Hidden asymptomatic breakthrough infection in a proof-of-concept longitudinal study on SARS-CoV-2 vaccine recipients

Background Individuals with asymptomatic SARS-CoV-2 infection can unknowingly transmit the virus, yet identifying such subclinical infections in post-vaccinated populations remains challenging. Methods We conducted a longitudinal study of 129 infection-naïve vaccine recipients immunized with various combinations of SARS-CoV-2 spike (S) protein vaccine platforms. Sera were collected before the first dose ( v 1), at 2 weeks ( v 7) and 6 months ( v 8) after the third dose. Taiwan’s first major COVID-19 outbreak occurred between v 7 and v 8. We measured anti-nucleocapsid (anti-N) and anti-S IgG antibody titers by ELISA and assessed virus-neutralizing activity using live virus and pseudovirus assays. Results By developing an iterative serial screening method, we identified asymptomatic breakthrough (post-vaccination) infections among unconfirmed cases. Our v 7- v 8 paired cohort resolved into three distinct groups: confirmed cases (21%), asymptomatic breakthrough infections (17%), and uninfected subjects (62%). In normalized v 8 sera, confirmed cases exhibited an anti-S +++ (high) /anti-N +++ (high) phenotype, while uninfected subjects showed an anti-S + (low)/anti-N + (baseline) phenotype. Statistical analysis validated a distinct asymptomatic group characterized by an antibody profile anti-S ++ (intermediate) /anti-N + (baseline). Conclusions This approach may enable more accurate estimates of vaccine efficacy and infection prevalence. In a spike-vaccinated population, anti-N antibody is more a potential specific marker for COVID-19 symptomatic disease than an ideal marker for SARS-CoV-2 infection. To our knowledge, this is the first preliminary report of identification of asymptomatic breakthrough infection from a well-vaccinated population using self-matched longitudinal pairs of serum samples.