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1.
PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-334671

ABSTRACT

The within-host viral kinetics of SARS-CoV-2 infection and how they relate to a person's infectiousness are not well understood. This limits our ability to quantify the impact of interventions on viral transmission. Here, we develop data-driven viral dynamic models of SARS-CoV-2 infection and estimate key within-host parameters such as the infected cell half-life and the within-host reproductive number. We then develop a model linking VL to infectiousness, showing that a person's infectiousness increases sub-linearly with VL. We show that the logarithm of the VL in the upper respiratory tract (URT) is a better surrogate of infectiousness than the VL itself. Using data on VL and the predicted infectiousness, we further incorporated data on antigen and reverse transcription polymerase chain reaction (RT-PCR) tests and compared their usefulness in detecting infection and preventing transmission. We found that RT-PCR tests perform better than antigen tests assuming equal testing frequency;however, more frequent antigen testing may perform equally well with RT-PCR tests at a lower cost, but with many more false-negative tests. Overall, our models provide a quantitative framework for inferring the impact of therapeutics and vaccines that lower VL on the infectiousness of individuals and for evaluating rapid testing strategies. SIGNIFICANCE: Quantifying the kinetics of SARS-CoV-2 infection and individual infectiousness is key to quantitatively understanding SARS-CoV-2 transmission and evaluating intervention strategies. Here we developed data-driven within-host models of SARS-CoV-2 infection and by fitting them to clinical data we estimated key within-host viral dynamic parameters. We also developed a mechanistic model for viral transmission and show that the logarithm of the viral load in the upper respiratory tract serves an appropriate surrogate for a person's infectiousness. Using data on how viral load changes during infection, we further evaluated the effectiveness of PCR and antigen-based testing strategies for averting transmission and identifying infected individuals.

2.
PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-333823

ABSTRACT

Antibodies that potently neutralize SARS-CoV-2 target mainly the receptor-binding domain or the N-terminal domain (NTD). Over a dozen potently neutralizing NTD-directed antibodies have been studied structurally, and all target a single antigenic supersite in NTD (site 1). Here we report the 3.7 A resolution cryo-EM structure of a potent NTD-directed neutralizing antibody 5-7, which recognizes a site distinct from other potently neutralizing antibodies, inserting a binding loop into an exposed hydrophobic pocket between the two sheets of the NTD beta-sandwich. Interestingly, this pocket has been previously identified as the binding site for hydrophobic molecules including heme metabolites, but we observe their presence to not substantially impede 5-7 recognition. Mirroring its distinctive binding, antibody 5-7 retains a distinctive neutralization potency with variants of concern (VOC). Overall, we reveal a hydrophobic pocket in NTD proposed for immune evasion can actually be used by the immune system for recognition. HIGHLIGHTS: Cryo-EM structure of neutralizing antibody 5-7 in complex with SARS CoV-2 spike5-7 recognizes NTD outside of the previously identified antigenic supersite5-7 binds to a site known to accommodate numerous hydrophobic ligandsStructural basis of 5-7 neutralization tolerance to some variants of concern.

3.
PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-333778

ABSTRACT

Emergence of novel variants of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean neutralizing antibody titers of 14,000-21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within four days in 7 of 8 animals receiving 50 microg RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only ~2-fold relative to USA-WA1. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-like betacoronavirus vaccine development. SIGNIFICANCE STATEMENT: The emergence of SARS-CoV-2 variants of concern (VOC) that reduce the efficacy of current COVID-19 vaccines is a major threat to pandemic control. We evaluate a SARS-CoV-2 Spike receptor-binding domain ferritin nanoparticle protein vaccine (RFN) in a nonhuman primate challenge model that addresses the need for a next-generation, efficacious vaccine with increased pan-SARS breadth of coverage. RFN, adjuvanted with a liposomal-QS21 formulation (ALFQ), elicits humoral and cellular immune responses exceeding those of current vaccines in terms of breadth and potency and protects against high-dose respiratory tract challenge. Neutralization activity against the B.1.351 VOC within two-fold of wild-type virus and against SARS-CoV-1 indicate exceptional breadth. Our results support consideration of RFN for SARS-like betacoronavirus vaccine development.

4.
PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-333760

ABSTRACT

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine in nonhuman primates (NHPs). High-dose (50 micro g) SpFN vaccine, given twice within a 28 day interval, induced a Th1-biased CD4 T cell helper response and a peak neutralizing antibody geometric mean titer of 52,773 against wild-type virus, with activity against SARS-CoV-1 and minimal decrement against variants of concern. Vaccinated animals mounted an anamnestic response upon high-dose SARS-CoV-2 respiratory challenge that translated into rapid elimination of replicating virus in their upper and lower airways and lung parenchyma. SpFN's potent and broad immunogenicity profile and resulting efficacy in NHPs supports its utility as a candidate platform for SARS-like betacoronaviruses. ONE-SENTENCE SUMMARY: A SARS-CoV-2 Spike protein ferritin nanoparticle vaccine, co-formulated with a liposomal adjuvant, elicits broad neutralizing antibody responses that exceed those observed for other major vaccines and rapidly protects against respiratory infection and disease in the upper and lower airways and lung tissue of nonhuman primates.

5.
PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-333657

ABSTRACT

Understanding protective mechanisms of antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We discovered a new antibody, 910-30, that targets the SARS-CoV-2 ACE2 receptor binding site as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. We performed sequence and structural analyses to explore how antibody features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer revealed its binding interactions and ability to disassemble spike. Despite heavy chain sequence similarity, biophysical analyses of IGHV3-53/3-66 antibodies highlighted the importance of native heavy:light pairings for ACE2 binding competition and for SARS-CoV-2 neutralization. We defined paired heavy:light sequence signatures and determined antibody precursor prevalence to be ~1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These data reveal key structural and functional neutralization features in the IGHV3-53/3-66 public antibody class to accelerate antibody-based medical interventions against SARS-CoV-2. HIGHLIGHTS: A molecular study of IGHV3-53/3-66 public antibody responses reveals critical heavy and light chain features for potent neutralizationCryo-EM analyses detail the structure of a novel public antibody class member, antibody 910-30, in complex with SARS-CoV-2 spike trimerCryo-EM data reveal that 910-30 can both bind assembled trimer and can disassemble the SARS-CoV-2 spikeSequence-structure-function signatures defined for IGHV3-53/3-66 class antibodies including both heavy and light chainsIGHV3-53/3-66 class precursors have a prevalence of 1:44,000 B cells in healthy human antibody repertoires.

6.
Open Forum Infectious Diseases ; 8(SUPPL 1):S373, 2021.
Article in English | EMBASE | ID: covidwho-1746454

ABSTRACT

Background. Molnupiravir (MOV, MK-4482, EIDD-2801) is an orally administered prodrug of N-hydroxycytidine (NHC, EIDD-1931), a nucleoside with broad antiviral activity against a range of RNA viruses. MOV acts by driving viral error catastrophe following its incorporation by the viral RdRp into the viral genome. Given its mechanism of action, MOV activity should not be affected by substitutions in the spike protein present in SARS-CoV-2 variants of concern which impact efficacy of therapeutic neutralizing antibodies and vaccine induced immunity. We characterized MOV activity against variants by assessing antiviral activity in vitro and virologic response from the Phase 2/3 clinical trials (MOVe-In, MOVe-Out) for treatment of COVID-19. Methods. MOV activity against several SARS-CoV-2 variants, was evaluated in an in vitro infection assay. Antiviral potency of NHC (IC50) was determined in Vero E6 cells infected with virus at MOI ~0.1 by monitoring CPE. Longitudinal SARSCoV-2 RNA viral load measures in participants enrolled in MOVe-In and MOVe-Out were analyzed based on SARS-CoV-2 genotype. Sequences of SARS-CoV-2 from study participants were amplified from nasal swabs by PCR and NGS was performed on samples with viral genome RNA of >22,000 copies/ml amplified by primers covering full length genome with Ion Torrent sequencing to identify clades represented in trial participants. SARS-CoV-2 clades were assigned using clade.nextstrain.org. Results. In vitro, NHC was equally effective against SARS-CoV-2 variants B.1.1.7 (20I), B.1351 (20H), and P1 (20J), compared with the original WA1 (19B) isolate. In clinical trials, no discernable difference was observed in magnitude of viral response measured by change from baseline in RNA titer over time across all clades represented including 20A through 20E and 20G to 20I. No participants at the time of the study presented with 20F, 20J, or 21A. Conclusion. Distribution of clades in participants in MOVe-In and MOVe-Out was representative of those circulating globally at the time of collection (Oct 2020 -Jan 2021). Both in vitro and clinical data suggest that spike protein substitutions do not impact antiviral activity of MOV and suggest its potential use for the treatment of SARS-CoV-2 variants.

7.
Embase;
Preprint in English | EMBASE | ID: ppcovidwho-327009

ABSTRACT

The recently emerged B.1.1.529 (Omicron) SARS-CoV-2 variant has a highly divergent spike (S) glycoprotein. We compared the functional properties of B.1.1.529 S with those of previous globally prevalent SARS-CoV-2 variants, D614G and B.1.617.2. Relative to these variants, B.1.1.529 S exhibits decreased processing, resulting in less efficient syncytium formation and lower S incorporation into virus particles. Nonetheless, B.1.1.529 S supports virus infection equivalently. B.1.1.529 and B.1.617.2 S glycoproteins bind ACE2 with higher affinity than D614G S. The unliganded B.1.1.529 S trimer is less stable at low temperatures than the other SARSCoV-2 spikes, a property related to spike conformation. Upon ACE2 binding, the B.1.1.529 S trimer sheds S1 at 37 degrees but not at 0 degrees C. B.1.1.529 pseudoviruses are relatively resistant to neutralization by sera from convalescent COVID-19 patients and vaccinees. These properties of the B.1.1.529 spike glycoprotein likely influence the transmission, cytopathic effects and immune evasion of this emerging variant.

8.
Embase;
Preprint in English | EMBASE | ID: ppcovidwho-326938

ABSTRACT

The recently reported B.1.1.529 Omicron variant of SARS-CoV-2 includes 34 mutations in the spike protein relative to the Wuhan strain that initiated the COVID-19 pandemic, including 15 mutations in the receptor binding domain (RBD). Functional studies have shown omicron to substantially escape the activity of many SARS-CoV-2-neutralizing antibodies. Here we report a 3.1 Å resolution cryo-electron microscopy (cryo-EM) structure of the Omicron spike protein ectodomain. The structure depicts a spike that is exclusively in the 1-RBD-up conformation with increased mobility and inter-protomer asymmetry. Many mutations cause steric clashes and/or altered interactions at antibody binding surfaces, whereas others mediate changes of the spike structure in local regions to interfere with antibody recognition. Overall, the structure of the omicron spike reveals how mutations alter its conformation and explains its extraordinary ability to evade neutralizing antibodies.

9.
Embase;
Preprint in English | EMBASE | ID: ppcovidwho-326824

ABSTRACT

The Omicron (B.1.1.529) variant of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) was only recently detected in southern Africa, but its subsequent spread has been extensive, both regionally and globally1. It is expected to become dominant in the coming weeks2, probably due to enhanced transmissibility. A striking feature of this variant is the large number of spike mutations3 that pose a threat to the efficacy of current COVID-19 (coronavirus disease 2019) vaccines and antibody therapies4. This concern is amplified by the findings from our study. We found B.1.1.529 to be markedly resistant to neutralization by serum not only from convalescent patients, but also from individuals vaccinated with one of the four widely used COVID-19 vaccines. Even serum from persons vaccinated and boosted with mRNA-based vaccines exhibited substantially diminished neutralizing activity against B.1.1.529. By evaluating a panel of monoclonal antibodies to all known epitope clusters on the spike protein, we noted that the activity of 17 of the 19 antibodies tested were either abolished or impaired, including ones currently authorized or approved for use in patients. In addition, we also identified four new spike mutations (S371L, N440K, G446S, and Q493R) that confer greater antibody resistance to B.1.1.529. The Omicron variant presents a serious threat to many existing COVID-19 vaccines and therapies, compelling the development of new interventions that anticipate the evolutionary trajectory of SARS-CoV-2.

10.
Embase;
Preprint in English | EMBASE | ID: ppcovidwho-326792

ABSTRACT

The devastation caused by SARS-CoV-2 has made clear the importance of pandemic preparedness. To address future zoonotic outbreaks due to related viruses in the sarbecovirus subgenus, we identified a human monoclonal antibody, 10-40, that neutralized or bound all sarbecoviruses tested in vitro and protected against SARS-CoV-2 and SARS-CoV in vivo. Comparative studies with other receptor-binding domain (RBD)-directed antibodies showed 10-40 to have the greatest breadth against sarbecoviruses and thus its promise as an agent for pandemic preparedness. Moreover, structural analyses on 10-40 and similar antibodies not only defined an epitope cluster in the inner face of the RBD that is well conserved among sarbecoviruses, but also uncovered a new antibody class with a common CDRH3 motif. Our analyses also suggested that elicitation of this class of antibodies may not be overly difficult, an observation that bodes well for the development of a pan-sarbecovirus vaccine.

12.
Journal of Virology ; 95(14):16, 2021.
Article in English | Web of Science | ID: covidwho-1486522

ABSTRACT

We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identified compounds with broad 3CLpro-inhibitory activity. We also adapted the assay for use in compound screening and in doing so uncovered additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CLpro inhibitors. We observed strong concordance between data emerging from this assay and those obtained from live virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. We identified two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested, including 3CLpro enzymes from understudied zoonotic coronaviruses. IMPORTANCE Multiple coronavirus pandemics have occurred over the last 2 decades. This has highlighted a need to be proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus pandemics. We developed and validated a simplified cell-based assay for the identification of chemical inhibitors of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter free, does not require specialized biocontainment, and is optimized for performance in high-throughput screening. By testing reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence similarity, we identified compounds with broad activity against 3CL proteases and uncovered structural insights into features that contribute to their broad activity. Furthermore, we demonstrated that this assay is suitable for identifying chemical inhibitors of pro teases from families other than 3CL proteases.

14.
PubMed; 2020.
Preprint in English | PubMed | ID: ppcovidwho-6273

ABSTRACT

Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes. Funding: Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID). Support for this work was also provided by COVID-19 Fast Grants, the Jack Ma Foundation, the Self Graduate Fellowship Program, and NIH grants DP5OD023118, R21AI143407, and R21AI144408. Some of this work was performed at the Columbia University Cryo-EM Center at the Zuckerman Institute, and some at the Simons Electron Microscopy Center (SEMC) and National Center for Cryo-EM Access and Training (NCCAT) located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310). Conflict of Interest: The authors declare that they have no conflict of interest. Ethical Approval: Peripheral blood mononuclear cells (PBMCs) for B cell sorting were obtained from a convalescent SARS-CoV-2 patient (collected 75 days post symptom onset under an IRB approved clinical trial protocol, VRC 200 - ClinicalTrials.gov Identifier: NCT00067054) and a healthy control donor from the NIH blood bank pre-SARS-CoV-2 pandemic.

15.
PubMed; 2020.
Preprint in English | PubMed | ID: ppcovidwho-3030

ABSTRACT

Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19) from mild to severe stages including fatality, with pro-inflammatory macrophages as one of the main mediators of lung hyper-inflammation. Therefore, there is an urgent need to better understand the interactions among SARS-CoV-2 permissive cells, macrophage, and the SARS-CoV-2 virus, thereby offering important insights into new therapeutic strategies. Here, we used directed differentiation of human pluripotent stem cells (hPSCs) to establish a lung and macrophage co-culture system and model the host-pathogen interaction and immune response caused by SARS-CoV-2 infection. Among the hPSC-derived lung cells, alveolar type II and ciliated cells are the major cell populations expressing the viral receptor ACE2 and co-effector TMPRSS2, and both were highly permissive to viral infection. We found that alternatively polarized macrophages (M2) and classically polarized macrophages (M1) had similar inhibitory effects on SARS-CoV-2 infection. However, only M1 macrophages significantly up-regulated inflammatory factors including IL-6 and IL-18, inhibiting growth and enhancing apoptosis of lung cells. Inhibiting viral entry into target cells using an ACE2 blocking antibody enhanced the activity of M2 macrophages, resulting in nearly complete clearance of virus and protection of lung cells. These results suggest a potential therapeutic strategy, in that by blocking viral entrance to target cells while boosting anti-inflammatory action of macrophages at an early stage of infection, M2 macrophages can eliminate SARS-CoV-2, while sparing lung cells and suppressing the dysfunctional hyper-inflammatory response mediated by M1 macrophages.

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