Rapid decline of neutralizing antibodies against SARS-CoV-2 among infected healthcare workers

Healthcare workers (HCW) face high exposure to SARS-CoV-2 and potential risk of reinfection, yet data on the persistence of neutralizing antibodies (NAbs) after mild COVID-19 have been limited. This study investigates the durability and kinetics of humoral immunity—including IgG, IgA, and IgM specific to SARS-CoV-2 antigens (N, S, and RBD)—and functional neutralization activity over the first three months following symptom onset. The authors hypothesized that neutralizing activity would wane over time, potentially driven by declines in specific antibody isotypes, particularly IgA, with implications for ongoing susceptibility and vaccination strategies for HCW.

The authors note that their findings align with prior reports of high seroconversion rates and typical antibody kinetics in SARS-CoV-2–infected patients. For SARS-CoV-1, peak IgG levels were reported around four months post-symptom onset and remained stable for at least one year, suggesting longer durability than observed here for SARS-CoV-2 neutralizing activity. Neutralization is primarily mediated by antibodies to the spike (S) protein and its receptor-binding domain (RBD), given their role in ACE2-mediated entry. Prior studies indicate limited cross-neutralization between endemic human coronaviruses and SARS-CoV-2 and variable durability of humoral responses, reinforcing the need to track S/RBD-specific responses and NAb titers over time.

Design and cohort: Retrospective observational follow-up of 26 HCW with mild COVID-19. Sampling at approximately 3 weeks (D21), 2 months (M2), and 3 months (M3) after symptom onset. Median timing: D21 at 17.1 days [14–23.5], M2 at 59 days [57–61], M3 at 97 days [91–100]. Median time from symptom onset to positive RT-PCR: 2 days [1–3]. Virologic diagnosis used RT-PCR; reported Ct at diagnosis median 22 [18.7–24.2]. Participant characteristics included sex, age, occupation, exposure, and symptoms (Table 1). Assays: Six commercial immunoassays measured isotype-specific antibodies to SARS-CoV-2 antigens: N (nucleocapsid), S (spike), and RBD. Cutoffs: OD index ratio 0.8 for anti-N IgG; 1.0 for anti-S IgG/IgA and anti-N/anti-S IgM; and 5× the cutoff for anti-RBD IgG/IgA. Neutralization: Whole-virus replication virus neutralization test (VNT) quantified NAb titers as the highest serum dilution achieving 100% inhibition of cytopathic effect against a local clinical SARS-CoV-2 isolate (GenBank MW322968). Additional neutralization experiments employed S-Fuse cells to measure the half-maximal effective concentration (EC50) of purified IgA and IgG fractions from sera of three participants at D21 and M3. Specificity and cross-neutralization controls: Ten sera from individuals with recent infections by seasonal HCoVs (2 HKU1, 3 OC43, 3 NL63, 2 229E; 11–54 days post-infection) were tested in SARS-CoV-2 serology and neutralization assays to assess cross-reactivity and cross-neutralization. Statistics: Wilcoxon signed-rank tests were used for paired comparisons of antibody levels and NAb titers across time points; Spearman rank correlation assessed associations between NAb titers and antigen/isotype-specific antibody levels. Ethics: Conducted per the Declaration of Helsinki; retrospective non-interventional study with appropriate data protection procedures. Data availability: Source data provided; viral strain accession MW322968.

- Serology at D21: All HCW had detectable IgG to N, IgA to RBD, and IgA to S; 92.3% (24/26) had anti-RBD IgG; 80.8% (21/26) had anti-S IgG; 42.3% (11/26) had anti-N1 IgG. - Serology over time: At M2, anti-S IgA seropositivity declined to 88.2% (15/17) and anti-RBD IgA to 47% (8/17); anti-S IgG and anti-RBD IgG remained high (94.1% and 100%, respectively). At M3, anti-S IgA was 80.8% (21/26); anti-RBD IgG 92.3% (24/26); anti-RBD IgA 58.3% (14/24); anti-N1 IgG detected in 57.7% (15/26). Anti-S and anti-RBD IgA levels declined significantly from D21 to later time points (p < 0.001). Anti-S and anti-RBD IgG showed less change, with transient increase at M2 and decrease by M3; overall D21 to M3 decrease significant for IgG (p < 0.001). Anti-N1 IgG did not change significantly (D21 vs M2 p = 0.18; D21 vs M3 p = 0.95). - Neutralization kinetics: At D21, 100% of sera had NAbs with median titer 1.20 [1.10–1.40]. At M2, most remained neutralizing (median titer 1.20 [1.15–1.20]); by M3, 84.6% remained neutralizing with median titer 1.10 [1.05–1.20]. NAb titers decreased significantly D21→M2 (p = 0.03) and D21→M3 (p < 0.001). Sera that lost neutralizing capacity lacked detectable anti-RBD IgA at those later time points. - Correlates of neutralization: Strong associations between NAb titers and anti-RBD IgG (Spearman r = 0.73; 95% CI 0.58–0.82; p < 0.0001) and anti-RBD IgA (r = 0.64; 95% CI 0.47–0.77; p < 0.0001). Purified IgA fractions had greater neutralizing capacity than IgG at D21 (IgA EC50 < IgG EC50), whereas IgG surpassed IgA at M3. - Specificity: No cross-neutralization of SARS-CoV-2 by sera from recent seasonal HCoV infections; some serology cross-reactivity controls indicated specificity of assays.

The study demonstrates that mild SARS-CoV-2 infection in HCW induces robust early seroconversion and neutralizing activity by three weeks post-symptom onset. However, neutralizing activity wanes within two to three months, paralleling significant declines in anti-S and especially anti-RBD IgA. Early neutralization appears to be driven predominantly by IgA, consistent with mucosal/systemic IgA responses to respiratory pathogens. By three months, IgG increases in relative contribution and neutralizing potency (potentially due to affinity maturation), yet this does not fully compensate for the decline in IgA-mediated neutralization, resulting in reduced overall serum neutralizing activity and loss of neutralization in a subset of individuals. The strong correlation between NAb titers and anti-RBD antibodies underscores the central role of RBD-targeting responses in functional immunity. Lack of cross-neutralization from seasonal HCoV sera supports antigenic specificity of SARS-CoV-2 neutralization. Collectively, these results suggest a transient nature of humoral neutralization after mild infection, implying potential vulnerability to reinfection over time and emphasizing ongoing infection prevention measures for HCW.

Mild SARS-CoV-2 infection in HCW elicits early, robust neutralizing responses closely linked to anti-RBD antibodies, with IgA dominating early neutralization. Neutralizing titers decline significantly within two to three months, and some individuals lose detectable neutralization, concomitant with falling anti-RBD IgA. These findings support continued infection control in healthcare settings and suggest that booster vaccination may be needed to sustain protective humoral immunity. Future research should extend follow-up beyond three months, assess mucosal (secretory) IgA and cellular immunity, define protective thresholds for neutralization, and evaluate how vaccination and boosters modulate isotype dynamics and durability in previously infected and naïve HCW.

- Small, single-center cohort (n = 26), limiting generalizability. - Predominantly mild disease; findings may not extrapolate to severe cases. - Short follow-up (to 3 months); longer-term durability not assessed. - Incomplete sampling at M2 (n = 17), introducing potential attrition bias. - Use of multiple commercial immunoassays with specific cutoffs may affect comparability and sensitivity; some inconsistencies in reported seropositivity percentages across time points. - Neutralization assays used a single viral isolate and in vitro readouts; in vivo protection was not measured. - Limited cross-reactivity assessment with a small panel of seasonal HCoV sera.