ABSTRACT
Background: Development of neutralizing antibody (Nab) is crucial for protection from SARS-CoV-2 reinfection. The aim of the study was to analyze Nab titers (NabT) and kinetics over time in a cohort of 85 unselected not hospitalized Italian subjects (pts) with COVID-19 infection, with mild or no symptoms, tested after symptoms onset or for surveillance of healthcare workers. Methods: Two-fold serial dilutions of heat-inactivated sera were incubated with 100 TCID50 of SARS-CoV-2 virus (lineage B) at 37°C for 1 h in 96-wells plates. Then, pre-seeded 10,000 Vero E6 cell lines per well (ATCC CRL-1586) were treated with serum-virus mixtures and incubated at 37°C. After 72h, cell viability was determined through the commercial kit Cell-titer Glo 2.0 (Promega). The NabT was defined as the reciprocal value of the sample dilution that showed a 50% protection of virus cytopathic effect (ID50). NabT ≥5 ID50 were defined as SARS-CoV-2 positive and neutralizing. Chi squared, Wilcoxon, Fisher's exact test and Spearman's correlation coefficient were used. Results: Female were 57 (67.1%) and median age was 48 years. Pts were classified as early tested (ET, <60 days, n=40) and late tested (LT, >60 days, n=45). Overall, 30 (35.3%) pts had low (<10 ID50) NabT, 33 (38.8%) had intermediate NabT (ID50 11 to 50), and 22 (25.9%) had high NabT (ID50 >51, 9 of them >100). The frequency of each NabT class was comparable in ET and LT: low NabT was found in 11 (27.5%) and 19 (42.2%), intermediate in 16 (40%) and 18 (40%) and high in 13 (32.5%) and 8 pts (17.8%), respectively. However, no NabT higher than 200 ID50 was found in LT vs 5 in ET (p=0.04). No correlation between age and gender and NabT was found. At now, 23 pts had 2 NabT available during the follow up (T1 and T2);the interval between the two tests was 117 days (median). Almost all pts (20/23, 87%), showed NabT decrease: the median value of percentage decrease respect to the previous value was 63.2% (IQR 30.8-85.7) (p=0.0006). One pt had undetectable NabT at both times and two pts experienced an increase (Figure 1). No correlation between interval length, T2 value and percentage of decrease was observed. Conclusion: One third of pts had a very low level of NabT regardless of test timing. After 60 days from diagnosis a subset of pts had a value >100 but no >200, differently from the first 60 days, suggesting that NabT level could be a useful tool for dating past infections. The observed decrease of NabT has implications for reinfection and vaccine.