Potent antibody immunity to SARS‐CoV‐2 variants elicited by a third dose of inactivated vaccine

SARS-CoV-2 variants are still prevalent worldwide and continue to pose a challenge to the effectiveness of current vaccines. It remains unknown whether a third dose of inactivated vaccine elicits immune potential against SARS-CoV-2 variants. Here, we showed a significant decline in plasma neutralization against SARS-CoV-2 at seven months after a second dose of the inactivated vaccine in a large-scale cohort. However, we also found that a third vaccination with an inactivated vaccine largely increased plasma neutralization against variants including Beta, Delta, and Lambda. More importantly, the high-affinity anti-RBD memory B cells were also generated by the third vaccination, suggesting a more potent and longer protection. These findings highlighted the importance and effectiveness of a third dose of inactivated vaccine in conferring higher protection against the emerging variants in populations.


Dear Editor,
This study found that a third dose of inactivated vaccine largely increased plasma neutralization against SARS-CoV-2 variants including beta, delta, and lambda. More importantly, the high-affinity anti-RBD (receptor-binding domain) memory B cells were also generated by the third vaccination, suggesting a more potent and longer protection.
The coronavirus disease 2019 (COVID-19) pandemic has already lasted for nearly 2 years and continues to threaten human health and life. While several effective vaccines had been deployed to combat wild-type (WT) virus infection, [1][2][3] emerging SARS-CoV-2 variants with enhanced transmissibility significantly escaped the neutralization of vaccineelicited plasma. Breakthrough infections of variants after vaccination have occurred widely with a significant reduction in vaccine efficacy over time. [4][5][6] Currently, many researchers have asked whether a third vaccination is necessary to increase the titres of neutralizing antibodies (nAbs) and to better control the COVID-19 pandemic. 7,8 Here, we evaluated the antibody immunity to SARS-CoV-2 variants elicited by a third dose of inactivated vaccine in a cohort of volunteers.
In this study, 533 participants who received two or three doses of BBIBP-CorV were enrolled and followed up at five follow-up time points ( Figure 1A). The mean value of plasma IgG to SARS-CoV-2 WT RBD was significantly increased 10.41-fold at week 2 after the second vaccination. However, IgG values were gradually decreased to 41.8% at month 2 after the second vaccination and additionally dropped to 42.9% at month 7. Subsequently, we collected blood samples from a total of 176 individuals who accepted a third dose of BBIBP-CorV. Their mean IgG values were increased eightfold compared with those before the third vaccination and 1.44-fold compared with those after the second vaccination ( Figure 1B). RBD-specific IgM displayed a similar pattern of kinetics as IgG. However, the difference between IgM and IgG was that the third vaccination did not induce a strong IgM response ( Figure 1C suggesting that IgG may play a more important role in recalling to the SARS-CoV-2 vaccine. The results of 113 serially paired samples also confirmed the above dynamics of RBD-specific plasma IgG and IgM ( Figure 1D,E).
We further compared the differences in anti-RBD IgG levels between male and female donors. As shown in Figure 1F, male and female donors displayed similar levels of RBD-specific IgG after the third vaccination, although female donors had higher levels of IgG than males at week 2 after the second vaccination. Meanwhile, there were no obvious relationships between IgG values and ages at three different follow-up time points ( Figure 1G). These data showed that the third dose of inactivated vaccine induced robust binding antibodies to SARS-CoV-2 independent of gender and age.
To evaluate the ability of a third vaccination to fight against the infection of mutant viruses, we established the enzyme-linked immunosorbent assay and neutralization assay to detect binding antibodies and nAbs against WT, beta, delta, and lambda variants ( Figure S1). Similar to the binding response to WT RBD, mutated RBD-specific IgG was sharply decreased at month 7 after the second vaccination but was significantly increased by the third vaccination ( Figure 2A). The plasma neutralizing activities displayed the same patterns as their binding activities ( Figure 2B). At week 2 after the second vaccination, over 95% of plasma demonstrated effective neutralization against WT virus and also, to some extent, maintained neutralizing activities against three important variants. However, at month 7 after the second vaccination, nearly half of the plasma (49/113) lost their neutralizing activities, and the mean inhibition was decreased to 53.6% against the WT strain. Notably, the inhibitions of plasma against beta, delta and lambda variants had decreased to 34.4%, 40.4% and 44.8%, indicating their poor defences. After the third vaccination, the plasma inhibitions were significantly increased to 94.6%, 71.6%, 83.4% and 89.0% within 2 weeks, respectively. In addition, there were strong correlations between plasma inhibitions and their binding activities ( Figure 2C The memory B cell (MBC) response is another important type of immune protection, whose quantity and quality contribute to the speed and potency of the immune system responding to viral reinfection. 9 Therefore, we randomly selected 24 individuals before the third vaccination (at week 2 and month 7 after the second vaccination) and week 2 after the third vaccination to detect SARS-CoV-2 RBD-specific MBCs ( Figure S4). Although plasma anti-RBD IgG was gradually decreased over time, the percentage of RBD-specific MBCs maintained a similar level at month 7 as that at week 2 after the second vaccination. Notably, the percentage rapidly increased after the third vaccination, which was significantly higher than that at  17). (E) The end-point titres of binding IgG to SARS-CoV-2 WT, beta, delta, and lambda RBD proteins at week 2 after third vaccination in the high-affinity and moderate-affinity groups. (F) The geometric mean titres of nAbs against SARS-CoV-2 WT, beta, delta, and lambda pseudoviruses at week 2 after third vaccination in the high-affinity and moderate-affinity groups. A cut-off of 1:20 dilution is indicated by a horizontal dashed line. ID 50 , 50% inhibitory dilution. '+' indicates an increase. 'X' indicates fold change. *p < .05; **p < .01; ***p < .001; ****p < .0001; ns, not significant week 2 after the second vaccination and before the third vaccination. The mean fluorescence intensity (MFI) of RBD-binding MBCs was also significantly enhanced by the third vaccination (4799 vs. 2951 and 2680 in APC, 8894 vs. 4516 and 4352 in PE) ( Figure 3A). These data indicated that the third vaccination not only increased the proportion of MBCs but also enhanced the affinity of MBCs to RBD.
More importantly, we found that the third dose of vaccine-induced extremely high-affinity MBCs in seven individuals, whose MFIs of APC and PE on RBD-binding MBCs were both more than two-fold higher than before the third vaccination ( Figure 3B-D). After the third vaccination, the geometric mean end-point titres of the plasma IgG were significantly higher in the high-affinity group than those in the moderate-affinity group against WT and mutated RBD proteins ( Figure 3E; Figure S5). Similarly, the neutralizing activities of plasma were significantly higher in the high-affinity group ( Figure 3F; Figure S6). Therefore, the third vaccination indeed elicited more potent nAbs and high-affinity MBCs, suggesting a persistent antibody immunity to SARS-CoV-2 variants.
In conclusion, our data highlighted the challenges for vaccine recipients who have received complete immunization for more than 6 months. They are at risk of breakthrough infection by SARS-CoV-2 variants. Based on the cohort of volunteers with a long follow-up time, we emphasized the importance of the third dose of inactivated vaccine to confer higher neutralization against emerging variants. Long-term follow-up will evaluate the duration of the antibody response elicited by the third vaccination in future studies.