medRxiv preprint doi: https://doi.org/10.1101/2021.05.06.21256781; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 1 Characterizing cellular and molecular variabilities of peripheral immune cells in healthy 2 inactivated SARS-CoV-2 vaccine recipients by single-cell RNA sequencing 3 4 Renyang Tong1#, Jianmei Zhong1#, Ronghong Li1, Yifan Chen1, Liuhua Hu1, Zheng Li1, 5 Jianfeng Shi1, Guanqiao Lin1, Yuyan Lyu1, Li Hu1, Xiao Guo1, Qi Liu1, Tian Shuang1, Chenjie 6 Zhang1, Ancai Yuan1, Minchao Zhang2, Wei Lin1*, Jun Pu1* 7 8 1 State Key Laboratory for Oncogenes and Related Genes, Division of Cardiology, School of 9 Medicine, Renji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong University, Shanghai, 10 China. 11 2 Colortech (Suzhou) Biotechnology, Suzhou, China. 12 13 * To whom correspondence should be addressed: 14 Jun Pu, MD, Ph.D.; State Key Laboratory for Oncogenes and Related Genes, Division of 15 Cardiology, School of Medicine, Renji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong 16 University, Shanghai, China. E-mail: [email protected]. 17 Wei Lin, Ph.D.; State Key Laboratory for Oncogenes and Related Genes, Division of 18 Cardiology, School of Medicine, Renji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong 19 University, Shanghai, China. E-mail: [email protected]. 20 21 #These authors contributed equally to this work 22 23 24 25 26 27 28 29 30 31 32 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 33 medRxiv preprint doi: https://doi.org/10.1101/2021.05.06.21256781; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 1 Abstract 2 We systematically investigated the transcriptomes of the peripheral immune cells from 6 3 inactivated vaccine, BBIBP-CorV recipients at 4 pivotal time points using single-cell RNA-seq 4 technique. First, the significant variation of the canonical immune-responsive signals of both 5 humoral and cellular immunity, as well as other possible symptom-driver signals were 6 evaluated in the specific cell types. Second, we described and compared the common and 7 distinct variation trends across COVID-19 vaccination, disease progression, and flu vaccination 8 to achieve in-depth understandings of the manifestation of immune response in peripheral blood 9 under different stimuli. Third, the expanded T cell and B cell clones were correlated to the 10 specific phenotypes which allowed us to characterize the antigen-specific ones much easier in 11 the future. At last, other than the coagulopathy, the immunogenicity of megakaryocytes in 12 vaccination were highlighted in this study. In brief, our study provided a rich data resource and 13 the related methodology to explore the details of the classical immunity scenarios. 14 15 Keywords: COVID-19; Inactivated SARS-CoV-2 Vaccines; Peripheral Immune Cells; Single 16 Cell Sequencing 17 medRxiv preprint doi: https://doi.org/10.1101/2021.05.06.21256781; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 1 2 Introduction 3 As of May 2021, World Health Organization (WHO) has reported 148 million Coronavirus 4 disease 2019 (COVID-19) infections and 3.1 million deaths worldwide 5 (https://covid19.who.int/). Promotion and popularization of vaccine is vital to disease control, 6 with its safety and efficacy of the top priority. There are multiple SARS-CoV-2 vaccines 7 available and several vaccines under development, including the inactivated, genetically 8 engineered subunit, adenovirus and nucleic acid vaccine types. So far, a number of institutions 9 of different countries and regions have reported the results of safety and immunogenicity of 10 SARS-CoV-2 vaccine on phase III clinical trials or phase III interim analysis, such as, vaccines 11 BNT162b21 and mRNA-12732, adenovirus vaccines AZD12223, Ad5-nCoV4 and 12 inactivated vaccines BBIBP-CorV5 and CoronaVac6, 7, and protein subunit vaccine NVX-23738. 13 For example, Ugur Sahin et al.9 demonstrated that RNA vaccine (BNT162b2) not only elicited 14 adaptive humoral, but also stimulated the cellular immune response even at the low-dose 15 vaccine. The new vaccine types such as adenovirus-vectored vaccines and RNA vaccines have 16 emerged and fast developed with good efficacy in recent years, though severalserious adverse 17 events have been reported10, 11 12, 13. Their efficacy and long-term safety were yet to be reviewed 18 by a follow-up study. With the support of long clinical practice, traditional inactivated vaccines 19 play an irreplaceable role in the prevention and control of infectious disease. As a pattern of 20 inactivated SARS-CoV-2 vaccines, BBIBP-CorV, was manufactured by Sinopharm China 21 National Biotec Group (CNBG) and contained a SARS-CoV-2 strain inactivated inside Vero 22 Cells14, which was confirmed for its safety, tolerability, and immunogenicity in clinical trials of 23 healthy people in China5 and UAE, Bahrain, Egypt and Jordan (https://cdn.who.int/) . 24 Single-cell RNA-seq (scRNA-seq) technique has demonstrated its power in the research 25 of cell biology by deconvoluting the transcriptional signals in the multi-cellular samples of 26 heterogenous cellular compositions15,16. Other than characterizing the cell types in a 27 heterogeneous tissue, it enables us to detect the variabilities within a cell lineage. The recent 28 pandemic of COVID-19 has prompted the demand of single-cell studies in order to uncover the 29 characteristics immune responses of different immune cell types during the symptom 30 development of COVID-1917, 18, 19, 20. These published scRNA-seq datasets provide abundant 31 and detailed reference information of this disease, though, no results have been reported for any 32 COVID-19 vaccines yet. Moreover, a comprehensive comparison of the human immune 33 responsive signals across the transcriptome at single-cell resolution over the time becomes 34 practical and necessary. With no doubt, such detailed comparison facilitates the in-depth medRxiv preprint doi: https://doi.org/10.1101/2021.05.06.21256781; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 1 understanding of the pathogenesis, symptom development and immunization of COVID-19 2 which guides us to the better vaccination strategy. Therefore, we aimed to look through a 'sight 3 glass' of the human immunity system, a.k.a., peripheral blood mononuclear cells (PBMCs) to 4 monitor the manifestation of vaccine response of the canonical immune cells. In the meantime, 5 we could take the advantage of the recent published scRNA-seq datasets of COVID-19 patient 6 samples21 and Flu vaccination samples22 to achieve such a goal. Besides, the roles of 7 megakaryocytes/platelets in COVID-19 recently have attracted some attentions due to recent 8 reports of blood-clot side effect by AZD122212, 23. Megakaryocytes were also found to 9 contribute to the complication of COVID-19 patients and be correlated to the organ failure and 10 mortality24, 25, 26. We were therefore also interested in looking into the signals related to immune- 11 responsive and coagulopathy in this non-canonical immune cell type using the scRNA-seq 12 dataset. 13 In this study, we extensively investigated the PBMC samples of BBIBP-CorV vaccine 14 recipients. Our scRNA-seq data delineated a high-resolution transcriptomic landscape of 15 peripheral immune cells during the vaccination of a classical vaccine type. Meanwhile, we 16 integrated the PBMC scRNA-seq datasets of COVID-19 patients and flu vaccination in order 17 to identify the common and distinct variation trends among our data set and the two other data 18 sets. All these efforts could provide a rich data resource and demonstrate a methodology to 19 explore the human immunity in disease development and vaccination at great details. Moreover, 20 this work could facilitate the in-depth understandings of the manifestation of immune response 21 in peripheral blood under different stimuli and provide many clues to design the new assay for 22 the assessment of the immune protection and adverse effects after the vaccination or infection 23 recovery. 24 25 Results 26 Overview of immune responses and peripheral immune cells variations by scRNA-seq 27 To assess the immune cells content and the phenotypic alterations following the vaccination 28 with inactivated SARS-CoV-2 vaccine (BBIBP-CorV), six healthy young adults with 4 males 29 and 2 females from Pu Dong Cohort, China, were enrolled in this 2019-nCoV vaccine 30 observational study [NCT04871932] according to inclusion and exclusion criteria (Fig.1A). All 31 six volunteers received 2 doses of inactivated SARS-CoV-2 vaccine (BBIBP-CorV). Blood 32 samples were collected at 4 time points including the day before vaccination (BV), 7 days after 33 first dose (1V7), 7 days after second dose (2V7) and 14 days after second dose (2V14) (Fig. 34 1A). Then, serum and PBMCs were separated from all time points of samples within 2-hour of medRxiv preprint doi: https://doi.org/10.1101/2021.05.06.21256781; this version posted May 10, 2021.