Virus-Like Particles Containing the E2 Core Domain of Hepatitis C Virus Generate Broadly Neutralizing Antibodies in Guinea Pigs.

A vaccine to prevent hepatitis C virus (HCV) infection is urgently needed for use alongside direct acting antiviral drugs to achieve elimination targets. We have previously shown that a soluble recombinant form of the glycoprotein E2 ectodomain (residues 384-661), that lacks three variable regions (Δ123) is able to elicit a higher titer of broadly neutralizing antibodies (bnAbs) in comparison to the parental form (receptor-binding domain; RBD). In this study, we engineered a viral nanoparticle that displays HCV glycoprotein E2 on a duck hepatitis B virus (DHBV) small surface antigen (S) scaffold. Four variants of E2-S virus-like particles (VLPs) were constructed: Δ123-S and RBD-S, and Δ123A7-S and RBDA7-S in which 7 cysteines were replaced with alanines. While all four E2-S VLPs display E2 as a surface antigen, the Δ123A7-S and RBDA7-S VLPs were the most efficiently secreted from transfected mammalian cells, and displayed epitopes recognized by cross-genotype broadly neutralizing monoclonal antibodies (bnmAbs). Both Δ123A7-S and RBDA7-S VLPs were immunogenic in guinea pigs, generating high titers of antibodies reactive to native E2 and able to prevent the interaction between E2 and the cellular receptor CD81. Four out of eight animals immunized with Δ123A7-S elicited neutralizing antibodies (nAbs), with three of those animals generating bnAbs against 7 genotypes. Immune serum generated by animals with nAbs mapped to major neutralization epitopes located at residues 412-420 (epitope I) and antigenic region 3. VLPs that display E2 glycoproteins represent a promising vaccine platform for HCV and could be adapted to large-scale manufacturing in yeast systems. IMPORTANCE There is currently no vaccine to prevent hepatitis C virus infection, which affects more than 71 million people globally and is a leading cause of progressive liver disease including cirrhosis and cancer. Broadly neutralizing antibodies that recognise the E2 envelope glycoprotein can protect against heterologous viral infection and correlate with viral clearance in humans. However, broadly neutralizing antibodies are difficult to generate due to conformational flexibility of the E2 protein and epitope occlusion. Here we show that a VLP vaccine using the duck hepatitis B virus S antigen fused to HCV glycoprotein E2 assembles into virus like particles that display epitopes recognised by broadly neutralizing antibodies and elicit such antibodies in guinea pigs. This platform represents a novel HCV vaccine candidate amenable to large-scale manufacture at low cost.

Sequential immunization with SARS-CoV-2 RBD vaccine induces potent and broad neutralization against variants in mice

The current COVID-19 pandemic caused by constantly emerging SARS-CoV-2 variants still poses a threat to public health worldwide. Effective next-generation vaccines and optimized booster vaccination strategies are urgently needed. Here, we sequentially immunized mice with a SARS-CoV-2 wild-type inactivated vaccine and a heterologous mutant RBD vaccine, and then evaluated their neutralizing antibody responses against variants including Beta, Delta, Alpha, Iota, Kappa, and A.23.1. These data showed that a third booster dose of heterologous RBD vaccine especially after two doses of inactivated vaccines significantly enhanced the GMTs of nAbs against all SARS-CoV-2 variants we tested. In addition, the WT and variants all displayed good cross-immunogenicity and might be applied in the design of booster vaccines to induce broadly neutralizing antibodies.

Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies has been limited by the continuous emergence of viral variants, and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on Omicron variant RBD recognized by broadly neutralizing antibodies. Based on this finding, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant RBD as revealed by Cryo-EM structures. This inhalable antibody exhibited exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. The structures also deciphered an uncommon cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.

Single-chain variable fragments of broadly neutralizing antibodies prevent HIV cell-cell transmission

Broadly neutralizing antibodies (bNAbs) are able to prevent HIV infection following passive administration. Single-chain variable fragments (scFv) may have advantages over IgG as their smaller size permits improved diffusion into mucosal tissues. We have previously shown that scFv of bNAbs retain significant breadth and potency against cell-free viral transmission in a TZM-bl assay. However, scFv have not been tested for their ability to block cell-cell transmission, a model in which full-sized bNAbs lose potency. We tested 4 scFv (CAP256.25, PGT121, 3BNC117 and 10E8v4) compared to IgG, in free-virus and cell-cell neutralization assays in A3.01 cells, against a panel of seven heterologous viruses. We show that free-virus neutralization titers in the TZM-bl and A3.01 assays were not significantly different, and confirm that scFv show a 1 to 32-fold reduction in activity in the cell-free model, compared to IgG. However, whereas IgG show 3.4 to 19-fold geometric mean potency loss in cell-cell neutralization compared to free-virus transmission, scFv had more comparable activity in the two assays, with only a 1.3 to 2.3-fold reduction. Geometric mean IC 50 of scFv for cell-cell transmission ranged from 0.65 μg/ml (10E8v4) to 2.3 μg/ml (3BNC117) with IgG and scFv neutralization showing similar potency against cell-associated transmission. Therefore, despite the reduced activity of scFv in cell-free assays, their retention of activity in the cell-cell format may make scFv useful for the prevention of both modes of transmission in HIV prevention studies. Importance Broadly neutralizing antibodies (bNAbs) are a major focus for passive immunization against HIV, with the recently concluded HVTN AMP (Antibody Mediated Protection) trial providing proof of concept. Most studies focus on cell-free HIV, however cell-associated virus may play a significant role in HIV infection, pathogenesis and latency. Single-chain variable fragments (scFv) of antibodies may have increased tissue penetration, and reduced immunogenicity. We previously demonstrated that scFv of four HIV-directed bNAbs (CAP256-VRC26.25, PGT121, 3BNC117 and 10E8v4) retain significant potency and breadth against cell-free HIV. As some bNAbs have been shown to lose potency against cell-associated virus, we investigated the ability of bNAb scFv to neutralize this mode of transmission. We demonstrate that unlike IgG, scFv of bNAbs are able to neutralize cell-free and cell-associated virus with similar potency. These scFv, which show functional activity in the therapeutic range, may therefore be suitable for further development as passive immunity for HIV prevention.

A recurring YYDRxG pattern in broadly neutralizing antibodies to a conserved site on SARS-CoV-2, variants of concern, and related viruses

Studying the antibody response to SARS-CoV-2 informs on how the human immune system can respond to antigenic variants as well as other SARS-related viruses. Here, we structurally and functionally characterized a potent human antibody ADI-62113 that also neutralizes SARS-CoV- 2 variants of concern and cross-reacts with many other sarbecoviruses. A YYDRxG motif encoded by IGHD3-22 in CDR H3 facilitates targeting to a highly conserved epitope on the SARS-CoV-2 receptor binding domain. A computational search for a YYDRxG pattern in publicly available sequences identified many antibodies with broad neutralization activity against SARS-CoV-2 variants and SARS-CoV. Thus, the YYDRxG motif represents a common convergent solution for the human humoral immune system to counteract sarbecoviruses. These findings also suggest an epitope targeting strategy to identify potent and broadly neutralizing antibodies that can aid in the design of pan-sarbecovirus vaccines and antibody therapeutics.

Structure of the hepatitis C virus E1E2 glycoprotein complex

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma in humans, and afflicts more than 58 million people worldwide. The HCV envelope E1 and E2 glycoproteins are essential for viral entry and infection, and comprise the primary antigenic target for neutralizing antibody responses. The molecular mechanisms of E1E2 assembly, as well as how the E1E2 heterodimer binds broadly neutralizing antibodies, remains elusive. We present the cryo-electron microscopy (cryoEM) structure of the membrane-extracted full-length E1E2 heterodimer in complex with broadly neutralizing antibodies (bNAbs) AR4A, AT12009 and IGH505 at ~3.5 Å resolution. We resolve the long sought-after interface between the E1 and E2 ectodomains and reveal how it is stabilized by hydrophobic interactions and glycans. This structure deepens our understanding of the HCV fusion glycoprotein and delivers a blueprint for the rational design of novel vaccine immunogens and anti-viral drugs.