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.

Resurfaced ZIKV EDIII nanoparticle immunogens elicit neutralizing and protective responses in vivo.

Zika virus (ZIKV) is a flavivirus that can cause severe disease, but there are no approved treatments or vaccines. A complication for flavivirus vaccine development is the potential of immunogens to enhance infection via antibody-dependent enhancement (ADE), a process mediated by poorly neutralizing and cross-reactive antibodies. Thus, there is a great need to develop immunogens that minimize the potential to elicit enhancing antibodies. Here we utilized structure-based protein engineering to develop "resurfaced" (rs) ZIKV immunogens based on E glycoprotein domain III (ZDIIIs), in which epitopes bound by variably neutralizing antibodies were masked by combinatorial mutagenesis. We identified one resurfaced ZDIII immunogen (rsZDIII-2.39) that elicited a protective but immune-focused response. Compared to wild type ZDIII, immunization with resurfaced rsZDIII-2.39 protein nanoparticles produced fewer numbers of ZIKV EDIII antigen-reactive B cells and elicited serum that had a lower magnitude of induced ADE against dengue virus serotype 1 (DENV1) Our findings enhance our understanding of the structural and functional determinants of antibody protection against ZIKV.

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.

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.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Qualitative Immunoglobulin G Assays: The Value of Numeric Reporting.

CONTEXT.­: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) testing is used for serosurveillance and will be important to evaluate vaccination status. Given the urgency to release coronavirus disease 2019 (COVID-19) serology tests, most manufacturers have developed qualitative tests. OBJECTIVE.­: To evaluate clinical performance of 6 different SARS-CoV-2 IgG assays and their quantitative results to better elucidate the clinical role of serology testing in COVID-19. DESIGN.­: Six SARS-CoV-2 IgG assays were tested using remnant specimens from 190 patients. Sensitivity and specificity were evaluated for each assay with the current manufacturer's cutoff and a lower cutoff. A numeric result analysis and discrepancy analysis were performed. RESULTS.­: Specificity was higher than 93% for all assays, and sensitivity was higher than 80% for all assays (≥7 days post-polymerase chain reaction testing). Inpatients with more severe disease had higher numeric values compared with health care workers with mild or moderate disease. Several discrepant serology results were those just below the manufacturers' cutoff. CONCLUSIONS.­: Severe acute respiratory syndrome coronavirus 2 IgG antibody testing can aid in the diagnosis of COVID-19, especially with negative polymerase chain reaction. Quantitative COVID-19 IgG results are important to better understand the immunologic response and disease course of this novel virus and to assess immunity as part of future vaccination programs.

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.

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.

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 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.

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 expression of secreted glycoproteins. We show that ExpiCHO-S ™ cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural ( cryo-EM ) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). 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. 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.

A Comparison of Iron Oxide Particles and Silica Particles for Tracking Organ Recellularization.

Reseeding of decellularized organ scaffolds with a patient's own cells has promise for eliminating graft versus host disease. This study investigated whether ultrasound imaging or magnetic resonance imaging (MRI) can track the reseeding of murine liver scaffolds with silica-labeled or iron-labeled liver hepatocytes. Mesoporous silica particles were created using the Stöber method, loaded with Alexa Flour 647 fluorophore, and conjugated with protamine sulfate, glutamine, and glycine. Fluorescent iron oxide particles were obtained from a commercial source. Liver cells from donor mice were loaded with the silica particles or iron oxide particles. Donor livers were decellularized and reperfused with silica-labeled or iron-labeled cells. The reseeded livers were longitudinally analyzed with ultrasound imaging and MRI. Liver biopsies were imaged with confocal microscopy and scanning electron microscopy. Ultrasound imaging had a detection limit of 0.28 mg/mL, while MRI had a lower detection limit of 0.08 mg/mL based on particle weight. The silica-loaded cells proliferated at a slower rate compared to iron-loaded cells. Ultrasound imaging, MRI, and confocal microscopy underestimated cell numbers relative to scanning electron microscopy. Ultrasound imaging had the greatest underestimation due to coarse resolution compared to the other imaging modalities. Despite this underestimation, both ultrasound imaging and MRI successfully tracked the longitudinal recellularization of liver scaffolds.