key: cord-0328120-2nrmigo1 authors: Zhang, Yuwei; Guo, Xingyu; Li, Cunbao; Kou, Zengqiang; Lin, Lanfang; Yao, Mingxiao; Pang, Bo; Zhang, Xiaomei; Duan, Qing; Tian, Xueying; Xing, Yufang; Jiang, Xiaolin title: Transcriptome analysis of SARS-CoV-2 naïve and recovered individuals vaccinated with inactivated vaccine date: 2021-11-08 journal: bioRxiv DOI: 10.1101/2021.11.05.467537 sha: 618e72f96b207da700558ada76bf8ad04d6b394a doc_id: 328120 cord_uid: 2nrmigo1 The urgent approval of the use of the inactivated COVID-19 vaccine is essential to reduce the threat and burden of the epidemic on global public health, however, our current understanding of the host immune response to inactivated vaccine remains limited. Herein, we performed serum IgG antibody detection and transcriptomics analysis on 20 SARS-CoV-2 naïve individuals who received multiple doses of inactivated vaccine and 5 SARS-CoV-2 recovered individuals who received single dose of inactivated vaccine. Our research revealed the important role of many innate immune pathways after vaccination, identified a significant correlation with the third dose of booster vaccine and proteasome-related genes, and found that SARS-CoV-2 recovered individuals can produces a strong immune response to a single dose of inactivated vaccine. These results help us understand the reaction mechanism of the host’s molecular immune system to the inactivated vaccine, and provide a basis for the choice of vaccination strategy. Since December 2019, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has swept the world, causing a variety of clinical syndromes termed coronavirus disease 2019 (COVID-19)(1). The clinical manifestations of COVID-19 include fever, dry cough, fatigue, sore throat, pneumonia, diarrhea and other symptoms, and may even develop into severe pneumonia, acute respiratory distress syndrome (ARDS) or multiple organ failure (2) . The World Health Organization declared a pandemic in March 2020. COVID-19 has caused considerable impacts on the global economy and public health. Although for a long time, people have relied on social distancing, hygiene measures, and repurposed drugs to coping with it, now many researchers are committed to developing safe and effective vaccines to establish herd immunity to prevent SARS-CoV-2. In view of the turmoil caused by the COVID-19 pandemic and the urgent need for effective vaccine, vaccine development can be accelerated by combining originally requested phases. The vaccine does not go through a complete approval process, but may be approved for emergency use (3) . Early clinical trial results of the inactivated vaccines produced by Sinopharm and Sinovac showed a low incidence of adverse reactions and good immunogenicity (4) (5) (6) (7) (8) . On July 22, 2020, the above two candidate inactivated vaccines were approved for use (9) . These two vaccines are widely promoted and vaccinated, but the reaction mechanism of the host's molecular immune response to the inactivated vaccine is not yet fully understood, and the implementation of the third booster dose is also being actively discussed recently (10) (https://www.cdc.gov/coronavirus/2019-ncov/vaccines/booster-shot.html). In addition, the impact of prior SARS-CoV-2 infection status on vaccination response is also worthy of further analysis. These insights may provide a theoretical basis for the determination of vaccination strategies and the allocation of vaccine resources. The application of high-throughput technology to systematically scan the transcriptome response and evaluate changes in gene expression levels is very suitable for identifying immune response dynamics and gene regulatory networks. Previously, transcriptome analysis of Hantavax vaccine(11), influenza vaccine(12), VSV-EBOV vaccine (13) and BNT162b mRNA vaccine (14) have fully revealed the dynamics of the host immune response after vaccination. In this study, we characterized the PBMC transcriptome changes of SARS-CoV-2 recovered individuals receiving one dose of vaccine and healthy SARS-CoV-2 naïve individuals receiving one to three doses of vaccine respectively. This real-world study showed the changes of various cytokines and the regulation of immune pathways induced after vaccination, reveal the indispensable role of innate immune pathways, and reflect the key modules information of vaccine response in different individuals and different doses of vaccine. Key words COVID-19, inactivated vaccine, RNA-seq, transcriptome analysis From January 2021, through August 2021, we recruited five SARS-CoV-2 recovered and twenty healthy SARS-CoV-2-naïve individuals to participate in this study in Linyi City, Shandong Province. Under different doses of the COVID-19 inactivated vaccine, anticoagulant and procoagulant blood samples were collected from participants. This study was approved by the Ethical Approval Committee of Shandong Center for Disease Control and Prevention. The venous blood was collected from each participant to separate serum or isolate PBMCs. We separated sera by centrifugation at 2500 rpm/min for ten minutes and preserved at −80 °C until testing. PBMCs were isolated by density-gradient sedimentation using Lymphoprep™ density gradients (Axis-Shield, Norway), frozen in cell saving media and stored in liquid nitrogen. The IgG antibodies were detected using an indirect ELISA kit (Beijing Wantai Biological Pharmacy Enterprise Co, China) (15, 16) based on a recombinant nucleoprotein of SARS-CoV-2. The specific operation is carried out in strict accordance with the instructions. The cut-off value for IgG is the mean OD value of three negative controls (if the mean absorbance value for three negative calibrators is < 0.03, take it as 0.03) + 0.16. A serum sample with an OD value ≥cut-off OD value was considered to be an anti-N IgG antibody positive. Total RNA was extracted from PBMCs by using the RNeasy RNA-seq data were processed quality control (QC), trimming and mapping to the human reference genome hg38 using CLC Genomics Workbench. Gene expression level was measured based on the transcripts per million (TPM). We calculated normalization factors using iterative edgeR (17) and limma (18) package, and filtered DEGs based on a p-value < 0.05 and a 2^logFC_cutoff. DEGs are visualized in the form of heat maps through the pheatmap (https://CRAN.R-project.org/package=pheatmap) package in R software. The initial PPI network for the protein products of identified up and down regulated DEGs was constructed using the STRING Database(19) (STRING v11.5; https://string-db.org/), and then the network was visualized and analyzed with Cytoscape software (20) . The CentiScape and MCODE plugins in the Cytoscape software were used to extract the characteristic genes from the DEGs. To be aware of the prospective functions of characteristic genes identified by the PPI network analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified using CluGO from Cytoscape software. P<0.05, subjected to Bonferroni adjustment, were defined as the cut-off criterion. We constructed the co-expression network through WGCNA package (21) in R software. The best β value was confirmed with a scale-free fit index bigger than 0.85 as well as the highest mean connectivity by performing a gradient test from 1 to 30. Subsequently, we transformed the adjacency matrix into a topological overlap matrix (TOM). The TOM obtained was then clustered by dissimilarity between genes, and we performed hierarchical clustering to identify modules, each containing at least 30 genes (minModuleSize=30). Some modules were combined according to the correlation coefficient. We calculated the correlation between the modules and the sample characteristics to identify significant modules. The genes in significant modules were analyzed using cytoHubba and GeneMANIA in Cytoscape software. The specific details are shown in Fig. 1 . In order to study the antibody response of serum samples to SARS-CoV-2 after vaccination, we used a fully validated commercial diagnostic ELISA kit to qualitatively measure IgG antibodies against N in serum samples diluted 1:10. The critical value for judging that the antibody level is positive is OD>0. 19 . The IgG antibodies of unvaccinated healthy participants were all negative, and only one healthy participant who received one dose of the vaccine was positive, whereas the IgG antibody results of recovered patients who received one dose of vaccine and healthy participants who received two or three doses of vaccine were both positive (Fig. 2) . The transcriptome data of five groups of PBMCs under different vaccination conditions were analyzed by limma and edgeR, and a total of 613 DEGs were found, of which 304 DEGs were up-regulated and 309 DEGs were down-regulated in the vaccination group. These results are shown in heatmap which is a simple yet effective way to compare the content of multiple major gene lists (Fig. 3A -B ). The PPI network of these up-regulated and down-regulated DEGs were established using STRING database, and the node and edge relationships were imported into Cytoscape software for visualization, and then the DC evaluation was completed. The degree results of 304 up-regulated DEGs and 309 down-regulated DEGs were demonstrated by the size of the point and the thickness of the edge (Fig. 4A and 5A ). The first 61 up-regulated genes and 76 down-regulated genes were selected as characteristic genes ( Fig. 4B and 5B) as a focus for subsequent examination. Using MCODE's k-core decomposition function, a variety of sub-networks were obtained, and two new networks were created, in which 23 up-regulated and 35 down-regulated differentially expressed genes were retained, respectively ( Fig. 4C and 5C ). The results of DC and MCODE of up-regulated and down-regulated characteristic DEGs analysis are summarized in Tab. 1 and Tab. 2. The differential expression of these characteristic DEGs in the four vaccination groups is shown in the heatmap ( Fig. 4D and 5D ). The KEGG term enrichment analyses were performed using CluGO. There were 19 KEGG pathways with P<0.05 (including Bonferroni adjustment) identified within the analysis of the 23 characteristic up-regulated genes, including TNF signaling pathway, IL-17 signaling pathway, Viral protein interaction with cytokine and cytokine receptor, Cytokine-cytokine receptor interaction, Pertussis, NF-kappa B signaling pathway and other pathways (Fig. 4E ). In these pathways, genes that play important roles include chemokine, interleukin, macrophage colony-stimulating factor, interferon regulatory factor 1 and tumor necrosis factor alpha-induced protein 3. There were four KEGG pathways with P<0.05 (including Bonferroni adjustment) identified within the analysis of the 35 characteristic down-regulated genes, including Systemic lupus erythematosus, Alcoholism, Neutrophil extracellular trap formation, Viral protein interaction with cytokine and cytokine receptor (Fig. 5E ). Among these pathways, it was hypothesized that genes which serve a significant role include histone cluster, chemokine and interleukin-6 receptor subunit alpha (IL6R). Detailed enrichment information for the KEGG pathways is summarized in Tab. 3-4. To explore the impact of different vaccine doses and SARS-CoV-2 infection status on the transcriptome, we made an analysis of the transcriptome using WGCNA. In this study, the power of β = 5(scale-free R 2 = 0.85) was selected as the soft-thresholding parameter to ensure a scale-free network (Fig. 6A-B) . A total of 37 modules were identified through hierarchical clustering, and a dendrogram of all DEGs was clustered based on a dissimilarity measure (1-TOM) (Fig. 6C-D) . The correlation between the sample characteristics and the co-expression module is shown in Fig. 6E , where the Brown (eigengene value = 0.69) module is significantly positively correlated with the vaccine dose. The darkorange (eigengene value = 0.91), yellow (eigengene value = 0.81) and steelblue (eigengene value = 0.71) modules are significantly correlated with the previous SARS-CoV-2 infection status (Fig. 6F-I) . In order to analyze the network in the corresponding modules and determine the importance and function of genes, we combined CytoHubba and GeneMANIA in Cytoscape to analyze and identify the hub genes of the above four modules. The functions of the hub gene of the brown module mainly focus on activation of innate immune response, response to interleukin-1, and cellular response to tumor necrosis factor, etc. The hub genes of darkorange module act on regulation of tyrosine phosphorylation of STAT protein, tyrosine phosphorylation of STAT protein, pattern specification process, postsynaptic neurotransmitter receptor activity, neurotransmitter receptor activity pathway. The hub gene of the yellow module performs adaptive immune response, regulation of T cell activation, and B cell mediated immunity, etc. The hub gene of steelblue module can participate in humoral immune response, detection of biotic stimulus, antimicrobial humoral response, glycosaminoglycan binding, peptidyl-arginine modification and other pathways (Fig. 7A-D) . The SARS-CoV-2 pandemic poses an imminent threat to humans, and the development and application of vaccines have brought hope to the effective prevention and control of the epidemic. In research on inactivated influenza vaccines, the expression of IL-17 and NF-κB pathway genes and related innate immune activation have been observed (12) ; Rubella vaccine significantly affects the host's antigen presentation and innate/inflammatory genome (22) ; Hantavax vaccine can mediate Th17 cell differentiation, antigen processing and presentation, NF-kappa B signalling pathway, phenylalanine metabolism, phagosome, Fc gamma R-mediated phagocytosis (11) . In the study of BNT162b2 mRNA vaccine, it was found that vaccination not only stimulated antiviral and interferon response modules, but also induced a broader innate immune response such as Toll-like receptor signalling, monocyte and neutrophil modules(23). The above research results emphasize the key role of innate immune response in vaccination. The characteristic genes that were significantly down-regulated in the vaccinated group included Histone cluster, chemokine and IL6R that participated in Systemic lupus erythematosus, Alcoholism, Neutrophil extracellular trap formation, Viral protein interaction with cytokine and cytokine receptor pathways. In a systems biology analysis of PBMC transcriptome data from COVID-19 patients, it was also found that key gene modules are not only involved in infection-related pathways, but also significantly enriched in Systemic lupus erythematosus and Alcoholism (24) . This coincidental consistency suggests that the role of these two pathways in COVID-19 needs to be further explored. In a study on Live-Attenuated Francisella tularensis vaccine, it was found that while a variety of immune pathways were activated early, some innate immune signaling pathways were inhibited, especially cytokine-cytokine receptor interactions. This result is related to the host escape mechanism of Francisella tularensis (25) . Recent investigations suggested that in addition to the virus itself, dysregulated host immune response to SARS-CoV-2 may also contribute to the pathogenesis of COVID-19 (26) (27) (28) . The inhibition of (29) . It is the catalytic core of the 26S proteasome and is distributed throughout eukaryotic cells at a high concentration (30) . An essential function of a modified proteasome is the processing of class I MHC peptides. It can continue to decompose intracellular proteins that may appear in the process of viral infection, and produce antigen peptides, which can be combined with MHC1 molecules in the endoplasmic reticulum and transport the complex to the cell surface to play a role in antigen presentation (31) . Obviously, the function of the proteasome in the immune system is significant, and the third booster vaccine is beneficial and harmless. (14, 35, 36) . The current results show that in the case of limited vaccine supply, we can consider reducing the number of vaccines for SARS-CoV-2 recovered individuals. However, in view of the relatively short duration of this study, future studies will be necessary to evaluate the impact of multiple doses of vaccine on the long-term immune response of recovered individuals. Our data provides quantitative and global insights into the dynamic changes of immune-related genes, reveals the differential expression of cytokines and inflammatory factors, as well as the regulation of antigen presentation and innate immune pathways, and helps us explain the effects of molecular immune mechanism induced by inactivated vaccines. The transcriptome characteristics of individuals with different SARS-CoV-2 infection and different doses may provide a new reference for the determination of vaccination strategies in the future. However, the results of this study rely on bioinformatics analysis, the sample size is small, and the long-term evaluation of the inoculation effect is lacking. We intend to further expand the sample size and conduct long-term follow-up to explore comprehensive insights into the inactivated COVID-19 vaccine. reviewed and edited the manuscript; X.L.J. and Y.W.Z. have shared responsibility for the decision to submit for publication. All authors reviewed and approved the final manuscript. The authors declare that they have no competing interests. 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