Design of a native-like secreted form of the hepatitis C virus E1E2 heterodimer

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Hepatitis C virus (HCV) is a global disease burden, with an estimated 71 million people infected worldwide (1, 2). Roughly 75% of HCV infections become chronic (3–5) and in severe cases can result in cirrhosis or hepatocellular carcinoma (6). Viral infection can be cured at high rates by direct-acting antivirals, but multiple public health and financial barriers (7, 8), along with the possibility of reinfection or continued disease progression (7, 9, 10), have resulted in a continued rise in HCV infections. An HCV vaccine remains essential to proactively protect against viral spread, yet vaccine developments against the virus have been unsuccessful to date (11, 12). The challenges posed by HCV sequence diversity (12, 13), glycan shielding (14, 15), immunodominant nonneutralizing epitopes (16–19), and preparation of a homogeneous E1E2 antigen all contribute to the difficulty in generating protective B cell immune responses. Although multiple studies in chimpanzees and humans have used E1E2 formulations to induce a humoral immune response, their success in generating high titers of broadly neutralizing antibody (bnAb) responses has been limited (20). Optimization of E1E2 to improve its immunogenicity and elicitation of bnAbs through rational design may lead to an effective B cell-based vaccine (21).HCV envelope glycoproteins E1 and E2 form a heterodimer on the surface of the virion (22–24). Furthermore, E1E2 assembly has been proposed to form a trimer of heterodimers (25) mediated by hydrophobic C-terminal transmembrane domains (TMDs) (24, 26, 27) and interactions between E1 and E2 ectodomains (28–30). These glycoproteins are necessary for viral entry and infection, as E2 attaches to the CD81 and SR-B1 coreceptors as part of a multistep entry process on the surface of hepatocytes (31–34). Neutralizing antibody responses to HCV infection target epitopes in E1, E2, or the E1E2 heterodimer (18, 35–40). Structural knowledge of bnAb antibody–antigen interactions, which often target E2 epitopes in distinct antigenic domains B, D, or E (18, 41, 42), can inform vaccine design efforts to induce bnAb responses against flexible HCV epitopes (43–45). E1E2 bnAbs, including AR4A, AR5A (46), and others recently identified (38), are not only among the most broadly neutralizing (35) but also represent E1E2 quaternary epitopes unique to antibody recognition of HCV.Although much is known about bnAb responses to E1E2 glycoproteins, induction of B cell-based immunity with an E1E2-based vaccine immunogen (47–49) has remained difficult. The inherent hydrophobicity of E1 and E2 TMDs (24, 50) may impede uniform production of an immunogenic E1E2 heterodimer that could be utilized for both vaccine development and E1E2 structural studies. Although partial E1 and E2 structures have been determined (39, 51–54), many other enveloped viruses have structures of a complete and near-native glycoprotein assembly (55–59), providing a basis for rational vaccine design (60–62). Viral glycoproteins of influenza hemagglutinin (63), respiratory syncytial virus (RSV) (55), severe acute respiratory syndrome coronavirus 2 (64), and others (65, 66) have been stabilized in soluble form using a C-terminal attached foldon trimerization domain to facilitate assembly. HIV gp120–gp41 proteins have been designed as soluble SOSIP trimers in part by introducing a furin cleavage site to facilitate native-like assembly when cleaved by the enzyme (56, 67). Previously described E1E2 glycoprotein designs include covalently linked E1 and E2 ectodomains (68, 69), E1E2 with TMDs intact and an immunoglobulin G (IgG) Fc tag for purification (70), as well as E1 and E2 ectodomains with a cleavage site (68), which presented challenges for purification either due to intracellular expression or to high heterogeneity. Two recently described scaffolded E1E2 designs, while promising, have not been shown to engage monoclonal antibodies (mAbs) that recognize the native E1E2 assembly, though they were engaged by E1-specific and E2-specific mAbs, as well as coreceptors that recognize E2 (71). Therefore, these presentations of E1E2 glycoproteins may not represent a native and immunogenic heterodimeric assembly, and thus their potential as vaccine candidates remains unclear.Here, we describe the design of a secreted E1E2 glycoprotein (sE1E2) that mimics both the antigenicity in vitro and the immunogenicity in vivo of the native heterodimer through the scaffolding of E1E2 ectodomains. In testing our designs, we found that both replacing E1E2 TMDs with a leucine zipper scaffold and inserting a furin cleavage site between E1 and E2 enabled secretion and native-like sE1E2 assembly. We assessed the size, heterogeneity, antigenicity, and immunogenicity of this construct (identified as sE1E2.LZ) in comparison with full-length membrane-bound E1E2 (mbE1E2). sE1E2.LZ binds a broad panel of bnAbs to E2 and E1E2, as well as coreceptor CD81, providing evidence of assembly into a native-like heterodimer. An immunogenicity study indicated that sera of mice injected with sE1E2.LZ neutralize HCV pseudoparticles at levels comparable to sera from mice immunized with mbE1E2. This sE1E2 design is a form of the native E1E2 heterodimer that both improves upon current designs and represents a platform for structural characterization and engineering of additional HCV vaccine candidates.