Publications by authors named "Jason S McLellan"

119 Publications

Local computational methods to improve the interpretability and analysis of cryo-EM maps.

Nat Commun 2021 02 23;12(1):1240. Epub 2021 Feb 23.

Departmento de Óptica, Universidad Complutense de Madrid, Madrid, Spain.

Cryo-electron microscopy (cryo-EM) maps usually show heterogeneous distributions of B-factors and electron density occupancies and are typically B-factor sharpened to improve their contrast and interpretability at high-resolutions. However, 'over-sharpening' due to the application of a single global B-factor can distort processed maps causing connected densities to appear broken and disconnected. This issue limits the interpretability of cryo-EM maps, i.e. ab initio modelling. In this work, we propose 1) approaches to enhance high-resolution features of cryo-EM maps, while preventing map distortions and 2) methods to obtain local B-factors and electron density occupancy maps. These algorithms have as common link the use of the spiral phase transformation and are called LocSpiral, LocBSharpen, LocBFactor and LocOccupancy. Our results, which include improved maps of recent SARS-CoV-2 structures, show that our methods can improve the interpretability and analysis of obtained reconstructions.
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http://dx.doi.org/10.1038/s41467-021-21509-5DOI Listing
February 2021

Prolonged evolution of the human B cell response to SARS-CoV-2 infection.

Sci Immunol 2021 02;6(56)

Adimab LLC, Lebanon, NH 03766, USA.

A comprehensive understanding of the kinetics and evolution of the human B cell response to SARS-CoV-2 infection will facilitate the development of next-generation vaccines and therapies. Here, we longitudinally profiled this response in mild and severe COVID-19 patients over a period of five months. Serum neutralizing antibody (nAb) responses waned rapidly but spike (S)-specific IgG memory B cells (MBCs) remained stable or increased over time. Analysis of 1,213 monoclonal antibodies (mAbs) isolated from S-specific MBCs revealed a primarily de novo response that displayed increased somatic hypermutation, binding affinity, and neutralization potency over time, providing evidence for prolonged antibody affinity maturation. B cell immunodominance hierarchies were similar across donor repertoires and remained relatively stable as the immune response progressed. Cross-reactive B cell populations, likely re-called from prior endemic beta-coronavirus exposures, comprised a small but stable fraction of the repertoires and did not contribute to the neutralizing response. The neutralizing antibody response was dominated by public clonotypes that displayed significantly reduced activity against SARS-CoV-2 variants emerging in Brazil and South Africa that harbor mutations at positions 501, 484 and 417 in the S protein. Overall, the results provide insight into the dynamics, durability, and functional properties of the human B cell response to SARS-CoV-2 infection and have implications for the design of immunogens that preferentially stimulate protective B cell responses.
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http://dx.doi.org/10.1126/sciimmunol.abg6916DOI Listing
February 2021

A glycan gate controls opening of the SARS-CoV-2 spike protein.

bioRxiv 2021 Feb 16. Epub 2021 Feb 16.

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded (down) to an exposed (up) state in order to bind the human ACE2 receptor and infect cells. While snapshots of the up and down states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 200 μs of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways. Together with biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408, and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.
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http://dx.doi.org/10.1101/2021.02.15.431212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899456PMC
February 2021

Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates.

bioRxiv 2021 Feb 11. Epub 2021 Feb 11.

The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor-binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.
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http://dx.doi.org/10.1101/2021.02.10.430696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885918PMC
February 2021

The impacts of COVID-19 vaccine timing, number of doses, and risk prioritization on mortality in the US.

medRxiv 2021 Jan 20. Epub 2021 Jan 20.

As COVID-19 vaccination begins worldwide, policymakers face critical trade-offs. Using a mathematical model of COVID-19 transmission, we find that timing of the rollout is expected to have a substantially greater impact on mortality than risk-based prioritization and adherence and that prioritizing first doses over second doses may be life saving.
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http://dx.doi.org/10.1101/2021.01.18.21250071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836125PMC
January 2021

Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody.

Science 2021 02 25;371(6531):823-829. Epub 2021 Jan 25.

Adimab, LLC, Lebanon, NH 03766, USA.

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.
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http://dx.doi.org/10.1126/science.abf4830DOI Listing
February 2021

Molecular determinants and mechanism for antibody cocktail preventing SARS-CoV-2 escape.

Nat Commun 2021 01 20;12(1):469. Epub 2021 Jan 20.

Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, 77030, USA.

Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. The determinants for selecting antibody combinations and the mechanism that antibody cocktails prevent viral escape remain unclear. We compared the critical residues in the receptor-binding domain (RBD) used by multiple neutralizing antibodies and cocktails and identified a combination of two antibodies CoV2-06 and CoV2-14 for preventing viral escape. The two antibodies simultaneously bind to non-overlapping epitopes and independently compete for receptor binding. SARS-CoV-2 rapidly escapes from individual antibodies by generating resistant mutations in vitro, but it doesn't escape from the cocktail due to stronger mutational constraints on RBD-ACE2 interaction and RBD protein folding requirements. We also identified a conserved neutralizing epitope shared between SARS-CoV-2 and SARS-CoV for antibody CoV2-12. Treatments with CoV2-06 and CoV2-14 individually and in combination confer protection in mice. These findings provide insights for rational selection and mechanistic understanding of antibody cocktails as candidates for treating COVID-19.
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http://dx.doi.org/10.1038/s41467-020-20789-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817669PMC
January 2021

SARS-CoV-2 escape from a highly neutralizing COVID-19 convalescent plasma.

bioRxiv 2020 Dec 28. Epub 2020 Dec 28.

To investigate the evolution of SARS-CoV-2 in the immune population, we co-incubated authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for 7 passages, but after 45 days, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed at day 80 by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom and South Africa of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.

One Sentence Summary: Three mutations allowed SARS-CoV-2 to evade the polyclonal antibody response of a highly neutralizing COVID-19 convalescent plasma.
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http://dx.doi.org/10.1101/2020.12.28.424451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781313PMC
December 2020

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma.

bioRxiv 2020 Dec 21. Epub 2020 Dec 21.

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq ) to the spike ectodomain (S-ECD ) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing , we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 Å ). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2020.12.20.423708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781304PMC
December 2020

Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions.

bioRxiv 2020 Dec 20. Epub 2020 Dec 20.

The continual emergence of novel coronavirus (CoV) strains, like SARS-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. Coronavirus spike (S) proteins share common structural motifs that could be vulnerable to cross-reactive antibody responses. To study this phenomenon in human coronavirus infection, we applied a high-throughput sequencing method called LIBRA-seq (Linking B cell receptor to antigen specificity through sequencing) to a SARS-CoV-1 convalescent donor sample. We identified and characterized a panel of six monoclonal antibodies that cross-reacted with S proteins from the highly pathogenic SARS-CoV-1 and SARS-CoV-2 and demonstrated a spectrum of reactivity against other coronaviruses. Epitope mapping revealed that these antibodies recognized multiple epitopes on SARS-CoV-2 S, including the receptor binding domain (RBD), N-terminal domain (NTD), and S2 subunit. Functional characterization demonstrated that the antibodies mediated a variety of Fc effector functions and mitigated pathological burden . The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies that may be useful for preventing potential future coronavirus outbreaks.
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http://dx.doi.org/10.1101/2020.12.20.414748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781301PMC
December 2020

An Engineered Antibody with Broad Protective Efficacy in Murine Models of SARS and COVID-19.

bioRxiv 2020 Nov 17. Epub 2020 Nov 17.

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. Here, we employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with remarkable potency. Structural and biochemical studies demonstrate that ADG-2 employs a unique angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In murine models of SARS-CoV and SARS-CoV-2 infection, passive transfer of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate for the treatment and prevention of SARS-CoV-2 and future emerging SARS-like CoVs.
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http://dx.doi.org/10.1101/2020.11.17.385500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685319PMC
November 2020

Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein.

ACS Cent Sci 2020 Oct 23;6(10):1722-1734. Epub 2020 Sep 23.

Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, United States.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 28,000,000 infections and 900,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). Similar to many other viral fusion proteins, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of the glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biological data. Multiple microsecond-long, all-atom molecular dynamics simulations were used to provide an atomistic perspective on the roles of glycans and on the protein structure and dynamics. We reveal an essential structural role of -glycans at sites N165 and N234 in modulating the conformational dynamics of the spike's receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry experiments, which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift toward the "down" state. Additionally, end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of the SARS-CoV-2 S protein, which may be exploited in the therapeutic efforts targeting this molecular machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development.
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http://dx.doi.org/10.1021/acscentsci.0c01056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523240PMC
October 2020

Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area.

mBio 2020 10 30;11(6). Epub 2020 Oct 30.

Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute, Houston Methodist Hospital, Houston, Texas, USA

We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotype and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein-the primary target of global vaccine efforts-are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR3022. Our report represents the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution. There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves.
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http://dx.doi.org/10.1128/mBio.02707-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642679PMC
October 2020

Continuous flexibility analysis of SARS-CoV-2 spike prefusion structures.

IUCrJ 2020 Sep 29;7(Pt 6). Epub 2020 Sep 29.

Centro Nacional de Biotecnologia-CSIC, Calle Darwin 3, 28049 Cantoblanco, Madrid, Spain.

Using a new consensus-based image-processing approach together with principal component analysis, the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state have been analysed. These studies revealed concerted motions involving the receptor-binding domain (RBD), N-terminal domain, and subdomains 1 and 2 around the previously characterized 1-RBD-up state, which have been modeled as elastic deformations. It is shown that in this data set there are not well defined, stable spike conformations, but virtually a continuum of states. An ensemble map was obtained with minimum bias, from which the extremes of the change along the direction of maximal variance were modeled by flexible fitting. The results provide a warning of the potential image-processing classification instability of these complicated data sets, which has a direct impact on the interpretability of the results.
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http://dx.doi.org/10.1107/S2052252520012725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553147PMC
September 2020

Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area.

medRxiv 2020 Sep 29. Epub 2020 Sep 29.

Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, 6565 Fannin Street, Houston, Texas 77030.

We sequenced the genomes of 5,085 SARS-CoV-2 strains causing two COVID-19 disease waves in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston, and an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotypes and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein - the primary target of global vaccine efforts - are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR30022. Our study is the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves, and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution.
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http://dx.doi.org/10.1101/2020.09.22.20199125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536878PMC
September 2020

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

bioRxiv 2020 Oct 1. Epub 2020 Oct 1.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection.

One Sentence Summary: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2020.09.30.318972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536866PMC
October 2020

Suptavumab for the Prevention of Medically Attended Respiratory Syncytial Virus Infection in Preterm Infants.

Clin Infect Dis 2020 Sep 8. Epub 2020 Sep 8.

Regeneron Pharmaceuticals, Inc, Tarrytown, New York, USA.

Background: Respiratory syncytial virus (RSV) is a major cause of childhood medically attended respiratory infection (MARI).

Methods: We conducted a randomized, double-blind, placebo-controlled phase 3 trial in 1154 preterm infants of 1 or 2 doses of suptavumab, a human monoclonal antibody that can bind and block a conserved epitope on RSV A and B subtypes, for the prevention of RSV MARI. The primary endpoint was proportion of subjects with RSV-confirmed hospitalizations or outpatient lower respiratory tract infection (LRTI).

Results: There were no significant differences between primary endpoint rates (8.1%, placebo; 7.7%, 1-dose; 9.3%, 2-dose). Suptavumab prevented RSV A infections (relative risks, .38; 95% confidence interval [CI], .14-1.05 in the 1-dose group and .39 [95% CI, .14-1.07] in the 2-dose group; nominal significance of combined suptavumab group vs placebo; P = .0499), while increasing the rate of RSV B infections (relative risk 1.36 [95% CI, .73-2.56] in the 1-dose group and 1.69 [95% CI, .92-3.08] in the 2-dose group; nominal significance of combined suptavumab group vs placebo; P = .12). Sequenced RSV isolates demonstrated no suptavumab epitope changes in RSV A isolates, while all RSV B isolates had 2-amino acid substitution in the suptavumab epitope that led to loss of neutralization activity. Treatment emergent adverse events were balanced across treatment groups.

Conclusions: Suptavumab did not reduce overall RSV hospitalizations or outpatient LRTI because of a newly circulating mutant strain of RSV B. Genetic variation in circulating RSV strains will continue to challenge prevention efforts.

Clinical Trials Registration: NCT02325791. https://clinicaltrials.gov/ct2/show/NCT02325791.
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http://dx.doi.org/10.1093/cid/ciaa951DOI Listing
September 2020

Specificity and effector functions of non-neutralizing gB-specific monoclonal antibodies isolated from healthy individuals with human cytomegalovirus infection.

Virology 2020 09 22;548:182-191. Epub 2020 Jul 22.

Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, USA. Electronic address:

Human cytomegalovirus (HCMV) is the most common congenital infection. A glycoprotein B (gB) subunit vaccine (gB/MF59) is the most efficacious clinically tested to date, having achieved 50% protection against primary infection of HCMV-seronegative women. We previously identified that gB/MF59 vaccination primarily elicits non-neutralizing antibody responses, with variable binding to gB genotypes, and protection associated with binding to membrane-associated gB. We hypothesized that gB-specific non-neutralizing antibody binding breadth and function are dependent on epitope and genotype specificity, and ability to interact with membrane-associated gB. We mapped twenty-four gB-specific monoclonal antibodies (mAbs) from naturally HCMV-infected individuals for gB domain specificity, genotype preference, and ability to mediate phagocytosis or NK cell activation. gB-specific mAbs were primarily specific for Domain II and demonstrated variable binding to gB genotypes. Two mAbs facilitated phagocytosis with binding specificities of Domain II and AD2. This investigation provides novel understanding on the relationship between gB domain specificity and antigenic variability on gB-specific antibody effector functions.
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http://dx.doi.org/10.1016/j.virol.2020.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447913PMC
September 2020

SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness.

Nature 2020 10 5;586(7830):567-571. Epub 2020 Aug 5.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant SARS-CoV-2 as well as CD8 T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.
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http://dx.doi.org/10.1038/s41586-020-2622-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581537PMC
October 2020

Recognition of a highly conserved glycoprotein B epitope by a bivalent antibody neutralizing HCMV at a post-attachment step.

PLoS Pathog 2020 08 3;16(8):e1008736. Epub 2020 Aug 3.

Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, Texas, United States of America.

Human cytomegalovirus (HCMV) is one of the main causative agents of congenital viral infection in neonates. HCMV infection also causes serious morbidity and mortality among organ transplant patients. Glycoprotein B (gB) is a major target for HCMV neutralizing antibodies, yet the underlying neutralization mechanisms remain largely unknown. Here we report that 3-25, a gB-specific monoclonal antibody previously isolated from a healthy HCMV-positive donor, efficiently neutralized 14 HCMV strains in both ARPE-19 cells and MRC-5 cells. The core epitope of 3-25 was mapped to a highly conserved linear epitope on antigenic domain 2 (AD-2) of gB. A 1.8 Å crystal structure of 3-25 Fab in complex with the peptide epitope revealed the molecular determinants of 3-25 binding to gB at atomic resolution. Negative-staining electron microscopy (EM) 3D reconstruction of 3-25 Fab in complex with de-glycosylated postfusion gB showed that 3-25 Fab fully occupied the gB trimer at the N-terminus with flexible binding angles. Functionally, 3-25 efficiently inhibited HCMV infection at a post-attachment step by interfering with viral membrane fusion, and restricted post-infection viral spreading in ARPE-19 cells. Interestingly, bivalency was required for HCMV neutralization by AD-2 specific antibody 3-25 but not the AD-4 specific antibody LJP538. In contrast, bivalency was not required for HCMV binding by both antibodies. Taken together, our results reveal the structural basis of gB recognition by 3-25 and demonstrate that inhibition of viral membrane fusion and a requirement of bivalency may be common for gB AD-2 specific neutralizing antibody.
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http://dx.doi.org/10.1371/journal.ppat.1008736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425986PMC
August 2020

Structure-based design of prefusion-stabilized SARS-CoV-2 spikes.

Science 2020 09 23;369(6510):1501-1505. Epub 2020 Jul 23.

Department of Molecular Biosciences, University of Texas, Austin, TX 78712, USA.

The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo-electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
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http://dx.doi.org/10.1126/science.abd0826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402631PMC
September 2020

Continuous flexibility analysis of SARS-CoV-2 Spike prefusion structures.

bioRxiv 2020 Jul 8. Epub 2020 Jul 8.

Centro Nacional de Biotecnologia-CSIC, C/ Darwin, 3, 28049, Cantoblanco, Madrid, Spain.

With the help of novel processing workflows and algorithms, we have obtained a better understanding of the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state. We have re-analyzed previous cryo-EM data combining 3D clustering approaches with ways to explore a continuous flexibility space based on 3D Principal Component Analysis. These advanced analyses revealed a concerted motion involving the receptor-binding domain (RBD), N-terminal domain (NTD), and subdomain 1 and 2 (SD1 & SD2) around the previously characterized 1-RBD-up state, which have been modeled as elastic deformations. We show that in this dataset there are not well-defined, stable, spike conformations, but virtually a continuum of states moving in a concerted fashion. We obtained an improved resolution ensemble map with minimum bias, from which we model by flexible fitting the extremes of the change along the direction of maximal variance. Moreover, a high-resolution structure of a recently described biochemically stabilized form of the spike is shown to greatly reduce the dynamics observed for the wild-type spike. Our results provide new detailed avenues to potentially restrain the spike dynamics for structure-based drug and vaccine design and at the same time give a warning of the potential image processing classification instability of these complicated datasets, having a direct impact on the interpretability of the results.
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http://dx.doi.org/10.1101/2020.07.08.191072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359526PMC
July 2020

Structure-Based Design of Nipah Virus Vaccines: A Generalizable Approach to Paramyxovirus Immunogen Development.

Front Immunol 2020 11;11:842. Epub 2020 Jun 11.

Viral Pathogenesis Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States.

Licensed vaccines or therapeutics are rarely available for pathogens with epidemic or pandemic potential. Developing interventions for specific pathogens and defining generalizable approaches for related pathogens is a global priority and inherent to the UN Sustainable Development Goals. Nipah virus (NiV) poses a significant epidemic threat, and zoonotic transmission from bats-to-humans with high fatality rates occurs almost annually. Human-to-human transmission of NiV has been documented in recent outbreaks leading public health officials and government agencies to declare an urgent need for effective vaccines and therapeutics. Here, we evaluate NiV vaccine antigen design options including the fusion glycoprotein (F) and the major attachment glycoprotein (G). A stabilized prefusion F (pre-F), multimeric G constructs, and chimeric proteins containing both pre-F and G were developed as protein subunit candidate vaccines. The proteins were evaluated for antigenicity and structural integrity using kinetic binding assays, electron microscopy, and other biophysical properties. Immunogenicity of the vaccine antigens was evaluated in mice. The stabilized pre-F trimer and hexameric G immunogens both induced serum neutralizing activity in mice, while the post-F trimer immunogen did not elicit neutralizing activity. The pre-F trimer covalently linked to three G monomers (pre-F/G) induced potent neutralizing antibody activity, elicited responses to the greatest diversity of antigenic sites, and is the lead candidate for clinical development. The specific stabilizing mutations and immunogen designs utilized for NiV were successfully applied to other henipaviruses, supporting the concept of identifying generalizable solutions for prototype pathogens as an approach to pandemic preparedness.
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http://dx.doi.org/10.3389/fimmu.2020.00842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300195PMC
June 2020

Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes.

bioRxiv 2020 May 30. Epub 2020 May 30.

Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712.

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to ~10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.
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http://dx.doi.org/10.1101/2020.05.30.125484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302215PMC
May 2020

Shielding and Beyond: The Roles of Glycans in SARS-CoV-2 Spike Protein.

bioRxiv 2020 Jun 11. Epub 2020 Jun 11.

Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, USA.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 7,000,000 infections and 400,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARSCoV-2 spike (S) protein, which mediates the host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). In the context of vaccine design, similar to many other viruses, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biological data. Multiple microsecond-long, all-atom molecular dynamics simulations were used to provide an atomistic perspective on the glycan shield and the protein structure, stability, and dynamics. End-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of SARS-CoV-2 S protein, which can be harnessed for vaccine development. In addition, a dynamic analysis of the main antibody epitopes is provided. Finally, beyond shielding, a possible structural role of N-glycans at N165 and N234 is hypothesized to modulate and stabilize the conformational dynamics of the spike's receptor binding domain, which is responsible for ACE2 recognition. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, which may be exploited by therapeutic efforts targeting this essential molecular machine.
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http://dx.doi.org/10.1101/2020.06.11.146522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302197PMC
June 2020

SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness.

bioRxiv 2020 Jun 11. Epub 2020 Jun 11.

Vaccine Research Center; National Institute of Allergy and Infectious Diseases; National Institutes of Health; Bethesda, Maryland, 20892; United States of America.

A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.
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http://dx.doi.org/10.1101/2020.06.11.145920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301911PMC
June 2020

Broad neutralization of SARS-related viruses by human monoclonal antibodies.

Science 2020 08 15;369(6504):731-736. Epub 2020 Jun 15.

Adimab LLC, Lebanon, NH 03766, USA.

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines.
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http://dx.doi.org/10.1126/science.abc7424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299279PMC
August 2020

Broad sarbecovirus neutralizing antibodies define a key site of vulnerability on the SARS-CoV-2 spike protein.

bioRxiv 2020 May 16. Epub 2020 May 16.

Adimab LLC, Lebanon, NH 03766, USA.

Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pan-sarbecovirus vaccines.
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http://dx.doi.org/10.1101/2020.05.15.096511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241100PMC
May 2020

Site-specific analysis of the SARS-CoV-2 glycan shield.

bioRxiv 2020 Mar 28. Epub 2020 Mar 28.

School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK.

The emergence of the betacoronavirus, SARS-CoV-2 that causes COVID-19, represents a significant threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, that mediates cell entry and membrane fusion. SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in immune evasion and occluding immunogenic protein epitopes. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.
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http://dx.doi.org/10.1101/2020.03.26.010322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239077PMC
March 2020