A Saliva-Based Serological and Behavioral Analysis of SARS-CoV-2 Antibody Prevalence in Howard County, Maryland.

The objective of the study was to estimate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence in the Howard County, Maryland, general population and demographic subpopulations attributable to natural infection or coronavirus disease 2019 (COVID-19) vaccination and to identify self-reported social behaviors that may affect the likelihood of recent or past SARS-CoV-2 infection. A cross-sectional, saliva-based serological study of 2,880 residents of Howard County, Maryland, was carried out from July through September 2021. Natural SARS-CoV-2 infection prevalence was estimated by inferring infections among individuals according to anti-nucleocapsid immunoglobin G levels and calculating averages weighted by sample proportions of various demographics. Antibody levels between BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) recipients were compared. Antibody decay rate was calculated by fitting exponential decay curves to cross-sectional indirect immunoassay data. Regression analysis was carried out to identify demographic factors, social behaviors, and attitudes that may be linked to an increased likelihood of natural infection. The estimated overall prevalence of natural infection in Howard County, Maryland, was 11.9% (95% confidence interval, 9.2% to 15.1%), compared with 7% reported COVID-19 cases. Antibody prevalence indicating natural infection was highest among Hispanic and non-Hispanic Black participants and lowest among non-Hispanic White and non-Hispanic Asian participants. Participants from census tracts with lower average household income also had higher natural infection rates. After accounting for multiple comparisons and correlations between participants, none of the behavior or attitude factors had significant effects on natural infection. At the same time, recipients of the mRNA-1273 vaccine had higher antibody levels than those of BNT162b2 vaccine recipients. Older study participants had overall lower antibody levels compared with younger study participants. The true prevalence of SARS-CoV-2 infection is higher than the number of reported COVID-19 cases in Howard County, Maryland. A disproportionate impact of infection-induced SARS-CoV-2 positivity was observed across different ethnic/racial subpopulations and incomes, and differences in antibody levels across different demographics were identified. Taken together, this information may inform public health policy to protect vulnerable populations. IMPORTANCE We employed a highly innovative noninvasive multiplex oral fluid SARS-CoV-2 IgG assay to ascertain our seroprevalence estimates. This laboratory-developed test has been applied in NCI's SeroNet consortium, possesses high sensitivity and specificity according to FDA Emergency Use Authorization guidelines, correlates strongly with SARS-CoV-2 neutralizing antibody responses, and is Clinical Laboratory Improvement Amendments-approved by the Johns Hopkins Hospital Department of Pathology. It represents a broadly scalable public health tool to improve understanding of recent and past SARS-CoV-2 exposure and infection without drawing any blood. To our knowledge, this is the first application of a high-performance salivary SARS-CoV-2 IgG assay to estimate population-level seroprevalence, including identifying COVID-19 disparities. We also are the first to report differences in SARS-CoV-2 IgG responses by COVID-19 vaccine manufacturers (BNT162b2 [Pfizer-BioNTech] and mRNA-1273 [Moderna]). Our findings demonstrate remarkable consistency with those of blood-based SARS-CoV-2 IgG assays in terms of differences in the magnitude of SARS-CoV-2 IgG responses between COVID-19 vaccines.

Persistent SARS-CoV-2-specific immune defects in kidney transplant recipients following third mRNA vaccine dose.

Kidney transplant recipients (KTRs) show poorer response to SARS-CoV-2 mRNA vaccination, yet response patterns and mechanistic drivers following third doses are ill-defined. We administered third monovalent mRNA vaccines to n = 81 KTRs with negative or low-titer anti-receptor binding domain (RBD) antibody (n = 39 anti-RBDNEG; n = 42 anti-RBDLO), compared with healthy controls (HCs, n = 19), measuring anti-RBD, Omicron neutralization, spike-specific CD8+%, and SARS-CoV-2-reactive T cell receptor (TCR) repertoires. By day 30, 44% anti-RBDNEG remained seronegative; 5% KTRs developed BA.5 neutralization (vs 68% HCs, P < .001). Day 30 spike-specific CD8+% was negative in 91% KTRs (vs 20% HCs; P = .07), without correlation to anti-RBD (rs = 0.17). Day 30 SARS-CoV-2-reactive TCR repertoires were detected in 52% KTRs vs 74% HCs (P = .11). Spike-specific CD4+ TCR expansion was similar between KTRs and HCs, yet KTR CD8+ TCR depth was 7.6-fold lower (P = .001). Global negative response was seen in 7% KTRs, associated with high-dose MMF (P = .037); 44% showed global positive response. Of the KTRs, 16% experienced breakthrough infections, with 2 hospitalizations; prebreakthrough variant neutralization was poor. Absent neutralizing and CD8+ responses in KTRs indicate vulnerability to COVID-19 despite 3-dose mRNA vaccination. Lack of neutralization despite CD4+ expansion suggests B cell dysfunction and/or ineffective T cell help. Development of more effective KTR vaccine strategies is critical. (NCT04969263).

Methodological approaches to optimize multiplex oral fluid SARS-CoV-2 IgG assay performance and correlation with serologic and neutralizing antibody responses.

BACKGROUND: Oral fluid (hereafter, saliva) is a non-invasive and attractive alternative to blood for SARS-CoV-2 IgG testing; however, the heterogeneity of saliva as a matrix poses challenges for immunoassay performance. OBJECTIVES: To optimize performance of a magnetic microparticle-based multiplex immunoassay (MIA) for SARS-CoV-2 IgG measurement in saliva, with consideration of: i) threshold setting and validation across different MIA bead batches; ii) sample qualification based on salivary total IgG concentration; iii) calibration to U.S. SARS-CoV-2 serological standard binding antibody units (BAU); and iv) correlations with blood-based SARS-CoV-2 serological and neutralizing antibody (nAb) assays. METHODS: The salivary SARS-CoV-2 IgG MIA included 2 nucleocapsid (N), 3 receptor-binding domain (RBD), and 2 spike protein (S) antigens. Gingival crevicular fluid (GCF) swab saliva samples were collected before December 2019 (n = 555) and after molecular test-confirmed SARS-CoV-2 infection from 113 individuals (providing up to 5 repeated-measures; n = 398) and used to optimize and validate MIA performance (total n = 953). Combinations of IgG responses to N, RBD and S and total salivary IgG concentration (µg/mL) as a qualifier of nonreactive samples were optimized and validated, calibrated to the U.S. SARS-CoV-2 serological standard, and correlated with blood-based SARS-CoV-2 IgG ELISA and nAb assays. RESULTS: The sum of signal to cutoff (S/Co) to all seven MIA SARS-CoV-2 antigens and disqualification of nonreactive saliva samples with ≤15 µg/mL total IgG led to correct classification of 62/62 positives (sensitivity [Se] = 100.0%; 95% confidence interval [CI] = 94.8%, 100.0%) and 108/109 negatives (specificity [Sp] = 99.1%; 95% CI = 97.3%, 100.0%) at 8-million beads coupling scale and 80/81 positives (Se = 98.8%; 95% CI = 93.3%, 100.0%] and 127/127 negatives (Sp = 100%; 95% CI = 97.1%, 100.0%) at 20-million beads coupling scale. Salivary SARS-CoV-2 IgG crossed the MIA cutoff of 0.1 BAU/mL on average 9 days post-COVID-19 symptom onset and peaked around day 30. Among n = 30 matched saliva and plasma samples, salivary SARS-CoV-2 MIA IgG levels correlated with corresponding-antigen plasma ELISA IgG (N: ρ = 0.76, RBD: ρ = 0.83, S: ρ = 0.82; all p < 0.001). Correlations of plasma SARS-CoV-2 nAb assay area under the curve (AUC) with salivary MIA IgG (N: ρ = 0.68, RBD: ρ = 0.78, S: ρ = 0.79; all p < 0.001) and with plasma ELISA IgG (N: ρ = 0.76, RBD: ρ = 0.79, S: ρ = 0.76; p < 0.001) were similar. CONCLUSIONS: A salivary SARS-CoV-2 IgG MIA produced consistently high Se (> 98.8%) and Sp (> 99.1%) across two bead coupling scales and correlations with nAb responses that were similar to blood-based SARS-CoV-2 IgG ELISA data. This non-invasive salivary SARS-CoV-2 IgG MIA could increase engagement of vulnerable populations and improve broad understanding of humoral immunity (kinetics and gaps) within the evolving context of booster vaccination, viral variants and waning immunity.

Neutralizing activity and 3-month durability of tixagevimab and cilgavimab prophylaxis against Omicron sublineages in transplant recipients.

Neutralizing antibody (nAb) responses are attenuated in solid organ transplant recipients (SOTRs) despite severe acute respiratory syndrome-coronavirus-2 vaccination. Preexposure prophylaxis (PrEP) with the antibody combination tixagevimab and cilgavimab (T+C) might augment immunoprotection, yet in vitro activity and durability against Omicron sublineages BA.4/5 in fully vaccinated SOTRs have not been delineated. Vaccinated SOTRs, who received 300 + 300 mg T+C (ie, full dose), within a prospective observational cohort submitted pre and postinjection samples between January 31, 2022, and July 6, 2022. The peak live virus nAb was measured against Omicron sublineages (BA.1, BA.2, BA.2.12.1, and BA.4), and surrogate neutralization (percent inhibition of angiotensin-converting enzyme 2 receptor binding to full length spike, validated vs live virus) was measured out to 3 months against sublineages, including BA.4/5. With live virus testing, the proportion of SOTRs with any nAb increased against BA.2 (47%-100%; P < .01), BA.2.12.1 (27%-80%; P < .01), and BA.4 (27%-93%; P < .01), but not against BA.1 (40%-33%; P = .6). The proportion of SOTRs with surrogate neutralizing inhibition against BA.5, however, fell to 15% by 3 months. Two participants developed mild severe acute respiratory syndrome-coronavirus-2 infection during follow-up. The majority of fully vaccinated SOTRs receiving T+C PrEP achieved BA.4/5 neutralization, yet nAb activity commonly waned by 3 months postinjection. It is critical to assess the optimal dose and interval of T+C PrEP to maximize protection in a changing variant climate.

SARS-CoV-2 Antibody Prevalence among Industrial Livestock Operation Workers and Nearby Community Residents, North Carolina, 2021 to 2022.

Industrial livestock operations (ILOs), particularly processing facilities, emerged as centers of coronavirus disease 2019 (COVID-19) outbreaks in spring 2020. Confirmed cases of COVID-19 underestimate true prevalence. To investigate the prevalence of antibodies against SARS-CoV-2, we enrolled 279 participants in North Carolina from February 2021 to July 2022: 90 from households with at least one ILO worker (ILO), 97 from high-ILO intensity areas (ILO neighbors [ILON]), and 92 from metropolitan areas (metro). More metro (55.4%) compared to ILO (51.6%) and ILON participants (48.4%) completed the COVID-19 primary vaccination series; the median completion date was more than 4 months later for ILO compared to ILON and metro participants, although neither difference was statistically significant. Participants provided a saliva swab we analyzed for SARS-CoV-2 IgG using a multiplex immunoassay. The prevalence of infection-induced IgG (positive for nucleocapsid and receptor binding domain) was higher among ILO (63%) than ILON (42.9%) and metro (48.7%) participants (prevalence ratio [PR], 1.38; 95% confidence interval [CI], 1.06 to 1.80; reference category ILON and metro combined). The prevalence of infection-induced IgG was also higher among ILO participants than among an Atlanta health care worker cohort (PR, 2.45; 95% CI, 1.80 to 3.33) and a general population cohort in North Carolina (PRs, 6.37 to 10.67). The infection-induced IgG prevalence increased over the study period. Participants reporting not masking in public in the past 2 weeks had higher infection-induced IgG prevalence (78.6%) than participants reporting masking (49.3%) (PR, 1.59; 95% CI, 1.19 to 2.13). Lower education, more people per bedroom, Hispanic/Latino ethnicity, and more contact with people outside the home were also associated with higher infection-induced IgG prevalence. IMPORTANCE Few studies have measured COVID-19 seroprevalence in North Carolina, especially among rural, Black, and Hispanic/Latino communities that have been heavily affected. Antibody results show high rates of COVID-19 among industrial livestock operation workers and their household members. Antibody results add to evidence of health disparities related to COVID-19 by socioeconomic status and ethnicity. Associations between masking and physical distancing with antibody results also add to evidence of the effectiveness of these prevention strategies. Delays in the timing of receipt of COVID-19 vaccination reinforce the importance of dismantling vaccination barriers, especially for industrial livestock operation workers and their household members.

IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function.

BackgroundSome clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.MethodsIn an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.ResultsAnti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38-42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6-21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.ConclusionWe identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDINGBill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891).

Prognostic Significance of Urinary Biomarkers in Patients Hospitalized With COVID-19.

RATIONALE & OBJECTIVE: Acute kidney injury (AKI) is common in patients with coronavirus disease 2019 (COVID-19) and associated with poor outcomes. Urinary biomarkers have been associated with adverse kidney outcomes in other settings and may provide additional prognostic information in patients with COVID-19. We investigated the association between urinary biomarkers and adverse kidney outcomes among patients hospitalized with COVID-19. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: Patients hospitalized with COVID-19 (n=153) at 2 academic medical centers between April and June 2020. EXPOSURE: 19 urinary biomarkers of injury, inflammation, and repair. OUTCOME: Composite of KDIGO (Kidney Disease: Improving Global Outcomes) stage 3 AKI, requirement for dialysis, or death within 60 days of hospital admission. We also compared various kidney biomarker levels in the setting of COVID-19 versus other common AKI settings. ANALYTICAL APPROACH: Time-varying Cox proportional hazards regression to associate biomarker level with composite outcome. RESULTS: Out of 153 patients, 24 (15.7%) experienced the primary outcome. Twofold higher levels of neutrophil gelatinase-associated lipocalin (NGAL) (HR, 1.34 [95% CI, 1.14-1.57]), monocyte chemoattractant protein (MCP-1) (HR, 1.42 [95% CI, 1.09-1.84]), and kidney injury molecule 1 (KIM-1) (HR, 2.03 [95% CI, 1.38-2.99]) were associated with highest risk of sustaining primary composite outcome. Higher epidermal growth factor (EGF) levels were associated with a lower risk of the primary outcome (HR, 0.61 [95% CI, 0.47-0.79]). Individual biomarkers provided moderate discrimination and biomarker combinations improved discrimination for the primary outcome. The degree of kidney injury by biomarker level in COVID-19 was comparable to other settings of clinical AKI. There was evidence of subclinical AKI in COVID-19 patients based on elevated injury biomarker level in patients without clinical AKI defined by serum creatinine. LIMITATIONS: Small sample size with low number of composite outcome events. CONCLUSIONS: Urinary biomarkers are associated with adverse kidney outcomes in patients hospitalized with COVID-19 and may provide valuable information to monitor kidney disease progression and recovery.

Differential Performance of CoronaCHEK SARS-CoV-2 Lateral Flow Antibody Assay by Geographic Origin of Samples.

We assessed the performance of the CoronaCHEK lateral flow assay on samples from Uganda and Baltimore to determine the impact of geographic origin on assay performance. Plasma samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR-positive individuals (Uganda, 78 samples from 78 individuals, and Baltimore, 266 samples from 38 individuals) and from prepandemic individuals (Uganda, 1,077, and Baltimore, 532) were evaluated. Prevalence ratios (PR) were calculated to identify factors associated with a false-positive test. After the first positive PCR in Ugandan samples, the sensitivity was 45% (95% confidence interval [CI], 24,68) at 0 to 7 days, 79% (95% CI, 64 to 91) at 8 to 14 days, and 76% (95% CI, 50 to 93) at >15 days. In samples from Baltimore, sensitivity was 39% (95% CI, 30 to 49) at 0 to 7 days, 86% (95% CI, 79 to 92) at 8 to 14 days, and 100% (95% CI, 89 to 100) at 15 days after positive PCR. The specificity of 96.5% (95% CI, 97.5 to 95.2) in Ugandan samples was significantly lower than that in samples from Baltimore, 99.3% (95% CI, 98.1 to 99.8; P < 0.01). In Ugandan samples, individuals with a false-positive result were more likely to be male (PR, 2.04; 95% CI, 1.03,3.69) or individuals who had had a fever more than a month prior to sample acquisition (PR, 2.87; 95% CI, 1.12 to 7.35). Sensitivity of the CoronaCHEK was similar in samples from Uganda and Baltimore. The specificity was significantly lower in Ugandan samples than in Baltimore samples. False-positive results in Ugandan samples appear to correlate with a recent history of a febrile illness, potentially indicative of a cross-reactive immune response in individuals from East Africa.

Magnetofluidic immuno-PCR for point-of-care COVID-19 serological testing.

Serological tests play an important role in the fight against Coronavirus Disease 2019 (COVID-19), including monitoring the dynamic immune response after vaccination, identifying past infection and determining community infection rate. Conventional methods for serological testing, such as enzyme-linked immunosorbent assays and chemiluminescence immunoassays, provide reliable and sensitive antibody detection but require sophisticated laboratory infrastructure and/or lengthy assay time. Conversely, lateral flow immunoassays are suitable for rapid point-of-care tests but have limited sensitivity. Here, we describe the development of a rapid and sensitive magnetofluidic immuno-PCR platform that can address the current gap in point-of-care serological testing for COVID-19. Our magnetofluidic immuno-PCR platform automates a magnetic bead-based, single-binding, and one-wash immuno-PCR assay in a palm-sized magnetofluidic device and delivers results in ∼30 min. In the device, a programmable magnetic arm attracts and transports magnetically-captured antibodies through assay reagents pre-loaded in a companion plastic cartridge, and a miniaturized thermocycler and a fluorescence detector perform immuno-PCR to detect the antibodies. We evaluated our magnetofluidic immuno-PCR with 108 clinical serum/plasma samples and achieved 93.8% (45/48) sensitivity and 98.3% (59/60) specificity, demonstrating its potential as a rapid and sensitive point-of-care serological test for COVID-19.

The Clinical Review Committee: Impact of the Development of In Vitro Diagnostic Tests for SARS-CoV-2 Within RADx Tech.

The NIH Rapid Acceleration of Diagnostics (RADxSM) Tech Program was created to speed the development, validation, and commercialization of innovative point-of-care (POC) and home-based tests, and to improve clinical laboratory tests, that can directly detect SARS-CoV-2. Leveraging the experience of the Point-of-Care Technologies Research Network, a Clinical Review Committee (CRC) composed of clinicians, bioengineers, regulatory experts, and laboratorians was created to provide structured feedback to SARS-CoV-2 diagnostic innovators. The CRC convened 53 meetings with 49 companies offering SARS-CoV-2 tests in POC and reference laboratory formats as well as collection materials. The CRC identified common barriers to device design finalization including biosafety, workflow, result reporting, regulatory requirements, sample type, supply chain, limit of detection, lack of relevant validation data, and price-performance-use mismatch. Feedback from companies participating was positive.

Demographic and clinical correlates of acute and convalescent SARS-CoV-2 infection among patients of a U.S. emergency department.

BACKGROUND: Emergency Departments (EDs) have served as critical surveillance sites for infectious diseases. We sought to determine the prevalence and temporal trends of acute (by PCR) and convalescent (by antibody [Ab]) SARS-CoV-2 infection during the earliest phase of the pandemic among patients in an urban ED in Baltimore City. METHODS: We tested remnant blood samples from 3255 unique ED patients, collected between March 16th and May 31st 2020 for SARS-CoV-2 Ab. PCR for acute SARS-CoV-2 infection from nasopharyngeal swabs was obtained on any patients based on clinical suspicion. Hospital records were abstracted and factors associated with SARS-CoV-2 infection were assessed. RESULTS: Of 3255 ED patients, 8.2% (95%CI: 7.3%, 9.2%) individuals had evidence of SARS-CoV-2 infection; 155 PCR+, 78 Ab+, and 35 who were both PCR+ and Ab+. Prevalence of disease increased throughout the study period, ranging from 3.2% (95%CI: 1.8%, 5.2%) PCR+ and 0.6% (95%CI: 0.1%, 1.8%) Ab+ in March, to 6.2% (95%CI: 5.1%, 7.4%) PCR+ and 4.2% (95%CI: 3.3%, 5.3%) Ab+ in May. The highest SARS-CoV-2 prevalence was found in Hispanic individuals who made up 8.4% (95%CI: 7.4%, 9.4%) of individuals screened, but 35% (95%CI: 29%, 41%) of infections (PCR and/or Ab+). Demographic and clinical factors independently associated with acute infection included Hispanic ethnicity, loss of smell or taste, subjective fever, cough, muscle ache and fever. Factors independently associated with convalescent infection were Hispanic ethnicity and low oxygen saturation. CONCLUSIONS: The burden of COVID-19 in Baltimore City increased dramatically over the 11-week study period and was disproportionately higher among Hispanic individuals. ED-based surveillance methods are important for identifying both acute and convalescent SARS-CoV-2 infections and provides important information regarding demographic and clinical correlates of disease in the local community.

SARS-CoV-2 Antibody Avidity Responses in COVID-19 Patients and Convalescent Plasma Donors.

BACKGROUND: Convalescent plasma therapy is a leading treatment for conferring temporary immunity to COVID-19-susceptible individuals or for use as post-exposure prophylaxis. However, not all recovered patients develop adequate antibody titers for donation and the relationship between avidity and neutralizing titers is currently not well understood. METHODS: SARS-CoV-2 anti-spike and anti-nucleocapsid IgG titers and avidity were measured in a longitudinal cohort of COVID-19 hospitalized patients (n = 16 individuals) and a cross-sectional sample of convalescent plasma donors (n = 130). Epidemiologic correlates of avidity were examined in donors by linear regression. The association of avidity and a high neutralizing titer (NT) were also assessed in donors using modified Poisson regression. RESULTS: Antibody avidity increased over duration of infection and remained elevated. In convalescent plasma donors, higher levels of anti-spike avidity were associated with older age, male sex, and hospitalization. Higher NTs had a stronger positive correlation with anti-spike IgG avidity (Spearman ρ = 0.386; P < .001) than with anti-nucleocapsid IgG avidity (Spearman ρ = 0.211; P = .026). Increasing levels of anti-spike IgG avidity were associated with high NT (≥160) (adjusted prevalence ratio = 1.58 [95% confidence interval = 1.19-2.12]), independent of age, sex, and hospitalization. CONCLUSIONS: SARS-CoV-2 antibody avidity correlated with duration of infection and higher neutralizing titers, suggesting a potential alternative screening parameter for identifying optimal convalescent plasma donors.

Comparative Performance of Five Commercially Available Serologic Assays To Detect Antibodies to SARS-CoV-2 and Identify Individuals with High Neutralizing Titers.

Accurate serological assays to detect antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to characterize the epidemiology of SARS-CoV-2 infection and identify potential candidates for coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) donation. This study compared the performances of commercial enzyme immunoassays (EIAs) with respect to detection of IgG or total antibodies to SARS-CoV-2 and neutralizing antibodies (nAbs). The diagnostic accuracy of five commercially available EIAs (Abbott, Euroimmun, EDI, ImmunoDiagnostics, and Roche) for detection of IgG or total antibodies to SARS-CoV-2 was evaluated using cross-sectional samples from potential CCP donors who had prior molecular confirmation of SARS-CoV-2 infection (n = 214) and samples from prepandemic emergency department patients without SARS-CoV-2 infection (n = 1,099). Of the 214 potential CCP donors, all were sampled >14 days since symptom onset and only a minority (n = 16 [7.5%]) had been hospitalized due to COVID-19; 140 potential CCP donors were tested by all five EIAs and a microneutralization assay. Performed according to the protocols of the manufacturers to detect IgG or total antibodies to SARS-CoV-2, the sensitivity of each EIA ranged from 76.4% to 93.9%, and the specificity of each EIA ranged from 87.0% to 99.6%. Using a nAb titer cutoff value of ≥160 as the reference representing a positive test result (n = 140 CCP donors), the empirical area under the receiver operating curve for each EIA ranged from 0.66 (Roche) to 0.90 (Euroimmun). Commercial EIAs with high diagnostic accuracy to detect SARS-CoV-2 antibodies did not necessarily have high diagnostic accuracy to detect high nAb titers. Some but not all commercial EIAs may be useful in the identification of individuals with high nAb titers among convalescent individuals.

COVID-19 Serology at Population Scale: SARS-CoV-2-Specific Antibody Responses in Saliva.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of an ongoing pandemic that has infected over 36 million and killed over 1 million people. Informed implementation of government public health policies depends on accurate data on SARS-CoV-2 immunity at a population scale. We hypothesized that detection of SARS-CoV-2 salivary antibodies could serve as a noninvasive alternative to serological testing for monitoring of SARS-CoV-2 infection and seropositivity at a population scale. We developed a multiplex SARS-CoV-2 antibody immunoassay based on Luminex technology that comprised 12 CoV antigens, mostly derived from SARS-CoV-2 nucleocapsid (N) and spike (S). Saliva and sera collected from confirmed coronavirus disease 2019 (COVID-19) cases and from the pre-COVID-19 era were tested for IgG, IgA, and IgM to the antigen panel. Matched saliva and serum IgG responses (n = 28) were significantly correlated. The salivary anti-N IgG response resulted in the highest sensitivity (100%), exhibiting a positive response in 24/24 reverse transcription-PCR (RT-PCR)-confirmed COVID-19 cases sampled at >14 days post-symptom onset (DPSO), whereas the salivary anti-receptor binding domain (RBD) IgG response yielded 100% specificity. Temporal kinetics of IgG in saliva were consistent with those observed in blood and indicated that most individuals seroconvert at around 10 DPSO. Algorithms employing a combination of the IgG responses to N and S antigens result in high diagnostic accuracy (100%) by as early as 10 DPSO. These results support the use of saliva-based antibody testing as a noninvasive and scalable alternative to blood-based antibody testing.

Evaluation of Serological SARS-CoV-2 Lateral Flow Assays for Rapid Point-of-Care Testing.

Rapid point-of-care tests (POCTs) for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies vary in performance. A critical need exists to perform head-to-head comparisons of these assays. The performances of 15 different lateral flow POCTs for the detection of SARS-CoV-2-specific antibodies were compared on a well-characterized set of 100 samples. Of these, 40 samples from known SARS-CoV-2-infected, convalescent individuals (collected an average of 45 days after symptom onset) were used to assess sensitivity. Sixty samples from the prepandemic era (negative control) that were known to represent infections with other respiratory viruses (rhinoviruses A, B, and C and/or coronavirus 229E, HKU1, and NL63 OC43) were used to assess specificity. The timing of seroconversion was assessed using five lateral flow assays (LFAs) and a panel of 272 longitudinal samples from 47 patients for whom the time since symptom onset was known. Among the assays that were evaluated, the sensitivity and specificity for any reactive band ranged from 55% to 97% and from 78% to 100%, respectively. Assessing the performance of the IgM and the IgG bands alone, sensitivity and specificity ranged from 0% to 88% and 80% to 100% for IgM and from 25% to 95% and 90% to 100% for IgG, respectively. Longitudinal testing revealed that the median times after symptom onset to a positive result were 7 days (interquartile range [IQR], 5.4 to 9.8) for IgM and 8.2 days (IQR, 6.3 to 11.3) for IgG. The testing performances differed widely among LFAs, with greatest amount of variation related to the sensitivity of the assays. The IgM band was the band most likely to misclassify prepandemic samples. The appearances of IgM and IgG bands occurred almost simultaneously.

Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity.

Understanding humoral responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for improving diagnostics, therapeutics, and vaccines. Deep serological profiling of 232 coronavirus disease 2019 (COVID-19) patients and 190 pre-COVID-19 era controls using VirScan revealed more than 800 epitopes in the SARS-CoV-2 proteome, including 10 epitopes likely recognized by neutralizing antibodies. Preexisting antibodies in controls recognized SARS-CoV-2 ORF1, whereas only COVID-19 patient antibodies primarily recognized spike protein and nucleoprotein. A machine learning model trained on VirScan data predicted SARS-CoV-2 exposure history with 99% sensitivity and 98% specificity; a rapid Luminex-based diagnostic was developed from the most discriminatory SARS-CoV-2 peptides. Individuals with more severe COVID-19 exhibited stronger and broader SARS-CoV-2 responses, weaker antibody responses to prior infections, and higher incidence of cytomegalovirus and herpes simplex virus 1, possibly influenced by demographic covariates. Among hospitalized patients, males produce stronger SARS-CoV-2 antibody responses than females.

Evaluation of Serological SARS-CoV-2 Lateral Flow Assays for Rapid Point of Care Testing.

BACKGROUND: Rapid point-of-care tests (POCTs) for SARS-CoV-2-specific antibodies vary in performance. A critical need exists to perform head-to-head comparison of these assays. METHODS: Performance of fifteen different lateral flow POCTs for the detection of SARS-CoV-2-specific antibodies was performed on a well characterized set of 100 samples. Of these, 40 samples from known SARS-CoV-2-infected, convalescent individuals (average of 45 days post symptom onset) were used to assess sensitivity. Sixty samples from the pre-pandemic era (negative control), that were known to have been infected with other respiratory viruses (rhinoviruses A, B, C and/or coronavirus 229E, HKU1, NL63 OC43) were used to assess specificity. The timing of seroconversion was assessed on five POCTs on a panel of 272 longitudinal samples from 47 patients of known time since symptom onset. RESULTS: For the assays that were evaluated, the sensitivity and specificity for any reactive band ranged from 55%-97% and 78%-100%, respectively. When assessing the performance of the IgM and the IgG bands alone, sensitivity and specificity ranged from 0%-88% and 80%-100% for IgM and 25%-95% and 90%-100% for IgG. Longitudinal testing revealed that median time post symptom onset to a positive result was 7 days (IQR 5.4, 9.8) for IgM and 8.2 days (IQR 6.3 to 11.3). CONCLUSION: The testing performance varied widely among POCTs with most variation related to the sensitivity of the assays. The IgM band was most likely to misclassify pre-pandemic samples. The appearance of IgM and IgG bands occurred almost simultaneously.