Pre-pandemic Samples Research Articles

College, Interrupted: Profiles in First-Year College Students Responses to the COVID-19 Pandemic Across One Year.

First-year college students in the 2019-2020 academic year are at risk of having their mental health, identity work, and college careers derailed as a result of the COVID-19 pandemic. To assess emerging and evolving impacts of the pandemic on mental health/well-being, identity development, and academic resilience, we collected data from a racially, ethnically, geographically, and economically diverse group of 629 students at four universities across the US within weeks of lockdown, and then followed up on these students’ self-reported mental health, identity, and academic resilience three times over the following year. Our findings suggest that: 1) students’ mental health, identity development, and academic resilience were largely negatively impacted compared to pre-pandemic samples; 2) these alterations persisted and, in some cases, worsened as the pandemic wore on; and 3) patterns of change were often worse for students indicating more baseline COVID-related stressors.

Electrochemical Immunosensors Based on Zinc Oxide Nanorods for Detection of Antibodies Against SARS-CoV-2 Spike Protein in Convalescent and Vaccinated Individuals.

Even after over 2 years of the COVID-19 pandemic, research on rapid, inexpensive, and accurate tests remains essential for controlling and avoiding the global spread of SARS-CoV-2 across the planet during a potential reappearance in future global waves or regional outbreaks. Assessment of serological responses for COVID-19 can be beneficial for population-level surveillance purposes, supporting the development of novel vaccines and evaluating the efficacy of different immunization programs. This can be especially relevant for broadly used inactivated whole virus vaccines, such as CoronaVac, which produced lower titers of neutralizing antibodies. and showed lower efficacy for specific groups such as the elderly and immunocompromised. We developed an impedimetric biosensor based on the immobilization of SARS-CoV-2 recombinant trimeric spike protein (S protein) on zinc oxide nanorod (ZnONR)-modified fluorine-doped tin oxide substrates for COVID-19 serology testing. Due to electrostatic interactions, the negatively charged S protein was immobilized via physical adsorption. The electrochemical response of the immunosensor was measured at each modification step and characterized by scanning electron microscopy and electrochemical techniques. We successfully evaluated the applicability of the modified ZnONR electrodes using serum samples from COVID-19 convalescent individuals, CoronaVac-vaccinated with or without positive results for SARS-CoV-2 infection, and pre-pandemic samples from healthy volunteers as controls. ELISA for IgG anti-SARS-CoV-2 spike protein was performed for comparison, and ELISA for IgG anti-RBDs of seasonal coronavirus (HCoVs) was used to test the specificity of immunosensor detection. No cross-reactivity with HCoVs was detected using the ZnONR immunosensor, and more interestingly, the sensor presented higher sensitivity when compared to negative ELISA results. The results demonstrate that the ZnONRs/spike-modified electrode displayed sensitive results for convalescents and vaccinated samples and shows excellent potential as a tool for the population’s assessment and monitoring of seroconversion and seroprevalence.

SARS-CoV-2-cross-reactive T cells identified in pre-pandemic lower airway samples.

SARS-CoV-2-cross-reactive T cells identified in pre-pandemic lower airway samples.

Salivary and serum IgA and IgG responses to SARS-CoV-2-spike protein following SARS-CoV-2 infection and after immunization with COVID-19 vaccines.

Background: Secretory immunoglobulin A (sIgA) plays an important role in antiviral protective immunity. Although salivary testing has been used for many viral infections, including severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS), its use has not yet been well established with the SARS coronavirus 2 (SARS-CoV-2). Quantification of salivary IgA and IgG antibodies can elucidate mucosal and systemic immune responses after natural infection or vaccination. Here, we report the development and validation of a rapid enzyme-linked immunosorbent assay (ELISA) for anti-SARS-CoV-2 salivary IgA and serum IgG antibodies, and present quantitative results for immunized subjects both prior to or following COVID-19 infections. Objective: Total and serum SARS-CoV-2 spike-specific IgG responses were compared with salivary spike-specific IgA and IgG responses in samples obtained from patients recently infected with SARS-CoV-2 and from subjects recently immunized with COVID-19 vaccines. Methods: A total of 52 paired saliva and serum samples were collected from 26 study participants: 7 subjects after COVID-19 infection and 19 subjects who were uninfected. The ELISA results from these samples were compared with five prepandemic control serum samples. Total IgG and SARS-CoV-2 spike-specific IgG in the serum samples from the subjects who were infected and vaccinated were also measured in a commercial laboratory with an enzyme immunoassay. Results: A wide variation in antibody responses was seen in salivary and serum samples measured by both methods. Three groups of serum total and IgG spike-specific SARS-CoV-2 antibody responses were observed: (1) low, (2) intermediate, and (3) high antibody responders. A correlational analysis of salivary IgA (sIgA) responses with serum IgG concentrations showed a statistical correlation in the low and intermediate antibody responder groups but not in the high group (which we believe was a result of saturation). Conclusion: These preliminary findings suggest measuring salivary and serum IgG and IgA merit further investigation as markers of current or recent SARS-CoV-2 infections.

NET Formation in Systemic Lupus Erythematosus: Changes during the COVID-19 Pandemic.

The severity of the coronavirus disease in 2019 (COVID-19) is strongly linked to a dysregulated immune response. This fuels the fear of severe disease in patients with autoimmune disorders continuously using immunosuppressive/immunomodulating medications. One complication of COVID-19 is thromboembolism caused by intravascular aggregates of neutrophil extracellular traps (NETs) occluding the affected vessels. Like COVID-19, systemic lupus erythematosus (SLE) is characterized by, amongst others, an increased risk of thromboembolism. An imbalance between NET formation and clearance is suggested to play a prominent role in exacerbating autoimmunity and disease severity. Serologic evidence of exposure to SARS-CoV-2 has a minor impact on the SLE course in a Swedish cohort reportedly. Herein, we assessed NET formation in patients from this cohort by neutrophil elastase (NE) activity and the presence of cell-free DNA, MPO-DNA, and NE-DNA complexes and correlated the findings to the clinical parameters. The presence of NE-DNA complexes and NE activity differed significantly in pre-pandemic versus pandemic serum samples. The latter correlated significantly with the hemoglobin concentration, blood cell counts, and complement protein 3 and 4 levels in the pre-pandemic but only with the leukocyte count and neutrophil levels in the pandemic serum samples. Taken together, our data suggest a change, especially in the NE activity independent of exposure to SARS-CoV-2.

Validation of a Novel Fluorescent Lateral Flow Assay for Rapid Qualitative and Quantitative Assessment of Total Anti-SARS-CoV-2 S-RBD Binding Antibody Units (BAU) from Plasma or Fingerstick Whole-Blood of COVID-19 Vaccinees.

Background: Limited commercial LFA assays are available to provide a reliable quantitative measurement of the total binding antibody units (BAU/mL) against the receptor-binding domain of the SARS-CoV-2 spike protein (S-RBD). Aim: This study aimed to evaluate the performance of the fluorescence LFA FinecareTM 2019-nCoV S-RBD test along with its reader (Model No.: FS-113) against the following reference methods: (i) the FDA-approved GenScript surrogate virus-neutralizing assay (sVNT); and (ii) three highly performing automated immunoassays: BioMérieux VIDAS®3, Ortho VITROS®, and Mindray CL-900i®. Methods: Plasma from 488 vaccinees was tested by all aforementioned assays. Fingerstick whole-blood samples from 156 vaccinees were also tested by FinecareTM. Results and conclusions: FinecareTM showed 100% specificity, as none of the pre-pandemic samples tested positive. Equivalent FinecareTM results were observed among the samples taken from fingerstick or plasma (Pearson correlation r = 0.9, p < 0.0001), suggesting that fingerstick samples are sufficient to quantitate the S-RBD BAU/mL. A moderate correlation was observed between FinecareTM and sVNT (r = 0.5, p < 0.0001), indicating that FinecareTM can be used for rapid prediction of the neutralizing antibody (nAb) post-vaccination. FinecareTM BAU results showed strong correlation with VIDAS®3 (r = 0.6, p < 0.0001) and moderate correlation with VITROS® (r = 0.5, p < 0.0001) and CL-900i® (r = 0.4, p < 0.0001), suggesting that FinecareTM can be used as a surrogate for the advanced automated assays to measure S-RBD BAU/mL.

Evaluation of a Lateral Flow Immunoassay COVIDTECH® SARS-CoV-2 IgM/IgG Antibody Rapid Test.

The new coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for severe respiratory illness (i.e., COVID-19). RT-PCR of respiratory samples is the gold standard for COVID-19 diagnosis, and serological tests may contribute to the detection of post-infection and post-vaccination immunity and enable seroprevalence studies. The lateral flow immunoassay (LFIA) COVIDTECH® SARS-CoV-2 IgM/IgG antibody rapid test that detects anti-SARS-CoV-2 IgM and IgG using an S protein recombinant antigen has been independently evaluated in two laboratories. The specificity evaluated for 65 pre-pandemic samples was 100% for IgM/IgG. An analysis of samples from patients with RT-PCR-confirmed infection revealed that IgM/IgG antibodies were detected in 18/26 (69%) samples before day 13 and in 58/58 (100%) samples from day 14 post-symptom onset. Before day 14 post-symptom onset, the COVIDTECH Test was less sensitive than other LFIA method (BIOSYNEX COVID-19 BSS IgM/IgG) and a chemiluminescent immunoassay (LIAISON® SARS-CoV-2 TrimericS IgG assay). Overall, this LFIA method is suitable for SARS-CoV-2 serological diagnosis for patients after > 14 days since the onset of symptoms.

Cross-reactive immunity against SARS-CoV-2 N protein in Central and West Africa precedes the COVID-19 pandemic

Early predictions forecasted large numbers of severe acute respiratory syndrome coronavirus (SARS-CoV-2) cases and associated deaths in Africa. To date, Africa has been relatively spared. Various hypotheses were postulated to explain the lower than anticipated impact on public health in Africa. However, the contribution of pre-existing immunity is yet to be investigated. In this study, the presence of antibodies against SARS-CoV-2 spike (S) and nucleocapsid (N) proteins in pre-pandemic samples from Africa, Europe, South and North America was examined by ELISA. The protective efficacy of N specific antibodies isolated from Central African donors was tested by in vitro neutralization and in a mouse model of SARS-CoV-2 infection. Antibodies against SARS-CoV-2 S and N proteins were rare in all populations except in Gabon and Senegal where N specific antibodies were prevalent. However, these antibodies failed to neutralize the virus either in vitro or in vivo. Overall, this study indicates that cross-reactive immunity against SARS-CoV-2 N protein was present in Africa prior to the pandemic. However, this pre-existing humoral immunity does not impact viral fitness in rodents suggesting that other human immune defense mechanisms could be involved. In Africa, seroprevalence studies using the N protein are over-estimating SARS-CoV-2 circulation.

High SARS-CoV-2 seroprevalence in Karaganda, Kazakhstan before the launch of COVID-19 vaccination.

COVID-19 exposure in Central Asia appears underestimated and SARS-CoV-2 seroprevalence data are urgently needed to inform ongoing vaccination efforts and other strategies to mitigate the regional pandemic. Here, in a pilot serologic study we assessed the prevalence of SARS-CoV-2 antibody-mediated immunity in a multi-ethnic cohort of public university employees in Karaganda, Kazakhstan. Asymptomatic subjects (n = 100) were recruited prior to their first COVID-19 vaccination. Questionnaires were administered to capture a range of demographic and clinical characteristics. Nasopharyngeal swabs were collected for SARS-CoV-2 RT-qPCR testing. Serological assays were performed to detect spike (S)-reactive IgG and IgA and to assess virus neutralization. Pre-pandemic samples were used to validate the assay positivity thresholds. S-IgG and -IgA seropositivity rates among SARS-CoV-2 PCR-negative participants (n = 100) were 42% (95% CI [32.2-52.3]) and 59% (95% CI [48.8-69.0]), respectively, and 64% (95% CI [53.4-73.1]) of the cohort tested positive for at least one of the antibodies. S-IgG titres correlated with virus neutralization activity, detectable in 49% of the tested subset with prior COVID-19 history. Serologically confirmed history of COVID-19 was associated with Kazakh ethnicity, but not with other ethnic minorities present in the cohort, and self-reported history of respiratory illness since March 2020. Overall, SARS-CoV-2 exposure in this cohort was ~15-fold higher compared to the reported all-time national and regional COVID-19 prevalence, consistent with recent studies of excess infection and death in Kazakhstan. Continuous serological surveillance provides important insights into COVID-19 transmission dynamics and may be used to better inform the regional public health response.

Population structure of Mycobacterium tuberculosis from referral clinics in Western Siberia, Russia: Before and during the Covid-19 pandemic.

The dramatic change in global health imposed by the Covid-19 pandemic has also impacted TB control. The TB incidence decreased dramatically not because of the improved situation but due to undertesting, reduced resources, and ultimately, substantially reduced detection rate. We hypothesized that multiple and partly counteracting factors could influence changes in the local Mycobacterium tuberculosis population. To test this hypothesis, we analyzed M. tuberculosis isolates collected in Western Siberia, Russia, before and during the Covid-19 pandemic. A total of 269 M. tuberculosis isolates from patients admitted at referral clinics were studied. The pre-pandemic and pandemic collections included 179 and 90 isolates, respectively. Based on genotyping, both pre-pandemic and pandemic samples are heavily dominated by the Beijing genotype isolates (95% and 88%) that were mostly MDR (80 and 68%). The high proportion of MDR isolates is due to the specific features of the studied collections biased towards patients with severe TB admitted at the National referral center in Novosibirsk. While no dramatic change was observed in the M. tuberculosis population structure in the survey area in Western Siberia during the Covid-19 pandemic in 2020–2021 compared to the pre-pandemic collection, still we note a certain decrease of the Beijing genotype and an increase in the proportion and diversity of the non-Beijing isolates. However, the transmissible and MDR Beijing B0/W148 did not increase its prevalence rate during the pandemic. More generally, the high prevalence rate of the Beijing genotype and its strong association with MDR both before and during the pandemic are alarming features of this region in Western Siberia, Russia.

SARS-CoV-2-specific T cells in unexposed adults display broad trafficking potential and cross-react with commensal antigens.

The baseline composition of T cells directly impacts later response to pathogens, but the complexity of precursor states remains poorly defined. Here, we examined the baseline state of SARS-CoV-2-specific T cells in unexposed individuals. SARS-CoV-2-specific CD4+ T cells were identified in pre-pandemic blood samples by class II peptide-MHC tetramer staining and enrichment. Our data revealed a substantial number of SARS-CoV-2-specific T cells that expressed memory phenotype markers. Integrated phenotypic analyses demonstrated diverse pre-existing memory states that included cells with distinct polarization states and trafficking potential to barrier tissues. T cell clones generated from tetramer-labeled cells cross-reacted with antigens from commensal bacteria in the skin and gastrointestinal tract. Direct ex vivo tetramer staining for one spike-specific population showed a similar level of cross-reactivity to sequences from endemic coronavirus and commensal bacteria. These data highlight the complexity of precursor T cell repertoire and implicate non-infectious exposures to common microbes as a key factor that shapes human pre-existing immunity to SARS-CoV-2.

Development of an in-house quantitative ELISA for the evaluation of different Covid-19 vaccines in humans

Reliable serological assays are needed to understand the real impact of COVID-19. In order to compare the efficiency of different COVID-19 vaccines used in the National Vaccination Program in Tunisia, we have developed a quantitative in-house ELISA. The ELISA is based on the ectodomain of the SARS-CoV-2 Spike Baculovirus recombinant protein. We used a panel of 145 COVID-19 RT-PCR positive serum samples and 116 pre-pandemic serum samples as a negative panel. The validation was carried out by comparison to four commercial techniques (Vidas SARS-CoV-2 IgG anti-RBD Biomérieux, Elecsys Anti-Nucleocapsid of SARS-CoV-2 Roche, cPass GenScript and the quantitative Elecsys Anti-RBD of SARS-CoV-2, Roche). For the evaluation of the National Vaccination campaign, we have included 115 recipients who received one of the approved vaccines. The qualitative performances of the developed ELISA gave 96% sensitivity, 97.5% specificity and 0.968 accuracy. For the evaluation of the different brand of vaccines in recipients not previously infected with SARS-CoV-2, it seems that mRNA vaccine of Pfizer/BioNTech has shown a higher efficacy compared to inactivated virus vaccines. COVID-19 convalescent individuals have generated poor antibody responses. Nevertheless, when they are vaccinated with any brand of the COVID-19 vaccines, many of them mounted an exponential increase of the induced immune responses, qualified as a “hybrid vigor immunity”. Our developed in-house ELISA seems to be very efficient in evaluating the effectiveness of anti-COVID-19 vaccination. Platforms based on mRNA vaccine are better performing than those based on inactivated virus.

Metallomic and Untargeted Metabolomic Signatures of Human Milk from SARS-CoV-2 Positive Mothers.

ScopeLack of information about the impact of maternal severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection on the elemental and metabolomic profile of human milk (HM).Methods and resultsAn observational study on HM from mothers with COVID‐19 is conducted including a prepandemic control group. Maternal–infant clinical records and symptomatology are recorded. The absolute quantification of elements and untargeted relative metabolomic profiles are determined by inductively coupled plasma mass spectrometry and gas chromatography coupled to mass spectrometry, respectively. Associations of HM SARS‐CoV‐2 antibodies with elemental and metabolomic profiles are studied. COVID‐19 has a significant impact on HM composition. COVID‐19 reduces the concentrations of Fe, Cu, Se, Ni, V, and Aluminium (Al) and increases Zn compared to prepandemic control samples. A total of 18 individual metabolites including amino acids, peptides, fatty acids and conjugates, purines and derivatives, alcohols, and polyols are significantly different in HM from SARS‐CoV‐2 positive mothers. Aminoacyl‐tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine, and linoleic acid pathways are significantly altered. Differences are obtained depending on COVID‐19 symptomatic and asymptomatic status.ConclusionsThis study provides unique insights about the impact of maternal SARS‐CoV‐2 infection on the elemental and metabolomic profiles of HM that warrants further research due the potential implications for infant health.

Manufacturer Signal-to-Cutoff Threshold Underestimates Cumulative Incidence of SARS-CoV-2 Infection: Evidence from the Los Angeles Firefighters Study.

BackgroundThe objective of this analysis was to compare the performance sensitivity and specificity of manufacturer-recommended signal-to-cutoff (S/Co) thresholds with modified S/Co values to estimate the prevalence of SARS-CoV-2-specific antibodies in a cohort of firefighters with a known infection history.MethodsPlasma venipuncture samples were used for serologic analysis of firefighters in Los Angeles, CA, USA, in October 2020. Seropositivity was assessed using the manufacturer’s recommended S/Co (≥1.4 IgG) and modified S/Co thresholds based on measured antibody levels in 178 negative control patients who had blood drawn prior to the emergence of COVID-19. Optimal S/Co threshold was determined by receiver operating characteristic (ROC) curve analysis.ResultsOf 585 firefighters included in the study, 52 (8.9%) reported having a PCR-positive test history prior to antibody testing. Thirty-five (67.3%) firefighters with a previous PCR-positive test were seropositive based on the manufacturer S/Co thresholds, consistent with an estimated 67.3% sensitivity and 100% specificity. After evaluating multiple modified S/Co thresholds based on pre-pandemic negative samples, a modified S/Co of 0.36 was found to yield optimal sensitivity (88.5%) and specificity (99.4%) by ROC curve analysis. This modified threshold improved serostatus classification accuracy by 21.2%.ConclusionsS/Co thresholds based on known negative samples significantly increase seropositivity and more accurately estimate cumulative incidence of disease compared to manufacturer-based thresholds.

Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: Antibody dynamics and linkage to severity of COVID-19 in hospital inpatients

Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: Antibody dynamics and linkage to severity of COVID-19 in hospital inpatients

Immunogenic epitope panel for accurate detection of non-cross-reactive T cell response to SARS-CoV-2.

The ongoing COVID-19 pandemic calls for more effective diagnostic tools. T cell response assessment serves as an independent indicator of prior COVID-19 exposure while also contributing to a more comprehensive characterization of SARS-CoV-2 immunity. In this study, we systematically assessed the immunogenicity of 118 epitopes with immune cells collected from multiple cohorts of vaccinated, convalescent, healthy unexposed, and SARS-CoV-2–exposed donors. We identified 75 immunogenic epitopes, 24 of which were immunodominant. We further confirmed HLA restriction for 49 epitopes and described association with more than 1 HLA allele for 14 of these. Exclusion of 2 cross-reactive epitopes that generated a response in prepandemic samples left us with a 73-epitope set that offered excellent diagnostic specificity without losing sensitivity compared with full-length antigens, and this evoked a robust cross-reactive response. We subsequently incorporated this set of epitopes into an in vitro diagnostic Corona-T-test, which achieved a diagnostic accuracy of 95% in a clinical trial. In a cohort of asymptomatic seronegative individuals with a history of prolonged SARS-CoV-2 exposure, we observed a complete absence of T cell response to our epitope panel. In combination with strong reactivity to full-length antigens, this suggests that a cross-reactive response might protect these individuals.

Seroprevalence of anti-SARS-CoV-2 antibodies in Thai adults during the first three epidemic waves.

This study determined the presence of anti-SARS-CoV-2 antibodies in 4964 individuals, comprising 300 coronavirus disease-19 (COVID-19) prepandemic serum samples, 142 COVID-19 patients, 2113 individuals at risk due to their occupations, 1856 individuals at risk due to sharing workplaces or communities with COVID-19 patients, and 553 Thai citizens returning after spending extended periods of time in countries with a high disease prevalence. We recruited participants between May 2020 and May 2021, which spanned the first two epidemic waves and part of the third wave of the COVID-19 outbreaks in Thailand. Their sera were tested in a microneutralization and a chemiluminescence immunoassay for IgG against the N protein. Furthermore, we performed an immunofluorescence assay to resolve discordant results between the two assays. None of the prepandemic sera contained anti-SARS-CoV-2 antibodies, while antibodies developed in 88% (15 of 17) of the COVID-19 patients at 8-14 days and in 94-100% of the patients between 15 and 60 days after disease onset. Neutralizing antibodies persisted for at least 8 months, longer than IgG antibodies. Of the 2113 individuals at risk due to their occupation, none of the health providers, airport officers, or public transport drivers were seropositive, while antibodies were present in 0.44% of entertainment workers. Among the 1856 individuals at risk due to sharing workplaces or communities with COVID-19 patients, seropositivity was present in 1.9, 1.5, and 7.5% of the Bangkok residents during the three epidemic waves, respectively, and in 1.3% of the Chiang Mai people during the first epidemic wave. The antibody prevalence varied between 6.5 and 47.0% in 553 Thai people returning from high-risk countries. This serosurveillance study found a low infection rate of SARS-CoV-2 in Thailand before the emergence of the Delta variant in late May 2021. The findings support the Ministry of Public Health's data, which are based on numbers of patients and contact tracing.

Development and Validation of an Enzyme Immunoassay for Detection and Quantification of SARS-CoV-2 Salivary IgA and IgG.

Oral fluids offer a noninvasive sampling method for the detection of Abs. Quantification of IgA and IgG Abs in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG Abs against the prefusion-stabilized form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein expressed in suspension-adapted HEK-293 cells. Normalization against total Ab isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 prepandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA, 95.5%; IgG, 89.7%) without compromising specificity (IgA, 99%; IgG, 97%). No cross-reactivity with endemic coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/ml and 0.30 ng/ml, respectively. Salivary IgA and IgG Abs were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary Ab titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 wk in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

A Novel Method to Reduce ELISA Serial Dilution Assay Workload Applied to SARS-CoV-2 and Seasonal HCoVs.

Assays using ELISA measurements on serially diluted serum samples have been heavily used to measure serum reactivity to SARS-CoV-2 antigens and are widely used in virology and elsewhere in biology. We test a method using Bayesian hierarchical modelling to reduce the workload of these assays and measure reactivity of SARS-CoV-2 and HCoV antigens to human serum samples collected before and during the COVID-19 pandemic. Inflection titers for SARS-CoV-2 full-length spike protein (S1S2), spike protein receptor-binding domain (RBD), and nucleoprotein (N) inferred from 3 spread-out dilutions correlated with those inferred from 8 consecutive dilutions with an R2 value of 0.97 or higher. We confirm existing findings showing a small proportion of pre-pandemic human serum samples contain cross-reactive antibodies to SARS-CoV-2 S1S2 and N, and that SARS-CoV-2 infection increases serum reactivity to the beta-HCoVs OC43 and HKU1 S1S2. In serial dilution assays, large savings in resources and/or increases in throughput can be achieved by reducing the number of dilutions measured and using Bayesian hierarchical modelling to infer inflection or endpoint titers. We have released software for conducting these types of analysis.

The IgA in milk induced by SARS-CoV-2 infection is comprised of mainly secretory antibody that is neutralizing and highly durable over time

Approximately 10% of infants infected with SARS-CoV-2 will experience COVID-19 illness requiring advanced care. A potential mechanism to protect this population is passive immunization via the milk of a previously infected person. We and others have reported on the presence of SARS-CoV-2-specific antibodies in human milk. We now report the prevalence of SARS-CoV-2 IgA in the milk of 74 COVID-19-recovered participants, and find that 89% of samples are positive for Spike-specific IgA. In a subset of these samples, 95% exhibited robust IgA activity as determined by endpoint binding titer, with 50% considered high-titer. These IgA-positive samples were also positive for Spike-specific secretory antibody. Levels of IgA antibodies and secretory antibodies were shown to be strongly positively correlated. The secretory IgA response was dominant among the milk samples tested compared to the IgG response, which was present in 75% of samples and found to be of high-titer in only 13% of cases. Our IgA durability analysis using 28 paired samples, obtained 4-6 weeks and 4-10 months after infection, found that all samples exhibited persistently significant Spike-specific IgA, with 43% of donors exhibiting increasing IgA titers over time. Finally, COVID-19 and pre-pandemic control milk samples were tested for the presence of neutralizing antibodies; 6 of 8 COVID-19 samples exhibited neutralization of Spike-pseudotyped VSV (IC50 range, 2.39-89.4ug/mL) compared to 1 of 8 controls. IgA binding and neutralization capacities were found to be strongly positively correlated. These data are highly relevant to public health, not only in terms of the protective capacity of these antibodies for breastfed infants, but also for the potential use of such antibodies as a COVID-19 therapeutic, given that secretory IgA is highly in all mucosal compartments.