Long-Term Humoral Immune Dynamics in Healthcare Workers with Hybrid and Vaccine-Induced Immunity: The VaCoMRI Study

Objectives: The VaCoMRI study tracked a cohort of healthcare workers from 2020 through June 2024 to characterize the long-term sustainability of hybrid and vaccine-induced SARS-CoV-2 immunity. Methods: An initial anti-nucleocapsid (N) IgG screening of 4554 health-care workers in early 2020 identified participants who had been infected during the first wave. Anti-N-positive and anti-N-negative individuals then received BNT162b2 through the institutional vaccination program, yielding groups with hybrid and vaccine-induced immunity. Both groups were monitored regularly for nearly four years for breakthrough infections (BTI). Serum collected regularly after vaccination and after BTI was analyzed for anti-N, quantitative anti-spike (anti-S) IgG, and surrogate viral neutralizing antibody (sVNT) titers. Results: Over a median follow-up of 1180 days, 142 healthcare workers contributed longitudinal samples; 66.2% experienced one BTI and 14.1% a second. In initially seronegative individuals, anti-N titers decreased 2.3-fold between 3 and 6 months after the first BTI, with seropositivity dropping from 82% to 40.4% (median time to seronegativity: 179 days). Prior anti-N seropositivity was associated with 4.5-fold higher anti-N IgG levels after BTI, with elevated levels persisting through 9 months. In both groups, the third vaccine dose significantly enhanced anti-S antibody persistence after 9 months compared to the second dose. In infection-naïve individuals, anti-S IgG titers remained 4.2-fold and sVNT titers 7.2-fold higher. Hybrid immunity was similarly durable: two vaccine doses after prior anti-N seropositivity yielded anti-S IgG and sVNT levels comparable to three-dose vaccination. Conclusions: This study demonstrates a rapid waning of anti-N IgG that severely limits its reliability for retrospective serosurveillance of SARS-CoV-2 exposure in occupational settings. The combination of vaccination and infection induced robust and durable spike-directed antibody responses. However, these were determined using assays based on the ancestral spike protein. This may limit the prediction of protection from SARS-CoV-2 variants, which usually cause BTIs.