Safety and superior immunogenicity of heterologous boosting with an RBD-based SARS-CoV-2 mRNA vaccine in Chinese adults

Homologous and heterologous booster with COVID-19 mRNA vaccines represent the most effective strategy to prevent the ongoing Omicron pandemic. The additional protection from these prototype SARS-CoV-2 S-targeting vaccine was attributed to the increased RBD-specific memory B cells with expanded potency and breadth. Herein, we show the safety and immunogenicity of heterologous boosting with the RBD-targeting mRNA vaccine AWcorna (also term ARCoV) in Chinese adults who have received two doses inactivated vaccine. The superiority over inactivated vaccine in neutralization antibodies, as well as the safety profile, support the use of AWcorna as heterologous booster in China.

pregnancy test results were excluded from this study. Participants with a medical history of 23 convulsion, serious acute hypersensitive reaction to vaccines, acute febrile diseases or 24 infectious diseases, congenital or acquired angioedema, asplenia or functional asplenia, 25 thrombocytopenia or other coagulation disorders, anti-allergy therapy, or blood products within 26 3 months were also excluded. 27 28

Randomization 29
Each participant was assigned a unique subject ID by authorized assigners successively 30 according to a Prespecified allocation kit, which was generated by an independent 31 randomization statistician from Beijing Key Tech Statistical Consulting Co., Ltd. via SAS 32 software (SAS® Institute, Cary, North Carolina, USA) with the ratio of 2:1 to the AWcorna and 33 CoronaVac groups. Since the different appearances of the two kinds of vaccines, inoculators 34 could not keep in blind when vaccines had been used. And hence, staff who were assigned to 35 inoculate would not be involved in any other research jobs, especially for subjects' safety 36 follow-up procedures. Participants would be masked when receiving the jab by a special curtain 37 in the injection room to avoid the identification of he or she, and disclosure of the allocated 38 group. Other investigators, laboratory staff, and outcome assessors were kept blinded also. After injection, subjects received an in-site 30-minutes safety observation conducted by 49 research staff to confirm if any immediate reactions occurred. Any adverse events (AEs) 50 discovered or any relevant concomitant medications declared within 28 days after vaccination 51 were recorded by subjects with the help of the daily cards and connections cards which were 52 prepared and managed by the study team. For each subject, serious adverse events (SAEs), 53 adverse events of interest (AESI), and pregnancy were collected from the enrollment till 12 54 months after his/her booster dose. Up to 6 ml of blood sample was collected from each 55 participant at baseline pre-booster vaccination and on day 14 and day 28 after receiving the 56 booster dose. 57 58

Endpoints 59
The primary endpoints for safety were: 1) The incidence rates of adverse reactions/adverse 60 events within 30 minutes, during Day 0-14 and Day 0-28 after vaccination; and 2) The 61 incidence rates of adverse reactions/adverse events with a severity of grade 3 and above within 62 30 minutes and during Day 0-14 and Day 0-28 after vaccination. The primary immunogenicity 63 endpoint was the titers of neutralizing antibody against wild type SARS-CoV-2 as measured by 64 live virus neutralization assay 14 days post booster.

Sample size 99
The sample size was determined based on the hypothesis that the booster vaccination of mRNA 100 vaccine following the two-dose inactivated vaccine regimen be non-inferior to that of the 101 booster of inactivated vaccine in neutralizing antibody. It was assumed that the pooled standard 102 deviation of log10-transformed neutralizing data was 0.5 and equal GMT in both the mRNA 103 vaccine group and CoronaVac group. 200 participants in the mRNA group and 100 participants 104 in the CoronaVac group could have at least 80% power to observe that the lower limit of the 105 95% confidence interval of GMT ratio between the two groups was greater than the non-106 inferiority margin (2/3), with the one-sided significance level of 2.5%. 107 108

Statistical analysis 109
The geometric mean titer (GMT) and 95% confidence interval (CI) were used to describe 110 neutralizing results in the mRNA vaccine group and CoronaVac group after booster vaccination, 111 and the GMT ratio between the two groups and 95% CI were estimated. The non-inferiority 112 result would be concluded if the lower bound of 95%CI was larger than 2/3. When the non-113 inferiority conclusion was concluded, the superiority would be considered sequentially if the 114 lower bound was larger than 1. The seroconversion rate and Clopper-Pearson 95%CI after 115 booster vaccination were estimated as well, and the difference between the two groups was 116 calculated using the Miettinen-Nurminen method. 117

118
We assessed the number and proportion of participants with adverse reactions 0-28 days after 119 the booster dose. For fever, besides the NMPA standard, we also derived the oral temperature 120 by adding 0.2℃ to the collected auxiliary temperature, and then re-graded the adverse reaction 121 based on the FDA standard to provide more comparable results with marketed vaccines. We 122 used the χ2 test or Fisher's exact test to analyze categorical data, the t-test to analyze the log-123 transformed antibody titers, and the Wilcoxon rank-sum test for data not following a normal 124 distribution. The correlation between concentrations of log-transformed neutralizing antibody 125 and binding antibody levels was analyzed using Pearson's correlation. The primary analysis 126 was performed based on the per-protocol population. Statistical analyses were performed using 127 SAS (version 9.4).  The geometric mean of titer (GMT) and geometric mean of increase against the pre-booster level (GMI) are presented with corresponding 2-sided 95% confidence 161 interval (CI) respectively in AWcorna and CoronaVac groups, and the ratios of GMT or GMI between the two groups and corresponding 95% CI are accordingly 162 showed. 163 The non-inferiority result would be concluded if the lower bound of 95% CI of the ratio between groups (AWcorna/CoronaVac) is larger than 2/3 When the non-164 inferiority conclusion is concluded, the superiority would be considered sequentially if the lower bound was larger than 1. 165 The seroconversion rate and the 4-fold Increase rate with their corresponding Clopper-Pearson 95% CI are shown, and the difference between the two groups were 166 calculated using Miettinen-Nurminen method. 167 N=the number of participants included the per-protocol population. The p values are the results of comparison between the AWcorna and CoronaVac groups. 168 *Seropositive defined as the status that the nAb level of a subject achieved the threshold of 1:8 (Limit of Detection, LOD). 169 **Seroconversion due to booster dose at a subject level is defined as a change from below the LOD to equal or above LOD, or for those who above the LOD pre-170 booster experienced at least a 4-fold rise in terms of nAbs. The geometric mean of titer (GMT) and geometric mean of increase against the pre-booster level (GMI) are presented with corresponding 2-sided 95% confidence 174 interval (CI) respectively in AWcorna and CoronaVac groups, and the ratios of GMT or GMI between the two groups and corresponding 95% CI are accordingly 175

showed. 176
The seroconversion rate and the 4-fold Increase rate with their corresponding Clopper-Pearson 95% CI are shown, and the difference between the two groups were 177 calculated using Miettinen-Nurminen method. 178 N=the number of subjects included the per-protocol population. The p values are the results of comparison between the AWcorna and CoronaVac groups. 179 *Seropositive defined as the status that the nAb level of a subject achieved the threshold of 1:10 (Limit of Detection, LOD). 180 ** Seroconversion due to booster dose at a subject level is defined as a change from below the LOD to equal or above LOD, or for those who above the LOD pre-181 booster experienced at least a 4-fold rise in terms of nAbs. 182