Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus

Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation.Hepatitis C virus (HCV) is a major cause of liver failure and infects more than 170 million people worldwide. HCV is a member of the Flaviviridae family and contains a 9.6-kb positive-strand RNA genome. The genome is translated into a single polypeptide that is cleaved by viral and cellular proteases into at least nine different proteins. The major HCV surface glycoproteins, E1 and E2, form a noncovalent heterodimer on the virion surface (23) and are believed to mediate viral entry via a complex set of poorly understood interactions with cellular coreceptors, including CD81 (28), claudin-1 (8), occludin (29), scavenger receptor class B type I (30), and others (38). The E2 glycoprotein has been shown to interact directly with receptors (38); currently, no function has been assigned to E1, although it is known to be required for viral infection. These viral glycoproteins provide an obvious target for neutralizing monoclonal antibodies (MAbs).Isolation of potently neutralizing HCV-specific MAbs has been complicated by the lack of an in vitro cell culture system to study the full infection cycle of the virus. Recently, systems have been developed that allow for the generation of infectious viral particles, highlighting the importance of E1 and E2 in viral binding and entry. A novel in vitro infection system employs HCV pseudotyped viral particles (HCVpp) generated from a lentivirus that are devoid of native glycoproteins and engineered to contain HCV glycoproteins E1 and E2 (4, 15). HCVpp specifically infect cell lines derived from human liver cells and can be neutralized by polyclonal and MAbs directed against the HCV envelope glycoproteins.HCVpp have allowed the identification of antibodies that can neutralize HCV infection in cell culture. E1 has proven to be a difficult target for MAb-mediated neutralization, possibly because it appears to have low immunogenicity (32), has no identified binding proteins on the cell surface, and has an undefined role in cell entry. Despite this challenge, two groups have identified HCV neutralizing MAbs directed to E1: these MAbs are H-111, which has moderate neutralizing activity (17), and the recently isolated IGH505 and IGH526, which neutralize numerous HCV genotypes (1a, 1b, 2a, 4a, 5a, and 6a but not 2b and 3a) (22). Although they are predicted to inhibit viral binding or fusion, the mechanism by which these E1-directed MAbs neutralize HCV infection is unclear.A diverse group of mouse anti-E2 antibodies, recognizing both linear and discontinuous epitopes, has been generated. Many of these MAbs showed broad neutralization of multiple HCV genotypes, but not surprisingly, several HCV isolates were refractory to neutralization. In contrast, AP33, a mouse MAb that largely recognizes a highly conserved linear epitope in the N terminus of E2 (amino acids 412 to 423), was identified as a broadly cross-reactive antibody that neutralized strains from all genotypes tested (1a, 1b, 2a, 2b, 3a, 4, 5, and 6), with the exception of one genotype 5 virus (UKN5.14.4; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY894682","term_id":"58220837","term_text":"AY894682"}}AY894682) (24). Recently, several cross-reactive neutralizing MAbs have been identified that are of human origin and have the capacity to neutralize a significant fraction of the genotypes tested (1, 5, 12, 13, 27, 31) or to neutralize all genotypes tested (16, 20, 25). As with the vast majority of previously described human MAbs (HuMAbs), these MAbs recognize conformation-dependent epitopes of E2. One broadly neutralizing human antibody, AR3B, was tested in a mouse model of infection and showed significant protection from viremia (20). Given the known function of the E2 envelope glycoprotein, the high level of immunogenicity, the surface vulnerability, and the abundance of data pertaining to E2 and HCV neutralization, E2 provides a promising target for the development of fully human neutralizing antibodies.Liver deterioration due to HCV infection is the leading reason for liver transplantation in the United States. Unfortunately, it is highly likely that the transplanted liver will also become infected with HCV, and 10 to 25% of these patients develop cirrhosis within 5 years of transplant (9, 40). Here we describe the characterization of HuMAbs directed against the HCV E2 envelope glycoprotein, generated using transgenic mice. Based on epitope conservation and broad neutralization capacity, HuMAbs HCV1 and 95-2 provide excellent candidates for prevention of graft reinfection of HCV-infected individuals undergoing liver transplantation.