Biology subjects

Torres, J. L.

Publications and source records attributed to Torres, J. L..

36 records · Page 2Linked to original sources

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

One dose of COVID-19 nanoparticle vaccine REVC-128 provides protection against SARS-CoV-2 challenge at two weeks post immunization

A COVID-19 vaccine with capability to induce early protection is needed to efficiently eliminate viral spread. Here, we demonstrate the development of a nanoparticle vaccine candidate, REVC-128, in which multiple trimeric spike ectodomain subunits with glycine (G) at position 614 were multimerized onto a nanoparticle. In-vitro characterization of this vaccine confirms its structural and antigenic integrity. In-vivo immunogenicity evaluation in mice indicates that a single dose of this vaccine induces potent serum neutralizing antibody titer at two weeks post immunization, which is significantly higher than titer induced by trimeric spike protein without nanoparticle presentation. The comparison of serum binding to spike subunits between animals immunized by spike with and without nanoparticle presentation indicates that nanoparticle prefers the display of spike RBD (Receptor-Binding Domain) over S2 subunit, likely resulting in a more neutralizing but less cross-reactive antibody response. Moreover, a Syrian golden hamster in-vivo model for SARS-CoV-2 virus challenge was implemented at two weeks post a single dose of REVC-128 immunization. The results show that vaccination protects hamsters against SARS-CoV-2 virus challenge with evidence of steady body weight, suppressed viral loads and alleviation of tissue damage (lung and nares) for protected animals, compared with ~10% weight loss, higher viral loads and tissue damage in unprotected animals. Furthermore, the data show that vaccine REVC-128 is thermostable at up to 37{degrees}C for at least 4 weeks. These findings, along with a long history of safety for protein vaccines, suggest that the REVC-128 is a safe, stable and efficacious single-shot vaccine candidate to induce the earliest protection against SARS-CoV-2 infection.

microbiology↗

bioRxiv2021

Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

The emergence of SARS-CoV-2 variants that threaten the efficacy of existing vaccines and therapeutic antibodies underscores the urgent need for new antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells of COVID-19 patients. The three most potent antibodies targeted distinct regions of the RBD, and all three neutralized the SARS-CoV-2 variants B.1.1.7 and B.1.351. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the ACE2 receptor, and has limited contact with key variant residues K417, E484 and N501. We designed bispecific antibodies by combining non-overlapping specificities and identified five ultrapotent bispecific antibodies that inhibit authentic SARS-CoV-2 infection at concentrations of <1 ng/mL. Through a novel mode of action three bispecific antibodies cross-linked adjacent spike proteins using dual NTD/RBD specificities. One bispecific antibody was >100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a 2.5 mg/kg dose. Notably, six of nine bispecific antibodies neutralized B.1.1.7, B.1.351 and the wild-type virus with comparable potency, despite partial or complete loss of activity of at least one parent monoclonal antibody against B.1.351. Furthermore, a bispecific antibody that neutralized B.1.351 protected against SARS-CoV-2 expressing the crucial E484K mutation in the hamster model. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.

immunology↗

bioRxiv2020

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

The HIV-1 envelope glycoprotein trimer is poorly immunogenic because it is covered by a dense glycan shield. As a result, recombinant Env glycoproteins generally elicit inadequate antibody levels that neutralize clinically-relevant, neutralization-resistant (Tier-2) HIV-1 strains. Multivalent antigen presentation on nanoparticles is an established strategy to increase vaccine-driven immune responses. However, due to nanoparticle instability in vivo, the display of non-native Env structures, and the inaccessibility of many neutralizing antibody (NAb) epitopes, the effects of nanoparticle display have been modest for Env trimers. Here, we generated two-component self-assembling protein nanoparticles presenting twenty SOSIP trimers of the clade C Tier-2 genotype 16055. An immunization study in rabbits demonstrated that these nanoparticles induced 60-fold higher autologous Tier-2 NAb titers than the corresponding SOSIP trimers. Epitope mapping revealed that nanoparticle presentation focused antibody responses to an immunodominant apical epitope. Thus, these nanoparticles are a promising platform to improve the immunogenicity of Env trimers with apex-proximate NAb epitopes.

microbiology↗

bioRxiv2020

Cross-reactive serum and memory B cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection

Pre-existing immune responses to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, either induced in natural infection or through vaccination. Such consequences are well established in the influenza and flavivirus fields. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. We compared serum antibody and memory B cell responses to coronavirus spike (S) proteins from pre-pandemic and SARS-CoV-2 convalescent donors using a series of binding and functional assays. We found weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we found stronger evidence of pre-existing cross-reactive memory B cells that were activated on SARS-CoV-2 infection. Monoclonal antibodies (mAbs) isolated from the donors showed varying degrees of cross-reactivity with betacoronaviruses, including SARS and endemic coronaviruses. None of the cross-reactive mAbs were neutralizing except for one that targeted the S2 subunit of the S protein. The results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.

immunology↗

bioRxiv2020

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

Vaccine efforts against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the current COVID-19 pandemic are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. Here, we performed cryo-EM and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax based on a full-length spike protein formulated in polysorbate 80 (PS 80) detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared to published spike ectodomain structures. Interestingly, we also observed novel 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.

immunology↗

bioRxiv2020

Cross-neutralization of a SARS-CoV-2 antibody to a functionally conserved site is mediated by avidity

Most antibodies isolated from COVID-19 patients are specific to SARS-CoV-2. COVA1-16 is a relatively rare antibody that also cross-neutralizes SARS-CoV. Here we determined a crystal structure of COVA1-16 Fab with the SARS-CoV-2 RBD, and a negative-stain EM reconstruction with the spike glycoprotein trimer, to elucidate the structural basis of its cross-reactivity. COVA1-16 binds a highly conserved epitope on the SARS-CoV-2 RBD, mainly through a long CDR H3, and competes with ACE2 binding due to steric hindrance rather than epitope overlap. COVA1-16 binds to a flexible up conformation of the RBD on the spike and relies on antibody avidity for neutralization. These findings, along with structural and functional rationale for the epitope conservation, provide a blueprint for development of more universal SARS-like coronavirus vaccines and therapies.

microbiology↗

bioRxiv2020

An alternative binding mode of IGHV3-53 antibodies to the SARS-CoV-2 receptor binding domain

IGHV3-53-encoded neutralizing antibodies are commonly elicited during SARS-CoV-2 infection and target the receptor-binding domain (RBD) of the spike (S) protein. Such IGHV3-53 antibodies generally have a short CDR H3 due to structural constraints in binding the RBD (mode A). However, a small subset of IGHV3-53 antibodies to the RBD contain a longer CDR H3. Crystal structures of two IGHV3-53 neutralizing antibodies here demonstrate that a longer CDR H3 can be accommodated in a different binding mode (mode B). These two classes of IGHV3-53 antibodies both target the ACE2 receptor binding site, but with very different angles of approach and molecular interactions. Overall, these findings emphasize the versatility of IGHV3-53 in this common antibody response to SARS-CoV-2, where conserved IGHV3-53 germline-encoded features can be combined with very different CDR H3 lengths and light chains for SARS-CoV-2 RBD recognition and virus neutralization.

microbiology↗

bioRxiv2020

SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.

microbiology↗

bioRxiv2020

A strain-specific inhibitor of receptor-bound HIV-1 targets a pocket near the fusion peptide and offers a template for drug design

Disruption of viral fusion represents a viable, albeit under-explored, target for HIV therapeutics. While studying the receptor-bound envelope glycoprotein conformation by cryo-EM, we identified a pocket near the base of the trimer containing a bound detergent molecule and performed in silico drug screening using a library of drug-like and commercially available molecules. After down-selection, we solved cryo-EM structures that validated 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 demonstrated low micromolar inhibition of the autologous virus by utilizing 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 a new 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.

biophysics↗

bioRxiv2020

Mapping the immunogenic landscape of near-native HIV-1 envelope trimers in non-human primates

The induction of broad and potent immunity by vaccines is the key focus of research efforts aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins have shown promise as vaccine candidates as they can induce potent autologous neutralizing responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and have characteristics akin to several human-derived broadly neutralizing antibodies.

immunology↗

bioRxiv2019

HIV-1 Envelope and MPER antibody structures in lipid assemblies

Structural and functional studies of HIV Env as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Thus, most structural studies have utilized soluble forms where the regions C-terminal to the ectodomain are deleted. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstituted a full-length Env clone into a nanodisc with MSP1D1 scaffold, complexed it with an MPER targeting antibody 10E8, and structurally defined the full quaternary epitope of 10E8 consisting of lipid, MPER and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapted the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with peptidisc scaffold.

immunology↗

bioRxiv2019

Visualization of the HIV-1 Env Glycan Shield Across Scales

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 inter-glycan 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 CHO cell line used for GMP 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 pre-fusion trimer, suggesting the glycan shield may function beyond immune evasion. Significance StatementThe HIV-1 Env "glycan shield" masks the surface of the protein from immune recognition, yet intrinsic heterogeneity defies a typical structure-function description. Using a complementary approach of cryo-EM, computational modeling, and mass-spectrometry we show how heterogeneity and dynamics affect glycan shield structure across scales. Our combined approach facilitated the development of new cryo-EM data analysis methods and allowed for validation of models against experiment. Comparison of Env across a range of glycosylation states revealed how subtle differences in composition impact glycan shield structure and affect the accessibility of epitopes on the surface. Finally, time-resolved cryo-EM experiments uncovered how highly connected glycan clusters help stabilize the pre-fusion trimer, suggesting the glycan shield may function beyond immune evasion.

biophysics↗

bioRxiv2018

Similarities and differences between native HIV-1 envelope glycoprotein trimers and stabilized soluble trimer mimetics

The HIV-1 envelope glycoprotein (Env) trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env trimer mimetics, such as SOSIP trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length native Env trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP trimers. Antigenically, the full-length and SOSIP trimers were comparable, but in contrast to the full-length trimer, the SOSIP trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP trimer. Furthermore, the glycan composition of full-length and SOSIP trimers was similar overall, but the SOSIP trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env trimers as immunogens.

immunology↗

bioRxiv2018

Cryo-EM structure of the circumsporozoite protein of Plasmodium falciparum with a vaccine-elicited antibody reveals maturation of inter-antibody contacts

The circumsporozoite protein (CSP) on the surface of Plasmodium falciparum sporozoites is important for parasite development, motility, and host hepatocyte invasion. However, intrinsic disorder of the NANP repeat sequence in the central region of CSP has hindered its structural and functional characterization. Here, the cryo-EM structure at [~]3.4 [A] resolution of a recombinant shortened CSP construct (rsCSP) with the variable domains (Fabs) of a highly protective monoclonal antibody reveals an extended spiral conformation of the central NANP repeat region surrounded by antibodies. This unusual structure appears to be stabilized and/or induced by interaction with an antibody where contacts between adjacent Fabs are somatically mutated and enhance the interaction. Such maturation in non-antigen contact residues may be an effective mechanism for antibodies to target tandem repeat sequences and provide novel insights into malaria vaccine design.\n\nSummaryAn unusual spiral conformation is formed for the NANP repeat region in Plasmodium falciparum circumsporozoite protein (CSP) in complex with antibodies generated by the RTS,S vaccine and is stabilized by affinity-matured inter-Fab interactions.

immunology↗