Background: Although inactivated COVID-19 vaccines are proven to be safe and effective in the general population, the dynamic response and duration of antibodies after vaccination in the real world should be further assessed.

Methods: We enrolled 1067 volunteers who had been vaccinated with one or two doses of CoronaVac in Zhejiang Province, China. Another 90 healthy adults without previous vaccinations were recruited and vaccinated with three doses of CoronaVac, 28 days and 6 months apart. Serum samples were collected from multiple timepoints and analyzed for specific IgM/IgG and neutralizing antibodies (NAbs) for immunogenicity evaluation. Antibody responses to the Delta and Omicron variants were measured by pseudovirus-based neutralization tests.

Results: Our results revealed that binding antibody IgM peaked 14–28 days after one dose of CoronaVac, while IgG and NAbs peaked approximately 1 month after the second dose and then declined slightly over time. Antibody responses had waned by month 6 after vaccination and became undetectable in the majority of individuals at 12 months. Levels of NAbs to live SARS-CoV-2 were correlated with anti-SARS-CoV-2 IgG and NAbs to pseudovirus, but not IgM. Homologous booster around 6 months after primary vaccination activated anamnestic immunity and raised NAbs 25.5-fold. The neutralized fraction subsequently rose to 36.0% for Delta (p=0.03) and 4.3% for Omicron (p=0.004), and the response rate for Omicron rose from 7.9% (7/89) to 17.8% (16/90).

Conclusions: Two doses of CoronaVac vaccine resulted in limited protection over a short duration. The inactivated vaccine booster can reverse the decrease of antibody levels to prime strain, but it does not elicit potent neutralization against Omicron; therefore, the optimization of booster procedures is vital.

Study design and participants

The cross-sectional investigation was conducted in five counties of Zhejiang Province, mainland China (Xihu, Yuecheng, Shangyu, Kaihua, and Longyou Districts), after nationwide COVID-19 vaccinations from May to October 2021. Potential participants aged 18–59 years who had no prior vaccinations or were vaccinated with one or two doses of CoronaVac (Sinovac Life Sciences, Beijing, China) were recruited from the community. Individuals with a history of infection with SARS-CoV-2 (based on epidemic surveillance system) or the use of blood products or immunosuppressive drugs were excluded. We randomly enrolled 1067 volunteers, including those on day 0 (V-0, no vaccination), day 14 ± 2 (V1-14d), and day 28 ± 3 (V1-28d) after the first vaccine dose, and day 30 ± 3 (V2-1m), day 90 ± 7 (V2-3m), day 180 ± 14 (V2-6m), day 270 ± 14 (V2-9m), and day 365 ± 30 (V2-12m) after the second dose and collected their venous blood samples (3–5 ml) to detect serum antibody levels (Figure 1A). This was not a longitudinal survey, as different subjects were enrolled at each point in time. We employed a questionnaire survey at blood drawing visits to gather demographic information.

In the prospective cohort study, we recruited 90 healthy adults aged 18–80 years from Jiaxing city, Zhejiang, in June 2021. The main exclusion criteria included previous or later SARS-CoV-2 infection; allergy to any ingredient included in the vaccine; those who had received any blood products or any research medicines or vaccines in the past month; those who had uncontrolled epilepsy or other serious neurological diseases, acute febrile disease, acute onset of chronic diseases, or uncontrolled severe chronic diseases; and those who were unable to comply with the study schedule. Subjects were administered 4 µg/0.5 mL of CoronaVac following a 3-shot vaccine schedule 28 days and 6 months apart. Following that, venous blood (3–5 ml) was collected from recipients at five time points: day 0 (Pre-V, before vaccination), day 30 ± 3 (V2-1m), day 90 ± 7 (V2-3m), and day 180 ± 14 (V2-6m) after the second dose, and day 30 ± 3 (V3-1m) after the third dose (Figure 1B).

SARS-CoV-2-specific IgG and IgM assay

The commercial detection kit iFlash-2019-nCoV NAb assay (Shenzhen YHLO Biotech Co. Ltd., Shenzhen, China) was employed to measure the levels of IgG and IgM against SARS-CoV-2 spike glycoprotein (S) and nucleocapsid protein (N) by chemiluminescence immunoassay. Briefly, serum samples were allowed to form a complex with SARS-CoV-2 S- and N-protein antigen-coated paramagnetic microparticles, then an acridinium-ester-labeled ACE2 conjugate was added to competitively combine with the particles, forming another reaction mixture. The analyzer converted relative light units (RLUs) into an antibody titer (AU/mL) through a two-point calibration curve. An inverse relationship existed between the amount of SARS-CoV-2 NAb in the sample and the RLUs detected by the iFlash optical system. According to the manufacturer, titers of ≥10.0 AU/mL and ≥1.0 AU/mL are considered positive (or reactive) for IgG and IgM, respectively. IgG and IgM against SARS-CoV-2 receptor binding domain (RBD) were detected using a commercial ELISA kit (Bioscience Biotech Co. Ltd., Chongqing, China). The positive cutoff values for RBD-specific IgG and IgM antibodies were defined as titers of ≥1.0 AU/mL. All tests were performed according to the manufacturer’s protocols (Chan et al. 2021; Li et al. 2021).

Live virus neutralization antibody assays

The levels of neutralizing antibodies to live SARS-CoV-2 were assessed by the reduction in the cytopathic effect (CPE) in Vero cells with infectious SARS-CoV-2 strain 19nCoVCDC-Tan-HB01 (HB01) in a BSL-3 laboratory(Zhang et al. 2021). Briefly, serum samples were heat-inactivated for 30 min at 56°C and successively diluted from 1:4 to the required concentration in a 2-fold series. An equal volume of challenge solution containing 100 TCID50 virus was added. After neutralization in a 37°C incubator for 2 h, a 1.5–2.5 × 10⁵/ml cell suspension was added to the wells. The CPE (cytopathic effect) on VeroE6 cells was analyzed at 4-days post-infection. NT50 (50% neutralization titer, the reciprocal of the highest dilution protecting 50% of the cells from virus challenge) was used to show the neutralization titers. NT50 above 1:4 was defined as positive.

Pseudovirus-based neutralization test

Serum samples were also quantified for their content of SARS-CoV-2-neutralizing antibodies to wildtype (Wuhan), Delta (B.1.617.2), and Omicron (B.1.1.529) using the pseudovirus-based virus neutralization test (Nie et al. 2020). Briefly, serum samples and a positive or negative reference sample were each diluted 50 times with phosphate-buffered saline combined with 50 µl of pseudovirus diluent per well in a 96-well plate. The mixed sample/pseudovirus was incubated at 37°C and 5% CO₂ for 1 h. A 2 × 10⁵/ml BHK-21-ACE2 cell suspension was added to each well of the plate containing the sample/pseudovirus mixture, then the plate was incubated in a 37°C and 5% CO₂ cell incubator for 48 h. Finally, the number of green-fluorescence-protein-positive cells per well was read with a porous plate imager (Tecan, Shanghai, SparkCyto). The results were determined by comparing the neutralized fraction using the following calculation: (1 – (fluorescence value of each well/average virus control value)) × 100%(Karaba et al. 2022). At least 4 wells were left blank for calibration to 0% inhibition. The conversion of neutralized fraction to serum titer was shown in Table S2.

Statistical analysis

Sex, age, BMI, and other clinical characteristics were collected for each vaccination recipient. We used the medians and interquartile ranges (IQR) for age, and numbers (percentages) for categorical variables. Specific binding antibodies against SARS-CoV-2 (IgG, IgM) and neutralized fraction of SARS-CoV-2-neutralizing antibodies are presented as mean ± standard error (SEM). Neutralizing antibodies are presented as geometric mean titers (GMT), and their 95% confidence interval (CI) was calculated with Student’s t distribution on log-transformed data and then back-transformed. Comparisons of titer-level differences between the two groups were performed using the paired Student's t-test. One-way analysis of variance (one-way ANOVA) was used to analyze the differences between the mean values at different timepoints. Correlations between NAb titers, neutralized fraction, and IgG/IgM levels were evaluated by Pearson’s correlation coefficient. Statistical tests were two-sided, and we considered p-values of less than 0.05 as statistically significant. All statistical analyses were conducted in SPSS 18.0 (IBM Corporation, Armonk, NY, USA) and GraphPad Prism 9 (San Diego CA, USA).