key: cord-0721096-dsrik76v
authors: Seki, Yohei; Yoshihara, Yasuo; Nojima, Kiyoko; Momose, Haruka; Fukushi, Shuetsu; Moriyama, Saya; Wagatsuma, Ayumi; Numata, Narumi; Sasaki, Kyohei; Kuzuoka, Tomoyo; Yato, Yoshiyuki; Takahashi, Yoshimasa; Maeda, Ken; Suzuki, Tadaki; Mizukami, Takuo; Hamaguchi, Isao
title: Safety and immunogenicity of the Pfizer/BioNTech SARS-CoV-2 mRNA third booster vaccine dose against the SARS-CoV-2 BA.1 and BA.2 Omicron variants
date: 2022-04-26
journal: Med (N Y)
DOI: 10.1016/j.medj.2022.04.013
sha: d7781359498f60f382fc8669845edc35e9ed3b18
doc_id: 721096
cord_uid: dsrik76v

Background The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified in Japan in November 2021. This variant contains up to 36 mutations in the spike protein, the target of neutralizing antibodies, and can escape vaccine-induced immunity. A booster vaccination campaign began with healthcare workers and high-risk groups. Safety and immunogenicity of the three-dose vaccination against Omicron remain unknown. Methods A total of 272 healthcare workers were initially evaluated for long-term vaccine safety and immunogenicity. We further established a vaccinee panel to evaluate the safety and immunogenicity against variants of concern (VOCs), including the Omicron variants, using a live virus microneutralization assay. Findings Two-dose vaccination induced robust anti-spike antibodies and neutralization titers (NTs) against the ancestral strain WK-521, whereas NTs against VOCs were significantly lower. Within 93–247 days of the second vaccine dose, NTs against Omicron were completely abolished in up to 80% of individuals in the vaccinee panel. Booster dose induced a robust increase in anti-spike antibodies and NTs against the WK-521, Delta, and Omicron variants. There were no significant differences in the neutralization ability of sera from boosted individuals among the Omicron subvariants BA.1, BA.1.1, and BA.2. Boosting increased the breadth of humoral immunity and cross-reactivity with Omicron without changes in cytokine signatures and adverse event rate. Conclusions The third vaccination dose is safe and increases neutralization against Omicron variants. Funding This study was supported by grants from AMED (grant numbers JP21fk0108104 and JP21mk0102146).

J o u r n a l P r e -p r o o f in Wuhan, China, and rapidly spread worldwide. 1 The disease was declared a pandemic by the World 74

Health Organization (WHO) in March 2020. 2 In Japan, the first coronavirus 2019 case 75 was reported in January 3 and the second in February 2020, after which a large number of COVID-19 76 cases were reported from the Diamond Princess cruise ship at Yokohama port near Tokyo, Japan. 4 The 77 number of infections increased slightly within cities, but the first major wave of infection of more than 78 10,000 cases per day occurred in April 2020. The Japanese government declared its first state of 79 emergency in major cities, including Tokyo, and implemented restrictive measures. As of January 19, 80 2022, the Japanese government has declared a state of emergency four times following the five 81 pandemic waves in Japan. 5 82

During the second wave of the pandemic, the SARS-CoV-2 strain presenting the D614G 83 mutation in the spike region (which affects its transmissibility 6 and infectivity 7,8 ) emerged and rapidly 84 increased the percentage of total SARS-CoV-2 infections. The newly emerged Alpha strain (B.1.1.7) 85 was isolated from the UK and contained an additional E484K mutation, which gradually appeared to 86 increase and replace the dominant endemic virus. 9 Simultaneously, the Beta strain was isolated from 87

South Africa with a new K417 mutation, along with three of the mutations observed in the Alpha 88 variant (E484K, N501Y, and D614G in the spike protein), enabling the virus to escape vaccine-induced 89 antibodies. 10 The Gamma (P.1) variant with a new K417T mutation and three previously identified 90 mutations observed in Alpha was also isolated in Brazil. 11 91

In late summer 2020, many countries began reviewing and authorizing new types of 92 vaccines, such as the Pfizer/BioNTech SARS-COV-2 mRNA vaccine, 12 Moderna mRNA vaccine, 13 93 Janssen/Johnson & Johnson adenovirus vector DNA vaccine, 14 and AstraZeneca adenovirus vector 94 DNA vaccine. 15 In December 2020, the US and EU countries approved these vaccines under 95

Emergency Use Authorization. These SARS-CoV-2 vaccines have remarkably reduced the number of 96 COVID-19 infections, hospitalizations, and deaths in clinical trials in many countries. 16 The Japanese 97 government first approved the two mRNA vaccines in February 2021, after which vaccination of 98 healthcare workers and high-risk people aged over 65 years was initiated. The National Hospital 99 mRNA vaccine (Comirnaty) from Japanese healthcare workers (LT-SI study, Figure 1 ). Anti-SAb 146 titers were measured using the Roche Elecsys ® Anti-SARS-CoV-2 (RUO), and NTs were measured 147 using the live virus for the original ancestral vaccine strain, WK-521, of SARS-CoV-2, as described 148

previously. 31-33 Although anti-SAb and NTs showed a weak correlation at the second vaccination in 149 our preliminary analysis (Figure 2A) , each anti-SAb titer was normally distributed in the NT, x20, 150 x40, x80, x160, x320, and x640 groups. We selected 10-14 representative samples from around the 151 mean anti-SAb value for each NT ( Figure 2B) . Details on the participants in the LT-SI study are listed 152

in Table 1 , and the characteristics of the selected vaccinee panel study (VP-SI study) are described in 153 Table S1 ; briefly, we selected 34-38 samples from a population that reflected the original LT-SI study. 154

We also included individuals who developed adverse events, including fever, headache, fatigue, and 155 injection site pain, matching the frequency of these events initially detected in the LT-SI study 12 days 156 after the second (2nd VAX) and third vaccinations (3rd VAX). There was no significant difference in 157 the anti-SAb and NT distributions between the LT-SI and VP-SI groups ( Figure 2C and D) . 158

In our VP-SI study, anti-SAb levels were significantly increased (x46) after the second and third 162

Pfizer/BioNTech SARS-CoV-2 mRNA dose ( Figure 3A ) compared the titers in response to the first 163 dose. Although anti-SAb levels slightly decreased at 97 (2nd VAX+3M) and 243 days ("pre3rd" VAX) 164 after the second vaccination, they remained significantly higher than those after the 1st VAX. A similar 165 trend was observed for NT ( Figure 3B ), which significantly increased (18.5x) after the 2nd VAX and 166 decreased in the following months. The third dose of the mRNA vaccine dramatically and significantly 167 increased both anti-Sab levels (61.6x) and NTs (43.9x) compared to that of the pre3rd VAX. 168 169 Second dose of vaccination was effective for some variants but not for Omicron 170

The Pfizer mRNA vaccine shows 95% effectiveness against the ancestral strain, WK-521. 12 171 using a heatmap of the Spearman's rank correlation coefficient ( Figure 4B ). We found that NTs against 182 the ancestral strain from individuals administered their second vaccination dose correlated with cross-183 neutralizations against Alpha, Beta, Gamma, R.1, Kappa, and Delta variants, and weakly correlated 184 with cross-neutralizations against two Omicron variant. These data suggest that the two-dose 185 vaccination induced high NTs against the vaccine strain WK-521 but not against the Omicron variant. 186 187 Cytokine signature was stable during vaccinations 1-3 188

The Pfizer/BioNtech mRNA vaccine is a new modality used in Japan and other countries. In this 189

LT-SI study, we collected information on adverse events and summarized vaccine safety data at each 190 time point after vaccination (Table S1) . Briefly, there were no serious adverse events associated with 191 this vaccine compared with those in previous phase II/III clinical studies in Japan and other countries. 192

In addition, we collected 2nd VAX, 2nd VAX+3M, and 3rd VAX samples to measure 48 cytokine 193 profiles. During vaccination, enhanced cytokine production, including that of some inflammatory 194 cytokines, was not observed at any time point (Supplemental Figure 3) . The cytokine levels of 195 eotaxin in the 3rd VAX group were significantly higher than those in the 2nd VAX and 2nd VAX+3M 196 groups. The cytokine levels of CTACK, a cutaneous T cell-attracting chemokine, in the 2nd VAX+3M 197 group were significantly lower than those in the 1st and 2nd VAX groups. The cytokine levels of 198 macrophage inhibitory factor and macrophage inhibitory protein-1β in the 3rd VAX group were 199 significantly lower than those in the 1st VAX group. 200

We divided the subjects into adverse event-positive and adverse event-negative groups, namely 201 high fever (≥39 °C), low fever (≥37 °C), and no fever groups, and observed no differences in the 202 cytokine signatures among these groups (Supplemental Figure 4) . There was no correlation between 203 body temperature and cytokine levels in the 2nd and 3rd VAX groups. In addition, we analyzed the 204 correlation between anti-SAb and each cytokine level and found that only the eotaxin level was weakly 205 correlated with the anti-SAb titer in the 3rd VA X group. 206 207

In Japan, the third booster vaccination was initiated in November 2021 for healthcare workers 210 only. We collected samples before ("pre3rd VAX") and two weeks after ("3rd VAX") a third vaccine 211 dose from the same participants in our panel. We first analyzed NTs against the original vaccine strain, 212 WK-521. NTs against WK-521 were significantly higher than after second dose ( Figure 5A ). Some 213 participants showed NTs that exceeded 2,560, and the geometric mean of the cohort was 476 after the 214 third dose. Samples collected after the third dose showed a 43.9-fold NT increase compared to the 215 third dose samples. In the most recently identified VOC, Omicron, NTs after two doses was 6, which 218 decreased to 3 three months later. These values are both lower than those of the WK-521 and Delta 219 variants ( Figure 5C ). Most NTs in the pre3rd VAX were below the detection limit of 2.5;. however, 220 they increased 33.3-fold after third dose administration, leading to higher NTs than those detected 221 against WK-521 after the second dose. A similar result was obtained using a commercially available 222 multiplex SARS-CoV-2 neutralization antibody detection assay kit. Compared to the response after 223 second dose, neutralizing antibodies against wild-type S1, Alpha S1, Beta S1, and D614G S1 were 224 significantly increased by 1.96-2.98-fold and neutralizing antibodies against the wild-type RBD, 225

Gamma RBD, Kappa RBD, Epsilon RBD, N501Y RBD, K417RBD, and E484K RBD significantly 226 increased by 2.35-3.15-fold ( Figure 5D ). 227 228

To characterize the neutralization patterns in individuals who were booster-vaccinated with the 231

Pfizer mRNA vaccine, we directly compared wild-type NTs with those developed against the Delta 232 and Omicron strains ( Figure 5E -G). We found that NTs against the ancestral strain from individuals 233 administered their second dose vaccination were weakly correlated with Delta variant cross-234 neutralization and not correlated with TY38-871 or TY38-873 Omicron variants cross-neutralization 235 in the 2nd VAX and pre3rd VAX groups. In contrast, wild-type neutralization of boosted individuals 236 correlated with Delta and Omicron variant cross-neutralization. These data suggest that booster 237 vaccination induces not only higher NTs against the vaccine strain WK-521, but also increases the 238 breadth of humoral immunity and cross-reactivity against the highly mutated SARS-CoV-2 Omicron 239 variant. 240 241

Since the genetic sequence of the SARS-CoV-2 Omicron variant was first isolated and determined, 244 subvariant strains have been isolated in many countries. At present, Omicron is composed of several 245 sublineages, such as BA.1, BA.1.1 (or Nextstrain clade 21 K), BA.2 (or Nextstrain clade 21 L), and 246 BA.2. Studies have shown that BA.2 has a growth advantage over BA.1, and some data suggest that 247 BA.2 is inherently more transmissible than BA.1. Some studies have shown that reinfection with BA.2 248 following infection with BA.1 can occur 40 . Thus, comparing the cross-neutralization ability among 249

the Omicron subvariants such as BA.1, BA.1.1, and BA.2, is crucially important to see the merit of 250 three-dose vaccination. Thus, we compared NTs against five SARS-COV-2 Omicron subvariants in 251 samples collected from individuals who had received three vaccine doses. Although the NTs against these Omicron subvariants ( Figure 6A) . We compared NTs against ancestral and Omicron subvariants 254 using a heatmap of the Spearman's rank correlation coefficient and found that booster vaccination 255 increased the correlation coefficient between the ancestral strain (WK-521) and Omicron subvariants; 256 in addition, there were no differences in cross-reactivity among Omicron subvariants ( Figure 6B) . 257 258

We first determined the changes in anti-SAb and NTs against Omicron variants in Japanese 260 healthcare workers receiving a three-dose vaccination with the Pfizer/BioNTech SARS-CoV-2 mRNA 261 vaccine. The second vaccine dose dramatically increased both anti-SAb and anti-NT against the 262 vaccine strain WK-521. The effectiveness of this vaccine has already been established, 12 preventing 263 more than 95% of COVID-19 cases in phase II/III clinical studies. In this study, we compared NTs 264 against VOCs after the second vaccination dose. Our results agree with those of a previous study 265

showing that NTs were severely reduced for Beta and Gamma strains, which exhibit three-vaccine 266 escape mutations in the spike RBD domain. 19 Similarly, NTs against Delta and Gamma were slightly 267 decreased compared to those against the ancestral strain, WK-521. In November 2021, the SARS- After the first isolation of the SARS-CoV-2 Omicron variant, a subvariant strain has also been 300 isolated in many countries. BA.2 is almost dominant in Denmark 22 and has increased its prevalence in 301

Omicron infections detected in the US 23 and the UK 24 . In our study, we found that a three-dose 302 vaccination increases neutralizing antibodies against Omicron subvariants and observed no significant 303 differences in neutralization ability among the Omicron subvariants. 304

In addition to immunogenicity, safety is a critical issue, as some people consider that emergency 305 use authorization means that complete safety research has not been performed, resulting in vaccine 306 hesitancy. A phase II/III study suggested that the booster shot does not change the adverse event rate 307 or specific adverse events in short-term analysis. 50 After 1-2 weeks after the 3rd vaccination dose, 308 abnormal or prolonged cytokine production was not observed in healthy participants (Supplemental 309 Eotaxin, a chemokine ligand for CCR3, is a chemoattractant for eosinophils, basophils, and Th2 312 lymphocytes, and is released from endothelial and epithelial cells. Eotaxin recruits eosinophils to the 313 site of inflammation and releases reactive oxygen species, causing tissue damage during chronic 314 inflammatory responses. Several studies have shown that eotaxin levels increased after the smallpox 315 vaccination correlating to adverse event presentation. 51 Thus, eotaxin levels may be related to adverse 316 events such as injection site pain and redness after the third vaccine dose. However, damage-associated 317 inflammatory cytokine levels induced by eosinophils were unchanged in our samples, as were key 318 pathways involving eotaxin, such as interferon-gamma and interleukin-4. These data suggest that 319 eotaxin alone is not an indicator of serious adverse events. Multiple pathway analyses are required to 320 determine the relationship between cytokine levels and adverse events. Overall, the cytokine data 321 suggest no signs of adverse events. 322

Taken together, booster vaccination induced not only an increase in the anti-Spike IgG titer, but 323 also an increase in the breadth of humoral immunity and cross-reactivity against newly emerged production. 326

This study has several limitations. First, the number of samples evaluated in this study for the analysis 329 of cross-reactivity of neutralizing antibodies against several SARS-CoV-2 variants was small 330 compared to the original survey. Thirty samples were selected as representative of the original survey 331 (N = 272) based on the correlations in anti-S antibodies and neutralizing antibodies to the ancestral 332 strain, WK-521, and the distribution of sex, age, and frequency of adverse events after the second 333 vaccination. Second, we could not take gender, age, ethnicity, and socioeconomic status into 334 consideration when enrolling the participants because our study was initiated upon the Government's 335 directive that COVID-19 vaccinations be first administered only to healthcare workers, the aged, and 336

high-risk persons. The sample reflects variations in the sex, ethnicity, and socioeconomic status of 337 hospital healthcare workers. We did not set typical exclusion criteria for enrollment. Vaccination-338 responsible doctors were able to decide who to exclude, but no one was excluded in this study, except 339 participants delivering a positive PCR test after the second vaccination. Third, vaccine sera used in 340 this study were collected at only one time point, 1-2 weeks after the first, second, and third 341 vaccinations. Thus, we could not analyze the differences in anti-SAb and neutralizing antibody titers 342 in the short term, especially in the early phase after vaccination. In the same way, we could not analyze 343 the early phases of inflammation status after vaccination. We focused only on the late-phase effects 344 after vaccination. In addition, we could not collect mononuclear cells from each participant; thus, we 345 could not analyze the cellular immunity against SARS-CoV-2 variants. T-cell immunity against SARS-346

CoV-2 variants, including Omicron variants, is important for protection against SARS-COV-2. were isolated from VeroE6/TMPRSS2 cells using respiratory specimens collected from individuals 503 screened at an airport quarantine facility in Japan at NIID with ethical approval from the Medical 504

Research Ethics Committee of NIID for the use of human subjects (#1178). All isolated viruses were 505 sequenced at NIID. TY38-871 was found to contain an additional R346K mutation. 506 507

The anti-spike antibody titer was measured using the Elecsys anti-SARS-CoV-2 S assay (Roche 510 Diagnostics International Ltd., Basel, Switzerland), which is an electrochemiluminescence 511 immunoassay with a double-antigen sandwich design used to detect immunoglobulins in the RBD of 512 the spike protein. This kit primarily detects IgG, IgA, and IgM. Serum samples were prepared 513

according to the manufacturer's instructions and analyzed using the Roche Cobas e411 platform. 514

According to the manufacturer's guidelines, sample values of ≥0.8 AU/mL were classified as positive 515

for anti-SARS-CoV-2 antibodies. 516 517

Live virus neutralization assays were performed as previously described. 31-33 Briefly, serum 519 samples were serially diluted (two-fold dilution starting from 1:5) in high-glucose Dulbecco's 520 VeroE6/TMPRSS2 cells (JCRB1819) seeded in 96-well plates and cultured at 37 °C with 5% CO2 for 527

Multiplex SARS-CoV-2 neutralization antibodies detection assay 539 SARS-CoV-2 neutralizing antibodies in serum samples were analyzed using the Bio-Plex Pro 540

Human SARS-CoV-2 Variant Neutralization Antibody, 11-plex (Bio-Rad) according to the 541 manufacturer's instructions and a previous report. 34 Briefly, after determination of the optimal 542 concentration of some typical serum samples, 25-µL serum samples were mixed with SARS-CoV-2 543 antigen-coupled beads in a 96-well plate. After incubation on a shaker at 850 rpm for 30 min at room 544 temperature, 25 µL of biotinylated detection angiotensin-converting enzyme 2 receptor was added and 545 the mixture was incubated at 850 rpm for 30 min. After washing, 50 µL of streptavidin-phycoerythrin 546 was added, and the mixture was incubated on a shaker at 850 rpm for 10 min at room temperature. 547

The beads were washed and resuspended in 125 µL of assay buffer. Fluorescence intensities were 548 measured using a Bio-Plex MAGPIX multiplex reader. Data were analyzed using Bio-Plex Manager 549

Software version 6.0 (Bio-Rad). 550 551

To monitor abnormal and prolonged cytokine production, the cytokine levels in serum samples 553 1-2 weeks after the 2nd and 3rd vaccination doses were analyzed using the Bio-Plex Pro Human 554

Cytokine Screening Panel 48-plex (#12007283) (Bio-Rad) according to the manufacturer's 555 instructions (Supplemental Figure 3 and 4) . Briefly, 50 µL of the serum sample was mixed with capture 556 antibody-coupled beads in a 96-well plate and incubated on a shaker at 850 rpm for 1 h at room 557 temperature. The beads were washed, mixed with 50 µL of biotinylated detection antibodies, and 558 incubated on a shaker at 850 rpm for 30 min at room temperature. After washing, 50 µL of streptavidin-559 phycoerythrin reporter dye was added and incubated on a shaker at 850 rpm for 10 min at room 560 temperature. The beads were washed and resuspended in 125 µL assay buffer. Fluorescence intensities 561

were measured using a Bio-Plex MAGPIX multiplex reader (Bio-Rad) and the data were analyzed 562 

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89/89, 89/89, 74/89) 1. Neutralization titers (NTs) against Omicron decreased after two-dose mRNA vaccination 2. Three-dose vaccination increased anti-Spike antibody and NTs against Omicron variants. 3

The cytokine signature remained unchanged after three-dose vaccination

report that a three-dose Pfizer/BioNTech mRNA vaccination induced a robust increase in anti-spike antibodies and neutralization titers against the WK-521, Delta, and Omicron variants

Immunogenicity against Omicron subvariants, including BA.1, BA.1.1, and three different BA

We thank Dr. Noriyo Nagata, Shinji Watanabe, Masaki Ochiai, and Seiichiro Fujisaki of the 350

The SARS-CoV-2 Omicron variant, later named BA.1, has emerged as a highly transmissible variant due to the 36 mutations in its spike protein, which is the target of neutralizing antibodies; it can therefore escape vaccine-induced immunity. The Omicron subvariant, BA.2, was recently identified and has rapidly become a major variant of concern in many countries, including Japan. This study found that anti-spike antibody levels and neutralization ability decreased gradually 6-9 months after the second vaccination. A third dose dramatically increased the response against multiple Omicron variants. These results show that a booster shot increases neutralization antibodies against SARS-CoV-