key: cord-1049370-w6bbdu9f
authors: Guo, Wanshen; Duan, Kai; Zhang, Yuntao; Yuan, Zhiming; Zhang, Yan-Bo; Wang, Zejun; Zhao, Dongyang; Zhang, Huajun; Xie, Zhiqiang; Li, Xinguo; Peng, Cheng; Zhang, Wei; Yang, Yunkai; Chen, Wei; Gao, Xiaoxiao; You, Wangyang; Wang, Xue-Wei; Shi, Zhengli; Wang, Yanxia; Yang, Xu-Qin; Zhang, Lianghao; Huang, Lili; Wang, Qian; Lu, Jia; Yang, Yong-Li; Guo, Jing; Zhou, Wei; Wan, Xin; Wu, Cong; Wang, Wenhui; Du, Jianhui; Nian, Xuanxuan; Li, Xing-Hang; Huang, Shihe; Shen, Shuo; Xia, Shengli; Pan, An; Yang, Xiaoming
title: Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18 years or older: A randomized, double-blind, placebo-controlled, phase 1/2 trial
date: 2021-07-07
journal: EClinicalMedicine
DOI: 10.1016/j.eclinm.2021.101010
sha: 3edf4f65b08f3d7c70c7262d2979a955362f74d4
doc_id: 1049370
cord_uid: w6bbdu9f

BACKGROUND: We aimed to assess the safety and immunogenicity of an inactivated vaccine against COVID-19 in Chinese adults aged ≥18 years. METHODS: This is an ongoing randomized, double-blind, placebo-controlled, phase 1/2 clinical trial among healthy adults aged ≥18 years in Henan Province, China. Participants (n = 336 in 18–59 age group and n = 336 in ≥60 age group) were enrolled between April 12 and May 17 2020, and were equally randomized to receive vaccine or placebo (aluminum hydroxide adjuvant) in a three-dose schedule of 2·5, 5, or 10 µg on days 0, 28, and 56. Another 448 adults aged 18–59 years were equally allocated to four groups (a one-dose schedule of 10 µg, and two-dose schedules of 5 µg on days 0 and 14/21/28) and received vaccine or placebo (ratio 3:1 within each group). The primary outcomes were 7-day post-injection adverse reactions and neutralizing antibody titres on days 28 and 90 after the whole-course vaccination. Trial registration: www.chictr.org.cn #ChiCTR2000031809. FINDINGS: The 7-day adverse reactions occurred in 4·8% to 32·1% of the participants in various groups, and most adverse reactions were mild, transient, and self-limiting. Twenty participants reported 68 serious adverse events which were judged to be unrelated to the vaccine. The 90-day post-injection geometric mean titres of neutralizing antibody ranged between 87 (95% CI: 61–125) and 129 (99–169) for three-dose schedule among younger and older adults; 20 (14–27), 53 (38–75), and 44 (32–61) in 5 µg days 0 and 14/21/28 groups, respectively, and 7 (6–9) in one-dose 10 µg group. There were no detectable antibody responses in all placebo groups. INTERPRETATION: The inactivated vaccine against COVID-19 was well tolerated and immunogenic in both younger and older adults. The two-dose schedule of 5 µg on days 0 and 21/28 and three-dose schedules on days 0, 28, and 56 could be further evaluated for long-term safety and efficacy in the phase 3 trials.

The Coronavirus Disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to affect everyone's life and is responsible for more than 3¢7 million deaths worldwide as of June 9, 2021 [1] . Unprecedented efforts have been made to rapidly develop vaccines against the COVID-19 by pharmaceutical companies, academic researchers, and government agencies [2] . According to the draft landscape of COVID-19 candidate vaccines by the World Health Organizations (WHO), there were over 185 candidate vaccines in preclinical evaluations and 102 undergoing clinical evaluations in different phases till June 8 [2] . Among them, the results of phase 1/2 trials of 24 candidate vaccines have been published or made available on preprint servers, including seven protein subunit [3À9], six inactivated [10À15], six recombinant, [16À21] three mRNA, [22À24] one DNA vaccine, [25] and one virus-like particle vaccine [26] . All 24 vaccines demonstrated acceptable safety profiles and immunogenicity. Among them, seven also published the interim results of the phase 3 trial [27À33].

Since age has been identified as an independent risk factor for critical illness or death of COVID-19 and there is a possibility of lower immune response [34, 35] , the safety and immunogenicity of the vaccines should be particularly assessed in older people. Previous studies indicated that the safety profile of vaccines against the COVID-19 was similar or even better among older adults compared with younger adults [4,12,14,18À20,24,29,36À38] . As for antibody responses, several studies reported lower antibody responses among older adults [4, 19, 24, 38] , while others reported similar antibody responses between older and younger adults [12, 14, 18, 36, 37] . Therefore, it is necessary to evaluate the safety and immunogenicity of each vaccine against COVID-19 in both young and old adults.

We have previously reported interim results of an ongoing phase 1/2 trial of an inactivated vaccine against COVID-19 among healthy adults aged 18À59 years old for the 28-day post-vaccination adverse events and 14-day post-vaccination humoral immunogenicity [13] .

The report included participants who received three doses of 2¢5 mg, 5 mg, or 10 mg in the phase 1 trial and two doses of 5 mg on days 0 and 14 or days 0 and 21 in the phase 2 trial [13] . In the current analysis, we included all adult groups that were designed in the original protocol (Supplementary Appendix Section 1) of the phase 1/2 trial including studies among those aged 60 years or older, and updated the antibody response till 90 days after the whole-course vaccination as well as serious adverse events till March 5, 2021 with a maximum follow-up of 327 days since the first injection.

The phase 1/2, randomized, double-blind, placebo-controlled trial was designed by the Wuhan Institute of Biological Products Co. Ltd. and Henan Province center for Disease Control and Prevention (CDC), and the study protocol was approved by the institutional review board of Henan CDC. All participants provided written informed consent before enrollment. Investigators at the Wuzhi county CDC, Henan Province, performed the ongoing trial and collected data since April 12, 2020 . An independent data and safety monitoring board (DSMB) monitored the safety data and evaluated risks among the participants during the trial. The trial was registered with www. chictr.org.cn (ChiCTR2000031809) and included participants aged 6 years; however, interventions among children and adolescents were only initiated after evaluating the 28-day post-vaccination safety and immunogenicity results among adults. Therefore, the trial among children and adolescents is still ongoing and data are not yet available. The current analysis included only adults aged 18 years and above.

Healthy, non-pregnant adults aged 18 years or older who had no known history of SARS-CoV-1 (via on-site inquiry) or SARS-CoV-2 infection (via serological and nucleic acid test); had not been to Hubei province, regions outside China, or other regions with confirmed COVID-19 cases; and had not contacted with confirmed or suspected cases were assessed for eligibility. Key exclusion criteria included respiratory diseases or symptoms within 14 days before vaccination; abnormalities in laboratory examinations; history of severe allergic reaction or allergy to ingredients in the vaccine; taking certain treatments or medications or other vaccines before the study as specified in Supplementary Appendix Section 1; and history of severe comorbidities that may affect the adherence. Details of the inclusion and exclusion criteria are described in the previous publication [13] and Supplementary Appendix Section 1. All the authors had full access to all the data in the study.

Sequential computer-generated randomization numbers were assigned to participants, and stratified block randomization by age and

Evidence before this study By May 10, 2021, results of the clinical trials in different phases of 24 SARS-CoV-2 vaccines (seven protein subunit, six inactivated, six recombinant, three mRNA, one DNA, and one viruslike particle vaccine) have been reported. For the inactivated vaccine developed by the Sinopharm (Wuhan, China), we have previously reported the interim results of the phase 1 trial of three-dose groups and phase 2 trial of days 0 and 14/21 groups among adults aged 18À59 years. It was reported that 6¢0% to 25¢0% of the participants had adverse reactions, and neutralizing antibody titres ranged between 121 and 316 on day 14 after the whole-course vaccination.

This study reported safety profile at 292À327 days after the first injection of an inactivated SARS-CoV-2 vaccine and antibody responses at 90 days after the whole-course vaccination, which showed that the vaccine was safe, tolerable, and immunogenic in healthy adults. Only 15% of participants experienced adverse reactions, which were mostly mild-to-moderate and self-limiting, and no severe adverse reaction was reported. Two-dose schedules of 5 mg on days 0 and 21/28 and three-dose schedules of 2¢5 mg, 5 mg, and 10 mg on days 0, 28, and 56 induced neutralizing antibodies in over 87% and 71% of recipients on days 28 and 90 after the whole-course vaccination, respectively.

The inactivated SARS-CoV-2 vaccine had a good safety profile and demonstrated immunogenicity among adults. Two-dose schedules of 5 mg on days 0 and 21/28 and three-dose schedules of 2¢5 mg, 5 mg, and 10 mg on days 0, 28, and 56 are potential options for phase 3 clinical trials and future applications in the real world. Extended follow-ups and phase 3 clinical trials are still warranted to evaluate the long-term safety, immunogenicity, and efficacy of the inactivated vaccine.. doses was adopted (block size 8). Within each randomization block, the ratio of vaccine to placebo (aluminum hydroxide adjuvant) was 3:1.

In the phase 1 trial, participants were stratified by 18À59 (younger adults) and 60 years old (older adults) and randomly allocated to receive the inactivated SARS-CoV-2 vaccine at 2¢5 mg, 5 mg, or 10 mg, or placebo on days 0, 28, and 56 via intramuscular injections (24 participants in each group). To accelerate the development of vaccine and provide relevant data for the phase 3 trial, the phase 2 trial was designed at the same time with the phase 1 trial, and same group assignment approach (i.e., vaccine at 2¢5 mg, 5 mg, and 10 mg, and placebo on days 0, 28, and 56 among younger and older adults, respectively) was used with a larger sample size for each group (60 participants in each group). With the purpose of quickly increasing vaccine coverage in the population, four more groups in those aged 18À59 years old were added in the phase 2 trial to explore whether less doses or shorter between-dose intervals could also be immunogenic, i.e., two-dose schedule of 5 mg on days 0 and 14, on days 0 and 21, or on days 0 and 28, or a one-dose schedule of 10 mg on day 0 (vaccine:placebo = 3:1 in each group).

Participants were sequentially enrolled and received injections in an age-and dose-escalating manner. The first group of participants aged 18À59 years in the phase 1 trial received the inactivated vaccine at 2¢5 mg first, and adverse events were monitored for 7 days after the first injection. If no severe adverse events were observed, the second group of participants aged 18À59 years in the phase 1 trial received the inactivated vaccine at 5 mg; meanwhile, a group of participants aged 60 years in the phase 1 trial and participants aged 18À59 years in the phase 2 trial received the inactivated vaccine at 2¢5 mg. Similar sequences were used for 5 mg and 10 mg groups (Supplementary Appendix, Fig. 1 ). Participants, investigators, and laboratory personnel were blinded to the intervention allocation.

Details of the vaccine development and production were reported previously [13, 39] . Briefly, a SARS-CoV-2 strain (WIV04 strain, GenBank accession number MN996528) was isolated from a patient in the Jinyintan Hospital, Wuhan. The virus strains were cultivated in qualified Vero cell lines for proliferation, followed by purification and b-propanolide inactivation (1:4000 vol/vol) at 2À8°C for 48 h. A second inactivation was subsequently performed after clarification of cell debris and ultrafiltration, then the vaccine was adsorbed to 0¢5 mg aluminum hydroxide and packed into pre-filled syringes in 0¢5 mL of sterile phosphate-buffered saline without preservative. The placebo only contained 0¢5 mg aluminum hydroxide in 0¢5 mL sterile phosphate-buffered saline. All the vaccines and placebos were approved by the National Institutes for Food and Drug Control of China, and provided in coded, identical-appearing, single-dose vials.

Participants were requested to record any injection-site-specific adverse events (e.g., pain, redness, and swelling), systemic adverse events (e.g., fever, headache, and fatigue), and unsolicited adverse events on diary cards (within 7 days) or contact cards (8 days and after) of each injection. After each dose, participants were observed for 30 min to record any adverse events. Within 7 days after the first dose, researchers visited the participants on a daily basis and guided the participants to correctly report the adverse events on the diary card. After the second and third dose, researchers made a phone call within 24 h, a face-to-face visit within 1À3 days, a phone call within 4À7 days, a weekly phone call between days 8 and 28, and a monthly phone call after 28 days following the whole-course vaccination to collect information on the adverse events. The participants could also actively report adverse events to the researchers at any time. The grading of adverse events and relationships between vaccinations and adverse events were decided by investigators according to the standard guidelines issued by the National Medical Products Administration of China, and details are shown in Supplementary Appendix Section 1. The 7-day adverse reactions were the primary safety outcome, while all adverse events within 28 days and serious adverse events after 28 days, regardless of relations with the vaccine, were considered as the secondary safety outcome. A total of 13 trained investigators were involved in adjudications of the relations of the serious adverse events with vaccine and determined the severity according to medical records. To assess any acute toxic effects after vaccination, laboratory safety tests (including blood routine tests, total bilirubin, liver enzymes, urea nitrogen, creatinine, and urine sugar, protein, and occult blood) were also performed before and on day 4 after each injection in the phase 1 trial and were considered as secondary safety outcomes.

The primary humoral immunogenicity outcomes were neutralizing antibody titres and specific IgG binding antibody titres measured on days 28 and 90 after the whole-course vaccination. Antibody titres at the same time point in the phase 1 and 2 trials were combined and analysed together. In the phase 1 trial, blood samples were also collected before each dose, on day 4 after each injection, and on day 14 after the first and second injections, and neutralizing antibody titres and specific IgG binding antibody titres measured at these time points were secondary immunogenicity outcomes. The schedule of blood collections is described in Supplementary Appendix Fig. 2 . Plaque reduction neutralization test (PRNT) was used to measure the neutralization capacity induced by the vaccine against live SARS-CoV-2 (BetaCoV/Wuhan/AMMS01/2020 activated), and the PRNT 50 values were used to determine the maximal dilution factors when SARS-CoV-2 plaque formation could be reduced by 50%. An in-house developed ELISA kit was used to measure specific IgG-binding antibody titres with the inactivated whole SARS-CoV-2 as coating antigen. The specificity, recovery, and inter-assay and intra-assay coefficients of variation of the ELISA assay were 100%, 96¢0À105¢3%, and 6¢4À10¢2%, respectively. Detailed immunogenicity assays have been reported previously [13] . The low detection limit was 5 for the neutralizing antibody titre and 10 for the specific IgG-binding antibody titre, and those below the detection limit were respectively assigned to 5 or 10 for further analysis. Seroconversion rate on days 28 and 90 after the whole-course vaccination was defined as at least a four-fold increase of antibody titres over baseline (i.e., 20 for neutralizing and 40 for IgG-binding antibody titres) and was considered as a secondary immunogenicity outcome.

The phase 1 and 2 trials were designed concurrently, thus the sample size was not calculated according to the statistical power calculation. Instead, the sample size was determined according to recommendations from the National Medical Products Administration of China, i.e., a minimum of 20À30 participants in a phase 1 vaccine trial and a roughly 3-fold sample size in a phase 2 vaccine trial. The sample size in the current study was in the similar range compared to other clinical trials of COVID-19 vaccines [12À14, 19, 40, 41] .

The safety analysis was conducted in all participants receiving at least one injection. The immunogenicity analysis was conducted in participants receiving at least one injection and having results of antibody titre measurements at the designated time points. Rates of adverse reactions, unsolicited adverse events, abnormal laboratory indicators, and seroconversion between vaccine and placebo groups and among different dose groups were compared using x 2 test or Fisher's exact test (for sparse outcomes). Log 10 -transformed antibody titres between vaccine and placebo groups were compared using Student's t-test or Mann-Whitney U test (for non-normally distributed data). We also made post-hoc comparisons of antibody titres across 2¢5 mg, 5 mg, and 10 mg days 0, 28, and 56 groups as well as across two-dose and one-dose group through ANOVA and Student-Newman-Keuls Tests. Results were presented by different age groups (18À59 and 60 years old) as specified as in Supplementary Appendix Section 1, and post-hoc stratified analyses of the immune responses were conducted by sex and body mass index (BMI; <24 kg/m 2 versus 24 kg/m 2 according to the Chinese criterion for determining overweight/obesity) [42] . Analyses were performed using SPSS software, version 25¢0 (IBM SPSS Inc., Chicago, IL), and hypothesis testing was two-sided with an alpha value of 0¢05. The results of secondary endpoints and post-hoc analyses should be interpreted cautiously given the potential type 1 error due to multiple comparisons.

The China National Biotec Group Company Limited and the Wuhan Institute of Biological Products Co. Ltd. were the study sponsors and designed the trial, provided the study product, and oversaw all trial operations. The sponsors used contract clinical research organizations to coordinate interactions with regulatory authorities and oversee clinical site operations. Data were collected by the clinical site research staff, managed by a blinded contract research organization data management team, monitored by a contract research organization, and overseen by the sponsor and an independent data and safety monitoring board. The analyses were performed after the data were checked and locked before unblinding by an independent statistician who was not involved in the trial. Manuscript preparation was performed by the study authors and the decision to submit the manuscript for publication was made by the study authors. All the authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Between April 12 and May 17, 2020, a total of 1120 volunteers aged 18À59 years old (younger adults) and 608 volunteers aged 60 years old (older adults) were screened, and 784 and 336, respectively, were finally enrolled in the phase 1 and 2 trials (Fig. 1) . All participants completed the first injection while five did not complete the second or third injection. All participants provided blood samples before the first injection, while a total of 8 and 9 participants did not provide blood samples on days 28 and 90, respectively, after the whole-course vaccination. Data in the three-dose schedule groups in both phase 1 and 2 trials were combined, and the baseline characteristics of the participants stratified by age groups (18À59 and 60 years old), dose groups (2¢5 mg, 5 mg, 10 mg, and placebo) and schedule groups (three doses on days 0, 28, and 56, two doses on days 0 and 14/21/28, and one dose on day 0) are shown in Table 1 . The mean (standard deviation) age was 43¢1 (9¢6) years in participants aged 18À59 years and 66¢7 (4¢3) in those aged 60 years (79¢2% aged 60À69 years); and there were 462 (58¢9%) and 136 (40¢5%) women in the two age groups, respectively.

Injection-site and systemic reaction symptoms (according to solicited reports) are shown in Table 2 . Within 7 days after each injection, the number of participants reporting adverse reactions was 12 (14¢3%), 18 (21¢4%), 21 (25¢0%), and 14 (16¢7%) in the 2¢5 mg, 5 mg, 10 mg, and placebo groups among younger adults with three doses, respectively; and the corresponding number was 4 (4¢8%), 13 (15¢5%), 6 (7¢1%), and 14 (16¢7%) in the age group of 60 years. The proportion of adverse reactions was similar between vaccine and Fig. 1 . Screening, randomization, and inclusion in safety and immunogenicity analyses stratified by age groups *The comorbidities in the exclusion criteria included cardiovascular disease, cancer, respiratory disease, autoimmune disease, tuberculosis, severe liver disease, congenital malformation, mental illness, nervous system diseases, uncontrolled hypertension and diabetes, severe malnutrition, fever within 14 days, and other diseases that could affect participation and compliance in the trial as judged by the investigators. y Laboratory tests included routine blood tests, liver enzymes, total bilirubin, creatinine, urea nitrogen, urine protein, urine sugar, and urinary occult blood. Details of the list and definition of abnormal values are provided in Supplementary Appendix Section 1. z One participant aged 18À59 years who received placebo on days 0, 28, and 56 in the phase 2 trial did not provide blood sample on days 28/90 after the third dose for antibody measures. One participant aged 18À59 years who received vaccines at 5 mg on days 0 and 21 in the phase 2 trial received two injections but did not provide blood sample on day 90 after the second dose. One participant aged 18À59 years who received vaccines at 5 mg on days 0 and 28 in the phase 2 trial did not provide blood sample on days 28/90 after the second dose. One participant aged 18À59 years who was allocated to receive placebo on days 0 and 28 in the phase 2 trial did not receive the second dose because of positive pregnancy test and thus did not provide blood sample on days 28/90 after the second dose. One participant aged 60 years who was allocated to receive vaccines at 5 mg on days 0, 28, and 56 in the phase 1 trial withdrew from the study before the second dose. One participant aged 60 years who was allocated to receive vaccines at 10 mg on days 0, 28, and 56 in the phase 1 trial withdrew from the study before the second dose. One participant aged 60 years who received vaccines at 5 mg on days 0, 28, and 56 in the phase 1 trial did not provide blood sample on days 28/90 after the third dose for antibody measures. Two participants aged 60 years who were allocated to receive vaccines at 10 mg on days 0, 28, and 56 in phase 2 trial withdrew from the study before the third dose. placebo groups (P >0¢099), except that the proportion of total adverse reactions was larger among those receiving placebo compared with 2¢5 mg on days 0, 28, and 56 among older adults (P = 0¢042). Besides, the proportion of adverse reactions was higher in 5 mg group compared with 2¢5 mg or 10 mg group among older adults receiving three doses (P = 0¢012), and no statistically significant differences were observed for other comparisons among different dose groups (P >0¢056). In the phase 2 trial among younger adults, the number of participants reporting adverse reactions was 5 (6¢0%), 4 (14¢3%), 16 (19¢1%), 5 (17¢9%), 17 (20¢2%), 9 (32¢1%), 9

(10¢7%), and 3 (10¢7%) in 5 mg days 0 and 14, placebo days 0 and 14, 5 mg days 0 and 21, placebo days 0 and 21, 5 mg days 0 and 28, placebo days 0 and 28, 10 mg day 0, and placebo day 0 groups, respectively. No statistically significant differences of incident adverse reactions were observed between vaccine and placebo groups (P > 0¢099); however, incidence rates of total and injection-site adverse reactions were lower among 5 mg days 0 and 14 group compared with 5 mg days 0 and 21/28 groups (P < 0¢013).

The most common adverse reaction was injection-site pain (126 out of 1120 participants), followed by fever (26 out of 1120 participants). Most adverse reactions were mild (grade 1/2), transient and self-limiting. Unsolicited adverse events with 28 days (regardless of relations with the vaccination) are shown in the Supplementary Appendix Tables 1 and 2. Up to March 5, 2021, a total of 68 severe adverse events from 20 participants occurred during the follow-up (median 306 days after the first injection, range 292À327 days; Supplementary Appendix, Table 3 ), but none were judged to be related to the vaccination.

In the phase 1 trial, there were 4 (16¢7%; 4¢7%À37¢4%; among older adults receiving vaccines at 2¢5 mg) to 10 (41¢7%; 22¢1%À63¢4%; among older adults receiving vaccines at 10 mg) participants in each group having abnormalities in blood biochemical tests or urinalysis tests before and on day 4 after each injection (Supplementary Appendix, Table 4 ), and all abnormalities were transient which needed no special treatment. A similar pattern was also found in younger adults and was reported in our previous publication [13] .

None of the participants had any measurable neutralizing antibody response at baseline, and none in the placebo group had any measurable neutralizing antibody response during the follow-up. Differences of neutralizing antibody tires and specific binding antibody titres on days 28 and 90 after the whole-course vaccination between vaccine and placebo groups were statistically significant (P 0¢0004). Among younger adults with three injections, the Age, years * Data are mean (SD) or n (%). y Differences between vaccine and placebo group were statistically insignificant (p>0¢05), which were tested using t-test for continuous variables and x 2 test for categorical variables. Table 2 7-day adverse reactions among study participants who received at least one dose in the phase 1 and 2 trials*. * Data are n (%; 95% confidence interval), and 95% confidence interval was calculated by the Clopper-Pearson (exact) method. Data from the phase 1 and 2 trials were pooled. y Difference between vaccine and placebo groups was statistically significant (P = 0¢042), all other P values>0¢085. z Difference among different dose groups was statistically significant (P = 0¢012), and all other P values>0¢056. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2. * Geometric mean titres are shown with 95% confidence intervals. Seroconversion rates are n (%; 95% confidence interval), and 95% confidence interval was calculated by the Clopper-Pearson (exact) method. Data from the same immunization procedure groups in the phase 1 and 2 trials were pooled. The antibodies were not measurable at baseline for all groups and days 28 and 90 after the whole-course vaccination for the alum-only group, and thus data were not shown. All T tests showed that the differences of neutralizing antibody tires and specific binding antibody titres between vaccine and placebo groups were statistically significant (P 0¢0004). Seroconversion rate was defined as at least a four-fold increase of antibody titres over baseline (i.e., 20 for neutralizing and 40 for IgG-binding antibody titres). Results of ANOVA, Student-Newman-Keuls Test, and Fisher's exact test are reported in the apendix (p 8).

geometric mean titre (GMT) of neutralizing antibody at day 28 after the third injection was 149 (95% CI, 110À202), 238 (188À302), and 216 (181À257) in the 2¢5 mg, 5 mg, and 10 mg group, respectively ( Table 3 ). The GMT for older adults was 161 (118À219), 161 (118À221), and 187 (138À253) in the 2¢5 mg, 5 mg, and 10 mg group, respectively. In the phase 2 trial among younger adults, the GMT was 39 (29À53) in the 5 mg days 0 and 14 group, 134 (104À174) in the 5 mg days 0 and 21 group, 91 (71À115) in the 5 mg days 0 and 28 group, and 24 (17À34) in 10 mg day 0 group. Among participants receiving three doses, seroconversion rates of neutralizing antibody on day 28 after the whole-course vaccination ranged between 90¢5% (82¢1%À95¢8%) and 100% (95¢7%À100¢0%) among younger adults and ranged between 87¢8% (78¢7%À94¢0%) and 90¢1% (83¢6%À96¢6%) among older adults. In the phase 2 trial among younger adults, the seroconversion rate was 72¢6% (61¢8%À81¢8%) in 5 mg days 0 and 14 group, 90¢5% (82¢1%À95¢8%) in 5 mg days 0 and 21 group, 91¢6% (83¢4%À96¢5%) in 5 mg days 0 and 28 group, and 51¢2% (40¢0%À62¢3%) in 10 mg day 0 group.

The GMT of neutralizing antibody titres on day 90 after the wholecourse vaccination were 87 (95% CI, 61À125). 128 (95À173), and 129 (99À169) in the 2¢5 mg, 5 mg, and 10 mg groups among younger adults, respectively, and the corresponding figures were 99 (70À139), 99 (69À144), and 120 (84À172) among older adults; in the phase 2 trial among younger adults, the GMT was 20 (14À27) in the 5 mg days 0 and 14 group, 53 (38À75) in the 5 mg days 0 and 21 group, 44 (32À61) in the 5 mg days 0 and 28 group, and 7 (6À9) in the 10 mg day 0 group. The GMT of neutralizing antibody titres declined by 35¢8% (10 mg days 0, 28, and 56 group among older adults) to 70¢8% (10 mg day 0 group) from day 28 to day 90 after the whole-course vaccination; however, we did not observe statistically different seroconversion rate of neutralizing antibody on day 90 after the whole-course vaccination among those receiving three doses at different dosages, and the seroconversion rates were 79¢8% (69¢6%À87¢8%) to 91¢7% (83¢6%À96¢6%) among younger adults and 78¢1% (67¢5%À86¢4%) to 82¢7% (72¢7%À90¢2%) among older adults. Among four exploratory 2-dose or 1-dose groups, seroconversion rates were 71¢1% (60¢1%À80¢5%) in 5 mg days 0 and 21/28 groups, which were higher than 50¢0% (38¢9%À61¢1%) in 5 mg days 0 and 14 group and 14¢3% (7¢6%À23¢6%) in 10 mg day 0 group (Supplementary Appendix, Table 5 ). A similar pattern was found for the specific IgGbinding antibody levels.

The dynamic changes of the antibody responses in the 0/28/56 groups in the phase 1 and 2 trials were further examined (Fig. 2) . The antibody responses with more time points in the phase 1 trial among younger adults were shown before [13] , and Supplementary Appendix Table 6 shows the results for older adults. Most participants Data from the phase 1 and 2 trials were combined. Placebo groups are not shown since no measurable antibody responses could be detected in these groups at any time. There were 84 participants in each group. Among participants aged 60 years in the medium-dose group, 1, 1, and 2 participants did not provide blood samples before the second dose, before the third dose, and on days 28 and 90 after the whole-course vaccination. Among participants aged 60 years in the high-dose group, 1, 1, and 3 participants did not provide blood samples before the second dose, before the third dose, and on days 28 and 90 after the whole-course vaccination. The dots represent individual participant values. The boxes and horizontal bars denote interquartile range (IQR) and median value. The whisker's upper endpoint equaled to 75th percentile + 15 £ IQR, and the whisker's lower endpoint equaled to 25th percentile -15 £ IQR. The numbers below the boxes indicate the number of participants with values below the detection limit. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2. started to generate antibody responses after the second injection, which remained at high levels 28 days after the third injection, but antibody titres declined 90 days after the whole-course vaccination. Results of subgroup analyses by sex and BMI groups are reported in Supplementary Appendix Figs. 3 and 4 , and all subgroups could generate neutralizing antibody and specific IgG-binding antibody on days 28 and 90 after the whole-course vaccination.

In this phase 1/2 clinical trial among healthy adults, the SARS-CoV-2 inactivated vaccine was safe and well-tolerated in all age and dose groups, and 15¢1% of participants experienced adverse reactions. The most common injection-site adverse reaction was injection-site pain, and the most common systemic adverse reaction was fever, which were mostly mild-to-moderate and self-limiting. No serious adverse reactions related to the vaccine occurred. The inactivated vaccine could effectively elicit humoral responses, and the GMT of neutralizing antibody titres on days 28 and 90 after the third injection ranged from 149 to 238 and 87 to 129, respectively among participants receiving three doses of vaccines. In general, the antibody response was weaker in 5 mg days 0 and 14 group and 10 mg day 0 group. Given that large-scale production of the inactivated vaccine would be achievable and the vaccine could be stored at 2À8°C, the inactivated vaccine could be an optimal option for many developing countries and regions where cold storage and distribution capacity was relatively poor.

The incidence rates of adverse reactions to the inactivated vaccine were generally comparable across different vaccine groups with no clear dose-response associations and similar when compared to the placebo group, indicating that the adverse reactions may not be entirely due to the vaccine but may be because of intramuscular injections or the aluminum hydroxide adjuvant that was contained in both the vaccines and placebos. The incidence rates of adverse reactions were similar to what we have reported before in selected groups with a smaller sample size [13] , as well as recent reports of other wholevirus inactivated vaccine candidates [10À12, 14, 40] . In the current analysis, we found similar incidence rates of adverse reactions among older adults (11¢0%) compared with younger adults (19¢3%) who received three injections on days 0, 28 and 56. This pattern is consistent with reports of another inactivated vaccine candidate [12, 40] . Nevertheless, the current studies could not provide firm evidence of the superiority and inferiority in terms of safety profile among different vaccines or between older and younger adults, because of small sample sizes in phase 1 and 2 trials, short follow-up duration, and varied populations, settings, and methodologies across trials. Phase 3 trials with long-term follow-up as well as larger sample size are still needed to provide the full safety profile of the vaccines.

The vaccine-enhanced diseases, i.e., antibody-dependent enhancement (ADE) and vaccine-associated enhanced respiratory disease (VAERD) [43, 44] , have been frequently raised as concerns for the COVID-19 vaccine candidates, particularly for inactivated vaccines. However, current phase 1/2 trials are not designed to assess these outcomes given that none of the participants was infected with SARS-CoV-2 after vaccination. Although published phase 3 trials of other vaccines did not report ADE and VAERD [27À33], the safety of the inactivated vaccine still need to be closely monitored in the subsequent follow-up visits as well as in the phase 3 trial and real-world observations.

We have previously reported the interim results of antibody responses for some groups from the phase 1 (2¢5 mg, 5 mg, and 10 mg with three injections) and phase 2 (5 mg on days 0 and 14/21) trials up to 14 days after the whole-course vaccination. The current analysis had an expanded sample size (e.g., 84 in each group rather than 24 in different dose groups with three injections), longer follow-up (90 rather than 14 days post injection), additional age groups (i.e., those aged 60 years old), and more schedule groups (i.e., 5 mg days 0 and 28 group and 10 mg day 0 group). Neutralizing antibody responses were generally similar among younger and older adults in our study, and the GMTs of neutralizing antibody on day 90 after three doses were 87À129 and 99À120 among younger and older adults, respectively, which was also observed in other vaccines [12, 14, 18, 36, 37] . Again, the efficacy in different age groups needs to be tested in the phase 3 clinical trials. The GMT of neutralizing antibody level was lower for those receiving vaccines at 5 mg on days 0 and 14/21 on days 28 and 90 compared to day 14 after the whole-course vaccination.

Although seroconversion rates of neutralizing antibody on day 90 after the whole-course vaccination were over 70% among participants receiving 2¢5 mg, 5 mg, or 10 mg on days 0, 28, and 56 or those receiving 5 mg on days 0 and 21/28, the results still raised a concern whether the antibody levels would decline over time, and longer follow-up is still needed to confirm the durability of antibody against SARS-CoV-2 and the efficacy of the vaccine. Moreover, current epidemiological evidence regarding the temporal change of antibody level against SARS-CoV-2 after infection remained debatable, and most were from studies with relatively small sample sizes. Some studies reported stable neutralizing or specific binding antibody over 6 months, while others found the antibody titre significantly declined and even turned to be undetectable in some participants [45À50] . Besides, two studies reported antibody titres on days 90 or 180 after the whole-course vaccination. The phase 1 trial of an inactivated vaccine, the BBV152 vaccine, did not observe declining antibody titres between days 14 and 90 after the whole-course vaccination, and seroconversion rates of neutralizing antibody on day 90 after the whole-course vaccination ranged between 73¢1 and 81¢1% among those receiving vaccines [51] . Another study in 33 adults found specific binding antibody titres induced by mRNA-1273 vaccine remained high in 6 months after vaccination, and nearly all participants had measurable neutralizing antibody titres [52] . Accordingly, future studies are still warranted to test the durability of humoral and cellular immune induced by different vaccines against SARS-CoV-2. The antibody titres on day 90 after the whole-course vaccination were low among those receiving 10 mg on day 0, and the seroconversion rates were only 32¢1% for specific IgG binding antibody and 14¢3% for neutralizing antibody, indicating the poor performance of this approach. Our results also indicated that a relatively longer interval (e.g., 21 or 28 days) between injections would be preferred, because the 5 mg days 0 and 14 group had lower antibody titres and seroconversion rates (60¢7% for specific IgG binding antibody and 50¢0% for neutralizing antibody). Nevertheless, the efficacy of the three-dose and two-dose schedules is unclear, and the optimal or minimum antibody titres that could protect people from COVID-19 are yet to be established. Besides, the sample size of current phase 2 trial is still limited, and the comparison among groups of different immune procedures might be underpowered, and thus the results should be interpreted cautiously. Subgroup analyses by intervals between injections should be performed in a study with a larger sample size to further verify the current results. Another concern is that whether current vaccines could protect against infection of emerging SARS-CoV-2 variants, such as the B. [56] . neutralizing capacity against emerging SARS-CoV-2 variants of this investigated inactivated vaccine is unavailable now but is planned to be tested to facilitate the understanding of its effects on different variants. This study has several limitations. First, the long-term safety and immunogenic responses are still unclear and extended follow-up visits are underway. Second, the results from subgroup and secondary analyses should be interpreted cautiously due to the limited sample size. Third, cellular immune responses and antigen-specific antibody binding responses were not measured in the current study, and data of neutralizing antibody titres against emerging variants of SARS-CoV-2 are unavailable. Fourth, data from older adults were mainly from those aged 60À69, and evidence from the oldest old is still warranted. Besides, the trial among children and adolescents is still ongoing and data are not yet available. Fifth, since we did not collect and measure antibody titres in convalescent sera as a comparator in this study, it is difficult to compare the humoral immune responses induced by the inactivated vaccine versus natural infection. Given varied PRNT and ELISA assays across epidemiological studies and clinical trials, it was also inappropriate to directly compare antibody titres in our study with other studies. However, a previous study of the phase 1/2 trials of BBV152, another inactivated vaccine, showed that the neutralizing antibody titres generated by vaccine were similar to the titres in human convalescent serum [11] .

In conclusion, the preliminary results of the phase 1/2 trial indicated that the inactivated vaccine against SARS-CoV-2 was safe and immunogenic among adults, including those aged 60 years or older.

The results also suggested that the two-dose schedule of 5 mg on days 0 and 21/28 could be further tested for efficacy in phase 3 trial given the better immunogenicity profile compared to a single high dose (10 mg on days 0) and the two-dose schedule with a shorter betweendose interval (5 mg on days 0 and 14). A third boost dose could generate even higher antibody titres as expected, but this may reduce the number of people who can get vaccinated and cause inadequate vaccine supplies. Extended follow-up is ongoing, and phase 3 trials have been launched in multiple countries using the two-dose schedule of 5 mg with 21À28 days of interval, which would provide additional evidence on the long-term safety and efficacy of the inactivated vaccine.

Xiaoming Yang and Shengli Xia are the principal investigators. Xiaoming Yang, Shengli Xia, Shuo Shen, Wanshen Guo, Kai Duan, Yuntao Zhang, Zhiming Yuan, Xinguo Li, Wei Chen, Cong Wu, Lianghao Zhang, and Yunkai Yang designed the trial and study protocol. An Pan, Yan-Bo Zhang, and Zejun Wang contributed to the literature search. All authors had access to data and Xiaoming Yang and Shengli Xia verified the data. Yong-Li Yang, Zejun Wang, Yan-Bo Zhang, and An Pan contributed to the analyses. Yan-Bo Zhang, An Pan, and Shihe Huang wrote the first draft of the manuscript. All authors contributed to the data interpretation and revision of the manuscript. Xiaoming Yang and Shengli Xia monitored the trial. Wei Chen and Zejun Wang were responsible for the laboratory analysis.

The study was funded by the National Program on Key Research Project of China (2020YFC0842100) and Major Science and Technology Project of the National New Drug Development of China (2018ZX09734-004).

The phase 1/2 trials are still ongoing, and the trial data will be made available 6 months after the trial is complete upon reasonable request of the corresponding author and approval by the institutional review board. Jianhui Du and Xing-Hang Li report being students of Wuhan Institute of Biological Products Co Ltd.

The vaccine was developed and the study was sponsored by the China National Biotec Group Company Limited and the Wuhan Institute of Biological Products Co. Ltd. We are grateful for all investigators at the Henan Center for Disease Control and Prevention and Wuzhi county Center for Disease Control and Prevention who contributed to the site work of the trials. We appreciate the contributions of the members in the data and safety monitoring board, including Dr. Xiaoju Zhang and Dr. Qiuxing Zhang from the Henan Province People's Hospital, and Dr. Guoli Yan from the Henan University of Chinese Medicine. They received remuneration for their work. We thank all the participants in the trial for their altruism and dedication to the trial.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.eclinm.2021.101010.

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