Development and Characterization of a Quantitative ELISA to Detect Anti-SARS-CoV-2 Spike Antibodies (preprint)

A novel clinical assay for the detection and quantitation of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was adapted from an in-house, research-based enzyme-linked immunosorbent assay (ELISA). Development and validation were performed under regulatory guidelines, and the test obtained emergency use authorization (EUA) from the New York State Department of Health (NYSDOH) and the Food and Drug Administration (FDA). The Mount Sinai COVID-19 antibody assay is an orthogonal, quantitative direct ELISA test which detects antibodies reactive to the receptor binding domain (RBD) and the spike protein of the novel SARS-CoV-2. The assay is performed on 96-well plates coated with either SARS-CoV-2 recombinant RBD or spike proteins. The test is divided into two stages, a qualitative screening assay against RBD and a quantitative assay against the full-length spike protein. The test uses pooled high titer serum as a reference standard. Negative pre-COVID-19 and positive post-COVID-19, PCR-confirmed specimens were incorporated in each ELISA test run, and the assays were performed independently at two different locations. The Mount Sinai COVID-19 serology performed with high sensitivity and specificity, 92.5% (95% CI: 0.785 – 0.980) and 100% (CI: 0.939 – 1.000) respectively. Between-run precision was assessed with a single run repeated over 22 days; and within-run precision was assessed with 10 replicates per day over 22 days. Both were within reported acceptance criteria (CV ≤20%). This population-based study reveals the applicability and reliability of this novel orthogonal COVID-19 serology test for the detection and quantitation of antibodies against SARS-CoV-2, allowing a broad set of clinical applications, including the broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different population subsets.Funding: This work was partially supported by the JPB foundation, the Open Philanthropy Project (#2020-215611) and other philanthropic donations.Declaration of Interests: Mount Sinai has licensed serological assays to commercial entities and has filed for patent protection for serological assays. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to the COVID-19 serological assay (“Serology Assay”) and NDV-based SARS-CoV-2 vaccines which list Florian Krammer (“Serology Assay”, vaccines), Daniel Stadlbauer (“Serology Assay”), Damodara Rao Mendu (“Serology Assay”), and Carlos Cordon-Cardo (“Serology Assay”) as co-inventors. The foundational “Serology Assay” intellectual property (IP) was licensed by the Icahn School of Medicine at Mount Sinai to commercial entities including Kantaro Biosciences, a company in which Mount Sinai has a financial interest. Florian Krammer consulted for Merck, Curevac, and Pfizer in the past (before 2020) and is currently consulting for Pfizer, Seqirus, and Avimex. The Krammer laboratory is collaborating with Pfizer on animal models of SARS-CoV-2.Patient Informed Consent Statement: Informed consent was obtained from all individuals included in this study.

Safety of administering biologics to IBD patients at an outpatient infusion center In New York City during the COVID-19 pandemic: Sars-CoV-2 seroprevalence and clinical and social characteristics (preprint)

Patients with immune-mediated inflammatory diseases (IMIDs) and acquired and genetic immunodeficiencies receiving therapeutic infusions are considered high risk for SARS-CoV-2 infection. However, the seroprevalance in this group and the safety of routine administrations at outpatient infusion centers are unknown. To determine the infection rate and clinical-social factors related to SARS-CoV-2 in asymptomatic patients with IMIDs and immunodeficiencies receiving routine non-cancer therapeutic infusions, we conducted a seroprevalence study at our outpatient infusion center. We report the first prospective SARS-CoV-2 sero-surveillance of 444 IBD/IMID, immunodeficiency, and immune competent patients at an outpatient infusion center in the U.S. showing lower seroprevalence in patients compared with the general population and provide clinical and social characteristics associated with seroprevalence in this group. These data suggest that patients can safely continue infusions at outpatient centers.

Longitudinal analysis of SARS-CoV-2 seroprevalence using multiple serology platforms (preprint)

Serological tests are important tools helping to determine previous infection with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) and to monitor immune responses. The current tests are based on spike (S), the receptor binding domain (RBD), or the nucleoprotein (NP) as substrate. Here, we used samples from a high seroprevalence cohort of health care workers (HCWs) to perform a longitudinal analysis of the antibody responses using three distinct serological assays. 501 serum samples were tested using: a) a research-grade RBD and spike based tandem enzyme-linked immunosorbent assay (MS-RBD ELISA, MS-spike ELISA), b) a commercial RBD and spike based tandem ELISA (Kantaro-RBD, -spike), and c) a commercial NP-based chemiluminescent microparticle immunoassay (CMIA, Abbott Architect). Seroprevalence ranged around 28% during the early stage of the pandemic (a: 28.4% positives; b: 28.1%; c: 27.3%). Good correlation was observed between the MS and Kantaro RBD ELISAs and between the MS and Kantaro spike ELISAs. By contrast, modest correlations were observed between the Abbott Architect and both RBD and spike-based assays. A proportion of HCWs (n=178) were sampled again 3-5 months after the first time point. Although antibody levels declined in most of the positive individuals, the overall seroprevalence measured by RBD-spike based assays remained unchanged. However the seroprevalence of NP-reactive antibodies significantly declined. Lastly, we tested six samples of individuals who received two doses of SARS-CoV-2 mRNA vaccine and found that seroconversion was detected by the RBD-spike assays but, of course as expected, not the NP based assay. In summary, our results consolidate the strength of different serological assays to assess the magnitude and duration of antibodies to SARS-CoV-2.

SARS-CoV-2 infection induces robust, neutralizing antibody responses that are stable for at least three months (preprint)

SARS-CoV-2 has caused a global pandemic with millions infected and numerous fatalities. Questions regarding the robustness, functionality and longevity of the antibody response to the virus remain unanswered. Here we report that the vast majority of infected individuals with mild-to-moderate COVID-19 experience robust IgG antibody responses against the viral spike protein, based on a dataset of 19,860 individuals screened at Mount Sinai Health System in New York City. We also show that titers are stable for at least a period approximating three months, and that anti-spike binding titers significantly correlate with neutralization of authentic SARS-CoV-2. Our data suggests that more than 90% of seroconverters make detectible neutralizing antibody responses and that these titers are stable for at least the near-term future.

Neutralizing Antibody Responses in COVID-19 Convalescent Sera (preprint)

Passive transfer of antibodies from COVID-19 convalescent patients is being used as an experimental treatment for eligible patients with SARS-CoV-2 infections. The United States Food and Drug Administrations (FDA) guidelines for convalescent plasma recommends target antibody titers of 160. We evaluated SARS-CoV-2 neutralizing antibodies in sera from recovered COVID-19 patients using plaque reduction neutralization tests (PRNT) at low (PRNT50) and high (PRNT90) stringency thresholds. We found that neutralizing activity increased with time post symptom onset (PSO), reaching a peak at 31-35 days PSO. At this point, the number of sera having neutralizing titers of at least 160 was [~]93% (PRNT50) and [~]54% (PRNT90). Sera with high SARS-CoV-2 antibody levels (>960 ELISA titers) showed maximal activity, but not all high titer sera contained neutralizing antibody at FDA recommended levels, particularly at high stringency. These results underscore the value of serum characterization for neutralization activity.

An inflammatory cytokine signature helps predict COVID-19 severity and death (preprint)

The COVID-19 pandemic caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to more than 100,000 deaths in the United States. Several studies have revealed that the hyper-inflammatory response induced by SARS-CoV-2 is a major cause of disease severity and death in infected patients. However, predictive biomarkers of pathogenic inflammation to help guide targetable immune pathways are critically lacking. We implemented a rapid multiplex cytokine assay to measure serum IL-6, IL-8, TNF-, and IL-1{beta} in hospitalized COVID-19 patients upon admission to the Mount Sinai Health System in New York. Patients (n=1484) were followed up to 41 days (median 8 days) and clinical information, laboratory test results and patient outcomes were collected. In 244 patients, cytokine measurements were repeated over time, and effect of drugs could be assessed. Kaplan-Meier methods were used to compare survival by cytokine strata, followed by Cox regression models to evaluate the independent predictive value of baseline cytokines. We found that high serum IL-6, IL-8, and TNF- levels at the time of hospitalization were strong and independent predictors of patient survival. Importantly, when adjusting for disease severity score, common laboratory inflammation markers, hypoxia and other vitals, demographics, and a range of comorbidities, IL-6 and TNF- serum levels remained independent and significant predictors of disease severity and death. We propose that serum IL-6 and TNF- levels should be considered in the management and treatment of COVID-19 patients to stratify prospective clinical trials, guide resource allocation and inform therapeutic options. We also propose that patients with high IL-6 and TNF- levels should be assessed for combinatorial blockade of pathogenic inflammation in this disease.

Convalescent plasma treatment of severe COVID-19: A matched control study (preprint)

Background Since December 2019, Coronavirus Disease 2019 (COVID-19) has become a global pandemic, causing mass morbidity and mortality. Prior studies in other respiratory infections suggest that convalescent plasma transfusion may offer benefit to some patients. Here, the outcomes of thirty-nine hospitalized patients with severe to life-threatening COVID-19 who received convalescent plasma transfusion were compared against a cohort of retrospectively matched controls. Methods Plasma recipients were selected based on supplemental oxygen needs at the time of enrollment and the time elapsed since the onset of symptoms. Recipients were transfused with convalescent plasma from donors with a SARS-CoV-2 (severe acute respiratory disease coronavirus 2) anti-spike antibody titer of [≥]1:320 dilution. Matched control patients were retrospectively identified within the electronic health record database. Supplemental oxygen requirements and survival were compared between plasma recipients and controls. Results Convalescent plasma recipients were more likely than control patients to remain the same or have improvements in their supplemental oxygen requirements by post-transfusion day 14, with an odds ratio of 0.86 (95% CI: 0.75~0.98; p=0.028). Plasma recipients also demonstrated improved survival, compared to control patients (log-rank test: p=0.039). In a covariates-adjusted Cox model, convalescent plasma transfusion improved survival for non-intubated patients (hazard ratio 0.19 (95% CI: 0.05 ~0.72); p=0.015), but not for intubated patients (1.24 (0.33~4.67); p=0.752). Conclusions Convalescent plasma transfusion is a potentially efficacious treatment option for patients hospitalized with COVID-19; however, these data suggest that non-intubated patients may benefit more than those requiring mechanical ventilation.