key: cord-0775870-nhwsj99h authors: Assawakosri, S.; Kanokudom, S.; Suntronwong, N.; Auphimai, C.; Nilyanimit, P.; Vichaiwattana, P.; Thongmee, T.; Duangchinda, T.; Chantima, W.; Pakchotanon, P.; Srimuan, D.; Thatsanatorn, T.; Klinfueng, S.; Yorsaeng, R.; Sudhinaraset, N.; Wanlapakorn, N.; Mongkolsapaya, J.; Honsawek, S.; Poovorawan, Y. title: Neutralizing Activities against the Omicron Variant after a Heterologous Booster in Healthy Adults Receiving Two Doses of CoronaVac Vaccination date: 2022-01-29 journal: nan DOI: 10.1101/2022.01.28.22269986 sha: 92e78cf4a5755f5be1668fbbea096480b6329463 doc_id: 775870 cord_uid: nhwsj99h Background. The use of an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine (CoronaVac) against SARS-CoV-2 is implemented worldwide. However, waning immunity and breakthrough infections have been observed. Therefore, we hypothesized that the heterologous booster might improve the protection against the delta and omicron variants. Methods. A total of 224 individuals who completed the two-dose CoronaVac for six months were included. We studied reactogenicity and immunogenicity following a heterologous booster with the inactivated vaccine (BBIBP), the viral vector vaccine (AZD1222), and the mRNA vaccine (both BNT162B2 and mRNA-1273). We also determined immunogenicity at 3- and 6-months boosting intervals. Results. The solicited adverse events (AEs) were mild to moderate and well-tolerated. Total RBD immunoglobulin (Ig), anti-RBD IgG, focus reduction neutralization test (FRNT50) against delta and omicron variants, and T cell response were highest in the mRNA-1273 group followed by the BNT162b2, AZD1222 and BBIBP groups, respectively. We also witnessed a higher total Ig anti-RBD in the long-interval than in the short-interval groups. Conclusions. All four booster vaccines significantly increased binding and NAbs in individuals immunized with two doses of CoronaVac. The present evidence may benefit vaccine strategies development to thwart variants of concern, including the omicron variant. Keywords. COVID-19; Third dose; heterologous booster; omicron; mRNA-1273; BNT162b2; AZD1222; NAbs; T cells. As of January 2022, coronavirus disease 2019 (COVID-19) has spread across 200 56 countries with 318 million confirmed cases, and over 5.5 million deaths worldwide [1] . 57 Vaccination is considered one of the best tools to prevent the spread of COVID-19. Several 58 Participants were separated into four groups by conveniently sampling of 50-60 125 participants each and were assigned to receive one dose of the following vaccines: Participants were observed under the supervision of medical professionals after 131 vaccination to prevent acute and severe side effects. AEs were recorded, including injection site 132 pain, induration, redness, fever, headache, muscle pain, and others. The participants then 133 received an AE record form for further self-monitoring within 7 days after vaccination. 134 Peripheral venous blood samples were collected to measure the immune response, 136 including the total Ig against RBD, IgG against RBD, and IgG against nucleocapsid (N) as 137 previously described [24] . 138 To measure NAbs against the SARS-CoV-2 variants, the cPass TM SARS-CoV-2 140 surrogate virus neutralization test Kit was used (GenScript Biotech, New Jersey, USA), 141 consisting of recombinant RBD from alpha, beta, delta, and omicron variants as previously 142 described [24] . 143 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 29, 2022. ; https://doi.org/10.1101/2022.01.28.22269986 doi: medRxiv preprint The neutralization ability of antibodies in vaccinated serum was measured using the 145 FRNT50. Briefly, serum was heated for 30 min at 56°C and then diluted serially (1:10-1:7,290). 146 The serum was mixed with the SARS-CoV-2 variant (delta and omicron) and incubated for 1 147 hour at 37°C. The mixtures were then transferred to a 96-well plate containing confluent Vero 148 cell monolayers and incubated for 2 hours, then 1.5% semi-solid carboxymethyl cellulose was 149 added to limit virus diffusion and cells were incubated further at 37°C, 5% CO2. Next, Vero 150 cells were fixed with 3.7% formaldehyde/PBS and permeabilized with 2% Triton X-100. Anti-151 NP human mAb was then added, followed by peroxidase-conjugated goat anti-human IgG. 152 Finally, the infected cells were visualized after adding TrueBlue Peroxidase Substrate. The foci 153 of virus infected cells were counted by CTL ImmunoSpot S6 analyzer. The percentage of focus 154 reduction was calculated and IC50 was determined by PROBIT software. The detection limit is 155 1:20 [29] . 156 Interferon-gamma release assay. 157 QuantiFERON (QFN) SARS-CoV-2 RUO (Qiagen, Hilden, Germany) interferon-158 gamma (IFN-γ) release assay was performed to measure T cell response as previously described 159 [24]. The seropositivity rate was calculated using IFN-γ levels from the stimulated tube minus 160 the negative control tube. An IFN-γ level above 0.15 IU/mL was defined as positive. 161 All statistical analyses were conducted using GraphPad Prism v9.0 (GraphPad Software, 163 San Diego, CA, USA), the Statistical package for the social sciences (SPSS) v.22 (SPSS Inc., 164 Chicago, IL, USA), and R v4.1.2. Software (R Foundation for Statistical Computing, Vienna, 165 Austria). The normality of the data was tested using the Kolmogorov-Smirnov test. Statistical 166 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 1A-1D ). The analysis of AEs revealed that pain at the injection site, 193 swelling, redness, myalgia, joint pain, and chilling in individuals who received BBIBP were 194 significantly less frequent than the other vaccine platforms (Supplementary Figure 2A- At the baseline visit, all participants presented seropositivity for total RBD Ig and anti-200 RBD IgG. There were no significant differences in the geometric mean titer (GMT) of the total 201 RBD Ig among all groups at baseline. Interestingly, the COVID-19 vaccines administered as 202 the booster dose significantly elicited higher total RBD Ig levels at 14 days post-vaccination, 203 with GMTs of 1740, 12260, 31793, and 51979 U/mL (p<0.001). Subsequently, the total RBD 204 Ig was slightly reduced to 1295, 12111, 21053, and 33519 U/mL (p<0.001) in the BBIBP, 205 AZD1222, BNT162b2, and mRNA-1273 groups, at 28 days post-vaccination, respectively 206 ( Figure 2A ). Comparable trends were observed with anti-RBD IgG levels ( Figure 2B ). 207 Following the administration of the third booster, the mRNA-1273-boosted individuals 208 possessed the highest total Ig and anti-RBD IgG levels. However, there was no significant 209 difference in the responses between the two mRNA vaccines. After boosting with BBIBP, the 210 total RBD Ig and anti-RBD IgG levels were significantly lower than the levels achieved by 211 viral-vector and mRNA groups (Supplementary Table 2) . Anti-N IgG levels were also 212 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 29, 2022. ; https://doi.org/10.1101/2022.01.28.22269986 doi: medRxiv preprint measured at three different time points, as shown in Figure 2C . At baseline, the median level 213 (IQR) of anti-N IgG was 0.13 (0.07-0.31). The results were comparable among the four 214 groups, which could be interpreted that none of 224 participants had been infected by SARS-215 CoV-2. After the booster dose, the median anti-N IgG level increased significantly to 2.78 216 (1.84-5.30) in those who received BBIBP (p<0.001). On the contrary, no change in anti-N 217 IgG levels was observed after the AZD1222 or mRNA vaccination because only the 218 inactivated vaccine contained the SARS-CoV-2 N protein in the formulation ( Figure 2C ). 219 The surrogate virus neutralization assay was performed to compare the functionality of 221 NAbs specific to the wild-type and SARS-CoV-2 variant strains, including alpha, beta, delta, 222 and omicron. Overall, the seropositivity rate of individuals who received two doses of 223 The NAbs titers in sera from participants were determined using a live virus neutralization 234 assay, FRNT50. For all participants, undetectable (titer<20) NAbs at baseline were observed in 235 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 29, 2022. Total interferon-gamma response following immunization 243 In addition to the serological responses, we evaluated the presence of T cell responses by 244 measuring the total IFN-γ level using the QFN SARS-CoV-2-ELISA assay. The seropositivity 245 rates for the IFN-γ CD4+/IFN-γ CD4+ and CD8+ levels observed in most participants after the 246 third booster with 86%/93% for AZD1222, 96%/100% for BNT162b2, and 90%/93% for 247 mRNA-1273 ( Figure 5A and 5B). In contrast, the seropositivity rate was 43%/47% for BBIBP, 248 suggesting a lower T-cell response was elicited. Consistent with the total RBD Ig levels, the T-249 cell-related IFN-γ response was significantly elevated at 14 days compared to baseline and 250 gradually declined at 28 days after vaccination (Supplementary Table 4 ). Our result suggests that 251 a single booster dose with AZD1222, mRNA-1273, or BNT162b2 could rapidly induce a T-cell 252 response in individuals vaccinated with CoronaVac. 253 We compared GMT levels of total RBD Ig between short-and long-interval groups. As 255 shown in Figure 6A -6C, the long-interval group elicited higher total RBD Ig than the short-256 interval group at 14 days post-booster vaccination. Compared to the pre-booster level, the 257 BBIBP vaccine significantly induced a 30.2-fold increase in total RBD Ig titer in the short-258 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 29, 2022. indicating that the heterologous third booster dose has a good safety profile. 279 Consistent with a previous study of CoronaVac, we observed that the baseline total IgG 280 levels were reduced at six months after the primary series of CoronaVac [8]. The data indicated 281 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 29, 2022. ; https://doi.org/10.1101/2022.01.28.22269986 doi: medRxiv preprint that the immunogenicity from CoronaVac is waning over time. As expected, the heterologous 282 booster with BBIBP, AZD1222, BNT162b2, or mRNA-1273 significantly elicited an immune 283 response at 14 days compared to the baseline level. Our data suggested that a single-shot booster 284 after a two-dose of CoronaVac may be sufficient to restore the B cell memory response [30] . 285 Regarding the total Ig RBD and anti-RBD IgG response, some differences were observed among 286 the four boosters. Following the administration of the inactivated vaccine, there was a lower 287 antibody response post-booster similar to that observed in a previous study from Brazil [31] . Our findings showed that the reduction of NAbs levels against omicron was much lower 301 relative to delta variants. Consistent with a previous study, the neutralizing titer against the 302 omicron variant was 4.9-fold less than that for the delta variant with the BBIBP-CorV/ZF2001 303 heterologous booster [38] and was 24.5-fold lower than that for the delta variant in sera from 304 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 29, 2022. Moreover, a long interval between the primary and the boosters could significantly 317 stimulate a better immune response than a shorter interval. A similar previous study using the 318 extended interval also suggested that antibody responses after receiving the third booster 319 AZD1222 and BNT162b2 vaccine were higher in extended interval vaccinations [45] . In 320 summary, these data indicated that a longer interval might allow more time for enhancing 321 immune memory responses. 322 An early estimate of vaccine effectiveness (VE) in Chile, the study found that the 323 adjusted VE against symptomatic, hospitalization, and COVID-19 related deaths due to the delta 324 variant was increased after a heterologous booster shot with a viral-vectored and mRNA vaccine 325 following a complete primary series of CoronaVac vaccines [46] . However, there were limited 326 data in that study on the VE against omicron infection following a heterologous booster. Our 327 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 29, 2022. ; https://doi.org/10.1101/2022.01.28.22269986 doi: medRxiv preprint 16 results highlight that the viral vector and mRNA vaccines, but not inactivated vaccines, were 328 promising booster vaccines that could elicit a strong immune response to prevent the omicron 329 variant infection. 330 This study had some limitations. First, the study did not examine the phenotype of CD4+ 331 and CD 8+ T cells. Moreover, the total IFN-γ level and the surrogate virus neutralization test 332 reached the upper limit of detection assay in some samples. Thus, we could not estimate the 333 exact cellular immune response following the third booster vaccine. Further study using more 334 advanced techniques included activation-induced marker assays may resolve this issue. 335 Moreover, long-term studies are needed to determine the durability of the booster vaccination. 336 In summary, we report a robust total RBD Ig response and acceptable safety profile after 337 implementing heterologous third booster vaccines. However, neutralizing activities against 338 omicron variants and the T-cell response were reduced in BBIBP-boosted individuals compared 339 to AZD1222, BNT162b2, and mRNA 1273-boosted individuals. 340 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The interval between the First and Third visits All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 29, 2022. ; https://doi.org/10.1101/2022.01.28.22269986 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 29, 2022. ; https://doi.org/10.1101/2022.01.28.22269986 doi: medRxiv preprint Figure 6 . Compare the total RBD Ig (U/mL) between the short interval (triangle) and the 545 long interval (circle). The total RBD Ig (U/mL) at 14 days following the third booster of 546 inactivated vaccine, BBIBP (Panel A), the viral-vector vaccine, AZD1222 (Panel B), or the 547 mRNA vaccine, and BNT162b2 (Panel C). ns indicates no significant difference; *, p<0.05, **, 548 p<0.01, ***, p<0.001 (***). 549 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 29, 2022. ; https://doi.org/10.1101/2022.01.28.22269986 doi: medRxiv preprint World Health Organization. WHO Coronavirus (COVID-19) Dashboard Progress of the COVID-19 365 vaccine effort: viruses, vaccines and variants versus efficacy, effectiveness and escape China's COVID vaccines have been crucial -now immunity is waning Waning Immunity after the BNT162b2 Vaccine in Israel Emerging SARS-CoV-2 variants of concern and potential 388 intervention approaches Structural and 390 functional insights into the spike protein mutations of emerging SARS-CoV-2 variants The Global Epidemic of the SARS-CoV-2 delta variant, 393 key spike mutations and immune escape Delta spike P681R mutation enhances SARS-CoV-2 395 fitness over Alpha variant. bioRxiv CoronaVac induces lower 398 neutralising activity against variants of concern than natural infection Reduced neutralization of SARS-CoV-2 B.1.617 401 by vaccine and convalescent serum Omicron escapes the majority of existing SARS-CoV-2 403 neutralizing antibodies Omicron (B. 1.1. 529) variant of concern and its global perspective All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted January 29, 2022. and variants of concern (Alpha and Delta) in infected and vaccinated individuals. Cell (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. swelling and redness were graded by measuring the diameter area as mild (<5 cm), moderate (5 502 to <10 cm), and severe (≥10 cm). Fever was graded as mild (38°C to <38.5°C), moderate 503 (38.5°C to <39°C), and severe (≥39°C). The other events were graded as mild (no limitation on 504 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted January 29, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted January 29, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted January 29, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted January 29, 2022. ; https://doi.org/10.1101/2022.01.28.22269986 doi: medRxiv preprint