Biology subjects

Schmidt, A. G.

Publications and source records attributed to Schmidt, A. G..

27 records · Page 2Linked to original sources

bioRxiv2021

Rationally designed immunogens enable immune focusing to the SARS-CoV-2 receptor binding motif

Eliciting antibodies to surface-exposed viral glycoproteins can lead to protective responses that ultimately control and prevent future infections. Targeting functionally conserved epitopes may help reduce the likelihood of viral escape and aid in preventing the spread of related viruses with pandemic potential. One such functionally conserved viral epitope is the site to which a receptor must bind to facilitate viral entry. Here, we leveraged rational immunogen design strategies to focus humoral responses to the receptor binding motif (RBM) on the SARS-CoV-2 spike. Using glycan engineering and epitope scaffolding, we find an improved targeting of the serum response to the RBM in context of SARS-CoV-2 spike imprinting. Furthermore, we observed a robust SARS-CoV-2-neutralizing serum response with increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses and represents an adaptable design approach for targeting conserved epitopes on other viral glycoproteins. One Sentence SummarySARS-CoV-2 immune focusing with engineered immunogens

immunology↗

bioRxiv2021

Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with an unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded monoclonal antibodies (mAbs) from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found 7 major mAb competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of mAb-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. mAbs that competed for binding the original S isolate bound differentially to S variants, suggesting the protective importance of otherwise-redundant recognition. The results furnish a global atlas of the S-specific memory B cell repertoire and illustrate properties conferring robustness against emerging SARS-CoV-2 variants.

immunology↗

bioRxiv2021

Naive human B cells can neutralize SARS-CoV-2 through recognition of its receptor binding domain

Exposure to a pathogen elicits an adaptive immune response aimed to control and eradicate. Interrogating the abundance and specificity of the naive B cell repertoire contributes to understanding how to potentially elicit protective responses. Here, we isolated naive B cells from 8 seronegative human donors targeting the SARS-CoV-2 receptor-binding domain (RBD). Single B cell analysis showed diverse gene usage with no restricted complementarity determining region lengths. We show that recombinant antibodies engage SARS-CoV-2 RBD, circulating variants, and pre-emergent coronaviruses. Representative antibodies signal in a B cell activation assay and can be affinity matured through directed evolution. Structural analysis of a naive antibody in complex with spike shows a conserved mode of recognition shared with infection-induced antibodies. Lastly, both naive and affinity-matured antibodies can neutralize SARS-CoV-2. Understanding the naive repertoire may inform potential responses recognizing variants or emerging coronaviruses enabling the development of pan-coronavirus vaccines aimed at engaging germline responses. One Sentence SummaryIsolation of antibody germline precursors targeting the receptor binding domain of coronaviruses.

immunology↗

bioRxiv2021

Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates

The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.

immunology↗

bioRxiv2020

Engineered receptor binding domain immunogens elicit pan-coronavirus neutralizing antibodies

Effective countermeasures are needed against emerging coronaviruses of pandemic potential, similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Designing immunogens that elicit broadly neutralizing antibodies to conserved viral epitopes on the major surface glycoprotein, spike, such as the receptor binding domain (RBD) is one potential approach. Here, we report the generation of homotrimeric RBD immunogens from different sarbecoviruses using a stabilized, immune-silent trimerization tag. In mice, we find that a cocktail of these homotrimeric sarbecovirus RBDs elicits antibodies to conserved viral epitopes outside of the ACE2 receptor binding motif (RBM). Importantly, these responses neutralize all sarbecovirus components even in context of prior SARS-CoV-2 imprinting. We further show that a substantial fraction of the neutralizing antibodies elicited after vaccination in humans also engages non-RBM epitopes on the RBD. Collectively, our results suggest a strategy for eliciting broadly neutralizing responses leading to a pan-sarbecovirus vaccine. Author summaryImmunity to SARS-CoV-2 in the human population will be widespread due to natural infection and vaccination. However, another novel coronavirus will likely emerge in the future and may cause a subsequent pandemic. Humoral responses induced by SARS-CoV-2 infection and vaccination provide limited protection against even closely related coronaviruses. We show immunization with a cocktail of trimeric coronavirus receptor binding domains induces a neutralizing antibody response that is broadened to related coronaviruses with pandemic potential. Importantly, this broadening occurs in context of an initial imprinted SARS-CoV-2 spike immunization showing that preexisting immunity can be expanded to recognize other related coronaviruses. Our immunogens focused the serum antibody response to conserved epitopes on the receptor binding domain outside of the ACE2 receptor binding motif; this contrasts with current SARS-CoV-2 therapeutic antibodies, which predominantly target the receptor binding motif.

immunology↗

bioRxiv2020

Rapid generation of potent antibodies by autonomous hypermutation in yeast

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, has poor compatibility with certain antigens (e.g., integral membrane proteins), and suffers from self-tolerance and immunodominance, which limit the functional spectrum of antibodies that can be obtained. Here, we describe Autonomous Hypermutation yEast surfAce Display (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. In AHEAD, antibody fragments are encoded on an error-prone orthogonal DNA replication system, resulting in Saccharomyces cerevisiae populations that continuously mutate surface-displayed antibody repertoires. Simple cycles of yeast culturing and enrichment for antigen binding drive the evolution of high-affinity antibody clones in a readily parallelizable process that takes as little as 2 weeks. We applied AHEAD to generate nanobodies against the SARS-CoV-2 S glycoprotein, a GPCR, and other targets. The SARS-CoV-2 nanobodies, concurrently evolved from an open-source naive nanobody library in 8 independent experiments, reached subnanomolar affinities through the sequential fixation of multiple mutations over 3-8 AHEAD cycles that saw [~]580-fold and [~]925-fold improvements in binding affinities and pseudovirus neutralization potencies, respectively. These experiments highlight the defining speed, parallelizability, and effectiveness of AHEAD and provide a template for streamlined antibody generation at large with salient utility in rapid response to current and future viral outbreaks.

bioengineering↗

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

Mercury methylation trait dispersed across diverse anaerobic microbial guilds in a eutrophic sulfate-enriched lake

Mercury (Hg) methylation is a microbially mediated process that converts inorganic Hg into the bioaccumulative neurotoxin methylmercury (MeHg). Exploring the diversity and metabolic potential of the dominant Hg-methylating microorganisms can provide insights into how biogeochemical cycles and water quality parameters underlie MeHg production. However, our understanding of the ecophysiology of methylators in natural ecosystems is still limited. Here, we used shotgun metagenomics paired with biogeochemical data to identify likely hotspots for MeHg production in a lake with elevated sulfate levels and characterize the microbial methylators and the flanking microbial community. Identified putative methylators were dominated by hgcA sequences divergent from those in canonical, experimentally confirmed methylators. Using genome-resolved metagenomics, these sequences were identified within genomes associated with Bacteroidetes and the recently described phylum Kiritimatiellaeota. Over half of the hgcA abundance comes from genomes corresponding to obligately fermentative organisms, many of which have a large number of glucoside hydrolases for polysaccharide degradation. Sulfate-reducing genomes encoding hgcA were also identified, but only accounted for 22% of the abundance of hgcA+ genomes. This work highlights the diverse dispersal of the methylation trait across the microbial anoxic food web.

genomics↗

bioRxiv2019

Structure-guided molecular grafting of a complex broadly neutralizing viral epitope

Antigenic variation and viral evolution have thwarted traditional influenza vaccination strategies. The broad protection afforded by a "universal" influenza vaccine will come from immunogens that elicit humoral immune responses targeting conserved epitopes on the viral hemagglutinin (HA), such as the receptor-binding site (RBS). Here, we engineered candidate immunogens that use non-circulating, avian influenza HAs as molecular scaffolds to present the broadly neutralizing RBS epitope from historical, circulating H1 influenzas. These "resurfaced" HAs (rsHAs) remove epitopes potentially targeted by strain-specific responses in immune-experienced individuals. Through structure-guided optimization we improved two antigenically different scaffolds to bind a diverse panel of pan-H1 and H1/H3 cross-reactive bnAbs with high affinity. Subsequent serological analyses from murine prime-boost immunizations show that the rsHAs are both immunogenic and can enrich for RBS-directed antibodies. Our structure-guided, RBS grafting approach provides candidate immunogens for selectively presenting a conserved viral epitope.

immunology↗