A Diverse Panel of Hepatitis C Virus Glycoproteins for Use in Vaccine Research Reveals Extremes of Monoclonal Antibody Neutralization Resistance

Abstract

Despite significant advances in the treatment of hepatitis C virus (HCV) infection, the need to develop preventative vaccines remains. Identification of the best vaccine candidates and evaluation of their performance in preclinical and clinical development will require appropriate neutralization assays utilizing diverse HCV isolates. We aimed to generate and characterize a panel of HCV E1E2 glycoproteins suitable for subsequent use in vaccine and therapeutic antibody testing. Full-length E1E2 clones were PCR amplified from patient-derived serum samples, cloned into an expression vector, and used to generate viral pseudoparticles (HCVpp). In addition, some of these clones were used to generate cell culture infectious (HCVcc) clones. The infectivity and neutralization sensitivity of these viruses were then determined. Bioinformatic and HCVpp infectivity screening of approximately 900 E1E2 clones resulted in the assembly of a panel of 78 functional E1E2 proteins representing distinct HCV genotypes and different stages of infection. These HCV glycoproteins differed markedly in their sensitivity to neutralizing antibodies. We used this panel to predict antibody efficacy against circulating HCV strains, highlighting the likely reason why some monoclonal antibodies failed in previous clinical trials. This study provides the first objective categorization of cross-genotype patient-derived HCV E1E2 clones according to their sensitivity to antibody neutralization. It has shown that HCV isolates have clearly distinguishable neutralization-sensitive, -resistant, or -intermediate phenotypes, which are independent of genotype. The panel provides a systematic means for characterization of the neutralizing response elicited by candidate vaccines and for defining the therapeutic potential of monoclonal antibodies.IMPORTANCE Hepatitis C virus (HCV) has a global burden of more than 170 million people, many of whom cannot attain the new, expensive, direct-acting antiviral therapies. A safe and effective vaccine that generates both T cell responses and neutralizing antibodies is required to eradicate the disease. Regions within the HCV surface glycoproteins E1 and E2 are essential for virus entry and are targets for neutralizing antibodies. Screening of vaccine candidates requires suitable panels of glycoproteins that represent the breadth of neutralization resistance. Use of a standard reference panel for vaccine studies will ensure comparability of data sets, as has become routine for HIV-1. Here, we describe a large panel of patient-derived HCV glycoproteins with an assessment of their neutralization sensitivity to defined monoclonal antibodies, which has enabled us to predict their likely efficacy in the wider HCV-infected population. The panel could also be important for future selection of additional therapeutic antibodies and for vaccine design.

Hepatitis C virus resistance to broadly neutralizing antibodies measured using replication-competent virus and pseudoparticles

“…Our work supports prior more limited studies showing that HCVcc and HCVpp with identical E1E2 have similar relative neutralization resistance (Bailey et al, 2015a;Fofana et al, 2012;Swann et al, 2016;Urbanowicz et al, 2015). Importantly, to our knowledge, this is the first study to show this correlation using a diverse panel of human bNAbs targeting distinct epitopes across E1E2 and a large panel of diverse natural E1E2 variants, suggesting that these results are broadly applicable to other E1E2 variants and other antibodies.…”

“…Importantly, to our knowledge, this is the first study to show this correlation using a diverse panel of human bNAbs targeting distinct epitopes across E1E2 and a large panel of diverse natural E1E2 variants, suggesting that these results are broadly applicable to other E1E2 variants and other antibodies. This work also increases the impact of prior studies showing in both HCVcc and HCVpp model systems that different E1E2 variants show very different sensitivities to antibody neutralization (Keck et al, 2012;Urbanowicz et al, 2015;Wasilewski et al, 2016).…”

“…Neutralization results obtained using HCVcc and HCVpp have generally been similar (Fofana et al, 2012;Meunier et al, 2005;Scheel et al, 2008;Swann et al, 2016;Urbanowicz et al, 2015), but comparisons of specific infectivity and neutralization of HCVpp and HCVcc expressing genetically identical E1E2 variants have previously been performed only on a fairly limited basis, and to our knowledge neutralization of E1E2-matched HCVpp and HCVcc has never been compared using a diverse panel of bNAbs targeting distinct epitopes.…”

“…HCVpp can be used to quantitate single round entry and neutralization in a high-throughput format. Recently, large panels of HCVpp have been produced that encompass more of the diversity of circulating HCV variants, which is critical for accurate measurement of antibody neutralizing breadth (Osburn et al, 2014;Urbanowicz et al, 2015). While significantly fewer replication-competent HCVcc variants are available, progress has been made in expanding the number of variants (Mathiesen et al, 2014;McClure et al, 2016;Urbanowicz et al, 2015).…”

“…Recently, large panels of HCVpp have been produced that encompass more of the diversity of circulating HCV variants, which is critical for accurate measurement of antibody neutralizing breadth (Osburn et al, 2014;Urbanowicz et al, 2015). While significantly fewer replication-competent HCVcc variants are available, progress has been made in expanding the number of variants (Mathiesen et al, 2014;McClure et al, 2016;Urbanowicz et al, 2015). E1E2 on surface of HCVcc may resemble more closely the envelopes of viruses circulating in vivo, given incorporation of human apolipoproteins into HCVcc virions (Hishiki et al, 2010;Jiang et al, 2012;Meunier et al, 2008).…”