Single-Cell Sequencing and Immune Function Assays of Peripheral Blood Samples Demonstrate Positive Responses of an Inactivated SARS-CoV-2 Vaccine

Background: Safe and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to halt the spread of coronavirus disease 2019 (COVID-19) pandemic. As a promising inactivated SARS-CoV-2 vaccine, CoronaVac (developed by Sinovac Life Sciences, Beijing, China) is currently undergoing its clinical trials in several countries and showing good results. We investigated the immune mechanisms underlying CoronaVac from the perspective of single-cell gene expressions and immune function features. Methods: In this controlled study, 13 healthy participants aged 21–54 years were recruited in the vaccination group and intramuscularly injected with two doses of CoronaVac (3 μg / 0·5 mL per dose, at Day00 and Day28, right after blood sampling), with peripheral blood samples collected at Day00, Day14, Day28, Day35, and Day45 to monitor dynamic changes. As positive control, 12 participants aged 28–75 years that had recovered from COVID-19 for approximately eight months were recruited in the recovery group with peripheral blood samples collected. Considering the high cost of sequencing, 11 samples from two healthy participants (H1 and H2) with four timepoints (Day00, Day14, Day28, and Day35) and three recovered participants (R1, R2, and R3) with one timepoint (Month8) were randomly chosen for single-cell RNA sequencing of peripheral blood mononuclear cells (PBMC). The transcriptomics of PBMC and its constituent cells were analyzed as gene expression responses. For samples from all 25 participants, the serum neutralizing antibody titer to live SARS-CoV-2 and the in vitro cytokine release activity of CD4+T cells were assayed as immune function responses. Findings: Single-cell RNA sequencing showed that the PBMC transcriptomics after vaccination resembled the COVID-19 recovery control more than that before vaccination, which also applied to its constituent cells such as B cells, T cells, NK cells, and myeloid cells. Gradual transitions of PBMC transcriptomics were observed from Day14 to Day28 and Day35 in vaccination groups, which finally approached the recovery control. The B cell levels in PBMC increased after each vaccination, while the T cell levels mainly increased within four weeks after the first vaccination and peaked at Day28. The serum neutralizing antibody titer to live SARS-CoV-2 was low within four weeks after the first vaccination, but the second vaccination could induce significantly higher serum neutralizing antibody titers due to the immune memory. For both vaccinated participants and recovered participants, the upregulated JUN/FOS network and tuned expressions of immunoglobulin fragments such as IGHV3-30 and IGLV2-23 were observed in B cells. By purifying CD4+T cells from PBMC and re-stimulating them with CoronaVac in vitro, five cytokines were significantly released, including Th1 cytokines (IFN-γ and IL-2) and Th2 cytokines (IL-4, IL-6, and IL-10). Th1 cytokines were mainly activated at Day14, Day28, and Day35, supporting cellular immune responses at early stages when the serum neutralizing antibody titer was low. Th2 cytokines were mainly activated at Day28, Day35, and Day45, supporting humoral immune responses at later stages, especially after the second vaccination. Interpretation: Two vaccinations of CoronaVac (3 µg per dose, with an interval of 28 days) could make positive gene expression and immune responses in healthy participants, as revealed by single-cell PBMC transcriptomics and immune function assays. Funding Statement: China’s National Science and Technology Major Projects for Major New Drugs Innovation and Development. Declaration of Interests: QG, YH, XM, and SZ are employees of Sinovac Life Sciences, Beijing, China. The other authors declare no competing interests. Ethics Approval Statement: This study was approved by the Peking University Biomedical Ethics Committee.