Determinants used to evaluate vaccine-mediated protection include measurement of antigen-specific antibodies; quality of such antibodies such as avidity, specificity or neutralization capacity; long-term persistence of these antibodies above the protective thresholds; and maintenance of immune memory cells that can respond rapidly in case of subsequent microbial exposure. In the current study, we determined the ASSA levels from the 14^th day post-first vaccine dose to three months post-second vaccine dose. We have shown that the ASSA levels peak on the 14^th day post-second vaccine dose and decline within three months thereafter in the CN group. Overall, the responder rate to Covishield was 83% on day 14, 98% on day 28 after the first dose, and 100% on day 14 after the second dose. Our results were similar to other studies after the first dose of ChAdOx1 [8,9].

In our study, the ASSA responses in the CN HCWs (GM – 1206 U/ml) at 14 days after the second dose were 6-fold higher than the PCPs (GM – 209 U/ml). The ASSA response after a natural infection is much less than that shown in other studies [10]. After the second dose, there was a 27-fold increase in the ASSA levels on the 14^th day in the CN group. Thus the first dose of Covishield elicited an antibody response as good as that of the natural infection among the South Indian population. Our findings also clearly indicate that the ASSA levels declined by 4-folds within three months after the second dose. The increase after the second dose was significantly greater when the second vaccine dose was taken 10-14 weeks after the first dose rather than four weeks. Our data support the current national recommendation for the time gap between the first and second doses. The longer time gap allows the activated cells to come back to the resting stage. A second dose at this time will work as a booster. On the other hand, if the second dose is given when the ASSA levels are high, it would result in anergy leading to negligible or small increase in antibody levels.

In the previously SARS-CoV2 infected mild (MCP) and asymptomatic (PEP) groups, the first vaccine dose boosted very high responses on the 14^th day. These responses only declined steadily up to the three months after the second dose. Throughout the longitudinal study period, the ASSA levels were 1.2 to 1.5-fold higher in the MCP group (mild) compared to the PEP group (asymptomatic). This indicates that the boosted ASSA responses were higher when the previous disease severity was higher. Similar to our findings, Velasco et al showed that the booster effect of the second dose was inversely proportional to the disease severity prior to the vaccination, and the booster effect was lower in the groups with a stronger response to the first prime dose [10]. These findings together suggest that in the previously infected groups the first vaccine dose worked as a robust booster of the memory cells and the second dose probably resulted in anergy or activation induced cell death due to the continuous challenge of the same immunogen within a short period of time. On the 14^th day after the second dose even though the ASSA levels declined they were still 8-fold and 11-fold higher in the PEP and MCP groups compared to the CN group. This probably suggests that a single vaccine dose works as a sufficient booster in the previously infected asymptomatic or mild groups. Additionally, checking the ASSA levels prior to the second dose might be beneficial to decide on the time gap between the two doses.

Additionally, in our study, we had 14% of BTI, most of which were after 28 days of the second vaccine dose when the GM of the ASSA levels was 1007 U/ml. This ASSA GM was almost 5-fold higher than the convalescent levels in the PCP group. The GM of ASSA levels at three months increased by 15-folds in the BTI group. However, all the HCWs had only mild symptoms and were managed as out-patients. Few other studies have shown similar BTI that are asymptomatic or mildly symptomatic after ChAd0X1 and the BNT1262b2, in spite of high antibody responses [1,10,11]. This suggests that the presence of ASSA at the time of BTI reduced the clinical severity of the disease.

In conclusion, our study delineates the longitudinal trend of the ASSA responses in vaccinees over a period of three months post-second dose both in CN and previously exposed HCWs. There is a clear benefit in giving the booster dose at least 3-4 months after the first priming dose. In the previously infected population, we have clearly shown that the first dose already acted as a booster. Since a number of HCWs could have been exposed asymptomatically to SARS-CoV2, it might be beneficial to check the ASSA levels before deciding on the time of the second dose. The ASSA levels declined considerably within three months suggesting that the ASSA are short-lived. However, future studies are warranted to determine further decline in later time points like six and 12 months that are crucial to understand the level of secondary and memory B cell responses to this vaccine. Additionally, it is also important to understand whether these ASSA would be present locally in the salivary and nasal glands to provide immediate protection against the invading SARS-CoV2.