Biology subjects

Stalls, V.

Publications and source records attributed to Stalls, V..

17 recordsLinked to original sources

bioRxiv2023

Broadly neutralizing antibody induction by non-stabilized SARS-CoV-2 Spike mRNA vaccination in nonhuman primates

Immunization with mRNA or viral vectors encoding spike with diproline substitutions (S-2P) has provided protective immunity against severe COVID-19 disease. How immunization with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike elicits neutralizing antibodies (nAbs) against difficult-to-neutralize variants of concern (VOCs) remains an area of great interest. Here, we compare immunization of macaques with mRNA vaccines expressing ancestral spike either including or lacking diproline substitutions, and show the diproline substitutions were not required for protection against SARS-CoV-2 challenge or induction of broadly neutralizing B cell lineages. One group of nAbs elicited by the ancestral spike lacking diproline substitutions targeted the outer face of the receptor binding domain (RBD), neutralized all tested SARS-CoV-2 VOCs including Omicron XBB.1.5, but lacked cross-Sarbecovirus neutralization. Structural analysis showed that the macaque broad SARS-CoV-2 VOC nAbs bound to the same epitope as a human broad SARS-CoV-2 VOC nAb, DH1193. Vaccine-induced antibodies that targeted the RBD inner face neutralized multiple Sarbecoviruses, protected mice from bat CoV RsSHC014 challenge, but lacked Omicron variant neutralization. Thus, ancestral SARS-CoV-2 spike lacking proline substitutions encoded by nucleoside-modified mRNA can induce B cell lineages binding to distinct RBD sites that either broadly neutralize animal and human Sarbecoviruses or recent Omicron VOCs. One Sentence SummaryNon-stabilized SARS-CoV-2 Spike mRNA vaccination activated B cells that target either conserved epitopes on SARS-CoV-2 Omicron variants of concern, or cross-neutralizing epitopes on pre-emergent Sarbecoviruses.

immunology↗

bioRxiv2023

Engineering immunogens that select for specific mutations in HIV broadly neutralizing antibodies

Vaccine development targeting rapidly evolving pathogens such as HIV-1 requires induction of broadly neutralizing antibodies (bnAbs) with conserved paratopes and mutations, and, in some cases, the same Ig-heavy chains. The current trial-and-error search for immunogen modifications that improve selection for specific bnAb mutations is imprecise. To precisely engineer bnAb boosting immunogens, we used molecular dynamics simulations to examine encounter states that form when antibodies collide with the HIV-1 Envelope (Env). By mapping how bnAbs use encounter states to find their bound states, we identified Env mutations that were predicted to select for specific antibody mutations in two HIV-1 bnAb B cell lineages. The Env mutations encoded antibody affinity gains and selected for desired antibody mutations in vivo. These results demonstrate proof-of-concept that Env immunogens can be designed to directly select for specific antibody mutations at residue-level precision by vaccination, thus demonstrating the feasibility of sequential bnAb-inducing HIV-1 vaccine design.

immunology↗

bioRxiv2023

Vaccine induction of CD4-mimicking broadly neutralizing antibody precursors in macaques

The CD4 binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the CD4bs is occluded by glycans, immunogen expansion of appropriate naive B cells, and selection of functional antibody mutations. Here, we demonstrate immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAbs bound to HIV-1 Env demonstrated binding angles similar to human bnAbs and heavy chain second complementarity determining region-dependent binding characteristic of all known human CD4-mimicking bnAbs. Macaque nAbs were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAbs. This vaccine study initiated the B cells from which derive CD4bs bnAbs in primates, accomplishing the key first step in development of an effective HIV-1 vaccine.

immunology↗

bioRxiv2022

Mutation-Guided Vaccine Design: A Strategy for Developing Boosting Immunogens for HIV Broadly Neutralizing Antibody Induction

A major goal of HIV-1 vaccine development is induction of broadly neutralizing antibodies (bnAbs). While success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knock-in mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof-of concept and milestone towards development of an HIV vaccine. One-Sentence SummaryA vaccine strategy for selecting key rare antibody mutations is shown to induce HIV broadly neutralizing antibodies in mice.

immunology↗

bioRxiv2022

Structural basis for breadth development in a HIV-1 neutralizing antibody

Antibody affinity maturation enables adaptive immune responses to a wide range of pathogens. In some individuals broadly neutralizing antibodies develop to recognize rapidly mutating pathogens with extensive sequence diversity. Vaccine design for pathogens such as HIV-1 and influenza have therefore focused on recapitulating the natural affinity maturation process. Here, we determined structures of antibodies in complex with HIV-1 Envelope for all observed members and ancestral states of a broadly neutralizing HIV-1 antibody clonal B cell lineage. These structures track the development of neutralization breadth from the unmutated common ancestor and define affinity maturation at high spatial resolution. By elucidating contacts mediated by key mutations at different stages of antibody development we have identified sites on the epitope-paratope interface that are the focus of affinity optimization. Thus, our results identify bottlenecks on the path to natural affinity maturation and reveal solutions for these that will inform immunogen design aimed at eliciting a broadly neutralizing immune response by vaccination. SummarySomatic hypermutation drives affinity maturation of germline-encoded antibodies leading to the development of their pathogen neutralization function1. Rational vaccine design efforts that aim to recapitulate affinity maturation rely on information from antibodies elicited and matured during natural infection. High-throughput next generation sequencing and methods for tracing antibody development have allowed close monitoring of the antibody maturation process. Since maturation involves both affinity-enhancing and affinity-independent diversification, the precise effect of each observed mutation, their role in enhancing affinity to antigens, and the order in which the mutations accumulated are often unclear. These gaps in knowledge most acutely hinder efforts directed at difficult targets such as pan-HIV, pan-Influenza, and pan-Coronavirus vaccines. In HIV-1 infection, antibody maturation over several years is required to achieve neutralization breadth. Here, we determined structures of antibodies in complex with HIV-1 Envelope trimers for all observed members and ancestral states of a broadly neutralizing HIV-1 antibody clone to examine affinity maturation as neutralization breadth developed from the unmutated common ancestor. Structural determination of epitope-paratope interfaces revealed details of the contacts evolving over a timescale of several years. Structures along different branches of the clonal lineage elucidated differences in the branch that led to broad neutralization versus off-track paths that culminated in sub-optimal neutralization breadth. We further determined structures of the evolving Envelope revealing details of the virus-antibody co-evolution through visualization of how the virus constructs barriers to evade antibody-mediated neutralization and the mechanisms by which the developing antibody clone circumvents these barriers. Together, our structures provide a detailed time-resolved imagery of the affinity maturation process through atomic level descriptions of virus-antibody co-evolution leading to broad HIV neutralization. While the findings from our studies have direct relevance to HIV-1, the principles of affinity optimization and breadth development elucidated in our study should have broad relevance to other pathogens.

immunology↗

bioRxiv2022

Cryo-EM structures of SARS-CoV-2 Omicron BA.2 spike

The BA.2 sub-lineage of the SARS-CoV-2 Omicron variant has gained in proportion relative to BA.1. As differences in spike (S) proteins may underlie differences in their pathobiology, here we determine cryo-EM structures of a BA.2 S ectodomain and compare these to previously determined BA.1 S structures. BA.2 Receptor Binding Domain (RBD) mutations induced remodeling of the internal RBD structure resulting in its improved thermostability and tighter packing within the 3-RBD-down spike. In the S2 subunit, the fusion peptide in BA.2 was less accessible to antibodies than in BA.1. Pseudovirus neutralization and spike binding assays revealed extensive immune evasion while defining epitopes of two RBD-directed antibodies, DH1044 and DH1193, that bound the outer RBD face to neutralize both BA.1 and BA.2. Taken together, our results indicate that stabilization of the 3-RBD-down state through interprotomer RBD-RBD packing is a hallmark of the Omicron variant, and reveal differences in key functional regions in the BA.1 and BA.2 S proteins.

biochemistry↗

bioRxiv2022

Structural diversity of the SARS-CoV-2 Omicron spike

Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor binding domain (RBD) and neutralizing antibody epitope presentation affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.

biochemistry↗

bioRxiv2021

Protective transfer: Maternal passive immunization with a rotavirus-neutralizing dimeric IgA protects against rotavirus disease in suckling neonates

Breast milk secretory IgA antibodies provide a first line of defense against enteric infections. Despite this and an effective vaccine, human rotaviruses (RVs) remain the leading cause of severe infectious diarrhea in children in low- and middle-income countries (LMIC) where vaccine efficacy is lower than that of developed nations. Therapeutic strategies that deliver potently neutralizing antibodies into milk could provide protection against enteric pathogens such as RVs. We developed a murine model of maternal protective-transfer using systemic administration of a dimeric IgA (dIgA) monoclonal antibody. We confirmed that systemically-administered dIgA passively transferred into milk and stomach of suckling pups in a dose-dependent manner. We then demonstrated that systemic administration of an engineered potent RV-neutralizing dIgA (mAb41) in lactating dams protected suckling pups from RV-induced diarrhea. This maternal protective-transfer immunization platform could be an effective strategy to improve infant mortality against enteric infections, particularly in LMIC with high rates of breastfeeding. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/461116v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1af34dcorg.highwire.dtl.DTLVardef@1b78cb2org.highwire.dtl.DTLVardef@1760a62org.highwire.dtl.DTLVardef@a3cea2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Effect of natural mutations of SARS-CoV-2 on spike structure, conformation and antigenicity

New SARS-CoV-2 variants that have accumulated multiple mutations in the spike (S) glycoprotein enable increased transmission and resistance to neutralizing antibodies. Here, we study the antigenic and structural impacts of the S protein mutations from four variants, one that was involved in transmission between minks and humans, and three that rapidly spread in human populations and originated in the United Kingdom, Brazil or South Africa. All variants either retained or improved binding to the ACE2 receptor. The B.1.1.7 (UK) and B.1.1.28 (Brazil) spike variants showed reduced binding to neutralizing NTD and RBD antibodies, respectively, while the B.1.351 (SA) variant showed reduced binding to both NTD- and RBD-directed antibodies. Cryo-EM structural analyses revealed allosteric effects of the mutations on spike conformations and revealed mechanistic differences that either drive inter-species transmission or promotes viral escape from dominant neutralizing epitopes. HighlightsO_LICryo-EM structures reveal changes in SARS-CoV-2 S protein during inter-species transmission or immune evasion. C_LIO_LIAdaptation to mink resulted in increased ACE2 binding and spike destabilization. C_LIO_LIB.1.1.7 S mutations reveal an intricate balance of stabilizing and destabilizing effects that impact receptor and antibody binding. C_LIO_LIE484K mutation in B.1.351 and B.1.1.28 S proteins drives immune evasion by altering RBD conformation. C_LIO_LIS protein uses different mechanisms to converge upon similar solutions for altering RBD up/down positioning. C_LI

microbiology↗

bioRxiv2021

The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-{gamma} (Fc{gamma}R)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated Fc{gamma}R-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.

immunology↗

bioRxiv2020

D614G mutation alters SARS-CoV-2 spike conformational dynamics and protease cleavage susceptibility at the S1/S2 junction

The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we analyze the D614G mutation in the context of a soluble S ectodomain construct. Cryo-EM structures, antigenicity and proteolysis experiments suggest altered conformational dynamics resulting in enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage altered the conformational dynamics of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on the RBD dynamics triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational dynamics and allostery, and have implications for vaccine design. HighlightsO_LISARS-CoV-2 S ectodomains with or without the K986P, V987P mutations have similar structures, antigenicity and stability. C_LIO_LIThe D614G mutation alters S protein conformational dynamics. C_LIO_LID614G enhances protease cleavage susceptibility at the S protein furin cleavage site. C_LIO_LICryo-EM structures reveal allosteric effect of changes at the S1/S2 junction on RBD dynamics. C_LI

biophysics↗

bioRxiv2020

Mapping the SARS-CoV-2 spike glycoprotein-derived peptidome presented by HLA class II on dendritic cells

Understanding and eliciting protective immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an urgent priority. To facilitate these objectives, we have profiled the repertoire of human leukocyte antigen class II (HLA-II)-bound peptides presented by HLA-DR diverse monocyte-derived dendritic cells pulsed with SARS-CoV-2 spike (S) protein. We identify 209 unique HLA-II-bound peptide sequences, many forming nested sets, which map to sites throughout S including glycosylated regions. Comparison of the glycosylation profile of the S protein to that of the HLA-II-bound S peptides revealed substantial trimming of glycan residues on the latter, likely introduced during antigen processing. Our data also highlight the receptor-binding motif in S1 as a HLA-DR-binding peptide-rich region. Results from this study have application in vaccine design, and will aid analysis of CD4+ T cell responses in infected individuals and vaccine recipients.

immunology↗

bioRxiv2020

Cold sensitivity of the SARS-CoV-2 spike ectodomain

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible "up" or receptor-inaccessible "down" conformations. Here, we report that the commonly used stabilized S ectodomain construct "2P" is sensitive to cold temperature, and that this cold sensitivity is resolved in a "down" state stabilized spike. Our results will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.

biochemistry↗

bioRxiv2020

A glycan cluster on the SARS-CoV-2 spike ectodomain is recognized by Fab-dimerized glycan-reactive antibodies

SummaryThe COVID-19 pandemic caused by SARS-CoV-2 has escalated into a global crisis. The spike (S) protein that mediates cell entry and membrane fusion is the current focus of vaccine and therapeutic antibody development efforts. The S protein, like many other viral fusion proteins such as HIV-1 envelope (Env) and influenza hemagglutinin, is glycosylated with both complex and high mannose glycans. Here we demonstrate binding to the SARS-CoV-2 S protein by a category of Fab-dimerized glycan-reactive (FDG) HIV-1-induced broadly neutralizing antibodies (bnAbs). A 3.1 Å resolution cryo-EM structure of the S protein ectodomain bound to glycan-dependent HIV-1 bnAb 2G12 revealed a quaternary glycan epitope on the spike S2 domain involving multiple protomers. These data reveal a new epitope on the SARS-CoV-2 spike that can be targeted for vaccine design.HighlightsFab-dimerized, glycan-reactive (FDG) HIV-1 bnAbs cross-react with SARS-CoV-2 spike.3.1 Å resolution cryo-EM structure reveals quaternary S2 epitope for HIV-1 bnAb 2G12.2G12 targets glycans, at positions 709, 717 and 801, in the SARS-CoV-2 spike.Our studies suggest a common epitope for FDG antibodies centered around glycan 709.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Fab-dimerized glycan-reactive antibodies neutralize HIV and are prevalent in humans and rhesus macaques

SummaryThe HIV-1 envelope (Env) is comprised by mass of over 50% glycans. A goal of HIV-1 vaccine development is the induction of Env glycan-reactive broadly neutralizing antibodies (bnAbs). The 2G12 bnAb recognizes an Env glycan cluster using a unique variable heavy (VH) domain-swapped conformation that results in fragment antigen-binding (Fab) dimerization. Here we describe Fab-dimerized glycan (FDG)-reactive antibodies without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques that neutralized heterologous HIV-1 isolates. FDG precursors were boosted by vaccination in macaques, and were present in HIV-1-naïve humans with an average estimated frequency of one per 340,000 B cells. These data demonstrate frequent HIV-1 Env glycan-reactive bnAb B cell precursors in macaques and humans and reveal a novel strategy for their induction by vaccination.HighlightsDiscovery of Fab-dimerized HIV-1 glycan-reactive antibodies with a non-domain-swapped architectureFab-dimerized antibodies neutralize heterologous HIV-1 isolates.Antibodies with this architecture can be elicited by vaccination in macaques.Fab-dimerized antibodies are found in HIV-1 naïve humans.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Glycans on the SARS-CoV-2 Spike Control the Receptor Binding Domain Conformation

The glycan shield of the beta-coronavirus (β-CoV) Spike (S) glycoprotein provides protection from host immune responses, acting as a steric block to potentially neutralizing antibody responses. The conformationally dynamic S-protein is the primary immunogenic target of vaccine design owing to its role in host-cell fusion, displaying multiple receptor binding domain (RBD) ‘up’ and ‘down’ state configurations. Here, we investigated the potential for RBD adjacent, N-terminal domain (NTD) glycans to influence the conformational equilibrium of these RBD states. Using a combination of antigenic screens and high-resolution cryo-EM structure determination, we show that an N-glycan deletion at position 234 results in a dramatically reduced population of the ‘up’ state RBD position. Conversely, glycan deletion at position N165 results in a discernable increase in ‘up’ state RBDs. This indicates the glycan shield acts not only as a passive hinderance to antibody meditated immunity but also as a conformational control element. Together, our results demonstrate this highly dynamic conformational machine is responsive to glycan modification with implications in viral escape and vaccine design.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Controlling the SARS-CoV-2 Spike Glycoprotein Conformation

The coronavirus (CoV) viral host cell fusion spike (S) protein is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, we implemented a structure-based vector analysis of available {beta}-CoV S-protein structures. We found that despite overall similarity in domain organization, different {beta}-CoV strains display distinct S-protein configurations. Based on this analysis, we developed two soluble ectodomain constructs in which the highly immunogenic and mobile receptor binding domain (RBD) is locked in either the all-RBDs down position or is induced to display a previously unobserved in SARS-CoV-2 2-RBDs up configuration. These results demonstrate that the conformation of the S-protein can be controlled via rational design and provide a framework for the development of engineered coronavirus spike proteins for vaccine applications.

immunology↗