Publications by authors named "Leonidas Stamatatos"

110 Publications

A single dose of the SARS-CoV-2 vaccine BNT162b2 elicits Fc-mediated antibody effector functions and T cell responses.

Cell Host Microbe 2021 Jun 4. Epub 2021 Jun 4.

Centre de Recherche du CHUM, Montréal, QC H2X 0A9, Canada; Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, QC H2X 0A9, Canada.

While the standard regimen of the BNT162b2 mRNA vaccine for SARS-CoV-2 includes two doses administered 3 weeks apart, some public health authorities are spacing these doses, raising concerns about efficacy. However, data indicate that a single dose can be up to 90% effective starting 14 days post-administration. To assess the mechanisms contributing to protection, we analyzed humoral and T cell responses three weeks after a single BNT162b2 dose. We observed weak neutralizing activity elicited in SARS-CoV-2 naive individuals but strong anti-receptor binding domain and spike antibodies with Fc-mediated effector functions and cellular CD4 T cell responses. In previously infected individuals, a single dose boosted all humoral and T cell responses, with strong correlations between T helper and antibody immunity. Our results highlight the potential role of Fc-mediated effector functions and T cell responses in vaccine efficacy. They also provide support for spacing doses to vaccinate more individuals in conditions of vaccine scarcity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2021.06.001DOI Listing
June 2021

Germinal center-dependent and -independent memory B cells produced throughout the immune response.

J Exp Med 2021 Aug 9;218(8). Epub 2021 Jun 9.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY.

Memory B cells comprise a heterogenous group of cells that differ in origin and phenotype. During the early phases of the immune response, activated B cells can differentiate into IgM-expressing memory cells, short-lived plasma cells, or seed germinal centers (GCs). The memory compartment is subsequently enriched by B cells that have been through several rounds of division and selection in the GC. Here, we report on the use of an unbiased lineage-tracking approach to explore the origins and properties of memory B cell subsets in mice with an intact immune system. We find that activated B cells continue to differentiate into memory B cells throughout the immune response. When defined on the basis of their origins, the memory B cells originating from activated B cells or GCs differ in isotype and overall gene expression, somatic hypermutation, and their affinity for antigen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20202489DOI Listing
August 2021

Development of a VRC01-class germline targeting immunogen derived from anti-idiotypic antibodies.

Cell Rep 2021 May;35(5):109084

Fred Hutchinson Cancer Research Center, Vaccines and Infectious Diseases Division, Seattle, WA 98109, USA; University of Washington, Department of Global Health, Seattle, WA 98195, USA. Electronic address:

An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs). Broad and potent VRC01-class bNAbs have been isolated from multiple infected individuals, suggesting that they could be reproducibly elicited by vaccination. Several HIV-1 envelope-derived germline-targeting immunogens have been designed to engage naive VRC01-class precursor B cells. However, they also present off-target epitopes that could hinder development of VRC01-class bNAbs. We characterize a panel of anti-idiotypic monoclonal antibodies (ai-mAbs) raised against inferred-germline (iGL) VRC01-class antibodies. By leveraging binding, structural, and B cell sorting data, we engineered a bispecific molecule derived from two ai-mAbs; one specific for VRC01-class heavy chains and one specific for VRC01-class light chains. The bispecific molecule preferentially activates iGL-VRC01 B cells in vitro and induces specific antibody responses in a murine adoptive transfer model with a diverse polyclonal B cell repertoire. This molecule represents an alternative non-envelope-derived germline-targeting immunogen that can selectively activate VRC01-class precursors in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2021.109084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127986PMC
May 2021

Designed proteins assemble antibodies into modular nanocages.

Science 2021 04;372(6537)

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

Multivalent display of receptor-engaging antibodies or ligands can enhance their activity. Instead of achieving multivalency by attachment to preexisting scaffolds, here we unite form and function by the computational design of nanocages in which one structural component is an antibody or Fc-ligand fusion and the second is a designed antibody-binding homo-oligomer that drives nanocage assembly. Structures of eight nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage, respectively, closely match the corresponding computational models. Antibody nanocages targeting cell surface receptors enhance signaling compared with free antibodies or Fc-fusions in death receptor 5 (DR5)-mediated apoptosis, angiopoietin-1 receptor (Tie2)-mediated angiogenesis, CD40 activation, and T cell proliferation. Nanocage assembly also increases severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc-angiotensin-converting enzyme 2 (ACE2) fusion proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.abd9994DOI Listing
April 2021

Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy.

bioRxiv 2021 Mar 22. Epub 2021 Mar 22.

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We visualized sequential spread of virus from the nasal cavity to the lungs followed by systemic spread to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days of infection. In addition to direct neutralization, efficacy required Fc effector functions of NAbs, with contributions from monocytes, neutrophils and natural killer cells, to dampen inflammatory responses and limit immunopathology. Thus, our study highlights the requirement of both Fab and Fc effector functions for an optimal efficacy afforded by NAbs against SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.03.22.436337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010726PMC
March 2021

Isolation and Characterization of Cross-Neutralizing Coronavirus Antibodies from COVID-19+ Subjects.

bioRxiv 2021 Mar 24. Epub 2021 Mar 24.

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterized 198 antibodies isolated from four COVID19+ subjects and identified 14 SARS-CoV-2 neutralizing antibodies. One targeted the NTD, one recognized an epitope in S2 and twelve bound the RBD. Three anti-RBD neutralizing antibodies cross-neutralized SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency rather than the antibody epitope specificity regulates the protective potential of anti-SARS-CoV-2 antibodies. The anti-S2 antibody also neutralized SARS-CoV-1 and all four cross-neutralizing antibodies neutralized the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.03.23.436684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010719PMC
March 2021

mRNA vaccination boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection.

Science 2021 Mar 25. Epub 2021 Mar 25.

Fred Hutchinson Cancer Research Center, Vaccine and Infectious Disease Division, Seattle, WA, USA.

Emerging SARS-CoV-2 variants have raised concerns about resistance to neutralizing antibodies elicited by previous infection or vaccination. We examined whether sera from recovered and naïve donors collected prior to, and following immunizations with existing mRNA vaccines, could neutralize the Wuhan-Hu-1 and B.1.351 variants. Pre-vaccination sera from recovered donors neutralized Wuhan-Hu-1 and sporadically neutralized B.1.351, but a single immunization boosted neutralizing titers against all variants and SARS-CoV-1 by up to 1000-fold. Neutralization was due to antibodies targeting the receptor binding domain and was not boosted by a second immunization. Immunization of naïve donors also elicited cross-neutralizing responses, but at lower titers. Our study highlights the importance of vaccinating both uninfected and previously infected persons to elicit cross-variant neutralizing antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.abg9175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8139425PMC
March 2021

A single mRNA immunization boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection.

medRxiv 2021 Mar 10. Epub 2021 Mar 10.

Emerging SARS-CoV-2 variants have raised concerns about resistance to neutralizing antibodies elicited by previous infection or vaccination. We examined whether sera from recovered and naïve donors collected prior to, and following immunizations with existing mRNA vaccines, could neutralize the Wuhan-Hu-1 and B.1.351 variants. Pre-vaccination sera from recovered donors neutralized Wuhan-Hu-1 and sporadically neutralized B.1.351, but a single immunization boosted neutralizing titers against all variants and SARS-CoV-1 by up to 1000-fold. Neutralization was due to antibodies targeting the receptor binding domain and was not boosted by a second immunization. Immunization of naïve donors also elicited cross-neutralizing responses, but at lower titers. Our study highlights the importance of vaccinating both uninfected and previously infected persons to elicit cross-variant neutralizing antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.02.05.21251182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987032PMC
March 2021

A single BNT162b2 mRNA dose elicits antibodies with Fc-mediated effector functions and boost pre-existing humoral and T cell responses.

bioRxiv 2021 Mar 18. Epub 2021 Mar 18.

The standard dosing of the Pfizer/BioNTech BNT162b2 mRNA vaccine validated in clinical trials includes two doses administered three weeks apart. While the decision by some public health authorities to space the doses because of limiting supply has raised concerns about vaccine efficacy, data indicate that a single dose is up to 90% effective starting 14 days after its administration. We analyzed humoral and T cells responses three weeks after a single dose of this mRNA vaccine. Despite the proven efficacy of the vaccine at this time point, no neutralizing activity were elicited in SARS-CoV-2 naïve individuals. However, we detected strong anti-receptor binding domain (RBD) and Spike antibodies with Fc-mediated effector functions and cellular responses dominated by the CD4 T cell component. A single dose of this mRNA vaccine to individuals previously infected by SARS-CoV-2 boosted all humoral and T cell responses measured, with strong correlations between T helper and antibody immunity. Neutralizing responses were increased in both potency and breadth, with distinctive capacity to neutralize emerging variant strains. Our results highlight the importance of vaccinating uninfected and previously-infected individuals and shed new light into the potential role of Fc-mediated effector functions and T cell responses in vaccine efficacy. They also provide support to spacing the doses of two-vaccine regimens to vaccinate a larger pool of the population in the context of vaccine scarcity against SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.03.18.435972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987016PMC
March 2021

Evaluation of SARS-CoV-2 neutralization assays for antibody monitoring in natural infection and vaccine trials.

medRxiv 2020 Dec 8. Epub 2020 Dec 8.

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays to be utilized in concert with clinical trials to establish correlates of risk and protection. This need has led to the appearance of a variety of neutralization assays used by different laboratories and companies. Using plasma samples from COVID-19 convalescent individuals with mild-to-moderate disease from a localized outbreak in a single region of the western US, we compared three platforms for SARS-CoV-2 neutralization: assay with live SARS-CoV-2, pseudovirus assay utilizing lentiviral (LV) and vesicular stomatitis virus (VSV) packaging, and a surrogate ELISA test. Vero, Vero E6, HEK293T cells expressing human angiotensin converting enzyme 2 (hACE2), and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 (TMPRSS2) were evaluated. Live-virus and LV-pseudovirus assay with HEK293T cells showed similar geometric mean titers (GMTs) ranging 141-178, but VSV-pseudovirus assay yielded significantly higher GMT (310 95%CI 211-454; p < 0.001). Fifty percent neutralizing dilution (ND50) titers from live-virus and all pseudovirus assay readouts were highly correlated (Pearson = 0.81-0.89). ND50 titers positively correlated with plasma concentration of IgG against SARS-CoV-2 spike and receptor binding domain (RBD) ( = 0.63-0.89), but moderately correlated with nucleoprotein IgG ( = 0.46-0.73). There was a moderate positive correlation between age and spike (Spearman's rho=0.37, p=0.02), RBD (rho=0.39, p=0.013) and nucleoprotein IgG (rho=0.45, p=0.003). ND80 showed stronger correlation with age than ND50 (ND80 rho=0.51 (p=0.001), ND50 rho=0.28 (p=0.075)). Our data demonstrate high concordance between cell-based assays with live and pseudotyped virions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.12.07.20245431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743084PMC
December 2020

Designed proteins assemble antibodies into modular nanocages.

bioRxiv 2020 Dec 1. Epub 2020 Dec 1.

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

Antibodies are widely used in biology and medicine, and there has been considerable interest in multivalent antibody formats to increase binding avidity and enhance signaling pathway agonism. However, there are currently no general approaches for forming precisely oriented antibody assemblies with controlled valency. We describe the computational design of two-component nanocages that overcome this limitation by uniting form and function. One structural component is any antibody or Fc fusion and the second is a designed Fc-binding homo-oligomer that drives nanocage assembly. Structures of 8 antibody nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage match the corresponding computational models. Antibody nanocages targeting cell-surface receptors enhance signaling compared to free antibodies or Fc-fusions in DR5-mediated apoptosis, Tie2-mediated angiogenesis, CD40 activation, and T cell proliferation; nanocage assembly also increases SARS-CoV-2 pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc-ACE2 fusion proteins. We anticipate that the ability to assemble arbitrary antibodies without need for covalent modification into highly ordered assemblies with different geometries and valencies will have broad impact in biology and medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.12.01.406611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724662PMC
December 2020

Antibody Binding to SARS-CoV-2 S Glycoprotein Correlates with but Does Not Predict Neutralization.

Viruses 2020 10 26;12(11). Epub 2020 Oct 26.

Centre de Recherche du CHUM, Montréal, QC H2X 0A9, Canada.

Convalescent plasma from SARS-CoV-2 infected individuals and monoclonal antibodies were shown to potently neutralize viral and pseudoviral particles carrying the S glycoprotein. However, a non-negligent proportion of plasma samples from infected individuals, as well as S-specific monoclonal antibodies, were reported to be non-neutralizing despite efficient interaction with the S glycoprotein in different biochemical assays using soluble recombinant forms of S or when expressed at the cell surface. How neutralization relates to the binding of S glycoprotein in the context of viral particles remains to be established. Here, we developed a pseudovirus capture assay (VCA) to measure the capacity of plasma samples or antibodies immobilized on ELISA plates to bind to membrane-bound S glycoproteins from SARS-CoV-2 expressed at the surface of lentiviral particles. By performing VCA, ELISA, and neutralization assays, we observed a strong correlation between these parameters. However, while we found that plasma samples unable to capture viral particles did not neutralize, capture did not guarantee neutralization, indicating that the capacity of antibodies to bind to the S glycoprotein at the surface of pseudoviral particles is required but not sufficient to mediate neutralization. Altogether, our results highlight the importance of better understanding the inactivation of S by plasma and neutralizing antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v12111214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692607PMC
October 2020

Structural basis for potent neutralization of SARS-CoV-2 and role of antibody affinity maturation.

Nat Commun 2020 10 27;11(1):5413. Epub 2020 Oct 27.

Fred Hutchinson Cancer Research Center, Vaccines and Infectious Diseases Division, Seattle, WA, USA.

SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determine the X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient infected with SARS-CoV-2, in complex with the receptor binding domain. The structure reveals that CV30 binds to an epitope that overlaps with the human ACE2 receptor binding motif providing a structural basis for its neutralization. CV30 also induces shedding of the S1 subunit, indicating an additional mechanism of neutralization. A germline reversion of CV30 results in a substantial reduction in both binding affinity and neutralization potential indicating the minimal somatic mutation is needed for potently neutralizing antibodies against SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-19231-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591918PMC
October 2020

HIV-1 VRC01 Germline-Targeting Immunogens Select Distinct Epitope-Specific B Cell Receptors.

Immunity 2020 10;53(4):840-851.e6

Fred Hutchinson Cancer Research Center, Vaccines and Infectious Diseases Division, Seattle, WA, USA; University of Washington, Department of Global Health, Seattle, WA, USA. Electronic address:

Activating precursor B cell receptors of HIV-1 broadly neutralizing antibodies requires specifically designed immunogens. Here, we compared the abilities of three such germline-targeting immunogens against the VRC01-class receptors to activate the targeted B cells in transgenic mice expressing the germline VH of the VRC01 antibody but diverse mouse light chains. Immunogen-specific VRC01-like B cells were isolated at different time points after immunization, their VH and VL genes were sequenced, and the corresponding antibodies characterized. VRC01 B cell sub-populations with distinct cross-reactivity properties were activated by each immunogen, and these differences correlated with distinct biophysical and biochemical features of the germline-targeting immunogens. Our study indicates that the design of effective immunogens to activate B cell receptors leading to protective HIV-1 antibodies will require a better understanding of how the biophysical properties of the epitope and its surrounding surface on the germline-targeting immunogen influence its interaction with the available receptor variants in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2020.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735217PMC
October 2020

Antibody binding to SARS-CoV-2 S glycoprotein correlates with, but does not predict neutralization.

bioRxiv 2020 Sep 8. Epub 2020 Sep 8.

Convalescent plasma from SARS-CoV-2 infected individuals and monoclonal antibodies were shown to potently neutralize viral and pseudoviral particles carrying the S glycoprotein. However, a non-negligent proportion of plasma samples from infected individuals as well as S-specific monoclonal antibodies were reported to be non-neutralizing despite efficient interaction with the S glycoprotein in different biochemical assays using soluble recombinant forms of S or when expressed at the cell surface. How neutralization relates to binding of S glycoprotein in the context of viral particles remains to be established. Here we developed a pseudovirus capture assay (VCA) to measure the capacity of plasma samples or antibodies immobilized on ELISA plates to bind to membrane-bound S glycoproteins from SARS-CoV-2 expressed at the surface of lentiviral particles. By performing VCA and neutralization assays we observed a strong correlation between these two parameters. However, while we found that plasma samples unable to capture viral particles did not neutralize, capture did not guarantee neutralization, indicating that the capacity of antibodies to bind to the S glycoprotein at the surface of viral particles is required but not sufficient to mediate neutralization. Altogether, our results highlights the importance of better understanding the inactivation of S by plasma and neutralizing antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.09.08.287482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491507PMC
September 2020

Structural basis for potent neutralization of SARS-CoV-2 and role of antibody affinity maturation.

bioRxiv 2020 Jun 12. Epub 2020 Jun 12.

Fred Hutchinson Cancer Research Center, Vaccines and Infectious Diseases Division, Seattle, WA, USA.

SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determined the X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient infected with SARS-CoV-2, in complex with the receptor binding domain (RBD). The structure reveals CV30's epitope overlaps with the human ACE2 receptor binding site thus providing the structural basis for its neutralization by preventing ACE2 binding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.06.12.148692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301900PMC
June 2020

Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation.

Immunity 2020 07 8;53(1):98-105.e5. Epub 2020 Jun 8.

Fred Hutchinson Cancer Research Center, Vaccines and Infectious Disease Division, Seattle, WA, USA; University of Washington, Department of Global Health, Seattle, WA, USA. Electronic address:

Antibody responses develop following SARS-CoV-2 infection, but little is known about their epitope specificities, clonality, binding affinities, epitopes, and neutralizing activity. We isolated B cells specific for the SARS-CoV-2 envelope glycoprotein spike (S) from a COVID-19-infected subject 21 days after the onset of clinical disease. 45 S-specific monoclonal antibodies were generated. They had undergone minimal somatic mutation with limited clonal expansion, and three bound the receptor-binding domain (RBD). Two antibodies neutralized SARS-CoV-2. The most potent antibody bound the RBD and prevented binding to the ACE2 receptor, while the other bound outside the RBD. Thus, most anti-S antibodies that were generated in this patient during the first weeks of COVID-19 infection were non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 S-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive and/or therapeutic potential and can serve as templates for vaccine design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2020.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7276322PMC
July 2020

Characterization of neutralizing antibodies from a SARS-CoV-2 infected individual.

bioRxiv 2020 May 12. Epub 2020 May 12.

Fred Hutchinson Cancer Research Center, Vaccines and Infectious Diseases Division, Seattle, WA, USA.

B cells specific for the SARS-CoV-2 S envelope glycoprotein spike were isolated from a COVID-19-infected subject using a stabilized spike-derived ectodomain (S2P) twenty-one days post-infection. Forty-four S2P-specific monoclonal antibodies were generated, three of which bound to the receptor binding domain (RBD). The antibodies were minimally mutated from germline and were derived from different B cell lineages. Only two antibodies displayed neutralizing activity against SARS-CoV-2 pseudo-virus. The most potent antibody bound the RBD in a manner that prevented binding to the ACE2 receptor, while the other bound outside the RBD. Our study indicates that the majority of antibodies against the viral envelope spike that were generated during the first weeks of COVID-19 infection are non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 spike-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive/therapeutic potential and can serve as templates for vaccine-design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.05.12.091298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241105PMC
May 2020

Overcoming Steric Restrictions of VRC01 HIV-1 Neutralizing Antibodies through Immunization.

Cell Rep 2019 12;29(10):3060-3072.e7

Vaccines and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Global Health, University of Washington, Seattle, WA, USA. Electronic address:

Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1-202 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.10.071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936959PMC
December 2019

Anti-idiotypic antibodies elicit anti-HIV-1-specific B cell responses.

J Exp Med 2019 10 25;216(10):2316-2330. Epub 2019 Jul 25.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA

Human anti-HIV-1 broadly neutralizing antibodies (bNAbs) protect against infection in animal models. However, bNAbs have not been elicited by vaccination in diverse wild-type animals or humans, in part because B cells expressing the precursors of these antibodies do not recognize most HIV-1 envelopes (Envs). Immunogens have been designed that activate these B cell precursors in vivo, but they also activate competing off-target responses. Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody, iv8, that selects for naive human B cells expressing immunoglobulin light chains with 5-amino acid complementarity determining region 3s, a key feature of anti-CD4 binding site (CD4bs)-specific VRC01-class antibodies. In mice, iv8 induced target cells to expand and mature in the context of a polyclonal immune system and produced serologic responses targeting the CD4bs on Env. In summary, the results demonstrate that an anti-idiotypic antibody can specifically recognize and expand rare B cells that express VRC01-class antibodies against HIV-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20190446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6780999PMC
October 2019

Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes.

J Exp Med 2019 10 25;216(10):2331-2347. Epub 2019 Jul 25.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA

Many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest. In light of this, there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes. Here, we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus 1 (HIV-1) broadly neutralizing antibody b12 (iglb12). We determined the crystal structure of two anti-idiotypes in complex with iglb12 and used these anti-idiotypes to identify rare naive human B cells expressing B cell receptors with similarity to iglb12. Immunization with a multimerized version of this anti-idiotype induced the proliferation of transgenic murine B cells expressing the iglb12 heavy chain in vivo, despite the presence of deletion and anergy within this population. Together, our data indicate that anti-idiotypes are a valuable tool for the study and induction of potentially protective antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20190164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6780997PMC
October 2019

Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques.

Nature 2019 06 29;570(7762):468-473. Epub 2019 May 29.

Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1250-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657810PMC
June 2019

HIV-specific humoral immune responses by CRISPR/Cas9-edited B cells.

J Exp Med 2019 06 11;216(6):1301-1310. Epub 2019 Apr 11.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY

A small number of HIV-1-infected individuals develop broadly neutralizing antibodies to the virus (bNAbs). These antibodies are protective against infection in animal models. However, they only emerge 1-3 yr after infection, and show a number of highly unusual features including exceedingly high levels of somatic mutations. It is therefore not surprising that elicitation of protective immunity to HIV-1 has not yet been possible. Here we show that mature, primary mouse and human B cells can be edited in vitro using CRISPR/Cas9 to express mature bNAbs from the endogenous locus. Moreover, edited B cells retain the ability to participate in humoral immune responses. Immunization with cognate antigen in wild-type mouse recipients of edited B cells elicits bNAb titers that neutralize HIV-1 at levels associated with protection against infection. This approach enables humoral immune responses that may be difficult to elicit by traditional immunization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20190287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547862PMC
June 2019

Inference of the HIV-1 VRC01 Antibody Lineage Unmutated Common Ancestor Reveals Alternative Pathways to Overcome a Key Glycan Barrier.

Immunity 2018 12 11;49(6):1162-1174.e8. Epub 2018 Dec 11.

Duke Human Vaccine Institute, Duke University, Durham, NC, USA; Department of Medicine, Duke University, Durham, NC, USA; Department of Immunology, Duke University, Durham, NC, USA. Electronic address:

Elicitation of VRC01-class broadly neutralizing antibodies (bnAbs) is an appealing approach for a preventative HIV-1 vaccine. Despite extensive investigations, strategies to induce VRC01-class bnAbs and overcome the barrier posed by the envelope N276 glycan have not been successful. Here, we inferred a high-probability unmutated common ancestor (UCA) of the VRC01 lineage and reconstructed the stages of lineage maturation. Env immunogens designed on reverted VRC01-class bnAbs bound to VRC01 UCA with affinity sufficient to activate naive B cells. Early mutations defined maturation pathways toward limited or broad neutralization, suggesting that focusing the immune response is likely required to steer B cell maturation toward the development of neutralization breadth. Finally, VRC01 lineage bnAbs with long CDR H3s overcame the HIV-1 N276 glycan barrier without shortening their CDR L1, revealing a solution for broad neutralization in which the heavy chain, not CDR L1, is the determinant to accommodate the N276 glycan.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2018.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303191PMC
December 2018

Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core.

Elife 2018 11 7;7. Epub 2018 Nov 7.

Department of Biochemistry, University of Washington, Seattle, United States.

VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC01 in complex with a modified trimeric 426c Env by cryo-electron microscopy and a 426c gp120 core by X-ray crystallography, biolayer interferometry, immunoprecipitation, and glycoproteomics. Our results show VRC01 germline antibodies interacted with a wild-type 426c core lacking variable loops 1-3 in the presence and absence of a glycan at position Asn276, with the latter form binding with higher affinity than the former. Interactions in the presence of an Asn276 oligosaccharide could be enhanced upon carbohydrate shortening, which should be considered for immunogen design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.37688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237438PMC
November 2018

HIV-1 envelope glycan modifications that permit neutralization by germline-reverted VRC01-class broadly neutralizing antibodies.

PLoS Pathog 2018 11 5;14(11):e1007431. Epub 2018 Nov 5.

Department of Surgery, Duke University Medical Center, Durham, NC, United States of America.

Broadly neutralizing antibody (bnAb) induction is a high priority for effective HIV-1 vaccination. VRC01-class bnAbs that target the CD4 binding site (CD4bs) of trimeric HIV-1 envelope (Env) glycoprotein spikes are particularly attractive to elicit because of their extraordinary breadth and potency of neutralization in vitro and their ability to protect against infection in animal models. Glycans bordering the CD4bs impede the binding of germline-reverted forms of VRC01-class bnAbs and therefore constitute a barrier to early events in initiating the correct antibody lineages. Deleting a subset of these glycans permits Env antigen binding but not virus neutralization, suggesting that additional barriers impede germline-reverted VRC01-class antibody binding to functional Env trimers. We investigated the requirements for functional Env trimer engagement of VRC01-class naïve B cell receptors by using virus neutralization and germline-reverted antibodies as surrogates for the interaction. Targeted deletion of a subset of N-glycans bordering the CD4bs, combined with Man5 enrichment of remaining N-linked glycans that are otherwise processed into larger complex-type glycans, rendered HIV-1 426c Env-pseudotyped virus (subtype C, transmitted/founder) highly susceptible to neutralization by near germline forms of VRC01-class bnAbs. Neither glycan modification alone rendered the virus susceptible to neutralization. The potency of neutralization in some cases rivaled the potency of mature VRC01 against wildtype viruses. Neutralization by the germline-reverted antibodies was abrogated by the known VRC01 resistance mutation, D279K. These findings improve our understanding of the restrictions imposed by glycans in eliciting VRC01-class bnAbs and enable a neutralization-based strategy to monitor vaccine-elicited early precursors of this class of bnAbs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1007431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237427PMC
November 2018

Live rubella vectors can express native HIV envelope glycoproteins targeted by broadly neutralizing antibodies and prime the immune response to an envelope protein boost.

Vaccine 2018 08 20;36(34):5166-5172. Epub 2018 Jul 20.

Lab of Immunoregulation, DVP, Office of Vaccines, Center for Biologics, FDA, Bldg 72, Room 1212, White Oak Campus, 10903 New Hampshire Ave., Silver Spring, MD 20993, USA. Electronic address:

Following HIV infection, most people make antibodies to gp120 and gp41, yet only a few make broadly neutralizing antibodies that target key antigenic sites on the envelope glycoproteins. The induction of broadly neutralizing antibodies by immunization remains a major challenge of HIV vaccine research. Difficulties include: variable protein sequence, epitopes that depend on the native conformation, glycosylation that conceals key antigenic determinants, and the assembly of Env trimers that mimic viral spikes. In addition, more potent immunogens may be needed to initiate the response of germline antibody precursors and drive B cell maturation toward antibodies with broad neutralizing activity. We have expressed HIV Env glycoproteins by incorporation into live attenuated rubella viral vectors. The rubella vaccine strain RA27/3 has demonstrated its safety and potency in millions of children. As a vector, it has elicited potent and durable immune responses in macaques to SIV Gag vaccine inserts. We now find that rubella/env vectors can stably express Env core derived glycoproteins ranging in size up to 363 amino acids from HIV clade C strain 426c. The expressed Env glycoproteins bind broadly neutralizing antibodies that target the native CD4 binding site. The vectors grew well in rhesus macaques, and they elicited a vaccine "take" in all animals, as measured by anti-rubella antibodies. By themselves, the vectors elicited modest antibody titers to the Env insert. But the combination of rubella/env prime followed by a homologous protein boost gave a strong response. Neutralizing antibodies appeared gradually after multiple vaccine doses. The vectors will be useful for testing new vaccine inserts and immunization strategies under optimized conditions of vector growth and protein expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vaccine.2018.07.010DOI Listing
August 2018

B cell clonal lineage alterations upon recombinant HIV-1 envelope immunization of rhesus macaques.

PLoS Pathog 2018 06 22;14(6):e1007120. Epub 2018 Jun 22.

Fred Hutchinson Cancer Research Center, Vaccines and Infectious Diseases Division, Seattle, Washington, United States of America.

Broadly neutralizing HIV-1 antibodies (bNAbs) isolated from infected subjects display protective potential in animal models. Their elicitation by immunization is thus highly desirable. The HIV-1 envelope glycoprotein (Env) is the sole viral target of bnAbs, but is also targeted by binding, non-neutralizing antibodies. Env-based immunogens tested so far in various animal species and humans have elicited binding and autologous neutralizing antibodies but not bNAbs (with a few notable exceptions). The underlying reasons for this are not well understood despite intensive efforts to characterize the binding specificities of the elicited antibodies; mostly by employing serologic methodologies and monoclonal antibody isolation and characterization. These approaches provide limited information on the ontogenies and clonal B cell lineages that expand following Env-immunization. Thus, our current understanding on how the expansion of particular B cell lineages by Env may be linked to the development of non-neutralizing antibodies is limited. Here, in addition to serological analysis, we employed high-throughput BCR sequence analysis from the periphery, lymph nodes and bone marrow, as well as B cell- and antibody-isolation and characterization methods, to compare in great detail the B cell and antibody responses elicited in non-human primates by two forms of the clade C HIV Env 426c: one representing the full length extracellular portion of Env while the other lacking the variable domains 1, 2 and 3 and three conserved N-linked glycosylation sites. The two forms were equally immunogenic, but only the latter elicited neutralizing antibodies by stimulating a more restricted expansion of B cells to a narrower set of IGH/IGK/IGL-V genes that represented a small fraction (0.003-0.02%) of total B cells. Our study provides new information on how Env antigenic differences drastically affect the expansion of particular B cell lineages and supports immunogen-design efforts aiming at stimulating the expansion of cells expressing particular B cell receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1007120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033445PMC
June 2018

Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen-binding affinity.

Proc Natl Acad Sci U S A 2018 05 16;115(18):4743-4748. Epub 2018 Apr 16.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065-6322;

The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody-based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination and instead would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin (Ig) knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain superphysiologic numbers of bNAb precursor-expressing B cells, and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center (GC) B cell recruitment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the GC when there are as few as 10 such cells per mouse. However, at low precursor frequencies, the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to GCs is variable and dependent on recirculation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1803457115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939114PMC
May 2018