Global mapping of antibody recognition of the hepatitis C virus E2 glycoprotein: Implications for vaccine design

Abstract

The E2 envelope glycoprotein is the primary target of human neutralizing antibody response against hepatitis C virus (HCV), and is thus a major focus of vaccine and immunotherapeutics efforts. There is emerging evidence that E2 is a highly complex, dynamic protein with residues across the protein that are modulating antibody recognition, local and global E2 stability, and viral escape. To comprehensively map these determinants, we performed global E2 alanine scanning with a panel of 16 human monoclonal antibodies (hmAbs), resulting in an unprecedented dataset of the effects of individual alanine substitutions across the E2 protein (355 positions) on antibody recognition. Analysis of shared energetic effects across the antibody panel identified networks of E2 residues involved in antibody recognition and local and global E2 stability, as well as predicted contacts between residues across the entire E2 protein. Further analysis of antibody binding hotspot residues defined groups of residues essential for E2 conformation and recognition for all 14 conformationally dependent E2 antibodies and subsets thereof, as well as residues that enhance antibody recognition when mutated to alanine, providing a potential route to engineer E2 vaccine immunogens. By incorporating E2 sequence variability, we found a number of E2 polymorphic sites that are responsible for loss of neutralizing antibody binding. These data and analyses provide fundamental insights into antibody recognition of E2, highlighting the dynamic and complex nature of this viral envelope glycoprotein, and can serve as a reference for development and rational design of E2-targeting vaccines and immunotherapeutics.alanine scanning | immune recognition | HCV | clustering | hotspots H epatitis C virus (HCV) infects ∼185 million of the world's population, with 3-4 million new infections each year. Infection often leads to chronic hepatitis, cirrhosis, and hepatocellular carcinoma, and is a leading reason for liver transplantation (1). Despite recently developed direct-acting antiviral agents, there is a major need for a preventive HCV vaccine, because of the high cost of treatment therapies-which limit their clinical use-a high rate of asymptomatic and untreated infected individuals (over 95% of the infected population) (2, 3), concern of viral resistance to direct-acting antiviral agents (4), and that treatment-induced cure in patients with established cirrhosis does not eliminate the risk of hepatocellular carcinoma (5).A major obstacle to HCV vaccine development efforts is the extreme diversity of the virus and its high rate of mutation, which allows it to actively evade the immune response in infected individuals. Critical to the development of an effective vaccine is the identification and characterization of conserved epitopes associated with viral neutralization. The antibody response to HCV is directed primarily against the E2 glycoprotein because E2 directly interacts with the HCV coreceptors, scavenger receptor class B type 1 (SR-B1) (6) and the tetraspan...

Complete mapping of viral escape from neutralizing antibodies

“…However, there are ∼10 4 single amino-acid mutants to a 500-residue viral protein, so individually creating and testing all of them is a daunting task. Therefore, even the most ambitious such studies limit themselves to a small fraction of the possible point mutations, such as by only testing mutations to alanine [13–15]. But as the current work will underscore, the antigenic effect of mutating a residue to one amino acid can be poorly predictive of the effects of mutating the same residue to another amino acid.…”

Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization

“…Another design strategy tested in this study was hyperglycosylation, through structure-based addition of N-glycan sequons to mask antigenic domain A, which is associated with nonneutralizing antibodies (25,26,28,42). The concept of down-modulating immunity to this region was based on the observation that this region is highly immunogenic and may divert antibody responses to bNAb epitopes of lower immunogenicity.…”

“…We selected one of these substitutions, H445P, that is adjacent to core contact residues for domain D located at aa 442-443 (32) for subsequent experimental characterization, due to its position in a region with no secondary structure, and location between residues Y443 and K446 which both make key antibody contacts in domain D antibody complex structures (31,32). This also represents a distinct region of the epitope from a substitution that we previously described and tested (A439P) (28).…”

“…Based on the intrinsic flexibility of the neutralizing face of E2 (27), which includes antigenic domain D, and on the locations of bNAb epitopes to this domain (24), we identified a structure-based design substitution to reduce the mobility of that region and preferentially form the bnAbbound conformation. We also tested several substitutions to hyperglycosylate and mask antigenic domain A located in a unique region on the back layer of E2, as determined by fine epitope mapping (28), which represents an approach that has been applied to mask epitopes in influenza (29) and HIV (30) glycoproteins. Designs were tested for antigenicity using a panel of monoclonal antibodies (mAbs), and selected designs were tested individually and as combinations for in vivo immunogenicity.…”

Complete mapping of viral escape from neutralizing antibodies

“…However, there are *10 4 single amino-acid mutants to a 500-residue viral protein, so individually creating and testing all of them is a daunting task. Therefore, even the most ambitious such studies limit themselves to a small fraction of the possible point mutations, such as by only testing mutations to alanine [13][14][15]. But as the current work will underscore, the antigenic effect of mutating a residue to one amino acid can be poorly predictive of the effects of mutating the same residue to another amino acid.…”