Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

Abstract

Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world's population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen-deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8°C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.hepatitis C virus | E2 | CD81-binding site | conformational flexibility | protein dynamics H epatitis C virus (HCV) is a leading cause of liver cirrhosis and hepatocellular carcinoma, infecting more than 2% of the world's population (1). HCV infection is highly prevalent in developing countries and among marginalized populations, such as injection drug users (IDUs) and prisoners in developed countries (2). Effective direct-acting antiviral (DAA) drugs have been developed recently to curb the advance of HCV (3, 4). Nevertheless, new infections are on the rise among young IDUs in developed countries and along drug-trafficking routes (2, 5, 6). Because DAA treatment is prohibitively expensive and does not prevent reinfection (7,8), an effective vaccine is essential for management of the global HCV epidemic (9-11).Despite the significant public health burden, no effective prophylactic vaccine against HCV has been developed. High genetic variability of the virus is a major barrier, particularly in the E1 and E2 envelope glycoproteins that are the primary neutralizing antibody (NAb) targets. E2, the receptor-binding protein, mediates cell entry by interacting with the tetraspanin CD81 and several other cell surface molecules (12, 13). The crystal structure of the E2 core domain (E2c) consists of a central β-sandwich flanked by "front" and "back" layers (14) (Fig. 1). The CD81-binding site (CD81bs) has been mapped by mutagenesis and electron microscopy (EM) to various elements: a conserved N-terminal...

An Optimized Hepatitis C Virus E2 Glycoprotein Core Adopts a Functional Homodimer That Efficiently Blocks Virus Entry

“…The broad conservation of the Δ123 protein across diverse isolates of HCV as well as its more accessible or optimal CD81-LEL binding site configuration in the absence of the variable loops is predicted to favor a protective response against infection. The reduced conformational flexibility of the Δ123 ectodomain proposed here may further limit the presentation of nonneutralizing epitopes, as a remarkable degree of structural flexibility recently reported within the CD81-binding site has been proposed to account for nonneutralizing antibody responses to this otherwise conserved, immunogenic face of E2 (27). The Δ123 protein may also limit the presentation of nonneutralizing domain A epitopes which are exposed at post-CD81 binding stages in virus entry and represents an antigenic cluster accounting for a substantial portion of isolate-specific antibody responses to E2 (13).…”

“…Hypervariable region 2 (HVR2; residues 460 to 485) and the intergenotypic variable region (igVR; residues 570 to 580) display 20% and 0% amino acid identity between HCV genotypes, respectively, yet each retains a highly conserved N-linked glycosylation site (25).We have also recently reported that the igVR is under considerable in vivo immune selection pressure, by using a longitudinal analysis of glycoprotein sequence evolution in genotype 3a-infected individuals (26). In addition to amino acid variability, a high degree of structural flexibility recently reported within the variable regions as well as the conserved CD81-binding site has been predicted to account for a significant proportion of the nonneutralizing antibody response to E2 (27).…”

Synergistic anti-HCV broadly neutralizing human monoclonal antibodies with independent mechanisms

“…These contrasting results may be the result of greater spatial separation between E2-bound HEPC98 and HEPC74 relative to E2-bound H77.16 and HC-11. This model is plausible, since recent studies have suggested that this region of E2 is highly flexible (39,40), so NAbs with overlapping epitopes might bind to different epitope conformations or bind with different angles of approach. The structural relationship between these adjacent neutralizing epitopes certainly warrants further investigation.…”

The core domain of hepatitis C virus glycoprotein E2 generates potent cross‐neutralizing antibodies in guinea pigs

“…The N‐terminal hypervariable region (HVR) 1 (HVR1) elicits type‐specific NAbs that drive immune escape, occludes NAb epitopes, and confers resistance to neutralization by chronic immune sera and NMAbs . HVR2 appears to be in a surface‐exposed flexible region, and the intergenotypic variable region (igVR or VR3) is located within a disulfide‐constrained loop and is also highly flexible . Deletion of all three variable regions from a recombinant subdomain of E2 (residues 384‐661) results in the expression of a soluble E2 core domain (Δ123; Fig.…”