key: cord-0756503-0xdij9tv
authors: Che, Yanchun; Liu, Xiaoqiang; Pu, Yi; Zhou, Meijian; Zhao, Zhimei; Jiang, Ruiju; Yin, Zhifang; Xu, Mingjue; Yin, Qiongzhou; Wang, Jianfeng; Pu, Jing; Zhao, Heng; Zhang, Ying; Wang, Lichun; Jiang, Ya; Lei, Jin; Zheng, Yan; Liao, Yun; Long, Runxiang; Yu, Li; Cui, Pingfang; Yang, Huijuan; Zhang, Yuehui; Li, Jingyu; Chen, Weiwu; He, Zhanlong; Ma, Kaili; Hong, Chao; Li, Dandan; Jiang, Guorun; Liu, Donglan; Xu, Xingli; Fan, Shengtao; Cheng, Chen; Zhao, Hongling; Yang, Jianbo; Li, Yan; Zou, Yanxiang; Zhu, Youshuai; Zhou, Yaling; Guo, Yingqiu; Yang, Ting; Chen, Hongbo; Xie, Zhongping; Li, Changgui; Li, Qihan
title: Randomized, double-blinded and placebo-controlled phase II trial of an inactivated SARS-CoV-2 vaccine in healthy adults
date: 2020-11-09
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1703
sha: 81ea49826e2935011bbf0c4fa901a84d6d68cc2b
doc_id: 756503
cord_uid: 0xdij9tv

BACKGROUND: We evaluated an inactivated SARS-CoV-2 vaccine for immunogenicity and safety in adults aged 18-59 years. METHODS: In this randomized, double-blinded and controlled trial, healthy adults received a medium (MD) or a high dose (HD) of the vaccine at an interval of either 14 days or 28 days. Neutralizing antibody (NAb) and anti-S and anti-N antibodies were detected at different times, and adverse reactions were monitored for 28 days after full immunization. RESULTS: A total of 742 adults were enrolled in the immunogenicity and safety analysis. Among subjects in the 0, 14 procedure, the seroconversion rates of NAb in MD and HD groups were 89% and 96% with GMTs of 23 and 30, respectively, at day 14 and 92% and 96% with GMTs of 19 and 21, respectively at day 28 after immunization. Anti-S antibodies had GMTs of 1883 and 2370 in MD and 2295 and 2432 in HD group. Anti-N antibodies had GMTs of 387 and 434 in MD group and 342 and 380 in HD group. Among subjects in the 0, 28 procedure, seroconversion rates for NAb at both doses were both 95% with GMTs of 19 at day 28 after immunization. Anti-S antibodies had GMTs of 937 and 929 for MD and HD group, and anti-N antibodies had GMTs of 570 and 494 for MD and HD group, respectively. No serious adverse events were observed during the study period. CONCLUSION: Adults vaccinated with inactivated SARS-CoV-2 vaccine had NAb as well as anti-S/N antibody, and had a low rate of adverse reactions. CLINICAL TRIALS REGISTRATION: NCT04412538.

M a n u s c r i p t 5 The global pandemic of coronavirus disease 2019 (COVID-19) induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a new member of the coronavirus family, caused active responses from governments and organizations worldwide [1] [2] [3] [4] and led to projects for the development of approximately 300 candidate vaccines (assessed at ClinicalTrials.gov as of Sep. 29, 2020). Overall, more than 10 different types of vaccines have entered into clinical trials [5] . However, without a systematic understanding of this viral pathogen and its pathogenesis and immunologic features [6, 7] , evaluations of the immunity and safety of these vaccines, which were developed through various technologies, are more dependent upon an analysis of serologic detection and adverse reaction data collected from subjects at different stages of clinical trials. Our previous work developed an inactivated SARS-CoV-2 vaccine with S-antigen and N-antigen exposure that elicited not only neutralizing antibodies but also antibodies against N-antigen that are associated with specific protective effects in a rhesus model under viral challenge. This vaccine entered clinical testing (Number: 2020L00020 from the Chinese State Food and Drug Administration (SFDA); ClinicalTrials registration number NCT04412538), and a phase I trial was successfully completed with recognized safety of the vaccine. Low rates of mild adverse reactions and unchanged levels of 48 cytokines in the serum of immunized subjects were observed, and the antibody response was higher than 95% at medium and high doses (manuscript in submission). In the work presented here, we further evaluated the immunity and safety of this vaccine in a phase II trial in which we enrolled 750 healthy adults 18-59 years of age for immunization at an interval of either 14 days (0, 14 procedure) or 28 days (0, 28 procedure), and for each procedure, either a medium or a high dose was administered. The trial was a A c c e p t e d M a n u s c r i p t 6 randomized, double-blinded and placebo-controlled trial that showed that two doses of the vaccine elicited effective antibody responses, including neutralizing antibodies, with a low rate of mild adverse reactions.

The SARS-CoV-2 inactivated vaccine was developed by the Institute of Medical Biology 

This randomized, double-blinded and adjuvant (Al(OH) 3 days between two inoculations, respectively. Furthermore, the subjects in each procedure were randomly assigned at a 2:2:1 ratio to receive a medium dose (containing 100 EU viral antigen) or a high dose (containing 150 EU viral antigen) of the SARS-CoV-2 vaccine or the placebo as a control. Blood samples were collected from the enrolled participants at days 0 (baseline), 14 and 28 (0, 14 procedure), and day 28 (0, 28 procedure) following the boost immunization to evaluate the potential immunogenicity of the vaccine at different time A c c e p t e d M a n u s c r i p t 8 points. The solicited and unsolicited adverse events (AEs), if any, were recorded within a period of 0-7 days following the 1st inoculation and 0-28 days after the second inoculation.

Study staff visited participants on site to monitor their health status and determine whether the participants needed to seek medical care when they returned to submit their safety records or provide blood samples.

The primary endpoints were the seroconversion rates of the anti-SARS-CoV-2 neutralizing antibody and ELISA IgG antibody at days 14 (0, 14 procedure) and 28 (0, 28 procedure), respectively, after the boost immunization. There were 2 secondary endpoints: (1) the total AE rate from 0 to 7 days following each inoculation and from 0 to 28 days after the boost immunization, and (2) the geometric mean titer (GMT) profiles of the anti-SARS-CoV-2 neutralizing antibody and ELISA IgG antibody at days 14 and 28 (0, 14 schedule) and day 28 (0, 28 schedule) following the boost immunization.

AEs included local and systemic reactogenicity signs and symptoms from 0 to 7 days following each inoculation and from 0 to 28 days after the boost immunization. Serious 

The neutralizing antibody assay was performed via microtitration in 96-well plates. Briefly, heat-inactivated serum was diluted and coincubated with live virus (100 lgCCID 50 /well) for 2 h at 37°C, followed by the addition of Vero cells (10 5 /mL), and the mixture was incubated at 37°C in 5% CO 2 for 7 days. The cytopathic effects (CPEs) were observed and assessed to determine the neutralizing antibody titer. The GMTs of neutralizing antibodies were measured. Antibody titers of ≥4 were considered positive. 

A total of 1130 adults were recruited for this phase II trial in two counties, Gejiu and Mile, and 750 were eligible for participation ( Fig. 1) , with 375 in Gejiu for the 0, 14 procedure and 375 in Mile for the 0, 28 procedure. Eligible participants from each county were randomly assigned to the medium-and high-dose groups and the placebo control group at a ratio of 2:2:1 (Fig. 1) . In June, immunization with the vaccine was performed at two sites, followed by clinical observation for 28 days and the collection of blood samples at days 14 and 28 for the 0, 14 procedure or at day 28 only for the 0, 28 procedure. A total of 374 subjects who underwent the 0, 14 procedure were entered into the full analysis set (FAS), and 372 were entered into the per protocol set (PPS), with 148 (98.7%) in the medium-dose group and 149 in the high-dose group. A total of 371 and 370 subjects who underwent the 0, 28 procedure were entered into the FAS and PPS, respectively (Fig. 1) . The demographic characteristics of the PPS are described in Table 1 .

The primary immunologic outcome of this phase II trial of an inactivated SARS-CoV-2 vaccine was the neutralizing antibody response against SARS-CoV-2 in the two dose groups with different immunization procedures. The results suggested that in the immunization procedure with an interval of 14 days, the compared to those in the placebo control group A c c e p t e d M a n u s c r i p t 11 seroconversion rates in the medium-and high-dose groups were 89% and 96%, respectively, with GMTs of 23 and 30, respectively, at day 14 after immunization (Fig. 2a) , and 92% and 96% with GMTs of 19 and 21, respectively, at day 28 after immunization (Fig. 2a) . In the immunization procedure with an interval of 28 days, the seroconversion rates in the medium-and high-dose groups were both 95%, with GMTs of 19, at day 28 after immunization (Fig. 2b) . The secondary immunologic outcome of this study was the ELISA antibody response elicited by the inactivated vaccine designed by our specific technical strategy for presenting viral S-and N-antigens. The results of detection using the plate coated with S protein suggested that anti-S antibody showed seroconversion rates that were similar to those of neutralizing antibody, with GMTs of 1883 and 2370 in the mediumand high-dose groups, respectively, for the 0, 14 procedure at day 14 after immunization and higher seroconversion rates with GMTs of 2295 and 2432 at day 28 after immunization ( Fig. 2a) . The detection of anti-N antibody suggested approximately 60% seroconversion with GMTs of 387 and 434 at day 14 for the 0, 14 procedure and approximately 50% seroconversion with GMTs of 342 and 380 at day 28 (Fig. 2a) . Further anti-S antibody detection for the medium and high doses in the 0, 28 procedure showed seroconversion rates of 89% and 93%, respectively, with GMTs of 937 and 929, respectively, at day 28 after immunization (Fig. 2b) ; anti-N antibody detection showed seroconversion rates of 68% and 78% with GMTs of 570 and 494, respectively (Fig. 2b) . These data suggested that the neutralizing antibody was induced by the vaccine in more than 90% of individuals in this adult population and that the elicited antibody response included anti-S and anti-N antibodies.

A c c e p t e d M a n u s c r i p t 12

As one of the indicators in the phase II trial, safety monitoring in this study focused on the clinical adverse reactions that occurred 7 days after each inoculation and 28 days after two immunizations. Observation suggested that with the 0, 14 procedure, adverse reactions occurred in 24%, 27.3% and 17.3% of all individuals in the medium-dose, high-dose and placebo groups, respectively, within 7 days after the first and second injections ( Table 2) ; with the 0, 28 procedure, these values were 26.7%, 19.3% and 12%, respectively ( Table 2 ).

Most of these reactions were slight pain, itching and redness at the injection site (Table 2) .

Induced systemic adverse reactions 7 days after the first and second immunizations, mainly including slight fatigue and fever, were reported in 10%, 13% and 14.7% of individuals in the medium-dose, high-dose and placebo groups, respectively, who received the 0, 14 procedure ( Table 2 ) and in 13.3%, 8% and 9.3% of individuals who received the 0, 28 procedure ( Table 2) . Overall adverse reaction rates during the 28 days after immunization were 24%, 27.3%, and 17.3% and 27.3%, 19.3%, and 12% in the medium-dose, high-dose and placebo groups, respectively, in the 0, 14 procedure and the 0, 28 procedure, respectively ( 

After concerns regarding the safety of our inactivated SARS-CoV-2 vaccine in a phase I trial, this study targeted the immunity elicited by the vaccine through the detection of neutralizing antibodies and ELISA antibodies against not only the S-antigen but also the Nantigen. The use of medium and high doses was based upon the detection of neutralizing antibodies in a phase I trial, in which the GMTs in subjects immunized with low (50 EU), medium (100 EU) and high (150 EU) doses reached 18, 54 and 37, respectively (manuscript in submission). Based on these data, medium and high doses were evaluated in this study in two immunization procedures with intervals of 14 and 28 days. The results indicated that vaccine immunization in this population showed a tendency toward a dose-effect relationship for either seroconversion or GMTs of neutralizing antibodies in the two procedures. However, the fact that the 0, 14 procedure presented a seroconversion rate of 96% with GMTs of 30 and 21 in the high-dose group at days 14 and 28 after immunization, respectively, which were higher than the values of the 0, 28 procedure, suggested that a high dose with the 0, 14 procedure could be a better choice for further phase III trials of this vaccine. This result is also supported by the detection of ELISA antibodies against the Sprotein and N protein, in which the high-dose group subjected to the 0, 14 procedure showed higher seroconversion rates and GMTs of anti-S and anti-N antibodies than the medium-dose and placebo groups. These data suggested not only that the immunogenicity of this vaccine induced a neutralizing antibody response in 95% of the adult population aged 18-59 years but also that the vaccine had the capacity to elicit anti-N and anti-S antibodies in the ELISA. Although recent reports of SARS-CoV-2 vaccines have provided much data on the neutralizing antibody response, obtaining an understanding of an integrated immune A c c e p t e d M a n u s c r i p t 14 response that shows protective efficacy still requires more data on the roles played by antibodies against various viral antigens in antiviral immunity. We still wanted to know whether the roles of cytotoxic T lymphocytes (CTLs) and antibody-dependent cellular cytotoxicity (ADCC), related to the antibodies to S-antigen, N-antigen and other viral antigens, could be involved in controlling and eliminating the virus during infection.

Certainly, safety was still a concern in this study of 750 subjects, and our observations M a n u s c r i p t A c c e p t e d M a n u s c r i p t 

Novel 2019-coronavirus on new year's Eve

WHO International Health Regulations Emergency Committee for the COVID-19 outbreak

Epidemiology of COVID-19

Central nervous system manifestations of COVID-19: A systematic review

Current Clinical Trials Protocols and the Global Effort for Immunization against SARS-CoV-2. Vaccines (Basel) 2020

Immune response to SARS-CoV-2 and mechanisms of immunopathological changes in COVID-19

A perspective on potential antibody-dependent enhancement of SARS-CoV-2

We appreciate the contributions of all investigators at Yunnan Center for Disease Control 

A c c e p t e d M a n u s c r i p t 16