According to the World Health Organization (WHO), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is estimated to have caused 446.5 million infections and more than 6.0 million deaths over the past 2 years (1). Vaccines are expected to be the most effective and economical means to prevent and control coronavirus disease 2019 (COVID-19) (2). Immunity to SARS-CoV-2 induced through either natural infection or vaccination has been shown to provide some degree of protection against reinfection/infection and reduce the risk of clinical fatality (3). Mass vaccination has played an important role in the effective control of the SARS-CoV-2 epidemic worldwide and protected from infection and severe disease (4–8). The CoronaVac vaccine is an inactivated vaccine and has been approved for emergency use in several countries and has been crucial for curbing the pandemic (9). A recent real-world study conducted in Guangzhou (China) showed that the protection rate of two doses of an inactivated vaccine against delta variant infection exceeded 50% (8).

However, the decline in vaccine efficacy over time remains a major concern (10), as the efficacy of mRNA vaccine was shown to obviously decline gradually over a six-month follow-up period (4). In our previous study, the half-lives of the neutralizing antibody, anti-RBD total antibody, anti-Spike IgG and anti-Spike IgM were 35.61 days, 36.46 days, 30.33 days and 13.54 days, respectively, and the neutralizing antibody seropositive rate dropped to only 19.67% at 160 days after vaccination (11).The decay of vaccine-induced anti-Spike IgG and anti-Spike IgA were faster than that those reported after natural SARS-CoV-2 infection (12). In addition, recently emerged SARS-CoV-2 variants may pose a threat to immunity. Lineage B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) significantly escaped natural infection-mediated neutralization, with average live virus neutralization titer reductions of 4.1-fold, 1.8-fold, and 3.2-fold, respectively (13, 14). For B.1.1.529 (Omicron), serum from vaccinees also showed to significant reductions in the geometric mean neutralization titers decreasing from 16.56 to 1.11 after receipt of the second vaccine dose (15). Improving immune responses may be beneficial for combating future SARS-CoV-2 variants (16–18).

Due to the effects of decay and emergence of variants, a three-dose schedule of inactivated vaccine in which a booster dose is administered at least 6 months after the second dose is of constant concern. It is still unknown whether a booster dose of an inactivated vaccine can quickly evoke and activate immunity and produce sufficient and durable immune protection. Here, we conducted a prospective cohort study to longitudinally profile the dynamic responses and decays of five subsets of anti-SARS-CoV-2 antibodies (neutralizing antibody, anti-RBD total antibody, anti-Spike IgG, anti-Spike IgM, and anti-Spike IgA) at 18 serial time points from 32 volunteers over 368 days, and specifically assessed the efficacy and characteristics of the third dose.

With the emergence of SARS-CoV-2 variants and decay of immunity, vaccine efficacies have weakened, thus requiring a booster dose (20–23). The efficacy of the booster dose is the focus of controlling the COVID-19 epidemic worldwide. In our study, the neutralizing antibody was only 4.75 IU/mL with a serum positivity rate of 3.12% 248 days after the second dose, but a rate of 96.88% was rapidly reached within one week of the third dose. After the third dose, the neutralizing antibody and anti-RBD total antibody levels rapidly increased to 773.60 IU/mL and 639.30 AU/mL, with 4.22-fold and 3.71-fold increases compared to those after the second dose, respectively. On the other hand, decay of the antibody was obvious. The half-lives of the neutralizing antibody, anti-RBD total antibody, and anti-Spike IgG were 56.26 (95% CI, 46.81 to 70.49) days, 66.37 (95% CI, 54.90 to 83.88) days, and 82.91 (95% CI, 63.65 to 118.89) days, respectively. Compared to the half-lives after the second dose, these values increased by 1.71-fold, 2.00-fold, and 2.93-fold. Our results showed that the immune response was more intense and that the decay was slower after the third dose.

Neutralizing antibody levels are highly predictive of immune protection (24). The threshold of the neutralizing antibody level for 50% protection was considered to be 54.00 IU/mL (3). In our study, the neutralizing antibody serum positive conversion rate reached 100% two weeks after the second dose and third doses. After booster vaccination, the majority of individuals reproduced neutralizing antibodies to prevent infection. Due to the limitations of anti-SARS-CoV-2 antibody decay and virus variant emergence, improving immune responses may be beneficial (25–27). The focus was on whether the third dose could quickly evoke the immune memory and increase response. With the BNT162b2 mRNA COVID-19 vaccine, one month after the third dose, neutralization geometric mean titers (GMTs) against wild-type virus increased to more than 7.00-fold higher than the GMTs one month after the second dose (28). In our study, the neutralizing antibody level quickly increased two weeks after the third dose, which was similar to the second dose, suggesting that immune memory was quickly evoked. In addition, the seroconversion rate rapidly reached 100% within two weeks from a baseline of 3.12%. Moreover, the peak level of the neutralizing antibody markedly increased from 209.70 IU/mL after the second dose to 773.60 IU/mL after the third dose, which was a 4.22-fold increase. This result was similar to the report that neutralization GMTs increased from 42.9 on day 28 after the second dose to 158.5 on day 28 following the third dose in inactivated COVID-19 vaccine, which was a 3.69-fold increase (21). A recent real-world study conducted in Guangzhou (China) showed that the protection rate of two doses of an inactivated vaccine against delta variant infection exceeded 50% (8). Given that the level of neutralizing antibody after the third dose was significantly higher than that after the second dose, we believe that the third dose can improve immunity and increase the level of neutralizing antibodies against reinfection/infection.

On the other hand, the duration of immunity after vaccination is vital and used to estimate the protective effects of vaccination. Recent studies have revealed a gradual decrease in the neutralization titer for up to 8 months after SARS-CoV-2 infection (11, 12, 29, 30). In our study, the neutralizing antibody seropositivity rate was only 3.12% 248 days after the second dose, which was lower than that at 132 days (19.67%) in our previous study (11), and the ability to protect against infection worsened. Goel, R. R. reported that recall responses to vaccination in individuals with preexisting immunity primarily increased antibody levels without substantially altering the antibody decay rates (18). To the best of our knowledge, the decay rate of the third dose has not been evaluated. In our study, the half-lives of the neutralizing antibody, anti-RBD total antibody, and anti-Spike IgG were increased 1.71-fold, 2.00-fold and 2.93-fold, respectively, compared with those after the second dose. Our findings supported that the recall responses to boost doses in individuals with preexisting immunity primarily increased antibody levels and substantially altered antibody decay rates. However, in terms of immunity persistence, the half-lives of the antibodies remained unsatisfactory. Due to decay, a fourth dose, or even annual revaccination, might be considered in the SARS-CoV-2 vaccination management strategy.

It is generally accepted that IgM antibodies provide an early-stage response during viral infections prior to the maturation of the class-switched, high affinity IgG response for long-term immunity and immunological memory (31). There are limited data on the kinetics of the appearance of IgM after vaccination and its association with virus neutralizing activity (32). With the BNT162b2 mRNA COVID-19 vaccine, the positive conversion rate for anti-Spike IgM after the second dose was 63.82% (33). In our study, the positive conversion rate after the second dose was 65.63%. Notably, the positive conversion rate was decreased to 9.38% after the third dose. It suggested that response of anti-Spike IgM may be influenced by pre-existing immunity.

To the best of our knowledge, this is the first prospective cohort study of five subsets of anti-SARS-CoV-2 antibodies to evaluate immunity after three-dose vaccination. However, this study has several limitations that need to be addressed. First, only 32 uninfected volunteers were enrolled, thus making the number of participants relatively limited. The results from this study can be applied to this cohort perhaps on cohort where the same vaccination strategy was used, but it cannot be really generalized. Second, immune cells, neutralizing titers against SARS-CoV-2 variants and heterologous vaccination were not investigated in the study. Third, this work lacks a functional correlate, such as antibody-mediated neutralizing activity. Finally, the study span was only 92 days after the third dose.

In conclusion, our results showed that the third dose of vaccine could dramatically increase antibody levels and prolonged the decay time. However, the half-life of neutralizing antibody remained very unsatisfactory. Due to a decline in the immune response, a fourth dose, and even annual revaccination, might be considered in the SARS-CoV-2 vaccination strategy in the future. These findings provide a meaningful basis for the understanding the effects of a booster dose on COVID-19.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Institutional Ethics Committee of Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

T-CY had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. T-CY, L-LL, L-RL, and X-ML supported funding. X-ML, Q-YX, and T-CY contributed to the protocol and design of the study. All authors designed and conducted the statistical analysis and accessed and verified the underlying data. X-ML, Q-YX, and T-CY drafted the manuscript. All authors critically revised the manuscript for important intellectual content. Z-JJ, M-JW, and Y-YL managed participants. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation [grant numbers 81973104, 81772260, and 82003512], the Key Projects for Science and Technology Program of Fujian Province [grant numbers 2021J02055 and 2020J011208], the project for Xiamen Science and Technology Program of Fujian [grant number 3502Z20184057], and the project for Xiamen Medical and Health Guidance [grant number 3502Z20214ZD1037]. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors ZJ-J, MJ-W and Y-YL are employed by Xiamen Boson Biotech Co., Ltd.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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