Publications by authors named "Pamela J Bjorkman"

165 Publications

Broad and potent neutralizing human antibodies to tick-borne flaviviruses protect mice from disease.

J Exp Med 2021 May;218(5)

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

Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III (EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC50s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV.
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http://dx.doi.org/10.1084/jem.20210236DOI Listing
May 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.
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http://dx.doi.org/10.1101/2021.03.22.436337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010726PMC
March 2021

Cryo-EM structures of HIV-1 trimer bound to CD4-mimetics BNM-III-170 and M48U1 adopt a CD4-bound open conformation.

Nat Commun 2021 03 29;12(1):1950. Epub 2021 Mar 29.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.

Human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, impacts millions of people. Entry into target cells is mediated by the HIV-1 envelope (Env) glycoprotein interacting with host receptor CD4, which triggers conformational changes allowing binding to a coreceptor and subsequent membrane fusion. Small molecule or peptide CD4-mimetic drugs mimic CD4's Phe43 interaction with Env by inserting into the conserved Phe43 pocket on Env subunit gp120. Here, we present single-particle cryo-EM structures of CD4-mimetics BNM-III-170 and M48U1 bound to a BG505 native-like Env trimer plus the CD4-induced antibody 17b at 3.7 Å and 3.9 Å resolution, respectively. CD4-mimetic-bound BG505 exhibits canonical CD4-induced conformational changes including trimer opening, formation of the 4-stranded gp120 bridging sheet, displacement of the V1V2 loop, and formation of a compact and elongated gp41 HR1C helical bundle. We conclude that CD4-induced structural changes on both gp120 and gp41 Env subunits are induced by binding to the gp120 Phe43 pocket.
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http://dx.doi.org/10.1038/s41467-021-21816-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007822PMC
March 2021

Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.

Nature 2021 Mar 25. Epub 2021 Mar 25.

Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-19. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-19. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.
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http://dx.doi.org/10.1038/s41586-021-03461-yDOI Listing
March 2021

Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies.

bioRxiv 2021 Mar 8. Epub 2021 Mar 8.

Antibodies elicited in response to infection undergo somatic mutation in germinal centers that can result in higher affinity for the cognate antigen. To determine the effects of somatic mutation on the properties of SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibodies, we analyzed six independent antibody lineages. As well as increased neutralization potency, antibody evolution changed pathways for acquisition of resistance and, in some cases, restricted the range of neutralization escape options. For some antibodies, maturation apparently imposed a requirement for multiple spike mutations to enable escape. For certain antibody lineages, maturation enabled neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.
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http://dx.doi.org/10.1101/2021.03.07.434227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987023PMC
March 2021

Mutational escape from the polyclonal antibody response to SARS-CoV-2 infection is largely shaped by a single class of antibodies.

bioRxiv 2021 Mar 18. Epub 2021 Mar 18.

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasma, including plasma from individuals from whom some of the antibodies were isolated. The plasma-escape maps most closely resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is dominated by a single class of antibodies targeting an epitope that is already undergoing rapid evolution.
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http://dx.doi.org/10.1101/2021.03.17.435863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987015PMC
March 2021

characterization of engineered red blood cells as viral traps against HIV-1 and SARS-CoV-2.

Mol Ther Methods Clin Dev 2021 Jun 10;21:161-170. Epub 2021 Mar 10.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

Engineered red blood cells (RBCs) expressing viral receptors could be used therapeutically as viral traps, as RBCs lack nuclei and other organelles required for viral replication. However, expression of viral receptors on RBCs is difficult to achieve since mature erythrocytes lack the cellular machinery to synthesize proteins. Herein, we show that the combination of a powerful erythroid-specific expression system and transgene codon optimization yields high expression levels of the HIV-1 receptors CD4 and CCR5, as well as a CD4-glycophorin A (CD4-GpA) fusion protein in erythroid progenitor cells, which efficiently differentiated into enucleated RBCs. HIV-1 efficiently entered RBCs that co-expressed CD4 and CCR5, but viral entry was not required for neutralization, as CD4 or CD4-GpA expression in the absence of CCR5 was sufficient to potently neutralize HIV-1 and prevent infection of CD4 T cells due to the formation of high-avidity interactions with trimeric HIV-1 Env spikes on virions. To facilitate continuous large-scale production of RBC viral traps, we generated erythroblast cell lines stably expressing CD4-GpA or ACE2-GpA fusion proteins, which produced potent RBC viral traps against HIV-1 and SARS-CoV-2. Our results suggest that this approach warrants further investigation as a potential treatment against acute and chronic viral infections.
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http://dx.doi.org/10.1016/j.omtm.2021.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944778PMC
June 2021

Intestinal Host Response to SARS-CoV-2 Infection and COVID-19 Outcomes in Patients With Gastrointestinal Symptoms.

Gastroenterology 2021 Mar 4. Epub 2021 Mar 4.

Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, New York; The Dr. Henry D. Janowitz Division of Gastroenterology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York. Electronic address:

Background & Aims: Given that gastrointestinal (GI) symptoms are a prominent extrapulmonary manifestation of COVID-19, we investigated intestinal infection with SARS-CoV-2, its effect on pathogenesis, and clinical significance.

Methods: Human intestinal biopsy tissues were obtained from patients with COVID-19 (n =19) and uninfected control individuals (n = 10) for microscopic examination, cytometry by time of flight analyses, and RNA sequencing. Additionally, disease severity and mortality were examined in patients with and without GI symptoms in 2 large, independent cohorts of hospitalized patients in the United States (n = 634) and Europe (n = 287) using multivariate logistic regressions.

Results: COVID-19 case patients and control individuals in the biopsy cohort were comparable for age, sex, rates of hospitalization, and relevant comorbid conditions. SARS-CoV-2 was detected in small intestinal epithelial cells by immunofluorescence staining or electron microscopy in 14 of 16 patients studied. High-dimensional analyses of GI tissues showed low levels of inflammation, including down-regulation of key inflammatory genes including IFNG, CXCL8, CXCL2, and IL1B and reduced frequencies of proinflammatory dendritic cells compared with control individuals. Consistent with these findings, we found a significant reduction in disease severity and mortality in patients presenting with GI symptoms that was independent of sex, age, and comorbid illnesses and despite similar nasopharyngeal SARS-CoV-2 viral loads. Furthermore, there was reduced levels of key inflammatory proteins in circulation in patients with GI symptoms.

Conclusions: These data highlight the absence of a proinflammatory response in the GI tract despite detection of SARS-CoV-2. In parallel, reduced mortality in patients with COVID-19 presenting with GI symptoms was observed. A potential role of the GI tract in attenuating SARS-CoV-2-associated inflammation needs to be further examined.
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http://dx.doi.org/10.1053/j.gastro.2021.02.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931673PMC
March 2021

Construction, characterization, and immunization of nanoparticles that display a diverse array of influenza HA trimers.

PLoS One 2021 4;16(3):e0247963. Epub 2021 Mar 4.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, United States of America.

Current influenza vaccines do not elicit broadly protective immune responses against multiple strains. New strategies to focus the humoral immune response to conserved regions on influenza antigens are therefore required for recognition by broadly neutralizing antibodies. It has been suggested that B-cells with receptors that recognize conserved epitopes would be preferentially stimulated through avidity effects by mosaic particles presenting multiple forms of a variable antigen. We adapted SpyCatcher-based platforms, AP205 virus-like particles (VLPs) and mi3 nanoparticles (NPs), to covalently co-display SpyTagged hemagglutinin (HA) trimers from group 1 and group 2 influenza A strains. Here we show successful homotypic and heterotypic conjugation of up to 8 different HA trimers to both VLPs and NPs. We characterized the HA-VLPs and HA-NPs by cryo-electron tomography to derive the average number of conjugated HAs and their separation distances on particles, and compared immunizations of mosaic and homotypic particles in wild-type mice. Both types of HA particles elicited strong antibody responses, but the mosaic particles did not consistently elicit broader immune responses than mixtures of homotypic particles. We conclude that covalent attachment of HAs from currently-circulating influenza strains represents a viable alternative to current annual influenza vaccine strategies, but in the absence of further modifications, is unlikely to represent a method for making a universal influenza vaccine.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247963PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7932532PMC
March 2021

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

Nature 2021 Feb 10. Epub 2021 Feb 10.

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

Here we report on the antibody and memory B cell responses of a cohort of 20 volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-2. Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection. However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors. However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 S in the presence of the monoclonal antibodies elicited by the vaccines. Together, these results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid a potential loss of clinical efficacy.
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http://dx.doi.org/10.1038/s41586-021-03324-6DOI Listing
February 2021

Intractable COVID-19 and Prolonged SARS-CoV-2 Replication in a CAR-T-cell Therapy Recipient: A Case Study.

Clin Infect Dis 2021 Jan 28. Epub 2021 Jan 28.

Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

A CAR-T-cell recipient developed severe COVID-19, intractable RNAemia, and viral replication lasting >2 months. Pre-mortem endotracheal aspirate contained 2x10 10 SARS-CoV-2 RNA copies/mL and infectious virus. Deep sequencing revealed multiple sequence variants consistent with intra-host virus evolution. SARS-CoV-2 humoral and cell-mediated immunity were minimal. Prolonged transmission from immunosuppressed patients is possible.
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http://dx.doi.org/10.1093/cid/ciab072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929077PMC
January 2021

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

bioRxiv 2021 Jan 19. Epub 2021 Jan 19.

To date severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected nearly 100 million individuals resulting in over two million deaths. Many vaccines are being deployed to prevent coronavirus disease-2019 (COVID-19) including two novel mRNA-based vaccines . These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known . Here we report on the antibody and memory B cell responses in a cohort of 20 volunteers who received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines. Consistent with prior reports, 8 weeks after the second vaccine injection volunteers showed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S), receptor binding domain (RBD) binding titers . Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection . However, activity against SARS-CoV-2 variants encoding E484K or N501Y or the K417N:E484K:N501Y combination was reduced by a small but significant margin. Consistent with these findings, vaccine-elicited monoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting a number of different RBD epitopes epitopes in common with mAbs isolated from infected donors. Structural analyses of mAbs complexed with S trimer suggest that vaccine- and virus-encoded S adopts similar conformations to induce equivalent anti-RBD antibodies. However, neutralization by 14 of the 17 most potent mAbs tested was reduced or abolished by either K417N, or E484K, or N501Y mutations. Notably, the same mutations were selected when recombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was cultured in the presence of the vaccine elicited mAbs. Taken together the results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid potential loss of clinical efficacy.
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http://dx.doi.org/10.1101/2021.01.15.426911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836122PMC
January 2021

Bispecific antibody prevents SARS-CoV-2 escape and protects mice from disease.

bioRxiv 2021 Jan 22. Epub 2021 Jan 22.

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19) . We developed a bispecific, IgG1-like molecule based on two antibodies derived from COVID-19 convalescent donors, C121 and C135 . CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, completely prevents S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 recognizes a broad panel of RBD variants and neutralizes SARS-CoV-2 and the escape mutants generated by the single monoclonals at sub-nanomolar concentrations. In a novel model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.
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http://dx.doi.org/10.1101/2021.01.22.427567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836104PMC
January 2021

Evolution of antibody immunity to SARS-CoV-2.

Nature 2021 03 18;591(7851):639-644. Epub 2021 Jan 18.

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

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models. Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.
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http://dx.doi.org/10.1038/s41586-021-03207-wDOI Listing
March 2021

Structural biology in the fight against COVID-19.

Nat Struct Mol Biol 2021 01;28(1):2-7

Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.

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http://dx.doi.org/10.1038/s41594-020-00544-8DOI Listing
January 2021

Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice.

Science 2021 02 12;371(6530):735-741. Epub 2021 Jan 12.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles with four to eight distinct RBDs). Mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic RBD nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs relative to sera from immunizations with homotypic SARS-CoV-2-RBD nanoparticles or COVID-19 convalescent human plasmas. Moreover, after priming, sera from mosaic RBD-immunized mice neutralized heterologous pseudotyped coronaviruses as well as or better than sera from homotypic SARS-CoV-2-RBD nanoparticle immunizations, demonstrating no loss of immunogenicity against particular RBDs resulting from co-display. A single immunization with mosaic RBD nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.
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http://dx.doi.org/10.1126/science.abf6840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928838PMC
February 2021

Mosaic RBD nanoparticles elicit neutralizing antibodies against SARS-CoV-2 and zoonotic coronaviruses.

bioRxiv 2020 Nov 17. Epub 2020 Nov 17.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

Protection against SARS-CoV-2 and SARS-related zoonotic coronaviruses with pandemic potential is urgently needed. To evaluate immunization strategies, we made nanoparticles displaying the receptor-binding domain (RBD) of only SARS-CoV-2 (homotypic nanoparticles) or co-displaying the SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles; 4-8 distinct RBDs). Mice immunized with RBD-nanoparticles, but not soluble antigen, elicited cross-reactive antibody binding and neutralization responses, confirming increased immunogenicity from multimerization. Mosaic-RBD-nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs compared to sera from immunizations with homotypic SARS-CoV-2-RBD-nanoparticles or antibodies from COVID-19 convalescent human plasmas. Moreover, sera from mosaic-RBD- immunized mice neutralized heterologous pseudotyped coronaviruses equivalently or better after priming than sera from homotypic SARS-CoV-2-RBD-nanoparticle immunizations, demonstrating no loss of immunogenicity against any particular RBD resulting from co-display. Thus, a single immunization with mosaic-RBD-nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.
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http://dx.doi.org/10.1101/2020.11.17.387092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685334PMC
November 2020

Evolution of Antibody Immunity to SARS-CoV-2.

bioRxiv 2020 Nov 5. Epub 2020 Nov 5.

SARS-CoV-2 has infected 47 million individuals and is responsible for over 1.2 million deaths to date. Infection is associated with development of variable levels of antibodies with neutralizing activity that can protect against infection in animal models. Antibody levels decrease with time, but the nature and quality of the memory B cells that would be called upon to produce antibodies upon re-infection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection. We find that IgM, and IgG anti-SARS-CoV-2 spike protein receptor binding domain (RBD) antibody titers decrease significantly with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by five-fold in pseudotype virus assays. In contrast, the number of RBD-specific memory B cells is unchanged. Memory B cells display clonal turnover after 6.2 months, and the antibodies they express have greater somatic hypermutation, increased potency and resistance to RBD mutations, indicative of continued evolution of the humoral response. Analysis of intestinal biopsies obtained from asymptomatic individuals 3 months after COVID-19 onset, using immunofluorescence, electron tomography or polymerase chain reaction, revealed persistence of SARS-CoV-2 in the small bowel of 7 out of 14 volunteers. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.
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http://dx.doi.org/10.1101/2020.11.03.367391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654855PMC
November 2020

De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2.

Science 2020 12 5;370(6521):1208-1214. Epub 2020 Nov 5.

Neoleukin Therapeutics Inc., Seattle, WA, USA.

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo-electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.
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http://dx.doi.org/10.1126/science.abe0075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920261PMC
December 2020

A broadly neutralizing macaque monoclonal antibody against the HIV-1 V3-Glycan patch.

Elife 2020 10 21;9. Epub 2020 Oct 21.

Laboratory of Molecular Immunology, The Rockefeller University, New York, United States.

A small fraction of HIV-1- infected humans develop broadly neutralizing antibodies (bNAbs) against HIV-1 that protect macaques from simian immunodeficiency HIV chimeric virus (SHIV). Similarly, a small number of macaques infected with SHIVs develop broadly neutralizing serologic activity, but less is known about the nature of simian antibodies. Here, we report on a monoclonal antibody, Ab1485, isolated from a macaque infected with SHIVAD8 that developed broadly neutralizing serologic activity targeting the V3-glycan region of HIV-1 Env. Ab1485 neutralizes 38.1% of HIV-1 isolates in a 42-pseudovirus panel with a geometric mean IC50 of 0.055 µg/mLl and SHIVAD8 with an IC50 of 0.028 µg/mLl. Ab1485 binds the V3-glycan epitope in a glycan-dependent manner. A 3.5 Å cryo-electron microscopy structure of Ab1485 in complex with a native-like SOSIP Env trimer showed conserved contacts with the N332gp120 glycan and gp120 GDIR peptide motif, but in a distinct Env-binding orientation relative to human V3/N332gp120 glycan-targeting bNAbs. Intravenous infusion of Ab1485 protected macaques from a high dose challenge with SHIVAD8. We conclude that macaques can develop bNAbs against the V3-glycan patch that resemble human V3-glycan bNAbs.
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http://dx.doi.org/10.7554/eLife.61991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577740PMC
October 2020

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies.

Nature 2020 12 12;588(7839):682-687. Epub 2020 Oct 12.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.

The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein show promise therapeutically and are being evaluated clinically. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to 'up' RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and 'down' RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2.
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http://dx.doi.org/10.1038/s41586-020-2852-1DOI Listing
December 2020

Structural classification of neutralizing antibodies against the SARS-CoV-2 spike receptor-binding domain suggests vaccine and therapeutic strategies.

bioRxiv 2020 Aug 30. Epub 2020 Aug 30.

The COVID-19 pandemic presents an urgent health crisis. Human neutralizing antibodies (hNAbs) that target the host ACE2 receptor-binding domain (RBD) of the SARS-CoV-2 spike show therapeutic promise and are being evaluated clincally . To determine structural correlates of SARS-CoV-2 neutralization, we solved 8 new structures of distinct COVID-19 hNAbs in complex with SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed classification into categories: (1) VH3-53 hNAbs with short CDRH3s that block ACE2 and bind only to up RBDs, (2) ACE2-blocking hNAbs that bind both up and down RBDs and can contact adjacent RBDs, (3) hNAbs that bind outside the ACE2 site and recognize up and down RBDs, and (4) Previously-described antibodies that do not block ACE2 and bind only up RBDs . Class 2 comprised four hNAbs whose epitopes bridged RBDs, including a VH3-53 hNAb that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking spike into a closed conformation. Epitope/paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally-occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects, suggesting combinations for clinical use, and providing insight into immune responses against SARS-CoV-2.
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http://dx.doi.org/10.1101/2020.08.30.273920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457611PMC
August 2020

Lower respiratory tract myeloid cells harbor SARS-CoV-2 and display an inflammatory phenotype.

medRxiv 2020 Aug 14. Epub 2020 Aug 14.

SARS-CoV-2 pneumonia may induce an aberrant immune response with brisk recruitment of myeloid cells into the lower respiratory tract, which may contribute to morbidity and mortality. We describe endotracheal aspirate samples from seven patients with SARS-CoV-2 pneumonia requiring mechanical ventilation. We note SARS-CoV-2 virions within lower respiratory tract myeloid cells shown by electron tomography, immunofluorescence confocal imaging, and immuno-electron microscopy. Endotracheal aspirates are primarily composed of mononuclear and polymorphonuclear leukocytes. These myeloid cells that harbor virus are frequently positive for CD14 and/or CD16 and most display an inflammatory phenotype marked by expression of IL-6 and tissue factor mRNA transcript and protein expression.
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http://dx.doi.org/10.1101/2020.08.11.20171967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430612PMC
August 2020

Electron tomography visualization of HIV-1 fusion with target cells using fusion inhibitors to trap the pre-hairpin intermediate.

Elife 2020 07 22;9. Epub 2020 Jul 22.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States.

Fusion of HIV-1 with the membrane of its target cell, an obligate first step in virus infectivity, is mediated by binding of the viral envelope (Env) spike protein to its receptors, CD4 and CCR5/CXCR4, on the cell surface. The process of viral fusion appears to be fast compared with viral egress and has not been visualized by EM. To capture fusion events, the process must be curtailed by trapping Env-receptor binding at an intermediate stage. We have used fusion inhibitors to trap HIV-1 virions attached to target cells by Envs in an extended pre-hairpin intermediate state. Electron tomography revealed HIV-1 virions bound to TZM-bl cells by 2-4 narrow spokes, with slightly more spokes present when evaluated with mutant virions that lacked the Env cytoplasmic tail. These results represent the first direct visualization of the hypothesized pre-hairpin intermediate of HIV-1 Env and improve our understanding of Env-mediated HIV-1 fusion and infection of host cells.
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http://dx.doi.org/10.7554/eLife.58411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394545PMC
July 2020

Nanoparticles presenting clusters of CD4 expose a universal vulnerability of HIV-1 by mimicking target cells.

Proc Natl Acad Sci U S A 2020 08 20;117(31):18719-18728. Epub 2020 Jul 20.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125;

CD4-based decoy approaches against HIV-1 are attractive options for long-term viral control, but initial designs, including soluble CD4 (sCD4) and CD4-Ig, were ineffective. To evaluate a therapeutic that more accurately mimics HIV-1 target cells compared with monomeric sCD4 and dimeric CD4-Ig, we generated virus-like nanoparticles that present clusters of membrane-associated CD4 (CD4-VLPs) to permit high-avidity binding of trimeric HIV-1 envelope spikes. In neutralization assays, CD4-VLPs were >12,000-fold more potent than sCD4 and CD4-Ig and >100-fold more potent than the broadly neutralizing antibody (bNAb) 3BNC117, with >12,000-fold improvements against strains poorly neutralized by 3BNC117. CD4-VLPs also neutralized patient-derived viral isolates that were resistant to 3BNC117 and other bNAbs. Intraperitoneal injections of CD4-CCR5-VLP produced only subneutralizing plasma concentrations in HIV-1-infected humanized mice but elicited CD4-binding site mutations that reduced viral fitness. All mutant viruses showed reduced sensitivity to sCD4 and CD4-Ig but remained sensitive to neutralization by CD4-VLPs in vitro. In vitro evolution studies demonstrated that CD4-VLPs effectively controlled HIV-1 replication at neutralizing concentrations, and viral escape was not observed. Moreover, CD4-VLPs potently neutralized viral swarms that were completely resistant to CD4-Ig, suggesting that escape pathways that confer resistance against conventional CD4-based inhibitors are ineffective against CD4-VLPs. These findings suggest that therapeutics that mimic HIV-1 target cells could prevent viral escape by exposing a universal vulnerability of HIV-1: the requirement to bind CD4 on a target cell. We propose that therapeutic and delivery strategies that ensure durable bioavailability need to be developed to translate this concept into a clinically feasible functional cure therapy.
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http://dx.doi.org/10.1073/pnas.2010320117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414181PMC
August 2020

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies.

Cell 2020 08 24;182(4):828-842.e16. Epub 2020 Jun 24.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA. Electronic address:

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1 and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.
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http://dx.doi.org/10.1016/j.cell.2020.06.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311918PMC
August 2020

Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies.

bioRxiv 2020 May 29. Epub 2020 May 29.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.

Neutralizing antibody responses to coronaviruses focus on the trimeric spike, with most against the receptor-binding domain (RBD). Here we characterized polyclonal IgGs and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their degree of focus on RBD epitopes, recognition of SARS-CoV, MERS-CoV, and mild coronaviruses, and how avidity effects contributed to increased binding/neutralization of IgGs over Fabs. Electron microscopy reconstructions of polyclonal plasma Fab-spike complexes showed recognition of both S1 and RBD epitopes. A 3.4Å cryo-EM structure of a neutralizing monoclonal Fab-S complex revealed an epitope that blocks ACE2 receptor-binding on "up" RBDs. Modeling suggested that IgGs targeting these sites have different potentials for inter-spike crosslinking on viruses and would not be greatly affected by identified SARS-CoV-2 spike mutations. These studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from and similarity to a SARS-CoV antibody, providing criteria for evaluating vaccine-elicited antibodies.
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http://dx.doi.org/10.1101/2020.05.28.121533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302198PMC
May 2020

Convergent antibody responses to SARS-CoV-2 in convalescent individuals.

Nature 2020 08 18;584(7821):437-442. Epub 2020 Jun 18.

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

During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-2. Here we report on 149 COVID-19-convalescent individuals. Plasma samples collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres; titres were less than 50 in 33% of samples, below 1,000 in 79% of samples and only 1% of samples had titres above 5,000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concentrations (IC values) as low as 2 ng ml. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.
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http://dx.doi.org/10.1038/s41586-020-2456-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442695PMC
August 2020

Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals.

bioRxiv 2020 May 22. Epub 2020 May 22.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.

During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-2. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (ICs) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.
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http://dx.doi.org/10.1101/2020.05.13.092619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263513PMC
May 2020