Assessment of antibody titer and side effects after third doses of COVID-19 mRNA vaccination in healthy volunteers

Objectives: Third dose of SARS-CoV-2 vaccination was started from December 1, 2021 in Japan. However, data on the precise analysis of the side effects after third vaccination, remain scarce. Here, we examined the side effects and the levels of SARS-CoV-2 IgG antibody in healthy volunteers who underwent BNT162b2 vaccination. Methods: Forty-one healthy volunteers were assessed for the side effects of the vaccination for the third dose, and samples were used for themeasurement of SARS-CoV-2 IgG antibody with chemiluminescent assays against the Receptor Binding Domain (RBD) of the virus. Results: We analyzed the humoral responses and found that the IgG levels showed clear declining trends with age. Commonly reported side effects in the participants after the third dose were similar to those in second dose, such as, generalized weakness/fatigue (65.9%), headache (58.5%), and sore arm/pain (87.8%). The frequency of the fever was slightly less (39.0%), compared to the second dose (57.5%), but localized symptoms, such as itching (14.6%) and lymphadenopathy (14.6%)were not negligible, whichwere not seen at the second dose. The number of side effects were tended to be decreased with age. Conclusions: The production of IgG after the third doses of BNT162b2 vaccination decreases age-dependently. The number of side effects were tended to be decreased with age. The high frequencies of generalized weakness/fatigue, fever, and sore arm/pain were not negligible, after the third dose.


Introduction
As of the end of April 2022, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 509 million individuals worldwide and caused more than 6.22 million deaths. In the year of 2021, a number of groups have reported humoral responses and side effects after two doses of BNT162b2 vaccinations have been reported [1][2][3]. Third dose of SARS-CoV-2 vaccination have just started from December 1, 2021 in Japan. Therefore, the side effects after third vaccination, still remain scarce. Our group have recently reported antibody titers and side effects after two doses of BNT162b2 vaccination [4], and subsequent study of antibody decline 6 months after first vaccination [5] and a potent increment of the IgG antibody titer (average, 702.9 AU/mL [SD 402.9], 40.6-fold increase) after third vaccination [6]. In the current study, we examined side effects and the relations between IgG antibody titer among healthy volunteers at Tohoku Medical and Pharmaceutical University Hospital, before and after vaccination with the Pfizer/BioNTech BNT162b2 mRNA vaccine for the third time.

Materials and methods Participants
Vaccines (30 µg of BNT162b2/Comirnaty; Pfizer/BioNTech, New York, NY, USA) were administered at Tohoku Medical and Pharmaceutical University Hospital starting on 15 March 2021. A total of 41 volunteer healthcare workers (31 women and 10 men, mean ± standard deviation [SD] age 40.1 ± 12.7 years) were enrolled in the study [4,5]. Participants received a first dose of BNT162b2 in March or April 2021, followed by a second identical dose 21 days later. Third vaccination was held on average day 265, December 2021 or January 2022, which is approximately 8 months after the second vaccination. Sera were collected at 14, 35, 180, 264 (1 day before third vaccination) and 279 days after the first vaccination. Results for the 264 and 279 day time point are presented in this report.

Antibody assays
Antibody titers were evaluated using a newly established, highly sensitive, fully automated chemiluminescent enzyme immunoassay (CLEIA) designed to specifically detect IgG against the SARS-CoV-2 spike protein receptor-binding domain (RBD) as described previously [4,5].

Safety assessments
Safety assessments were monitored by solicited local and systemic adverse events, collected by a questionnaire, as described previously [4]. The questionnaire was sent by e-mail to the participants enrolled in the study, and responses were collected until the end of January 2022. Responses were obtained from all of the 41 healthy healthcare volunteers (Table 1) who received third doses of the BNT162b2 vaccine. For analysis of the questionnaire data, the responses were displayed as both percentage and number.

Results
Of 41 volunteers who received two doses of BNT162b2 at our hospital, all completed 9 months of follow-up after the first dose. At the time of writing, all 41 participants have completed this period, and none experienced SARS-CoV-2 infections prior to third vaccination or during post-third vaccination follow-up. First, we analyzed the humoral responses and found that a potent increment of the IgG (40.6 fold) titer after third vaccination, which was already reported in the previous paper [6]. In the current study, we revealed that the IgG levels before (at day 264) and after (day 279) vaccination showed clear declining trends with age ( Figure 1). We further analyzed the differences between males and females, but no significant difference in IgG titer was observed (data not shown).
We next assessed the side effects after the third vaccination ( Figure 2). Throughout the study, almost all participants (90% after the first dose; 97.5% after the second dose; 97.5% after the third dose) had symptoms ( Figure 2A). The total number of side effects was approximately the same after the third vaccination, compared with the second vaccination ( Figure 2B). The number of the side effects were significantly increased in 3rd dose, compared to the first dose (p<0.0001), however, there were no significant difference between 2nd and 3rd doses ( Figure 2B). We further analyzed whether there were any differences in the numbers of side effects and age, and found that, although not statistically significant (p=0.0991), the number of side effects were tended to be decreased with age ( Figure 2C). Summary of the side effects of the BNT 162b2 vaccine after the third doses of the vaccine is shown in Figure 3.
Commonly reported side effects in the participants after the third dose were similar to those in second dose, such as, generalized weakness/fatigue (65.9%), headache (58.5%), and sore arm/pain (87.8%). The frequency of the fever was slightly less (39.0%), compared to the second dose (57.5%), but localized symptoms, such as itching (14.6%) and lymphadenopathy (14.6%) was not negligible, were not seen at the second dose. We further examined whether there were any relationships between the common side effects and antibody titers. As a result, we observed that there were no differences in IgG levels with or without side effects (Supplementary Figure 1).

Discussion
In the current study, we observed that the age is inversely related to the production of IgG level, which is consistent with several other studies from 2nd vaccination [7,8]. However, reports of the third dose of BNT162b2 vaccination is still scarce. Several studies about this issue, has been reported, such as, analysis in immunocompromised host [9,10], aged population [11,12], or studies in vaccination proceeding country, Israel [12][13][14]. Recently, the antibody titers before and after a third dose of the SARS-Cov-2 BNT162b2 vaccine in adults aged more that 60 years (n=97) have been published [11,12]. In Israel, Bar-On et al. [12] have extracted from the Israeli Ministry of Health database regarding 1,137,804 persons who were 60 years of age or older and had received two doses of BNT162b2 at least 5 months earlier. They reported that the rates of confirmed COVID-19 and severe illness were substantially lower among those who received a booster third dose of the BNT162b2 vaccine. Shapiro Ben David et al. [14] performed retrospective cohort study in these participants and a total of 17,820 were participated in this survey. As a result, 67.3% of immunocompromised and 62% of seniors reported experiencing a better or a similar response to the third dose, comparted to the second [14], like we observed in the current study.
From the retrospective cohort study, Saciuk et al. [15], concluded that the third dose provides added protection against SARS-CoV-2 infection for those vaccinated 6 months   Psychological and/or psychiatric symptom/s Decreased feelings of joy/relief/gratitude Brain fogging or reduced mental clarity/attention/ concentration Similarly, from Israel, Barda et al. [13], recently demonstrated that using data from mandatory health-care coverage for over half of the Israeli population. They concluded that admission to hospital, severe disease and death is significantly reduced in the population vaccinated with three doses [13]. In February 2022, Wilfredo et al. have reported the effect of booster dose for neutralizing immunity against SARS-CoV-2 omicron variant [16]. They reported that individuals boosted with mRNA vaccines exhibited potent neutralization of Omicron, suggesting enhanced cross-reactivity of neutralizing antibody responses. The long-term efficacy of BNT162b2 vaccination remains to be determined. Our current study may have limitations, such as small sample size. However, based on our observations of moderate side effects after third vaccination, third doses of BNT 162b2 is tolerable and therefore, efficient to protect against SARS-Co-V2.