Third-Dose BNT162b2 Vaccination Elicits Markedly High-Level SARS-CoV-2–Neutralizing Antibodies in Vaccinees Who Responded Poorly to a Second Dose in Japan

To the Editor—We read with great interest the article by Saciuk et al demonstrating that, in a retrospective cohort study in Israel, an additional dose of BNT162b2 vaccine 6 months after initial 2-dose vaccination bolsters protection against infection, with a vaccine effectiveness of 89%, as assessed during August–October 2021, when the majority of infections were due to the Delta variant [1]. Recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) studies using pseudovirus have shown that a third dose of BNT162b2 well elicits neutralizing antibodies against VOC (variants of concern) including Omicron (B.1.1.529, BA.1) [2–4]. However, Cheng et al very recently reported a single-source outbreak of Omicron BA.2 sublineage in Hong Kong, which indicates high transmissibility of Omicron/BA.2 [5] and poses further concerns on the efficacy of anti–SARS-CoV-2 vaccines.

In the present prospective study, enrolling 225 health care workers (see demographic characteristics in Supplementary Table 1), who received 3 doses of BNT162b2 in Japan, we consecutively determined SARS-CoV-2 neutralizing activity (50% neutralizing titer, NT₅₀) of their sera using VeroE6^TMPRSS2 cells and its kinetics/profiles over 300 days following the first dose; this is a continuation of our previous study [6]. We also determined NT₅₀ of selected sera using VeroE6^TMPRSS2 and HeLa^{hACE2-TMPRSS2} cells against infectious VOC, including Delta and Omicrons (BA.1 and BA.2), whose emergence has been associated with a steep increase in coronavirus disease 2019 (COVID-19) cases and hospitalizations (experimental details are provided in the “Methods” section of Supplementary Materials).

There was significant neutralizing activity on day 28 after the first dose (NT₅₀ = 501, 1 week after the second dose), while there was a continual decrease until day 280. NT₅₀ values further decreased to 51 by day 280 when approximately 85% of the participants had NT₅₀ values of <100 and approximately 36% had less than 20 NT₅₀ or undetectable (Supplementary Figure 1). However, 2 weeks after administration of the third dose (205 participants [91.1%] remained in the cohort on day 300), there was a substantial rise in neutralizing activity, achieving an average NT₅₀ of 3531. There was a concern that individuals who poorly responded to the second dose might again fail to produce sufficient neutralizing antibodies. Therefore, we specifically determined neutralization activity in vaccinees, who had achieved the lowest 10% level of neutralization following the second dose (n = 22, average-NT₅₀ = 110 on day 28; inset in Supplementary Figure 1). Notably, by day 300, all these low responders achieved markedly greater levels of neutralizing activity with an average NT₅₀ of 2341 (range 482–9113 in VeroE6^TMPRSS2; Table 1). In HeLa^{hACE2 + TMPRSS2} cells, sera from these low responders substantially neutralized SARS-CoV-2^05-2N, Alpha, Beta, Gamma, and Delta (geometric mean [gMean] NT₅₀ = 1777, 1350, 480, 1015, and 959, respectively), but had only marginal activity against Omicron/BA.1 with a gMean-NT₅₀ of 52 (range ≤ 20–197; Table 1). The same sera had similar neutralization profiles in VeroE6^TMPRSS2 cells. On the other hand, sera from participants who achieved the highest 10% level of neutralization on day 300 (n = 22, average NT₅₀ = 10 885 in VeroE6^TMPRSS2) had high neutralizing activity against SARS-CoV-2^05-2N, Alpha, Beta, Gamma, and Delta (gMean NT₅₀ = 9774, 4906, 2279, 3271, and 3377, respectively) in HeLa^{hACE2 + TMPRSS2} cells and good neutralizing activity against Omicron/BA.1 (gMean NT₅₀ = 500, range 171–979). Notably, all day 280 sera of the highest 10% of participants failed to neutralize both Omicron/BA.1 and BA.2 (NT₅₀ values ≤20; Supplementary Figure 2); however, these participants’ sera on day 300 also neutralized Omicron/BA.2 well (gMean-NT₅₀ = 702, range 262–1653; Table 1).

Table 1.

Neutralization Activity of Highest and Lowest 10% of Responders’ Sera against SARS-CoV-2s, Including the Wuhan Strain and 6 VOC

	SARS-CoV-205-2N, Wuhan	SARS-CoV-2QK002, Alpha	SARS-CoV-2TY8-612, Beta	SARS-CoV-2TY7-501, Gamma	SARS-CoV-21734, Delta	SARS-CoV-2NCGM-929-1N, Omicron/BA.1	SARS-CoV-22037, Omicron/BA.2, in VeroE6TMPRSS2a
gMean NT50 of highest 10% of sera in HeLahACE2 + TMPRSS2 cells	9774 (3745–27 921)	4906 (1926–11 018)	2279 (844–8996)	3271 (1723–8849)	3377 (1157–8053)	500 (171–979)	702 (262–1653)
gMean NT50 of lowest 10% of sera in
VeroE6TMPRSS2 cells	1654 (482–9113)	1544 (458–4335)	483 (114–3486)	928 (278–2877)	1014 (371–4246)	130 (≤20–487)	115 (≤20–649)
HeLahACE2+ TMPRSS2cells	1777 (410–7608)	1350 (429–4337)	480 (96–2861)	1015 (528–2410)	959 (386–5905)	52 (≤20–197)	ND

gMean NT₅₀ titers of day 300 sera of lowest 10% of responders (n = 22) and highest 10% of responders (n = 22) against SARS-CoV-2^05-2N and 6 VOC were determined in cell-based assays using each SARS-CoV-2 strain and VeroE6^TMPRSS2 cells or HeLa^{hACE2 + TMPRSS2} cells. The numbers in parentheses denote ranges of NT₅₀ values determined for each strain.

Abbreviations: gMean, geometric mean; ND, not determined; NT₅₀, 50% neutralizing titer; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; VOC, variant of concern.

^aOnly VeroE6^TMPRSS2 cells were used for SARS-CoV-2²⁰³⁷ (Omicron/BA.2) because SARS-CoV-2²⁰³⁷ did not propagate well in HeLa^{hACE2 + TMPRSS2} cells.

The present data clearly show that a third dose of BNT162b2 elicits high-level SARS-CoV-2-neutralizing antibodies even in those who poorly responded to the second dose, although low responders to the vaccines may be vulnerable to infection with Omicron sublineages BA.1 and BA.2. Of note, however, in terms of the effectiveness of a third dose of BNT162b2 against Omicron sublineages, the morbidity and mortality have yet to be determined between individuals who received the third dose but contracted symptomatic Omicron-related COVID-19 and those not receiving the third dose who contracted symptomatic Omicron-related COVID-19.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Author contributions . M. A. and H. M. had access to all data in this study and hold all responsibility for the integrity of the data and the accuracy of the data analysis. M. A. and H. M. contributed the concept and design, and wrote the original draft. M. A., K. M., and K. T. contributed acquisition, analysis, and/or interpretation of data. S. S. provided administrative and material support. All authors contributed to writing and reviewing the manuscript.

Acknowledgments . The authors greatly appreciate Dr Shinichiro Hattori, Dr Yuki Takamatsu, Dr Yukari Uemura, Dr Yosuke Shimizu, Ms Sachiko Otsu, Ms Mariko Kato, and Ms Asuka Fujiwara for their dedicated experimental supports and valuable discussion. The authors also thank Ms Yasuko Ichikawa and Ms Mari Kinoshita for handling blood samples, and Dr Kenji Sadamasu for providing SARS-CoV-2²⁰³⁷ (Omicron/BA.2).

Financial support . This work was supported by the Japan Agency for Medical Research and Development (grant numbers JP20fk0108260 and 20fk0108502 to K. M., and 20fk0108502 to H. M.); Ministry of Health, Labor and Welfare, Japan, Research on Emerging and Re-emerging Infectious Diseases and Immunization Program (grant number JPMH20HA1006 to K. M.); the National Center for Global Health and Medicine, Intramural Research Program (grant numbers 19A3001 to H. M. and 20A2003D to K. M.); and the Center for Research, National Cancer Institute, National Institutes of Health, Intramural Research Program (to H. M.).