Biology subjects

Maynard, J. A.

Publications and source records attributed to Maynard, J. A..

11 recordsLinked to original sources

bioRxiv2026

Broad Sarbecovirus Neutralization by an S2-Directed Plasma Antibody Defines a New Site of Vulnerability in SARS-like Viruses

The repeated emergence of pathogenic coronaviruses highlights the continual threat of zoonotic spillover events and the need to understand conserved regions of the viral spike protein that influence immune recognition. Here, we employ high-resolution proteomic analysis of circulating immunoglobulins to characterize the antibody response to the SARS-CoV-2 spike protein in vaccinated and infected individuals. We recombinantly expressed abundant plasma IgG lineages and identified nine antibodies targeting the highly conserved S2 spike subunit. Most of these S2-reactive plasma mAbs (7 of 9) exhibited neutralizing activity against pangolin coronavirus, while one mAb (SC45) demonstrated potent (IC50 < 1 {micro}g/mL) neutralizing activity against both pangolin and bat sarbecoviruses RsSHC014 and WIV1. Additionally, SC45 exhibited potent prophylactic efficacy in the K18-hACE2 mouse model challenged with Pangolin-Guangdong CoV MP789 (Pg-CoV), significantly reducing lung viral replication at day 4 post-infection. The cryo-EM structure of SC45 bound to the prefusion-stabilized S2 domain of Pg-CoV reveals that SC45 targets a novel, conserved epitope in the connector domain. Strikingly, even though SC45 was the most abundant antibody ([~]13%) of anti-spike plasma IgG in an infected patient and binds to both SARS-CoV-2 and Pg-CoV spike with comparable affinities, it fails to neutralize early (D614G and Omicron BA.1) SARS-CoV-2 variants. Together, these findings identify a conserved site of vulnerability in the spike S2 subunit which informs on potential mechanisms of antibody immunity to zoonotic coronaviruses.

immunology↗

bioRxiv2026

Mouse Fc-FcγRIV structure guides Fc engineering for cross-species FcγR recognition

Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism of action for many FDA-approved therapeutic antibodies that is driven by interactions between the antibody Fc and Fc{gamma} receptors (Fc{gamma}Rs) on immune effector cells. Murine models used for preclinical antibody evaluation currently have limited predictive value for clinical ADCC performance due to interspecies differences in Fc-Fc{gamma}R interactions. The molecular determinants governing Fc-Fc{gamma}R engagement in mice remain poorly defined, complicating the interpretation of murine ADCC data and its clinical relevance. To address this, we present the high-resolution crystal structure of the receptor that regulates Fc-mediated cytotoxicity in mice, mouse Fc{gamma}RIV, alone and in complex with mouse IgG2a Fc. This complex preserves key features of the human IgG1 Fc-human Fc{gamma}RIIIa interface which mediates ADCC in humans including salt bridges, hydrogen bonds, and a proline sandwich. However, subtle variations in receptor orientation, Fc-Fc{gamma}R electrostatics, and glycan positions reduce human IgG1 Fc- mouse Fc{gamma}RIV binding affinity, resulting in species-restricted Fc-Fc{gamma}R mediated immune responses. Modeling of human IgG1 Fc interactions with mouse Fc{gamma}RIV predicted steric clashes, suggesting opportunities to modulate the interaction. One structure-guided substitution variant of human IgG1, Fchumo, maintains comparable human Fc{gamma}RIIIa engagement with enhanced binding to and activation of mouse Fc{gamma}RIV, relative to human IgG1 Fc. This study provides proof-of-concept for engineering human Fc domains for cross-species Fc{gamma}R recognition and provides a strategic framework to improve the predictive power of in vivo preclinical models.

biochemistry↗

bioRxiv2026

Treatment of murine autoimmune myocarditis with a novel monoclonal antibody that targets multiple inflammatory pathways

Severe forms of inflammation-induced acute and chronic myocarditis have a poor prognosis. Promising therapeutic efforts focused on monoclonal antibodies (mAbs) inhibiting inflammation-inducing molecules. However, most mAbs target only one or a limited number of such molecules. Since inflammation involves multiple redundant pathways, we postulated that an mAb inhibiting multiple inflammatory pathways would be a potent therapeutic agent. We initially tested the commercially available anti-natural killer (NK) cell mAb (anti-NK1.1), which binds a receptor expressed on NK cells and depletes them. Since NK cells are key cellular orchestrators of inflammation, by reducing their number, we aimed to inhibit multiple inflammatory pathways. Our initial studies demonstrated that administration of this antibody significantly improved myocardial outcomes in mouse models of acute myocardial infarction and of heart failure. Since NK1.1 is not expressed in human cells, we built on these promising preclinical results by developing a novel mAb targeting CD160 on human NK cells for evaluation as an immunosuppressive therapy. We found that the anti-CD160 mAb depletes both murine and human NK cells. We also found that, while CD160+ cells were largely present in the NK population, they also occurred among CD8+ and {gamma}/{delta} T cell subsets in human cells. Anti-CD160 therapy entirely prevented the deterioration of the myocardial function of mice with autoimmune-induced acute myocarditis. This outcome suggests our novel approach for inhibiting multiple inflammatory pathways may provide a potent strategy for improving outcomes of inflammation-driven myocarditis, as well as of other inflammation-driven diseases. Key PointsO_ST_ABSQuestionC_ST_ABSCan the depletion of CD160+ cells prevent autoimmune-induced myocarditis? FindingsIn this study we found that CD160 is expressed by mouse and human natural killer cells and other subtypes of cytotoxic T cells, and that a monoclonal antibody targeting CD160 depletes NK cells. In a preclinical model of experimental autoimmune myocarditis, administration of the anti-CD160 monoclonal antibody prevented myocardial dysfunction and systemic inflammation. MeaningOur results are compatible with the hypothesis that early autoimmune-induced myocardial dysfunction is promoted by CD160+ cells, which elevate inflammation-induced circulating factors (or factors released by tissue-resident cytotoxic immune cells) that cause myocardial dysfunction in the absence of myocardial necrosis or fibrosis, and further, that targeting CD160+cells with a mAb that depletes NK cells (and probably CD160 expressing cytotoxic T cells) entirely prevents the deterioration of myocardial function in such mice. This outcome suggests our novel approach for inhibiting multiple inflammatory pathways may provide a potent strategy for improving outcomes of inflammation-driven myocarditis, as well as of other inflammation-driven diseases.

systems biology↗

bioRxiv2025

Antibodies blocking PlGF or VEGF interactions with the NRP1 receptormediate anti-proliferative effects

Antibodies blocking the function of vascular endothelial growth factor A (VEGFA) remain a promising therapeutic strategy, especially when combined with check-point inhibitors, but their efficacy is limited by tumor resistance. This can occur via multiple mechanisms, including upregulation of placental growth factor 2 (PlGF-2), an alternative ligand for VEGF receptor 1 (VEGFR1) and neuropilin receptor 1 (NRP1). Activity of both growth factors is mediated by interactions with multiple receptors and extra-cellular matrix components, which complicates efforts to understand their contributions to cancer progression. To complement existing antibodies, we discovered those blocking interactions between PlGF-2 or VEGFA and their shared NRP1 receptor in the presence of heparin. Limiting angiogenesis to promote vascular normalization is one mechanism of anti-VEGF protection; here, anti-VEGFA antibodies blocking interactions with VEGFR1 and NRP1 reduced HUVEC tube formation in a physiological angiogenesis model. By contrast, antibodies binding PlGF-2 or VEGFA to block NRP1 significantly reduced proliferation of Caki-I kidney carcinoma cells in vitro, indicating this receptor mediates additional effects. Interestingly, one antibody exhibited dual-reactive binding to VEGFA and PlGF-2, suggesting a novel therapeutic strategy to prevent PlGF-driven VEGF-resistance. Overall, these antibodies define new mechanisms to disrupt PlGF activity and support a role for NRP1 in cell proliferation. Key ResultsO_LINew antibodies binding VEGFA or PlGF selectively block NRP1-receptor interactions. C_LIO_LIAntibody blockade of VEGFA binding to NRP1 reduced HUVEC angiogenesis. C_LIO_LIBlocking growth factor interactions with NRP1 reduced Caki-I renal carcinoma proliferation. C_LIO_LIIdentified an antibody with dual-reactive binding to VEGFA and PlGF. C_LI

biochemistry↗

bioRxiv2025

Selective decoupling of IgG1 binding to viral Fc receptorsrestores antibody-mediated NK cell activation against HCMV-infected cells

A key mechanism of antiviral antibodies is to bind cell-surface viral antigens and activate cellular immunity to clear infected cells, yet antibodies targeting human cytomegalovirus (HCMV) have exhibited limited efficacy. This appears due to HCMVs multiple immune evasion mechanisms, including viral receptors (vFc{gamma}Rs) which bind human IgG Fc domains to co-operatively inhibit Fc activation of host Fc{gamma} receptors and impair Fc-mediated effector functions. We biochemically characterized and evaluated the functions of two highly conserved vFc{gamma}Rs, gp34 and gp68, and mapped their binding epitopes on the Fc domain. Based on this information, we then engineered Fc variants that retain binding to CD16A, which is essential for NK activation, and to FcRn but have markedly attenuated binding to gp34 and gp68. IgG1 antibodies targeting the gB fusogen with engineered Fc domains were not internalized by infected cells, mediated enhanced CD16A activation and limited viral spread in HCMV-infected fibroblasts more effectively than wild-type Fc. Together, this work demonstrates a strategy to enhance the efficacy of antibody therapies to clear HCMV infections. HighlightsO_LIHost and HCMV FcR compete for IgG1 binding but engage different residues. C_LIO_LIFc-engineering abrogates viral FcR antagonism while retaining CD16A activation. C_LIO_LIAntibodies that resist vFcR capture promote superior ADCC against infected cells. C_LIO_LIDesigner Fc domains complement Fabs to create enhanced disease-specific therapies. C_LI

immunology↗

bioRxiv2024

Structural Basis for Antibody Neutralization of Pertussis Toxin

SUMMARY/ABSTRACTPertussis toxin (PT) is a key protective antigen in vaccine- and natural immunity-mediated protection from Bordetella pertussis infection. Despite its importance, no PT-neutralizing epitopes have been characterized structurally. To define neutralizing epitopes and identify key structural elements to preserve during PT antigen design, we determined a 3.6 [A] cryo-electron microscopy structure of genetically detoxified PT (PTg) bound to hu11E6 and hu1B7, two potently neutralizing anti-PT antibodies with complementary mechanisms: disruption of toxin adhesion to cells and intracellular activities, respectively. Hu11E6 bound the paralogous S2 and S3 subunits of PTg via a conserved epitope, but surprisingly did not span the sialic acid binding site implicated in toxin adhesion. High-throughput glycan array analysis showed that hu11E6 specifically prevents PTg binding to sialylated N-glycans, while a T cell activation assay showed that hu11E6 blocks PTg mitogenic activities to define the neutralizing mechanism. Hu1B7 bound a quaternary epitope spanning the S1 and S5 subunits, although functional studies of hu1B7 variants suggested that S5 binding is not involved in its PT neutralization mechanism. These results are the first to structurally define neutralizing epitopes on PT, improving our molecular understanding of immune protection from B. pertussis and providing key information for the future development of PT immunogens. SIGNIFICANCEAntibodies neutralizing pertussis toxin (PT) prevent the severe clinical symptoms associated with infection by Bordetella pertussis. However, the molecular basis of effective PT-targeted immunity is poorly understood. To gain insight into PT-inhibitory mechanisms, we determined the cryo-electron microscopy structure of genetically detoxified PT (PTg) with two potently neutralizing antibodies to precisely define their epitopes. Carbohydrate-binding studies show that the hu11E6-binding surface on PT interacts with N-linked glycans and that blocking these interactions prevents PTs T cell mitogenic activities. Hu1B7 binds an epitope near the S1 active site that includes S5 contacts but these do not appear important for neutralization. This work identifies PT-neutralizing epitopes and supports inclusion of the hu1B7 and hu11E6 epitopes in next-generation vaccines and PT-based immunogens.

biochemistry↗

bioRxiv2024

Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been FDA-approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 [A] resolution cryo-electron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses. TeaserA structure-based design campaign leads to stabilization of the class III viral fusion protein from HCMV in a prefusion-like conformation.

molecular biology↗

bioRxiv2021

The SARS-CoV-2 spike reversibly samples an open-trimer conformation exposing novel epitopes

Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen deuterium exchange mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-EM, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation--an open trimer-- contains easily accessible RBDs. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines. One Sentence SummaryAn alternative conformation of SARS-CoV-2 spike ectodomain modulated by temperature, binding, and sequence variants.

biophysics↗

bioRxiv2021

Identification of a conserved neutralizing epitope present on spike proteins from highly pathogenic coronaviruses

Three pathogenic human coronaviruses have emerged within the last 20 years, with SARS-CoV-2 causing a global pandemic. Although therapeutic antibodies targeting the SARS-CoV-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report an antibody binding an epitope conserved in the pre-fusion core of MERS-CoV, SARS-CoV and SARS-CoV-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes spike from SARS-CoV, SARS-CoV-2 and variants of concern in in vitro pseudovirus assays. Hydrogen-deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, suggesting 3A3 prevents the S2 conformational rearrangements required for conversion to the spike post-fusion state and virus-host cell fusion. This work defines a conserved vulnerable site on the SARS-CoV-2 S2 domain and guides the design of pan-protective spike immunogens.

biochemistry↗

bioRxiv2020

Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to [~]10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 [A]-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.

microbiology↗

bioRxiv2019

Characterizing the innate and adaptive responses of immunized mice to Bordetella pertussis infection using in vivo imaging and transcriptomic analysis

Bordetella pertussis (B. pertussis) is the causative agent of pertussis (whooping cough). Since the 1990s, pertussis has re-emerged in the United States despite an estimated 95% vaccine coverage. Our goal was to characterize neutrophil responses and gene expression profiles of murine lungs in the context of vaccination and B. pertussis challenge. We utilized a bioluminescent neutrophil mouse model (NECre luc) to track neutrophil recruitment. NECre luc mice were immunized with whole cell vaccine (WCV), acellular vaccine (ACV), or a truncated adenylate cyclase toxoid (RTX) vaccine. Neutrophil recruitment was measured in live mice across time and corroborated by flow cytometry and other data. WCV immunized mice showed signs of neutrophilia in response to B. pertussis challenge. Mice immunized with either ACV or WCV cleared the challenge infection; however immunization with RTX alone was not protective. RNA sequencing revealed distinctive gene expression profiles for each immunization group. We observed an increase in expression of genes associated with responses to infection, and changes in expression of distinct genes in each vaccine group, providing a complex view of the immune response to B. pertussis infection in mice. This study suggests that combination of immunological analysis with transcriptomic profiling can facilitate discovery of pre-clinical correlates of protection for vaccine development.

immunology↗