key: cord-1006822-w7fc3ohn
authors: Naaber, P.; Tserel, L.; Kangro, K.; Sepp, E.; Jurjenson, V.; Karner, J.; Haljasmagi, L.; Haljasorg, U.; Kuusk, M.; Gerhold, J. M.; Planken, A.; Ustav, M.; Kisand, K.; Peterson, P.
title: Protective antibodies and T cell responses to Omicron variant three months after the booster dose of BNT162b2 vaccine
date: 2022-03-07
journal: nan
DOI: 10.1101/2022.03.04.22271890
sha: d3a81185494332992a58258e74e1656a0c62ca39
doc_id: 1006822
cord_uid: w7fc3ohn

The high number of mutations in the Omicron variant of SARS-CoV-2 cause its immune escape when compared to the earlier variants of concern (VOC). At least three vaccine doses are required for the induction of Omicron neutralizing antibodies and further reducing the risk for hospitalization. However, most of the studies have focused on the immediate response after the booster vaccination while the duration of immune response is less known. We here studied longitudinal serum samples from the vaccinated individuals up to three months after their third dose of the BNT162b2 vaccine for their capacity to produce protective antibodies and T cell responses to Wuhan and Omicron variants. After the second dose, the antibody levels to the unmutated spike protein were significantly decreased at three months, and only 4% of the individuals were able to inhibit Omicron spike interaction compared to 47%, 38%, and 14% of individuals inhibiting wild-type, delta, and beta variants spike protein. Nine months after the second vaccination, the antibody levels were similar to the levels before the first dose and none of the sera inhibited SARS-CoV-2 wild-type or any of the three VOCs. The booster dose remarkably increased antibody levels and their ability to inhibit all variants. Three months after the booster the antibody levels and the inhibition activity were trending lower but still up and not significantly different from their peak values at two weeks after the third dose. Although responsiveness towards mutated spike peptides was lost in less than 20 % of vaccinated individuals, the wild-type spike-specific CD4+ and CD8+ memory T cells were still present at three months after the booster vaccination in the majority of studied individuals. Our data show that two doses of the BNT62b2 vaccine are not sufficient to protect against the Omicron variant, however, the spike-specific antibodies and T cell responses are strongly elicited and well maintained three months after the third vaccination dose.

The Omicron variant of SARS-CoV-2 has caused breakthrough infections worldwide among previously values measured 6 months earlier at 3mA2D. Administration of the third dose (2wA3D) increased these 147 percentages to 100% and 90%. The frequency of S-specific T cells was significantly higher in 2wA3D group 148 among CD4+ (median/IQR: 0.49/0.3 -0.82) compared to 9mA2D (0.18/0.06 -0.38; p<0.0001) and CD8+ T cells 149 (median/IQR: 0.31/0.13 -0.45 vs 0.04/0 -0.24; p<0.0001). At 3mA3D 97% of the vaccinees still harboured S-150 specific CD4+ T cells (median/IQR: 0.34/0.23 -0.61) and 79% had AIM+ CD8+ T cells (median/IQR: 0.11/0.04 151 -0.27) that was not significantly different from the previous time point for CD4+ T cells, but slightly decreased 152 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 As the Omicron variant has significantly fewer fully conserved spike epitopes for CD4+ and CD8+ T cells than 155 the previous VOCs 21 , T cell responses induced by mRNA vaccines coding Wuhan spike, could be also affected. 156

To check this scenario, we performed in parallel T cell stimulation with the peptide pool containing only 157 mutated peptides from Omicron spike (SARS-CoV-2 Prot_S B.1.1.529 Mutation Pool from Miltenyi Biotec) at 158 3mA3D. We found that only 17% and 15% of the vaccinated individuals with AIM+ T cells towards Wuhan 159 spike, had lost responsiveness against the mutated epitopes by CD4+ and CD8+ T cells respectively. 160

Collectively, these results showed that the majority of the vaccinated individuals developed efficient SARS-161

CoV-2 virus-specific memory T cell responses after the second dose, the response was boosted after the third 162 dose, and that both CD4+ and CD8+ memory T cells to the viral antigen persisted at three months after the 163 booster, albeit with slightly lower T cell response against the Omicron variant. 164

Predictors of vaccine response. Altogether 97% of participants reported adverse effects after the third dose. 165

The frequency and severity of side effects after the third dose were similar to those after the second dose 166 (total score median/IQR: 7/3 -12 vs 6/2 -12; p=0.7). The common side effects after the third dose were pain 167 or swelling at the injection site (in 90%), fatigue (69%), myalgia (51%), malaise (47%), headache (47%) and 168 chills (36%), and resembled those after the second dose. 169

We found a significant correlation of severity of side effects after the third and the second dose (r= 0·48, 170 p=0·0003). Thus, the individuals with pronounced side effects after the second dose also tended to have these 171 after the third one. The total score of adverse effects after the third dose significantly correlated with the 172 antibody response to S-RBD (r= 0.53, p=0.001), similar to the effect that we previously reported after the 173 second vaccine dose 20 . 174 175 176 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted March 7, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 dynamics of neutralizing antibodies to Omicron, Beta and Delta VOCs, and original wild-type virus, and studied 179 samples of the vaccinated cohort for their capacity to inhibit spike protein interaction with ACE2. Among the 180 emerged VOCs, the Beta and Omicron variants have a greater ability to escape from neutralizing antibodies 181 and Omicron in particular can escape from the neutralizing antibodies in those who have received two mRNA 182 vaccine doses. 183 Consistent with previous reports 2,8,11 , we found decreased capacity to block Omicron in vaccinated serum 184 samples. This was particularly obvious after the second vaccine dose when less than half of the vaccinated 185 sera were able to inhibit the reaction, whereas the Beta variant was blocked by most (80%), and wild-type 186 virus and Delta VOC by more than 95% of the samples. However, the blocking efficiency declined over time 187

and at 9 months after the second dose, all serum samples had lost their blocking activity. Together with the 188 significant waning of the S-RBD binding, this result suggests a remarkable decline of protective antibodies at 189 9 months after the initial two BNT62b2 vaccine doses. However, two weeks after the booster dose, the serum 190 samples regained their blocking activity, which was in line with the elevated antibody levels to Given the weaker serum activity generated against Omicron's spike protein, it was important to determine 192 whether the blocking antibody response stays durable in individuals who received the third dose of mRNA 193 vaccine. For this, we measured the antibodies to S-RBD and VOCs three months after the third dose. After 194 their peak values immediately after the third dose, the antibodies to spike RBD were lower three months later. 195

The decline was not significant, however, and was in their median values almost equal to the levels at 1.5 196 months after the second dose (median 12752 AU/mL, IQR 8209-7538 for 6wA2D vs median 12729 AU/mL, IQR 197 628-19883 for 3mA3D). The result indicates that over time the antibodies tend to decline after the booster 198 dose albeit more slowly than after the second dose. Interestingly the antibody blocking efficiency after the 199 third dose remained on the same level for wild-type but was slightly declining with VOCs. Nevertheless, 200 individual serum samples retained a strong correlation between S-RBD levels and the blocking capacity of wild 201 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 type and the variants, including Omicron. This is in contrast to the inhibition activity that we saw after the 202 second dose, where protection against Omicron did not correlate with S-RBD IgG antibody levels and was 203 discordant from the inhibition of wild-type, Beta, and Delta VOCs. The additional booster dose may elicit 204

Omicron-neutralizing memory B cell subsets and expand the repertoire of the broadly neutralizing antibodies 205 22 . In this case, it is plausible that the Omicron-neutralizing clones were acquired as a result of affinity 206 maturation of those with neutralizing activity to previously recognized wild-type spike vaccine antigen. 207

The waning of antibody levels over time and the emergence of immune escape mutants have turned the 208 focus towards antigen-specific T cells as important players in preventing serious COVID-19 23 . The durability 209 of cellular response is one of the key parameters to consider in planning future booster vaccination 210 strategies. mRNA vaccines have shown potent induction of Spike-specific CD4+ and CD8+ responses by the 211 first two vaccine doses but significant contraction of the number of circulating antigen-specific T cells during 212 the first 3 months that stabilize thereafter 19, 24 . This was evident also in our study: the percentages of AIM+ 213 CD4+ and CD8+ T cells were stable between 3 and 9 months after the second dose. However, we 214 demonstrated here that the booster dose of BNT162b2 increased the percentage of both subtypes of Spike-215 reactive T cells. This is in line with recent reports that have measured immune responses shortly after the 216 booster dose 25 . Importantly, we found that Spike-specific CD4+ T cell percentages were still elevated 3 217 months after the third dose of BNT162b2, and AIM+ CD8+ T cells showed only a moderate decline in 218 comparison to 2wA3D time point. 219

Another important concern about the mRNA vaccine induced T cell response is its cross-reactivity towards 220

VOCs. The Omicron variant has multiple mutations in its spike protein and in comparison to previous VOCs is 221 associated with the fewest fully conserved spike epitopes (72% for CD4+ and 86% for CD8+) 21 . This means 222 that nevertheless the majority of T cell epitopes are unaffected by the Omicron spike mutations. Indeed, 223 several studies have already confirmed that T-cell reactivity to the Omicron variant is rather well preserved in 224 the majority of vaccinated individuals 16, 17, 21, 25, 26 . At 3mA3D we stimulated PBMC samples in parallel with 225 overlapping peptide pools spanning the entire spike protein sequence of the Wuhan strain or a selected pool 226 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022.

of peptides mutated in the Omicron spike. It has been predicted that approximately 70% of the mutated 227

Omicron sequences retain HLA class I binding capacity 21 , e.g. due to conservative substitutions or changes 228 that don't impact HLA binding. Our findings are in line with these estimations as only less than 20% of the 229 studied individuals had lost responsiveness towards the mutation pool. 230

In sum, our results suggest increased durability of humoral and cellular immune responses towards SARS-CoV-231 2 spike protein after the booster dose of BNT62b2 vaccine and confirm the notion of increase in the breadth 232 of antibody response and preservation of T cell responses towards the Omicron variant. 233

Recruitment, sample, and data collection. The study group and the blood sample collection procedures were 235 reported in our previous study 20 . Briefly, SYNLAB Estonia employees volunteering to be vaccinated (2 to 3 236 doses) with COVID-19 mRNA BNT162b2 (Comirnaty Pfizer-BioNTech) vaccine were invited to participate in the 237 study. Starting January 2021, the first two vaccine doses were given three weeks apart, and the third dose was 238 administered nine months after the second dose. The samples were taken before the first dose (B1D), before 239 the second dose (B2D), one week after the second dose (1wA2D), six weeks after the second dose (6wA2D), 240 three months after the second dose (3mA2D), 6 months after the second dose (6mA2D), 9 months after the 241 second dose (9mA2D), two weeks after the third dose (2wA3D) and 3 months after the third dose (3mA3D). 242

The information about the presence of side effects after the third dose was collected as reported previously 20 . 243

The study has been approved by the Research Ethics Committee of the University of Tartu on February 15, 244 2021 (No 335/T-21). Participants signed informed consent before the recruitment into the study. The study 245 was performed in accordance with Helsinki Declaration and followed Good Laboratory Practice. 246

Description of study group is presented previously 20 . We excluded the participants diagnosed with COVID-19 247 before or during the study. The number of participants of each time point are presented in Table 1 . 248 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101/2022.03.04.22271890 doi: medRxiv preprint immunoassay (CLIA) on ARCHITECT i2000SR analyzer (Abbott Laboratories) as described previously 20 . 251 ACE2-Spike interaction blocking assay. The serum capacity to block the angiotensin-converting enzyme 2 252 (ACE2) receptor interaction with SARS-CoV-2 trimeric S protein receptor-binding domain (RBD) was tested 253 using IVD-CE SARS-CoV-2 Neutralizing Antibody ELISA kit (Icosagen) as described previously 20 capacities to block the interaction of ACE2 receptor and Spike protein of wild type, Beta, Delta and Omicron 307 variants before the first dose (B1D), at 1 week (1wA2D), 3 months (3mA2D) and 9 months after the second 308 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 dose, and 2 weeks (2wA3D) and 3 months (3mA3D) after the third dose of vaccinations. The dotted line 309 indicates the relative OD value of 0.75, which is a threshold for sufficient blocking of ACE2 binding. The 310 matched data analysis was performed with the Friedman test with Dunn's multiple testing correction; p-311 values >0.0001 are reported as exact numbers. The percentage of samples that were able to reach the 312 threshold of blocking activity is shown below each graph. 313 dots correspond to the percentage of T cells that responded to peptide pool consisting selectively from 332 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Omicron mutated sequences. The data were analysed with the Kruskal-Wallis test with Dunn's multiple 333 testing correction. The percentage of samples that had spike responsive T cells is shown below each graph. 334 335 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101/2022.03.04.22271890 doi: medRxiv preprint B1D -before the first dose, B2D -before the second dose, 1wA2D -one week after the second dose, 6wA2D -337 six weeks after the second dose, 3mA2D -three months after the second dose, 6mA2D -6 months after the 338 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 second dose, 9mA2D -9 months after the second dose, 2wA3D -two weeks after the third dose, 3mA3D -3 339 months after the third dose 340 341 . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 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 March 7, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 7, 2022. ; https://doi.org/10.1101/2022.03.04.22271890 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted March 7, 2022. ; https://doi.org/10.1101/2022.03.04.22271890 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted March 7, 2022. 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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mRNA-based COVID-19 vaccine boosters 392 induce neutralizing immunity against SARS-CoV-2 Omicron variant

Activity of convalescent and vaccine serum against 396 SARS-CoV-2 Omicron

SARS-CoV-2 variants of concern and variants under investigation in England (B.1.1.529)

Three exposures to the spike protein of SARS-CoV-2 by either 405 infection or vaccination elicit superior neutralizing immunity to all variants of concern

mRNA vaccines induce durable immune 422 memory to SARS-CoV-2 and variants of concern