ABSTRACT
Background: Sero-studies of SARS-CoV-2 have used antibody (Ab) responses to spike (S) and nucleocapsid (N) antigens to differentiate mRNA vaccinated (S+/N-) from infected (S+/N+) individuals. We performed testing on wellcharacterized subjects to determine how repeated vaccination or infection, and time from those exposures, influence these Ab levels. Method(s): Samples from individuals with known infection status: prepandemic negative controls n=462;first-time infected n=237 (~45 days post);vaccinated after infection n= 34 (~40 days post-vaccination and ~180 days post-infection);fully vaccinated n=158 (~50 days post);boosted n=31 (~30 days post);breakthrough n=18 (~14 days post-infection);reinfected n=10 (varied). Longitudinal samples (n=51) from subjects with evidence of reinfection (symptoms and/or positive rapid antigen test), were tested to determine the impact of the order of infection and/or vaccination on the magnitude of the anti-S and anti-N IgG Ab detected in the blood. Testing was performed with MesoScale Diagnostics (Gaithersburg, MD) assay. Outcomes are presented in WHO International Binding Antibody Units (BAU/mL). The cutoff for a positive result was 18 BAU for S and 12 BAU for N. Result(s): The median amount of Ab (IQR) in BAU for each group (Figure A) was: pre-pandemic negative controls S 0.53(0.27,1.03), N 0.55(0.18,1.67);first-time infected S 114(51,328), N 70(29,229);vaccinated after infection S 4367(2479,4837), N 15(7,35);fully vaccinated S 998(586,1529), N 0.31(0.16,0.68);boosted S 2988(1768,3522), N 0.59(0.32,1.03);breakthrough S 2429(2032,3413), N 2.5(0.93,8.6);reinfected S 1533(486,4643), N 7.8(2.6,62). For the breakthrough and second infections 17% and 40% were seropositive to N, respectively. Longitudinal analysis (Figure B) of those with multiple infections showed that all those with a positive rapid antigen test for their second infection had an increase in N Ab. Conclusion(s): The prevalence of antibodies to nucleocapsid cannot be used to determine the proportion of individuals infected to SARS-CoV-2 in a vaccinated population. Booster, repeated, and breakthrough infections are associated with IgG Ab levels to S >400 BAU/mL. A majority of breakthrough infections did not elicit an Ab response to N. For those with repeated infection, a minority elicited antibody responses to N. This could be related to misdiagnosis or the burden of infection, as only those who were positive by rapid antigen assay (indicative of a high viral load) had an increase in N Ab.
ABSTRACT
Purpose: Kidney transplant recipients taking belatacept (KTR-B) have poor immune response to two-dose SARS-CoV-2 vaccination. We sought to characterize the impact of an additional vaccine dose on plasma neutralizing capacity and cellular responses as compared to that of KTRs controls (KTR-C) not taking belatacept. Method(s): Within an observational cohort, we tested 26 KTR-Bs and 27 KTR-Cs for anti-spike antibody responses before and after a third SARS-CoV-2 vaccine dose (D3) using two clinical assays (Roche Elecsys anti-S Ig and EUROIMMUN anti-S1 IgG). For a subset of 5 KTR-Bs and for all KTR-Cs we used a research assay (Meso Scale Diagnostics V-Plex [MSD]) to further assess anti-spike and RBD IgG, as well as surrogate plasma neutralizing activity (% ACE2 inhibition) versus the ancestral and delta variants. For 3 KTR-Bs, post D3 T cell response was assessed via IFN-y ELISpot and deemed positive if spot forming units > 20 per million PBMC and stimulation index > 3. Result(s): KTR-Bs had significant lower clinical anti-spike seroconversion than KTR-Cs (31% vs 74%, p=0.001) after D3 despite similar demographics, clinical factors, and vaccines administered (Table 1). No KTR-B (0/5) was seropositive by MSD anti-spike or anti-RBD IgG (Figure 1). % ACE2 inhibition versus the ancestral variant was significantly lower in KTR-Bs than in KTR-Cs (Median [IQR] 5.2 [2.8, 6.5] vs 12.5 [7.7, 23.9], p<0.01);all KTR-Bs were below a level consistent with detectable neutralizing antibody. All tested KTR-Bs (3/3) had a negative ELISpot, consistent with negligible cellular response. Conclusion(s): These results suggest minimal humoral or cellular immunogenicity of additional vaccine doses for KTR-Bs and indicates the need for alternative strategies to improve vaccine response such as immunosuppression alteration or use of passive immunoprophylaxis with monoclonal anti-spike antibody to improve protection versus SARS-CoV-2.
ABSTRACT
Purpose: Solid organ transplant recipients (SOTRs) are at increased risk for severe COVID-19 and exhibit lower antibody responses to SARS-CoV-2 vaccines. This study aimed to determine if pre-vaccination cytokine levels are associated with antibody response to SARS-CoV-2 vaccination. Method(s): A cross-sectional study was performed among 58 SOTRs before and after two-dose mRNA vaccine series, 35 additional SOTRs before and after a third vaccine dose, with comparison to 16 healthy controls (HCs). Anti-spike antibody was assessed using the IgG Euroimmun ELISA. Electrochemiluminescence detectionbased multiplexed sandwich immunoassays were used to quantify plasma cytokine and chemokine concentrations (n=20 analytes). Concentrations between SOTRs and HCs, stratified by ultimate antibody response to the vaccine, were compared using Wilcoxon-rank-sum test with false discovery rates (FDR) computed to correct for multiple comparisons. Result(s): In the study population, 100% of HCs, 59% of SOTRs after two doses and 63% of SOTRs after three doses had a detectable antibody response. Multiple baseline cytokines were elevated in SOTRs versus HCs. There was no significant difference in cytokine levels between SOTRs with high vs low-titer antibodies after two doses of vaccine. However, as compared to poor antibody responders, SOTRs who went on to develop a high-titer antibody response to a third dose of vaccine had significantly higher pre-third dose levels of several innate immune cytokines including IL-17, IL-2Ra, IL-6, IP-10, MIP-1alpha, and TNF-alpha (FDR <0.05). Conclusion(s): A specific inflammatory profile or immune state may identify which SOTRs are likely to develop stronger sero-response and possible protection after a third dose of SARS-CoV-2 vaccine.
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Purpose: The impact of antigenic imprinting, when immune memory of one antigen influences the response to subsequent similar antigens, on the antibody response in solid organ transplant recipients (SOTRs) after SARS-CoV-2 vaccination is currently unknown. This study examines the relationship between seasonal coronaviruses (sCoV) and SARS-CoV-2 antibody levels pre- and post-vaccination in SOTRs. Method(s): Plasma from 52 SOTRs pre- and post-SARS-CoV-2 vaccination (2 doses, mRNA) was analyzed using the Meso Scale Diagnostic Coronavirus Panel 3 (an electrochemiluminescence detection-based multiplexed sandwich immunoassay) for IgG antibodies against alpha sCoVs (229E, NL63), beta sCoVs (HKU1, OC43), and SARS-CoV-2 spike proteins. Changes in IgG titers were determined by paired Wilcoxon rank-sum tests. Spearman correlation analysis was used to determine associations between pre-vaccination anti-sCoVs and post-vaccination anti-SARS-CoV-2 IgG. Result(s): Vaccination increased both anti-SARS-CoV-2 (fold change (FC) 1.9, p<0.001) and anti-beta sCoV (HKU1 [FC 0.05, p<0.001], OC43 [FC 0.8, p<0.001]) IgG titers in SOTRs, but did not increase anti-alpha sCoV IgG. Furthermore, prevaccination anti-beta sCoV (HKU1 [rho= -0.3, p=0.03], OC43 [rho= -0.3, p<0.03]) IgG titers were negatively correlated with post-vaccination anti-SARS-CoV-2 IgG. Conclusion(s): These exploratory findings suggest that prior exposure to seasonal betacoronaviruses may lead to antigenic imprinting in SOTRs that negatively impacts the antibody response to vaccination against the novel pandemic betacoronavirus, SARS-CoV-2.
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Purpose: While SARS-CoV-2 vaccination has dramatically reduced COVID-19 severity in the general population, fully vaccinated solid organ transplant recipients (SOTRs) demonstrate reduced seroconversion and increased breakthrough infection rates. Furthermore, a third vaccine dose only increases antibody and T cell responses in a proportion of SOTRs. We sought to investigate the underlying mechanisms resulting in varied humoral responses in SOTRs. Method(s): Within a longitudinal prospective cohort of SOTRs, anti-spike IgG, total and spike-specific B cells were evaluated in 44 SOTR participants before and after a third vaccine dose using high dimensional flow cytometry to assess immunologic and metabolic phenotypes. B cell phenotypes were compared to those of 10 healthy controls who received a standard two-dose mRNA series. Result(s): Notably, even in the absence anti-spike antibody after two doses, spikespecific B cells were detectable in most SOTRs (76%). While 15% of participants were seropositive before the third dose, 72% were seropositive afterward. B cells, however, were differentially skewed towards non-class switched B cells in SOTRs as compared to healthy control B cells. Expansion of spike-specific class-switched B cells in SOTRs following a third vaccine dose correlated with increased classswitched (IgG) antibody titers. Antibody response to a third vaccine dose was associated with expanded populations of germinal center-like (CD10+CD27+) B cells, as well as CD11c+ alternative lineage B cells with specific upregulation of CPT1a, the rate limiting enzyme of fatty acid oxidation and a preferred energy source of germinal center B cells. Conclusion(s): This analysis defines a distinct B cell phenotype in SOTRs who respond to a third SARS-CoV-2 vaccine dose, specifically identifying fatty acid oxidation as pathway that could be targeted to improve vaccine response such as through targeted immunosuppressive modulation. (Figure Presented).
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Background: Recent studies have shown that vaccinated individuals harbor cross-reactive T cell responses that can cross-recognize SARS-CoV-2 and endemic human common cold coronaviruses (HCoVs). However, it is still unknown whether CD4+ T cells from vaccinated individuals recognize peptides from bat coronaviruses that may have the potential of causing future pandemics. In this study, we identified a SARS-CoV-2 spike protein epitope (S815-827) that is conserved in coronaviruses from different genera and subgenera including SARS-CoV, MERS-CoV, multiple bat coronaviruses and a feline coronavirus. We hypothesized that S815-827 is recognized by vaccinated individuals, and that S815-827-reactive T cells can cross-recognize homologues bat coronaviruses. Methods: To evaluate CD4+ T cell responses, we isolated CD8 depleted PBMCs from COVID-19 vaccinated individuals and performed IFN-γ ELISPOT assays. To assess T cell cross-reactivity, S815-827-reactive T cell lines were re-stimulated with homologous coronavirus peptides and cytokine production was assessed with flow cytometry. Additionally, the Vira-FEST assay (which utilizes TCR Vβ CDR3 sequencing) was performed to identify cross-reactive CD4+ T cell clones. Statistical comparisons were done using Mann-Whitney test, Wilcoxon matched-pairs signed rank test or Friedman test with Dunn's multiple comparison as appropriate. Results: Our results show that 16 out of 38 (42%) of vaccinated participants in our study who received the Pfizer-BioNTech (BNT162b2) or Moderna (mRNA-1273) COVID-19 vaccines had robust CD4+ T cell responses to S815-827. All responders also recognized homologous peptides from at least 2 other coronaviruses, and 8 out of 11 responders recognized peptides from at least 6 out of the 9 other coronaviruses tested. To determine T cell cross-reactivity, we re-stimulated S815-827 specific T cell lines with homologous coronavirus peptides. We found that S815-827 specific T cells had a robust increase in IFN-γ+ TNF-α+ expression upon re-stimulation with other peptides. We next used the Vira-FEST assay to confirm cross-reactivity by assessing if the same CD4+ T receptor clonotypes recognize both S815-827 and homologous bat coronavirus peptides. In all 3 participants tested, we identified multiple cross-reactive T cell receptors that recognize both S815-827 and homologous bat coronavirus peptides. Conclusion: Our results suggest that current mRNA vaccines elicit T cell responses that can cross-recognize bat coronaviruses, and thus might induce protection.
ABSTRACT
Background: The prevalence of vaccinated, previously infected, and individuals at risk of SARS-CoV-2 infection is important for epidemiologic studies and public health interventions. Asymptomatic infections and reluctance to disclose vaccination status hinder accurate assessments of the current state of the epidemic. Since COVID-19 vaccines generate immune responses to spike (S1), but not nucleocapsid (N), it is possible to differentiate between vaccinated, infected, and unexposed individuals by comparing antibody reactivity to each antigen. The MSD V-Plex SARS-CoV-2 IgG assay can potentially differentiate each state in one test by simultaneously evaluating IgG reactivity to the S1, receptor binding domain (RBD), and N proteins. Methods: The MSD assay was validated with three sample sets: known vaccination with no previous infection (n=158);known infected and not vaccinated (n=157);and samples collected prior to the COVID-19 pandemic in 2016 (n=144). Of the previously infected individuals, 15 (9.6%) were hospitalized;sample collection occurred a median of 48 days after a PCR-positive result. Using an algorithm, samples with positive results on both S1 and RBD but negative on N were classified as vaccinated. Samples with a positive result on all three proteins were considered to be infected with the possibility of subsequent vaccination. Any other result was classified as unexposed. Sensitivity and specificity for each state were calculated. Results: Reactivity to each antigen is shown in the figure. 100% (95% confidence interval [CI] 97.7-100), 92% (95% CI 86.3-95.5), and 0.7% (95% CI 0.02-3.8) of vaccinated, infected, and unexposed samples were positive for S1. 100% (95% CI 97.7-100.0%), 91% (95% CI 85.5-95.0%), and 0.7% (95% CI 0.02-3.8%) of vaccinated, infected and unexposed samples were positive for RBD. 0% (95% CI 0-2.3), 86% (95% CI 79.6-91.0), and 2.1% (95% CI 0.4-6.0) of vaccinated, infected and unexposed samples were positive for N. Algorithm sensitivity and specificity for classification of vaccinated samples were 100% (95% CI 97.7-100) and 96.7 (95% CI 94-98.4). For the classification of samples from previously infected individuals, sensitivity and specificity were 83.4% (95% CI 76.7-88.9) and 100% (95% CI 98.8-100). Conclusion: This study establishes the sensitivity and specificity for a high-throughput assay ideal for SARS-CoV-2 seroprevalence studies. Future research should focus on applying this assay in health care settings to guide practice and policy to mitigate the pandemic.