ANTIBODIES TO SARS-CoV-2 VARY DUE TO ORDER AND FREQUENCY OF VACCINATION OR INFECTION

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.

Immunogenicity of a 3rd Dose of Mrna Covid-19 Vaccine in Kidney Transplant Recipients Who Failed to Respond to 2 Prior Doses

Purpose: Kidney transplant recipients (KTRs) have diminished immune response and protection after 2-dose mRNA COVID-19 vaccination. It is unknown if additional doses improve neutralization of variants of concern (VOC) in KTRs with prior poor seroresponse. Method(s): Adult KTRs with negative (<0.8 U/mL) or low (<=50 U/ml) anti-RBD Ig (Roche Elecsys anti-SARS-CoV-2-S) after 2-dose mRNA series were given a homologous 3rd dose (D3). Anti-RBD and VOC surrogate neutralization (%ACE2i) were measured 30 days post D3;responses were stratified by baseline anti-RBD. Reactogenicity, serial SARS-CoV-2 swabs, and donor-specific antibody (DSA) were assessed. Result(s): 81 KTRs (50% negative anti-RBD) received D3 (72% BNT162b2, 28% mRNA-1273) at median 167 days post D2 (Table). Median (IQR) anti-RBD increase was 410 (8-2309) U/mL with 69% (40% negative vs 98% low anti-RBD) achieving day 30 anti-RBD >50 U/ml (Fig1a). 22% remained seronegative. Non-response was associated with lower baseline lymphocyte count (median 770 vs 1160 cells/ uL;p=0.05) and IgG (median 779 vs 979 mg/dL;p<0.01), but not demographics, vaccine, or immunosuppressives. Median (IQR) delta variant %ACE2i increased from 6% (3-7) to 10% (4-22) (p<0.001), a 1% (0-5) increase in negative vs 13% (5-25) in low anti-RBD. %ACE2i was linearly associated with anti-RBD >=100 U/ mL (all VOC shown in Fig1b);64% of KTRs with anti-RBD >=250 U/mL had delta %ACE2i >20. There were 3 cases of mild-moderate COVID-19 >=7 days post-D3, with pre-infection anti-RBD <0.4, 22, 76 U/mL and delta %ACE2i 6, 9, and 16, respectively. There was no acute rejection, nor increased or de novo DSA. Conclusion(s): A 3rd mRNA vaccine dose increased anti-RBD and VOC neutralization in KTRs without inducing clinical alloimmunity, yet 45% with negative baseline anti-RBD remained seronegative without delta variant neutralization. Trials are ongoing to test immune response augmentation in this subgroup via temporary immunosuppression reduction or heterologous boosting.

Humoral and Cellular Immune Response to a Third Dose of SARSCoV- 2 Vaccine in Kidney Transplant Recipients on Belatacept

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.

Higher Proinflammatory Cytokines Are Associated with Increased Antibody Titer After a Third Dose of SARS-CoV-2 Vaccine in Solid Organ Transplant Recipients

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.

Pre-Vaccine Antibodies Against Seasonal Betacoronaviruses Are Associated with Decreased Antibody Response to SARS-CoV-2 mRNA Vaccination in Solid Organ Transplant Recipients

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.

Effect of a Fourth COVID-19 Vaccine Dose on Omicron Variant Neutralization in Solid Organ Transplant Recipients

Purpose: Humoral response to COVID-19 vaccines is attenuated in many solid organ transplant recipients (SOTRs), necessitating additional primary and booster vaccinations. The omicron variant demonstrates substantial immune evasion, and it is not known if boosters increase neutralizing capacity versus omicron among SOTRs. We therefore investigated SOTR antibody response and neutralization versus variants of concern (VOC) including omicron to a 4th vaccine dose (D4). Method(s): Within a national, prospective observational cohort, 25 SOTRs underwent anti-SARS-CoV-2 spike and receptor binding domain (RBD) IgG testing using the Meso Scale Discovery platform before and 2-4 weeks after D4. Surrogate neutralization (%ACE2 inhibition [%ACE2i], range 0-100% with >20% correlating with live virus neutralization), was measured versus full spike proteins of the ancestral ("vaccine") strain and 5 VOCs including delta and omicron. Change in IgG level and %ACE2i were compared using paired Wilcoxon rank-sum testing. Result(s): Demographics are outlined in Table 1, including median (IQR) age 59 (45- 55) years, 64% kidney recipients, and D4 receipt (60% Moderna, 40% Pfizer) median (IQR) 93 days (28-134) post D3. Two participants had SARS-CoV-2 exposure per anti-nucleocapsid testing, including one incident infection. Overall, anti-RBD (92%- >100%) and anti-spike (84%->92%) seropositivity increased after D4, as did median (IQR) anti-spike IgG 42.3 (4.9-134.2)->228.9 (115.4-655.8) WHO binding antibody units (p<0.05). Median (IQR) %ACE2i significantly increased after D4 vs the vaccine strain 5.8% (0-16.8)->20.6% (5.8-45.9) and delta variant 9.1% (4.9-12.8)->17.1% (10.3-31.7) (both p<0.001). In contrast, no SOTR showed neutralization vs omicron before or after D4: median (IQR) %ACE2i 4.1% (0-6.9)->0.5% (0-5.7) (p=0.11). Conclusion(s): Although a 4th vaccine dose increased anti-spike IgG and neutralizing capacity vs some VOC, there was no omicron variant neutralization among SOTRs. SOTRs may remain at high risk for SARS-CoV-2 infection despite boosting, thus additional protective interventions should be urgently explored. (Figure Presented).

B Cells Fit for Germinal Center Activity Predict Response to a Third Dose of SARS-CoV-2 Vaccine in Solid Organ Transplant Recipients

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).

MSD V-PLEX SARS-CoV-2 ASSAY to IDENTIFY VACCINATED and INFECTED INDIVIDUALS

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.