Characterizing the cellular and molecular variabilities of peripheral immune cells in healthy recipients of BBIBP-CorV inactivated SARS-CoV-2 vaccine by single-cell RNA sequencing

ABSTRACT Over 3 billion doses of inactivated vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been administered globally. However, our understanding of the immune cell functional transcription and T cell receptor (TCR)/B cell receptor (BCR) repertoire dynamics following inactivated SARS-CoV-2 vaccination remains poorly understood. Here, we performed single-cell RNA and TCR/BCR sequencing on peripheral blood mononuclear cells at four time points after immunization with the inactivated SARS-CoV-2 vaccine BBIBP-CorV. Our analysis revealed an enrichment of monocytes, central memory CD4+ T cells, type 2 helper T cells and memory B cells following vaccination. Single-cell TCR-seq and RNA-seq comminating analysis identified a clonal expansion of CD4+ T cells (but not CD8+ T cells) following a booster vaccination that corresponded to a decrease in the TCR diversity of central memory CD4+ T cells and type 2 helper T cells. Importantly, these TCR repertoire changes and CD4+ T cell differentiation were correlated with the biased VJ gene usage of BCR and the antibody-producing function of B cells post-vaccination. Finally, we compared the functional transcription and repertoire dynamics in immune cells elicited by vaccination and SARS-CoV-2 infection to explore the immune responses under different stimuli. Our data provide novel molecular and cellular evidence for the CD4+ T cell-dependent antibody response induced by inactivated vaccine BBIBP-CorV. This information is urgently needed to develop new prevention and control strategies for SARS-CoV-2 infection. (ClinicalTrials.gov Identifier: NCT04871932).

Single-cell RNA sequencing (scRNA-seq) and singlecell T cell receptor (TCR)/B cell receptor (BCR) sequencing (scTCR/BCR-seq) techniques are powerful in cell biology research at single-cell resolution [16]. The recent pandemic of COVID-19 has prompted the demand for single-cell studies to uncover the immune response characteristics of different immune cells during symptom development [17]. Recent studies explored the individual immune responses to mRNA-based vaccines (BNT162b2) [18] and adenovirus vaccines (Ad5-nCoV) [19] across the transcriptome at single-cell resolution. These published scRNA-seq datasets indicated various individual immune dynamics induced by different vaccines. Along with the support of clinical practice, traditional inactivated vaccines play an important role in the prevention and control of infectious diseases [20]. Two types of inactivated SARS-CoV-2 vaccines, BBIBP-CorV and CoronaVac, have been developed, with BBIBP-CorV offering a higher protection rate against severe disease compared with CoronaVac in clinical trials [13]. A recent scRNA-seq study that focused on the CoronaVac vaccine reported potential CoronaVac-related side effects and systemic inflammation [21]. However, the functional transcription and TCR/BCR repertoire dynamics of immune cells induced by the SARS-CoV-2 vaccine, and the underlying cellular and molecular mechanism of the host adaptive immune response to the inactivated vaccine have not been well defined.
Here, by combined analysis of scRNA-seq and scTCR/BCR-seq, we revealed the unique dynamic changes in the immune response at different time points after immunization with the inactivated SARS-CoV-2 vaccine BBIBP-CorV. We also compared the transcription and repertoire dynamics of immune cells from peripheral blood mononuclear cells (PBMCs) during BBIBP-CorV vaccination and COVID-19 progression at single-cell resolution. Our study provides novel insights illustrating the possible cellular and molecular mechanisms of the post-vaccination response and facilitates an in-depth understanding of the immune responses under different stimuli.

Participants and ethics
All human samples used in this study were processed under Institutional Review Board approved protocols at Shanghai Jiao Tong University. All study participants were recruited after providing informed consent and with the approval of the Ethics Committee of Ren Ji Hospital (KY2021-046), School of Medicine, Shanghai Jiao Tong University, and the study was conducted according to the criteria set by the Declaration of Helsinki (2013) [22]. This study was registered with ClinicalTrials.gov (NCT04871932). Written informed consent letters were routinely obtained from all participants in the study and the investigators complied with all relevant ethical regulations regarding human research participants. Nine healthy, non-frail, individuals who had not received any vaccines over the past year were recruited, including five males and four females (aged 27-66 years). All participants tested negative for SARS-CoV-2 infection by RT-qPCR tests, and tested negative for serum SARS-CoV-2specific IgM/IgG-antibody by an ELISA before vaccination. The participants had no history of epidemiological contact with patients with COVID-19. All participants were free of clinical symptoms including fever, cough, headache, sore throat, malaise, loss of smell, runny nose, abdominal pain, diarrhea, joint pain, wheezing or dyspnea for the 28 days prior to vaccination. Participants were also free of any steroid usage, chronic diseases, pregnancy, lactation and obvious allergies to any known ingredients contained in the inactivated vaccine.

Vaccine and vaccination protocol
For the inactivated SARS-CoV-2 vaccine (Vero cells), a single-dose schedule of 4 μg/0.5 ml was supported by the Beijing Institute of Biological Products (Beijing, China). Vaccine recipients received two doses of BBIBP-CorV via the intramuscular route, with 2-4 weeks between each injection, in accordance with the manufacturer's instructions.

Sample collection, preparation and storage
Peripheral venous blood samples were collected in Vacutainer CPT (362761, BD, San Diego, CA, US) tubes and coagulation promoting tubes at four time points, including the day before vaccination (BV), 7 days after the first dose (1V7), 7 days after the second dose (2V7) and 14 days after the second dose (2V14). The blood samples were processed in 2 h after collection. The PBMCs were isolated using Vacutainer CPT tubes according to the manufacturer's protocol [23]. The viability of PBMCs in each sample was confirmed to be >90% by Trypan Blue staining. PBMCs were immediately used for scRNA library construction and sequencing, or were cryopreserved at −80°C in 10% dimethylsulfoxide in fetal bovine serum. Blood samples in the coagulation promoting tubes were incubated upright for 20 min at 4°C. Then, the coagulation promoting tubes were centrifuged at 1000 × g for 10 min at 4°C. The supernatant serum in each tube was extracted and stored at −80°C for future assays.

scRNA-seq library construction and sequencing
Using a Single Cell 5 ′ Library and Gel Bead kit (10× Genomics, 1000006) and Chromium Single Cell A Chip kit (10× Genomics, 120236), the cell suspensions (600-1000 living cells per microliter, as determined by Count Star) were loaded onto a Chromium Single Cell Controller (10× Genomics) to generate single-cell gel beads in emulsion, following the manufacturer's protocol. Approximately 12,000 cells were added to each channel and approximately 7,000 target cells were recovered. Single-cell RNA libraries were prepared using the Chromium Single Cell 5 ′ Reagent (10× Genomics, PN-1000263), according to the manufacturer's instructions. Each sequencing library was generated with a unique sample index. The libraries were sequenced using the Illumina platform with a sequencing depth of at least 100,000 reads per cell and 150 bp (PE150) paired-end reads (performed by GENE SHINE, Shanghai, China).

scRNA-seq data processing
The matrices of unique molecular identifier (UMI) counts were generated for each sample by the Cell Ranger (10× Genomics, Version 4.0.0) Pipeline coupled with the human reference version GRCh38 (10× Genomics, Version 3.0.0.) using STAR (version 2.5.1b). The raw matrix data of 17 PBMC samples from COVID-19 patients (aged 15-62 years) were downloaded from the Gene Expression Omnibus (GEO) Data Center (accession GSE15805521) [17]. The blood of these patients with confirmed COVID-19 was sampled before September 2020. These patients were infected with wild-type SARS-CoV-2 according to World Health Organization reports. Then, the expression matrix was analyzed by R software (v.3.6.0) with the DoubletFinder package (version 2.0.2), Harmony package (v.0.1.0) and Seurat package (v.3.0.0) for filtering, data normalization, dimensionality reduction and clustering. The scRNA-seq data were analyzed via the following steps: 1) To remove low-quality cells, cells with fewer than 400 genes and a mitochondrial gene ratio of greater than 10% were excluded. Genes with at least one count in more than three cells were used for the following analysis. 2) To remove potential doublets, cells with UMI counts above the mean ± 2SD were filtered out. Additionally, we applied the DoubletFinder package, with the default parameters, to identify potential doublets. 3) For each cell, we normalized the gene expression profiles using the "NormalizeData" function with the default parameters. 4) Variable genes for each sample were selected using the "FindVariableFeatures" function with the default parameters. All ribosomal, mitochondrial and immunoglobulin genes were then removed from the list. 5) Dimension reduction was performed using the "RunPCA" function to obtain the first 50 principal components, followed by integration using Harmony to correct the batch effects. 6) A shared nearest neighbour graph was constructed based on the Euclidean distance in the low-dimensional subspace spanning the selected significant principal components. Cells were clustered using the "FindClusters" function at an appropriate resolution. 7) Cells were visualized using the UMAP algorithm with the "RunUMAP" function. Using the above pipeline, we processed the scRNAseq data of 263,624 high-quality cells from 44 samples.
Identification of differential expression genes and functional enrichment analysis Differential gene expression testing was performed using the "FindMarkers" function in Seurat with the default parameter "test.use = wilcox" and the Benjamini-Hochberg method was used to estimate the adjusted p value. Differential expression genes (DEGs) were filtered using a minimum log 2 (foldchange) of 0.4 and a maximum adjusted p value of 0.05. Gene ontology biological process (GO:BP) functional enrichment analysis of the upregulated DEGs in the 2V7 and 2V14 groups was conducted using the R package clusterProfiler, with the default parameters. Gene Set Enrichment Analysis (GSEA) between the 2V7 and 2V14 groups and the BV group was also conducted using the R package clusterProfiler, with the default parameters.

TCR and BCR V(D)J sequencing and analysis
TCR and BCR clonal types were determined using the CellRanger V(D)J pipeline (10× Genomics, Version 4.0.0). The TCR and BCR repertoire sequencing data were filtered according to the following criteria: 1) productive is "True"; 2) high_confidence is "True"; 3) umis ≥1; and 4) raw_clonotype_id is not "None". To identify the TCR clonotype for each T cell, only cells with at least one TCR α chain and one TCR β chain were used. For a given T cell, if there were two or more TCR α or TCR β chains assembled, the highest expressed (UMI or reads) TCR α or TCR β chains were regarded as the dominant TCR α or TCR β chain in the cell. Each unique TCR α(s)-TCR β(s) pair (CDR3 nucleotide sequences and rearranged VJ genes included) was defined as a clonotype. BCR clonotypes were identified in a similar manner to TCR clonotypes. Only cells with at least one productive heavy chain (IGH) and one productive light chain (IGL or IGK) were kept for further analysis. Each unique IGH-IGL/IGK pair was defined as a clonotype. For a given B cell, if there were two or more IGH or IGL/IGK assembled, the highest expressed (UMI or reads) IGH or IGL/IGK chain was defined as the dominant IGH or IGL/IGK chain in the cell. If one TCR α-TCR β pair or one IGH-IGL/IGK pair was present in at least two cells, cells harbouring this clonotype were considered to be clonal expanded and the number of cells with the same clonotype indicated the degree of cellular clonal expansion. Cells expressing the same clonotype were counted. The counts were classified into eight classes to identify the cellular clonal expansion status, i.e. > 100, 51-100, 31-50, 21-30, 11-20, 6-10, 2-5, and a unique clone without expansion. For CD4 + T cells, CD8 + T cells and B/plasma cells, the clonal expansion status percentage was computed for each group. The percentage of the TCR type count to the total cell number was used to measure TCR diversity.

Transcription factor analysis
After arranging the input by the gene expression raw matrix, the SCENIC package in R facilitated evaluation of transcription factors among immune cell subsets using the default parameters. The pheatmap and ggplot2 packages in R were adopted to visualize the expression profile of transcription factors.

Definition of cell state scores
For assessing the immune state, the "AddModule-Score" function in Seurat was used to calculate the modular score of pathways with default settings. The following gene sets, positive regulation of type I interferon production (GO:0032481), T cell receptor signalling pathway (GO:0050852), immunoglobulin production (GO:0002377), interleukin-6 production (GO:0032635), tumour necrosis factor production (GO:0032640) and positive regulation of T cell cytokine production (GO:0002726) were used to define type I interferon production, T cell receptor signalling, immunoglobulin production, interleukin-6 production, tumour necrosis factor production and T cell cytokine production, respectively. The expression score for each pathway was calculated based on the average expression of genes from the abovementioned gene sets.

Statistical analysis
Statistical analyses were performed using the R software package (version 3.6.0) and involved the Shapiro-Wilk test, F-test, Levene's test, unpaired Mann-Whitney U-test and Kruskal-Wallis test as previously reported [25]. Data normality was determined by the Shapiro-Wilk test. The F-test was used to examine the homogeneity of variance for two group comparisons. The Levene's test was used to examine homogeneity of variance for the Kruskal-Wallis rank sum test. We performed unpaired Mann-Whitney U-tests on the levels of cytokines in serum, cell subset proportions, the fraction of cells expressing cytokine genes and immunoglobulin heavy chain genes, the module score of gene sets, TCR diversity and the usage of BCR and TCR genes between pre-and post-vaccination samples. When comparing the usage of paired TCR and BCR genes among the four time points, the Kruskal-Wallis rank sum test was used, followed by Benjamini and Hochberg adjustment. The p value significance thresholds used for each set of data are described in the corresponding methods and figure legends.

Data and code availability
The processed transcriptomic data reported in this study has been deposited in China National Center for Bioinformation, National Genomics Data Center under the accession code HRA004001, OMIX003038 and PRJCA004747. During our analysis, which proceeded as described in the methods section, we did not use any special code.

Overview of the immunological changes after BBIBP-CorV vaccination
To comprehensively assess the immune responses following vaccination with inactivated SARS-CoV-2 vaccine (BBIBP-CorV), nine healthy donors (HDs) were enrolled in this 2019-nCoV vaccine observational study [NCT04871932] according to the inclusion and exclusion criteria ( Figure 1A and Supplementary Table 1). First, peripheral blood samples from the nine HDs were collected before vaccination (BV group, n = 9). Then, six of the nine HDs received two doses of BBIBP-CorV vaccine. Peripheral blood samples from all two-dose-vaccinated participants were collected 7 days after the first vaccination (1V7 group, n = 6), 7 days after the second vaccination (2V7 group, n = 6) and 14 days after the second vaccination (2V14 group, n = 6) ( Figure 1A). Serum and PBMCs were separated from all peripheral blood samples, and 2019-nCoV Spike receptor binding domain (RBD) antibodies and cytokines in serum were measured. After isolating the PBMCs, we profiled the V(D)J repertoires of T and B cells integrated with 5' Gene Expression using a droplet-based single-cell sequencing method (10×Genomics) ( Figure 1A). In addition, to reveal differences in the immune response between vaccination and SARS-CoV-2 infection, we collected 17 published PBMCs data [17] with single-cell RNA and TCR/BCR sequencing from patients with confirmed COVID-19 of both sexes. Among these 17 samples, 6 were from COVID-19-infected patients with moderate symptoms and 11 were from recovered convalescent patients ( Figure 1A). Finally, 44 samples were classified according to six clinical conditions: before vaccination (BV group, n = 9), 7 days after the first dose (1V7 group, n = 6), 7 days after the second dose (2V7 group, n = 6), 14 days after the second dose (2V14 group, n = 6), symptomatic COVID-19 patients (n = 6) and convalescent patients (conv; n = 11) ( Figure 1A). The detailed clinical information is listed in Supplementary Table 1.
After filtering out low-quality droplets, 263,624 cells were obtained from 44 PBMC samples. The filtered data were integrated, dimension reduced and clustered in an unsupervised method. Based on the unsupervised clustering and expression of signature genes for each cluster (Supplementary Table 2), 12 each sample across four groups (n = 9 in BV group, n = 6 in the 1V7, 2V7 and 2V14 groups). All box plots display the median, 25th and 75th percentiles, and whiskers extending to the maximum and minimum data points. Data were analyzed by an unpaired Mann-Whitney U-test. A p value < 0.05 was considered statistically significant. E. Measurement of anti-recombinant S-RBD neutralizing antibodies across the four groups (n = 6 in the BV, 1V7, 2V7 and 2V14 groups). Serum was diluted 55 times for IgG. S-RBD, receptor binding domain of spike protein. F. Fraction of cells expressing IL2 and IL4 in BV group (n = 9), 1V7 group (n = 6), 2V7 group (n = 6) and 2V14 group (n = 6). All box plots display the median, 25th and 75th percentiles, and whiskers extending to the maximum and minimum data points. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. Conv, convalescent patients. Mega, megakaryocytes. S-RBD, Spike receptor binding domain. BV group, before vaccination. 1V7 group, 7 days after the first vaccination. 2V7 group, 7 days after the second vaccination. 2V14 group, 14 days after the second vaccination.
To reveal the dynamics of the cell composition at different time points before and after vaccination, we calculated the relative percentage of all cell types in the PBMCs of each sample ( Figure 1D). We found that CD14 + monocytes were significantly increased in the 1V7, 2V7 and 2V14 groups compared with the BV group ( Figure 1D), which was consistent with the BBIBP-CorV vaccination study using flow cytometry [26]. The relative percentage of CD16 + monocytes was significantly increased in the 1V7 group compared with the BV group ( Figure 1D). Both CD14 + monocytes and CD16 + monocytes have great potential for cytokine production [27], which plays a role in humoral immunity against extracellular pathogen invasion [28]. The proportion of proliferative cells was significantly increased in the 2V7 and 2V14 groups compared with the BV group ( Figure  1D). The relative abundance of plasma cells was increased in the 1V7 group ( Figure 1D). By contrast, the relative percentage of B cells (excluding plasma cells) decreased after the second dose ( Figure 1D). CD8 + T cells were decreased in the 2V14 group compared with the BV group.
To further validate the vaccination efficacy in our participants, 2019-nCoV Spike RBD IgG levels and neutralizing antibodies were measured using indirect enzyme-linked immunosorbent assay (ELISA) kits. Anti-S-RBD IgG levels and neutralizing antibodies were significantly increased after the second dose vaccination ( Figure 1E and S1C). Next, we calculated the fraction of cells expressing cytokine genes including IL1B, IL2, IL4, IL5, IL6, IL10, TNF and IFNG, at single-cell resolution. Compared with the BV group, the fraction of cells expressing IL2 (a T cell growth factor involved in the clonal expansion of antigen-specific T cells [29]) in PBMCs was significantly increased in the 2V7 and 2V14 groups ( Figure 1F). The fraction of cells expressing IL4 (a crucial immune factor for the differentiation of naïve T cells into type 2 helper T cells [30]) in PBMCs was significantly increased in the 2V14 group ( Figure 1F). By contrast, the fraction of cells expressing cytokine gene IL6 in PBMCs was significantly decreased after vaccination ( Figure   S1D). Whereas, the serum levels of circulating proinflammatory cytokines, such as IL2, IL-6 and TNFα, did not significantly change after vaccination ( Figure S1E).

Features of innate immune cells after BBIBP-CorV vaccination
To further investigate the transcriptomic changes in innate immune cells (Figure 2A, B) after BBIBP-CorV vaccination, we compared the gene expression profiles of monocytes and NK cells pre-and post-vaccination. An immune response against the virus is generally achieved 2 weeks after booster vaccination [31]. We found that anti-S-RBD IgG levels and neutralizing antibodies were significantly increased after the second dose vaccination ( Figure 1E and S1C). Thus, we focused the 2V7 and 2V14 groups for further analyses. We identified DEGs in the CD14 + monocytes and CD16 + monocytes of the 2V7 and 2V14 groups compared with the BV group ( Figure 2C-F). Functional enrichment analysis showed that the upregulated DEGs in CD14 + monocytes in the 2V7 and 2V14 groups were enriched in "interlukin-1 production", "positive regulation of cytokine production", "cellular response to interferon-gamma" and "type I interferon production" pathways ( Figure 2C, D). Interestingly, the IL-1 family cytokines are potential mucosal vaccine adjuvants and can induce antigenspecific immune responses against pathogens such as influenza virus [32]. The upregulated DEGs in CD16 + monocytes in the 2V7 and 2V14 groups were enriched in "type I interferon production", "positive regulation of cytokine production", "response to interferon gamma", "toll-like receptor signaling pathway" and "antigen processing and exogenous antigen" pathways ( Figure 2E, F). Notably, type I interferon signalling promotes humoral immunity following vaccination, including vaccine-induced antibody, B cell and follicular helper T (Tfh) cell responses [33]. Additionally, we analyzed the expression of type I interferon production-related pathway in innate immune cells across four time points. We found that type I interferon production was uniformly and significantly upregulated in monocytes, mDCs and NK cells in the 2V7 and 2V14 groups ( Figure 2G).
In addition, we identified DEGs in NK cells, including CD56 low and CD56 high NK cells, by comparing the 2V7 and 2V14 groups with the BV group ( Figure S2A, B). Functional enrichment analysis showed that upregulated DEGs in the 2V7 and 2V14 groups were enriched in the "response to virus", "lymphocyte differentiation" and "response to interferon-gamma" pathways, which had previously been reported in COVID-19 patients [34]. Interesting BP terms are highlighted in red. G. Box plots showing the expression of the type I interferon production pathway across innate immune cells derived from BV group (n = 9), 1V7 group (n = 6), 2V7 group (n = 6) and 2V14 group (n = 6). Groups are shown in different colours. All box plots display the median, 25th and 75th percentiles, and whiskers extending to the maximum and minimum data points. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. DEGs, differentially expressed genes. FC, fold change. BP, biological process. BV group, before vaccination. 1V7 group, 7 days after the first vaccination. 2V7 group, 7 days after the second vaccination. 2V14 group, 14 days after the second vaccination. Interesting BP terms are highlighted in red. F. Box plots showing the expression of the T cell receptor signalling pathway across CD4 + T cell subsets derived from BV group (n = 9), 1V7 group (n = 6), 2V7 group (n = 6) and 2V14 group (n = 6). Groups are shown in different colours. All box plots display the median, 25th and 75th percentiles, and whiskers extending to the maximum and minimum data points. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. G. GSEA enrichment plots showing two upregulated gene sets in CD4 + T cells of the 2V14 group compared with those in the BV group. NES, normalized enrichment score. A p value < 0.05 was considered statistically significant. DEGs, differentially expressed genes. FC, fold change. BP, biological process. BV group, before vaccination. 1V7 group, 7 days after the first vaccination. 2V7 group, 7 days after the second vaccination. 2V14 group, 14 days after the second vaccination.

Characterization of T cells after BBIBP-CorV vaccination
To characterize changes in T cell subsets, we subclustered T cells from PBMCs and obtained 15 subsets according to the expression of T cell signature genes ( Figure 3A , B and Supplementary Table 2, see  Methods). To gain insight into the features in T cell subsets before and after vaccination, we calculated the relative percentage of each T cell subset against the total T cell population of each sample ( Figure 3C). The proportion of CD4_Tem_ANAX1 cells was significantly increased in the 1V7 group compared with the BV group ( Figure 3C). Whereas, CD4_Th_GATA3 and CD4_Tcm_AQP3 cells expanded in the 1V7, 2V7 and 2V14 groups compared with the BV group ( Figure  3C). CD4_Th_GATA3 cells harboured Th2 cell features with high expression of GATA3 ( Figure 3B), which was involved in humoral immunity against extracellular pathogens by producing cytokines such as IL-4, IL-5 and IL-13 [35,36]. The increased central memory CD4 + T cells (CD4_Tcm_AQP3) produce cytokines associated with rapid T cell secondary expansion [37], such as IL-2. In addition, the proportion of CD8_Effctor_GNLY cells was significantly increased in the 1V7 group compared with the BV group, and returned to basal levels after the second dose vaccination ( Figure 3C).
To further investigate the transcriptomic changes in T cells after the booster vaccination, we compared the gene expression profiles of the 2V7 and 2V14 groups with that of the BV group in CD4 + T and CD8 + T cells. For CD4 + T cells, functional enrichment analysis showed that the upregulated DEGs in the 2V7 and 2V14 groups were enriched in "T cell activation", "leukocyte cell-cell adhesion", "T cell differentiation", "T cell receptor signaling pathway" and "B cell activation" pathways ( Figure 3D, E and S3A, B). In CD4 + T cells, the expression of IFN-related gene IFITM3 [38] and broad-spectrum anti-retroviral factor SAMHD1 [39] was significantly upregulated in the 2V7 and 2V14 groups compared with the BV group ( Figure 3D and  S3A, B). These results indicated enhanced anti-viral immunity after the booster vaccination. In addition, we analyzed the expression of T cell receptor signalling pathway in memory and Th CD4 + T cell subsets across four time points. We found that expression of the T cell receptor signalling pathway was significantly upregulated in these cell subsets including memory CD4 + T cells (CD4_Tcm_ANXA1 and CD4_Tem_ANXA1), helper CD4 + T cells (CD4_Th_GATA3 and CD4_Th_STAT1) and regulatory CD4 + T cells (CD4_Treg_FOXP3) from the 2V7 and 2V14 groups ( Figure 3F). Furthermore, Gene Set Enrichment Analysis (GSEA) showed that the "positive regulation of humoral immune response" and "positive regulation of T cell cytokine production" pathways were significantly enriched in CD4 + T cells from the 2V7 and 2V14 groups ( Figure S3C). Additionally, the JAK-STAT signalling and PI3K-Akt signalling pathways were significantly enriched in CD4 + T cells from the 2V14 group ( Figure 3G). For CD8 + T cells, functional enrichment analysis showed that upregulated DEGs in CD8 + T cells in the 2V7 and 2V14 groups were enriched in "T cell activation", "leukocyte cellcell adhesion", "T cell differentiation", "T cell receptor signaling pathway" and "B cell activation" pathways ( Figure S3D, E). Remarkably, the expression of cytotoxicity-related genes (FCGR3A [40] and KLRD1 [41]) and anti-retroviral factor SAMHD1 in CD8 + T cells was significantly increased in the 2V7 and 2V14 groups compared with the BV group ( Figure S3D, E). The expression of granzyme-related genes (GZMA and GZMK) was upregulated in CD8 + T cells in the 2V7 group ( Figure S3D), whereas the expression of cytotoxicity gene PRF1 (encoding perforin 1) was significantly increased in CD8 + T cells in the 2V14 group compared with the BV group ( Figure S3E). Collectively, these highly expressed IFN-related genes and cytotoxic genes hinted that T cells may possess powerful cytotoxicity against SARS-CoV-2-infected cells, following booster vaccination.

Global profile of CDR3 length and TCR VJ genes distribution after BBIBP-CorV vaccination
To gain insight into the dynamics of the TCR repertoire during immune responses to BBIBP-CorV vaccine, we reconstructed full-length TCR sequences from the sequencing data. We first assessed the complementarity determining region 3 (CDR3) length distribution of the TCR α and TCR β chains across four time points. The results showed that the distribution of CDR3 length was not significantly changed before and after vaccination ( Figure 4A, B).
We then examined biases in TCR α and TCR β gene usage by comparing repertoire assignments before and after vaccination ( Figure 4C-F and  S4A). Within the TCR α chain ( Figure 4C, D), compared with pre-vaccination, the results showed that: 1) the expression of TRAV39 and TRAJ8 was increased in the 1V7, 2V7 and 2V14 groups; 2) the expression of TRAJ3 was increased in the 1V7 and 2V7 groups and returned to the basal level in the 2V14 group; 3) the expression of TRAV34, TRAJ15 and TRAJ24 was increased in the 2V7 group and returned to the basal level in the 2V14 group; and 4) the expression of TRAV36/DV7 was increased in the 2V14 group. Within the TCR β chain ( Figure 4E, F), compared with pre-vaccination, the results showed that: 1) the expression of TRBV16 was increased in the 1V7 and 2V7 groups and returned to the basal level in the 2V14 group; 2) the expression of TRBV12-5 was increased in the 1V7 group and returned to the basal level in the 2V7 and 2V14 groups; and 3) the expression of TRBV3-1 was increased in the 2V14 group. In addition, we compared the paired VJ frequencies before and after vaccination ( Figure 4G and S4B). The results showed that the frequencies of TRAV41/TRAJ53, TRAV26-1/TRAJ41 and TRAV8-4/TRAJ44 were increased in the 2V14 group ( Figure 4G and S4C). The selective usage of VJ genes suggested that different immunodominant epitopes may drive the molecular composition of the TCR and may be associated with the BBIBP-CorV-specific T cell response.

Clonal expansion of CD4 + T cells after BBIBP-CorV vaccination
To further investigate the T cell repertoire and related T cell functions post-BBIBP-CorV vaccination, we analyzed the features of T cell clonal expansion at different showing the distribution of CDR3 amino acid lengths for the TCR α chain (A) and TCR β chain (B) across the four groups (n = 9 in BV group, n = 6 in the 1V7, 2V7 and 2V14 groups). C-F. Bar plot showing the usage of some TRAV genes (C), TRAJ genes (D), TRBV genes (E) and TRBJ genes (F) across the four groups (n = 9 in BV group, n = 6 in the 1V7, 2V7 and 2V14 groups). Error bars represent ± s.e.m. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. G. Heatmap showing the usage of significantly differentially-expressed V-J gene pairs of the TCR α and TCR β chains across the four groups. Red, high expression; blue, low expression. CDR3, complementarity determining region 3. BV group, before vaccination. 1V7 group, 7 days after the first vaccination. 2V7 group, 7 days after the second vaccination. 2V14 group, 14 days after the second vaccination. Interesting BP terms are highlighted in red. E. GSEA enrichment plots showing five upregulated gene sets in the clonal expanded CD4 + T cells of 2V14 group compared with those in the BV group. NES, normalized enrichment score. A p value< 0.05 was statistically significant. F. Heatmap showing the inferred significantly differentially-expressed TF regulons between the 2V14 and BV groups. Numbers between brackets represent the regulon counts for respective transcription factors. Red, high expression; blue, low expression. G. Box plots showing the area under the curve of STAT1(940 g), STAT4 (14 g) and STAT6 (56 g) in the clonal expanded CD4 + T cells derived from BV group (n = 9), 1V7 group (n = 6), 2V7 group (n = 6) and 2V14 group (n = 6). Groups are shown in different colours. Numbers between brackets represent the regulon counts for respective transcription factors. All box plots display the median, 25th and 75th percentiles, and whiskers extending to the maximum and minimum data points. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. DEGs, differentially expressed genes. FC, fold change. BP, biological process. BV group, before vaccination. 1V7 group, 7 days after the first vaccination. 2V7 group, 7 days after the second vaccination. 2V14 group, 14 days after the second vaccination.
time points after vaccination. The percentage of TCR classification counts (the number of different TCR sequences in a sample) to the total cell number was used to indicate TCR diversity. Notably, clonal analysis showed that vaccination induced the expansion of small-sized clones (clone size: 2-5 cells) in CD4 + T cells in the 2V14 group ( Figure 5A, B); whereas, no obvious clonal expansion was observed in CD8 + T cells ( Figure S5A). We next calculated the TCR diversity for T cell subsets across four time points (Figure 5B and S5B). Compared with pre-vaccination, the results showed that: 1) the TCR diversity of CD4_Tcm_AQP3 cells was decreased in the 2V7 and 2V14 groups; 2) the TCR diversity of CD4_Th_GATA3, CD4_Treg_FOXP3 and CD8_Naive_LEF1 cells was decreased in the 2V14 group; and 3) the TCR diversity of CD4_Naive_CCR7 cells was decreased in the 1V7 and 2V14 groups ( Figure  5B and S5B).
Next, to reveal the functional features of CD4 + T cells with expanded TCR clonotypes (cell sizes ≥ 2) between pre-and post-vaccination, we performed DEG analysis on these clonal expanded CD4 + T cells, which may represent the most active CD4 + T cells against viral antigens. The DEG analysis showed that the expression of Th and memory T cell differentiation transcription factor (TF) genes TCF7 [42], LEF1 [43], STAT1 [44], STAT5B [45] and IKZF1 [46] was upregulated in the 2V7 and 2V14 groups compared with BV group (Figure 5C and S5C). Functional enrichment analysis showed that upregulated DEGs in the 2V7 and 2V14 groups were enriched in "T cell activation", "T cell differentiation", "T cell receptor signaling pathway" and "B cell activation" pathways ( Figure 5C, D and S5C, D). We next illustrated the properties of these clonal expanded CD4 + T cells before and after vaccination by GSEA analysis. FOXO signalling, JAK-STAT signalling, PI3K-Akt signalling, B cell activation and humoral response-related pathways were significantly enriched in these clonal expanded CD4 + T cells in the 2V14 group ( Figure 5E). To further investigate the transcriptional regulatory features of clonal expanded CD4 + T cells after the booster vaccination, we predicted the core TFs using SCENIC ( Figure 5F). Consistent with the DEG analysis ( Figure 5C), the expression of TF regulons LEF1(12 g), STAT1(940 g), STAT5B(174 g) and IKZF1(334 g) was significantly upregulated in the 2V14 group ( Figure 5F). Notably, the expression of TF regulon STAT1(940 g), associated with Th1 cell differentiation, was significantly upregulated after vaccination (1V7, 2V7 and 2V14 groups) ( Figure 5G). Whereas, the expression levels of Th1associated TF regulon STAT4(14 g) and Th2-associated TF regulon STAT6 (56 g) were significantly upregulated in the 2V7 and 2V14 groups ( Figure 5G). These results implied a potential role of these TFs in regulating the differentiation of CD4 + T cells during the vaccination process.
To gain insight into the features of B/plasma cell subsets, we evaluated the distribution of each subset during the vaccination process ( Figure 6C). Compared with the BV group, the proportion of memory B (Memory_B) cells was increased after vaccination (1V7, 2V7 and 2V14 groups) ( Figure 6C). The proportion of atypical memory B (Atypical_memory_B) cells was increased in the 1V7 and 2V14 groups ( Figure 6C). By contrast, a previous scRNA-seq study demonstrated that the proportion of memory B cells was decreased in patients with COVID-19 compared with healthy donors [31]. The proportion of Plasma_B_XBP1 cells was increased in the 1V7 group ( Figure 6C). By contrast, the proportion of naïve B cells was decreased after vaccination (1V7, 2V7 and 2V14 groups) ( Figure 6C), which was consistent with a recent study on COVID-19 mRNA vaccination involving scRNA-seq analysis [31].
To further investigate differential transcriptomic changes in B/plasma cells after the booster vaccination, we compared the expression profiles of B/ plasma cells in the 2V7 and 2V14 groups to those in the BV group ( Figure 6D and S6A, B). In comparison with the BV group, functional enrichment analysis showed that the upregulated DEGs in the 2V7 and 2V14 groups were enriched in "B cell activation", "B cell receptor signaling" and "toll-like receptor signaling pathway" pathways ( Figure 6E). Furthermore, we analyzed the expression of immunoglobulin production pathway in B/plasma cell subsets. Compared with the BV group, Naïve_B, Germinal_central_B, Memory_B and Atypical_memory_B cells showed higher enrichment of the immunoglobulin production pathway in the 2V7 and 2V14 groups ( Figure 6F).

Specific rearrangement of the VJ genes of BCR after BBIBP-CorV vaccination
Next, to understand the dynamics of the BCR repertoire during immune responses to BBIBP-CorV vaccine, we reconstructed BCR sequences from BCR sequencing data and analyzed the usage of VJ genes and BCR clonal expansion. We first assessed the length distribution of CDR3 amino acid (aa) sequences across four time points. The results showed that the frequency of 18 aa of heavy chain ( Figure 7A) and 13 aa of light chain ( Figure 7B) was increased in the 2V14 group compared with the BV group.
We then compared the usage of VJ genes across four time points after vaccination ( Figure 7C-F and showing the expression of the immunoglobulin production pathway across B cell subsets derived from BV group (n = 9), 1V7 group (n = 6), 2V7 group (n = 6) and 2V14 group (n = 6). Groups are shown in different colours. All box plots display the median, 25th and 75th percentiles, and whiskers extending to the maximum and minimum data points. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. DEGs, differentially expressed genes. FC, fold change. BP, biological process. BV group, before vaccination. 1V7 group, 7 days after the first vaccination. 2V7 group, 7 days after the second vaccination. 2V14 group, 14 days after the second vaccination. S7A). Notably, in comparison with the BV group, gene usage analysis showed that: 1) the expression of IGHV2-26 and IGHV3-73 was increased in the 2V7 and 2V14 groups ( Figure 7C); and 2) the expression of IGHV3-74 was increased in the 2V14 group. Furthermore, the expression of IGHJ1 and IGHJ4 was increased in the 1V7 and 2V14 groups ( Figure 7D). In addition, compared with the BV group, we observed that: 1) the expression of IGKV3-15 was increased after vaccination (1V7, 2V7 and 2V14 groups); 2) the expression of IGKV3-10 and IGLV10-54 was increased in the 2V7 and 2V14 groups; and showing the distribution of amino acid lengths of CDR3 for the BCR heavy chain (A) and BCR light chain (B) across the four groups (n = 9 in BV group, n = 6 in the 1V7, 2V7 and 2V14 groups). Error bars represent ± s.e.m. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. C-F. Bar plot showing the usage of IGHV genes (C), IGHJ genes (D), IGKV/LV genes (E) and IGKJ/LJ genes (F) across the four groups (n = 9 in BV group, n = 6 in the 1V7, 2V7 and 2V14 groups). Error bars represent ± s.e.m. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. G. Heatmap showing the usage of significantly differentially-expressed V-J gene pairs of the BCR heavy and light chains across the four groups (n = 9 in BV group, n = 6 in the 1V7, 2V7 and 2V14 groups). Red, high expression; blue, low expression. H. Box plots showing the UMI percentages of IGHD, IGHA, IGHG and IGHM in B/plasma cells across the four groups (n = 9 in BV group, n = 6 in the 1V7, 2V7 and 2V14 groups). All box plots display the median, 25th and 75th percentiles, and whiskers extending to the maximum and minimum data points. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. CDR3, complementarity determining region 3. BV group, before vaccination. 1V7 group, 7 days after the first vaccination. 2V7 group, 7 days after the second vaccination. 2V14 group, 14 days after the second vaccination.
Then, we evaluated the distribution of immunoglobulin heavy genes, IgA, IgD, IgG and IgM, in B cells across four time points. In comparison with the BV group, the abundance of IGHA and IGHG was increased in the 1V7 and 2V14 groups ( Figure 7H). Whereas, the abundance of IGHD and IGHM was decreased after vaccination (1V7, 2V7 and 2V14 groups) ( Figure 7H). Next, we assessed the clonal BCR expansion status before and after vaccination. The BCR clones of HD1, HD4 and HD5 expanded after the second-dose vaccination in the 2V7 and 2V14 groups ( Figure S7C). Taken together, these results revealed the dynamics of the BCR repertoire induced by booster BBIBP-CorV vaccination.

Comparison of immunological features following BBIBP-CorV vaccination and SARS-CoV-2 infection
To reveal the differences in the immune response between vaccination and SARS-CoV-2 infection, we first assessed key pathways that were reported to be associated with symptom development in patients [17] and were also enriched in BBIBP-CorV vaccine recipients ( Figure 2D, 2F, 6F and S3C), including the interleukin-6 production pathway, tumour necrosis factor production pathway, type I interferon production pathway, T cell cytokine production pathway and immunoglobulin production pathway. We found that the expression of pro-inflammatory cytokine production-related pathways in monocytes and mDCs (including the IL-6 and TNF-α production pathways) was upregulated in patients with moderate symptoms, whereas no significant changes were observed in individuals following vaccination ( Figure 8A, B). The expression of the T cell cytokine production-related genes was upregulated in T cells post-vaccination and in recovered convalescent patients compared with pre-vaccination ( Figure 8C), but no significant changes were observed in patients with moderate symptoms ( Figure 8C). The expression of the type I interferon production-related genes in monocytes and mDCs was upregulated after the booster vaccination, whereas it increased in the mDCs of patients with moderate symptoms and returned to normal levels in recovered convalescent patients ( Figure   S8A, B). In addition, compared with pre-vaccination, the expression of the immunoglobulin production pathway in B cells was upregulated after booster vaccination and in patients with moderate symptoms, and returned to normal levels in recovered convalescent patients ( Figure 8D). Together, these results revealed the differences and similarities in the immune response, as evidenced by the transcriptome, between vaccination and SARS-CoV-2 infection.
Next, we compared the expression of IGV genes following vaccination or SARS-CoV-2 infection with expression levels pre-vaccination. We found that the expression of IGHV2-26 increased in vaccinated participants and recovered convalescent patients compared with pre-vaccination participants ( Figure 8E). The expression of IGLV3-10 ( Figure 8F) and paired IGHV4-30-2/IGHJ4 ( Figure 8G) was upregulated in vaccinated participants, patients with moderate symptoms and recovered convalescent patients. Interestingly, a previous study reported that IGLV3-10 was involved in the production of N276-dependent HIV-1-neutralizing antibodies [47]. Our results revealed the changes in transcription and the repertoire dynamics of immune cells in response to BBIBP-CorV vaccination and COVID-19 progression.

Discussion
The establishment of immunity against SARS-CoV-2 by vaccination is a key strategy to reduce the spread and severity of COVID-19. Notably, recent clinical studies demonstrated that the effectiveness of different vaccines, including BBIBP-CorV, mRNA, Ad5-nCoV and CoronaVac, against COVID-19 was distinct [13,14]. Here, we extensively investigated the variability of the single-cell transcriptomes and TCR/BCR repertoires of PBMCs at four time points following vaccination with the inactivated vaccine BBIBP-CorV. We also compared the transcription and repertoire dynamics of immune cells in response to vaccination and SARS-CoV-2 infection. We observed temporally aligned alterations in cell type composition, gene expression and clonal expansion, providing novel insights into BBIBP-CorV-elicited immune responses. Moreover, we identified the similarities and differences in immunity induced by SARS-CoV-2 infection and vaccination.
The adaptive immune response is a major determinant of the clinical outcome following SARS-CoV-2 infection and underpins vaccine efficacy [48]. Notably, there is a direct association between the intensity of a vaccine-elicited T cell response and the capability of the immune system to eliminate virus [49]. Studies have provided evidence that effective prophylactic vaccines against replicating viruses should engage a proper T cell immune response [50]. The distribution analysis of T cell subsets showed significant increases in Th2 cells (CD4_Th_GATA3) and central memory CD4 + T (CD4 + Tcm) cells (CD4_Tcm_AQP3) after the primary and booster vaccinations. Th2 cells are critical for humoral immunity against extracellular pathogens by producing cytokines such as IL-4 and IL-5 [51]. CD4 + Tcm cells, with superior proliferation capacity, are required for long-lasting immunity and are induced by vaccination strategies, including those against influenza [52] and tuberculosis [53]. This is in line with a recent study of Ad5-nCoV vaccination, which demonstrated the expansion of CD4 + Tcm cells at day 28 following immunization [19]. Moreover, the TCR diversity of CD4 + Tcm cells, Th2 cells and CD4 + Treg cells decreased at 2 weeks after the booster vaccination, displaying increased cell numbers and expanded clones, which indicated the in CD14 + monocytes, CD16 + monocytes and mDCs derived from the BV group (n = 9), 1V7 group (n = 6), 2V7 group (n = 6), 2V14 group (n = 6), moderate group (n = 6) and conv group (n = 11). Groups are shown in different colours. All box plots display the median, 25th and 75th percentiles, and whiskers extending to the maximum and minimum data points. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. C. Box plots showing the expression of the T cell cytokine production pathway in CD4 + and CD8 + T cells derived from the BV group (n = 9), 1V7 group (n = 6), 2V7 group (n = 6), 2V14 group (n = 6), moderate group (n = 6) and conv group (n = 11). Groups are shown in different colours. All box plots display the median, 25th and 75th percentiles, and whiskers extending to the maximum and minimum data points. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. D. Box plots showing the expression of the immunoglobulin production pathway in B cells derived from the BV group (n = 9), 1V7 group (n = 6), 2V7 group (n = 6), 2V14 group (n = 6), moderate group (n = 6) and conv group (n = 11). Groups are shown in different colours. All box plots display the median, 25th and 75th percentiles, and whiskers extending to the maximum and minimum data points. Data were analyzed by an unpaired Mann-Whitney U-test. A p value< 0.05 was considered statistically significant. E. Venn diagrams showing the upregulated IGHV gene numbers for the post-vaccination and conv groups compared with the BV group. F. Venn diagrams similar to E, but for the IGKV/LV gene in the post-vaccination, moderate and conv groups. G. Venn diagrams similar to F, but for the V-J gene pairs for the BCR heavy and light chains. To be considered upregulated post-vaccination, the genes had to be upregulated in at least one of the three groups (1V7, 2V7 or 2V14). Conv, convalescent patients. BV group, before vaccination. 1V7 group, 7 days after the first vaccination. 2V7 group, 7 days after the second vaccination. 2V14 group, 14 days after the second vaccination. rapid response of these T cell types to SARS-Cov-2 infection. CD4 + Treg cells are a subset of T cells with the capacity to negatively regulate the immune response, maintaining homeostasis and preventing autoimmunity [54]. TCR specificity is determined through VJ gene rearrangement to generate more efficient clonal amplification, which is essential for a successful immune response [55,56]. In current study, some of highly used VJ genes in post vaccination group have been previously reported to be related to memory CD4 T cell activation. For example, one recent study revealed that TRAV39 expression was associated with memory CD4 T cell activation in Esophageal Squamous Cell Carcinoma [57]. Clinical evidence showed that deaths from COVID-19 disease were mainly caused by a cytokine storm [58], which is the result of an uncontrolled immune response. Of note, a recent study reported that individuals who received two doses of BBIBP-CorV were protected against the more serious consequences of infection [13]. The small clone expansion of CD4 + Treg cells might be involved in controlling the overactive immune response and cytokine storm induced by SARS-CoV-2 in severe cases, thus BBIBP-CorV vaccination reduces the risk of serious disease and death.
The presence of neutralizing antibodies is currently used as a surrogate indicator of immunity. Our data indicated that neutralizing antibody levels were significantly higher in the vaccinated groups. In addition, the number of memory B cells increased following the booster vaccination. Consistent with our results, recent studies using ELISA and flow cytometry also demonstrated that BBIBP-CorV vaccine induced neutralizing antibody production and spike-or RBDspecific memory B cells following the standard twodose vaccination regimen [26,59]. A high level of IgG effectively protects against virus infection [60], while IgA is the major mucosal antibody that constitutes an important first line of defense [61]. Compared with pre-vaccination, the abundance of IGHG and IGHA was increased after the booster vaccination, indicating the high efficacy of the B cell response following two doses of BBIBP-Cov2 vaccine.
Additionally, we found that there was an increase in BCR clonal expansion and skewed usage of VJ genes of BCR in the vaccinated individuals. Notably, some of those biased usage of VJ genes were reported to be associated with SARS-CoV-2-specific immune responses and antibodies. For example, IGLV3-10 and IGHV1-18/IGHJ6 are involved in the human B cell response to SARS-CoV-2 virus [62,63], and IGHV2-70 and paired IGHV1-24/IGHJ4 are involved in the production of anti-SARS-CoV-2 neutralizing antibodies [64,65]. Collectively, these data may help pave the way for the development of the next-generation precision vaccine.
Comparison of the immune responses elicited by SARS-CoV-2 infection and vaccination will help us to better understand how BBIBP-CorV protects against the risk of serious disease and death. The expression of genes associated with the production of pro-inflammatory cytokines, such as IL-6 and TNF-α, did not change significantly in BBIBP-CorV vaccination recipients, despite these genes being significantly upregulated in patients with moderate symptoms. The systemic distribution of TNF-α and IL-6 is a major trigger of the cytokine storm observed in some patients [66], as well as some of the side effects induced by the vaccine [67]. Moreover, CD8 + T cells are the mainly clonal expanded population of cells in patients recovering from SARS-CoV-2 infection [68] and those vaccinated with Ad5-nCoV [19]. Additionally, mRNA vaccines, including BNT162b2 and mRNA-1273, were shown to elicit CD8 + T cells clonal expansion and the Th1 cell responses without affecting Th2 cell responses [69]. However, our analysis of TCR repertoires revealed that CD4 + T cells (but not CD8 + T cells) displayed an expansion following BBIBP-CorV vaccination. These data suggested that the inactivated vaccines mainly induce a Th2-B cell immune response favouring the production of anti-SARS-Cov-2 antibodies [70].
Clinical studies of two types of inactivated SARS-CoV-2 vaccines, BBIBP-CorV and CoronaVac, showed that BBIBP-CorV exhibits a higher protection rate against severe cases and a lower infection rate with SARS-Cov-2 compared with CoronaVac [13]. A recent scRNA-seq study revealed that inactivated vaccine CoronaVac downregulated anti-viral signalling pathways (i.e. type I interferon responses) and elevated pro-inflammatory signalling pathways (i.e. NF-κB signalling) [21], which was associated with side effects and systemic inflammation. However, the functional transcription and TCR/BCR repertoire dynamics of immune cells had not been defined.
Although a recent scRNA-seq study indicated that BBIBP-CorV could induce an efficient humoral immune response, the cellular immune response, which is the pivot of humoral immune response, remained to be further investigated [71]. Our current study indicated that the inactivated vaccine BBIBP-CorV activated an anti-viral immune response with clonal expansion of Th2 cells, central memory CD4 + T cells and Treg cells. The distinct immune features induced by these two SARS-CoV-2 inactivated vaccines may contribute to the different protection rates against severe cases of SARS-CoV-2 infection.
Based on the abovementioned findings, we hypothesize that: 1) inflammatory monocytes induced by booster vaccination optimize immune mobilization against SASR-CoV-2 infection; 2) the Th2 cells and CD4 + Tcm cells induced by booster vaccination rapidly respond to SASR-CoV-2 infection, thereby preventing disease exacerbation; 3) post-booster vaccination, memory B cells rapidly produce neutralizing antibody with the help of CD4 + T cells; and 4) along with the immune response against SASR-CoV-2, expanded CD4 + Treg clones act as a brake, preventing an excessive immune response induced by inflammatory immune cells, and orchestrating appropriate immunity against SASR-CoV-2 in the vaccination recipients.
Taken together, our study provides novel insights into the cellular and molecular basis of the post-SARS-CoV-2 vaccination response at single-cell resolution, especially the CD4 + T cell-dependent antibody response induced by BBIBP-CorV. Our findings may facilitate the development of more potent, durable and safe prophylactic vaccines against SARS-CoV-2 infection. However, the present study is limited by its small sample size. Further studies involving a larger number of samples and longer follow-up times are required to understand the long-term cellular and humoral immune responses to SARS-CoV-2 vaccines. In addition, the combination of single-cell RNA and TCR/BCR sequencing analysis revealed that the BBIBP-CorV vaccination might induce antigenspecific expansion of T and B cells, while further studies with more direct evidence are needed to validate these observations.