Biology subjects

Giang, E.

Publications and source records attributed to Giang, E..

6 recordsLinked to original sources

bioRxiv2026

Distinct mechanisms of antibody-mediated HCV neutralization revealed by nanobody-guided epitope mapping

Hepatitis C virus (HCV) remains a major global health challenge despite the availability of highly effective antiviral therapies, underscoring the need for a broadly protective vaccine. The envelope glycoprotein E2 is the principal target of neutralizing antibodies, yet the full repertoire of vulnerable epitopes and mechanisms of antibody-mediated neutralization remains incompletely understood. Here, we exploited the unique binding properties of camelid nanobodies to probe the antigenic landscape of HCV E2 beyond the immunodominant human antibody response. We isolated a diverse panel of E2-specific nanobodies, including broadly neutralizing antibodies with high-affinity cross-reactivity toward genetically diverse HCV isolates. By combining cross-neutralization assays, competition binding experiments, and high-resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), we identified three mechanistically distinct classes of neutralizing epitopes. While one class targets the canonical E2 neutralization face, a second class recognizes antigenic region 1 (AR1), independently validating and extending recent evidence that this region represents a functional site of viral vulnerability. These findings demonstrate that broadly neutralizing antibody responses extend beyond the canonical neutralization face and establish a broader framework for understanding HCV neutralization. More broadly, our study illustrates how alternative antibody repertoires can reveal functionally important antigenic surfaces that are underrepresented in conventional human antibody responses, providing new opportunities for the rational design of next-generation HCV vaccine immunogens.

biochemistry↗

bioRxiv2025

The conserved bridging domain on HCV E1E2 glycoprotein complex is targeted by neutralizing antibodies from diverse lineages

The induction of potent and cross-reactive neutralizing antibody (nAb) responses remains a challenge in vaccine development against antigenically diverse viruses such as hepatitis C virus (HCV). The HCV E1E2 glycoprotein complex contains two major neutralizing sites: the neutralizing face (NF) and the less explored bridging domain (BD). Here, we characterized 25 BD-targeting nAbs isolated from infection or immunization. These antibodies arise from diverse B cell lineages but share convergent CDRH3 features. Epitope mapping by alanine scanning and negative-stain electron microscopy revealed overlapping epitopes on BD spanning antigenic regions AR4 and AR5, with variable back layer engagement. The crystal structure of a non-human primate BD nAb RM3-26 in complex with E2 uncovered a back layer-directed recognition mode analogous to that of the human nAb hcab40. Together, BD- and NF-directed nAbs exhibited additivity in their neutralization, highlighting BD as a conserved site of vulnerability on HCV and a valuable target for rational vaccine design.

immunology↗

bioRxiv2025

B cell transcriptomics reveals lasting dysregulation and rapid decline of protective memory after hepatitis C cure

O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/664545v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@d12426org.highwire.dtl.DTLVardef@9c54daorg.highwire.dtl.DTLVardef@186d559org.highwire.dtl.DTLVardef@120f2f3_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG HighlightsO_LIB cells in CHC patients retained dysregulated transcriptional profiles despite successful DAA treatment C_LIO_LIB cell dysregulation is marked by global B cell hyperactivation and antigen-specific atypical MBC expansion C_LIO_LISustained upregulation of TNF- signaling via NF-{kappa}B (TNF-/NF-{kappa}B) is a central driver of persistent B cell dysregulation and chronic inflammation C_LIO_LIViral clearance leads to restoration of IFN responses and IFN-stimulated gene (ISG) signatures in B cells but not TNF-/NF-{kappa}B C_LIO_LIHCV IGHV1-69 bnAb-producing B cells are enriched within the CD86hi IgG MBC subset before treatment C_LIO_LICD86hi IgG MBC subset contracts rapidly post-cure in parallel with the rapid decline of cross-nAb responses and loss of protective immune memory against HCV reinfection C_LI Chronic hepatitis C (CHC) disrupts host humoral immune response by impairing the timely generation of neutralizing antibody (nAb) and durable immune memory. However, the underlying mechanisms and their reversibility after viral clearance remain poorly defined. Here, through integrated single-cell transcriptomics and antibody repertoire characterization, we show that B cells from CHC patients retain transcriptional dysregulation even after successful antiviral therapy. Sustained TNF- signaling emerged as a central driver of chronic B cell hyperactivation, persistent dysregulation and unresolved inflammation following cure. Furthermore, a CD86hi memory B cell subset, responsible for an IGHV1-69-encoded multi-donor class recall nAb response, declined rapidly following viral clearance, compromising immune memory against reinfection. Together, these findings reveal how CHC imprints lasting B cell dysregulation, impairs nAb memory, and sustains inflammation in the B cell compartment, after viral clearance. The insights underscore the need for strategies aimed at restoring B cell homeostasis to achieve durable immune protection.

immunology↗

bioRxiv2025

Structural and genetic signatures of two classes of HCV E2 neutralizing face antibodies from non-human primates immunized with a recombinant E1E2

Hepatitis C continues to be a significant public health problem despite advancements in antiviral therapeutics. To eliminate this disease, an effective vaccine against new infections and re-infections is needed. However, to date only one Hepatitis C virus (HCV) envelope protein (E1E2) immunogen, developed by Chiron Inc., has been tested in a Phase I clinical trial (ClinicalTrials.gov identifier NCT00500747). To establish a benchmark for elicitation of broadly neutralizing antibodies (bnAbs) by E1E2, we previously immunized non-human primates (NHPs) with this immunogen and isolated monoclonal nAbs that exhibit neutralization potency comparable to human nAbs. Here we show that NHP nAbs, encoded by germline genes IGHV1-138*01 and IGHV4-NL_5*01 (homologs of human IGHV1-69*10 and IGHV4-59*12, respectively), recognize a relatively E2 conserved region (neutralizing face) proximal to antigenic region 3 (AR3). These NHP AR3-targeting nAbs share highly similar binding modes to human AR3-targeting nAbs, suggesting a similarity in human and NHP immune responses to the same HCV immunogen.

immunology↗

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↗

bioRxiv2019

Rational design of hepatitis C virus E2 core nanoparticle vaccines

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are critical for cell entry with E2 being the major target of neutralizing antibodies (NAbs). Here, we present a comprehensive strategy for B cell-based HCV vaccine development through E2 optimization and nanoparticle display. We redesigned variable region 2 in a truncated form (tVR2) on E2 cores derived from genotypes 1a and 6a, resulting in improved stability and antigenicity. Crystal structures of three optimized E2 cores with human cross-genotype NAbs (AR3s) revealed how the modified tVR2 stabilizes E2 without altering key neutralizing epitopes. We then displayed these E2 cores on 24- and 60-meric nanoparticles and achieved high yield, high purity, and enhanced antigenicity. In mice, these nanoparticles elicited more effective NAb responses than soluble E2 cores. Next-generation sequencing (NGS) defined distinct B-cell patterns associated with nanoparticle-induced antibody responses, which cross-neutralized HCV by targeting the conserved neutralizing epitopes on E2.\n\nOne Sentence SummaryAn HCV vaccine strategy is presented that displays redesigned E2 cores on nanoparticles as vaccine candidates for eliciting a broadly neutralizing B-cell response.

microbiology↗