Publications
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Title & Authors Journal Publication Date

The C3/465 glycan hole cluster in BG505 HIV-1 envelope is the major neutralizing target involved in preventing mucosal SHIV infection


Charles TP, Burton SL, Arunachalam PS, Cottrell CA, Sewall LM, Bollimpelli VS, Gangadhara S, Dey AK, Ward AB, Shaw GM, Hunter E, Amara RR, Pulendran B, van Gils MJ, Derdeyn CA.
PLoS Pathogens Feb. 8, 2021

Stabilized HIV-1 envelope (Env) trimers elicit tier 2 autologous neutralizing antibody (nAb) responses in immunized animals. We previously demonstrated that BG505 SOSIP.664.T332N gp140 (BG505 SOSIP) immunization of rhesus macaques (RM) provided robust protection against autologous intra-vaginal simian-human immunodeficiency virus (SHIV) challenge that was predicted by high serum nAb titers. Here, we show that nAb in these protected RM targeted a glycan hole proximal to residue 465 in gp120 in all cases. nAb also targeted another glycan hole at residues 241/289 and an epitope in V1 at varying frequencies. Non-neutralizing antibodies directed at N611-shielded epitopes in gp41 were also present but were more prevalent in RM with low nAb titers. Longitudinal analysis demonstrated that nAb broadened in some RM during sequential immunization but remained focused in others, the latter being associated with increases in nAb titer. Thirty-eight monoclonal antibodies (mAbs) isolated from a protected RM with an exceptionally high serum neutralization titer bound to the trimer in ELISA, and four of the mAbs potently neutralized the BG505 Env pseudovirus (PV) and SHIV. The four neutralizing mAbs were clonally related and targeted the 465 glycan hole to varying degrees, mimicking the serum. The data demonstrate that the C3/465 glycan hole cluster was the dominant neutralization target in high titer protected RM, despite other co-circulating neutralizing and non-neutralizing specificities. The isolation of a neutralizing mAb family argues that clonotype expansion occurred during BG505 SOSIP immunization, leading to high titer, protective nAb and setting a desirable benchmark for HIV vaccines.

The C3/465 glycan hole cluster in BG505 HIV-1 envelope is the major neutralizing target involved in preventing mucosal SHIV infection


Charles TP, Burton SL, Arunachalam PS, Cottrell CA, Sewall LM, Bollimpelli VS, Gangadhara S, Dey AK, Ward AB, Shaw GM, Hunter E, Amara RR, Pulendran B, van Gils MJ, Derdeyn CA.
PLoS Pathogens Feb. 8, 2021

Stabilized HIV-1 envelope (Env) trimers elicit tier 2 autologous neutralizing antibody (nAb) responses in immunized animals. We previously demonstrated that BG505 SOSIP.664.T332N gp140 (BG505 SOSIP) immunization of rhesus macaques (RM) provided robust protection against autologous intra-vaginal simian-human immunodeficiency virus (SHIV) challenge that was predicted by high serum nAb titers. Here, we show that nAb in these protected RM targeted a glycan hole proximal to residue 465 in gp120 in all cases. nAb also targeted another glycan hole at residues 241/289 and an epitope in V1 at varying frequencies. Non-neutralizing antibodies directed at N611-shielded epitopes in gp41 were also present but were more prevalent in RM with low nAb titers. Longitudinal analysis demonstrated that nAb broadened in some RM during sequential immunization but remained focused in others, the latter being associated with increases in nAb titer. Thirty-eight monoclonal antibodies (mAbs) isolated from a protected RM with an exceptionally high serum neutralization titer bound to the trimer in ELISA, and four of the mAbs potently neutralized the BG505 Env pseudovirus (PV) and SHIV. The four neutralizing mAbs were clonally related and targeted the 465 glycan hole to varying degrees, mimicking the serum. The data demonstrate that the C3/465 glycan hole cluster was the dominant neutralization target in high titer protected RM, despite other co-circulating neutralizing and non-neutralizing specificities. The isolation of a neutralizing mAb family argues that clonotype expansion occurred during BG505 SOSIP immunization, leading to high titer, protective nAb and setting a desirable benchmark for HIV vaccines.

Prominent Neutralizing Antibody Response Targeting the Ebolavirus Glycoprotein Subunit Interface Elicited by Immunization


Wang Y, Howell KA, Brannan J, Agans KN, Turner HL, Wirchnianski AS, Kailasan S, Fusco M, Galkin A, Chiang CI, Zhao X, Saphire EO, Chandran K, Ward AB, Dye JM, Aman MJ, Geisbert TW, Li Y.
Journal of Virology Feb. 3, 2021

The elicitation of sustained neutralizing antibody (nAb) responses against diverse ebolavirus strains remains a high priority for the vaccine field. The most clinically advanced rVSV-ZEBOV vaccine could elicit moderate nAb responses against only one ebolavirus strain, Zaire Ebola (EBOV), among the five ebolavirus strains, which last less than 6 months.

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection


Brouwer PJM, Brinkkemper M, Maisonnasse P, Dereuddre-Bosquet N, Grobben M, Claireaux M, de Gast M, Marlin R, Chesnais V, Diry S, Allen JD, Watanabe Y, Giezen JM, Kerster G, Turner HL, van der Straten K, van der Linden CA, Aldon Y, Naninck T, Bontjer I, Burger JA, Poniman M, Mykytyn AZ, Okba NMA, Schermer EE, van Breemen MJ, Ravichandran R, Caniels TG, van Schooten J, Kahlaoui N, Contreras V, Lemaître J, Chapon C, Fang RHT, Villaudy J, Sliepen K, van der Velden YU, Haagmans BL, de Bree GJ, Ginoux E, Ward AB, Crispin M, King NP, van der Werf S, van Gils MJ, Le Grand R, Sanders RW.
Cell Jan. 26, 2021

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication in the upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

Polyclonal epitope mapping reveals temporal dynamics and diversity of human antibody responses to H5N1 vaccination


Han J, Schmitz AJ, Richey ST, Dai YN, Turner HL, Mohammed BM, Fremont DH, Ellebedy AH, Ward AB.
Cell Reports Jan. 26, 2021

Novel influenza A virus (IAV) strains elicit recall immune responses to conserved epitopes, making them favorable antigenic choices for universal influenza virus vaccines. Evaluating these immunogens requires a thorough understanding of the antigenic sites targeted by the polyclonal antibody (pAb) response, which single-particle electron microscopy (EM) can sensitively detect. In this study, we employ EM polyclonal epitope mapping (EMPEM) to extensively characterize the pAb response to hemagglutinin (HA) after H5N1 immunization in humans. Cross-reactive pAbs originating from memory B cells immediately bound the stem of HA and persisted for more than a year after vaccination. In contrast, de novo pAb responses to multiple sites on the head of HA, targeting previously determined key neutralizing sites on H5 HA, expanded after the second immunization and waned quickly. Thus, EMPEM provides a robust tool for comprehensively tracking the specificity and durability of immune responses elicited by novel universal influenza vaccine candidates.

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


Antanasijevic A, Schulze AJ, Reddy VS, Ward AB.
PNAS Nexus Jan. 13, 2021

Non-polio enteroviruses (NPEVs) cause serious illnesses in young children and neonates, including aseptic meningitis, encephalitis, and inflammatory muscle disease, among others. While over 100 serotypes have been described to date, vaccine only exists for EV-A71. Efforts toward rationally designed pan-NPEV vaccines would greatly benefit from structural biology methods for rapid and comprehensive evaluation of vaccine candidates and elicited antibody responses. Toward this goal, we introduced a cryo-electron-microscopy-based approach for structural analysis of virus- or vaccine-elicited polyclonal antibodies (pAbs) in complex with whole NPEV virions. We demonstrated the feasibility using coxsackievirus A21 and reconstructed five structurally distinct pAbs bound to the virus. The pAbs targeted two immunodominant epitopes, one overlapping with the receptor binding site. These results demonstrate that our method can be applied to map broad-spectrum polyclonal immune responses against intact virions and define potentially cross-reactive epitopes.

Structure and immune recognition of the porcine epidemic diarrhea virus spike protein


Kirchdoerfer RN, Bhandari M, Martini O, Sewall LM, Bangaru S, Yoon KJ, Ward AB
Structure Dec. 22, 2020

Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus responsible for significant morbidity and mortality in pigs. A key determinant of viral tropism and entry, the PEDV spike protein is a key target for the host antibody response and a good candidate for a protein-based vaccine immunogen. We used electron microscopy to evaluate the PEDV spike structure, as well as pig polyclonal antibody responses to viral infection. The structure of the PEDV spike reveals a configuration similar to that of HuCoV-NL63. Several PEDV protein-protein interfaces are mediated by non-protein components, including a glycan at Asn264 and two bound palmitoleic acid molecules. The polyclonal antibody response to PEDV infection shows a dominance of epitopes in the S1 region. This structural and immune characterization provides insights into coronavirus spike stability determinants and explores the immune landscape of viral spike proteins.

Quantification of the Resilience and Vulnerability of HIV-1 Native Glycan Shield at Atomistic Detail


Chakraborty S, Berndsen ZT, Hengartner NW, Korber BT, Ward AB, Gnanakaran S.
iScience Dec. 18, 2020

Dense surface glycosylation on the HIV-1 envelope (Env) protein acts as a shield from the adaptive immune system. However, the molecular complexity and flexibility of glycans make experimental studies a challenge. Here we have integrated high-throughput atomistic modeling of fully glycosylated HIV-1 Env with graph theory to capture immunologically important features of the shield topology. This is the first complete all-atom model of HIV-1 Env SOSIP glycan shield that includes both oligomannose and complex glycans, providing physiologically relevant insights of the glycan shield. This integrated approach including quantitative comparison with cryo-electron microscopy data provides hitherto unexplored details of the native shield architecture and its difference from the high-mannose glycoform. We have also derived a measure to quantify the shielding effect over the antigenic protein surface that defines regions of relative vulnerability and resilience of the shield and can be harnessed for rational immunogen design.

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody


Wu NC, Yuan M, Bangaru S, Huang D, Zhu X, Lee CD, Turner HL, Peng L, Yang L, Burton DR, Nemazee D, Ward AB, Wilson IA.
PLoS Pathogens Dec. 4, 2020

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.

Diverse Antibody Responses to Conserved Structural Motifs in Plasmodium falciparum Circumsporozoite Protein


Pholcharee T, Oyen D, Torres JL, Flores-Garcia Y, Martin GM, González-Páez GE, Emerling D, Volkmuth W, Locke E, King CR, Zavala F, Ward AB, Wilson IA.
Journal of Molecular Biology Nov. 25, 2020

Malaria vaccine candidate RTS,S/AS01 is based on the central and C-terminal regions of the circumsporozoite protein (CSP) of P. falciparum. mAb397 was isolated from a volunteer in an RTS,S/AS01 clinical trial, and it protects mice from infection by malaria sporozoites. However, mAb397 originates from the less commonly used VH3-15 germline gene compared to the VH3-30/33 antibodies generally elicited by RTS,S to the central NANP repeat region of CSP. The crystal structure of mAb397 with an NPNA4 peptide shows that the central NPNA forms a type I β-turn and is the main recognition motif. In most anti-NANP antibodies studied to date, a germline-encoded Trp is used to engage the Pro in NPNA β-turns, but here the Trp interacts with the first Asn. This “conserved” Trp, however, can arise from different germline genes and be located in the heavy or the light chain. Variation in the terminal ψ angles of the NPNA β-turns results in different dispositions of the subsequent NPNA and, hence, different stoichiometries and modes of antibody binding to rsCSP. Diverse protective antibodies against NANP repeats are therefore not limited to a single germline gene response or mode of binding.

A Strain-Specific Inhibitor of Receptor-Bound HIV-1 Targets a Pocket near the Fusion Peptide


Ozorowski G, Torres JL, Santos-Martins D, Forli S, Ward AB.
Cell Reports Nov. 24, 2020

Disruption of viral fusion represents a viable, albeit under-explored, target for HIV therapeutics. Here, while studying the receptor-bound envelope glycoprotein conformation by cryoelectron microscopy (cryo-EM), we identify a pocket near the base of the trimer containing a bound detergent molecule and perform in silico drug screening by using a library of drug-like and commercially available molecules. After down-selection, we solve cryo-EM structures that validate the binding of two small molecule hits in very similar manners to the predicted binding poses, including interactions with aromatic residues within the fusion peptide. One of the molecules demonstrates low micromolar inhibition of the autologous virus by using a very rare phenylalanine in the fusion peptide and stabilizing the surrounding region. This work demonstrates that small molecules can target the fusion process, providing an additional target for anti-HIV therapeutics, and highlights the need to explore how fusion peptide sequence variations affect receptor-mediated conformational states across diverse HIV strains.

Cross-neutralization of influenza A viruses mediated by a single antibody loop


Ekiert DC, Kashyap AK, Steel J, Rubrum A, Bhabha G, Khayat R, Lee JH, Dillon MA, O'Neil RE, Faynboym AM, Horowitz M, Horowitz L, Ward AB, Palese P, Webby R, Lerner RA, Bhatt RR, Wilson IA
Nature Nov. 21, 2020

Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens. The crystal structure of an influenza antibody that recognizes a small, conserved site in the variable receptor-binding domain of HA is described; this antibody shows broad neutralization across multiple subtypes of influenza A virus through an antibody–antigen interaction dominated by a single heavy-chain complementarity-determining region 3 loop. This manuscript reports the identification and structural characterization of a novel anti-influenza antibody, C05, that recognizes a small conserved site in the variable receptor-binding domain of haemagglutinin. The antibody achieves broad neutralization by the insertion of a single loop of the heavy-chain complementarity-determining region 3 into the small conserved site amplified by the avidity of additional binding interactions. This finding highlights loop insertion into the receptor-binding pocket of haemagglutinin as a possible strategy to achieve broad neutralization of influenza by vaccines and therapeutic antibodies.

Visualization of the HIV-1 Env glycan shield across scales


Berndsen ZT, Chakraborty S, Wang X, Cottrell CA, Torres JL, Diedrich JK, López CA, Yates JR 3rd, van Gils MJ, Paulson JC, Gnanakaran S, Ward AB.
Proceedings of the National Academy of Sciences of the United States of America Oct. 22, 2020

The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the “glycan shield,” is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure–function analysis. Here, we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same cell line used for vaccine production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner, we found that highly connected glycan clusters are resistant to digestion and help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.

Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate


Bangaru S, Ozorowski G, Turner HL, Antanasijevic A, Huang D, Wang X, Torres JL, Diedrich JK, Tian JH, Portnoff AD, Patel N, Massare MJ, Yates JR 3rd, Nemazee D, Paulson JC, Glenn G, Smith G, Ward AB.
Science Oct. 20, 2020

Vaccine efforts to combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the current coronavirus disease 2019 (COVID-19) pandemic, are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. We performed cryo–election microscopy and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax, which is based on a full-length spike protein formulated in polysorbate 80 detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared with published spike ectodomain structures. We also observed interactions between the spike trimers, allowing formation of higher-order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.

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Title & Authors Journal Publication Date

Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM


Antanasijevic A, Sewall LM, Cottrell CA, Carnathan DG, Jimenez LE, Ngo JT, Silverman JB, Groschel B, Georgeson E, Bhiman J, Bastidas R, LaBranche C, Allen JD, Copps J, Perrett HR, Rantalainen K, Cannac F, Yang YR, Torrents de la Peña A, Froes Rocha R, Berndsen ZT, Baker D, King NP, Sanders RW, Moore JP, Crotty S, Crispin M, Montefiori DC, Burton DR, Schief WR, Silvestri G, Ward AB

Now Published: 10.1038/s41467-021-25087-4
bioRxiv Jan. 28, 2021

Canonical features of human antibodies recognizing the influenza hemagglutinin trimer interface


Zost SJ, Dong J, Gilchuk I, Gilchuk P, Thornburg NJ, Bangaru S, Kose N, Finn JA, Bombardi R, Soto C, Nargi R, Irving RP, Suryadevara N, Westover JB, Carnahan RH, Turner HL, Li S, Ward AB, Crowe JE

Now Published: 10.1172/jci146791
bioRxiv Dec. 31, 2020

Neutralizing antibodies induced by first-generation gp41-stabilized HIV-1 envelope trimers and nanoparticles


Kumar S, Lin X, Ngo T, Shapero B, Sou C, Allen JD, Copps J, Zhang L, Ozorowski G, He L, Crispin M, Ward AB, Wilson IA, Zhu J

Now Published: 10.1128/mbio.00429-21
bioRxiv Dec. 2, 2020

Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles


Brouwer PJM, Antanasijevic A, de Gast M, Allen JD, L. Bijl TP, Yasmeen A, Ravichandran R, Burger JA, Ozorowski G, Torres JL, LaBranche C, Montefiori DC, Ringe RP, van Gils MJ, Moore JP, Klasse PJ, Crispin M, King NP, Ward AB, Sanders RW

Now Published: 10.1038/s41541-021-00285-9
bioRxiv Nov. 30, 2020

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection


Brouwer PJM, Brinkkemper M, Maisonnasse P, Dereuddre-Bosquet N, Grobben M, Claireaux M, de Gast M, Marlin R, Chesnais V, Diry S, Allen JD, Watanabe Y, Giezen JM, Kerster G, Turner HL, van der Straten K, van der Linden CA, Aldon Y, Naninck T, Bontjer I, Burger JA, Poniman M, Mykytyn AZ, A. Okba NM, Schermer EE, van Breemen MJ, Ravichandran R, Caniels TG, van Schooten J, Kahlaoui N, Contreras V, Lemaître J, Chapon C, Ho Tsong Fang R, Villaudy J, Sliepen K, van der Velden YU, Haagmans BL, de Bree GJ, Ginoux E, Ward AB, Crispin M, King NP, van der Werf S, van Gils MJ, Le Grand R, Sanders RW

Now Published: 10.1016/j.cell.2021.01.035
bioRxiv Nov. 7, 2020

High-resolution mapping of the neutralizing and binding specificities of polyclonal rabbit serum elicited by HIV Env trimer immunization


Dingens AS, Pratap P, Malone K, Hilton SK, Ketas T, Cottrell CA, Overbaugh J, Moore JP, Klasse PK, Ward AB, Bloom JD

Now Published: 10.7554/eLife.64281
bioRxiv Oct. 21, 2020