Biology subjects

Graham, B. S.

Publications and source records attributed to Graham, B. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Elicitation of stem-directed antibodies in rhesus macaques by a conventional hemagglutinin immunogen

Because they can bind many strains of influenza, antibodies targeting the hemagglutinin (HA) stem have been attractive targets for vaccine development. Many monoclonal antibodies (mAbs) directed at the HA stem have been isolated from humans, and these mAbs have mediated broad protection in animal models. We describe here HA stem-directed mAbs isolated from rhesus macaques immunized with an "ordinary" H1 HA trimer. All immunized rhesus macaques developed high serum titers with broad reactivity to diverse H1N1 and H5N1 viruses, and 7 isolated mAbs strongly blocked canonical stem antibody CR6261 binding to H1. MAb DH726.1 robustly protected mice from lethal challenge with H1N1 and H5N1 viruses, and cryo-EM showed the binding footprint overlapped that of some human mAbs. These findings suggest that vaccination with the standard, trimeric HA immunogens may be sufficient to elicit stem antibodies at titers adequate to protect against zoonotic H5N1 influenza. In BriefEfforts to achieve broad influenza protection have largely emphasized increasingly sophisticated immunogen designs to redirect antibody responses toward conserved epitopes. Here, we show that a single, conventional HA immunogen readily elicits antibodies targeting the conserved HA stem, suggesting that routine influenza vaccination may provide broader protection than previously appreciated. HighlightsO_LIMonovalent HA immunization induces strong, stem-directed immune responses in rhesus macaques C_LIO_LIEpitope on the HA stem confirmed by Cryo-EM C_LIO_LIFc-mediated activation of immune effector cell by rhesus-derived stem mAb C_LIO_LIHA stem-directed antibody protects mice against H1N1 and H5N1 viruses C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/738984v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1535b59org.highwire.dtl.DTLVardef@192422dorg.highwire.dtl.DTLVardef@136b3d5org.highwire.dtl.DTLVardef@586fd4_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Human Coronavirus HKU1 Neutralizing Monoclonal Antibodies Target Diverse Epitopes Within and Around the TMPRSS2 Receptor Binding Site

Endemic human coronaviruses (HCoVs), such as HCoV-HKU1, account for [~]30% of common colds each year and can cause serious upper and lower respiratory infections, yet no licensed vaccines or therapeutic antibodies target HCoVs. Despite being endemic to the human population, little is known about the antigenic landscape of HCoV-HKU1. Here, we characterized key interactions between the HCoV-HKU1 spike (S) protein and monoclonal antibodies (mAbs) isolated from convalescent HCoV-HKU-1-positive peripheral blood mononuclear cells collected prior to the COVID-19 pandemic. We isolated 14 mAbs that bound distinct regions of S, including the receptor-binding domain (RBD), N-terminal domain, and S2 subunit. Structural and functional studies revealed three groups of RBD-specific mAbs targeting diverse footprints within and around the TMPRSS2 receptor-binding site: (1) H501-022, which recognizes the TMPRSS2-binding site and thereby blocks receptor engagement; (2) H501-008, which binds a distinct epitope outside the TMPRSS2-binding site that is shared with HCoV-OC43; and (3) H501-018, which recognizes both "up" and "down" RBD conformations at a distinct, non-overlapping epitope outside the TMPRSS2-binding site. H501-008 weakly neutralized live HCoV-OC43 in vitro and protected mice against lethal HCoV-OC43 challenge. Notably, the three RBD-specific mAbs potently neutralized HCoV-HKU1 pseudovirus in TMPRSS2-overexpressing cell lines, but exhibited limited neutralizing activity against authentic HCoV-HKU1 infection in primary human airway epithelial cells. Together, these findings structurally define the antigenic landscape within and around the HKU1 receptor-binding site and demonstrate that receptor-binding site recognition alone is insufficient to predict physiologically relevant antibody-mediated neutralization.

microbiology↗

bioRxiv2024

Structural serology of polyclonal antibody responses to mRNA-1273 and NVX-CoV2373 COVID-19 vaccines

Current COVID-19 vaccines are largely limited in their ability to induce broad, durable immunity against emerging viral variants. Design and development of improved vaccines utilizing existing platforms requires an in-depth understanding of the antigenic and immunogenic properties of available vaccines. Here we examined the antigenicity of two of the original COVID-19 vaccines, mRNA-1273 and NVX-CoV2373, by electron microscopy-based polyclonal epitope mapping (EMPEM) of serum from immunized non-human primates (NHPs) and clinical trial donors. Both vaccines induce diverse polyclonal antibody (pAb) responses to the N-terminal domain (NTD) in addition to the receptor-binding domain (RBD) of the Spike protein, with the NTD supersite being an immunodominant epitope. High-resolution cryo-EMPEM studies revealed extensive pAb responses to and around the supersite with unique angles of approach and engagement. NTD supersite pAbs were also the most susceptible to variant mutations compared to other specificities, indicating that ongoing Spike ectodomain-based vaccine design strategies should consider immuno-masking this site to prevent induction of these strain-specific responses.

immunology↗

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↗

bioRxiv2024

pr-independent biogenesis of infectious mature Zika virus particles

Flavivirus assembly at the endoplasmic reticulum is driven by the structural proteins envelope (E) and premembrane (prM). Here, contrary to the established paradigm for flavivirus assembly, we demonstrate that the biogenesis of flavivirus particles does not require an intact prM nor proteolytic activation. The expression of E preceded by a truncated version of prM (M-E) was sufficient for the formation of non-infectious Zika virus subviral particles and pseudo-infectious reporter virions. Subviral particles encoded by a ZIKV M-E DNA vaccine elicited a neutralizing antibody response that was insensitive to the virion maturation state, a feature of flavivirus humoral immunity shown to correlate with protection. M-E vaccines that uniformly present structural features shared with mature virions offer a higher quality and broadly applicable approach to flavivirus vaccination.

microbiology↗

bioRxiv2022

An optimized messenger RNA vaccine candidate protects non-human primates from Zika virus infection

Zika virus (ZIKV), an arbovirus transmitted by mosquitoes, was identified as a cause of congenital disease during a major outbreak in the Americas in 2016. Vaccine design strategies relied on limited available isolate sequence information due to the rapid response necessary. The first-generation ZIKV mRNA vaccine, mRNA-1325, was initially generated and, as additional strain sequences became available, a second mRNA vaccine, mRNA-1893, was developed. Herein, we compared the immune responses following mRNA-1325 and mRNA-1893 vaccination and reported that mRNA-1893 generated comparable neutralizing antibody titers to mRNA-1325 at 1/20th of the dose and provided complete protection from ZIKV challenge in non-human primates. In depth characterization of these vaccines indicated that the observed immunologic differences could be attributed to a single amino acid residue difference that compromised mRNA-1325 virus-like particle formation.

immunology↗

bioRxiv2022

A Multivalent Polyomavirus Vaccine Elicits Durable Neutralizing Antibody Responses in Macaques

In 2019, there were about 100,000 kidney transplants globally, with more than a quarter of them performed in the United States. Unfortunately, some engrafted organs are lost to polyomavirus-associated nephropathy (PyVAN) caused by BK and JC viruses (BKPyV and JCPyV). Transplant patients are routinely monitored for BKPyV viremia, which is an accepted hallmark of nascent nephropathy. If viremia is detected, a reduction in immunosuppressive therapy is standard care, but the intervention comes with increased risk of immune rejection of the engrafted organ. Recent reports have suggested that transplant recipients with high levels of polyomavirus-neutralizing antibodies are protected against PyVAN. Virus-like particle (VLP) vaccines, similar to approved human papillomaviruses vaccines, have an excellent safety record and are known to induce high levels of neutralizing antibodies associated and long-lasting protection from infection. In this study, we demonstrate that VLPs representing BKPyV genotypes I, II, and IV, as well as JCPyV genotype 2 produced in insect cells elicit robust antibody titers. In rhesus macaques, all monkeys developed neutralizing antibody titers above a previously proposed protective threshold of 10,000. A second inoculation, administered 19 weeks after priming, boosted titers to a plateau of [≥]25,000 that was maintained for almost two years. No vaccine-related adverse events were observed in any macaques. A multivalent BK/JC VLP immunogen did not show inferiority compared to the single-genotype VLP immunogens. Considering these encouraging results, we believe a clinical trial administering the multivalent VLP vaccine in patients waiting to receive a kidney transplant is warranted to evaluate its ability to reduce or eliminate PyVAN. HIGHLIGHTSO_LIRecombinant virus-like particle vaccine was safely administered to rhesus macaques C_LIO_LIVaccination generated high-titer neutralizing antibody responses C_LIO_LIMultivalent BK/JC polyomavirus vaccine was as effective as monovalent vaccines C_LIO_LIHigh neutralizing titers were sustained for 92 weeks without appreciable decline C_LI

immunology↗

bioRxiv2022

Structural basis for antibody recognition of vulnerable epitopes on Nipah virus F protein

SUMMARY/ABSTRACTNipah virus (NiV) is a pathogenic paramyxovirus that causes fatal encephalitis in humans. Two envelope glycoproteins, the attachment protein (G) and fusion protein (F), facilitate entry into host cells. Due to its vital role, NiV F presents an attractive target for developing vaccines and therapeutics. Several neutralization-sensitive epitopes on the NiV F apex have been described, however the antigenicity of most of the F proteins surface remains uncharacterized. Here, we immunize mice with prefusion-stabilized NiV F and isolate ten monoclonal antibodies that neutralize pseudotyped virus. Cryo-electron microscopy reveals eight neutralization-sensitive epitopes on NiV F, four of which have not previously been described. Novel sites span the lateral and basal faces of NiV F, expanding the known library of vulnerable epitopes. This work identifies new epitopes as targets for therapeutics, provides a molecular basis for NiV neutralization, and lays a foundation for development of new antibodies targeting NiV F.

molecular biology↗

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

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in nonhuman primates is coincident with an anamnestic antibody response in the lower airway

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. We immunized rhesus macaques at weeks 0 and 4 and assessed immune responses over one year in blood, upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody binding titers also decreased in bronchoalveolar lavage (BAL). Four days after challenge, virus was unculturable in BAL and subgenomic RNA declined [~]3-log10 compared to control animals. In nasal swabs, sgRNA declined 1-log10 and virus remained culturable. Anamnestic antibody responses (590-fold increase) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

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↗

bioRxiv2021

Durability of mRNA-1273-induced antibodies against SARS-CoV-2 variants

SARS-CoV-2 mutations may diminish vaccine-induced protective immune responses, and the durability of such responses has not been previously reported. Here, we present a comprehensive assessment of the impact of variants B.1.1.7, B.1.351, P.1, B.1.429, and B.1.526 on binding, neutralizing, and ACE2-blocking antibodies elicited by the vaccine mRNA-1273 over seven months. Cross-reactive neutralizing responses were rare after a single dose of mRNA-1273. At the peak of response to the second dose, all subjects had robust responses to all variants. Binding and functional antibodies against variants persisted in most subjects, albeit at low levels, for 6 months after the primary series of mRNA-1273. Across all assays, B.1.351 had the greatest impact on antibody recognition, and B.1.1.7 the least. These data complement ongoing studies of clinical protection to inform the potential need for additional boost vaccinations. One-Sentence SummaryMost mRNA-1273 vaccinated individuals maintained binding and functional antibodies against SARS-CoV-2 variants for 6 months.

immunology↗

bioRxiv2021

Infection and vaccine-induced neutralizing antibody responses to the SARS-CoV-2 B.1.617.1 variant

SARS-CoV-2 has caused a devastating global pandemic. The recent emergence of SARS-CoV-2 variants that are less sensitive to neutralization by convalescent sera or vaccine-induced neutralizing antibody responses has raised concerns. A second wave of SARS-CoV-2 infections in India is leading to the expansion of SARS-CoV-2 variants. The B.1.617.1 variant has rapidly spread throughout India and to several countries throughout the world. In this study, using a live virus assay, we describe the neutralizing antibody response to the B.1.617.1 variant in serum from infected and vaccinated individuals. We found that the B.1.617.1 variant is 6.8-fold more resistant to neutralization by sera from COVID-19 convalescent and Moderna and Pfizer vaccinated individuals. Despite this, a majority of the sera from convalescent individuals and all sera from vaccinated individuals were still able to neutralize the B.1.617.1 variant. This suggests that protective immunity by the mRNA vaccines tested here are likely retained against the B.1.617.1 variant. As the B.1.617.1 variant continues to evolve, it will be important to monitor how additional mutations within the spike impact antibody resistance, viral transmission and vaccine efficacy.

immunology↗

bioRxiv2021

Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a global pandemic. Safe and effective COVID-19 vaccines are now available, including mRNA-1273, which has shown 94% efficacy in prevention of symptomatic COVID-19 disease. However, the emergence of SARS-CoV-2 variants has led to concerns of viral escape from vaccine-induced immunity. Several variants have shown decreased susceptibility to neutralization by vaccine-induced immunity, most notably B.1.351 (Beta), although the overall impact on vaccine efficacy remains to be determined. Here, we present the initial evaluation in mice of 2 updated mRNA vaccines designed to target SARS-CoV-2 variants: (1) monovalent mRNA-1273.351 encodes for the spike protein found in B.1.351 and (2) mRNA-1273.211 comprising a 1:1 mix of mRNA-1273 and mRNA-1273.351. Both vaccines were evaluated as a 2-dose primary series in mice; mRNA-1273.351 was also evaluated as a booster dose in animals previously vaccinated with mRNA-1273. The results demonstrated that a primary vaccination series of mRNA-1273.351 was effective at increasing neutralizing antibody titers against B.1.351, while mRNA-1273.211 was effective at providing broad cross-variant neutralization. A third (booster) dose of mRNA-1273.351 significantly increased both wild-type and B.1.351-specific neutralization titers. Both mRNA-1273.351 and mRNA-1273.211 are being evaluated in pre-clinical challenge and clinical studies.

microbiology↗

bioRxiv2021

Antibodies with potent and broad neutralizing activity against antigenically diverse and highly transmissible SARS-CoV-2 variants

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50 <0.0006 to 0.0102 g/mL; IC80 < 0.0006 to 0.0251 g/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs. One Sentence SummaryUltrapotent antibodies from convalescent donors neutralize and mitigate resistance of SARS-CoV-2 variants of concern.

immunology↗

bioRxiv2021

Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization

The COVID-19 pandemic has ravaged the globe, and its causative agent, SARS-CoV-2, continues to rage. Prospects of ending this pandemic rest on the development of effective interventions. Single and combination monoclonal antibody (mAb) therapeutics have received emergency use authorization1-3, with more in the pipeline4-7. Furthermore, multiple vaccine constructs have shown promise8, including two with ~95% protective efficacy against COVID-199,10. However, these interventions were directed toward the initial SARS-CoV-2 that emerged in 2019. The recent emergence of new SARS-CoV-2 variants B.1.1.7 in the UK11 and B.1.351 in South Africa12 is of concern because of their purported ease of transmission and extensive mutations in the spike protein. We now report that B.1.1.7 is refractory to neutralization by most mAbs to the N-terminal domain (NTD) of spike and relatively resistant to a few mAbs to the receptor-binding domain (RBD). It is not more resistant to convalescent plasma or vaccinee sera. Findings on B.1.351 are more worrisome in that this variant is not only refractory to neutralization by most NTD mAbs but also by multiple individual mAbs to the receptor-binding motif on RBD, largely due to an E484K mutation. Moreover, B.1.351 is markedly more resistant to neutralization by convalescent plasma (9.4 fold) and vaccinee sera (10.3-12.4 fold). B.1.351 and emergent variants13,14 with similar spike mutations present new challenges for mAb therapy and threaten the protective efficacy of current vaccines.

immunology↗

bioRxiv2021

mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative infection of a global pandemic that has led to more than 2 million deaths worldwide. The Moderna mRNA-1273 vaccine has demonstrated ~94% efficacy in a Phase 3 study and has been approved under Emergency Use Authorization. The emergence of SARS-CoV-2 variants with mutations in the spike protein, most recently circulating isolates from the United Kingdom (B.1.1.7) and Republic of South Africa (B.1.351), has led to lower neutralization from convalescent serum by pseudovirus neutralization (PsVN) assays and resistance to certain monoclonal antibodies. Here, using two orthogonal VSV and lentivirus PsVN assays expressing spike variants of 20E (EU1), 20A.EU2, D614G-N439, mink cluster 5, B.1.1.7, and B.1.351 variants, we assessed the neutralizing capacity of sera from human subjects or non-human primates (NHPs) that received mRNA-1273. No significant impact on neutralization against the B.1.1.7 variant was detected in either case, however reduced neutralization was measured against the mutations present in B.1.351. Geometric mean titer (GMT) of human sera from clinical trial participants in VSV PsVN assay using D614G spike was 1/1852. VSV pseudoviruses with spike containing K417N-E484K-N501Y-D614G and full B.1.351 mutations resulted in 2.7 and 6.4-fold GMT reduction, respectively, when compared to the D614G VSV pseudovirus. Importantly, the VSV PsVN GMT of these human sera to the full B.1.351 spike variant was still 1/290, with all evaluated sera able to fully neutralize. Similarly, sera from NHPs immunized with 30 or 100g of mRNA-1273 had VSV PsVN GMTs of ~ 1/323 or 1/404, respectively, against the full B.1.351 spike variant with a ~ 5 to 10-fold reduction compared to D614G. Individual mutations that are characteristic of the B.1.1.7 and B.1.351 variants had a similar impact on neutralization when tested in VSV or in lentivirus PsVN assays. Despite the observed decreases, the GMT of VSV PsVN titers in human vaccinee sera against the B.1.351 variant remained at ~1/300. Taken together these data demonstrate reduced but still significant neutralization against the full B.1.351 variant following mRNA-1273 vaccination.

immunology↗

bioRxiv2021

mRNA-1273 efficacy in a severe COVID-19 model: attenuated activation of pulmonary immune cells after challenge

The mRNA-1273 vaccine was recently determined to be effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from interim Phase 3 results. Human studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibit more severe SARS-CoV-2 disease similar to hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and non-human primates, mRNA-1273- mediated immunity was non-sterilizing and coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high resolution analysis which is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a two-dose schedule and provides insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

immunology↗