Janus, B. M. | Search bioRxiv

bioRxiv2025

Native-like soluble E1E2 glycoprotein heterodimers on self-assembling protein nanoparticles for hepatitis C virus vaccine design

Hepatitis C virus (HCV) is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. E1E2-based HCV vaccine development has been hindered by the challenge of producing a soluble E1E2 (sE1E2) antigen that faithfully recapitulates the native glycoprotein heterodimer found on virions. Based on available cryo-electron microscopy (cryo-EM) structures, we rationally engineered sE1E2 for genotype 1a H77 by truncating the E1 and E2 stems (Cut1), removing a putative fusion peptide (pFP)-containing region in E1 (Cut2), and stabilizing the E1-E2 interface with diverse heterodimeric scaffolds. All H77 sE1E2.Cut1+2 scaffolds showed native-like E1-E2 association and robust binding to the broadly neutralizing antibody (bNAb) AR4A. A genotype 1a HCV-1 sE1E2.Cut1+2 variant scaffolded by a modified SpyTag/SpyCatcher (SPY{Delta}N) was selected for in vitro, structural, and immunogenic characterization. The structure of this sE1E2 scaffold in complex with bNAbs was analyzed by cryo-EM and negative-stain EM (nsEM), with an nsEM-based approach developed for antibody epitope mapping. HCV-1 sE1E2.Cut1+2.SPY{Delta}N was displayed on self-assembling protein nanoparticles (SApNPs) to enhance immunogenicity. HCV-1 sE1E2.Cut1+2.SPY{Delta}N heterodimer and SApNPs with wildtype and modified glycans were tested in mice, revealing the beneficial effects of multivalent display and oligomannose enrichment. Our study provides a rigorous foundation for next-generation HCV vaccine development. ONE-SENTENCE SUMMARYRational design, characterization, and in vivo assessment of HCV soluble E1E2 heterodimer and nanoparticles will inform vaccine development.

microbiology↗

bioRxiv2023

Cross-reactive macaque antibodies targeting marburgvirus glycoprotein induced by multivalent immunization

We utilized B cells from a Rhesus macaque immunized with a multivalent prime-boost regimen of filovirus antigens to isolate a novel panel of marburgvirus glycoprotein (GP)-specific monoclonal antibodies (mAbs). A heterologous marburgvirus GP probe was used to sort for B cells with cross-marburgvirus reactive breadth. 33 mAbs belonging to 28 unique lineages were expressed and experimentally characterized. Antibody specificities were assessed by binding competition and overlapping pepscan analyses, and were found to map to a previously characterized protective region on GP2 and a cross-filovirus reactive region on GP1, among others. A third of the lineages targeted the predicted receptor binding region (RBR), including two lineages with potent Marburg pseudovirus neutralization that were structurally analyzed and confirmed to recognize this region. Our study describes the discovery and characterization of a diverse panel of antibodies against marburgvirus GP induced by multivalent immunization and provides candidate immunotherapeutics for further study and development. Author SummaryMarburgviruses were the first filoviruses characterized to emerge in humans in 1967, and have led to multiple outbreaks since then with average case fatality rates of [~]50%. Although a vaccine and monoclonal antibody countermeasures have been approved for clinical use against the related Ebola viruses, these are ineffective against marburgviruses or other filoviruses. As such, gaps exist in the clinical toolkit against filoviruses, in particular marburgviruses. Here, we isolated and characterized a novel panel of monoclonal antibodies directed against the marburgvirus surface glycoprotein from an immunized Rhesus macaque. We utilized an antibody isolation method that ensured broad antibody recognition across multiple marburgvirus isolates. Functional and structural analyses revealed that roughly half of the antibodies in the panel mapped to regions on the glycoprotein shown previously to protect from infection, including the receptor binding domain and a protective region on the membrane-anchoring subunit, while a quarter of the antibodies did not fall into any known binding competition group indicating potential novel specificities. Our study advances the understanding of marburgvirus glycoprotein antigenicity and furthers efforts to develop candidate antibody countermeasures against these lethal viruses.

immunology↗