key: cord-0844733-oej4i1av
authors: Chen, Yuxin; Yin, Shengxia; Tong, Xin; Tao, Yue; Ni, Jun; Pan, Jie; Li, Ming; Wan, Yawen; Mao, Minxin; Xiong, Yali; Yan, Xiaomin; Yang, Yue; Huang, Rui; Shen, Han; Wu, Chao
title: Dynamic SARS-CoV-2 specific B cell and T cell responses following immunization of an inactivated COVID-19 vaccine
date: 2021-10-26
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2021.10.006
sha: e83e71a7677de3f243b83945aa464f0964740743
doc_id: 844733
cord_uid: oej4i1av

OBJECTIVE: The dynamic adaptive immune responses elicited by the inactivated virus vaccine, CoronaVac, remain elusive. METHODS: In a prospective cohort of 100 SARS-CoV-2 naïve healthcare professionals who received two doses of CoronaVac, we analyzed SARS-CoV-2-specific humoral and cellular responses at four different timepoints, including before vaccination (T1), 2 weeks after the first dose (T2), 2 weeks after the booster dose (T3), and 8-10 weeks post the booster dose (T4). SARS-CoV-2-specific antibodies, serum neutralizing activities, peripheral B cells, CD4(+) and CD8(+) T cells, and their memory subsets were simultaneously measured in this cohort. RESULTS: SARS-CoV-2 Spike-specific IgG responses reached the peak (geometric mean titer [GMT] 54827, 30969-97065) after two doses and rapidly declined (GMT 502, 212-1190) at T4, whereas suboptimal IgA responses were detected (GMT 5, 2-9). Spike-specific circulating B cells (0.60%, 0.46-0.73% of total B cells) and memory B cells (1.18%, 0.92-1.44% of total memory B cells) were effectively induced at T3 and sustained over time (0.33%, 0.23-0.43%; 0.87%, 0.05-1.67%, respectively). SARS-CoV-2-specific circulating CD4(+) T cells (0.57%, 0.47-0.66%) and CD8(+) T cells (1.29%, 1.04-1.54%) were detected at T3. At T4, 0.78% (0.43-1.20%) of memory CD4+ T cells and 0.68% (0.29-1.30%) of memory CD8+ T cells were identified as SARS-CoV-2 specific, while 0.62% (0.51-0.75%) of CD4(+) T cells and 0.47% (0.38-0.58%) of CD8(+) T cells were SARS-CoV-2 specific terminally differentiated effector memory cells. Furthermore, age and interval between doses affected the magnitude of CoronaVac induced immune responses. SARS-CoV-2 memory CD4(+) T cells was strongly associated with both RBD-specific memory B cells (r=0.87, p<0.0001) and SARS-CoV-2 specific memory CD8(+) T cells (r=0.48, p<0.0001). CONCLUSIONS: CoronaVac induced robust circulating and memory B cells and T cell responses. Our study offers new insight into the underlying immunobiology of inactivated virus vaccines in humans and may have implications for vaccine strategies in the future.

Vaccines are the cornerstone of the management of infectious disease outbreak and the surest mean to defuse pandemic risk. CoronaVac (Sinovac Biotech, China), a wholevirion chemically inactivated SARS-CoV-2 vaccine, has so far inoculated at least 243 million individuals from more than 45 countries. A large, observational study in Chile indicated that two doses of CoronaVac had vaccine effectiveness of 65.9% against coronavirus disease 2019 (COVID- 19) , 90.3% against intensive care unit admission and 86.3% against COVID-19 related death (1) . Nevertheless, few studies on CoronaVac recipients largely focused on binding and/or neutralizing antibodies (NAbs) as primary endpoints, while vaccine induced cellular immune responses remains elusive.

It is well established that three fundamental components of the adaptive immune system (B cells, CD4 + and CD8 + T cells) are essential to control SARS-CoV-2 infection (2) (3) (4) (5) (6) (7) .

Despite the immune correlates of protection remained unknown (8, 9) , antibodies and T cell responses are important for resolution of primary SARS-CoV-2 infection. Additionally, SARS-CoV-2 infection induced various immunological memory components displayed distinct kinetics (10) .

Recently, we conducted a prospective, observational cohort (NCT04729374) with 100 healthcare personnels in a tertiary hospital in Nanjing, China. Most sera elicited by twodose CoronaVac were capable of effectively neutralize the ancestral strain, Alpha and Epsilon variant, but not for Beta and Gamma variants bearing E484K mutation (11) . In this current study, we provided data from this cohort with new insights into the kinetics of J o u r n a l P r e -p r o o f 6 vaccine-induced humoral and cellular immune responses, including circulating antibodies, antigen-specific B cells, CD4 + and CD8 + T cells, as well as their memory subsets at four timepoints extending up to 8-10 weeks post 2-dose immunization. The impact of gender, age and interval between doses on the magnitude of vaccine responses were further analyzed. The interrelationships between antibody and cellular responses were also evaluated.

A total of 100 healthcare professional was enrolled in a prospective study (NCT04729374) from January to February 2021 in Nanjing Drum Tower Hospital. All participants were tested negative for SARS-CoV-2 infection at screening and provided written informed consent. The clinical trial protocol was approved by hospital ethics committee (2021-034-01). Two cohorts of COVID-19 convalescent patients were included, and their demographic characteristics provided in Figure 1 . In the first cohort, serum samples were collected from 26 convalescent patients on 4-week follow-up visit after hospital discharge, while peripheral blood mononuclear cells (PBMCs) from 12 convalescent patients were collected 16 months after COVID-19 infection in the second cohort.

The quantification of antigen-specific antibodies against SARS-CoV-2 and serum neutralization activities were performed as previously described (11, 12) . Fluorescent labeled ectodomain of Spike or RBD protein were used as probes to identify SARS-CoV-J o u r n a l P r e -p r o o f pools to measure antigen specific CD4 + and CD8 + T cells. The details of peptide pools, conjugation of antibodies, sample staining and statistical analysis are presented in the Supplementary Materials and Methods.

100 healthcare workers were enrolled in this study, ranging from 23 to 59 years of age 

At baseline, all participants had undetectable levels of IgM, IgG and IgA antibodies specific for ectodomain of Spike protein (Spike), nucleocapsid protein (NP) and RBD protein (Figure 2A-F and S1 ). Two doses of CoronaVac significantly boosted antibody responses achieving the peak level of humoral immunity, and 100% of participants were Figure 2G ). At T4, Spike-specific and NP-specific IgG responses were significantly declined, and vaccinee sera had significantly higher anti-Spike IgG titer but remarkable lower IgA responses compared to those in convalescent sera (Figure 2A-F) .

The first dose of CoronaVac induced a significant portion (0.32%, 0.27-0.38%) of Spikespecific B cells, which expanded after the second dose (0.60%, 0.46-0.73%) despite of no statistical differences, and slightly reduced at T4 (0.33%, 0.23-0.43%) ( Figure 3A) .

Similarly, the frequency of Spike-specific memory B cells at T3 has an average of 1.18% Immunoglobin (Ig) isotypes among antigen-specific memory B cell population shifted with time ( Figure 3A-B) . After primary immunization, ~23% of RBD-specific memory B cells were IgG+ and ~22% were IgM+. The frequency of IgG+ memory B cells surged to ~45% J o u r n a l P r e -p r o o f following the second dose, and slightly increased to ~55% after 8-10 weeks post full vaccination. RBD-specific IgA + memory B cell frequency was ~13% at both T2 and T3 and slightly increased to ~22% at T4. Our data suggested that CoronaVac effectively induced virus-specific memory CD4 + T cells and CD8+ T cells as well as effector population.

There were no relationships identified between gender and the magnitude of SARS-CoV-2 specific adaptive responses ( Figure 5A) . Consistent with previous report (15) , the participants between 20 to 40 years old had significantly higher neutralizing titers (GMT 42, 33-52) against ancestral strain, compared to the participants between 40 to 60 years old (GMT 26, 19-37) (Figure 5B and S6A) . Despite young participants had higher magnitude of serum neutralizing activities than elder individuals, both groups had a comparable level of anti-Spike IgG, suggesting potential qualitative differences in Spikespecific humoral immunity. There was no association between age and vaccine-induced cellular responses, including Spike-specific memory B cells, virus-specific CD4 + T cells and CD8 + T cells. Our data suggested that potentially relevant age-related changes in neutralizing activities but not virus-specific T cell or B cell responses.

Furthermore, interval between two doses is a critical factor that affects the magnitude of immune responses. The participants with dosing interval greater than 21 days had higher neutralizing antibody (NAb) titers against ancestral strain and B.1.617.1, compared to the group with the interval less than 21 days (Figure 5C ), which might be associated with the increased anti-Spike IgG responses. The interval greater than 21 days also induced higher percentage of Spike-specific B cells, SARS-CoV-2 specific memory CD4 + T cells and CD8 + T cells, compared to the group with interval less than 21 days. Consistently, the interval correlated with Spike-specific CD4 + T cell responses at T3 (Figure S6B ). 12 We also addressed the potential relationship between humoral immunity and cellular immune parameters. NAb responders had significantly higher level of anti-Spike IgG responses compared to NAb non-responders at T3 ( Figure 5D ). There is a trend that NAb responders generated higher Spike-specific memory B cells among total memory B cells than that in NAb non-responders. Of note, NAb non-responders generated comparable level of SARS-CoV-2 specific memory CD4 + and CD8 + T cells. Additionally, neutralization titers against B.1.1.7 correlated with SARS-CoV-2 specific CD4+ T cells at T3 (r=0.22, p=0.04), and Spike-specific memory B cells at T4 correlated with Spikespecific CD4 + T cells at T2 (r=0.29, p=0.03). SARS-CoV-2 memory CD4 + T cells at T4 was strongly associated with both RBD-specific memory B cells at T3 (r=0.87, p<0.0001) as well as SARS-CoV-2 specific memory CD8 + T cells at T4 (r=0.48, p<0.0001) ( Figure   5E ).

Here we provided an extensive characterization of adaptive immune responses specific to SARS-CoV-2 following SARS-CoV-2 inactivated vaccine. Our data is encouraging and fill our gaps of immune responses elicited by CoronaVac. First, we observed robust IgG responses specific to Spike, RBD and NP after each dose of CoronaVac. However, these antigen-specific IgG responses decayed rapidly within 6-8 weeks, consistent with observation in COVID-19 patients and vaccinees (12, 16) . Such waned antibody responses in COVID-19 patients might be caused by a lack of germinal centre (GC) reaction (17) , which is essential to generate long-lived and high affinity antibody IgA was considered as a critical component for anti-viral immune component (18, 19) .

Nevertheless, suboptimal SARS-CoV-2 specific IgA responses among most vaccinee recipients, suggesting that the formulation and delivery approach of next-generation COVID-19 vaccine might be further optimized to induce the mucosal immunity. Besides, the vaccinee sera showed reduced level of neutralization ability against B.1.617.1 and other circulating variants, highlighting the urgent necessities of booster doses beyond the conventional two-dose regimen.

We observed a notable expansion of long-lasting, isotype-switched IgG + memory B cells among virus specific memory B cells following vaccinations, lasting at least for 6-8 weeks.

Indeed, SARS-CoV-2 infection induced memory B cells are durable and long-lived for at least 8 months post disease onset (10, 20) . Our data indicated that sustained memory B cells might be important for durability of anti-SARS-CoV-2 immunity and potential recall responses to infection or future boost.

Beyond humoral responses, successful protection against infectious diseases can be accomplished by alternative adaptive immune responses, including CD4 + T cells, CD8 + T cells and their corresponding memory subsets (21, 22) . SARS-CoV-2 specific CD4 + T cells and CD8 + T cells were associated with reduced disease severity (4, 23) . Potent memory CD4 + and CD8 + T cell responses were also detected from vaccinees, and the magnitudes were comparable to those in convalescent patients. Further, a prominent J o u r n a l P r e -p r o o f 14 population of CD4 + and CD8 + memory T cells were biased toward TEMRA and TEM cells.

These favorable phenotypes were considered as cytotoxic and long-lived with the potential respond rapidly to eliminate the infected cells (13, 24) .

Age and interval might account for the heterogeneity of adaptive immune responses elicited by full vaccination of CoronaVac. As widely observed in COVID-19 patients, age correlated with COVID-19 disease severity which might be associated with low percentage of naïve CD4 + and CD8 + T cells (23) . Here we also observed a trend that the quality of vaccine-elicited immune response deteriorates with age, especially for neutralizing activities (25) . In addition, the dosing interval greater than 21 days was beneficial for robust SARS-CoV-2 specific adaptive responses. Consistently, extended interval vaccination for BNT162b2 could boost the peak antibody responses in elder individuals, which might be critical to further optimize vaccine regimen for provision of effective and sustained immunity (26) .

Very few published data sets compared antigen-specific antibody, B cell, CD8+ T cells and CD4+ T cells following vaccination in the same individuals. For those vaccinees who failed to generate neutralizing antibodies, robust Spike-specific memory B cells, SARS-CoV-2 memory CD4 + and CD8 + T cells were detected at a similar magnitude as those in NAb responders. Whether these specific CD4 + and CD8 + T cells could also serve as surrogates for protective immunity remains to be determined. Meanwhile, we also identified SARS-CoV-2 memory CD4+ T cells strongly associated with RBD-specific memory B cells as well as SARS-CoV-2 memory CD8+ T cells,indicating a convergent development of humoral and cellular adapative immunity.

The current study has some limitations. The follow-up observation time in our study was relative short, only extending up to 8-10 weeks post full vaccination. Besides, the alternative function of vaccine elicited antibody such as antibody dependent cell-mediated cytotoxicity (ADCC) (27) were not evaluated.

In summary, this study demonstrated multiple compartments of adaptive immunity elicited by an authorized inactivated vaccine in an integrated manner. Our study offers insight into the underlying immunobiology of inactivated virus vaccines in humans and may have implications for vaccine strategies in the future.

The authors have declared that no conflict of interest exists. In addition, the antigen-specific titers were also compared between sera collected from vaccinees at T4 timepoints and convalescent patient cohort 1 (8-10 weeks post symptom onset). Dotted lines indicate the limit of detection (LOD) for the assay. Statistics were calculated using Wilcoxon matched-pairs signed rank for comparison between timepoints and unpaired Wilcoxon test for comparison between vaccinees at T4 and convalescent patients from cohort 1. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. ns indicates no significant difference. (A-C) Serum titers that achieved 50% pseudovirus neutralization (pNT50) against ancestral strain, the P.1, the B.1.351, the B.1.617.1, anti-spike IgG titer, the frequency of spike-specific memory B cells, the frequency of SARS-CoV-2-specific memory CD4+ and CD8+ T cells compared with gender (A), age (B), interval between doses (C). (D) Anti-spike IgG titer, the frequency of spike-specific memory B cells, and the frequency of SARS-CoV-2-specific CD4+ and CD8+ memory T cells among neutralizing antibody (NAb) responders versus NAb non-responders. (E) Correlation analysis of pNT50 against B.1.1.7 and SARS-CoV-2 specific CD4+ T cells at T2 timepoint (left), correlation analysis of spike specific CD4+ T cells at T2 and spike+ specific B cells at T4 timepoint (middle), and correlation analysis of RBD-specific B cells at T3 and SARS-CoV-2-specific CD4+ memory T cells responses at T4 (right). Statistics were analyzed using unpaired Wilcoxon test between groups. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. ns indicates no significant difference.

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We thank all vaccine recipients for providing serum samples. This study was supported