Assessment of antibody titer and side effects after fourth doses of COVID-19 mRNA vaccination in 38 healthy volunteers

Introduction

As of the middle of November 2022, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 634 million individuals worldwide and caused more than 6.6 million deaths. Our group have recently reported antibody titers and side effects after two doses of BNT162b2 vaccination [1], and subsequent study of antibody decline 6 months after first vaccination [2] and a potent increment of the IgG antibody titer after third vaccination [3]. Additionally, we found that the production of IgG after third doses of BNT162b2 vaccination decreases age-dependently [4]. We further analyzed the duration of antibody titer after 6 months after the third doses of COVID-19 mRNA vaccination and found that the long-term durability of IgG titer by the third time BNT162b2 vaccination was significantly higher than the second time, and the reduction of IgG titer after the third time vaccination was inversely correlated with age [5].

The humoral effects of fourth doses of BNT162b2 vaccination have been reported, in booster dose required populations, such as, in older subjects [6–11], liver transplant recipients [12], patients with solid tumors on active treatment [13], systemic lupus erythematosus and rheumatoid arthritis [14], after heart transplantation [15] and dialysis patients [16]. Overall, these studies suggest that receipt of a fourth BNT162b2 dose confers some protection against infection. Especially, Bar-On et al. [9], have reported that protection against confirmed infection appeared short-lived, whereas protection against severe illness did not wane during the study period.

However, in healthy, and below middle age subjects, the effects of fourth vaccination is still scarce. 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 fourth time.

Materials and methods

Results

Of 41 volunteers who started vaccination of BNT162b2 at our hospital in March or April 2021, 30 completed 17 months of follow-up after the first dose. The overall sampling procedure is shown in Figure 1A. In addition, 8 participants were newly enrolled in this study who have received three times of BNT162b2, and vaccinated in August or September 2022 for the fourth time (Table 1).

Figure 1:

Vaccination procedures, sampling and anti-SARS-CoV-2 antibody levels following vaccination. (A) Schematic presentation of vaccination and sampling procedures. (B and C). Box plots of anti-SARS-CoV-2 antibody levels following vaccination with the BNT162b2 vaccine. (B) Anti-SARS-CoV-2 IgG titer 504 and 520 days after the first vaccination in all participants. Data are shown as boxplots representing the 25th percentile, 75th percentile, median, and 95 % confidence interval using Prism software (version 7.0; GraphPad Software Inc., La Jolla, CA, USA). Data at baseline, day 14, 35, 180, 264, 279 and 445 are shown in gray, presented for comparison. Statistical significance of each time points between the data at day 520 are shown in asterisks. In addition, statistical significance between day 504 and 264 is shown (^δδδp<0.001, ns, not significant). (C) Anti-SARS-CoV-2 IgM titer 504 and 520 days after the first vaccination in all participants. No significances were observed between day 504 and 520. Correlation of age and post-vaccination at day 504 (D) and day 520 (E) of anti-Spike IgG and IgM levels with superimposed the linear regression lines (with 95 % CI). The statistical methods of Spearman’s rank correlation coefficient were used, and p-value is indicated. Linear regression lines, blue.

As shown in Figure 1B, we found that the level of IgG at day 504 (average, 117.9 AU/mL [SD 76.9]), is significantly higher than at day 264 (average, 17.3 AU/mL [SD 13.1]) [4], which are 8 months after the third and second vaccination, respectively. The level of IgG was potently increased after fourth vaccination (average, 711.8 AU/mL [SD 361.9]), whereas IgM remained baseline level (Figure 1C). Interestingly, although we observed IgG declining trends with age until third vaccination [1, 4], however, the current study at fourth vaccination, there are no relationships between IgG nor IgM titer and age (Figure 1D and E).

We next assessed the side effects after the fourth vaccination (Table 2, Figures 2 and 3). Throughout the study, almost all participants (90 % after the first dose; 97.5 % after the second dose; 97.5 % after the third dose, 100 % after the fourth dose) had symptoms (Figure 2A). The number of side effects were similar to those at second to fourth dose (Figure 2A and B). We further analyzed whether there were any differences in the numbers of side effects and age, and found that, they were significantly decreased with age (p=0.0228) (Figure 2C). Summary of the side effects of the BNT 162b2 vaccine after the fourth doses of the vaccine is shown in Table 2 and Figure 3. Commonly reported side effects in the participants after the third dose were similar to those until third dose, such as, sore arm/pain (81.0 %), generalized weakness/fatigue (57.1 %), fever (54.8 %), and headache (42.9 %) (Table 2, Figure 3). We further examined whether there were any relationships between the common side effects and IgG antibody titers (Figure 4). As a result, although most of all side effect did not affect IgG antibody titers, we observed that participant with sore arm/pain had higher IgG titer (p=0.0007) (Figure 4G), and participant with lymphadenopathy had lower IgG (p=0.0371) (Figure 4I).

Figure 2:

Summary of the side effects of the BNT162b2 vaccine. (A) Percentages for the presence (symptoms) or absence (no symptoms) of the side effects of the BNT162b2 vaccine reported by the participants after the first (white columns), second (light gray columns), third (dark gray columns) and fourth (black columns) doses of the vaccine. The results for first, second (1), and third vaccinations (4) were shown, as a comparison. (B) Box plots of total numbers of side effects in individual participants after the first, second, third and fourth doses. The results for first, second (1), and third vaccinations (4) were shown, as a comparison. ***p<0.001. (C) Correlation of age and the number of side effects after the injection with superimposed the linear regression lines (with 95 % CI). The statistical methods of Spearman’s rank correlation coefficient were used. p-value (p=0.0035) is indicated. Linear regression lines, blue.

Figure 3:

Summary of the side effects of the BNT162b2 vaccine after the first (white bars), second (light gray bars), third (dark gray bars) and fourth (black bars) doses of the vaccine. The results for first, second (1), and third vaccinations (4) were shown, as a comparison. The data are shown as frequency (%).

Figure 4:

Bar graphs of anti-SARS-CoV-2 antibody titers at 520 days after the first vaccination. (A) Anti-SARS-CoV-2 IgG titers, categorized by the presence or absence of (A) generalized weakness/fatigue, (B) headache, (C) chills, (D) fever, (E) sweating, (F) Dizziness, (G) sore arm pain, (H) localized swelling at the injection site, (I) lymphadenopathy, (J) muscle pain/myalgia, (K) arthritis/joint pains, (L) whole body pain (M) nausea, (N) decreased sleep quality, (O) decreased appetite, (P) heat/cold intolerance, (Q) trouble to perform regular daily living activities temporarily, (R) required transient off from work. Bar graph display the median values with the interquartile range (lower and upper hinge) and ±1.5-fold the interquartile range from the first and third quartile (lower and upper whiskers). Data was analyzed using the unpaired t-test or Welch’s t-test and p-values were shown.

Discussion

In healthy, and below middle age subjects, the reports of the effects of fourth vaccination is scarce, however, several have been published so far. Cohen et al. [17], from vaccination proceeding country Israel, recently reported an important observation. Among a total of 29,611 Israeli health care workers who had received three vaccine doses, 5,331 (18 %) received fourth doses. Overall breakthrough infection rates were 368 of 5,331 (7 %) in the fourth dose group and 4,802 of 24,280 (20 %) in the third dose group (relative risk, 0.35; 95 % CI, 0.32–0.39), suggesting the reduced breakthrough infection rate among hospital staff after fourth dose group [17]. Magen et al. [11], analyzed data recorded by the largest health care organization in Israel, and found that a fourth dose of the BNT162b2 vaccine was effective in reducing the short-term risk of Covid-19-related outcomes among persons who had received a third dose at least 4 months earlier [11]. Another group have reported 112 health workers in Mexico [18]. After the first dose, subjects had a median IgG AU/mL of 122 that increased to 1875, 3,020, and 4,230, 21–28 days after the second, third, and fourth doses, respectively [18]. Hein et al. [19], have reported an important paper, which recently examined the quantity and quality of the booster vaccination (third and fourth dose). They found that even a repetitive booster vaccination based on the Wuhan isolate has a limited capacity to induce a long-lasting humoral immune response against a distant variant such as Omicron [19]. This indicates the urgent need for the development of a variant-adapted second generation of SARS-CoV-2 vaccines.

In the current study at fourth vaccination, there are no relationships between IgG titer and age, although we and others have observed IgG declining trends with age until third vaccination [1, 4]. Recently, we observed that the reduction in IgG from 180 days after the third vaccination showed a significant inverse correlation with age, and the higher antibody response in younger participants at 14 days after the third vaccination disappeared at 180 days after the third vaccination [5]. Since the studies about the fourth vaccination is still ongoing, so we should wait for the results from other studies. However, our results indicate that the effect of BNT162b2 vaccination after fourth time may not be affected by age.

We observed that participant with sore arm/pain had higher IgG titer, and participant with lymphadenopathy had lower IgG. We haven’t observed these relationships until third vaccination [1, 4]. In February in this year, Kodde et al. [20] have been published about the relationships between IgG titer and vaccination in healthy volunteers. Consistent with our results, they concluded that age, vaccination regimen, days since vaccination, and physical complaint influenced the antibody level. Our current study may have limitations, such as small sample size, and we are going to carefully observe these effects in the future study. However, the level of IgG was significantly higher in 8 months after the third, compared to the second, vaccination, and it was potently increased after fourth vaccination. Based on our observations of moderate side effects after fourth vaccination, fourth doses of BNT 162b2 is tolerable and therefore, efficient to protect against SARS-Co-V2.