The humoral and cellular immune response of a third dose adenoviral-based vectored vaccine after a single or 2 shots of inactivated COVID-19 vaccine in healthy adults

The coronavirus pandemic is a severe infectious respiratory disease which caused massive loss worldwide. Thailand was affected by the wild-type, and the Delta variant started in mid-2021. Due to the shortage of effective vaccines, the ministry of public health of Thailand suggested the heterologous vaccine scheme, which comprises inactivated COVID-19 vaccine (CoronaVac) and an adenoviral-based vectored vaccine (ChAdOx1). However, data on the humoral and cellular immune responses of the single or 2 shots of inactivated vaccine followed by the adenoviral-based vaccine are very limited. In this current study, the sera from participants who received either single or 2 shots of CoronaVac followed by the ChAdOx1 vaccine were evaluated for SARS-CoV-2 spike receptor-binding-domain (RBD) IgG. The cytokine level was also assessed using Luminex immunoassay. The PBMC were collected to evaluate spike-specific T-cell and B-cell responses. Participants who received 2 shots of CoronaVac followed by ChAdOx1 possessed significantly (P<0.0001) higher levels of spike RBD-specific IgG. They also exhibited a higher level of CD4+T-cell and IFN-gamma than those who received only 1 shot of CoronaVac followed by the ChAdOx1 vaccine. The volunteers who received two shots of CoronaVac followed by ChAdOx1 had a significantly (p<0.01) higher marginal B-cell response against wild-type SARS-CoV-2 S peptides than those who received only one shot of CoronaVac followed by ChAdOx1. Surprisingly, the class switch B-cell response to Delta variant SARS-CoV-2 S peptides of the volunteers who received 1 shot of CoronaVac followed by ChAdOx1 was significantly (p<0.01) higher than those who received 2 shots of CoronaVac. However, participants who received only a single shot of inactivated vaccine followed by an adenoviral-based vectored vaccine possessed a higher level of TNF-alpha and IL-6. This study indicated that boosting the ChAdOx1 as a third dose after completing 2 shots of CoronaVac induced strong humoral and cellular immune responses.

shots of CoronaVac followed by the ChAdOx1 vaccine were evaluated for SARS-CoV-2 22 spike receptor-binding-domain (RBD) IgG. The cytokine level was also assessed using 23 Luminex immunoassay. The PBMC were collected to evaluate spike-specific T-cell and 24 B-cell responses. Participants who received 2 shots of CoronaVac followed by ChAdOx1 25 possessed significantly (P<0.0001) higher levels of spike RBD-specific IgG. They also 26 exhibited a higher level of CD4+T-cell and IFN-gamma than those who received only 1 27 shot of CoronaVac followed by the ChAdOx1 vaccine. The volunteers who received two 28 shots of CoronaVac followed by ChAdOx1 had a significantly (p<0.01) higher marginal B-29 cell response against wild-type SARS-CoV-2 S peptides than those who received only 30 one shot of CoronaVac followed by ChAdOx1. Surprisingly, the class switch B-cell 31 response to Delta variant SARS-CoV-2 S peptides of the volunteers who received 1 shot 32 of CoronaVac followed by ChAdOx1 was significantly (p<0.01) higher than those who 33 received 2 shots of CoronaVac. However, participants who received only a single shot of 34 inactivated vaccine followed by an adenoviral-based vectored vaccine possessed a 35 higher level of TNF-alpha and IL-6. This study indicated that boosting the ChAdOx1 as a 36 6. T-cell restimulation with SAR-CoV2 peptide. 121 Cryotubes containing frozen PBMCs were placed at 37°C in a bath until thawed cells. 122 Cells were pipetted into 15 mL tubes and added 3 ml of warm RPMI-1640 media. Then, 123 cells were washed 2 times by centrifugation at 350×g for 5 min. After that, cells were 124 resuspended in complete RPMI-1640 supplemented with 5% human platelet lysate and 125 1% streptomycin-penicillin. Cell viability was checked with Trypan Blue. For stimulation, 126 2.5x10 5 cells were diluted with an equal volume of each of the diluted spike protein 127 peptide pools. The wild-type peptide pools were prepared in the complete RPMI medium 128 at the final concentration of 2 µg/mL.

IFN-gamma and IL-2 ELISpot assay 148
The ELISpot assay was performed using PVDF membrane 96-well microplates (R&D 149 Systems) following the manufacturer's guidelines. The isolated PBMCs were thawed and 150 rested in RPMI media for 1 hour. After that, 200 µl of 2 x 10 6 PBMC/mL cell suspension 151 (2.5 x 10 5 PBMC per well) were stimulated for 16-18 hours with either RPMI media 152 (negative control), Dyna bead (positive control), each SAR-CoV-2 peptides mixes (2 153 µg/mL of either wild-type or Delta variant SAR CoV-2 S peptides. Each sample was 154 performed in duplicate and then incubated at 37°C with 5% CO 2 . Cell viability of more 155 than 80% was used in this assay. 156 The number of SARS-CoV-2 specific IFN-gamma and IL-2-secreted T-cells per 2.5 x 157 10 5 PBMCs were determined using ImmunoSpot S6 Ultimate Analyzer (CTL, Cleveland, 158 USA) and presented as spot forming unit per 2.5 hundred thousand PBMCs 159 (SFU/2.5x10 5 ).

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To evaluate the circulating IgG-specific RBD of the SARS-CoV2 antibody, the 187 serums from volunteers were collected 4 weeks after immunization with ChAdOx1 and 188 measured the antibody level using CMIA. The average IgG antibody of the participants 189 who received either 1 or 2 shots of CoronaVac followed by the ChAdOx1 vaccine was 190 5,077 AU/mL and 14,008 AU/mL, respectively. Interestingly, the results showed that the 191 level of IgG-specific RBD of SARS-CoV2 antibody of participants who received 2 shots of 192 CoronaVac followed by ChAdOx1 vaccine was significant (P<0.0001) higher than 193 participants who received only 1 dose of CoronaVac before ChAdOx1 vaccination as 194 seen in Fig 1.  195

T-cell response to spike protein-peptide pools in wild-type
197 strains and variants of concern.
198 The human IFN-gamma/IL-2 Dual-Color ELISpot Kits evaluated the spike-specific 199 T-cell response. The responses of T-cells from volunteers who received 1 CoronaVac 200 followed by ChAdOx1 Vaccine (n=12) were compared to the volunteers who received 2 201 shots of CoronaVac followed by ChAdOx1 Vaccine (n=12). There was no significant 202 difference in IFN-gamma and IL-2 levels against wild-type and VOC following the 203 immunization with the 2nd or third dose with ChAdOx1, as shown in The immunostaining analysis of CD4+T-cell, CD8+T-cell, B-cell, and NK cell from 208 the whole blood samples were analyzed in volunteers who received 1 CoronaVac 209 followed by ChAdOx1 (n = 30) with the volunteers who received 2 doses of CoronaVac 210 followed by ChAdOx1 (n = 20). The amount of CD4+T-cell from volunteers who received 211 2 doses of CoronaVac followed by ChAdOx1 Vaccine was significantly (p<0.0001) higher 212 than the volunteers who received only 1 shot of CoronaVac followed by ChAdOx1 213 Vaccine. On the other hand, the level of CD4+T-cell from volunteers who received 2 214 doses of CoronaVac followed by ChAdOx1 Vaccine was significantly (p<0.0001) lower 215 than the volunteers who received only 1 dose of CoronaVac followed by ChAdOx1 216 Vaccine. There was no significant difference in the amount of B-cell and NK cells 217 between both groups, as shown in

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This experiment tested cytokine quantification of Th1/Th2 using 6 cytokine types, 221 including IFN-gamma, TNF-alpha, IL-4, IL-6, and IL-12p70, in response to T-cell 222 stimulated with pools of Spike peptides derived from SARS-CoV-2 wild type and Delta 223 variants of concern. The cytokine quantification was compared in volunteers who 224 received only 1 shot of CoronaVac vaccine followed by ChAdOx1 Vaccine (n=11) with 225 the volunteers who received 2 doses of CoronaVac vaccine followed by ChAdOx1 226 vaccine (n=11). The result was statistically significant in IL-6 and IL-12p70 (Fig 4). was significantly (p<0.01) higher than in the group that received 2 shots of CoronaVac 235 followed by ChAdOx1. When stimulated with Delta variant SARS-CoV-2 S peptides, 236 volunteers who received 1 dose of CoronaVac had significantly (p<0.01) higher TNF-237 alpha expression levels than those who received only 2 shots of CoronaVac followed by 238 ChAdOx1. Surprisingly, the expression of INF-gamma in volunteers who received two 239 doses of CoronaVac followed by ChAdOx1 was significantly (p<0.001) increased in 240 response to wild-type SARS-CoV-2 S peptides. The results were similar to those 241 stimulated with Delta SARS-CoV-2 S peptides. However, there was no significant 242 difference in intracellular IL-2 expression between 2 groups of people who received different types of vaccines stimulated with wild-type and Delta SARS-CoV-2 S peptides, 244 as shown in Fig 5.

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B-Cells responses were observed using stimulated PBMC with peptide variants as 246 described previously. As shown in Fig 6, the marginal B-cell response to wild-type 247 SARS-CoV-2 S peptides of the volunteers who received 2 shots of CoronaVac followed 248 by ChAdOx1 was significantly (p<0.01) higher than those who received only 1 shot of 249 CoronaVac followed by ChAdOx1. The responses of transitional B-cell and class switch 250 B-cell against wild-type SARS-CoV-2 S peptides were higher in volunteers who received 251 only one shot of CoronaVac followed by ChAdOx1, but this was not statistically 252 significant. Surprisingly, when stimulated with Delta variant SARS-CoV-2 S peptides, the 253 class switch B-cell of the volunteers who received 2 shots of CoronaVac followed by 254 ChAdOx1 was significantly (p<0.01) lower than those who received only 1 shot of 255 CoronaVac followed by ChAdOx1. The SAR-CoV-2 pandemic crisis is still ongoing in several parts of the world. The 262 mass vaccine is one of the best strategies to encounter this problem to create herd 263 immunity against SAR-CoV-2 infection [12]. The heterologous vaccination is currently 264 considered in developing countries due to the shortage of vaccines. In this study, we 265 investigated the IgG antibody response against receptor-binding domain (RBD) of S1 266 spike protein of SARS-CoV-2 and cellular immune response following the 1 or 2 doses of 267 inactivated SAR-CoV-2 vaccine CoronaVac followed by the adenoviral-based vectored 268 vaccine (ChAdOx1 AZD1222). We demonstrated the antibody level against the SAR-CoV-2 induced by either 1 or 2 shots of CoronaVac followed by ChAdOx1 at 4 weeks 270 after immunization. The antibody response induced by 2 doses of CoronaVac followed by 271 ChAdOx1 was greater up to almost 3-fold compared to only 1 dose of CoronaVac 272 followed by ChAdOx1 vaccination. These data suggested that a third dose of the COVID-273 19 vaccine elicits greater immunogenicity than 2 doses. Our results were similar to 274 previous research, which indicated that boosting ChAdOx1 after completing 2 shots of 275 CoronaVac elicited a higher number of RBD-specific IgG than those without the boosting 276 [5]. Several studies found that three doses of inactivated or mRNA vaccine resulted in a 277 more significant immunogenic response than two doses [13,14]. ChAOx1 demonstrated higher T-cell intracellular TNF-alpha against wild-type SAR CoV-295 2 and Delta variant than those who received 2 doses of CoronaVac followed by ChAOx1. 296 The previous study showed similar results: boosting with CoronaVac induced the 297 activation of CD4+T-cell and secretion of IFN-gamma against wild-type SAR-CoV-2, 298 Delta, and Omicron variants [17]. Goa and colleagues demonstrated that CD4+T-cell 299 response against wild-type strain is cross-reactive against Omicron strain in participants 300 who received BNT162b2 mRNA vaccine [18]. 301 The cytokines secretion 302 respectively) in response to T-cell stimulated with pools of Spike peptides derived from 303 SARS-CoV-2 wild type and Delta variant were also analyzed by Luminex immunoassay. 304 Surprisingly, IL-6 and IL-12p70 were significantly higher in participants immunized by a 305 single shot of CoronaVac followed by ChAdOx1 compared to those who were immunized 306 with 2 doses of CoronaVac 4 weeks after vaccination. Many types of vaccines increase 307 IL-6, such as influenza and the Bacillus Calmette-Guérin vaccine [19]. For the influenza 308 vaccine, the natural killer cell produces a high level of interferon-gamma, which 309 stimulates CD11b+ dendritic cells to release IL-6 [20]. A recent study has reported the 310 response of NK-cell followed by ChAdOx1, which may explain and support the increase 311 of IL-6 in this current study [21]. The current study has some limitations. We included a small number of 332 participants who received either 1 or 2 doses of inactivated vaccine followed by 1 shot of 333 adenoviral-based vaccine. Second, we did not measure the baseline IgG antibody 334 response against the receptor-binding domain (RBD) of S1 spike protein SARS-CoV-2 335 before and after the first vaccination. In addition, the samples for T-cell and B-cell 336 responses were low due to this study's limited time and budget. Furthermore, we only 337 included Wild type SAR CoV-2 and Delta variants because they were the dominant 338 strains during the study. The investigation of vaccine protection against emerging 339 variants should be considered. 340 In conclusion, boosting the ChAdOx1 as a third dose after completing 2 doses of 341