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.