Serological Assays for Differentiating Natural COVID-19 Infection from Vaccine Induced Immunity

Background: Natural SARS-CoV-2 infection may elicit antibodies to a range of viral proteins including non-structural protein ORF8. RNA, adenovirus vectored and sub-unit vaccines expressing SARS-CoV-2 spike would be only expected to elicit S-antibodies and antibodies to distinct domains of nucleocapsid (N) protein may reliably differentiate infection from vaccine-elicited antibody. However, inactivated whole virus vaccines may potentially elicit antibody to wider range of viral proteins, including N protein. We hypothesized that antibody to ORF8 protein will discriminate natural infection from vaccination irrespective of vaccine type.Methods: We optimized and validated the ORF8 and N C-terminal domain (N-CTD) ELISA assays using sera from pre-pandemic, RT-PCR confirmed natural infection sera and BNT162b2 (BNT)/CoronaVac vaccinees. We then applied these optimized assays to a cohort of blood donor sera collected in April-July 2022 with known vaccination and self-reported infection status.Results: We optimized cut-off values for the ORF8 and N-CTD IgG ELISA assays using receiver-operating-characteristic (ROC) curves. The sensitivity of the ORF8 and N-CTD ELISA for detecting past infection was 83.2% and 99.3%, respectively. Specificity of ORF8 ELISA was 96.8% vs. the pre-pandemic cohort or 93% considering the pre-pandemic and vaccine cohorts together. The N-CTD ELISA specificity of 98.9% in the pre-pandemic cohort, 93% in BNT vaccinated and only 4% in CoronaVac vaccinated cohorts. N-CTD antibody was longer-lived than ORF8 antibody after natural infection.Conclusions: N-CTD antibody assays provide good discrimination between natural infection and vaccination in BNT162b2 vaccinated individuals. ORF8 antibody can help discriminate infection from vaccination in either type of vaccine and help estimate infection attack rates (IAR) in communities.