Biology subjects

Gillespie, R. A.

Publications and source records attributed to Gillespie, R. A..

16 recordsLinked to original sources

bioRxiv2025

Stabilization of H5 highly pathogenic avian influenza hemagglutinin improves vaccine-elicited neutralizing antibody responses

Transmission of highly pathogenic avian influenza from H5 clade 2.3.4.4b has expanded in recent years to infect large populations of birds and mammals, heightening the risk of a human pandemic. Influenza viruses adapted to transmission in birds and some other animals tend to have a less stable hemagglutinin (HA) than seasonal influenza viruses, enabling membrane fusion at comparatively high pH levels. Here, we combine five mutations within H5 HA that dramatically increase its melting temperature and promote stable closure of the HA trimer. Structural analysis by cryo-electron microscopy revealed that the stabilizing mutations create several new hydrophobic interactions, while maintaining local HA structure. We found that vaccinating mice with stabilized H5 HA immunogens resulted in higher hemagglutination inhibition and neutralization titers than non-stabilized comparators. Epitope mapping of vaccine-elicited polyclonal antibody responses using negative stain electron microscopy and deep mutational scanning showed that site E on the side of the HA receptor binding domain was immunodominant across all groups; however, the stabilized immunogens shifted responses toward the receptor binding site (RBS), eliciting a higher proportion of neutralizing antibodies. These findings highlight that H5 HA-stabilizing mutations enhance the quality of antibody responses across different vaccine formats, underscoring their potential to improve pandemic preparedness vaccines targeting viruses from this widely circulating clade. One Sentence SummaryVaccinating with stabilized H5 HA increases the proportion of humoral responses against neutralizing RBS epitopes.

immunology↗

bioRxiv2025

Isolation of genetically diverse influenza antibodies highlights the role of IG germline gene variation and informs the design of population-comprehensive vaccine strategies

The regular emergence of influenza strains with pandemic potential creates a strong incentive to develop vaccines that stimulate protective responses across all human populations. A critical consideration is how variation in the human immunoglobulin (IG) loci influences B cell recognition of viral epitopes and elicitation of neutralizing antibodies. Here, we applied personalized IG germline genotyping and high-throughput sequencing of paired antibody chains from influenza A virus hemagglutinin (HA)-binding B cells to demonstrate that the response to HA is highly individual. We show that a germline-encoded polymorphism in IGHV2-70 alters the functionality of the LPAF-a class of neutralizing antibodies, and we describe HA stem-targeting broadly neutralizing antibodies (bNAbs) that use germline IGHV genes other than the population-restricted IGHV genes used by many previously known stem bNAbs. Our results demonstrate that the approach used here can be used to discover and avert population vulnerabilities arising from IG gene variation when designing HA-based influenza vaccines aimed for the global human population.

immunology↗

bioRxiv2025

Functional, Immunogenetic, and Structural Convergence in Influenza Immunity between Humans and Macaques

Human B cell immunity to the influenza hemagglutinin (HA) stem region, a universal influenza vaccine target, is often stereotyped and immunogenetically restricted, posing challenges for study outside humans. Here, we show that macaques vaccinated with a HA stem immunogen elicit human-like public B cell lineages targeting two major conserved sites of vulnerability, the central stem and anchor epitopes. Central stem antibodies were predominantly derived from VH1-138, the macaque homolog of human VH1-69, a VH-gene preferentially used in human central stem broadly neutralizing antibodies (bnAbs). Similarly, macaques produced anchor bnAbs with the human-like NWP motif. Both bnAb lineages were functionally and structurally analogous to their human counterparts, with recognition mediated largely by germline-encoded motifs. Thus the macaque immunoglobulin repertoire supports human-like public bnAb responses to influenza HA. Moreover, this underscores the utility of homologous germline-encoded immunity, suggesting that immune repertoires of macaques and humans may have been similarly shaped during evolution. HIGHLIGHTS[bullet] Functional human-like public antibody lineages can be elicited to HA stem supersites in macaques. [bullet]Macaque central stem bnAbs are predominantly derived from VH1-138, a VH-gene homologous to human VH1-69. [bullet]The human-like CDR L3 NWP anchor epitope-targeting lineage can be elicited in macaques. [bullet]Central stem and anchor bnAbs from humans and macaques engage their respective epitopes with atomic level similarity.

immunology↗

bioRxiv2025

Glycan-reactive antibodies isolated from human HIV-1 vaccine trial participants show broad pathogen cross-reactivity

HIV-1 continues to pose a significant global health challenge, requiring ongoing research into effective prevention and treatment strategies. Understanding the B cell repertoire that can be engaged upon vaccination in humans is crucial for the development of future preventive vaccines. In this study, PBMCs from HIV-negative participants in the multivalent HVTN124 human HIV-1 vaccine clinical trial were interrogated for HIV-reactive B cells using LIBRA-seq, a high-throughput B cell mapping technology. We report the discovery of glycan-reactive antibodies capable of neutralizing diverse heterologous HIV-1 virus strains. Further, isolated antibodies showed broad cross-reactivity against antigens from a variety of other pathogens, while remaining mostly negative on autoreactivity assays. The emerging class of glycan- reactive virus-neutralizing antibodies with exceptional breadth of pathogen cross- reactivity may present an effective target for vaccination at the population level.

immunology↗

bioRxiv2024

Generation of antigen-specific paired heavy-light chain antibody sequences using large language models

The traditional process of antibody discovery is limited by inefficiency, high costs, and low success rates. Recent approaches employing artificial intelligence (AI) have been developed to optimize existing antibodies and generate antibody sequences in a target-agnostic manner. In this work, we present MAGE (Monoclonal Antibody GEnerator), a sequence-based Protein Language Model (PLM) fine-tuned for the task of generating paired human variable heavy and light chain antibody sequences against targets of interest. We show that MAGE can generate novel and diverse antibody sequences with experimentally validated binding specificity against SARS-CoV-2, an emerging avian influenza H5N1, and respiratory syncytial virus A (RSV-A). MAGE represents a first-in-class model capable of designing human antibodies against multiple targets with no starting template.

bioinformatics↗

bioRxiv2024

Structural Convergence and Water-Mediated Substrate Mimicry Enable Broad Neuraminidase Inhibition by Human Antibodies

Influenza has been responsible for multiple global pandemics and seasonal epidemics and claimed millions of lives. The imminent threat of a panzootic outbreak of avian influenza H5N1 virus underscores the urgent need for pandemic preparedness and effective countermeasures, including monoclonal antibodies (mAbs). Here, we characterize human mAbs that target the highly conserved catalytic site of viral neuraminidase (NA), termed NCS mAbs, and the molecular basis of their broad specificity. Cross-reactive NA-specific B cells were isolated by using stabilized NA probes of non-circulating subtypes. We found that NCS mAbs recognized multiple NAs of influenza A as well as influenza B NAs and conferred prophylactic protections in mice against H1N1, H5N1, and influenza B viruses. Cryo-electron microscopy structures of two NCS mAbs revealed that they rely on structural mimicry of sialic acid, the substrate of NA, by coordinating not only amino acid side chains but also water molecules, enabling inhibition of NA activity across multiple influenza A and B viruses, including avian influenza H5N1 clade 2.3.4.4b viruses. Our results provide a molecular basis for the broad reactivity and inhibitory activity of NCS mAbs targeting the catalytic site of NA through substrate mimicry.

immunology↗

bioRxiv2023

Convergent Sequence Features of Antiviral B Cells

Throughout life, humans experience repeated exposure to viral antigens through infection and vaccination, building diverse antigen-specific antibody repertoires. In recent years, these repertoires have become an important source for novel antibody-based antiviral therapeutics, yet there is still limited understanding of the determinants of antibody-antigen specificity. Here, we generated a large dataset mapping antibody sequence to antigen specificity for thousands of B cells, by screening the repertoires of a set of healthy individuals against twenty viral antigens representing diverse pathogens of biomedical significance. Analysis revealed antigen-specific patterns in variable gene usage, gene pairing, and somatic hypermutation, as well as the presence of convergent antiviral signatures across multiple individuals. These results help define the characteristics of human antibody repertoires simultaneously against an unprecedented number and diversity of viral targets. Understanding the fundamental rules of antibody-antigen interactions can lead to transformative new approaches for the development of antibody therapeutics and vaccines against current and emerging viruses.

immunology↗

bioRxiv2023

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase I clinical trial investigating a ferritin nanoparticle displaying H2 hemagglutinin in H2-naive and H2-exposed adults. Therefore, we could perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited after H2 vaccination. We temporally map the epitopes targeted by serum antibodies after first and second vaccinations and show previous H2 exposure results in higher responses to the variable head domain of hemagglutinin while initial responses in H2-naive participants are dominated by antibodies targeting conserved epitopes. We use cryo-EM and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the hemagglutinin head including the receptor binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses. HighlightsO_LISerum Abs after first H2-F vaccination in H2-exposed donors bound variable HA head epitopes C_LIO_LISerum Abs after first H2-F vaccination in H2-naive donors bound conserved HA head and stem epitopes C_LIO_LIRBS-targeting VH1-69 cross-reactive antibodies were induced in H2-naive individuals C_LIO_LIThe medial junction is a previously uncharacterized conserved epitope on the HA head C_LI

immunology↗

bioRxiv2023

C1q Enables Influenza HA Stem Binding Antibodies to Block Viral Attachment and Broadens the Antibody Escape Repertoire

Broadly neutralizing, anti-hemagglutinin stem antibodies (Abs) are a promising universal influenza vaccine target. While anti-stem Abs are not believed to block viral attachment, we show that C1q confers attachment inhibition and boosts fusion and neuraminidase inhibition, greatly enhancing virus neutralization activity in vitro and in mice challenged with influenza virus via the respiratory route. These effects reflect increased steric interference and not increased Ab avidity. Remarkably, C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the hemagglutinin. Some substitutions cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-RBD anti-SARS-CoV-2 Spike Abs, an effect dependent on Spike density on the virion surface. Together, our findings show that first, Ab function must be considered in a physiological context and second, inferring the exact selection pressure for Ab-driven viral evolution is risky business, at best.

microbiology↗

bioRxiv2022

Vaccine-elicitation of cross-group neutralizing protective antibodies to influenza A viruses

Current influenza vaccines predominantly induce immunity to the hypervariable viral hemagglutinin (HA) head, requiring frequent vaccine reformulation. Conversely, antigenic sites on the conserved HA stem are subdominant and harbor a supersite which is targeted by broadly neutralizing antibodies (bnAbs), making it a prime target for universal vaccines. Here, we show that co-immunization of two stem immunogens derived from influenza A group 1 and 2 HAs elicits cross-group protective immunity and neutralizing antibody responses in mice, ferrets, and nonhuman primates (NHPs). Immunized mice were protected from multiple group 1 and 2 viruses, and all animal models showed broad serum neutralizing activity. A bnAb isolated from an immunized NHP broadly neutralized and conferred protection from viruses including H5N1 and H7N9. Genetic and structural analyses revealed a remarkable convergent evolution between macaque and human bnAbs, illustrating the biophysical constraints for acquiring immunoglobulins with cross-group specificity. Co-immunization of stem immunogens elicits not only group-specific protective immunity, but also cross-group bnAb responses, and represents a step towards broadly protective influenza vaccines.

immunology↗

bioRxiv2021

Structure-based design of stabilized recombinant influenza neuraminidase tetramers

Influenza virus neuraminidase (NA) is a major antiviral drug target and has recently reemerged as a key target of antibody-mediated protective immunity. Here we show that recombinant NAs across all non-bat subtypes adopt various tetrameric conformations, including a previously unreported "open" state that may help explain poorly understood variations in NA stability across viral strains and subtypes. We used homology-directed protein design to uncover the structural principles underlying these distinct tetrameric conformations and stabilize multiple recombinant NAs in the "closed" state. In addition to improving thermal stability, conformational stabilization improved affinity to protective antibodies elicited by viral infection, including antibodies targeting a quaternary epitope and the broadly conserved catalytic site. The stabilized NA proteins can also be integrated into viruses without affecting fitness. Our findings provide a deeper understanding of NA structure, stability, and antigenicity, as well as a roadmap towards structure-based discovery of NA-directed therapeutics and vaccines.

microbiology↗

bioRxiv2020

SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness

A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.

immunology↗

bioRxiv2020

Elicitation of broadly protective immunity to influenza by multivalent hemagglutinin nanoparticle vaccines

Influenza vaccines that confer broad and durable protection against diverse virus strains would have a major impact on global health. However, next-generation vaccine design efforts have been complicated by challenges including the genetic plasticity of the virus and the immunodominance of certain epitopes in its glycoprotein antigens. Here we show that computationally designed, two-component nanoparticle immunogens induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens display 20 hemagglutinin (HA) trimers in a highly immunogenic array, and their assembly in vitro enables precisely controlled co-display of multiple distinct HAs in defined ratios. Nanoparticle immunogens displaying the four HAs of licensed quadrivalent influenza vaccines (QIV) elicited hemagglutination inhibition and neutralizing antibody responses to vaccine-matched strains that were equivalent or superior to commercial QIV in mice, ferrets, and nonhuman primates. The nanoparticle immunogens--but not QIV--simultaneously induced broadly protective antibody responses to heterologous viruses, including H5N1 and H7N9, by targeting the subdominant yet conserved HA stem. Unlike previously reported influenza vaccine candidates, our nanoparticle immunogens can alter the intrinsic immunodominance hierarchy of HA to induce both potent receptor-blocking and broadly cross-reactive stem-directed antibody responses and are attractive candidates for a next-generation influenza vaccine that could replace current seasonal vaccines. One Sentence SummaryNanoparticle immunogens displaying four seasonal influenza hemagglutinins elicit neutralizing antibodies directed at both the immunodominant head and the conserved stem and confer broad protective immunity.

immunology↗

bioRxiv2020

A comprehensive influenza reporter virus panel for high-throughput deep profiling of neutralizing antibodies

A number of broadly neutralizing antibodies (bnAbs) to influenza virus have been isolated, characterized and developed as potential countermeasures for seasonal influenza epidemic and pandemic. Deep characterization of these bnAbs and polyclonal sera is critical to our understanding of influenza immunity and for desgining universal influenza vaccines. However, conventional influenza virus neutralization assays with live viruses require high-containment laboratories and are difficult to standardize and roboticize. Here, we built a panel of engineered influenza viruses carrying a fluorescent reporter gene to replace an essential viral gene. This restricts virus replication to cells expressing the missing viral gene in trans, allowing it to be manipulated in a biosafety level 2 environment. Using this system, we characterize the neutralization profile of a set of published and new bnAbs with a panel consisting of 55 viruses that spans the near complete antigenic evolution of human H1N1 and H3N2 viruses, as well as pandemic viruses such as H5N1 and H7N9. Our system opens opportunities to systematically characterize influenza immunity in greater depth, including the response directed at the viral hemagglutinin stem, a major target of universal influenza vaccines.

immunology↗

bioRxiv2020

Tailored Design of Protein Nanoparticle Scaffolds for Multivalent Presentation of Viral Glycoprotein Antigens

The adaptive immune system is highly sensitive to arrayed antigens, and multivalent display of viral glycoproteins on symmetric scaffolds has been found to substantially increase the elicitation of antigen-specific antibodies. Motivated by the considerable promise of this strategy for next-generation anti-viral vaccines, we set out to design new self-assembling protein nanoparticles with geometries specifically tailored to scaffold ectodomains of different viral glycoproteins. We first designed and characterized homo-trimers from designed repeat proteins with N-terminal helices positioned to match the C termini of several viral glycoprotein trimers. Oligomers found to experimentally adopt the designed configuration were then used to generate nanoparticles with tetrahedral, octahedral, or icosahedral symmetry. Examples of all three target symmetries were experimentally validated by cryo-electron microscopy and several were assessed for their ability to display viral glycoproteins via genetic fusion. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles display conformationally intact native-like HIV-1 Env, influenza hemagglutinin, and prefusion RSV F trimers in the predicted geometries. This work demonstrates that novel nanoparticle immunogens can be designed from the bottom up with atomic-level accuracy and provides a general strategy for precisely controlling epitope presentation and accessibility.

synthetic biology↗

bioRxiv2018

Design of nanoparticulate group 2 influenza hemagglutinin stem antigens that activate unmutated ancestor B cell receptors of broadly neutralizing antibody lineages

Influenza vaccines targeting the highly-conserved stem of the hemagglutinin (HA) surface glycoprotein have the potential to protect against pandemic and drifted seasonal influenza viruses not covered by current vaccines. While HA stem-based immunogens derived from group 1 influenza A have been shown to induce intra-group heterosubtypic protection, HA stem-specific antibody lineages originating from group 2 may be more likely to possess broad cross-group reactivity. We report the structure-guided development of mammalian cell-expressed candidate vaccine immunogens based on influenza A group 2 H3 and H7 HA stem trimers displayed on self-assembling ferritin nanoparticles using an iterative, multipronged approach involving helix stabilization, loop optimization, disulfide bond addition, and side chain repacking. These immunogens were thermostable, formed uniform and symmetric nanoparticles, were recognized by cross-group-reactive broadly neutralizing antibodies (bNAbs) with nanomolar affinity, and elicited protective, homosubtypic antibodies in mice. Importantly, several immunogens were able to activate B cells expressing inferred unmutated common ancestor (UCA) versions of cross-group-reactive human bNAbs from two multi-donor classes, suggesting they could initiate elicitation of these bNAbs in humans.

immunology↗