Biology subjects

Ward, A. B.

Publications and source records attributed to Ward, A. B..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Broad SARS-CoV-2 Neutralization by Monoclonal and Bispecific Antibodies Derived from a Gamma-infected Individual

The worldwide pandemic caused by SARS-CoV-2 has remained a human medical threat due to the continued evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants for therapeutic and prophylactic use. A stabilized autologous SARS-CoV-2 spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants of concern, with COVA309-35 being the most potent against the autologous virus, as well as against Omicron BA.1 and BA.2. When combining the COVA309 mAbs as cocktails or bispecific antibody formats, the breadth and potency was significantly improved against all tested variants. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.

immunology↗

bioRxiv2022

Structural basis of epitope selectivity and potent protection from malaria by PfCSP antibody L9

A primary objective in malaria vaccine design is the generation of high-quality antibody responses against the circumsporozoite protein of the malaria parasite, Plasmodium falciparum (PfCSP). To enable rational antigen design, we solved a cryo-EM structure of the highly potent anti-PfCSP antibody L9 in complex with recombinant PfCSP. We found that L9 Fab binds multivalently to the CSP minor (NPNV) repeats, which is stabilized by a novel set of affinity-matured homotypic, antibody-antibody contacts. Molecular dynamics simulations revealed a critical role of the L9 light chain in integrity of the homotypic interface, which likely impacts CSP affinity and protective efficacy. These findings reveal the molecular mechanism of the unique NPNV selectivity of L9 and emphasize the importance of anti-homotypic affinity maturation in protective immunity against P. falciparum. One sentence summaryThe L9 light chain is crucial for potency by conferring multivalent, high affinity binding to the NPNV minor repeats of PfCSP.

immunology↗

bioRxiv2022

Structural conservation of Lassa virus glycoproteins and recognition by neutralizing antibodies

SummaryLassa fever is an acute hemorrhagic fever caused by the zoonotic Lassa virus (LASV). The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target for neutralizing antibodies. Immunogen design is complicated by the metastable nature of recombinant GPCs and the antigenic differences amongst LASV lineages. Despite the sequence diversity of GPC, structures of most lineages are lacking. We present the development and characterization of prefusion-stabilized, trimeric GPCs of LASV lineages II, V, and VI, revealing structural conservation despite sequence diversity. High-resolution structures and biophysical characterization of GPC in complex with GP1-A antibodies reveal their neutralization mechanisms. Finally, we present the isolation and characterization of a novel trimer-preferring neutralizing antibody belonging to the GPC-B competition group with an epitope that spans adjacent protomers and includes the fusion peptide. Our work provides molecular detail information on LASV antigenic diversity and will guide efforts to design pan-LASV vaccines. HighlightsO_LIStructural characterization of soluble glycoproteins from four Lassa virus lineages. C_LIO_LIMAb 12.1F, belonging to the GP1-A cluster, inhibits matriglycan and LAMP-1 binding. C_LIO_LIGP1-A mAbs show glycan-dependence with 19.7E demonstrating lineage-dependent binding. C_LIO_LIA novel trimer-preferring NAb S370.7 targets the GPC-B epitope. C_LI

immunology↗

bioRxiv2022

LipIDens: Simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. The LipIDens code is open-source and embedded within a notebook format to assist automation and usability.

biophysics↗

bioRxiv2022

CD4 binding-site antibodies induced by a subtype B HIV-1 envelope trimer

In the last decade considerable advances have been made towards the design of HIV-1 vaccines that induce neutralizing antibodies (NAbs). Despite the progress, no vaccine is able to consistently elicited broadly neutralizing antibodies (bNAbs). Here we present a case study of a rabbit that was immunized with a subtype B native like envelope glycoprotein (Env) trimer, AMC016 SOSIP.v4.2, with a dense and intact glycan shield, followed by a trivalent combination of subtype B trimers. After the priming phase serum from this animal neutralized several heterologous subtype B neutralization resistant (tier 2) viruses. Subsequent immunization with the trivalent combination of subtype B trimers further increased the breadth and potency of the NAb response. EM based polyclonal epitope mapping revealed that a cross reactive CD4 binding-site (CD4bs) antibody response, that was present after priming with the monovalent trimer and boosting with the trivalent combination, was most likely responsible for the broad neutralization. While anecdotal, this study provides proof-of-concept that native-like Env trimers are capable of inducing CD4bs-directed bNAb responses and should guide efforts to improve the consistency with which such responses are generated.

immunology↗

bioRxiv2022

Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels

Otopetrin (OTOP) channels are proton-selective ion channels conserved among vertebrates and invertebrates and with no structural similarity to other ion channels. There are three vertebrate OTOP channels (OTOP1, OTOP2, and OTOP3), of which one (OTOP1), functions as a sour taste receptor. Whether OTOP channels are gated by, as well as permeating, protons was not known. Here, by comparing functional properties of the three vertebrate proton channels with patch-clamp recording and cytosolic pH microfluorimetry, we provide evidence that each is gated by external protons. OTOP1 and OTOP3 are both activated by extracellular protons, with a sharp threshold of pHe <6.0 and 5.5 respectively, while OTOP2 is negatively gated by protons, and more conductive at alkaline extracellular pH (>pH 9.0). Strikingly, we found that we could change pH-sensitive gating of OTOP2 and OTOP3 channels by swapping extracellular linkers that connect transmembrane domains. Swaps of linkers within the N domain changed the relative conductance at alkaline pH, while swaps within the C domain tended to change the rates of OTOP3 current activation. We conclude that members of the OTOP channel family are proton-gated (acid-sensitive) proton channels and that the gating apparatus is distributed across multiple extracellular regions within both the N and C domains of the channels. In addition to the taste system, OTOP channels are found in the vestibular and digestive systems, where pH sensitivity may be tuned to specific functions.

biophysics↗

bioRxiv2022

High-resolution structural analysis of enterovirus-reactive polyclonal antibodies in complex with whole virions

Non-polio enteroviruses (NPEVs) cause serious illnesses in young children and neonates including aseptic meningitis, encephalitis, neonatal sepsis and inflammatory muscle disease, among others. Over 100 serotypes have been described to date but except for the EV-A71, there are no available vaccines or therapeutics against NPEVs. Efforts towards rationally designed pan-NPEV vaccines would greatly benefit from structural biology methods for rapid and comprehensive evaluation of vaccine candidates and elicited antibody responses. Towards this goal, we tested if electron-microscopy-based polyclonal epitope mapping (EMPEM), a method where structural analysis is performed using serum-derived polyclonal antibodies (pAbs), can be applied to an NPEV. EMPEM was performed on immune complexes featuring CV-A21 viral particles and CV-A21-specific pAbs isolated from mice. Notably, this is the first example of structural analysis of polyclonal immune complexes comprising whole virions. We introduce a data processing workflow that allows reconstruction of different pAbs at near-atomic resolution. The analysis resulted in identification of several antibodies targeting two immunodominant epitopes, near the 3-fold and 5-fold axis of symmetry; the latter overlapping with the receptor binding site. These results demonstrate that EMPEM can be applied to map broad-spectrum polyclonal immune responses against intact virions and define potentially cross-reactive epitopes.

immunology↗

bioRxiv2022

Engineering SARS-CoV-2 neutralizing antibodies for increased potency and reduced viral escape

The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we use a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduced the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for anti-viral indications could benefit from in vitro affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.

bioengineering↗

bioRxiv2021

Molecular insights into antibody-mediated protection against the prototypic simian immunodeficiency virus

SIVmac239 infection of macaques is a favored model of human HIV infection. However, the SIVmac239 envelope (Env) trimer structure, glycan occupancy, and the targets and ability of neutralizing antibodies (nAbs) to protect against SIVmac239 remain unknown. Here, we report the isolation of SIVmac239 nAbs that recognize a glycan hole and the V1/V4 loop. A high-resolution structure of a SIVmac239 Env trimer-nAb complex shows many similarities to HIV and SIVcpz Envs, but with distinct V4 features and an extended V1 loop. Moreover, SIVmac239 Env has a higher glycan shield density than HIV Env that may contribute to poor or delayed nAb responses in SIVmac239-infected macaques. Passive transfer of a nAb protects macaques from repeated low dose intraveneous SIVmac239 challenge at serum titers comparable to those described for protection of humans against HIV infection. Our results provide structural insights for vaccine design and shed light on antibody-mediated protection in the SIV model.

immunology↗

bioRxiv2021

Long-lasting germinal center responses to a priming immunization with continuous proliferation and somatic mutation

Germinal centers (GCs) are the engines of antibody evolution. Using HIV Env protein immunogen priming in rhesus monkeys (RM) followed by a long period without further immunization, we demonstrate GC B cells (BGC) lasted at least 6 months (29 weeks), all the while maintaining rapid proliferation. A 186-fold BGC cell increase was present by week 10 compared to a conventional immunization. Single cell transcriptional profiling revealed that both light zone and dark zone GC states were sustained throughout the 6 months. Antibody somatic hypermutation (SHM) of BGC cells continued to accumulate throughout the 29 week priming period, with evidence of selective pressure. Additionally, Env-binding BGC cells were still 49-fold above baseline 29 weeks after immunization, suggesting that they could be active for significantly longer periods of time. High titers of HIV neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing significant immunodominance challenges for B cells, among other difficulties. Memory B cells (BMem) generated under these long priming conditions had higher levels of SHM, and both BMem cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning the >6-month GC period were identified, demonstrating continuous GC activity and selection for at least 191 days, with no additional antigen exposure. A long prime, adjuvanted, slow delivery (12-day) immunization approach holds promise for difficult vaccine targets, and suggests that patience can have great value for tuning GCs to maximize antibody responses.

immunology↗

bioRxiv2021

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 [A] 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.

immunology↗

bioRxiv2021

A public antibody class recognizes a novel S2 epitope exposed on open conformations of SARS-CoV-2 spike

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigated the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We found that [~]82% of SARS-CoV-2 S-reactive B cells show a naive phenotype, which represents an unusually high fraction of total human naive B cells ([~]0.1%). Approximately 10% of these naive S-reactive B cells shared an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. A proportion of memory B cells, comprising switched ([~]0.05%) and unswitched B cells ([~]0.04%), was also reactive with S and some of these cells were reactive with ADAMTS13, which is associated with thrombotic thrombocytopenia. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

immunology↗

bioRxiv2021

The pre-existing human antibody repertoire to computationally optimized influenza H1 hemagglutinin vaccines

The computationally optimized broadly reactive antigen (COBRA) approach has previously been used to generate hemagglutinin (HA) immunogens for several influenza subtypes that expand vaccine-elicited antibody breadth. As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination. We determined the epitope specificity and repertoire characteristics of pre-existing human B cells to H1 COBRA HA antigens. Cross-reactivity between wild type HA and H1 COBRA HA proteins were observed at both the oligoclonal B cell level and for a subset of isolated monoclonal antibodies (mAbs). The mAbs bound five distinct epitopes on the pandemic A/California/04/2009 head and stem domains, and the majority of the mAbs had HAI and neutralizing activity against pandemic H1 strains. Two head-directed mAbs, CA09-26 and CA09-45, had HAI and neutralizing activity against a pre-pandemic H1 strain. One mAb, P1-05, targets the stem region of H1 HA proteins, but does not compete with known stem-targeting H1 mAbs. We determined that mAb P1-05 recognizes a recently discovered membrane proximal epitope on HA, the anchor epitope, and we identified similar mAbs using B cell repertoire sequencing. In addition, the trimerization domain distance from HA was critical to recognition of this epitope by P1-05. Overall, these data indicate that seasonally vaccinated individuals possess a population of functional H1 COBRA HA- reactive B cells that target head, central stalk, and anchor epitopes, and demonstrate the importance of structure-based assessment of subunit protein vaccine candidates to ensure accessibility of optimal protein epitopes. SignificanceInfluenza imposes significant human and economic costs every year. The current seasonal vaccine elicits primarily strain-specific antibodies, and year to year vaccine effectiveness is variable. The COBRA approach could provide longer protection and obviate the requirement for annual vaccination. Whereas COBRA HAs have previously been evaluated in animal models, the pre-existing COBRA HA-reactive human B cell population has yet to be elucidated, and is important to identify specific B cells that may be recalled by H1 HA COBRA vaccination. This work demonstrates that seasonally vaccinated individuals possess a functional B cell population targeting both head and stem domains that could be recalled with COBRA HA immunogens.

immunology↗

bioRxiv2021

Structural mapping of antibody landscapes to human betacoronavirus spike proteins

Preexisting immunity against seasonal coronaviruses (CoV) represents an important variable in predicting antibody responses and disease severity to Severe Acute Respiratory Syndrome CoV-2 (SARS-2) infections. We used electron microscopy based polyclonal epitope mapping (EMPEM) to characterize the antibody specificities against {beta}-CoV spike proteins in sera from healthy donors (HDs) or SARS-2 convalescent donors (CDs). We observed that most HDs possessed antibodies specific to seasonal human CoVs (HCoVs) OC43 and HKU1 spike proteins while the CDs showed reactivity across all human {beta}-CoVs. Detailed molecular mapping of spike-antibody complexes revealed epitopes that were differentially targeted by antibodies in preexisting and convalescent serum. Our studies provide an antigenic landscape to {beta}-HCoV spikes in the general population serving as a basis for cross-reactive epitope analyses in SARS-2 -infected individuals. One-Sentence summaryWe present the epitope mapping of polyclonal antibodies against beta-coronavirus spike proteins in human sera.

immunology↗

bioRxiv2021

Structural insights of a highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody

As the coronavirus disease 2019 (COVID-19) pandemic continues, there is a strong need for highly potent monoclonal antibodies (mAbs) that are resistant against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants of concern (VoCs). Here, we evaluate the potency of a previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cryo-EM. We show that mAb J08 has low nanomolar affinity against VoCs, binds high on the receptor binding domain (RBD) ridge and is therefore unaffected by most mutations, and can bind in the RBD-up and -down conformations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy. One Sentence SummaryPotent neutralizing monoclonal antibody J08 binds SARS-CoV-2 spike independent of known escape mutations.

immunology↗

bioRxiv2021

Targeted isolation of panels of diverse human broadly neutralizing antibodies against SARS-like viruses

The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy 1-7. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed 8, 9. Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone 10-13. Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination 14-18. Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.

immunology↗

bioRxiv2021

Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting neutralizing antibody responses against multiple CoVs. Because of the phylogenetic similarity to humans, rhesus macaques are an animal model of choice for many virus-challenge and vaccine-evaluation studies, including SARS-CoV-2. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein generates potent receptor binding domain cross- neutralizing antibody (nAb) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to human infection or vaccination where responses are typically SARS-CoV-2-specific. Furthermore, the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Structural studies show that different immunodominant sites are targeted by the two primate species. Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site (RBS), whereas the macaque antibodies recognize a relatively conserved region proximal to the RBS that represents another potential pan-SARS-related virus site rarely targeted by human antibodies. B cell repertoire differences between the two primates appear to significantly influence the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines to SARS-related viruses intended for human use. ONE SENTENCE SUMMARYBroadly neutralizing antibodies to an unappreciated site of conservation in the RBD in SARS- related viruses can be readily induced in rhesus macaques because of distinct properties of the naive macaque B cell repertoire that suggest prudence in the use of the macaque model in SARS vaccine evaluation and design.

immunology↗

bioRxiv2021

Structural Insights into the Venus flytrap Mechanosensitive Ion Channel Flycatcher1

Flycatcher1 (FLYC1), a MscS homolog, has recently been identified as a candidate mechanosensitive (MS) ion channel involved in Venus flytrap prey recognition. FLYC1 is larger and its sequence diverges from previously studied MscS homologs, suggesting it has unique structural features that contribute to its function. Here, we characterized FLYC1 by cryo-electron microscopy, molecular dynamics simulations, and electrophysiology. Akin to bacterial MscS and plant MSL1 channels, we find that FLYC1 central core includes side portals in the cytoplasmic cage that regulate ion conduction, by identifying critical residues that modulate channel conductance. Topologically unique cytoplasmic flanking regions can adopt up or down conformations, making the channel asymmetric. Disruption of an up conformation-specific interaction severely delays channel deactivation by 40-fold likely due to stabilization of the channel open state. Our results illustrate novel structural features and likely conformational transitions that regulate mechano-gating of FLYC1.

biochemistry↗