Validation of dried blood spot sampling for detecting SARS-CoV-2 antibodies and total immunoglobulins in a large cohort of asymptomatic young adults.

BACKGROUND: Detecting antibody responses following infection with SARS-CoV-2 is necessary for sero-epidemiological studies and assessing the role of specific antibodies in disease, but serum or plasma sampling is not always viable due to logistical challenges. Dried blood spot sampling (DBS) is a cheaper, simpler alternative and samples can be self-collected and returned by post, reducing risk for SARS-CoV-2 exposure from direct patient contact. The value of large-scale DBS sampling for the assessment of serological responses to SARS-CoV-2 has not been assessed in depth and provides a model for examining the logistics of using this approach to other infectious diseases. The ability to measure specific antigens is attractive for remote outbreak situations where testing may be limited or for patients who require sampling after remote consultation. METHODS: We compared the performance of SARS-CoV-2 anti-spike and anti-nucleocapsid antibody detection from DBS samples with matched serum collected by venepuncture in a large population of asymptomatic young adults (N = 1070) living and working in congregate settings (military recruits, N = 625); university students, N = 445). We also compared the effect of self-sampling (ssDBS) with investigator-collected samples (labDBS) on assay performance, and the quantitative measurement of total IgA, IgG and IgM between DBS eluates and serum. RESULTS: Baseline seropositivity for anti-spike IgGAM antibody was significantly higher among university students than military recruits. Strong correlations were observed between matched DBS and serum samples in both university students and recruits for the anti-spike IgGAM assay. Minimal differences were found in results by ssDBS and labDBS and serum by Bland Altman and Cohen kappa analyses. LabDBS achieved 82.0% sensitivity and 98.2% specificity and ssDBS samples 86.1% sensitivity and 96.7% specificity for detecting anti-spike IgGAM antibodies relative to serum samples. For anti-SARS-CoV-2 nucleocapsid IgG there was qualitatively 100% agreement between serum and DBS samples and weak correlation in ratio measurements. Strong correlations were observed between serum and DBS-derived total IgG, IgA, and IgM. CONCLUSIONS: This is the largest validation of DBS against paired serum for SARS-CoV-2 specific antibody measurement and we have shown that DBS retains performance from prior smaller studies. There were no significant differences regarding DBS collection methods, suggesting that self-collected samples are a viable sampling collection method. These data offer confidence that DBS can be employed more widely as an alternative to classical serology.

Correlation between post-vaccination titres of anti-Spike antibodies and protection against breakthrough SARSCoV-2 infection in 7530 UK adults

Background: A robust correlate of vaccine-induced protection against SARS-CoV-2 infection has yet to be found. Aim(s): To explore whether post-vaccination combined IgG, IgA, and IgM responses to the SARS-CoV-2 trimeric spike glycoprotein (anti-S IgGAM) can predict protection against breakthrough SARS-CoV-2 infection. Method(s): In this prospective population-based study, we used dried blood spots to determine post-vaccination anti-S IgGAM responses in SARS-CoV-2-vaccinated UK adults. Using receiver operating characteristic (ROC) curve analysis, we assessed the ability of anti-S IgGAM titres (adjusted for days since vaccination) to predict postvaccination incident SARS-CoV-2 infection. After adjusting for household and behavioural factors reflecting risk of SARS-CoV-2 exposure, we compared the area under the ROC curve (AUROC) between minimally and fully adjusted models. Finding(s): Between Jan 12, 2021, and Jan 31, 2022, 300 (4.0%) of 7530 participants reported a breakthrough SARS-CoV-2 infection during 18 weeks of follow-up (220 [4.4%] ChAdOx1 nCoV-19 [ChadOx1] recipients and 75 [3.1%] BNT162b2 recipients). Anti-S IgGAM titres were modestly predictive of breakthrough infection (overall: AUROC 0.582 [95% CI 0.550-0.614];ChAdOx1: 0.564 [0.526-0.602];BNT162b2: 0.562 [0.488-0.636]). Adjustment for exposure factors significantly improved discrimination (overall: 0.666 [0.633-0.699], p<0.0001;ChAdOx1: 0.656 [0.617-0.695], p<0.0001;BNT162b1: 0.709 [0.649-0.769], p=0.0012). Conclusion(s): Anti-S IgGAM titres correlate with protection against SARS-CoV-2 infection in vaccinated adults, but exposure factors contribute significantly to risk.

Determinants of pre-vaccination SARS-CoV-2 seropositivity: a population-based longitudinal study (COVIDENCE UK)

Background: Prospective population-based studies investigating multiple determinants of pre-vaccination antibody responses to SARS-CoV-2 are lacking. Aim(s): To study factors associated with SARS-CoV-2 seropositivity. Method(s): We did a prospective population-based study in SARS-CoV-2 vaccine-naive UK adults recruited from May to November 2020, without a positive swab test result for SARS-CoV-2 prior to enrolment. Information on 88 potential sociodemographic, behavioural, nutritional, clinical, and pharmacological risk factors was obtained through online questionnaires, and combined IgG/IgA/IgM responses to SARS-CoV-2 spike glycoprotein were determined in dried blood spots. Result(s): 1696 (15.2%) of 11,130 participants were seropositive. Factors independently associated with higher risk of seropositivity included frontline health/care occupation (adjusted OR 1.86, 95% CI 1.48-2.33), international travel (1.20, 1.07-1.35), number of visits to shops and other indoor public places (>=5 vs 0/week: 1.29, 1.06-1.57, Ptrend=0.01), BMI >=25 vs <25 kg/m2 (1.24, 1.11-1.39), South Asian vs White ethnicity (1.65, 1.10-2.49), alcohol consumption >=15 vs 0 units/week (1.23, 1.04-1.46), sex hormone therapy (1.25, 1.02-1.52), and use of vitamin D supplements (1.16, 1.03-1.30). Postgraduate degree (vs primary or secondary level: 0.82, 0.67-0.99), light physical exercise (0.80, 0.70-0.93, for >=10 vs 0-4 h/week), passive smoking (0.59, 0.37-0.95), and prescribed paracetamol use (0.70, 0.52-0.96) were independently associated with lower risk. Conclusion(s): Our findings confirm ethnic, occupational, and lifestyle determinants of SARS-CoV-2 seropositivity and identify additional risk factors.

Determinants of antibody responses to primary courses and booster doses of SARS-CoV-2 vaccines in 9,101 UK adults

Background: Antibody responses to SARS-CoV-2 vaccines vary for reasons that are poorly understood. AIM: To determine factors modifying antibody responses to SARS-CoV-2 vaccination. Method(s): We tested for anti-Spike (S) antibodies before and after 2 doses of ChAdOx1 or BNT162b2 given to UK adults December 2020-July 2021. Participant characteristics and outcomes were captured by online questionnaires. Logistic regression was used to estimate odds of seronegativity after vaccination. For those who were seronegative after 2 vaccine doses, repeat testing was offered following a booster dose of BNT162b2 or mRNA-1273. Result(s): Anti-S antibodies were undetectable in 378/9101 (4.2%) participants after 2 vaccine doses. Increased risk of post-vaccination seronegativity associated with administration of ChAdOx1 vs BNT162b2 (aOR 7.0, 95% CI 4.411.2), shorter interval between vaccine doses (aOR 1.6, 1.2-2.1, 6-10 vs >10 weeks), poor vs excellent general health (aOR 3.3, 1.5-7.5), immunodeficiency (aOR 6.8, 2.6-17.4) and immunosuppressant use (aOR 3.8, 2.4-5.8). Odds of seronegativity were lower for participants who were SARS-CoV-2 seropositive pre-vaccination (aOR 0.2, 0.0-0.7) and for those taking vitamin D supplements (aOR 0.7, 0.5-0.9). Of 247 participants who were seronegative following 2 vaccine doses, 8 (3.2%) remained seronegative post-booster: all were immunosuppressed. Conclusion(s): We identify multiple determinants of antibody responses to SARS-CoV-2 vaccines, many of which are modifiable. Booster doses of BNT162b2 or mRNA-1273 were highly effective in achieving seroconversion in those who failed to mount anti-S responses following two doses of ChAdOx1 or BNT162b2.

Association between vitamin D deficiency and extended duration of COVID-19 symptoms

Introduction and ObjectivesVitamin D (VD) is involved in immunity and inflammation through mechanisms such as renin inhibition and inflammatory cytokine reduction. There is already evidence to suggest that VDD may increase COVID-19 infection susceptibility, however research assessing the impact of VDD on COVID-19 symptom duration is limited. The aim of this research was to determine whether VDD is a significant independent risk factor for extended durations of COVID-19 symptoms.MethodsThe study included 392 healthcare workers who isolated due to COVID-19 symptoms during the first wave of the pandemic (12th to 22nd May 2020) as part of the convalescent immunity (COCO) study. Data on 8 symptom types and duration of symptoms were collected, including patients’ demographics and co-morbidities. Anti-SARS-Cov-2 antibodies were measured using a combined IgG, IgA and IgM ELISA (The Binding Site). Vitamin D status was determined by measurement of serum 25(OH)D3 using the AB SCIEX Triple Quad 4500 mass spectrometry system. VDD was defined as serum 25(OH)D3 <30 nmol/L.ResultsThrough univariate analysis of VDD and non-VDD staff, we initially showed VDD to be significantly associated with longer durations of body aches (median 7 days, IQR 5–14 vs. median 5 days, IQR 3–7.5;p=0.0075) and fatigue (median 12 days, IQR 7–14 vs. median 7 days, IQR 4–14;p=0.0127). VDD did not influence the duration of the other 6 symptoms analysed, such as cough and fever. Using binary logistic regression models, we confirm that VDD is a significant independent risk factor for extended durations of fatigue (OR 2.089, 95% CI 1.087–4.011;p=0.027) and body aches (OR 3.069, 95% CI 1.538–6.124;p=0.001). Additionally, VDD staff experienced a significantly greater quantity of symptoms compared to non-VDD staff (median 5, IQR 4–7 versus median 4, IQR 3–6;p=0.0030).ConclusionsThis is one of the first studies to investigate the influence of VDD on COVID-19 symptom duration. Our results indicate that VDD is a significant independent risk factor for a longer duration of body aches and fatigue. Larger studies are required to confirm these results and determine if VD supplementation could shorten symptoms.

CLL-VR: A UK study of vaccine responses in patients with B-cell chronic lymphocytic leukaemia

B-cell chronic lymphocytic leukaemia (CLL) is associated with immune suppression and patients are at increased risk following SARS-CoV-2 infection. The Chronic Lymphocytic Leukaemia-Vaccine Response (CLL-VR) study was designed to assess immune responses following the introduction of Covid-19 vaccination in UK. Five hundred patients with CLL were recruited nationally through NHS and charity communications. Phlebotomy blood samples were taken from local patients ( n = 100) and dried blood spot samples were collected via post from participants across the UK ( n = 400). Ninety-six age-matched control subjects were also recruited locally. Samples were taken at 2-3 weeks following the first, second and third primary vaccine doses. Antibody and cellular responses against spike protein, and neutralising antibody titre to delta and omicron variant, were measured. Total serum immunoglobulin level was also determined. Responses were analysed according to clinical history, serum immunoglobulin level and vaccine type received. Donors with a clinical or serological history of prior natural infection were excluded from the analysis. Twenty percent (70/353) of participants developed a measurable antibody response after the first vaccination and this increased to 67% (323/486) following the second dose and 80% (202/254) after a third dose. The response rate in healthy controls plateaued at 100% after only two doses. The magnitude of the antibody response was also 3.7-fold lower following the second vaccine compared to controls ( n = 244;490 vs. 1821 U/ml, p < 0.0001) but increased markedly to 3114 U/ ml after third dose ( n = 51). No difference was observed in relation to the initial vaccine platform received. Multivariate analysis on 486 participants showed that BTKi therapy, history of recurrent infection and low serum antibody levels of IgA or IgM were independent prognostic markers for poor antibody response. Among participants with a detectable antibody response, a marked reduction in the ability to neutralise the delta and omicron variants of concern was noted compared to healthy controls following both the second and third dose of vaccine. Cellular responses were assessed following the second vaccine by IFN-g ELISPOT ( n = 91). Patients who had received the ChAdOx1 vaccine had similar levels to controls ( p = 0.39), while those who had received BNT162b2 had lower levels ( p < 0.0001). Five patients with poor spike-specific antibody responses to vaccination subsequently developed breakthrough infection with SARS-CoV-2 delta variant. Antibody responses and neutralisation remained poor following recovery from infection although T-cell responses were strong and only one patient required hospital admission. CLL-VR is the largest vaccine study conducted in patients with CLL and reveals diminished but comparable antibody responses to both the ChAdOx1 and BNT162b2 vaccines with some improvement following third primary dose of mRNA vaccine. In contrast T-cell responses following second dose are greater in those who received ChAdOx1 platform. Low neutralising activity against the delta and omicron variants highlights an ongoing risk for this vulnerable population despite repeated vaccination and reveals the need for alternative approaches to protection including prophylactic monoclonal antibody therapy..

Response to COVID-19 Vaccines in Patients Receiving Intensified Post-ASCT Therapy with Daratumumab, Lenalidomide, Bortezomib (Dara-VR) Due to Ultra-High Risk (UHiR) Newly Diagnosed Myeloma (NDMM) or Primary Plasma Cell Leukemia (pPCL): Exploratory Analysis of the UK Optimum/Muknine Trial

Background: Successful vaccination against SARS-CoV2 is highly effective in preventing serious COVID-19 illness and is particularly recommended for at risk populations including patients with multiple myeloma (MM). However, there is uncertainty to which extent modern intensified therapies targeting plasma cell features might attenuate vaccination responses;some early vaccination recommendations for MM have proposed extended treatment breaks of several weeks to maximise vaccination success. Such an approach can be challenging in UHiR MM and pPCL, where maintaining treatment intensity is hallmark for preventing rapid relapse of the aggressive tumor. To address this uncertainty, we measured post-vaccination serological responses in patients treated uniformly with intensified Dara-VR consolidation and Dara-R maintenance post-ASCT for UHiR NDMM or pPCL in the UK OPTIMUM/MUKnine trial (NCT03188172). Methods: Between Sep 2017 and Jul 2019, 107 patients with UHiR NDMM or pPCL were recruited to OPTIMUM and received intensified post-ASCT consolidation with Dara-VR(d) for 18 cycles followed by maintenance with Dara-R until progression. In an exploratory analysis, centrally stored serum samples available for patients with a completed and documented vaccination history of two doses of an anti-SARS-CoV2 vaccine were analyzed for serological vaccine responses Total IgG/IgA/IgM Anti-SARS-CoV-2 spike glycoprotein was measured by ELISA (MK654;The Binding Site). As per UK national guidance and local availability, patients received two vaccine doses 12 weeks apart of either tozinameran (Pfizer/Biontech) or vaxzevria (AstraZeneca);serum taken at least 3 weeks after patients received their second dose was analyzed. Results were correlated with baseline characteristics and annotated with treatment and response data. Patient with available matched serological and vaccination status data at time of data cut-off (09 JUL 2021) were included. Collection of vaccination status data is ongoing and updated results comprising additional patients enrolled in OPTIMUM, as well as antigen levels, will be presented. Data will also comprise longitudinal antibody level measurements for patient with available sequential material. Results: Serological vaccine response data was available for 40 OPTIMUM patients with documented completed double vaccination status. Median patient age was 58.5 years (range 39-70) and clinical and molecular tumor features were similar to the overall trial safety population. All patients had received their second dose before June 2021. Of the 40 patients, 42.5% had received tozinameran and 57.5% vaxzevria. Baseline characteristics of the two groups were comparable. At time of second vaccine dose, 55% of patients were receiving Dara-VR consolidation treatment and 45% Dara-R maintenance. There was no recommendation to pause trial treatment for purposes of vaccination and no extended times off treatment for this reason were reported. Overall, 72.5% of patients had a positive vaccine antibody level as per manufacturer cut-point for high specificity evidence of antigen exposure (infection or vaccine). The response rate was nominally higher for vaxzevria (91.3%) than for tozinameran (47.1%), a dysbalance that will be further investigated with ongoing extension of the cohort. Of note, 90% of patients analyzed had reached a complete response (CR) of their MM prior to being vaccinated, and the majority of patients not in CR had a positive vaccine response. Response rates were nominally slightly higher in patients in receipt of Dara-R maintenance at time of second dose with 77.8% compared to Dara-VR consolidation with 68.2%. Conclusions: These results show a high serological response rate to COVID-19 vaccination in UHiR MM patients receiving intensified post-ASCT consolidation and maintenance therapy in remission. Findings suggest that continuation of intensified post-ASCT therapy for patients with aggressive tumors and a high risk of relapse are compatible with serological responses to commonly used COVID-19 vaccines. Disclosures: Jen er: Janssen: Consultancy, Honoraria, Speakers Bureau;BMS/Celgene: Consultancy, Honoraria, Speakers Bureau;Takeda: Consultancy;Pfizer: Consultancy. Hall: BMS/Celgene: Research Funding;Janssen: Research Funding. Garg: University Hospital Leicester: Current Employment;Takeda Janssen Novartis Sanofi: Other: Travel Accommodations, Expenses;Amgen Janssen Novartis Sanofi Takeda: Honoraria. Jackson: J and J: Consultancy, Honoraria, Speakers Bureau;GSK: Consultancy, Honoraria, Speakers Bureau;takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau;amgen: Consultancy, Honoraria, Speakers Bureau;celgene BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau;oncopeptides: Consultancy;Sanofi: Honoraria, Speakers Bureau. Pratt: Binding Site: Consultancy;BMS/Celgene: Consultancy;Gilead: Consultancy;Janssen: Consultancy;Takeda: Consultancy;Amgen: Consultancy. Cook: Karyopharm: Consultancy;Sanofi: Consultancy;Takeda: Consultancy, Research Funding;Janssen: Consultancy, Research Funding;BMS/Celgene: Consultancy, Research Funding;Amgen: Consultancy. Drayson: Abingdon Health: Current holder of individual stocks in a privately-held company. Kaiser: BMS/Celgene: Consultancy, Other: Travel support, Research Funding;Janssen: Consultancy, Other: Educational support, Research Funding;GSK: Consultancy;Karyopharm: Consultancy, Research Funding;Pfizer: Consultancy;Amgen: Honoraria;Seattle Genetics: Consultancy;Takeda: Consultancy, Other: Educational support;AbbVie: Consultancy.

Vaccine Efficacy after Rituximab Exposure: First Interim Analysis of Virtue Project on Behalf of West Midlands Research Consortium, UK

[Formula presented] Background: Anti-CD20 B cell depleting agents are amongst the most commonly used immunotherapeutics employed in the treatment of haematological malignancy and autoimmune diseases. By inducing peripheral B cell aplasia, anti-CD20 depleting agents are hypothesised to significantly impair serological responses to neoantigens, including the SARS-CoV-2 spike glycoprotein within SARS-CoV-2 vaccines. Seropositivity following SARS-CoV-2 is the strongest, measurable correlate of protection from severe COVID-19. Understanding the kinetics of B cell reconstitution and vaccine responsiveness following exposure to B cell depleting agents is essential to maximise vaccine efficacy in patients vulnerable to severe COVID-19. Methods: 80 patients with underlying haematological malignancy and 38 patients with underlying rheumatological disease previously treated with anti-CD20 B cell depleting agents were studied following their second dose of a SARS-CoV-2 vaccine (median time to sampling: 46.5d, IQR: 33.8-63.3). Lymphocyte subset (CD4, CD8, CD19, CD56/16) enumeration was performed using 6 colour flow cytometry (BD Trucount). Total anti-SARS-CoV-2 spike glycoprotein antibodies were measured by enzyme-linked immunosorbent assay (The Binding Site, Human Anti-IgG/A/M SARS-CoV-2-ELISA). The relationship between immune reconstitution following B cell depletion and vaccine responsiveness was explored. Results: In the haematology cohort (median age 70y, IQR 60.3-76.0, 62.5% male), overall seropositivity following vaccination was 60.0%. Individuals on active chemotherapy had significantly lower seroprevalence than those vaccinated following the completion of chemotherapy (22.7% vs 74.1%, p<0.0001). In the rheumatology cohort (median age 65y, IQR 58.3-70.8, 39.9% male), overall seropositivity was 69.4%. In both cohorts, vaccine non-responders had significantly smaller populations of peripheral CD19+ B cells (haematology: 0.20 vs 0.02 x10 9/L, p=0.004, rheumatology: 0.07 vs 0.01 x10 9/L, p=0.03). The magnitude of the antibody response following vaccination did not differ between recipients of Tozinameran and Vaxzeveria in either cohort. Vaccine responsiveness was lower in the first 6 months following B cell depletion therapy;42.9% in the haematology cohort and 33.3% in the rheumatology cohort, increasing to 100% and 75% respectively in individuals receiving their second dose 6-12 months following B cell depletion (Figure 1). B cell reconstitution in the 7-12 month window following B cell depletion was faster in haematology compared to rheumatology patients (77.8% v 22.2% achieving normal B cell count, p=0.005) and associated with improved vaccine responsiveness. However, persistent immunodeficiency occurred in some haematology patients following completion of treatment: 25% of patients who had completed therapy at least 36 months previously failed to respond to vaccination. In this cohort of vaccine non-responders, 83.3% of individuals had B cell numbers within the normal range. These patients had all previously been treated for follicular lymphoma suggesting a specific mechanism for long-range secondary immunodeficiency in these patients. Conclusions: Serological responsiveness to SARS-CoV-2 vaccines is poor during active chemotherapy for haematological malignancy and in the first 6 months following B cell depletion, regardless of underlying disease. Vaccine responsiveness significantly improves in the 7-12 month window following B cell depletion. Compared to haematology patients, B cell reconstitution is slower in rheumatology patients and associated with reduced vaccine responsiveness, possibly due to the use of additional concurrent disease-modifying anti-rheumatic therapies. Furthermore, long-term secondary immunodeficiency occurs in a minority of haematology patients. To maximise the efficacy from SARS-CoV-2 booster vaccination and optimal utilisation of available vaccine doses, immunisations should be delivered at least 6 months following the administration of anti-CD20 depleting drugs. Figure 1: Kinetics of return f vaccine responsiveness following B cell depletion in haematology and rheumatology patients. [Formula presented] Disclosures: Paneesha: Roche: Honoraria;Janssen: Honoraria;Gilead: Honoraria;Bristol Myers Squibb: Honoraria;AbbVie: Honoraria;Celgene: Honoraria. Drayson: Abingdon Health: Current holder of individual stocks in a privately-held company.

Antibody responses after first and second Covid-19 vaccination in patients with chronic lymphocytic leukaemia.

B-cell chronic lymphocytic leukaemia (CLL) is associated with immunosuppression and patients are at increased clinical risk following SARS-CoV-2 infection. Covid-19 vaccines offer the potential for protection against severe infection but relatively little is known regarding the profile of the antibody response following first or second vaccination. We studied spike-specific antibody responses following first and/or second Covid-19 vaccination in 299 patients with CLL compared with healthy donors. 286 patients underwent extended interval (10-12 week) vaccination. 154 patients received the BNT162b2 mRNA vaccine and 145 patients received ChAdOx1. Blood samples were taken either by venepuncture or as dried blood spots on filter paper. Spike-specific antibody responses were detectable in 34% of patients with CLL after one vaccine (n = 267) compared to 94% in healthy donors with antibody titres 104-fold lower in the patient group. Antibody responses increased to 75% after second vaccine (n = 55), compared to 100% in healthy donors, although titres remained lower. Multivariate analysis showed that current treatment with BTK inhibitors or IgA deficiency were independently associated with failure to generate an antibody response after the second vaccine. This work supports the need for optimisation of vaccination strategy in patients with CLL including the potential utility of booster vaccines.

COVID-19: Seroprevalence and Vaccine Responses in UK Dental Care Professionals.

Dental care professionals (DCPs) are thought to be at enhanced risk of occupational exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, robust data to support this from large-scale seroepidemiological studies are lacking. We report a longitudinal seroprevalence analysis of antibodies to SARS-CoV-2 spike glycoprotein, with baseline sampling prior to large-scale practice reopening in July 2020 and follow-up postimplementation of new public health guidance on infection prevention control (IPC) and enhanced personal protective equipment (PPE). In total, 1,507 West Midlands DCPs were recruited into this study in June 2020. Baseline seroprevalence was determined using a combined IgGAM enzyme-linked immunosorbent assay and the cohort followed longitudinally for 6 mo until January/February 2021 through the second wave of the coronavirus disease 2019 pandemic in the United Kingdom and vaccination commencement. Baseline seroprevalence was 16.3%, compared to estimates in the regional population of 6% to 7%. Seropositivity was retained in over 70% of participants at 3- and 6-mo follow-up and conferred a 75% reduced risk of infection. Nonwhite ethnicity and living in areas of greater deprivation were associated with increased baseline seroprevalence. During follow-up, no polymerase chain reaction-proven infections occurred in individuals with a baseline anti-SARS-CoV-2 IgG level greater than 147.6 IU/ml with respect to the World Health Organization international standard 20-136. After vaccination, antibody responses were more rapid and of higher magnitude in those individuals who were seropositive at baseline. Natural infection with SARS-CoV-2 prior to enhanced PPE was significantly higher in DCPs than the regional population. Natural infection leads to a serological response that remains detectable in over 70% of individuals 6 mo after initial sampling and 9 mo from the peak of the first wave of the pandemic. This response is associated with protection from future infection. Even if serological responses wane, a single dose of the Pfizer-BioNTech 162b vaccine is associated with an antibody response indicative of immunological memory.

Validation of a combined ELISA to detect IgG, IgA and IgM antibody responses to SARS-CoV-2 in mild or moderate non-hospitalised patients.

BACKGROUND: Frequently SARS-CoV-2 results in mild or moderate disease with potentially lower concentrations of antibodies compared to those that are hospitalised. Here, we validated an ELISA using SARS-CoV-2 trimeric spike glycoprotein, with targeted detection of IgG, IgA and IgM (IgGAM) using serum and dried blood spots (DBS) from adults with mild or moderate disease. METHODS: Targeting the SARS-CoV-2 trimeric spike, a combined anti-IgG, IgA and IgM serology ELISA assay was developed using 62 PCR-confirmed non-hospitalised, mild or moderate COVID-19 samples, ≥14 days post symptom onset and 624 COVID-19 negative samples. The assay was validated using 73 PCR-confirmed non-hospitalised, mild or moderate COVID-19 samples, ≥14 days post symptom onset and 359 COVID-19 negative serum samples with an additional 81 DBSs. The assay was further validated in 226 PCR-confirmed non-hospitalised, mild or moderate COVID-19 samples, ≥14 days post symptom onset and 426 COVID-19 negative clinical samples. RESULTS: A sensitivity and specificity of 98.6% (95% CI, 92.6-100.0), 98.3% (95% CI, 96.4-99.4), respectively, was observed following validation of the SARS-CoV-2 ELISA. No cross-reactivities with endemic coronaviruses or other human viruses were observed, and no change in results were recorded for interfering substances. The assay was stable at temperature extremes and components were stable for 15 days once opened. A matrix comparison showed DBS to correlate with serum results. Clinical validation of the assay reported a sensitivity of 94.7% (95% CI, 90.9-97.2%) and a specificity of 98.4% (95% CI, 96.6-99.3%). CONCLUSIONS: The human anti-IgGAM SARS-CoV-2 ELISA provides accurate and sensitive detection of SARS-CoV-2 antibodies in non-hospitalised adults with mild or moderate disease. The use of dried blood spots makes the assay accessible to the wider community.