Prevalence of SARS-CoV-2 Viremia in Presymptomatic Blood Donors in the Delta and Omicron Variant Eras.

Presymptomatic plasma samples from 1596 donors reporting coronavirus disease 2019 infection or symptoms after blood donation were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and anti-S and anti-N antibodies. Prior infection and vaccination both protected from developing SARS-CoV-2 RNAemia and from symptomatic infection. RNAemia rates did not differ in the Delta and Omicron variant eras.

Monitoring SARS-CoV-2 incidence and seroconversion among university students and employees: a longitudinal cohort study in California, June-August 2020.

OBJECTIVES: To identify incident SARS-CoV-2 infections and inform effective mitigation strategies in university settings, we piloted an integrated symptom and exposure monitoring and testing system among a cohort of university students and employees. DESIGN: Prospective cohort study. SETTING: A public university in California from June to August 2020. PARTICIPANTS: 2180 university students and 738 university employees. PRIMARY OUTCOME MEASURES: At baseline and endline, we tested participants for active SARS-CoV-2 infection via quantitative PCR (qPCR) test and collected blood samples for antibody testing. Participants received notifications to complete additional qPCR tests throughout the study if they reported symptoms or exposures in daily surveys or were selected for surveillance testing. Viral whole genome sequencing was performed on positive qPCR samples, and phylogenetic trees were constructed with these genomes and external genomes. RESULTS: Over the study period, 57 students (2.6%) and 3 employees (0.4%) were diagnosed with SARS-CoV-2 infection via qPCR test. Phylogenetic analyses revealed that a super-spreader event among undergraduates in congregate housing accounted for at least 48% of cases among study participants but did not spread beyond campus. Test positivity was higher among participants who self-reported symptoms (incidence rate ratio (IRR) 12.7; 95% CI 7.4 to 21.8) or had household exposures (IRR 10.3; 95% CI 4.8 to 22.0) that triggered notifications to test. Most (91%) participants with newly identified antibodies at endline had been diagnosed with incident infection via qPCR test during the study. CONCLUSIONS: Our findings suggest that integrated monitoring systems can successfully identify and link at-risk students to SARS-CoV-2 testing. As the study took place before the evolution of highly transmissible variants and widespread availability of vaccines and rapid antigen tests, further research is necessary to adapt and evaluate similar systems in the present context.

Frequent detection but lack of infectivity of SARS-CoV-2 RNA in presymptomatic, infected blood donor plasma.

Respiratory viruses such as influenza do not typically cause viremia; however, SARS-CoV-2 has been detected in the blood of COVID-19 patients with mild and severe symptoms. Detection of SARS-CoV-2 in blood raises questions about its role in pathogenesis as well as transfusion safety concerns. Blood donor reports of symptoms or a diagnosis of COVID-19 after donation (post-donation information, PDI) preceded or coincided with increased general population COVID-19 mortality. Plasma samples from 2,250 blood donors who reported possible COVID-19-related PDI were tested for the presence of SARS-CoV-2 RNA. Detection of RNAemia peaked at 9%-15% of PDI donors in late 2020 to early 2021 and fell to approximately 4% after implementation of widespread vaccination in the population. RNAemic donors were 1.2- to 1.4-fold more likely to report cough or shortness of breath and 1.8-fold more likely to report change in taste or smell compared with infected donors without detectable RNAemia. No infectious virus was detected in plasma from RNAemic donors; inoculation of permissive cell lines produced less than 0.7-7 plaque-forming units (PFU)/mL and in susceptible mice less than 100 PFU/mL in RNA-positive plasma based on limits of detection in these models. These findings suggest that blood transfusions are highly unlikely to transmit SARS-CoV-2 infection.

Using sero-epidemiology to monitor disparities in vaccination and infection with SARS-CoV-2.

As SARS-CoV-2 continues to spread and vaccines are rolled-out, the "double burden" of disparities in exposure and vaccination intersect to determine patterns of infection, immunity, and mortality. Serology provides a unique opportunity to measure prior infection and vaccination simultaneously. Leveraging algorithmically-selected residual sera from two hospital networks in the city of San Francisco, cross-sectional samples from 1,014 individuals from February 4-17, 2021 were each tested on two assays (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 and Roche Elecsys Anti-SARS-CoV-2), capturing the first year of the epidemic and early roll-out of vaccination. We estimated, using Bayesian estimation of infection and vaccination, that infection risk of Hispanic/Latinx residents was five times greater than of White residents aged 18-64 (95% Credible Interval (CrI): 3.2-10.3), and that White residents over 65 were twice as likely to be vaccinated as Black/African American residents (95% CrI: 1.1-4.6). We found that socioeconomically-deprived zipcodes had higher infection probabilities and lower vaccination coverage than wealthier zipcodes. While vaccination has created a 'light at the end of the tunnel' for this pandemic, ongoing challenges in achieving and maintaining equity must also be considered.

How do we form and coordinate a national serosurvey of SARS-CoV-2 within the blood collection industry?

BACKGROUND: A national serosurvey of U.S. blood donors conducted in partnership with the Centers for Disease Control and Prevention (CDC) was initiated to estimate the prevalence of SARS-CoV-2 infections and vaccinations. METHODS: Beginning in July 2020, the Nationwide Blood Donor Seroprevalence Study collaborated with multiple blood collection organizations, testing labs, and leadership from government partners to capture, test, and analyze approximately 150,000 blood donation specimens per month in a repeated, cross-sectional seroprevalence survey. RESULTS: A CDC website (https://covid.cdc.gov/covid-data-tracker/#nationwide-blood-donor-seroprevalence) provided stratified, population-level results to public health professionals and the general public. DISCUSSION: The study adapted operations as the pandemic evolved, changing specimen flow and testing algorithms, and collecting additional data elements in response to changing policies on universal blood donation screening and administration of SARS-CoV-2 spike-based vaccines. The national serosurvey demonstrated the utility of serosurveillance testing of residual blood donations and highlighted the role of the blood collection industry in public-private partnerships during a public health emergency.

Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity had no antiviral effects but moderately reduced lung inflammation.

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable but low levels of antiviral antibodies after infusion. In comparison to the control animals, CCP-treated animals had similar levels of viral RNA in upper and lower respiratory tract secretions, similar detection of viral RNA in lung tissues by in situ hybridization, but lower amounts of infectious virus in the lungs. CCP-treated animals had a moderate, but statistically significant reduction in interstitial pneumonia, as measured by comprehensive lung histology. Thus overall, therapeutic benefits of CCP were marginal and inferior to results obtained earlier with monoclonal antibodies in this animal model. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses.

Use of US Blood Donors for National Serosurveillance of Severe Acute Respiratory Syndrome Coronavirus 2 Antibodies: Basis for an Expanded National Donor Serosurveillance Program.

BACKGROUND: The Recipient Epidemiology and Donor Evaluation Study-IV-Pediatric (REDS-IV-P) Epidemiology, Surveillance and Preparedness of the Novel SARS-CoV-2 Epidemic (RESPONSE) seroprevalence study conducted monthly cross-sectional testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in blood donors in 6 US metropolitan regions to estimate the extent of SARS-CoV-2 infections over time. METHODS: During March-August 2020, approximately ≥1000 serum specimens were collected monthly from each region and tested for SARS-CoV-2 antibodies using a well-validated algorithm. Regional seroprevalence estimates were weighted based on demographic differences compared with the general population. Seroprevalence was compared with reported coronavirus disease 2019 (COVID-19) case rates over time. RESULTS: For all regions, seroprevalence was <1.0% in March 2020. New York, New York, experienced the biggest increase (peak seroprevalence, 15.8% in May). All other regions experienced modest increases in seroprevalence (1%-2% in May-June to 2%-4% in July-August). Seroprevalence was higher in younger, non-Hispanic black, and Hispanic donors. Temporal increases in donor seroprevalence correlated with reported case rates in each region. In August, 1.3-5.6 estimated cumulative infections (based on seroprevalence data) per COVID-19 case were reported to the Centers for Disease Control and Prevention. CONCLUSIONS: Increases in seroprevalence were found in all regions, with the largest increase in New York. Seroprevalence was higher in non-Hispanic black and Hispanic than in non-Hispanic white blood donors. SARS-CoV-2 antibody testing of blood donor samples can be used to estimate the seroprevalence in the general population by region and demographic group. The methods derived from the RESPONSE seroprevalence study served as the basis for expanding SARS-CoV-2 seroprevalence surveillance to all 50 states and Puerto Rico.

Evaluation of Commercially Available High-Throughput SARS-CoV-2 Serologic Assays for Serosurveillance and Related Applications.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serosurveys can estimate cumulative incidence for monitoring epidemics, requiring assessment of serologic assays to inform testing algorithm development and interpretation of results. We conducted a multilaboratory evaluation of 21 commercial high-throughput SARS-CoV-2 serologic assays using blinded panels of 1,000 highly characterized specimens. Assays demonstrated a range of sensitivities (96%-63%), specificities (99%-96%), and precision (intraclass correlation coefficient 0.55-0.99). Durability of antibody detection was dependent on antigen and immunoglobulin targets; antispike and total Ig assays demonstrated more stable longitudinal reactivity than antinucleocapsid and IgG assays. Assays with high sensitivity, specificity, and durable antibody detection are ideal for serosurveillance, but assays demonstrating waning reactivity are appropriate for other applications, including correlation with neutralizing activity and detection of anamnestic boosting by reinfections. Assay performance must be evaluated in context of intended use, particularly in the context of widespread vaccination and circulation of SARS-CoV-2 variants.

Vaccination of COVID-19 convalescent plasma donors increases binding and neutralizing antibodies against SARS-CoV-2 variants.

BACKGROUND: COVID-19 convalescent plasma (CCP) was widely used as passive immunotherapy during the first waves of SARS-CoV-2 infection in the US. However, based on observational studies and randomized controlled trials, the beneficial effects of CCP were limited, and its use was virtually discontinued early in 2021, in concurrence with increased vaccination rates and availability of monoclonal antibody (mAb) therapeutics. Yet, as new variants of the SARS-CoV-2 spread, interest in CCP derived from vaccine-boosted CCP donors is resurging. The effect of vaccination of previously infected CCP donors on antibodies against rapidly spreading variants is still under investigation. STUDY DESIGN/METHODS: In this study, paired-samples from 11 CCP donors collected before and after vaccination was tested to measure binding antibody levels and neutralization activity against the ancestral Wuhan-Hu-1 and SARS-CoV-2 variants (Wuhan-Hu-1 with D614G, alpha, beta, gamma, delta, epsilon) on the Ortho Vitros Spike Total Ig and IgG assays, the MSD V-PLEX SARS-CoV-2 arrays for IgG binding and ACE2 inhibition, and variant-specific Spike Reporter Viral Particle Neutralization (RVPN) assays. RESULTS/FINDINGS: Binding and neutralizing antibodies were significantly boosted by vaccination, with several logs higher neutralization for all the variants tested post-vaccination compared to the pre-vaccination samples, with no difference found among the individual variants. DISCUSSION: Vaccination of previously infected individuals boosts antibodies including neutralizing activity against all SARS-CoV-2 variants.

Antibody profiles in COVID-19 convalescent plasma prepared with amotosalen/UVA pathogen reduction treatment.

BACKGROUND: COVID-19 convalescent plasma (CCP), from donors recovered from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, is one of the limited therapeutic options currently available for the treatment of critically ill patients with COVID-19. There is growing evidence that CCP may reduce viral loads and disease severity; and reduce mortality. However, concerns about the risk of transfusion-transmitted infections (TTI) and other complications associated with transfusion of plasma, remain. Amotosalen/UVA pathogen reduction treatment (A/UVA-PRT) of plasma offers a mitigation of TTI risk, and when combined with pooling has the potential to increase the diversity of the polyclonal SARS-CoV-2 neutralizing antibodies. STUDY DESIGN AND METHODS: This study assessed the impact of A/UVA-PRT on SARS-CoV-2 antibodies in 42 CCP using multiple complimentary assays including antigen binding, neutralizing, and epitope microarrays. Other mediators of CCP efficacy were also assessed. RESULTS: A/UVA-PRT did not negatively impact antibodies to SARS-CoV-2 and other viral epitopes, had no impact on neutralizing activity or other potential mediators of CCP efficacy. Finally, immune cross-reactivity with other coronavirus antigens was observed raising the potential for neutralizing activity against other emergent coronaviruses. CONCLUSION: The findings of this study support the selection of effective CCP combined with the use of A/UVA-PRT in the production of CCP for patients with COVID-19.

SARS-CoV-2 Infection of Rhesus Macaques Treated Early with Human COVID-19 Convalescent Plasma.

Human clinical studies investigating use of convalescent plasma (CP) for treatment of coronavirus disease 2019 (COVID-19) have produced conflicting results. Outcomes in these studies may vary at least partly due to different timing of CP administration relative to symptom onset. The mechanisms of action of CP include neutralizing antibodies but may extend beyond virus neutralization to include normalization of blood clotting and dampening of inflammation. Unresolved questions include the minimum therapeutic titer in the CP units or CP recipient as well as the optimal timing of administration. Here, we show that treatment of macaques with CP within 24 h of infection does not reduce viral shedding in nasal or lung secretions compared to controls and does not detectably improve any clinical endpoint. We also demonstrate that CP administration does not impact viral sequence diversity in vivo, although the selection of a viral sequence variant in both macaques receiving normal human plasma was suggestive of immune pressure. Our results suggest that CP, administered to medium titers, has limited efficacy, even when given very early after infection. Our findings also contribute information important for the continued development of the nonhuman primate model of COVID-19. These results should inform interpretation of clinical studies of CP in addition to providing insights useful for developing other passive immunotherapies and vaccine strategies. IMPORTANCE Antiviral treatment options for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain very limited. One treatment that was explored beginning early in the pandemic (and that is likely to be tested early in future pandemics) is plasma collected from people who have recovered from coronavirus disease 2019 (COVID-19), known as convalescent plasma (CP). We tested if CP reduces viral shedding or disease in a nonhuman primate model. Our results demonstrate that administration of CP 1 day after SARS-CoV-2 infection had no significant impact on viral loads, clinical disease, or sequence diversity, although treatment with normal human plasma resulted in selection of a specific viral variant. Our results demonstrate that passive immunization with CP, even during early infection, provided no significant benefit in a nonhuman primate model of SARS-CoV-2 infection.

Estimated US Infection- and Vaccine-Induced SARS-CoV-2 Seroprevalence Based on Blood Donations, July 2020-May 2021.

Importance: People who have been infected with or vaccinated against SARS-CoV-2 have reduced risk of subsequent infection, but the proportion of people in the US with SARS-CoV-2 antibodies from infection or vaccination is uncertain. Objective: To estimate trends in SARS-CoV-2 seroprevalence related to infection and vaccination in the US population. Design, Setting, and Participants: In a repeated cross-sectional study conducted each month during July 2020 through May 2021, 17 blood collection organizations with blood donations from all 50 US states; Washington, DC; and Puerto Rico were organized into 66 study-specific regions, representing a catchment of 74% of the US population. For each study region, specimens from a median of approximately 2000 blood donors were selected and tested each month; a total of 1 594 363 specimens were initially selected and tested. The final date of blood donation collection was May 31, 2021. Exposure: Calendar time. Main Outcomes and Measures: Proportion of persons with detectable SARS-CoV-2 spike and nucleocapsid antibodies. Seroprevalence was weighted for demographic differences between the blood donor sample and general population. Infection-induced seroprevalence was defined as the prevalence of the population with both spike and nucleocapsid antibodies. Combined infection- and vaccination-induced seroprevalence was defined as the prevalence of the population with spike antibodies. The seroprevalence estimates were compared with cumulative COVID-19 case report incidence rates. Results: Among 1 443 519 specimens included, 733 052 (50.8%) were from women, 174 842 (12.1%) were from persons aged 16 to 29 years, 292 258 (20.2%) were from persons aged 65 years and older, 36 654 (2.5%) were from non-Hispanic Black persons, and 88 773 (6.1%) were from Hispanic persons. The overall infection-induced SARS-CoV-2 seroprevalence estimate increased from 3.5% (95% CI, 3.2%-3.8%) in July 2020 to 20.2% (95% CI, 19.9%-20.6%) in May 2021; the combined infection- and vaccination-induced seroprevalence estimate in May 2021 was 83.3% (95% CI, 82.9%-83.7%). By May 2021, 2.1 SARS-CoV-2 infections (95% CI, 2.0-2.1) per reported COVID-19 case were estimated to have occurred. Conclusions and Relevance: Based on a sample of blood donations in the US from July 2020 through May 2021, vaccine- and infection-induced SARS-CoV-2 seroprevalence increased over time and varied by age, race and ethnicity, and geographic region. Despite weighting to adjust for demographic differences, these findings from a national sample of blood donors may not be representative of the entire US population.

Emergence of Multiple SARS-CoV-2 Antibody Escape Variants in an Immunocompromised Host Undergoing Convalescent Plasma Treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), harboring spike protein N-terminal domain (NTD) or receptor-binding domain (RBD) mutations, exhibit reduced in vitro susceptibility to convalescent-phase serum, commercial antibody cocktails, and vaccine neutralization and have been associated with reinfections. The accumulation of these mutations could be the consequence of intrahost viral evolution due to prolonged infection in immunocompromised hosts. In this study, we document the microevolution of SARS-CoV-2 recovered from sequential tracheal aspirates from an immunosuppressed patient on steroids and convalescent plasma therapy and identify the emergence of multiple NTD and RBD mutations. SARS-CoV-2 genomes from the first swab (day 0) and from three tracheal aspirates (days 7, 21, and 27) were compared at the sequence level. We identified a mixed viral population with five different S protein mutations (141 to 144 deletion, 243 to 244 deletion, E484K, Q493K, and Q493R) at the NTD or RBD region from the second tracheal aspirate sample (day 21) and a predominance of the S protein 141 to 144 LGVY deletion and E484K mutant on day 27. The neutralizing antibodies against various S protein lentiviral pseudovirus mutants, as well as the anti-SARS-CoV-2 total Ig and IgG, showed "U" shape dynamics, in support of the endogenous development of neutralizing antibodies. The patient's compromised immune status, the antirejection regiment, convalescent plasma treatment, and the development of neutralizing antibodies may have resulted in unique selective pressures on the intrahost genomic evolution, and this observation supports the hypotheses that VOCs can independently arise and that immunocompromised patients on convalescent plasma therapy are potential breeding grounds for immune escape mutants. IMPORTANCE Over a year of the COVID-19 pandemic, distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages have arisen in multiple geographic areas around the world. SARS-CoV-2 variants of concern (VOCs), i.e., B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), and B.1.617.2 (delta), harboring mutations and/or deletions in spike protein N-terminal domain (NTD) or receptor-binding domain (RBD) regions showed evidence of increased transmissibility and disease severity and possible reduced vaccine efficacy. In this study, we report the emergence of five different NTD and RBD mutations in an uncommon SARS-CoV-2 B.1.369 lineage from an immunosuppressed patient undergoing steroid and convalescent plasma therapy. The observation highlighted that VOCs can independently arise in immunocompromised populations undergoing anti-SARS-CoV-2 therapy, and enhanced measures will be required to reduce the transmission.

Characteristics of High-Titer Convalescent Plasma and Antibody Dynamics After Administration in Patients With Severe Coronavirus Disease 2019.

We characterized the antibody composition of coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) and the immunologic responses of hospitalized COVID-19 patients after receiving CCP or nonimmune fresh frozen plasma. Despite selection of CCP with significantly higher total immunoglobulin G than recipients, neutralizing antibody levels did not differ between donor plasma and CCP recipients.

SARS-CoV-2 antibody persistence in COVID-19 convalescent plasma donors: Dependency on assay format and applicability to serosurveillance.

BACKGROUND: Antibody response duration following severe acute respiratory syndrome coronavirus 2 infection tends to be variable and depends on severity of disease and method of detection. STUDY DESIGN AND METHODS: COVID-19 convalescent plasma from 18 donors was collected longitudinally for a maximum of 63-129 days following resolution of symptoms. All the samples were initially screened by the Ortho total Ig test to confirm positivity and subsequently tested with seven additional direct sandwich or indirect binding assays (Ortho, Roche, Abbott, Broad Institute) directed against a variety of antigen targets (S1, receptor binding domain, and nucleocapsid [NC]), along with two neutralization assays (Broad Institute live virus PRNT and Vitalant Research Institute [VRI] Pseudovirus reporter viral particle neutralization [RVPN]). RESULTS: The direct detection assays (Ortho total Ig total and Roche total Ig) showed increasing levels of antibodies over the time period, in contrast to the indirect IgG assays that showed a decline. Neutralization assays also demonstrated declining responses; the VRI RVPN pseudovirus had a greater rate of decline than the Broad PRNT live virus assay. DISCUSSION: These data show that in addition to variable individual responses and associations with disease severity, the detection assay chosen contributes to the heterogeneous results in antibody stability over time. Depending on the scope of the research, one assay may be preferable over another. For serosurveillance studies, direct, double Ag-sandwich assays appear to be the best choice due to their stability; in particular, algorithms that include both S1- and NC-based assays can help reduce the rate of false-positivity and discriminate between natural infection and vaccine-derived seroreactivity.

Minipool testing for SARS-CoV-2 RNA in United States blood donors.

BACKGROUND: SARS-CoV-2 RNA prevalence in blood donors from large geographic areas of high community transmission is limited. We tested residual donor plasma minipools (MPs) to determine SARS-CoV-2 RNAemia prevalence in six United States areas. STUDY DESIGN/METHODS: Blood donations collected from 7 March 2020 to 25 September 2020 were tested for SARS-CoV-2 RNA (vRNA) in MP of 6 or 16 donations using the Grifols Procleix SARS-CoV-2 research-use only (RUO) transcription-mediated amplification (TMA) assay. Reactive results were confirmed using an alternate target region TMA assay. Reactive MPs were tested by TMA after serial dilution to estimate viral load. Testing for anti-SARS-CoV-2 antibodies and infectivity was performed. RESULTS: A total of 17,995 MPs corresponding to approximately 258,000 donations were tested for vRNA. Three confirmed reactive MP16 were identified. The estimated prevalence of vRNA reactive donations was 1.16/100,000 (95% CI 0.40, 3.42). The vRNA-reactive samples were non-reactive for antibody, and the estimated viral loads of the (presumed single) positive donations within each MP ranged from <1000 to <4000 copies/ml. When tested, no infectivity was observed in inoculated permissive cell cultures. DISCUSSION: Blood donation MP-nucleic acid testing (NAT) indicated that SARS-CoV-2 RNAemia is infrequent and, when detected, the vRNA was at low concentrations. Only one RNA-reactive MP could be tested for infectivity for operational reasons and was not infectious in cell culture. These findings support current recommendations from international and national regulatory agencies to not screen donors by NAT.

Distinct SARS-CoV-2 antibody reactivity patterns in coronavirus convalescent plasma revealed by a coronavirus antigen microarray.

A coronavirus antigen microarray (COVAM) was constructed containing 11 SARS-CoV-2, 5 SARS-1, 5 MERS, and 12 seasonal coronavirus recombinant proteins. The array is designed to measure immunoglobulin isotype and subtype levels in serum or plasma samples against each of the individual antigens printed on the array. We probed the COVAM with COVID-19 convalescent plasma (CCP) collected from 99 donors who recovered from a PCR+ confirmed SARS-CoV-2 infection. The results were analyzed using two computational approaches, a generalized linear model (glm) and random forest (RF) prediction model, to classify individual specimens as either Reactive or non-reactive against the SARS-CoV-2 antigens. A training set of 88 pre-COVID-19 specimens (PreCoV) collected in August 2019 and102 positive specimens from SARS-CoV-2 PCR+ confirmed COVID-19 cases was used for these analyses. Results compared with an FDA emergency use authorized (EUA) SARS-CoV2 S1-based total Ig chemiluminescence immunoassay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and with a SARS-CoV-2 S1-S2 spike-based pseudovirus micro neutralization assay (SARS-CoV-2 reporter viral particle neutralization titration (RVPNT) showed high concordance between the three assays. Three CCP specimens that were negative by the VITROS CoV2T immunoassay were also negative by both COVAM and the RVPNT assay. Concordance between VITROS CoV2T and COVAM was 96%, VITROS CoV2T and RVPNT 93%, and RVPNT and COVAM 91%. The discordances were all weakly reactive samples near the cutoff threshold of the VITROS CoV2T immunoassay. The multiplex COVAM allows CCP to be grouped according to antibody reactivity patterns against 11 SARS-CoV-2 antigens. Unsupervised K-means analysis, via the gap statistics, as well as hierarchical clustering analysis revealed three main clusters with distinct reactivity intensities and patterns. These patterns were not recapitulated by adjusting the VITROS CoV2T or RVPNT assay thresholds. Plasma classified by COVAM reactivity patterns offers potential to improve CCP therapeutic efficacy CoV2T alone. The use of a SARS-CoV-2 antigen array can qualify CCP for administration as a treatment for acute COVID-19, and interrogate vaccine immunogenicity and performance in preclinical, clinical studies, and routine vaccination to identify antibody responses predictive of protection from infection and disease.

Selecting COVID-19 convalescent plasma for neutralizing antibody potency using a high-capacity SARS-CoV-2 antibody assay.

BACKGROUND: Efficacy of COVID-19 convalescent plasma (CCP) is hypothesized to be associated with the concentration of neutralizing antibodies (nAb) to SARS-CoV-2. High capacity serologic assays detecting binding antibodies (bAb) have been developed; nAb assays are not adaptable to high-throughput testing. We sought to determine the effectiveness of using surrogate bAb signal-to-cutoff ratios (S/Co) in predicting nAb titers using a pseudovirus reporter viral particle neutralization (RVPN) assay. METHODS: CCP donor serum collected by three US blood collectors was tested with a bAb assay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and a nAb RVPN assay. Prediction effectiveness of various CoV2T S/Co criteria was evaluated for RVPN nAb NT50 titers using receiver operating characteristics. RESULTS: Seven hundred and fifty-three CCPs were tested with median CoV2T S/Co and NT50 of 71.2 of 527.5. Proportions of donors with NT50 over target nAb titers were 86% ≥1:80, 76% ≥1:160, and 62% ≥1:320. Increasing CoV2T S/Co criterion reduced the sensitivity to predict NT50 titers, while specificity to identify those below increased. As target NT50 titers increase, the CoV2T assay becomes less accurate as a predictor with a decline in positive predictive value and rise in negative predictive value. CONCLUSION: Selection of a clinically effective nAb titer will impact availability of CCP. Product release with CoV2T assay S/Co criterion must balance the risk of releasing products below target nAb titers with the cost of false negatives. A two-step testing scheme may be optimal, with nAb testing on CoV2T samples with S/Cos below criterion.