Comparison of the reactogenicity and immunogenicity between two-dose mRNA COVID-19 vaccine and inactivated followed by an mRNA vaccine in children aged 5 - 11 years

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To compare the reactogenicity and immunogenicity between the two-dose mRNA 22 COVID-19 vaccine regimen and one or two doses of inactivated vaccine followed by an 23 mRNA vaccine regimen in healthy children between 5-11 years of age. 24

A prospective cohort study was performed at King Chulalongkorn Memorial 25 Hospital in Thailand between March to June 2022. Healthy children between 5-11 years of 26 age were enrolled and received the two-dose mRNA COVID-19 vaccine (BNT162b2) 27 regimen or the inactivated (CoronaVac) vaccine followed by the BNT162b2 vaccine regimen. 28 In addition, healthy children who received two doses of BBIBP-CorV between 1-3 months 29 prior were enrolled to receive a heterologous BNT162b2 as a third dose (booster). 30 Reactogenicity was assessed by a self-reported online questionnaire. Immunogenicity 31 analysis was performed to determine binding and surrogate neutralizing antibodies to SARS-32 CoV-2 wild-type and Omicron variants. 33

Overall, 166 eligible children were enrolled. Local and systemic AE which occurred 34 within 7 days after vaccination were mild to moderate and well-tolerated. At one-month, 35 post-two or post-three doses, children vaccinated with two-dose BNT162b2, 36 CoronaVac/BNT162b2, and two-dose BBIBP-CorV followed by BNT162b2 elicited similar 37 levels of anti-receptor-binding domain (RBD) IgG. However, the two-dose BNT162b2 and 38 two-dose BBIBP-CorV followed by BNT162b2 groups elicited higher neutralizing activities 39 against Omicron BA.2 variant than the CoronaVac/BNT162b2 group. 40

The heterologous, CoronaVac vaccine followed by the BNT162b2 vaccine, 41 regimen elicited lower neutralizing activities against the emerging Omicron BA.2 variant 42 than the two-dose mRNA regimen. A third dose (booster) mRNA vaccine should be 43 prioritized for this group. 44 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 3

Severe acute respiratory virus 2 (SARS-CoV-2); Omicron; mRNA; inactivated; 45 vaccine; booster 46 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 4 1. Introduction 47 The COVID-19 pandemic, caused by SARS-CoV-2, has caused more than 600 48 million infections and more than 6.5 million deaths worldwide as of October 2022.1 The first 49 COVID-19 vaccine that received emergency use authorization from the United States Food 50 and Drug Administration (FDA) in December 2020 was the BNT162b2 vaccine for persons 51 16 years of age or older.2, 3 Later on, several effective COVID-19 vaccines were developed, 52 tested, and initially distributed to the adult population, leaving children as a vulnerable 53 population. 54 During the early COVID-19 pandemic, children infected with SARS-CoV-2 often 55 developed mild symptoms.4 Nevertheless, the increase of symptomatic SARS-CoV-2 56 infection and hospitalization rate among children during the Delta (B.1.617.2) and Omicron 57 (B.1.1.529) variants era indicated the need to extend vaccination to the pediatric population. 58 Between October to December 2020, a phase 1/2 randomized controlled trial to assess the 59 safety and immunogenicity of the inactivated CoronaVac vaccine in children between 3-17 60 years was conducted in China.5 The results showed that 3.0 µg CoronaVac was safe and able 61 to elicit 100% seroconversion in children. In addition, another randomized controlled trial in 62 2020 demonstrated that the 4.0 µg inactivated BBIBP-CorV vaccine was safe for children 3-63 18 years and could elicit a robust humoral response after two doses.6 64 Apart from the inactivated vaccine, an mRNA vaccine has also been extended to the 65 pediatric population. On October 29, 2021, the BNT162b2 vaccine was approved as an 66 emergency use authorization for children 5-to-11-years old in the US based on the data in 67 phase 2/3 trial which demonstrated that two doses of the 10 µg BNT162b2 vaccine 68 administered 21 days apart showed an efficacy of 90.7% against the circulating SARS-CoV-2 69 variant.2 In December 2021, the Centers for Disease Control and Prevention (CDC) reviewed 70 adverse events of the BNT162b2 vaccine from the Vaccine Adverse Event Reporting System 71 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 5 (VAERS) and reported that myocarditis was associated with the mRNA-based COVID-19 72 vaccine.7 This rare but serious side effect raised concerns among the parents which could lead 73 to vaccine hesitancy. 74 Thailand has secured the inactivated CoronaVac vaccine since February 2021, the 75 inactivated BBIBP-CorV vaccine since June 2021, and the mRNA BNT162b2 vaccine since 76 August 2021. In December 2021, the BNT162b2 vaccine was approved by the Thai FDA as a 77 two-dose regimen for children aged between 5-11 years. In February 2022, the two-dose 78 CoronaVac and two-dose BBIBP-CorV vaccines were approved by the Thai FDA for 79 children aged between 6-11 years. The Ministry of Public Health and the Royal College of 80 Thai Pediatricians recommended a dosing interval of 8 weeks for the two-dose BNT162b2 81 regimen based on an immunogenicity study reporting that a long-interval vaccination 82 schedule induced higher antibody response compared to a short-interval vaccination 83 schedule.8 Besides, the Ministry of Public Health and the Royal College of Thai 84 Pediatricians also recommended that a heterologous inactivated (CoronaVac) vaccine 85 followed by the BNT162b2 vaccine administered 4 weeks apart could be an alternative 86 regimen for parents concerned about the adverse events following an mRNA vaccination. 87 Our previous immunogenicity study in Thai healthy adults vaccinated with a heterologous, 88 CoronaVac vaccine followed by the BNT162b2 vaccine regimen showed that this regimen 89 induced a higher antibody response compared to the homologous CoronaVac.9 90 Amidst the emergence of the Omicron variant, both two-dose mRNA or inactivated 91 vaccination regimens cannot prevent breakthrough infections.10, 11 A third dose is 92 recommended to obtain high immunity against the Omicron variant. Our previous studies in 93 adults have shown that a booster mRNA vaccine after inactivated (CoronaVac or BBIBP-94 CorV)-primed individuals elicited a high level of antibody responses against the Omicron 95 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 6 variant.12, 13 However, there was limited information about the safety and immunogenicity of 96 a heterologous mRNA booster in inactivated vaccine-primed children. 97 This study aims to compare the reactogenicity and immunogenicity between the 98 heterologous CoronaVac vaccine followed by the BNT162b2 vaccine regimen and the two-99 dose BNT162b2 vaccine regimen in children between 5-11 years of age. In addition, the 100 reactogenicity and immunogenicity of the heterologous BNT162b2 booster in BBIBP-CorV-101 primed children were evaluated. The results of this study will help guide the physician’s 102 decision on a mix-and-match vaccine strategy in certain circumstances and guide the booster 103 strategy in the two-dose BBIBP-CorV-primed children. 104 105 2. Materials and methods 106 2.1. Study design and participants 107 This prospective cohort study was conducted between March to June 2022 at the 108 clinical trial research unit at the Center of Excellence in Clinical Virology, Department of 109 Pediatrics, Faculty of Medicine, Chulalongkorn University in Bangkok, Thailand. The study 110 protocol was approved by the Institutional Review Board (IRB) of the Faculty of Medicine of 111 Chulalongkorn University (IRB 059/65) and was performed under the principles of the 112 Declaration of Helsinki. This trial was registered in the Thai Clinical Trials Registry 113 (TCTR20220212001). Written informed consent was obtained from the parents or the legal 114 guardians of participants prior to enrollment. Written assent was obtained from children aged 115 7 years and above. The inclusion criteria were immunocompetent children between 5-11 of 116 age with no or well-controlled underlying diseases, no previous COVID-19 vaccination, and 117 no previous SARS-CoV-2 infection from the medical history. The first group of participants 118 was enrolled to receive the two-dose BNT162b2 regimen administered 8 weeks apart. The 119 second group was enrolled to receive the CoronaVac followed by BNT162b2 vaccination 120 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 7 administered 4 weeks apart. In addition, the third group of participants who had previously 121 been immunized with two doses of the BBIBP-CorV vaccine between 1-3 months prior were 122 enrolled. Participants received two doses of the BBIBP-CorV vaccine in other hospitals but 123 need to provide immunization records in the electronic health record from the Ministry of 124 Public health, Thailand prior to enrollment. This group is consented to receiving the third 125 dose (booster) with BNT162b2. The inclusion criteria are the same as in the CoronaVac 126 followed by BNT162b2 and homologous two-dose BNT162b2 groups, except the criteria of 127 no previous COVID-19 vaccination. 128 2.2. Vaccine and blood collection 129 The CoronaVac vaccine from Sinovac Life Sciences, Beijing, China (hereafter 130 referred to as SV) is an inactivated SARS-CoV-2 vaccine (CZ02 strain). The dosage for the 131 pediatric population is 0.5 mL per dose containing 600 Spike Units (equal to 3 micrograms) 132 of inactivated SARS-CoV-2 whole virus as antigen. The BBIBP-CorV vaccine from 133 Sinopharm, Beijing, China (hereafter referred to as SP) is also an inactivated vaccine 134 developed from the whole SARS-CoV-2 stain HB02. The interval recommended for the SP 135 vaccine is two doses administered 4 weeks apart. The BNT162b2 from Pfizer-BioNTech, 136 NY, USA (hereafter referred to as PF) is a lipid nanoparticle containing mRNA encoding the 137 SARS-CoV-2 full-length spike of ancestral SARS-CoV-2 strain. The dosage for the pediatric 138 population is 0.2 mL (10 micrograms) per dose. The recommended interval for SV/PF and 139 PF/PF regimens are 4 and 8 weeks, respectively. 140 For participants in the SV/PF and PF/PF groups, blood samples were collected before 141 the first dose vaccination (V1, baseline or pre-dose 1), before the second dose vaccination 142 (V2, pre-dose 2), and one month after the second dose vaccination (V3, post-dose 2). 143 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 8 For participants in the SP/SP/PF group, blood samples were collected between 1-3 144 months after the second dose vaccination (V3, post-dose 2) and one month after the third 145 dose vaccination (V4, post-dose 3). 146 2.3. Safety assessments 147 Parents or guardians of the participants recorded both local and systemic adverse 148 events (AEs) after immunization within 7 days using self-administered online and paper 149 questionnaires. An explanation of data collection was given to participants by trained 150 investigators during the vaccination visit. Local and systemic AEs were classified as mild, 151 moderate, and severe as previously described.14 152 2.4. Laboratory assessments 153 Serum samples were tested for binding antibody specific to the receptor-binding 154 domain (RBD) of SARS-CoV-2, including total RBD immunoglobulin (Ig) and anti-RBD 155 IgG, and anti-nucleocapsid (N) IgG as previously described.15 Neutralizing activities against 156 wild-type (Euroimmun, Lubeck, Germany) and Omicron (BA.2) (GenScript Biotech, NJ, 157 USA) were analyzed using a surrogate virus neutralization test (sVNT) as previously 158 described.16 The seropositivity of sVNTs against wild-type and Omicron (BA.2) were 159 determined as ≥35% and ≥30% inhibition, respectively. Samples were tested at the end of the 160 study using the same batch of test kits. 161 2.5. Statistical analysis 162 The statistical differences in age between groups were performed using the Kruskal-163 Wallis test, followed by Dunn’s post hoc test with Bonferroni correction. Local and systemic 164 adverse events between different regimens after the first and second doses were compared by 165 risk differences with a 95% confidence interval (CI). Total RBD Ig and anti-RBD IgG were 166 presented as geometric mean titers (GMT) with a 95% CI. Percent inhibitions by the sVNT 167 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 9 assay were presented as a median with interquartile ranges (IQR). Differences in the 168 geometric mean ratio (GMR) of total RBD Ig and anti-RBD IgG between groups were 169 calculated by ANCOVA with Bonferroni’s adjustment. Differences in percentages of 170 inhibition between groups were calculated by the Kruskal-Wallis test with Dunn’s multiple 171 comparisons. A p-value of <0.05 was considered statistically significant. 172 3. Results 173 3.1 Demographic data and baseline characteristics 174 From March to June 2022, a total of 166 children were enrolled in the study. The 175 consort flow diagram of study participants was shown in Figure 1. There were 43 eligible 176 participants enrolled in SV/PF group. Among this group, there were 3 participants tested 177 positive for COVID-19 after the first or second dose vaccination (breakthrough infection), 11 178 participants who had anti-N IgG, total RBD IgG, or anti-RBD IgG positive at baseline, 179 presumably due to asymptomatic COVID-19 infection, and 2 participants who had evidence 180 of SARS-CoV-2 infection as determined by anti-N IgG seroconversion. Therefore, a total of 181 16 participants in the SV/PF groups were excluded from the final immunogenicity analysis. 182 There were 62 eligible participants enrolled in PF/PF group. Among this group, there 183 were 11 and 1 participant(s) tested positive for COVID-19 after the first and second dose 184 vaccination, respectively (breakthrough infection), 3 participants who had anti-N IgG, total 185 RBD IgG, or anti-RBD IgG positive at baseline, presumably due to asymptomatic COVID-19 186 infection, 12 participants who had evidence of SARS-CoV-2 infection as determined by anti-187 N IgG seroconversion, and 5 participants were lost to follow-up. Therefore, a total of 32 188 participants in the PF/PF groups were excluded from the final immunogenicity analysis. 189 There was 1 participant who skipped visit 2, received PF at a local hospital, and returned for 190 blood testing on visit 3. 191 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 10 There were 61 eligible participants enrolled in SP/SP/PF group. Among this group, 192 there was 1 participant who tested positive for COVID-19 after the third dose of vaccination, 193 1 participant who had evidence of SARS-CoV-2 infection as determined by anti-N IgG 194 seroconversion, and 3 participants who were lost to follow-up. Therefore, a total of 5 195 participants in the SP/SP/PF group were excluded for the final immunogenicity analysis. 196 The demographics and characteristics of the participants are shown in Table 1. There 197 were 27, 30, and 56 participants in the SV/PF, PF/PF, and SP/SP/PF groups who were 198 included in the final immunogenicity analysis, respectively. The number of female 199 participants per total (%) participants were similar among groups. Nevertheless, there was a 200 statistically significant difference in age between the PF/PF group (mean 6.2 years) and 201 SP/SP/PF group (mean 7.8 years) (P-value <0.001). The mean interval between doses 1 and 2 202 were 30.2, 57.4, and 22.9 days in the SV/PF, PF/PF, and SP/SP/PF groups, respectively. The 203 mean interval between doses 2 and 3 was 61.0 days in the SP/SP/PF group. 204 We also compared the antibody responses elicited by the different vaccine regimens 205 with those elicited by vaccine regimens plus natural infection (hybrid immunity). Hybrid 206 immunity groups refer to participants who had anti-N IgG seroconversion after receiving the 207 vaccine or participants who had pre-existing antibodies (anti-N IgG or total RBD IgG) at 208 baseline before the first vaccine as described in Table 1. 209 3.2 Safety and reactogenicity profile 210 The most common solicited local adverse reaction (AE) after vaccination was pain at 211 the injection site: SV/PF group (first dose 62.5%; second dose 68.6%), PF/PF group (first 212 dose 59.3%; second dose 57.8%), SP/SP/PF group (third dose; 85.2%). The most common 213 systemic AE was myalgia (20.0-28.6%) (Figure S1). Comparisons of AEs between SV/PF 214 and PF/PF regimens after the first or second dose vaccination showed no significant 215 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 11 differences in any of the local or systemic AEs (Figure 2). Most of the solicited local and 216 systemic AEs were mild (grade 1) or moderate (grade 2) and resolved within a few days post-217 vaccination (Figure S1). Frequencies of grade 3 local or systemic AEs after ranged between 218 1% to 3%. No serious AEs were reported. 219 3.3 Total RBD Ig and anti-RBD IgG responses in pediatric participants after different 220 regimens of COVID-19 vaccines. 221 The geometric mean titer (GMT) of total RBD immunoglobulin (Ig) which 222 predominantly included IgG, but also some amount of IgM and IgA, and anti-RBD IgG were 223 compared among groups vaccinated with different regimens using ANCOVA with 224 Bonferroni’s adjustment as shown in Figure 3. There were no differences in total RBD Ig and 225 anti-RBD IgG at pre-vaccination among the SV/PF and PF/PF groups at pre-dose 1 (V1). At 226 one-month post-dose 2 (V3), the PF/PF group had significantly higher total RBD Ig than the 227 SV/PF group (Fig 3A) (geometric mean ratio (GMR) 2.04). However, there were no 228 differences in anti-RBD IgG levels among the SV/PF and PF/PF groups (Fig 3B). In addition, 229 the three-dose SP/SP/PF group also possessed higher total RBD Ig than the two-dose SV/PF 230 and PF/PF groups (GMR 3.09 and 1.52, respectively), but there was no difference in the anti-231 RBD IgG levels among all groups. 232 3.4 Total RBD Ig and anti-RBD IgG responses between two-dose vaccination and 233 hybrid immunity 234 There was a subgroup of participants (n=11) in the SV/PF group found to have a pre-235 existing anti-N IgG, total RBD IgG, or anti-RBD IgG at baseline (before receiving the first 236 dose SV). The baseline characteristics of this subgroup was shown in Table 1. This group 237 was excluded from the immunogenicity analysis as they presumably had asymptomatic or 238 unrecognized COVID-19 infection prior to enrollment. However, all the immunogenicity 239 All rights reserved. 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were available at the end of the study and children in this group had received the two-240 dose SV/PF vaccine. Therefore, antibody levels in this SV/PF-vaccinated group who 241 possessed pre-existing immunity (refers to as pre-existing immunity group) were compared to 242 the SV/PF-vaccinated group without pre-existing immunity as shown in Figures 4A and B. 243 The results showed that the pre-existing immunity group elicited higher total RBD Ig at pre-244 dose 2 (V2) and post-dose 2 (V3) than those without pre-existing immunity. However, the 245 anti-RBD IgG at post-dose 2 (V3) was comparable between both groups. 246 Similarly, there was a subgroup of participants (n=12) in the PF/PF group found to 247 have been infected with SARS-CoV-2 between vaccination visits 1 and 2 as evidenced by the 248 seroconversion of anti-N IgG. The baseline characteristics of this subgroup was shown in 249 Table 1. This group was excluded from the immunogenicity analysis as they presumably had 250 asymptomatic or unrecognized COVID-19 infection after receiving the first dose of the 251 BNT162b2 vaccine. Similarly, all the immunogenicity results were available at the end of the 252 study and children in this group had received the second dose BNT162b2 vaccine. Antibody 253 levels in this PF/PF-vaccinated group who had a breakthrough infection (refers to as the 254 breakthrough group) were compared to the PF/PF-vaccinated group without breakthrough 255 infection as shown in Figure 4C and D. The results showed that the breakthrough group 256 possessed higher levels of total RBD Ig and anti-RBD IgG than the non-breakthrough group 257 at pre-dose 2 but not at post-dose 2. 258 3.5 Neutralizing activity against wild-type and Omicron BA.2 using sVNT 259 Neutralizing activities against wild-type SARS-CoV-2 after two-dose or three-dose 260 vaccination was above 99% and similar among all groups (Figure 5A). However, following 261 the second dose vaccination, the homologous PF/PF group possessed higher neutralizing 262 activities against the Omicron BA.2 variant compared to the heterologous SV/PF group (p < 263 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 13 0.001) (Figure 5B). In addition, the three-dose vaccinated group (SP/SP/PF) also elicited 264 higher neutralizing activities against the Omicron BA.2 variant compared to the heterologous 265 SV/PF group (p < 0.001). 266 4. Discussion 267 In this study, we evaluated the extent of binding and neutralizing antibody responses 268 to the two-dose BNT162b2 regimen and the heterologous CoronaVac followed by the 269 BNT162b2 vaccine regimen in children between 5-11 years. In addition, we also evaluated 270 the antibody response after a BNT162b2 vaccine as a booster dose in pediatric individuals 271 who had received two-dose vaccination (priming) with the inactivated BBIBP-CorV vaccine 272 regimen. Our study found that at one month post two-dose or three-dose vaccination, children 273 vaccinated with the two-dose BNT162b2 regimen, the heterologous regimen, and the two-274 dose BBIBP-CorV followed by BNT162b2 regimen elicited similar levels of anti-RBD IgG. 275 All vaccinated groups elicited neutralizing activities against wild-type and Omicron BA.2 276 variants of SARS-CoV-2 after completion of two-dose or three-dose vaccination. However, 277 neutralizing activities as determined by percent inhibitions against the Omicron BA.2 variant 278 were lower than that toward the ancestral strain. Furthermore, the two-dose BNT162b2 and 279 two-dose BBIBP-CorV followed by BNT162b2 groups elicited higher neutralizing activities 280 against Omicron BA.2 variant than the CoronaVac/BNT162b2 group. 281 Regarding the two-dose vaccine regimen in children, the real-world effectiveness of 282 the two-dose CoronaVac or BNT162b2 vaccine regimen amidst the Omicron variant outbreak 283 showed comparable results. A study in Chile demonstrated that the effectiveness of the two-284 dose CoronaVac regimen in children 3-5 years of age was 38.2% against symptomatic 285 COVID-19, 64.6% against hospitalization, and 69.0% against ICU admission.17 Similarly, 286 vaccine effectiveness against hospitalization during the Omicron predominance in children 5 287 to 11 years old elicited by two doses of BNT162b2 was 68%.18 Although the present study 288 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 14 did not evaluate the effectiveness, the immunogenicity results showed that neutralizing 289 activities against Omicron BA.2 variant and total RBD Ig in the two-doses BNT162b2 group 290 administered 8 weeks apart were higher than the heterologous CoronaVac/BNT162b2 291 administered at 4 weeks apart. Previous studies on adenoviral-vectored and mRNA COVID-292 19 vaccine showed that increased dosing intervals improved the vaccine immunogenicity and 293 effectiveness. 8, 19 Similarly, the extended dosing interval of the CoronaVac/BNT162b2 mix-294 and-match strategy also showed improved immunogenicity in adults.9 In Thailand, two doses 295 of the BNT162b2 vaccine were administered 8 weeks apart, instead of 3 weeks apart as 296 recommended by the Advisory Committee on Immunization Practices (ACIP),20 due to the 297 limited vaccine supply and improved immunogenicity. Nevertheless, the 298 CoronaVac/BNT162b2 regimen administered 4 weeks apart was recommended. Due to the 299 lower immunogenicity profile of CoronaVac/BNT162b2, it is worth noting that the 300 CoronaVac/BNT162b2-vaccinated group should be prioritized for the third dose booster. 301 Several studies in adults showed that heterologous boosters (mRNA vaccine as the 302 third dose in inactivated COVID-19 vaccine-primed individuals) could enhance antibody 303 response against the emerging Omicron variant.12, 21 In this study, we evaluated the 304 immunogenicity after the BNT162b2 booster in BBIBP-CorV-primed individuals 1-3 months 305 prior and found that the boosted children can elicit neutralizing antibody response against 306 Omicron BA.2 variant similar to those elicited by two-dose BNT162b2. Our previous study 307 found that longer interval between primary doses of CoronaVac and booster (i.e., 6 months) 308 could enhance the total Ig and anti-RBD IgG responses compared to the short interval booster 309 (i.e., 3 months).12 Nevertheless, the present study chose the short interval for BBIBP-CorV-310 primed individuals to receive a booster because Omicron-specific anti-RBD IgG elicited by 311 the two-dose inactivated vaccine were low at 3 months after second dose, and get boosted 312 significantly after an mRNA vaccine as the third (booster) dose.21 Thus, the heterologous 313 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 15 mRNA booster in children primed with inactivated vaccine regimen should be recommended 314 to increase protection against the emerging Omicron variant. although a longer interval 315 between the second and the third dose could stimulate higher antibody responses. 316 All COVID-19 vaccines administered as primary or booster doses in this study had 317 acceptable reactogenicity with mild to moderate AEs that generally resolved within a few 318 days after vaccination. The adverse events reported herein were similar to those of pain, 319 myalgia, and fever described in the previous studies.2, 22 320 Nearly one-third of the participants in the CoronaVac/BNT162b2 group had been 321 exposed to the SARS-CoV-2 virus prior to enrollment. This was detected by seropositivity of 322 anti-nucleocapsid (N) IgG or total RBD Ig at baseline. This study showed that total RBD Ig 323 are higher in previously infected vaccinees than in SARS-CoV-2 naïve subjects following the 324 first dose of the CoronaVac vaccination and at one month post-second dose (BNT162b2) 325 vaccination. Our findings are in line with a study in adults showing that previously infected 326 individuals who received a booster COVID-19 vaccine elicited higher neutralizing antibodies 327 compared to the infection alone or vaccine alone.23 Regarding the two-dose BNT162b2 328 groups, there was a long waiting time (8 weeks) between the first and second doses of the 329 BNT162b2 vaccine. Thus, nearly one-third of the participants in the two-dose BNT162b2 330 group had breakthrough infection after receipt of the first dose of the BNT162b2 vaccine as 331 determined by seroconversion of anti-N IgG. Similarly, at visit 2 (post one dose + natural 332 infection), breakthrough infection vaccinees had anti-RBD IgG and total RBD Ig higher than 333 in SARS-CoV-2 naïve subjects immunized with one-dose BNT162b2 vaccine, but this 334 difference disappeared after the second dose vaccine. This is also in agreement with a 335 previous study in adults which showed that previously infected vaccinees who received 2 336 doses mRNA vaccine elicited higher Omicron BA.1-specific neutralizing antibodies similar 337 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 16 to the triple-vaccinated subjects, but higher than SARS-CoV-2 naïve subjects who received 2 338 doses mRNA vaccine.24 339 Potential limitations of our study may be attributed to the loss of some participants 340 from the final immunogenicity analysis due to the peak of the SRAS-CoV-2 outbreak in 341 Bangkok during March to April 2022. The BNT162b2 vaccine was in multiple-dose vials 342 which needed to be administered in a short period. The time limit of keeping opened multi-343 dose vials made the randomization not feasible in our study. As of May 2022, there was a 344 recommendation from the Centers for Disease Control and Prevention that children who 345 receive two-dose BNT162b2 should get a BNT162b2 booster. Thus, additional studies on the 346 immunogenicity and efficacy of the third dose (booster) in two-dose BNT162b2 or 347 heterologous CoronaVac/BNT162b2-primed children should be further investigated. 348 5. Conclusions 349 Local and systemic AE which occurred within 7 days after vaccination were mild to 350 moderate and well-tolerated. The heterologous, CoronaVac vaccine followed by the 351 BNT162b2 vaccine, regimen elicited lower neutralizing activities against the emerging 352 Omicron BA.2 variant than the two-dose mRNA regimen. A third dose (booster) mRNA 353 vaccine should be prioritized for this group. 354 Acknowledgments: We would like to thank all Center of Excellence in Clinical Virology 355 personnel and all participants for contributing to and supporting this project. This research 356 was financially supported by the Health Systems Research Institute (HSRI), National 357 Research Council of Thailand (NRCT), the Center of Excellence in Clinical Virology, 358 Chulalongkorn University, and King Chulalongkorn Memorial Hospital, and partially 359 supported by the Second Century Fund (C2F) of Sitthichai Kanokudom, Chulalongkorn 360 University. 361 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 17 Author Contributions: Conceptualization, N.W., S.K. (Sitthichai Kanokudom), 362 N.S.(Nungruthai Suntronwong), P.N. and Y.P.; data curation, N.W., S.K. (Sitthichai 363 Kanokudom), N.S. (Nungruthai Suntronwong), S.A., R.Y., D.S., T.T. (Thaksaporn 364 Thatsanatorn) and N.S. (Natthinee Sudhinaraset); formal analysis, N.W., S.K. (Sitthichai 365 Kanokudom) and H.P.; methodology, S.K. (Sitthichai Kanokudom), J.C., P.V., S.K. (Sirapa 366 Klinfueng), T.T (Thanunrat Thongmee), R.A. and N.K.; project administration, Y.P.; 367 writing—original draft, N.W., S.K. (Sitthichai Kanokudom); writing—review and editing, 368 N.W., S.K. (Sitthichai Kanokudom) and Y.P. All authors have read and agreed to the 369 published version of the manuscript. 370 Funding: This research was financially supported by the Health Systems Research Institute 371 (HSRI), National Research Council of Thailand (NRCT), the Center of Excellence in Clinical 372 Virology, Chulalongkorn University, and King Chulalongkorn Memorial Hospital, and 373 partially supported by the Second Century Fund (C2F) of Sitthichai Kanokudom, 374 Chulalongkorn University. 375 Institutional Review Board Statement: The study protocol was approved by the 376 Institutional Review Board (IRB), Faculty of Medicine, Chulalongkorn University (IRB 377 number 059/65). This trial was registered in the Thai Clinical Trials Registry 378 (TCTR20220212001). 379 Informed Consent Statement: Informed consent was obtained from parents or legal 380 guardians before participant enrollment. The study was conducted according to the 381 Declaration of Helsinki and the Good Clinical Practice Guidelines (ICH-GCP) principles. 382 Data Availability Statement: The datasets generated and analyzed during the current study 383 are available from the corresponding author upon reasonable request. 384 Conflicts of Interest: The authors declare no conflict of interest. 385 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 18

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Cheng SMS, Mok CKP, Leung YWY, et al. Neutralizing antibodies against the 453 SARS-CoV-2 Omicron variant BA.1 following homologous and heterologous CoronaVac or 454 BNT162b2 vaccination. Nat Med. 2022;28:486-9. 455 24. Trombetta CM, Piccini G, Pierleoni G, et al. Immune response to SARS-CoV-2 456 Omicron variant in patients and vaccinees following homologous and heterologous 457 vaccinations. Commun Biol. 2022;5:903. 458 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 21 Table Legends 459 Table 1. Demographics and characteristics of the enrolled children (5-11 years). 460 Participants eligible for final immunogenicity analysis Participants who had pre- existing antibody Participants who had seroconversion of anti-N IgG Group SV/PF PF/PF SP/SP/PF SV/PF ¥ PF/PF § Total 27 30 56 11 12 Sex, Female/total (%) 12/27 (44.4) 16/30 (53.3) 27/56 (48.2) 6/11 (54.5) 10/12 (83.3) Mean age in year (SD) 7.4 (2.2) 6.2 (1.1) 7.8 (1.6) 6.3 (1.6) 6.8 (1.8) No comorbidity (%) Underlying diseases (%) Allergy Asthma Autism spectrum disorder Obstructive sleep apnea Thalassemia trait 24/27 (88.9) 2/27 (7.4) - - 1/27 (3.7) - 27/30 (90.0) 2/30 (6.7) 1/30 (3.3) - - - 48/56 (85.7) 6/56 (10.7) - 1/56 (1.8) - 1/56 (1.8) 11/11 (100) - - - - - 12/12 (100) - - - - - Time interval between first and second dose Mean (range), days 30.2 (28-41) 57.4 (57-67) 22.9 (21-82) 58.0 (57-67) 58.2 (57-67) Time interval between second dose and blood sampling Mean (range), days 29.3 (27-37) 31.0 (24-46) - 32.0 (27-36) 32.0 (27-36) Time interval between second dose and third dose Mean (range), days - - 61.0 (27-167) - - Time interval between third dose and blood sampling Median (range), days - - 31.0 (29-36) - - ¥ refers to the children who had pre-existing antibody before receipt of 1st dose of CoronaVac. 461 § refers to the children who had seroconversion of anti-N IgG before receipt of 2nd dose of BNT162b 462 463 464 465 466 467 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 22 Figure Legends 468 469 Figure 1. Study flow diagram of enrolled children (5-11 years) for final vaccine 470 immunogenicity analysis. Unvaccinated children were enrolled to receive either 471 heterologous CoronaVac/BNT162b2 (SV/PF) or homologous BNT162b2/BNT162b2 (PF/PF) 472 regimen. In addition, the two-dose BBIBP-CorV-primed children were enrolled to receive the 473 BNT162b2 as a third dose (SP/SP/PF). Sera were collected at pre-dose 1 (V1), pre-dose 2 474 (V2), post-dose 2 (V3), and post-dose 3 (V4), respectively. Children who contracted COVID-475 19 or had evidence of infection as determined by anti-N IgG seroconversion were excluded 476 from the final vaccine immunogenicity analysis. 477 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 23 478 Figure 2. Forest plot showing the percentages of solicited local and systemic adverse events 479 (AEs) and the risk differences with 95% confidence intervals (95% CI) in pediatric 480 participants with any grade AEs across 7 days after vaccination. The AEs of first dose 481 vaccination (A) and second dose vaccination (B) between heterologous SV/PF regimen and 482 homologous PF/PF regimen were compared. SV and PF refer to CoronaVac and BNT162b2, 483 respectively. 484 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 24 485 Figure 3. Binding antibody specific for SARS-CoV-2 in vaccinated children (5-11 years). 486 A) Total immunoglobulin specific to the receptor-binding domain (RBD) (Total RBD Ig) 487 (U/mL) and B) anti-RBD IgG (BAU/mL) of the heterologous CoronaVac/BNT1612b2 488 (referred to as SV/PF), homologous BNT1612b2 (referred to as PF/PF), and third dose of 489 BNT162b2 in two-dose BBIBP-CorV-primed (refers to SP/SP/PF) groups. For both graphs 490 the intervals of reported immunological values are as follows: on the day of the first dose 491 (V1; pre-dose 1), 4 and 8 weeks later for SV/PF and PF/PF groups, respectively (V2; pre-492 dose 2), 4 weeks after two-dose completion in the SV/PF and PF/PF groups and 1-3 months 493 after two-dose completion in the SP/SP/PF group (V3; post-dose 2), and 4 weeks after three-494 dose completion in the SP/SP/PF group (V4; Post-dose 3). Data points are the reciprocals of 495 the individual. The gray area indicates the seronegativity of total RBD Ig (<0.8 U/mL) or 496 anti-RBG IgG (<7.1 BAU/mL). Lines indicate geometric means and bars indicate 95% 497 confidence intervals (95%CI). A pairwise comparisons display geometric mean ratio (GMR) 498 and significant values including p <0.05 (*), p <0.001 (***) and no statistical significance 499 (ns). 500 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 25 501 Figure 4. Binding antibody specific for SARS-CoV-2 classified by immunity status. A) 502 Total immunoglobulin specific to the receptor-binding domain (RBD) (Total RBD Ig) 503 (U/mL) and B) anti-RBD IgG (BAU/mL) of the heterologous CoronaVac/BNT1612b2 504 (referred to as SV/PF) group classified by presence or absence of pre-existing immunity at 505 baseline. C) Total RBD Ig (U/mL) and D) Anti-RBD IgG (BAU/mL) of the homologous 506 BNT1612b2 (referred to as PF/PF) group classified by breakthrough or non-breakthrough 507 infection. For all graphs the intervals of reported immunological values are as follows: on the 508 day of the first dose (V1; pre-dose 1), 4 and 8 weeks later for SV/PF and PF/PF groups, 509 respectively, (V2; pre-dose 2), and 4 weeks after two-dose completion (V3; post-dose 2). 510 Data points are the reciprocals of the individual. The gray area indicates the seronegativity of 511 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint 26 total RBD Ig (<0.8 U/mL) or anti-RBG IgG (<7.1 BAU/mL). Lines indicate geometric 512 means and bars indicate 95% confidence intervals (95%CI). A pairwise comparisons display 513 geometric mean ratio (GMR) and significant values including p <0.01 (**), p <0.001 (***) 514 and no statistical significance (ns). 515 516 Figure 5. Neutralizing activity against the A) wild-type and B) BA.2 Omicron variant at one 517 month after a two-dose or three-dose vaccination. Lines indicate median percent inhibition 518 and the error bar indicates the interquartile range (IQR). The gray area indicates the 519 seronegativity of neutralizing activity of the wild-type (<35%) and BA.2 Omicron variant 520 (<30%). A pairwise comparisons display statistical significance, p <0.001 (***). 521 522 523 524 525 526 527 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted November 10, 2022. ; https://doi.org/10.1101/2022.11.07.22282028doi: medRxiv preprint