To the Editor: The vaccine-related adaptive immune systems, including humoral immunity induced by B cell reaction and cellular immunity induced by T cells, are crucial for controlling severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Therefore, it is vital to clarify the immune changes caused by inactivated vaccines, which are most used in China, for evaluating the protective effect of vaccines and formulating vaccination plans.
The study was approved by the Medical Research and Ethics Committee of Henan Provincial People's hospital (No. 2021-21). All participants provided written informed consent before enrollment. This was a prospective, single-arm cohort study conducted in Henan Provincial People's hospital from March to December 2021.
A total of 142 healthcare workers and neighborhood residents aged ≥18 years, vaccinated with two doses of inactivated vaccines (CoronaVac, Sinovac Life Sciences, Beijing, China; BBIBP-CorV, Sinopharm, Beijing, China) with three weeks apart were recruited. Patients with SARS-CoV-2 infection history, taking immunosuppressive drugs, allergic to vaccine ingredients, or pregnant were excluded. We collected information on comorbidities, vaccination (dosage, time, and adverse reactions), and blood samples at three scheduled time points (i.e., the time points 2-, 8-, and 24-week after the second vaccination), and divided participants into positive group and negative group according to anti-SARS-CoV-2 immunoglobulin G (IgG) antibody at 8 weeks after receiving the second dose of vaccine. The level of anti-SARS-CoV-2 IgG in the serum sample was tested with the magnetic chemiluminescence enzyme immunoassay (MCLIA) kit provided by the Bioscience company (Tianjin, China); and the white blood cell count (WBC), lymphocyte count (LYMPH), neutrophil count (NEUT), and the concentration of hemoglobin (HGB) were tested with Sysmex XN-9100 machine and its supporting reagents (Sysmex Corporation, Kobe, Japan); the concentration of interleukin 2 (IL-2), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), IL-4, IL-6, and IL-10 was tested by Agilent NovoCyte machine and its supporting reagents (Agilent Technologies, Inc. Beijing, China);finally, BD flow cytometry (Franklin Lake, New Jersey, USA) was used to count the memory B cells and detect the T-lymphocyte subsets stimulated by the SARS-CoV-2 specific ELIspot kit (AmyJet Scientific, Wuhan, China). Repeated measurement data (WBC, NEUT, LYMPH, HGB, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ) were compared using Friedman's M test and q test. Inter-group differences of the number of memory B cells, SARS-CoV-2 specific T cells, cluster of differentiation 4 positive (CD4+), and CD8+ T lymphocytes were compared using Mann-Whitney U test. Statistical significance was defined as P <0.05. All analysis was performed using SPSS software, version 26.0 (IMB, Chicago, IL, USA).
Among all participants, several participants had one or two ongoing diseases, and the most common comorbidities were hypertension (n = 29, 20.4%), hyperlipidemia (n = 16, 11.3%), diabetes mellitus (n = 10, 7.0%), respiratory disease (n = 8, 5.6%), and tumor (n = 7, 4.9%). Thirty-two (22.5%) participants had local adverse reactions after vaccination, with pain at the injection site being the most common. Ten (7.0%) participants had systemic adverse reactions, of which fatigue and muscle pain were the most common. All participants were negative for SARS-CoV-2 nucleic acid before inclusion in the study and at 2 -, 8 -, or 24-week after vaccination.
As all participants were identified no infection with SARS-CoV-2, they were considered negative for anti-SARS-CoV-2 IgG antibodies before vaccination (IgG <1 signal-to-cutoff [S/CO]). The seropositivity rates increased as high as 86.6% (n = 123), 72.5% (n = 103), and 50.7% (n = 72) at the 2-, 8-, and 24-week time points post-scond vaccination, respectively. Two weeks after the second dose, the average antibody titer was close to the maximum at 14.67 S/CO (95% confidence interval [CI], 2.04–25.10 S/CO); at eight weeks the average titer reduced to 5.78 S/CO (95% CI, 0.89–9.65 S/CO), and by 24 weeks, the average titer declined to 2.52 S/CO (95% CI, 0.40–3.45 S/CO).
Accordring to the anti-SARS-CoV-2 IgG antibody at 8 weeks, there were 103 participants in the positive group, and the negative group comprised 39 participants, including 19 participants without seroconversion and 20 participants who turned negative at 8 weeks (2-week [+] and 8-week [-]).
In patients with coronavirus infection, the peripheral blood cell count often changes, and several indicators can be used to predict the severity and prognosis of patients. In this study, we investigated the changes of the peripheral blood cell count and found that LYMPH, HGB, and WBC of the positive group were different at 2 weeks, 8 weeks and 24 weeks, while LYMPH, HGB, and WBC of the negative group and NEUT of the two groups were similar at the three time points. Specifically, LYMPH and WBC in the positive group at 24 weeks were significantly lower than those at 2 weeks (LYMPH: P <0.001; WBC: P =0.011 ), while the HGB in the positive group was lower at 8 weeks and 24 weeks than that at 2 weeks (P =0.040; P <0.001). Further comparative analysis of the two groups at the same time point found that the LYMPH of the positive group was significantly lower than that of the negative group at 24 weeks (P =0.006) [Supplementary Tables 1 and 2, https://links.lww.com/CM9/B562].
The abnormal increase of cytokines is another important feature of SARS-CoV-2 specific immune response. The evaluation of the characteristics of vaccine-induced cytokines can better characterize the vaccine-induced immune response, and the study of the cytokine response induced after inactivated vaccine inoculation is still rare. In this study, we investigated the profiles of cytokine changes and found none of the participants showed cytokine storm. Except for IL-4 of the negative group, the observed cytokines have fluctuated at 2, 8 and 24 weeks. Specifically, IL-2, TNF-α, IFN-γ, IL-6, and IL-10 in the two groups and IL-4 of the positive group were higher at 8 weeks than that at 2 weeks, TNF-α, IFN-γ, IL-6, and IL-10 of the two groups and IL-2, IL-4 in the positive group at 24 weeks were still higher than those at 2 weeks (all P <0.05, Supplementary Tables 1 and 2, https://links.lww.com/CM9/B562). Comparative analysis of the two groups at the same time point found that the IL-2, TNF-α, and IFN-γ of the positive group at 2 weeks was higher than the negative group (P <0.001; P =0.018; P <0.001). At 24 weeks, IL-2, TNF-α, IFN-γ, IL-6, and IL-10 in the positive group were higher than those in the negative group (IL-2: P <0.001; TNF-α: P <0.001; IFN-γ: P =0.003; IL-6: P =0.021; IL-10: P =0.004)[Supplementary Tables 1 and 2, https://links.lww.com/CM9/B562].
Then we evaluate the long-term immune protection function induced by the vaccine at 24 weeks. High proportion of IgG+ IgM- CD19+ memory B cells was found at 24 weeks in both groups. We further analyzed the difference of induced memory B lymphocytes between the two groups and found that the proportions of memory B lymphocyte subsets in the positive group were significantly higher than those in the negative group (P <0.001). ELIspot results showed that after stimulating by the SARS-CoV-2 peptide library, some participants retained a SARS-CoV-2 specific T lymphocyte immune response at 24 weeks. Induced expression of IFN-γ and TNF-α in CD4+ and CD8+ T lymphocytes was found 24 weeks after vaccination, with the expression in CD4+ T lymphocytes being more significant (P =0.042), and the number of SARS-CoV-2 specific T lymphocytes (CD4+ and CD8+ T cell) in the positive group was notably higher than that in the negative group at 24 weeks (P <0.001).
This research offers an insight into the immunogenicity of inactivated vaccine vaccination in China. We found that two weeks after the second administration, the participants had a strong humoral reaction, and the serum conversion rate reached 86.6%. The current study also shows that circulating antibodies would last at least 6 months after vaccination, although the peak levels have declined after the second injection. The patients infected with SARS-CoV-2 may show declined LYMPH, HGB, and WBC, and increased NEUT of different degrees. However, in our study, the NEUT did not change significantly after vaccination in both groups, and the LYMPH, HGB, and WBC of the positive group at the 24 weeks decreased compared with those at the 2 weeks, but still fluctuated within the normal range, which means that the inactivated vaccine is safe and reliable. The abnormal increase of cytokines is another important feature of SARS-CoV-2 infection specific immune response. Excessive production of cytokines not only plays a vital role in resisting viruses, but also leads to autoimmune inflammation and subsequent organ failure, even death. In our study, there is no cytokine storm after the vaccination. Vaccination can effectively induce the rising of IL-2, TNF-α, IFN-γ, IL-4, IL-6, and IL-10 and last for several months, and the concentration of cytokines produced in people with high or lasting antibody titers is higher than that of people with low antibody titers or those at turn negative in a short time. Antibodies play an important role in immune protection that mediates viral infection. However, when the antibody titer is low, long-lived memory B cells and T cells are also significant for effective immune response. We found that memory B cells and T lymphocytes can be detected at 24 weeks in both groups, but the number of above cells in negative group is far lower than that in positive group. Similarly, it has been shown in the past that memory B and T cells can be rapidly reactivated 24 weeks after immunization, thus strengthening the control of the initial replication of the virus and limiting the transmission of the virus in the host.[2,5] This also indicates that even if the anti-SARS-CoV-2 IgG titer is low or turns negative in a short time, similar T cell immunity is also induced and participants with longer-lasting or higher IgG titers may induce stronger T cell response.
This study did not calculate the protective threshold of anti SARS-CoV-2 IgG antibody, which is crucial for the guidance of vaccination booster and observation of the effective protection time of antibody after COVID-19 vaccination. In the future, it is necessary to study the incidence of vaccine breakthrough infection and the effects of multiple doses of booster vaccine on the human immune system.
In conclusion, inactivated vaccine can safely and effectively induce humoral immune response, cytokine response, and memory lymphocyte up to 24 weeks. The IgG antibody titer reaches the peak in the second week and gradually decreases with time, moreover, the cytokine concentration and number of memory lymphocytes in the positive group were higher than those in the negative group. We encourage the monitoring of immune response after vaccination and people with low humoral immune responses or who was vaccinated over six months before to receive a booster shot to.
This work was supported by a grant from the Emergency Scientific Research on Novel Coronavirus Prevention and Control of Henan Province (No. 221111311500)
Conflicts of interest
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