and a emitting immunoconjugates and antibody-conjugated nanoparticles. H. BiTES

Bi-specific T-cell engagers (BiTEs) are a class of artificial bispecific monoclonal antibodies that are investigated for the use as anti-cancer drugs. They direct a host's immune system, more specifically the T cells' cytotoxic activity, against infected cells. BiTE is a registered trademark of Micromet AG.

BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, and the other to an infected cell via a specific molecule.

Like other bispecific antibodies, and unlike ordinary monoclonal antibodies, BiTEs form a link between T cells and target cells. This causes T cells to exert cytotoxic/anti-viral activity on infected cells by producing proteins like perforin and granzymes, independently of the presence of MHC I or co-stimulatory molecules. These proteins enter infected cells and initiate the cell's apoptosis. This action mimics physiological processes observed during T cell attacks against infected cells. I. 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 et al., 1997).

By virtue of their ability to enter cells, intrabodies have additional uses that other types of antibodies may not achieve. In the case of the present antibodies, the ability to interact with the MUC1 cytoplasmic domain in a living cell may interfere with functions associated with the MUC1 CD, such as signaling functions (binding to other molecules) or oligomer formation. In particular, it is contemplated that such antibodies can be used to inhibit MUC1 dimer formation. J. 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 Hepatitis C virus Infection

A. Formulation and Administration

The present disclosure provides pharmaceutical compositions comprising anti- Hepatitis C 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, intra-rectal, vaginal, topical 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 Hepatitis C 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, by nebulizer, or via intrarectal or vaginal delivery. Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

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

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

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

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or fragments thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. By“antibody having increased/reduced antibody dependent cell-mediated cytotoxicity (ADCC)” is meant an antibody having increased/reduced ADCC as determined by any suitable method known to those of ordinary skill in the art.

As used herein, the term“increased/reduced ADCC” is defined as either an increase/reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or a reduction/increase in the concentration of antibody, in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The increase/reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example, the increase in ADCC mediated by an antibody produced by host cells engineered to have an altered pattern of glycosylation (e.g., to express the glycosyltransferase, GnTIII, or other glycosyltransferases) by the methods described herein, is relative to the ADCC mediated by the same antibody produced by the same type of non-engineered host cells. C. CDC

Complement-dependent cytotoxicity (CDC) is a function of the complement system. It is the processes in the immune system that kill pathogens by damaging their membranes without the involvement of antibodies or cells of the immune system. There are three main processes. All three insert one or more membrane attack complexes (MAC) into the pathogen which cause lethal colloid-osmotic swelling, i.e., CDC. It is one of the mechanisms by which antibodies or antibody fragments have an anti-viral effect. IV. Antibody Conjugates

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

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

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

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

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

Another type of antibody conjugate contemplated in the present disclosure are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.

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

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

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

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

In still further embodiments, the present disclosure concerns immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting Hepatitis C 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 antigens in viruses. Alternatively, the methods may be used to screen various antibodies for appropriate/desired reactivity profiles.

Other immunodetection methods include specific assays for determining the presence of Hepatitis C virus in a subject. A wide variety of assay formats are contemplated, but specifically those that would be used to detect Hepatitis C virus in a fluid obtained from a subject, such as saliva, blood, plasma, sputum, semen or urine. In particular, semen has been demonstrated as a viable sample for detecting Hepatitis C virus (Purpura et al., 2016; Mansuy et al., 2016; Barzon et al., 2016; Gornet et al., 2016; Duffy et al., 2009; CDC, 2016; Halfon et al., 2010; Elder et al.2005). The assays may be advantageously formatted for non-healthcare (home) use, including lateral flow assays (see below) analogous to home pregnancy tests. These assays may be packaged in the form of a kit with appropriate reagents and instructions to permit use by the subject of a family member.

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 Hepatitis C virus antibodies directed to specific parasite 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 Hepatitis C 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 Hepatitis C 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 Hepatitis C virus or antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the Hepatitis C 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 Hepatitis C 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 Hepatitis C virus or its antigens and contact the sample with an antibody that binds Hepatitis C 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 Hepatitis C virus or Hepatitis C 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 Hepatitis C virus or antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

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

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

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

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

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

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

In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the Hepatitis C virus or Hepatitis C 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-Hepatitis C 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-Hepatitis C 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 Hepatitis C virus or Hepatitis C virus antigen are immobilized onto the well surface and then contacted with the anti- Hepatitis C virus antibodies of the disclosure. After binding and washing to remove non- specifically bound immune complexes, the bound anti-Hepatitis C virus antibodies are detected. Where the initial anti-Hepatitis C 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-Hepatitis C 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 Hepatitis C 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 inventor proposes the use of labeled Hepatitis C virus monoclonal antibodies to determine the amount of Hepatitis C virus antibodies in a sample. The basic format would include contacting a known amount of Hepatitis C virus monoclonal antibody (linked to a detectable label) with Hepatitis C virus antigen or particle. The Hepatitis C virus antigen or organism is preferably attached to a support. After binding of the labeled monoclonal antibody to the support, the sample is added and incubated under conditions permitting any unlabeled antibody in the sample to compete with, and hence displace, the labeled monoclonal antibody. By measuring either the lost label or the label remaining (and subtracting that from the original amount of bound label), one can determine how much non-labeled antibody is bound to the support, and thus how much antibody was present in the sample. B. Western Blot

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

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

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

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

Lateral flow assays, also known as lateral flow immunochromatographic assays, are simple devices intended to detect the presence (or absence) of a target analyte in sample (matrix) without the need for specialized and costly equipment, though many laboratory-based applications exist that are supported by reading equipment. Typically, these tests are used as low resources medical diagnostics, either for home testing, point of care testing, or laboratory use. A widely spread and well-known application is the home pregnancy test.

The technology is based on a series of capillary beds, such as pieces of porous paper or sintered polymer. Each of these elements has the capacity to transport fluid (e.g., urine) spontaneously. The first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element (conjugate pad) in which the manufacturer has stored the so-called conjugate, a dried format of bio-active particles (see below) in a salt-sugar matrix that contains everything to guarantee an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle's surface. While the sample fluid dissolves the salt- sugar matrix, it also dissolves the particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure. In this way, the analyte binds to the particles while migrating further through the third capillary bed. This material has one or more areas (often called stripes) where a third molecule has been immobilized by the manufacturer. By the time the sample-conjugate mix reaches these strips, analyte has been bound on the particle and the third 'capture' molecule binds the complex. After a while, when more and more fluid has passed the stripes, particles accumulate and the stripe-area changes color. Typically there are at least two stripes: one (the control) that captures any particle and thereby shows that reaction conditions and technology worked fine, the second contains a specific capture molecule and only captures those particles onto which an analyte molecule has been immobilized. After passing these reaction zones, the fluid enters the final porous material – the wick– that simply acts as a waste container. Lateral Flow Tests can operate as either competitive or sandwich assays. Lateral flow assays are disclosed in U.S. Patent 6,485,982. D. Immunohistochemistry

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

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

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

In certain embodiments, the Hepatitis C virus antibody may be pre-bound to a solid support, such as a column matrix and/or well of a microtiter plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody. Further suitable immunodetection reagents for use in the present kits include the two- component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.

The kits may further comprise a suitably aliquoted composition of the Hepatitis C virus or Hepatitis C 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. F. Vaccine and Antigen Quality Control Assays

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 correct and 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.

Antibodies and fragments thereof as described in the present disclosure may also be used in a kit for monitoring the efficacy of vaccination procedures by detecting the presence of protective Hepatitis C virus antibodies. Antibodies, antibody fragment, or variants and derivatives thereof, as described in the present disclosure may also be used in a kit for monitoring vaccine manufacture with the desired immunogenicity. VI. 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 inventor 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

Study subjects. Plasma samples and PBMC were obtained from subjects in the BBAASH cohort (Cox et al., 2005).

Source of reference bNAbs. HC84.26 (Keck et al., 2012), and HC33.8 (Keck et al., 2013) were a gift of Steven Foung (Stanford University School of Medicine, Stanford, California). AR1A, AR2A, AR3A, AR3C (Law et al., 2008), AR4A, and AR5A (Giang et al., 2012) were a gift from Mansun Law (The Scripps Research Institute, La Jolla, California, USA).

HCV viral load and serology testing. HCV viral loads (International Unit (IU)/mL) were quantified using a process of RNA extraction and utilization of commercial real time reagents (Abbot HCV Real Time Assay) migrated onto a research based real time PCR platform (Roche 480 Lightcycler). HCV seropositivity was determined using the Ortho HCV Version 3.0 ELISA Test System (Ortho Clinical Diagnostics, Raritan, New Jersey).

Generation of human hybridomas secreting monoclonal antibodies (mAbs). Human hybridomas were generated as described previously (Flyak et al., 2015). In brief, cryopreserved PBMC samples from Subjects 117 and 110 were transformed with Epstein-Barr virus, CpG and additional supplements. After 7 days, cells from each well of the 384-well culture plates were expanded into four 96-well culture plates using cell culture medium containing irradiated heterologous human PBMCs and incubated for an additional four days. B cell culture supernatants were screened in enzyme-linked immunosorbent assays (ELISAs) with E1E2 cell lysates and in neutralization assays against autologous or heterologous HCV variants. Screening was performed against E1E2 variants 1a53 (autologous) and 1a154 (H77) (heterologous) E1E2 variants for Subject 117 and 1a53 (heterologous) and 1a38 (autologous) variants for Subject 110. Cells from wells with supernatants reacting with HCV antigens were fused with HMMA2.5 myeloma cells using an electrofusion technique (Yu et al., 2008). After fusion, hybridoma cell lines were cloned by limited dilutions and single-cell fluorescence- activated cell sorting and expanded in post-fusion medium as previously described (Flyak et al., 2015). HiTrap Protein G or HiTrap MabSelectSure columns were used to purify HCV- specific antibodies from filtered cell culture supernatants.

HCVpp production and neutralization assays. The panel of 19 heterologous genotype 1 HCVpp has been described previously (Osburn et al., 2014; Bailey et al., 2015). The HCVpp panels used to measure neutralizing breadth of plasma samples and mAbs were identical, except that two E1E2 clones used to screen plasma, 1b20 and 1a114, were replaced in mAb experiments by related clones 1b21 and 1a116, which gave more consistent HCVpp infectivity results. HCVpp were produced by lipofectamine-mediated transfection of HCV E1E2 and pNL4-3.Luc.R-E- plasmids into HEK293T cells as previously described (Hsu et al., 2003; Logvinoff et al., 2004). Neutralization assays were performed as described previously (Dowd et al., 2009). HCVpp were incubated for one hour with plasma at a 1:100 dilution or mAb at a final concentration of 50 or 10 µg/mL, then added in duplicate to Hep3B target cells for 5-6 hours before medium was changed. Infection was determined after 3 days by measurement of luciferase activity of cell lysates in relative light units (RLU). The majority of HCVpp used in neutralization assays produced RLU>2E6, or more than 100-fold above background produced by mock pseudoparticles lacking any E1E2. All HCVpp used in neutralization assays produced RLU of at least 200,000, which is at least 10-fold above background. Percent neutralization was calculated as (1-RLU_{test mAb}/RLU_{control IgG} )*100. Neutralization of MLV-pseudotyped particles was measured as a negative control.

HCVcc neutralization assays. Chimeric genotype 1-6 HCVcc constructs (Gottwein et al., 2009; Scheel et al., 2008) were a gift of Jens Bukh, (Copenhagen University Hospital, Copenhagen, Denmark). HCVcc neutralization assays were performed as described elsewhere (Wasilewski et al., 2016). Briefly, human hepatoma Huh7.5.1 cells (a gift of Jake Liang, NIH, Bethesda, Maryland, USA) were maintained in DMEM supplemented with 10% fetal bovine serum, 1% sodium pyruvate, and 1% l-glutamate.10,000 Huh7.5.1 cells per well were plated in flat bottom 96 well tissue culture plates and incubated overnight at 37°C. The following day, HCVcc were mixed with mAb (2.5-fold dilutions started at 50 mg/mL) then incubated at 37°C for 1 hour. Medium was removed from the cells and replaced with 50 µL of HCVcc/antibody mixture. The plates were placed in a CO2 incubator at 37°C overnight, after which the HCVcc were removed and replaced with 100 µL of Huh7.5.1 medium and incubated for 48 hours at 37°C. After 48 hours, medium was removed and cells were fixed and stained. Images were acquired and spot forming units were counted in the presence of mAb (HCVccSFUtest) or nonspecific IgG (HCV_ccSFU_control) using an AID iSpot Reader Spectrum operating AID ELISpot Reader version 7.0. Percent neutralization was calculated as 100% x [1-(HCVccSFUtest /HCV_ccSFU_control)].

HCV NS5A immunostaining. HCV NS5A immunostaining was conducted as described elsewhere (Wasilewski et al., 2016). Briefly, cells were fixed with 4% formaldehyde then stained for HCV NS5A using primary anti-NS5A antibody 9E10 (a gift of Charles Rice, The Rockefeller University, New York City, New York, USA) at a 1:10,000 dilution for 1 hour at room temperature. Cells were washed twice with PBS and stained using secondary antibody Alexa Daylight 488–conjugated goat anti-mouse IgG (Life Technologies) at a 1:500 dilution for 1 hour at room temperature. Cells were washed twice in PBS and then stored covered in 100 mL PBS at 4°C.

Shotgun mutagenesis epitope mapping. Comprehensive alanine scanning mutagenesis of an expression construct for HCV E1/E2 (genotype 1a, strain H77) changed each residue to alanine (with alanine residues changed to serine) to create a library of clones, each representing an individual point mutant, covering 552 of 555 target E1/E2 residues. Each mutation was confirmed by DNA sequencing, and clones were arrayed into 384-well plates, one mutant per well, transfected into HEK-293T cells, and allowed to express for 22 hours.

Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% (wt/vol) saponin (Sigma-Aldrich, St. Louis, MO) in PBS plus calcium and magnesium, and stained with MAbs diluted in 10% normal goat serum (NGS; Sigma), 0.1% w/v saponin, pH 9.0. Primary MAb concentrations were determined using an independent immunofluorescence titration curve against wild-type HCV E1/E2 to ensure that signals were within the linear range of detection. The cells were incubated with anti-HCV antibody for 1 hour at 20^oC, followed by a 30-minute incubation with Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Westgrove, PA) in 10% NGS. Cells were washed twice with PBS without calcium or magnesium and resuspended in Cellstripper (Cellgro, Manassas, VA) plus 0.1% BSA (Sigma-Aldrich, St. Louis, MO). Cellular fluorescence was detected using the Intellicyt high throughput flow cytometer (HTFC, Intellicyt, Albuquerque, NM). Background fluorescence was determined by fluorescence measurement of vector-transfected control cells. MAb reactivities against each mutant HCV E1/E2 clone were calculated relative to wild-type E1/E2 reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type HCV E1/E2 -transfected controls. Mutated residues within critical clones were identified as critical to the MAb epitope if they did not support reactivity of the test MAb but did support reactivity of other control anti- HCV MAbs. This counter-screen strategy facilitates the exclusion of E1/E2 mutants that are locally misfolded or that have an expression defect (Paes et al., 2009; Davidson and Doranz, 2014).

Hierarchical clustering of mAbs based on correlations between neutralization profiles. Neutralization of each of 19 HCVpp by each mAb were compared pairwise for all mAbs using spearman correlation. Spearman rho and p values then were used as input for hierarchical clustering as implemented in the “pvclust” package for R (cran.r- project.org/web/packages/pvclust/index.html) (Suzuki et al., 2006). Approximately unbiased values greater than 95 are considered strongly supported by the data. This clustering, depicted as a tree, was also used to order a matrix of correlation values produced using the“corrplot” package for R (cran.r-project.org/web/packages/corrplot/index.html) (Friendly et al., 2002).

HCV E1E2 ELISA. MAb binding to E1E2 was quantitated using an ELISA as previously described (Keck et al., 2009). Briefly, 293T cells were transfected with E1E2 expression constructs. Cell lysates were harvested at 48 h. Plates were coated with 500 ng of Galanthus nivalis lectin (Sigma) and blocked with PBS containing 0.5% Tween 20, 1% nonfat drymilk, 1% goat serum and E1E2–containing cell lysates were added. MAbs were assayed in duplicate at 5-fold serial dilutions, starting at 100 µg/mL and binding detected with HRP- conjugated anti-human IgG secondary antibody (BD-Pharmingen #555788). ELISA used for hybridoma screening followed the same protocol with the following modifications: 384-well ELISA plates were coated with Galanthus nivalis lectin (Sigma) at 5 µg/mL in Dulbecco’s PBS (DPBS). Plates were blocked with a blocking solution consisted of 10 g powdered milk, 10 mL of goat serum, 100 mL of 10× DPBS, and 0.5 mL of Tween-20 mixed to a 1 L final volume with distilled water. Autologous and heterologous E1E2 cell lysates were diluted in DPBS with 0.05% Tween-20 and added to plates. B cell culture supernatants from tissue- culture plates were transferred to ELISA plates coated with E1E2 lysates. For binding competitions ELISA, E1E2 protein-coated ELISA wells were pre-incubated with 20 µg/mL of blocking bNAbs, followed by biotinylated mAbs at 2 µg/mL, with binding of the biotinylated bNAb detected using streptavidin-horseradish peroxidase. A ratio of binding of each biotinylated bNAb in the presence of blocking bNAb divided by binding in the absence of blocking bNAb was calculated. For ELISA with denatured E1E2 protein, E1E2 was boiled for 5 min in Tris-buffered saline (TBS)-10% FCS containing 1.0% sodium dodecyl sulfate and 50 mM dithiothreitol.prior to addition to GNA-lectin-coated plates. Sequence analysis of antibody variable region genes. Total cellular RNA was extracted from clonal hybridomas that produced HCV antibodies, and RT-PCR reaction was performed using mixtures of primers designed to amplify all heavy-chain or light-chain antibody variable regions. Antibody variable gene sequence analysis was performed as previously described (Flyak et al., 2015). Heavy and light chain antibody variable region sequences were analyzed using the IMGT/V-Quest program (Brochet et al., 2008; Guidicelli et al., 2011).

HEPC3 mutagenesis and antibody expression. The genes encoding HEPC3 heavy- or light-chain variable regions were synthesized and cloned into a mammalian expression plasmid vector for full length IgG1 by GenScript. Site-directed mutagenesis to revert individual somatic mutations was performed using QuikChange Lightning Site-Directed Mutagenesis kit (Agilent). Transient expression of HEPC3 variants was done in Freestyle^TM 293-F cells (Thermo Fisher Scientific). Briefly, equal amounts of heavy and light chain DNA were mixed with polyethylenimine, PEI, (Polysciences, Inc.) at 2:1 ratio of PEI to DNA and the DNA-PEI complexes were added to Freestyle cells. 200 mL or 300 mL of culture was used for each variant, and supernatants were collected on day 6 after transfection. MAbs were harvested from the supernatant using HiTrap MabSelectSure columns (Life Technologies).

Biolayer interferometry based K_D measurements. HEPC3 variants binding affinities were determined using an Octet RED biosensor (ForteBio Menlo Park, CA). HEPC3 variants were diluted in 1x kinetic buffer to 10 µg/mL and immobilized onto anti-human IgG Fc Capture sensors (ForteBio). The kinetic experiments included five steps: (i) baseline (60 s); (ii) HEPC3 variants loading onto sensors (60 s); (iii) second baseline (60 s); (iv) association of J6 E2 (300 s); and (v) dissociation of J6 E2 (300 s). Fitting curves were constructed using ForteBio Data Analysis 7.0 software using 1:1 binding model and background subtraction was used for correction.

Single HCV genome amplification. HCV hemigenomes from plasma virus were amplified by RT-PCR after limiting dilution to ensure single genome amplification, using previously described methods (Li et al., 2012). PCR products were gel-extracted and directly Sanger-sequenced. E1E2 was PCR amplified from hemigenomic SGA amplicons of interest and cloned as previously described (Osburn et al., 2014). All E1E2 clones were Sanger sequenced to confirm that errors had not been introduced by the additional PCR step. Sequences have been submitted to GenBank, accession numbers (pending). Subject 117 E1E2 variants named 1a49-1a53 were amplified previously for another study (Dowd et al., 2009). E1E2 sequence analysis. Nucleotide sequences spanning E1E2 were trimmed and aligned using MUSCLE, with the alignment manually adjusted in BioEdit. The phylogenetic tree was inferred from nucleotide sequences using the Maximum Likelihood method based on the Tamura 3-parameter model (Tamura et al., 2002), gamma distributed. The tree with highest log likelihood is shown with branches drawn to scale. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. 500 bootstrap tests were performed. Analyses were implemented in the Mega6 program (world-wide-web megasoftware.net). Sliding window nonsynonymous/synonymous analysis was performed by the Nei-Gojobori method implemented in VarPlot (sray.med.som.jhmi.edu), with 20 codon windows and one codon steps. T/F genomes were inferred as previously described (Li et al., 2012; 2016). Highlighter plots were generated using aligned E1E2 amino acid sequences and the Highlighter tool at the Los Alamos HIV database (world-wide-web at LANL.gov).

Statistics. Neutralization curves for HCVcc and ELISA binding curves were fit by nonlinear regression in Graphpad Prism and mAb 50% inhibitory concentration (neutralization assay) or area under the curve (binding assay) calculated based on that curve. Log10 (area under the curve (AUC)) of binding to each of the 19 genotype 1 heterologous E1E2 variants were compared between HEPC3 and HEPC3 variant mAbs by paired one-way ANOVA analysis with correction for multiple comparisons. P values <0.05 were considered significant.

Study approval. The protocol was approved by the Institutional Review Board of the Johns Hopkins Hospital, and informed consent was obtained from all study participants.

Example 2– Results

Identification of subjects who spontaneously cleared HCV infection and possess broadly-neutralizing circulating antibodies. The inventors developed a diverse panel of 19 genotype 1a and 1b HCV pseudoparticles (HCVpp) that allowed us to screen donor plasma for HCV neutralizing breadth (Osburn et al.2014; Bailey et al., 2015). This panel comprises 94% of amino acid polymorphisms present at greater than 5% frequency in a reference panel of 643 genotype 1 HCV isolates from GenBank. In a previous study, the inventors used this panel to screen plasma samples isolated prior to viral clearance by 21 subjects who spontaneously cleared HCV infection or 42 duration-of-infection-matched plasma samples from subjects with persistent infection (Osburn et al., 2014). They identified two subjects who spontaneously cleared HCV and who possessed exceptionally broad plasma neutralizing antibody activity. Subject 117 was HCV plasma antibody-negative and HCV RNA negative on study enrollment before becoming infected with genotype 1a HCV. Subject 110 was initially viremic, also with genotype 1a virus, but was HCV serum antibody negative, indicating recent infection. Both subjects exhibited typical high viral loads during acute infection, followed by a subsequent viral load decline and eventual clearance of the infection (FIG.1A). Subject 117 was infected for a total of about 1 year prior to clearance. Subject 110 had a more unusual disease course with high viral loads for about 1 year followed by a drop in viral load, years of extremely low- level, intermittent viremia, and eventual viral clearance. Repeated sequencing of viral structural genes throughout infection did not reveal any large genetic shifts in either subject that would suggest superinfection or reinfection with a new viral strain. After clearance, each subject was followed for more than 5 years with frequent viral load testing, with no recurrence of viremia. Subject 117 plasma was tested for neutralizing antibody breadth at day 388 post- infection, the last time point prior to viral clearance. Subject 110 plasma was tested at day 401 post-infection, the time point immediately preceding a decline of 10⁷ International Units (IU)/mL in viral load. Using a threshold of at least 50% neutralization by a 1:100 dilution of plasma, the plasma of Subject 117 neutralized 14 of 19 HCVpp variants (74%), and the plasma of Subject 110 neutralized 17 of 19 variants (89%) (FIG. 1B). For comparison, the median percent neutralization by time-matched plasma samples from 42 subjects with persistent infection did not exceed 50% for any of the 19 HCVpp tested.

Isolation of neutralizing mAbs and measurement of neutralizing breadth. The inventors isolated peripheral blood mononuclear cells (PBMCs) from Subjects 117 and 110 after HCV clearance. Using previously established methods, the inventors transformed B cells in PBMC samples with Epstein-Barr virus (EBV) and screened the transformed B lymphoblastoid cell line culture supernatants for neutralizing activity against one autologous HCV variant and a second heterologous variant. Cells from wells with neutralizing activity against either variant were fused with myeloma cells to produce hybridomas, which then were cloned biologically using limiting dilution and flow cytometric sorting and repeated screening of culture supernatants for HCV E1E2 protein binding activity. The inventors isolated 15 anti- E1E2 mAbs from the two subjects. Purified mAbs were tested for intra-genotypic neutralizing breadth against the panel of 19 genotype 1 HCVpp, using a threshold of ³ 50% reduction as the definition of neutralization. The most broadly-neutralizing mAbs are shown in FIG.2, with the remaining mAbs shown in FIG. 9. For comparison, two previously described bNAbs, AR4A and AR3C (9, 17) were tested against the same viral panel. The most broadly- neutralizing mAbs isolated from subject 117, designated HEPC3 and HEPC84, neutralized 16 of 19 (84%) or 11 of 19 (58%) of variants, respectively. HEPC43 and HEPC74, the most broadly-neutralizing mAbs isolated from subject 110, each neutralized 17 of 19 (90%) variants. For comparison, AR3C and AR4A neutralized 16 of 19 (84%) and 12 of 19 (63%) variants, respectively. Next, the inventors measured cross-genotypic neutralizing titers of mAbs HEPC3, HEPC43, HEPC74, or HEPC84 against a standard panel of genotype 1-6 replication competent cell culture viruses (HCVcc) (FIG. 3). HEPC3, HEPC43, and HEPC74 each neutralized variants from 5 of 6 genotypes, and HEPC84 neutralized variants from 4 of 6 genotypes. None of the mAbs showed potent neutralizing activity against the genotype 3 isolate (S52), which, interestingly, is also true of many other previously-described broadly-neutralizing anti-HCV human mAbs (17). HEPC3, HEPC43, HEPC74, and HEPC84 each bound native E1E2 protein in an ELISA, but did not bind to denatured protein, indicating that they bind conformational epitopes on E1E2 (FIG.10).

Epitope mapping. MAb binding epitopes were mapped using comprehensive alanine scanning mutagenesis across E1E2. Six mAbs displayed adequate binding to the target variant, strain H77 E1E2, to allow mapping by this method (FIGS. 4A-D and FIG. 11). HEPC3, HEPC43, and HEPC74 displayed overlapping epitopes with multiple shared binding residues. Surprisingly, the binding epitope of these mAbs also overlapped with the epitope of the previously described bNAb, AR3C, which was determined by co-crystallization of AR3C Fab with HCV E2 core (Kong et al., 2012). Two of the binding residues shared by HEPC3, HEPC43, and HEPC74 (aa 530 and 535) also have been shown to be critical for interaction with the HCV receptor CD81, suggesting that escape mutations at these positions might adversely affect viral fitness (Oswianka et al., 2006). The inventors further mapped the binding epitopes of these mAbs by measuring competition for E1E2 binding among the mAbs or between the mAbs and a panel of previously characterized reference bNAbs. The six most broadly-neutralizing mAbs are shown in FIGS.4B-C, with all mAbs shown in FIGS.12A-B. HEPC3, HEPC43, and HEPC74 showed reciprocal competition for binding with each other, and HEPC82 reduced binding of HEPC84 and HEPC98. As expected from the alanine- scanning maps, reference mAb AR3C reduced binding of HEPC3, HEPC43, and HEPC74, suggesting that the mAbs compete for overlapping binding sites. Reference mAb AR4A, which binds only to complexed E1 and E2, reduced binding of the new mAbs HEPC82 and HEPC84. Reference mAb HC33.8 (Keck et al., 2013), which targets an epitope spanning amino acids 408-423, competed with mAb HEPC98, which has binding residues at amino acids 402, 405, and 408. The inventors also characterized relationships between the most broadly-neutralizing mAbs using a neutralization profiling method that has been used extensively for characterization of anti-HIV mAbs (Georgiev et al., 2013). They recently adapted this method to allow grouping of HCV mAbs into functionally-related clusters based upon relative neutralizing capacity across a genetically diverse panel of HCV variants (Bailey et al., 2015). In this analysis, the inventors compared neutralization values of the novel mAbs across the panel of 19 HCVpp to each other and to a large panel of previously described reference bNAbs, using spearman correlations (FIG.4D and FIG.13). HEPC3, HEPC43, and HEPC74 clustered most closely with each other and, of the 18 reference bNAbs tested, they clustered most closely with mAb AR3C, in agreement with the alanine scanning and competition-binding data. Consistent with the competition-binding results, HEPC84 clustered most closely with mAb AR4A, and HEPC98 clustered near mAbs HC33.4 and HC33.8. Taken together, these three mapping studies show remarkable similarity between the binding epitopes of mAbs HEPC3, HEPC43, and HEPC74, which were isolated from two different subjects who spontaneously cleared HCV infection, and AR3C, a previously described bNAb isolated from a patient with persistent HCV infection. Based upon competition-binding data and neutralization profiling, HEPC84 and the reference mAb AR4A also appear to bind to overlapping epitopes, as do HEPC98 and the reference mAb HC33.8.

Somatic mutations and antibody breadth. The inventors sequenced the heavy and light chain variable gene sequences of each of the mAbs. Surprisingly, despite developing in two different individuals, HEPC3, HEPC43, and HEPC74 are each encoded by the same antibody heavy chain variable gene segment, VH1-69 (Table A). Of note, mAb AR3C, which was isolated from a subject with chronic HCV infection, also uses V_H1-69 (17), as do some other anti-HCV bNAbs (Merat et al., 2016; Chan et al., 2001). Together with the epitope mapping data, these results suggest that V_H1-69 encodes antibodies with capacity to form a public B cell clonotype that favors binding to an immunodominant broadly-neutralizing HEPC3/AR3C epitope. Remarkably, these bNAbs were encoded by antibody genes with sparse somatic mutations. HEPC3 has 95% identity to its germline heavy chain VH-gene sequence, while HEPC43 has 95% and HEPC74 has 92% identity to germline. The light chains encoding these mAbs have 96-98% identity to their germline VL-genes. By comparison, mAb AR3C, isolated previously from a subject with chronic HCV infection, has only 86% identity to its VH-gene (17), meaning that the bNAbs isolated from subjects who spontaneously cleared infection have substantially fewer V_H-gene somatic mutations than AR3C. In addition, these mAbs have significantly fewer somatic mutations than most previously described anti-HIV bNAbs, including VRC01 (22), which has only 68% identity with germline.

TABLE A

To identify somatic mutations critical for bNAb breadth, the inventors mutated all 13 somatic mutations in the HEPC3 heavy chain, all 7 somatic mutations in the light chain, or both, to match the inferred germline sequence, generating HEPC3 reverted unmutated ancestor (RUA) variants designated H-RUA, L-RUA, and H,L-RUA, respectively. They measured the ability of mature HEPC3, H-RUA, L-RUA, and H,L-RUA antibodies to neutralize a diverse panel of genotype 1 E1E2 variants (FIG.5A). As in prior tests, mature HEPC3 neutralized the majority of variants in the panel. HEPC3 L-RUA showed nearly identical neutralization results, indicating that somatic mutations in the light chain are not critical for neutralizing breadth. Neutralizing breadth of both HEPC3 H-RUA and H,L-RUA was greatly attenuated, indicating that somatic mutations in the heavy chain are critical for HEPC3 neutralizing breadth. Consistent with the neutralization data, mature HEPC3 bound all heterologous E1E2 variant proteins in an ELISA (FIG. 5B). There was little reduction in binding of HEPC3 L-RUA relative to mature HEPC3. Binding of HEPC3 H-RUA and H,L-RUA to all E1E2s was significantly reduced, confirming that somatic mutations in heavy chain CDR1, 2, and/or 3 are critical for binding affinity and breadth of HEPC3.

To identify individual somatic mutations that are important for breadth of HEPC3, the inventors also performed site-directed mutagenesis to revert each heavy chain somatic mutation individually to the germline-encoded amino acid, without altering the other 12 somatic mutations in the sequence. They expressed the mAbs, and measured binding to the panel of heterologous E1E2 variants proteins (FIG. 5B). The inventors also measured the effect of simultaneous reversion of all somatic mutations in the sequence encoding HCDR1, HCDR2, or HCDR3. Reversion of all mutations in HCDR1 or HCDR3 significantly reduced binding across the E1E2 panel, suggesting that these somatic mutations are important for binding to most heterologous E1E2 variants. Reversion of all somatic mutations in HCDR2 reduced binding to a subset of E1E2 variants. On evaluation of individual somatic mutations, reversion of glutamic acid (E) 38 to alanine (A) in HCDR1 significantly decreased binding of HEPC3 across the genotype 1 E1E2 panel. Similarly, reversion of threonine (T) 65 to A in HCDR2 also significantly reduced binding across the panel, as did reversion of arginine (R) 112 to S in HCDR3. Interestingly, reversion of other individual somatic mutations had no detectable effect on binding to some E1E2 variants, but profoundly reduced binding to others. For example, as shown in FIG.6A, reversion of leucine (L) 30 to phenylalanine (F) in HCDR1 or re-insertion of the germline-encoded glycine (G) at the site of a deletion in HCDR2 (Del63G) had no effect on antibody binding to genotype 1a variants 1a09 or 1a157, but these reversions profoundly reduced binding to 1b variants 1b09 and 1b52. Overall, reversion of each somatic mutation reduced binding to one or more variants in the heterologous panel, and each single amino acid reversion, except framework mutation threonine (T) 87 to alanine (A), reduced median binding across the E1E2 panel relative to mature HEPC3. These results, together with the almost complete loss of binding of HEPC3 H-RUA to all E1E2 variants, suggests that most or all somatic mutations present in the heavy chain of HEPC3 contribute in combination to the breadth of E2 recognition by the bNAb.

The inventors also performed quantitative kinetic binding analysis with the panel of HEPC3 mAb variants and purified soluble J6 (genotype 2a) E2 protein (sE2) (FIG. 6B and FIG. 14). Individual reversion of 11 of 13 heavy chain somatic mutations slightly reduced HEPC3 binding affinity for E2, as did reversion of all mutations simultaneously in HCDR1, HCDR2, or HCDR3. Interestingly, the inventors observed large reductions in binding affinity with reversion of E38 to A and T66 to N, two reversions that also consistently reduced binding across the heterologous genotype 1 panel. Notably, they also observed a reduction in binding affinity after reversion of L30 to F in HCDR1 and Del63 to G in HCDR2, which supports the qualitatively similar effects of these reversions on antibody binding to genotype 1b variants 1b09 and 1b52 (FIG. 6A). Together, these data confirm that the majority of the heavy chain somatic mutations in HEPC3 are critical in combination for binding to heterologous E1E2 variants, and that L30 and Del63 somatic mutations are particularly important for binding to subtype 1b and 2a strains that are highly divergent from the genotype 1a virus infecting the HEPC3 donor.

Longitudinal evolution of autologous virus. To define the autologous E1E2 antigenic variants that favored selection and maturation of HEPC3, and investigate molecular mechanisms of viral clearance, the inventors performed extensive longitudinal sequencing of the HCV quasispecies of viruses present in plasma samples collected over time from Subject 117. Plasma was isolated at seven longitudinal time points, the first when HCV viremia initially was detected approximately 17 days after infection, and the last immediately prior to HCV clearance. Plasma RNA was isolated and RT-PCR performed with single genome amplification of 16-41 E1E2-spanning amplicons per time point in order to define the viral diversity at each time point and the viral evolution over time. As shown in FIG. 7, the subject was infected initially by at least three different transmitted/founder (T/F) viruses that differed from each other by 0.42-1.23% in E1E2 nucleotide sequence and 0.72-1.99% in E1E2 amino acid sequence. This estimate of three T/F genomes responsible for productive clinical infection is a minimum estimate whose accuracy and precision are based on the numbers of sequences determined at the earliest sampling time points. In this subject, the inventors determined 65 sequences in the initial 46 days of infection; previously described statistical power calculations (35) indicate that this provides a >95% probability of detecting minor variant sequences present at a frequency of at least 5%. Two of these viral lineages, T/F#1 and T/F#2, persisted and diversified over the next two sampling time points before they were apparently extinguished. The T/F#3 lineage persisted and evolved throughout the course of infection but exhibited a series of stringent population bottlenecks such that only a single sublineage of day 194 sequences, exemplified by clone D194-FD13, gave rise to the last detectable virus population at day 388. Remarkably, day 388 sequences were comprised of a homogeneous expansion from a single genome present at day 285, again indicative of a stringent population bottleneck. This lineage, D388-5A12, was last detected at the final sampling time point when HCV viral load had already declined to 754 International Units (I/U)/mL just prior to its extinction. The ratio of non-synonymous to synonymous changes was high in genes encoding E1E2, suggesting positive selection (FIG. 15). The majority of non-synonymous changes occurred in the hypervariable region 1 (HVR1) of E2. Outside of HVR1, 6 amino acid changes in E2 became fixed in the viral quasispecies over time (FIG.16). Notably, 4 of these amino acid changes fell in a region of E2 spanned by the HEPC3 binding epitope, indicating selective pressure at this locus. E1E2 variants representative of all viral clades observed throughout the course of infection were cloned and expressed for binding studies. Somatic mutations and recognition of autologous E1E2. The inventors measured binding with an ELISA of mature HEPC3, HEPC3 H-RUA, and HEPC3 H,L-RUA to each of the 21 longitudinal autologous E1E2 variants (FIG. 8). Remarkably, mature HEPC3 showed binding above background to all autologous variants, including the variants circulating immediately prior to viral clearance, suggesting that this mAb may have contributed to clearance of infection. HEPC3 H,L-RUA, with all somatic mutations in both heavy and light chain reverted to the germline-encoded sequence, lost detectable binding to 18 of 21 autologous variants, but retained binding to two of three T/F E1E2 variants and to a third variant also present at day 17 post-infection. HEPC3 H-RUA showed a very similar pattern of binding to HEPC3 H,L-RUA, consistent with testing against heterologous E1E2 showing that somatic mutations in the heavy chain are more important than light chain somatic mutations for the neutralizing breadth of this bNAb. Taken together, these data suggest that the HEPC3 lineage may have arisen through binding of the unmutated ancestor of HEPC3 to T/F virus present very early after infection, and B cell clones with somatic mutations necessary for neutralizing breadth were likely selected by more resistant E1E2 variants circulating later in infection. Example 3 - Discussion

These studies suggest that early emergence in diverse individuals of commonly occurring genetically-related B cell clones encoding HCV-specific bNAbs with sparse somatic mutations is associated with spontaneous viral clearance. The data suggest a rational epitope- based vaccine approach may be highly feasible for HCV. To study the immune mechanism associated with clearance, the inventors isolated and characterized bNAbs from subjects who spontaneously cleared HCV infection. They found that the intra- and cross-genotypic neutralizing breadth of these mAbs compares favorably with the most potent anti-HCV bNAbs described to date. They also found that the binding epitopes of three mAbs isolated from two different subjects were remarkably similar to each other and to the binding epitope of the well- characterized bNAb AR3C (from a third subject), suggesting that the V_H1-69 antibody gene segment commonly encodes a public B cell clonotype that favors early recognition of this AR3C/HEPC3 epitope. Remarkably, the antibody genes encoding these bNAbs were not extensively somatically mutated, but the few somatic mutations present were critical in combination for facilitating the neutralizing breadth of one of the most potent bNAbs, HEPC3, as well as recognition of autologous viral variants circulating prior to viral clearance. Finally, the inventors identified early T/F autologous envelopes that likely were responsible for selection of B cells expressing the HEPC3 unmutated ancestor antibody.

Multiple studies have now confirmed that several anti-HCV bNAbs are encoded by the V_H1-69 antibody gene segment (Merat et al., 2016; Chan et al., 2001). This study shows for the first time that VH1-69 commonly encodes a public B cell clonotype that can lead to bNAb development after a relatively brief period of infection, producing an antibody with recognition of an entire autologous viral quasispecies, thus providing the first potential mechanistic link between this B cell clonotype and spontaneous clearance of HCV. These data suggest that antigens selecting B cells expressing VH1-69-encoded antibodies may be highly desirable for vaccine development.

It is noteworthy that these bNAbs achieved broad neutralizing activity without extensive somatic mutation and after only a year of persistent viral infection. This finding stands in contrast to the typical evolution of bNAbs against HIV, which tend to require extensive somatic mutation that develops after years of chronic infection (Bhiman et al., 2015; Bonsignori et al., 2016). These results may suggest that, despite the enormous genetic variation of HCV, some E1E2 epitopes are relatively conserved and accessible to antibody binding. The degree of somatic mutation necessary to generate HEPC3, HEPC43, or HEPC74 (which exhibit 92-95% identity to inferred germline genes) is generally achievable with traditional vaccination strategies (Scherer et al., 2014; Wang et al., 2015), whereas the more extensive level of somatic mutation found in anti-HIV bNAbs or even the anti-HCV mAb AR3C is less likely to be achievable.

Elegant studies of longitudinal viral evolution have been performed in HIV-infected subjects who developed bNAbs, with the goal of informing sequential vaccination strategies Bhiman et al., 2015; Bonsignori et al., 2016). The results of these studies have been sobering, as they showed extensive and complex viral evolution driving the late development of bNAbs against HIV. This study suggests that the antigens required for stimulation of bNAbs against HCV may be less complex. Excluding HVR1, which is not the target of the bNAbs isolated in this study, HCV E1E2 of the most divergent E1E2 variant isolated from Subject 117 differed by only 7 out of 546 amino acids (~1 percent) from the T/F viruses that initially infected Subject 117. It is remarkable that the epitopes presented by the initial T/F strains along with these few mutations in E1E2 were adequate to facilitate selection of both the bNAb unmutated ancestor and the mature antibody, which is capable of binding and neutralizing HCV variants from multiple genotypes, which differ at approximately 30% of their amino acids. This study also suggests that subjects who spontaneously clear HCV infection have mAbs that are similar in potency, neutralizing breadth, and epitope recognition to mAbs developed later in infection by some subjects with persistent HCV infection. However, these mAbs developed early in those who cleared infection without requiring extensive somatic hypermutation, which may allow mAbs to contribute to clearance before viral diversity and antibody resistance becomes too extensive. Therefore, the timing of emergence of bNAbs may be a critical factor in their ability to contain infection and restrict the emergence of additional viral diversity. The concentration of HEPC3-like mAbs present in plasma of these subjects at the time of clearance is not known, so further studies are needed to confirm a direct contribution of these mAbs to clearance of infection. However, phylogenetic bottlenecks in the virus of subject 117, with selection of mutations in E1E2 conferring partial HEPC3 resistance, provides intriguing evidence that HEPC3-like mAbs were present at sufficient concentrations during infection to exert physiologically relevant pressure on the virus. The inventors observed some recognition of all autologous variants by HEPC3, but the other distinct bNAbs isolated from this donor likely also contributed to clearance. The mechanisms by which these bNAbs may contribute in combination to clearance of HCV infection warrants further investigation.

In summary, the inventors have isolated the first bNAbs from subjects with broadly neutralizing serum who spontaneously cleared HCV infection. Multiple bNAbs bind to the same epitope and use the same heavy chain V-gene allele, identifying a public B cell clonotype that favors early binding to a conserved neutralizing epitope. Remarkably, these bNAbs were encoded by antibody genes with few somatic mutations. These somatic mutations were critical for antibody neutralizing breadth and binding to autologous envelope variants circulating late in infection, but they were not required for binding of the HEPC3 unmutated ancestor to envelope proteins of early autologous T/F viruses. This study shows that anti-HCV bNAbs can achieve significant breadth with relatively few somatic mutations, and it identifies HCV envelope variants that were sufficient for selection and maturation of an anti-HCV bNAb in vivo. These data provide a roadmap to guide development of a vaccine capable of stimulating anti-HCV bNAbs with a physiologic number of somatic mutations characteristic of vaccine responses. The work also provides one of the first views of the molecular basis for antibody- mediated clearance of an antigenically diverse and evolving chronic viral infection in humans. TABLE 1– NUCLEOTIDE SEQUENCES FOR ANTIBODY VARIABLE REGIONS

Clone Variable Sequence Region SEQ

ID NO: HEPC‐3 caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaa 1 heavy ggcttctggaggcaccttgaacagctatgaaatcacctgggtgcgacaggcccctggacaagggcttg

agtggatgggagggataacccctatctttgaaacaacctacgcccagaagttccagggcagagtcac gattaccgcggacgaatccacgagcacaacctacatggagctgagcagcctgagacctgaggacacg gccgtgtattactgtgcgagagatggggtcagatattgtggtggtggtaggtgctacaactggttcgac ccctggggccagggaaccctggtcaccgtctcctca

HEPC‐3 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgc 2 light cgggcaggtcagaacattaacaactatttaaattggtatcaacaaaaaccagggaaagcccctaagg

tcctgatctacgctgcatccaatttgcaaagtggggtcccatcaaggttcagtggcagtggatctggga cagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgccaacagagtca cagtaccgtccggacgttcggccaagggaccaaggtggaaatcaaa

HEPC‐43 caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaa 3 heavy ggcttctggaggcaccttcagcagctttggtatcagctgggtgcgacaggcccctggacaagggcttga

gtggatgggagggatcatccctgtctttggtagagcaaaatacgcacagaagttccagggcagagtca ccattaccgcggacgaattcacgaccacagccgacatggaagtgaccagcctgagatttgaggacac ggccgtgtattactgtgcgataaaggtagtagcaccgcgagtgatcttttacggtatggacgtctgggg ccaagggaccctggtcaccgtctcctca

HEPC‐43 caggctgtggtgactcaggagccctcactgactgtgtccccaggagggacagtcactctcacctgtggc 4 light tccagcactggagctgtcaccagtggtcattatacatactggttccagcagaagtctggccaagccccc

aggacactgatttatgatacaagcaacaaacactcctggacccctgcccggttctcaggctccctcctt gggggcaaagctgccctgaccctttcgggtgcgcagcctgaggatgaggctgactattactgcttactc tcctatagtggtgctccgtcaggggtgttcggcggagggaccaaactgaccgtccta

HEPC‐46 caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaa 5 heavy ggcttctggttacatctttacgagccacggtatcagctgggtgcgacaggcccctggacaagggcttga

gtggatgggatggatcagcgtgtacaatggttacacaaactacgcacagaatctccagggcagagtc accatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgaca cggccgtgtatttctgtgcgagggcatctcaaattcgaggtgttgactactggggccagggaaccctgg tcaccgtctcctca

HEPC‐46 cagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctg 6 light gaagcagctccaacatcggaagtaattatgtatactggtaccaacagttcccaggaacggcccccaag

ctcctcatctatggcaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggca cctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagcctggg atgacagcctgagtggtccttgggtgttcggcggagggacccaggtsaccgtccta

HEPC‐50 caaatgcagctggtgcagtctgggcctgaggtgaagaagcctgggacctcagtgaaggtctcctgcaa 7 heavy ggcttctggattcacctttcctagttctactatgcagtgggtgcgacaggctcgtggacaacgccttgac tggataggcaggatcgtcgttggcagtggtaacacaaactacgcacagaagtttcaggaaagagtca ccattaccagggacatgtccacaagcacagcctacatggagctgagcagcctgagatccgaggacac ggccgtgtattactgtgcggcagcggtttctgaactatggttcggggactctccctaccactactacggt ttggacgtctggggccaagggaccctggtcaccgtctcctca

HEPC‐50 gacattgtgatgactcagtctcctctctccctgcccgtcacccctggagagccggcctccatctcctgca 8 light ggtctagtcagagcctcctgcatagtaatggatacaactatttggattggtacctgcagaagccagggc agtctccacaggtcctgatctatttgggttctaatcgggcctccggggtccctgacaggttcagtggcag tggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactg catgcaagctctacaaactcctcatacttttggccaggggaccaagctggagatcaaa

HEPC‐74 caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtaaaggtctcctgcac 9 heavy gacttctggaggcacctacatcaactatgctatcagctgggtgcgacaggcccctggacaagggcttg agtgggtgggagggatgagccctatctcaaatacaccgaagtatgcacagaagttccagggcagagt cacgattaccgcggacgagtccacaagcacaacctacatggaactgagcagcctgagacctgaggac acggccgtctattactgtgccagagacctgctgaaatattgtggtggtggtaactgccactctcttttagt cgacccctggggccagggaaccctggtcaccgtctcctca

HEPC‐74 gacatcgtgatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatctcttgcc 10 light gggccagtcagagcattagtagttggttggcctggtatcagcagaaaccagggagagcccctaaactc ctgatctataaggcgtctagtttagaaactggggtcccatcaaggttcagcggcagtggatctgggaca gaattcactctcaccatcagcagcctgcagcctgatgattttgcgacttattactgccaacattataata cttatttattcactttcggccctgggaccaaagtggatctcaaa

HEPC‐80 gaggtgcagctggtgcagtctgggggaggcttggtacagccagggcggtccctgagactctcctgtag 11 heavy agcttcaggatttacctttggtgaatatgctatgagctgggtccgccaggctccagggaagggactgga gtgggtaggtttcattagaagcaaaacttatggtgggacaacagattacgccgcgtctgtgacaggca gattcaccatctccagagatgattccaaaagcgtcgcctatctgcaaatgaacagcctgaaaaccgag gacacagccgtttattactgtactagagaggataatgattacatttgggggaccaatgcggcggacta ctggggccagggaaccctggtcaccgtctcctcc

HEPC‐80 gaaattgtgatgacccagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgc 12 light agggccagtcagagtattagcagctacttagcctggtaccaacagaaacctggccaggctcccaggct cctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggac agacttcactctcaccatcaccagcctagagcctgaagattttgcagtttattactgtcagcagcgtacc aactggcctccgggcactttcggcggagggaccacggtggagatcaaa

HEPC‐82 caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcac 13 heavy tgtctctggtggctccatcagcagtggtggttactactggagctggatccgccagcacccagggaatgc cctggagtggattgggtacatctattacagtgggaacacctactacaacccgtccctcaagagtcgagt taccatgtcagtagacacgtctaagaaccagttctccctgaaggtgagctccgtgactgccgcggaca cggccgtgtattactgtgcgagaggaggatactatgatagtagtggttatgccagcgactcctggggcc agggaaccctggtcaccgtctcctca

HEPC‐82 aattttatgctgactcagccccactctgtgtcggagtctccggggaagacggtaaccatctcctgcaccc 14 light gcagcagtggcagcattgccaccaactatgtgcattggtaccagcagcgcccgggcagttcccccacc actgtgatctgtgaggatgaccaaagaccctctggggtccctgatcggttctctggctccatcgacacct cctccaactctgcctccctcaccatctctggactgaggactgaggacgaggctgactactactgtcagt cttttgataacatcaatcactatgcgatattcggcggagggaccacgctgaccgtccta

HEPC‐84 gaggtgcagctggtgcagtctgggggaggcttggtacagccagggcggtccctgagactctcctgtac 15 heavy agcttctggattcaactttggtgattttgctatgaactgggtccgccaggctccagggagggggctgga gtgggtaggtatcattagaagcaatacttatggtggaacaacagaatacgccgcgtctgtaagaggca gattcagcctctcaagagaagatttcaaaagtatcgtccatctgcaaatgaacagcctgaataccgag gacacagccgtgtattactgtaccagagcgagggctccgattacaatgatatcagcggaactccagaa agcctacttctacaacggtttggacgtctggggccaagggaccctggtcaccgtctcctcc

HEPC‐84 gacatccagatgacccagtctccatcctccctgtctgcatctatgggagacagagtcaccatcacttgc 16 light cgggcaagtcagagcattgacagttacttaaattggtatcascagaaaccagggaaagcccctaaact cctgatctatgctgcatccaatttgcaaagtggggtcccatcaaggttcagtggcagtgattctgggac aactttcactctcaccatcatcagtctgcaacctgaagattttgcaacttattactgtcaacagacttaca ctaccccgctcacttttggcggagggaccaaagtggatatcaaa

HEPC‐85 caggtgcagctggtgcagtctgggggaggcgtagtccaccctgggagttccctgagactctcttgttca 17 heavy gcctctggattcaacttcagtgcctatggcatgcactgggtccgccaggctccaggcaaggggccgga gtgggtggcagctatttcatttgatggaagtcattacttctatgcagactccgtgaagggccgattcacc atctccagagacaattccaagaacaccttgtatttgctaacgaacagcgtgagaactgaggacacggc tctatatacatgtgcgaggcacactaattcttacagttggttcgacccctggggccagggaaccctggt caccgtctcctca

HEPC‐85 gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcttgca 18 light agtctagtcagagcctcctacatagtgatggaaagacgtatgtctattggtacctacagaagccaggc cagtttccacaactcctgatctatgaggtttccagccggttgtctggagtaccagctaggttcagtggca gcgggtcagggacagatttcacattgaaaatcagtcgggtggagactgaggatgttggggtttattact gcatgcaaggaatacaccttcccccgatcaccttcggccaagggacacgactggacattaaa

HEPC‐87 caggtgcagctgcaggagtcgggcccaggaatggtgaagccttcacagaccctgtccctcacctgcac 19 heavy tgtttctggtgcctccatcagccgcggtggttactactggacccggatccgccagcacccagggaaggg cctagagtggattgggtacatctattacaatgggcgcaccttatacaacccgtccctcaagagtcgagt taccatatcgtcagacacgtctgaggaccagttcttcctgaagctgacctctgtgactgccgcggacgc ggccgtgtattactgtgcgagaggacaaatgagcagcagctggtttgtggactactggggccagggaa ccctgggcaccgtctcctca

HEPC‐87 NOT AVAILABLE 20 light

HEPC‐90 caggtgcagctggtgcagtcggggggaggcgtagtccagcctgggaggtccctcagactctcctgtgc 21 heavy agcgtctggattcaccttcagtacctatggcatgcactgggtccgccaggctccaggcaaggggctgg agtgggtggcagtcatatggtatgatggatttgacaaatattatgcagagtccgtgaagggccgattca ccatctccagagacaattccaggaacttggtgtttctgcaaatgaacagcctgagagccgaggacacg gctgtgtattactgtgtgagagtccggtcggcaggtactatgatacgagcggaaagcgcgtcaaatgc ctttgatatctggggccaagggaccctggtcaccgtctcctca

HEPC‐90 gaaatagtgatgacgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgc 22 light agggccagtcagagtgttagcaacaacttagcctggtaccagcagagacctggccaggctcccaggc tcctcatctatgatgcatccaccagggccactgatatcccagccaggttcagtggcagtgggtctggga cagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttactactgtcagcaatataa taactggcccccgtggacgttcggccaagggaccaaggtggaaatcaaa

HEPC‐91 caggtgcagctggtgcagtctgggggaggcgtggtccagcctgggaggtccctgagactctcttgtgc 23 heavy aggctctggattcagctttggtgactttggcatccactgggtccgccaggctccaggcaaggggccgga atggctgtcaagtatttcaaatgatggcagtcatatattctatgcagactcagtggagggccgattcac cgtctccagagacaattccaagagtacggtctttctccagatggacaggctgatacctgacgactcggc tttttattactgtgcgagacacagtaacacttaccagtggttcgacccctggggccagggaaccctggt caccgtctcctca

HEPC‐91 gacattgtgatgacccagactccactctctctgtccgtcacacctgggcagccggcctccatctcatgca 24 light agtctagtcggagcctcctccacagtgatggaaagacctatgtgtactggtatttgcagaggccaggcc agttaccacagctcctcatctatgaagtttctagtcgactctctggagtgccagataggttcagtggcag cgggtcagggacagatttcacgctggaaatcagccgggtggaggctgaggatgttgggatttattact gcgtgcaaggtatacaccttcccccgatcaccttcggccaagggacacgactggatattaaa

HEPC‐96 caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcac 25 heavy tgtctctggtggctccatcagcagtggtgattactactggagttggatccgccagtccccagggaaggg cctggagtggattgggtacatccactccagtgggagcacctcctacaacccgtccctcaagagtccagt tattatattactagacacgtccaagaaccagctctccctggaactgagctctgtgactgccgcagacac ggccgtctattactgtgccaacttggactacagtgcctacgggtccccccgctggttcgactcctggggc cagggaaccctggtcaccgtctcctca

HEPC‐96 gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgc 26 light agggccagtcggagtgttggcagcaggtacttagcctggtaccagcagaaacctggccaggctccca ggctcctcatctatgatgcatcccgcagggccactggcatcccagacaggttcagtggcggtgggtctg ggacagacttcactctcaccgtcagcagactggagcctgaagattttgcagtgtattactgtcagcagt atggtagctcacctctcactttcggcggaggaacacgactggatattaaa

HEPC‐97 caggtgcagctacagcagtggggcgcaggtctgttgaagccttcggagaccctgtccctcacctgcgct 27 heavy gtctatggtgggtctctcagtggtcactactggagctggattcgccagtccccagggaaggggctggag tggattggagagatcaatcataatggaggcgccaattatggcccgtccctcaacagtcgagccaccat atcagttgacacgtctaagaatcagttcttcctgaacctgaggtctgtgaccgccgcggacacggctgt ctattactgtgctcgagccctacctccttatagctcctcgtcctggttcgacccctggggccagggaacc ctggtcaccgtctcctca

HEPC‐97 NOT AVAILABLE 28 light

HEPC‐98 caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaa 29 heavy ggcttctgaagggaccctcaacagttctgctgtcaactgggtgcgacaggcccctggacaagggcttg agtggattggaaggatcatccctatctttcgtgcaacaaactacgcacagaggttccaggacagagtc acaattaccgcggacgatttcacgaacacagcctacatggagatcaacagcctgagatctgaggaca cggccgtctattactgtgcgagaggaaccacggagatctggttcgactcttggggccagggaaccctg gtcaccgtctcctca

HEPC‐98 tcctatgagctgacacagccaccctcggtgtcagtgtccccaggacagacggccacgatcacttgctct 30 light ggagatgtattgccgcaacagtttacttattggtaccagcagaagtcaggccaggcccctgttttgctg atatatagagacattaagaggccgtcagggatcgctgagcgattctctgccttcacgtcagggacatc agtcaccttgatcatcagtggagtccaggcagaagatgaggctgactattactgtcaatcagcagaca tcagtggtactatggcggggttcggcggagggaccaagctgaccgtccta

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

NO: HEPC‐3 QVQLVQSGAEVKKPGSSVKVSCKASGGTLNSYEITWVRQAPGQGLEWMGGITP 31 heavy IFETTYAQKFQGRVTITADESTSTTYMELSSLRPEDTAVYYCARDGVRYCGGGRCY

NWFDPWGQGTLVTVSS

HEPC‐3 DIQMTQSPSSLSASVGDRVTITCRAGQNINNYLNWYQQKPGKAPKVLIYAASNL 32 light QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTVRTFGQGTKVEIK

HEPC‐43 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSFGISWVRQAPGQGLEWMGGIIP 33 heavy VFGRAKYAQKFQGRVTITADEFTTTADMEVTSLRFEDTAVYYCAIKVVAPRVIFYG

MDVWGQGTLVTVSS

HEPC‐43 QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGHYTYWFQQKSGQAPRTLIYDTS 34 light NKHSWTPARFSGSLLGGKAALTLSGAQPEDEADYYCLLSYSGAPSGVFGGGTKLT

VL

HEPC‐46 QVQLVQSGAEVKKPGASVKVSCKASGYIFTSHGISWVRQAPGQGLEWMGWISV 35 heavy YNGYTNYAQNLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYFCARASQIRGVDY

WGQGTLVTVSS

HEPC‐46 QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQFPGTAPKLLIYGNNQR 36 light PSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGPWVFGGGTQVT

VL

HEPC‐50 QMQLVQSGPEVKKPGTSVKVSCKASGFTFPSSTMQWVRQARGQRLDWIGRIV 37 heavy VGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAAAVSELWFG

DSPYHYYGLDVWGQGTLVTVSS

HEPC‐50 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQVLIYLG 38 light SNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPHTFGQGTKLEI

K

HEPC‐74 QVQLVQSGAEVKKPGSSVKVSCTTSGGTYINYAISWVRQAPGQGLEWVGGMSP 39 heavy ISNTPKYAQKFQGRVTITADESTSTTYMELSSLRPEDTAVYYCARDLLKYCGGGNC

HSLLVDPWGQGTLVTVSS

HEPC‐74 DIVMTQSPSTLSASVGDRVTISCRASQSISSWLAWYQQKPGRAPKLLIYKASSLET 40 light GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYNTYLFTFGPGTKVDLK

HEPC‐80 EVQLVQSGGGLVQPGRSLRLSCRASGFTFGEYAMSWVRQAPGKGLEWVGFIRS 41 heavy KTYGGTTDYAASVTGRFTISRDDSKSVAYLQMNSLKTEDTAVYYCTREDNDYIWG

TNAADYWGQGTLVTVSS

HEPC‐80 EIVMTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRAT 42 light GIPARFSGSGSGTDFTLTITSLEPEDFAVYYCQQRTNWPPGTFGGGTTVEIK

HEPC‐82 QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGNALEWIGYIYY 43 heavy SGNTYYNPSLKSRVTMSVDTSKNQFSLKVSSVTAADTAVYYCARGGYYDSSGYAS

DSWGQGTLVTVSS

HEPC‐82 NFMLTQPHSVSESPGKTVTISCTRSSGSIATNYVHWYQQRPGSSPTTVICEDDQR 44 light PSGVPDRFSGSIDTSSNSASLTISGLRTEDEADYYCQSFDNINHYAIFGGGTTLTVL

HEPC‐84 EVQLVQSGGGLVQPGRSLRLSCTASGFNFGDFAMNWVRQAPGRGLEWVGIIRS 45 heavy NTYGGTTEYAASVRGRFSLSREDFKSIVHLQMNSLNTEDTAVYYCTRARAPITMIS

AELQKAYFYNGLDVWGQGTLVTVSS

HEPC‐84 DIQMTQSPSSLSASMGDRVTITCRASQSIDSYLNWYXQKPGKAPKLLIYAASNLQ 46 light SGVPSRFSGSDSGTTFTLTIISLQPEDFATYYCQQTYTTPLTFGGGTKVDIK

HEPC‐85 QVQLVQSGGGVVHPGSSLRLSCSASGFNFSAYGMHWVRQAPGKGPEWVAAIS 47 heavy FDGSHYFYADSVKGRFTISRDNSKNTLYLLTNSVRTEDTALYTCARHTNSYSWFDP

WGQGTLVTVSS

HEPC‐85 DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYVYWYLQKPGQFPQLLIYEV 48 light SSRLSGVPARFSGSGSGTDFTLKISRVETEDVGVYYCMQGIHLPPITFGQGTRLDIK

HEPC‐87 QVQLQESGPGMVKPSQTLSLTCTVSGASISRGGYYWTRIRQHPGKGLEWIGYIYY 49 heavy NGRTLYNPSLKSRVTISSDTSEDQFFLKLTSVTAADAAVYYCARGQMSSSWFVDY

WGQGTLGTVSS

HEPC‐87 NOT AVAILBLE 50 light

HEPC‐90 QVQLVQSGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVI 51 heavy WYDGFDKYYAESVKGRFTISRDNSRNLVFLQMNSLRAEDTAVYYCVRVRSAGTM IRAESASNAFDIWGQGTLVTVSS

HEPC‐90 EIVMTQSPATLSVSPGERATLSCRASQSVSNNLAWYQQRPGQAPRLLIYDASTRA 52 light TDIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPWTFGQGTKVEIK

HEPC‐91 QVQLVQSGGGVVQPGRSLRLSCAGSGFSFGDFGIHWVRQAPGKGPEWLSSISN 53 heavy DGSHIFYADSVEGRFTVSRDNSKSTVFLQMDRLIPDDSAFYYCARHSNTYQWFDP

WGQGTLVTVSS

HEPC‐91 DIVMTQTPLSLSVTPGQPASISCKSSRSLLHSDGKTYVYWYLQRPGQLPQLLIYEVS 54 light SRLSGVPDRFSGSGSGTDFTLEISRVEAEDVGIYYCVQGIHLPPITFGQGTRLDIK

HEPC‐96 QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQSPGKGLEWIGYIHS 55 heavy SGSTSYNPSLKSPVIILLDTSKNQLSLELSSVTAADTAVYYCANLDYSAYGSPRWFD

SWGQGTLVTVSS

HEPC‐96 EIVLTQSPGTLSLSPGERATLSCRASRSVGSRYLAWYQQKPGQAPRLLIYDASRRA 56 light TGIPDRFSGGGSGTDFTLTVSRLEPEDFAVYYCQQYGSSPLTFGGGTRLDIK

HEPC‐97 QVQLQQWGAGLLKPSETLSLTCAVYGGSLSGHYWSWIRQSPGKGLEWIGEINH 57 heavy NGGANYGPSLNSRATISVDTSKNQFFLNLRSVTAADTAVYYCARALPPYSSSSWF

DPWGQGTLVTVSS

HEPC‐97 NOT AVAILABLE 58 light

HEPC‐98 QVQLVQSGAEVKKPGSSVKVSCKASEGTLNSSAVNWVRQAPGQGLEWIGRIIPIF 59 heavy RATNYAQRFQDRVTITADDFTNTAYMEINSLRSEDTAVYYCARGTTEIWFDSWG

QGTLVTVSS

HEPC‐98 SYELTQPPSVSVSPGQTATITCSGDVLPQQFTYWYQQKSGQAPVLLIYRDIKRPSG 60 Light IAERFSAFTSGTSVTLIISGVQAEDEADYYCQSADISGTMAGFGGGTKLTVL

* * * * * * * * * * * * * * * * * 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 Hepatitis C 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 Hepatitis C virus in said sample by binding of said antibody or antibody fragment to a Hepatitis C 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, semen, cervical or vaginal secretions, amniotic fluid, placental tissues, urine, exudate, transudate, tissue scrapings or feces.

4. The method of claims 1-3, wherein detection comprises ELISA, RIA, lateral flow assay 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 Hepatitis C 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 Hepatitis C virus, or reducing the likelihood of infection of a subject at risk of contracting Hepatitis C 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, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

20. The method of claims 13-19, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab ^)2 fragment, or Fv fragment.

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, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

22. The method of claims 13-19, wherein said antibody is a chimeric antibody or a bispecific antibody.

23. The method of claim 13-22, wherein said antibody or antibody fragment is

administered prior to infection or after infection.

24. The method of claim 13-23, wherein said subject is a pregnant female, a sexually active female, or a female undergoing fertility treatments.

25. The method of claim 13-24, wherein delivering comprises antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

26. A monoclonal antibody, 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 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 26-31, wherein said antibody is a chimeric antibody, or is bispecific antibody.

34. The monoclonal antibody of claim 26-33, 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, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

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 the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab ^)₂ fragment, or Fv fragment.

44. The hybridoma or engineered cell of claim 36-43, wherein said antibody is a chimeric antibody or a bispecific antibody.

45. The hybridoma or engineered cell of claim 36-43, 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, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

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 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.

54. The vaccine formulation of claims 47-52, wherein at least one of said antibodies is a chimeric antibody, or is bispecific antibody.

55. The vaccine formulation of claims 47-54, 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, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

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 vaccine formulation comprising one or more expression vectors encoding a first antibody or antibody fragment according to claims 26-34.

58. The vaccine formulation of claim 57, wherein said expression vector(s) is/are Sindbis virus or VEE vector(s).

59. The vaccine formulation of claims 57-58, formulated for delivery by needle injection, jet injection, or electroporation.

60. The vaccine formulation of claim 57, further comprising one or more expression vectors encoding for a second antibody or antibody fragment, such as a distinct antibody or antibody fragment of claims 26-34.

61. A method of protecting the health of a placenta and/or fetus of a pregnant a subject infected with or at risk of infection with Hepatitis C 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.

62. The method of claim 61, the antibody or antibody fragment is encoded by clone-paired light and heavy chain variable sequences as set forth in Table 1.

63. The method of claim 61-62, 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.

64. The method of claim 61-62, 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.

65. The method of claim 61, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

66. The method of claim 61, 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.

67. The method of claim 61, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

68. The method of claims 61-67, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab ^)₂ fragment, or Fv fragment.

69. The method of claims 61-68, 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, to increase half-life and/or increase therapeutic efficacy, such as a LALA, N297, GASD/ALIE, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.

70. The method of claims 61-67 wherein said antibody is a chimeric antibody or a bispecific antibody.

71. The method of claim 61-70, wherein said antibody or antibody fragment is

administered prior to infection or after infection.

72. The method of claim 61-71, wherein said subject is a pregnant female, a sexually active female, or a female undergoing fertility treatments.

73. The method of claim 61-72, wherein delivering comprises antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

74. The method of claim 61, wherein the antibody or antibody fragment increases the size of the placenta as compared to an untreated control.

75. The method of claim 61, wherein the antibody or antibody fragment reduces viral load and/or pathology of the fetus as compared to an untreated control.

76. A method of determining the antigenic integrity, correct conformation and/or correct sequence of a Hepatitis C virus 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, correct conformation and/or correct sequence of said antigen by detectable binding of said first antibody or antibody fragment to said antigen.

77. The method of claim 76, wherein said sample comprises recombinantly produced antigen.

78. The method of claim 76, wherein said sample comprises a vaccine formulation or vaccine production batch.

79. The method of claims 76-78, wherein detection comprises ELISA, RIA, western blot, a biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometric staining.

80. The method of claims 76-79, wherein the first antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1.

81. The method of claims 76-79, 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.

82. The method of claims 76-79 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.

83. The method of claims 76-79, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

84. The method of claims 76-79, 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.

85. The method of claims 76-79, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences having 95% identity to clone-paired sequences from Table 2.

86. The method of claims 76-85, wherein the first antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab ^)₂ fragment, or Fv fragment.

87. The method of claims 76-86, further comprising performing steps (a) and (b) a second time to determine the antigenic stability of the antigen over time.

88. The method of claims 76-87, further comprising: (c) contacting a sample comprising said antigen with a second 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 second antibody or antibody fragment to said antigen.

89. The method of claim 88, wherein the second antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1.

90. The method of claim 88, 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.

91. The method of claim 88, 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.

92. The method of claims 88, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.

93. The method of claim 88, 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.

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

95. The method of claim 88, wherein the second antibody fragment is a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab ^)2 fragment, or Fv fragment.

96. The method of claim 88, further comprising performing steps (c) and (d) a second time to determine the antigenic stability of the antigen over time.