Structural and mechanistic basis of neutralization by a pan-hantavirus protective antibody.

Emerging rodent-borne hantaviruses cause severe diseases in humans with no approved vaccines or therapeutics. We recently isolated a monoclonal broadly neutralizing antibody (nAb) from a Puumala virus-experienced human donor. Here, we report its structure bound to its target, the Gn/Gc glycoprotein heterodimer comprising the viral fusion complex. The structure explains the broad activity of the nAb: It recognizes conserved Gc fusion loop sequences and the main chain of variable Gn sequences, thereby straddling the Gn/Gc heterodimer and locking it in its prefusion conformation. We show that the nAb's accelerated dissociation from the divergent Andes virus Gn/Gc at endosomal acidic pH limits its potency against this highly lethal virus and correct this liability by engineering an optimized variant that sets a benchmark as a candidate pan-hantavirus therapeutic.

Immunogenicity, safety, and tolerability of a recombinant measles-vectored Lassa fever vaccine: a randomised, placebo-controlled, first-in-human trial.

BACKGROUND: Lassa fever is a substantial health burden in west Africa. We evaluated the safety, tolerability, and immunogenicity of a recombinant, live-attenuated, measles-vectored Lassa fever vaccine candidate (MV-LASV). METHODS: This first-in-human phase 1 trial-consisting of an open-label dose-escalation stage and an observer-blinded, randomised, placebo-controlled treatment stage-was conducted at a single site at the University of Antwerp, Antwerp, Belgium, and involved healthy adults aged 18-55 years. Participants in the dose-escalation stage were sequentially assigned to a low-dose group (two intramuscular doses of MV-LASV at 2 × 104 times the median tissue culture infectious dose) or a high-dose group (two doses at 1 × 105 times the median tissue culture infectious dose). Participants in the double-blinded treatment stage were randomly assigned in a 2:2:1 ratio to receive low dose, high dose, or placebo. The primary endpoint was the rate of solicited and unsolicited adverse events up to study day 56 and was assessed in all participants who received at least one dose of investigational product. The trial is registered with ClinicalTrials.gov, NCT04055454, and the European Union Drug Regulating Authorities Clinical Trials Database, 2018-003647-40, and is complete. FINDINGS: Between Sept 26, 2019, and Jan 20, 2020, 60 participants were enrolled and assigned to receive placebo (n=12) or MV-LASV (n=48). All 60 participants received at least one study treatment. Most adverse events occurred during the treatment phase, and frequencies of total solicited or unsolicited adverse events were similar between treatment groups, with 96% of participants in the low-dose group, 100% of those in the high-dose group, and 92% of those in the placebo group having any solicited adverse event (p=0·6751) and 76% of those in the low-dose group, 70% of those in the high-dose group, and 100% of those in the placebo group having any unsolicited adverse event (p=0·1047). The only significant difference related to local solicited adverse events, with higher frequencies observed in groups receiving MV-LASV (24 [96%] of 25 participants in the low-dose group; all 23 [100%] participants in the high-dose group) than in the placebo group (6 [50%] of 12 participants; p=0·0001, Fisher-Freeman-Halton test). Adverse events were mostly of mild or moderate severity, and no serious adverse events were observed. MV-LASV also induced substantial concentrations of LASV-specific IgG (geometric mean titre 62·9 EU/ml in the low-dose group and 145·9 EU/ml in the high-dose group on day 42). INTERPRETATION: MV-LASV showed an acceptable safety and tolerability profile, and immunogenicity seemed to be unaffected by pre-existing immunity against the vector. MV-LASV is therefore a promising candidate for further development. FUNDING: Coalition for Epidemic Preparedness Innovations.

Genomic surveillance of SARS-CoV-2 during the first year of the pandemic in the Bronx enabled clinical and epidemiological inference.

The Bronx was an early epicenter of the COVID-19 pandemic in the USA. We conducted temporal genomic surveillance of 104 SARS-CoV-2 genomes across the Bronx from March October 2020. Although the local structure of SARS-CoV-2 lineages mirrored those of New York City and New York State, temporal sampling revealed a dynamic and changing landscape of SARS-CoV-2 genomic diversity. Mapping the trajectories of mutations, we found that while some became 'endemic' to the Bronx, other, novel mutations rose in prevalence in the late summer/early fall. Geographically resolved genomes enabled us to distinguish between cases of reinfection and persistent infection in two pediatric patients. We propose that limited, targeted, temporal genomic surveillance has clinical and epidemiological utility in managing the ongoing COVID pandemic.

A Powassan virus domain III nanoparticle immunogen elicits neutralizing and protective antibodies in mice.

Powassan virus (POWV) is an emerging tick borne flavivirus (TBFV) that causes severe neuroinvasive disease. Currently, there are no approved treatments or vaccines to combat POWV infection. Here, we generated and characterized a nanoparticle immunogen displaying domain III (EDIII) of the POWV E glycoprotein. Immunization with POWV EDIII presented on nanoparticles resulted in significantly higher serum neutralizing titers against POWV than immunization with monomeric POWV EDIII. Furthermore, passive transfer of EDIII-reactive sera protected against POWV challenge in vivo. We isolated and characterized a panel of EDIII-specific monoclonal antibodies (mAbs) and identified several that potently inhibit POWV infection and engage distinct epitopes within the lateral ridge and C-C' loop of the EDIII. By creating a subunit-based nanoparticle immunogen with vaccine potential that elicits antibodies with protective activity against POWV infection, our findings enhance our understanding of the molecular determinants of antibody-mediated neutralization of TBFVs.

Induction of SARS-CoV-2 neutralizing antibodies by CoronaVac and BNT162b2 vaccines in naïve and previously infected individuals.

BACKGROUND: A major challenge of the SARS-CoV-2 pandemic is to better define "protective thresholds" to guide the global response. We aimed to characterize the longitudinal dynamics of the antibody responses in naturally infected individuals in Chile and compared them to humoral responses induced after immunization with CoronaVac-based on an inactivated whole virus -or the BNT162b2- based on mRNA-vaccines. We also contrasted them with the respective effectiveness and efficacy data available for both vaccines. METHODS: We determined and compared the longitudinal neutralizing (nAb) and anti-nucleocapsid (anti-N) antibody responses of 74 COVID-19 individuals (37 outpatient and 37 hospitalized) during the acute disease and convalescence. We also assessed the antibody boosting of 36 of these individuals who were immunized after convalescence with either the CoronaVac (n = 30) or the BNT162b2 (n = 6) vaccines. Antibody titres were also measured for 50 naïve individuals immunized with two doses of CoronaVac (n = 35) or BNT162b2 (n = 15) vaccines. The neutralizing level after vaccination was compared to those of convalescent individuals and the predicted efficacy was estimated. FINDINGS: SARS-CoV-2 infection induced robust nAb and anti-N antibody responses lasting >9 months, but showing a rapid nAb decay. After convalescence, nAb titres were significantly boosted by vaccination with CoronaVac or BNT162b2. In naïve individuals, the calculated mean titre induced by two doses of CoronaVac or BNT162b2 was 0·2 times and 5.2 times, respectively, that of convalescent individuals, which has been proposed as threshold of protection. CoronaVac induced no or only modest anti-N antibody responses. Using two proposed logistic models, the predicted efficacy of BNT162b2 was estimated at 97%, in close agreement with phase 3 efficacy studies, while for CoronaVac it was ∼50% corresponding to the lowest range of clinical trials and below the real-life data from Chile (from February 2 through May 1, 2021 during the predominant circulation of the Gamma variant), where the estimated vaccine effectiveness to prevent COVID-19 was 62·8-64·6%. INTERPRETATION: The decay of nAbs titres in previously infected individuals over time indicates that vaccination is needed to boost humoral memory responses. Immunization of naïve individuals with two doses of CoronaVac induced nAbs titres that were significantly lower to that of convalescent patients, and similar to vaccination with one dose of BTN162b2. The real life effectiveness for CoronaVac in Chile was higher than estimated; indicating that lower titres and additional cellular immune responses induced by CoronaVac might afford protection in a highly immunized population. Nevertheless, the lower nAb titre induced by two doses of CoronaVac as compared to the BTN162b2 vaccine in naïve individuals, highlights the need of booster immunizations over time to maintain protective levels of antibody, particularly with the emergence of new SARS-CoV-2 variants. FUNDING: FONDECYT 1161971, 1212023, 1181799, FONDECYT Postdoctorado 3190706 and 3190648, ANID Becas/Doctorado Nacional 21212258, PIA ACT 1408, CONICYT REDES180170, Centro Ciencia & Vida, FB210008, Financiamiento Basal para Centros Científicos y Tecnológicos de Excelencia grants from the Agencia Nacional de Investigación y Desarrollo (ANID) of Chile; NIH-NIAD grants U19AI135972, R01AI132633 and contracts HHSN272201400008C and 75N93019C00051; the JPB Foundation, the Open Philanthropy Project grant 2020-215611 (5384); and by anonymous donors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Longitudinally monitored immune biomarkers predict the timing of COVID-19 outcomes.

The clinical outcome of SARS-CoV-2 infection varies widely between individuals. Machine learning models can support decision making in healthcare by assessing fatality risk in patients that do not yet show severe signs of COVID-19. Most predictive models rely on static demographic features and clinical values obtained upon hospitalization. However, time-dependent biomarkers associated with COVID-19 severity, such as antibody titers, can substantially contribute to the development of more accurate outcome models. Here we show that models trained on immune biomarkers, longitudinally monitored throughout hospitalization, predicted mortality and were more accurate than models based on demographic and clinical data upon hospital admission. Our best-performing predictive models were based on the temporal analysis of anti-SARS-CoV-2 Spike IgG titers, white blood cell (WBC), neutrophil and lymphocyte counts. These biomarkers, together with C-reactive protein and blood urea nitrogen levels, were found to correlate with severity of disease and mortality in a time-dependent manner. Shapley additive explanations of our model revealed the higher predictive value of day post-symptom onset (PSO) as hospitalization progresses and showed how immune biomarkers contribute to predict mortality. In sum, we demonstrate that the kinetics of immune biomarkers can inform clinical models to serve as a powerful monitoring tool for predicting fatality risk in hospitalized COVID-19 patients, underscoring the importance of contextualizing clinical parameters according to their time post-symptom onset.

Genotype-specific features reduce the susceptibility of South American yellow fever virus strains to vaccine-induced antibodies.

The resurgence of yellow fever in South America has prompted vaccination against the etiologic agent, yellow fever virus (YFV). Current vaccines are based on a live-attenuated YF-17D virus derived from a virulent African isolate. The capacity of these vaccines to induce neutralizing antibodies against the vaccine strain is used as a surrogate for protection. However, the sensitivity of genetically distinct South American strains to vaccine-induced antibodies is unknown. We show that antiviral potency of the polyclonal antibody response in vaccinees is attenuated against an emergent Brazilian strain. This reduction was attributable to amino acid changes at two sites in central domain II of the glycoprotein E, including multiple changes at the domain I-domain II hinge, which are unique to and shared among most South American YFV strains. Our findings call for a reevaluation of current approaches to YFV immunological surveillance in South America and suggest approaches for updating vaccines.

Genomic surveillance of SARS-CoV-2 during the first year of the pandemic in the Bronx enabled clinical and epidemiological inference.

The Bronx was an early epicenter of the COVID-19 pandemic in the USA. We conducted temporal genomic surveillance of SARS-CoV-2 genomes across the Bronx from March-October 2020. Although the local structure of SARS-CoV-2 lineages mirrored those of New York City and New York State, temporal sampling revealed a dynamic and changing landscape of SARS-CoV-2 genomic diversity. Mapping the trajectories of variants, we found that while some became 'endemic' to the Bronx, other, novel variants rose in prevalence in the late summer/early fall. Geographically resolved genomes enabled us to distinguish between cases of reinfection and persistent infection in two pediatric patients. We propose that limited, targeted, temporal genomic surveillance has clinical and epidemiological utility in managing the ongoing COVID pandemic. One sentence summary: Temporally and geographically resolved sequencing of SARS-CoV-2 genotypes enabled surveillance of novel genotypes, identification of endemic viral variants, and clinical inferences, in the first wave of the COVID-19 pandemic in the Bronx.

Functional convalescent plasma antibodies and pre-infusion titers shape the early severe COVID-19 immune response.

Transfer of convalescent plasma (CP) had been proposed early during the SARS-CoV-2 pandemic as an accessible therapy, yet trial results worldwide have been mixed, potentially due to the heterogeneous nature of CP. Here we perform deep profiling of SARS-CoV-2-specific antibody titer, Fc-receptor binding, and Fc-mediated functional assays in CP units, as well as in plasma from hospitalized COVID-19 patients before and after CP administration. The profiling results show that, although all recipients exhibit expanded SARS-CoV-2-specific humoral immune responses, CP units contain more functional antibodies than recipient plasma. Meanwhile, CP functional profiles influence the evolution of recipient humoral immunity in conjuncture with the recipient's pre-existing SARS-CoV2-specific antibody titers: CP-derived SARS-CoV-2 nucleocapsid-specific antibody functions are associated with muted humoral immune evolution in patients with high titer anti-spike IgG. Our data thus provide insights into the unexpected impact of CP-derived functional anti-spike and anti-nucleocapsid antibodies on the evolution of SARS-CoV-2-specific response following severe infection.

A Combination of Receptor-Binding Domain and N-Terminal Domain Neutralizing Antibodies Limits the Generation of SARS-CoV-2 Spike Neutralization-Escape Mutants.

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150, and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent-phase sera were reduced by 4- to 16-fold against rVSV-SARS2 bearing Y145D, K150E, or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs did not enhance their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. IMPORTANCE The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19. These MAb therapeutics are solely targeting the receptor-binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent-phase COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor-binding and N-terminal domains may be beneficial to combat the emergence of virus variants.

Long-lasting neutralizing antibody responses in SARS-CoV-2 seropositive individuals are robustly boosted by immunization with the CoronaVac and BNT162b2 vaccines.

The durability of circulating neutralizing antibody (nAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and their boosting by vaccination remains to be defined. We show that outpatient and hospitalized SARS-CoV-2 seropositive individuals mount a robust neutralizing antibody (nAb) response that peaks at days 23 and 27 post-symptom onset, respectively. Although nAb titers remained higher in hospitalized patients, both study groups showed long-lasting nAb responses that can persist for up to 12 months after natural infection. These nAb responses in previously seropositive individuals can be significantly boosted through immunization with two doses of the CoronaVac (Sinovac) or one dose of the BNT162b2 (BioNTech/Pfizer) vaccines, suggesting a substantial induction of B cell memory responses. Noteworthy, three obese previously seropositive individuals failed to mount a booster response upon vaccination, warranting further studies in this population. Immunization of naïve individuals with two doses of the CoronaVac vaccine or one dose of the BNT162b2 vaccine elicited similar levels of nAbs compared to seropositive individuals 4.2 to 13.3 months post-infection with SARS-CoV-2. Thus, this preliminary evidence suggests that both, seropositive and naïve individuals, require two doses of CoronaVac to ensure the induction of robust nAb titers.

Single-Dilution COVID-19 Antibody Test with Qualitative and Quantitative Readouts.

The coronavirus disease 2019 (COVID-19) global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to place an immense burden on societies and health care systems. A key component of COVID-19 control efforts is serological testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior SARS-CoV-2 infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this sensitive test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test make it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.IMPORTANCE Serological surveillance has become an important public health tool during the COVID-19 pandemic. Detection of protective antibodies and seroconversion after SARS-CoV-2 infection or vaccination can help guide patient care plans and public health policies. Serology tests can detect antibodies against past infections; consequently, they can help overcome the shortcomings of molecular tests, which can detect only active infections. This is important, especially when considering that many COVID-19 patients are asymptomatic. In this study, we describe an enzyme-linked immunosorbent assay (ELISA)-based qualitative and quantitative serology test developed to measure IgG and IgA antibodies against the SARS-CoV-2 spike glycoprotein. The test can be deployed using commonly available laboratory reagents and equipment and displays high specificity and sensitivity. Furthermore, we demonstrate that IgG titers in patient samples can be estimated from a single measurement, enabling the assay's use in high-throughput clinical environments.

Functional Antibodies in COVID-19 Convalescent Plasma.

In the absence of an effective vaccine or monoclonal therapeutic, transfer of convalescent plasma (CCP) was proposed early in the SARS-CoV-2 pandemic as an easily accessible therapy. However, despite the global excitement around this historically valuable therapeutic approach, results from CCP trials have been mixed and highly debated. Unlike other therapeutic interventions, CCP represents a heterogeneous drug. Each CCP unit is unique and collected from an individual recovered COVID-19 patient, making the interpretation of therapeutic benefit more complicated. While the prevailing view in the field would suggest that it is administration of neutralizing antibodies via CCP that centrally provides therapeutic benefit to newly infected COVID-19 patients, many hospitalized COVID-19 patients already possess neutralizing antibodies. Importantly, the therapeutic benefit of antibodies can extend far beyond their simple ability to bind and block infection, especially related to their ability to interact with the innate immune system. In our work we deeply profiled the SARS-CoV-2-specific Fc-response in CCP donors, along with the recipients prior to and after CCP transfer, revealing striking SARS-CoV-2 specific Fc-heterogeneity across CCP units and their recipients. However, CCP units possessed more functional antibodies than acute COVID-19 patients, that shaped the evolution of COVID-19 patient humoral profiles via distinct immunomodulatory effects that varied by pre-existing SARS-CoV-2 Spike (S)-specific IgG titers in the patients. Our analysis identified surprising influence of both S and Nucleocapsid (N) specific antibody functions not only in direct antiviral activity but also in anti-inflammatory effects. These findings offer insights for more comprehensive interpretation of correlates of immunity in ongoing large scale CCP trials and for the design of next generation therapeutic design.

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.

Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased protein expression. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high-quality S protein (nonaggregated, uniform material with appropriate biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM structures reveals conformation plasticity in the region composed of amino acids 614-642 and 828-854. Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome and report no novel binding partners and notably fail to validate the Spike:CD147 interaction. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural, and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

Treatment of severe COVID-19 with convalescent plasma in Bronx, NYC.

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as a treatment for coronavirus disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200 mL of CCP with a spike protein IgG titer ≥ 1:2430 (median 1:47,385) within 72 hours of admission with propensity score-matched controls cared for at a medical center in the Bronx, between April 13 and May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroid use, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared with matched controls, CCP recipients less than 65 years had 4-fold lower risk of mortality and 4-fold lower risk of deterioration in oxygenation or mortality at day 28. For CCP recipients, pretransfusion spike protein IgG, IgM, and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients less than 65 years, but data from controlled trials are needed to validate this finding and establish the effect of aging on CCP efficacy.

Treatment of Severe COVID-19 with Convalescent Plasma in the Bronx, NYC.

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as treatment for Coronavirus Disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200mL of CCP with a Spike protein IgG titer ≥1:2,430 (median 1:47,385) within 72 hours of admission to propensity score-matched controls cared for at a medical center in the Bronx, between April 13 to May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroids, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared to matched controls, CCP recipients <65 years had 4-fold lower mortality and 4-fold lower deterioration in oxygenation or mortality at day 28. For CCP recipients, pre-transfusion Spike protein IgG, IgM and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients <65 years, but data from controlled trials is needed to validate this finding and establish the effect of ageing on CCP efficacy.

Development, clinical translation, and utility of a COVID-19 antibody test with qualitative and quantitative readouts.

The COVID-19 global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to place an immense burden on societies and healthcare systems. A key component of COVID-19 control efforts is serologic testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test makes it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.

A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition.

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

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.

A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.