Vaccination against SARS-CoV-2 with BNT162b2 mRNA vaccine has undoubtedly proven to be extremely important for public health. Although BNT162b2 is quite effective against COVID-19, there is a time-dependent decrease in neutralizing antibodies (NAbs). Even 1 month after the second BNT162b2 injection, a slight decrease in antibody titers was observed, and the time since the second vaccine dose was associated with lower neutralizing antibody activity against SARS-CoV-2 variants and weaker protection against COVID-19, especially for variants like delta.^{1, 2} As a result, many public health agencies around the world are now recommending a third dose (booster), particularly after the appearance of variants of interest of SARS-CoV-2, such as delta and omicron. The aim of this study was to investigate the increase in neutralizing antibodies and anti-SARS-CoV-2 spike receptor-binding domain (anti-SRBD) IgGs in health professionals, 1 month after vaccination with the third dose of BNT162b2 mRNA vaccine. The possible role of gender and age was further investigated.

The participants were health workers from Alexandra General Hospital in Athens, Greece, who participated in a prospective study (NCT04743388) that evaluates the efficacy of vaccination for the prevention of COVID-19. Major inclusion criteria for participation in this study included: (i) age above 18 years; (II) ability to sign the informed consent form; and (iii) eligibility for vaccination, according to the national program for COVID-19 vaccination. Major exclusion criteria included the presence of: (i) autoimmune disorder under immunosuppressive therapy; (ii) active malignant disease; and (iii) end-stage renal disease.

Anti-spike-RBD IgG antibodies and NAbs against SARS-CoV-2 were measured using FDA approved methods, that is, the Elecsys Anti-SARS-CoV-2 S assay (Roche Diagnostics GmbH, Mannheim, Germany) and the cPass™ SARS-CoV-2 NAbs Detection Kit (GenScript, Piscataway, NJ, USA), respectively, as previously described.¹

Time points for blood collection and serum isolation were day 1 (D1; first BNT162b2 dose), D22 (before the second dose), and then 2 weeks, 1, 3, 6, and 9 months after the second dose and 1 month after the booster BNT162b2 dose (1MP3D). The booster dose was administered within 1 week after the blood sampling of the 9-month time point after the second dose. That was the time (October 2021) that the booster dose was approved for health employees in Greece. After vein puncture, serum was separated within 4 h from blood collection and stored at −80°C until the day of measurement.

Demographic data, comorbidities, and medications taken were obtained from patients after a personal interview. Body mass index (BMI) was calculated from each individual's weight and height data. Based on BMI, subjects were divided into groups: underweight with a BMI of less than 18.4; normal weight with a BMI of 18.5–24.9; overweight with a BMI of 25–29.9; and obese with a BMI of 30 or more. Participants' data were also divided into three age groups (to obtain an approximately equal number of participants), 20–40, 40–55, and ≥55 years, and the role of age in the immune response after the booster was explored.

Statistical analysis started with descriptive metrics such as mean, median, quartiles, and estimation of dispersion metrics. A normality test was performed before statistical comparison between two or more groups. To determine the normality of the data distribution, the Kolmogorov–Smirnov and Shapiro–Wilk tests and QQ plots were used. According to these tests, if the nominal normality hypothesis is rejected, the data are considered not to follow the normal distribution. It was found that the data deviated from the normal distribution in all situations of this study. Therefore, nonparametric approaches were used for the following statistical analysis. The Mann–Whitney U test was used to compare two independent groups, for example, to examine the gender effect or the influence of age groups (<50 and ≥50 years). The Wilcoxon signed-rank test was used for pairwise group comparisons, such as neutralizing antibody levels between two occasions. For the simultaneous comparison of many groups (e.g., age groups), the Kruskal–Wallis test was utilized. The significance level was set at 5% in all cases in this study, and a result was considered significant if the calculated p value (p) was below the significance level. Python v.3.9.2 was used for statistical analysis.

The study was approved by the respective Ethical Committee of Alexandra Hospital, in accordance with the Declaration of Helsinki and the International Conference on Harmonization for Good Clinical Practice. All participants provided informed consent before entering the study.

This paper reports the results of the first 150 (57M/93F) consecutive health professionals who received the booster vaccine dose out of 308 health workers, who initially entered the study and had received two vaccine doses. The median age of all individuals was 49.6 years, whereas the median age of men was 54 years, and the median age of women was 49 years. Overall, the median BMI was 25.9 for the overall sample, with male subjects having a BMI of 27 and female subjects having a BMI of 24.

Figure 1A depicts the percent inhibition of NAbs across the time period from 2 weeks after the second immunization to 1 month after the third dose (1MP3D). One month after the booster dose, the median inhibition percentage was 97.6% (mean 95.9%), which was considerably greater than any other time point tested (Wilcoxon p value <0.001). Only one person (0.67%) had NAbs that were less than 50%, whereas the vast majority (148 subjects, or 98.7%) had very high levels of protection. Therefore, it can be clearly stated that the booster dose causes a burst in neutralizing antibody titers when this increase in NAbs levels is contrasted with the levels observed at earlier time periods. Prior to the booster, the highest levels of NAbs are recorded 2 weeks following the second vaccination, and it is at this point that a consistent decline in NAbs becomes apparent, indicating that the vaccine is working. The median NAbs levels were 96.9% 2 weeks after the second immunization, and they subsequently fell to 96.1% and 55.5% 1 and 9 months later, respectively, after the second vaccination. When compared with the previously considered maximum levels (i.e., 2 weeks after the second vaccination), the NAbs levels at 1MP3D were 151% higher, which is plausible given that the injection triggers the development of anti-spike antibodies. High NAbs were described in convalescent COVID-19 patients after one vaccination dose,² due to a rapid and strong cytokine induction the day after their vaccine dose.³ This is possibly the reason for high NAbs after the booster dose. Similarly, the booster dose increases the NAbs titers even in patients with myeloma who did not respond to the two first vaccine doses.⁴

Anti-SRBD antibodies had similar findings (Figure 1B), with antibody levels reaching the upper limit of 2500 units/mL in 149 out of 150 participants. Anti-SRBD titers were statistically significantly greater at 1MP3D than at any prior measurement point (p value <.001), which was consistent with previous findings. A huge rise in anti-SRBDs of 804% was observed 1 month following the third immunization, when compared with baseline levels before the booster dose (i.e., at 9 months after the second dose). A recent study also described the high anti-S IgG antibodies in 97 Israeli individuals above the age of 65.⁵

In addition, the impact of a variety of additional parameters on NAbs and anti-SRBD titers has been examined. Several factors were examined to determine if they could influence antibody levels on each day or the reported increase in immune response as a result of the booster dosage, including age, gender, medical history (i.e., comorbidities), and BMI. For either NAbs or anti-SRBDs, no differences were identified between males and females; the Mann–Whitney p values for NAbs and anti-SRBDs were .803 and .670, respectively, for NAbs and anti-SRBDs. Furthermore, when the participants were divided into three age groups, no statistically significant differences were found for either NAbs (Kruskal–Wallis p value = .230) or anti-SRBDs (p = .779) antibodies.

A limitation of this study was the relatively small sample size, which may make it impossible to investigate specific pathophysiological situations. In addition, patients with severe concomitant diseases, such as cancer, were excluded from participation in the current study. The use of active immunosuppressive drugs has been shown to contribute significantly to poor humoral response after COVID-19 vaccination, but such patients were not included in this study. Regarding the role of gender, it should be emphasized that unequal sample sizes were available for men and women, which should be considered. Women are almost two times more represented in the workforce than men. Although unequal sample sizes in general can lead to biased results, this is not a problem in our situation, as the imbalance is not extreme but rather normal (33.8% versus 66.2%).

We conclude that a third vaccine dose, 9 months post-full vaccination with the BNT162b2 vaccine, produces very high NAbs and anti-SRBD IgG titers, irrespective of age and gender. This is possibly responsible for the lower COVID-19 rates in those who receive a third vaccine dose,⁶ or for the milder COVID-19 symptoms to those who are infected with omicron variant.

使用 BNT162b2 mRNA 疫苗接种针对 SARS-CoV-2 的疫苗无疑已被证明对公众健康极为重要。尽管 BNT162b2 对 COVID-19 非常有效，但中和抗体 (NAb) 会随时间减少。即使在第二次 BNT162b2 注射后 1 个月，也观察到抗体滴度略有下降，并且自第二次接种疫苗以来的时间与针对 SARS-CoV-2 变体的中和抗体活性较低和对 COVID-19 的保护较弱有关，尤其是对于delta 等变体。^{1, 2}因此，世界各地的许多公共卫生机构现在都建议使用第三剂（加强剂），尤其是在出现 SARS-CoV-2 感兴趣的变体（例如 delta 和 omicron）之后。本研究的目的是调查在接种第三剂 BNT162b2 mRNA 疫苗 1 个月后，卫生专业人员中中和抗体和抗 SARS-CoV-2 刺突受体结合域（抗 SRBD）IgG 的增加情况。进一步研究了性别和年龄的可能作用。

参与者是希腊雅典亚历山德拉综合医院的卫生工作者，他们参加了一项前瞻性研究 (NCT04743388)，该研究评估了疫苗接种对预防 COVID-19 的功效。参与本研究的主要纳入标准包括：(i) 18 岁以上；（二）签署知情同意书的能力；(iii) 根据国家 COVID-19 疫苗接种计划的疫苗接种资格。主要排除标准包括存在： (i) 免疫抑制治疗下的自身免疫性疾病；(ii) 活动性恶性疾病；(iii) 终末期肾病。

使用 FDA 批准的方法测量针对 SARS-CoV-2 的抗尖峰 RBD IgG 抗体和 NAb，即 Elecsys Anti-SARS-CoV-2 S 测定（Roche Diagnostics GmbH，曼海姆，德国）和 cPass™ SARS -CoV-2 NAbs 检测试剂盒（GenScript，Piscataway，NJ，USA），分别如前所述。¹

采血和血清分离的时间点是第 1 天（D1；第一次 BNT162b2 剂量）、D22（第二次剂量之前），然后是 2 周、第二次剂量后 1、3、6 和 9 个月以及治疗后 1 个月加强 BNT162b2 剂量 (1MP3D)。加强剂量在第二剂后 9 个月时间点的采血后 1 周内给药。那时（2021 年 10 月）加强剂量被批准用于希腊的卫生工作者。静脉穿刺后，在采血后 4 小时内分离血清，并在 -80°C 下储存直至测量当天。

人口统计数据、合并症和服用的药物是在个人访谈后从患者那里获得的。根据每个人的体重和身高数据计算体重指数（BMI）。根据 BMI，将受试者分为以下几组：体重不足且 BMI 小于 18.4；体重正常，BMI 为 18.5–24.9；体重指数为 25–29.9 的超重；体重指数为 30 或更高的肥胖者。参与者的数据也分为三个年龄组（以获得大致相等的参与者数量），20-40、40-55 和≥55 岁，并探讨了年龄在加强免疫反应中的作用。

统计分析从描述性指标开始，例如平均值、中位数、四分位数和离散度指标的估计。在两组或更多组之间进行统计比较之前进行正态性检验。为了确定数据分布的正态性，使用了 Kolmogorov-Smirnov 和 Shapiro-Wilk 检验和 QQ 图。根据这些检验，如果拒绝名义正态性假设，则认为数据不服从正态分布。发现在本研究的所有情况下，数据都偏离了正态分布。因此，非参数方法用于以下统计分析。Mann-Whitney U 检验用于比较两个独立组，例如，检查性别效应或年龄组（<50 岁和 ≥50 岁）的影响。Wilcoxon 符号秩检验用于成对组比较，例如两次之间的中和抗体水平。为了同时比较多个组（例如，年龄组），使用了 Kruskal-Wallis 检验。本研究中所有病例的显着性水平均设定为 5%，如果计算出的结果被认为是显着的p值 ( p ) 低于显着性水平。Python v.3.9.2 用于统计分析。

根据赫尔辛基宣言和良好临床实践协调国际会议，该研究得到了亚历山德拉医院各自的伦理委员会的批准。所有参与者在进入研究之前都提供了知情同意书。

本文报告了 308 名卫生工作者中前 150 名 (57M/93F) 连续接受加强疫苗剂量的卫生专业人员的结果，他们最初进入研究并接受了两剂疫苗。所有个体的中位年龄为 49.6 岁，而男性的中位年龄为 54 岁，女性的中位年龄为 49 岁。总体而言，整个样本的 BMI 中位数为 25.9，男性受试者的 BMI 为 27，女性受试者的 BMI 为 24。

图 1A 描绘了从第二次免疫后 2 周到第三次接种后 1 个月 (1MP3D) 期间 Nabs 的抑制百分比。加强剂量后 1 个月，中位抑制百分比为 97.6%（平均 95.9%），显着高于任何其他测试时间点（Wilcoxon p值 <0.001)。只有 1 人 (0.67%) 的 NAb 低于 50%，而绝大多数人（148 名受试者，或 98.7%）具有非常高的保护水平。因此，可以清楚地表明，当 NAbs 水平的这种增加与早期观察到的水平形成对比时，加强剂量会导致中和抗体滴度的爆发。在加强免疫之前，在第二次疫苗接种后 2 周记录到最高水平的 NAbs，此时 NAbs 的持续下降变得明显，表明疫苗正在发挥作用。第二次免疫后 2 周的中位 NAbs 水平为 96.9%，随后在第二次免疫后 1 个月和 9 个月后分别降至 96.1% 和 55.5%。与之前考虑的最高水平（即，第二次疫苗接种后 2 周），1MP3D 的 NAbs 水平高出 151%，鉴于注射引发抗尖峰抗体的发展，这是合理的。在一剂疫苗接种后，恢复期的 COVID-19 患者中描述了高 NAb，²由于在接种疫苗后的第二天迅速而强烈的细胞因子诱导。³这可能是加强剂量后 NAb 升高的原因。同样，加强剂量增加了 NAbs 滴度，即使在对前两次疫苗接种没有反应的骨髓瘤患者中也是如此。⁴

抗 SRBD 抗体也有类似的发现（图 1B），150 名参与者中有 149 名的抗体水平达到了 2500 单位/mL 的上限。1MP3D 时的抗 SRBD 滴度在统计学上显着高于任何之前的测量点（p值 <.001），这与之前的发现一致。在第三次免疫接种后 1 个月，与加强剂量前（即第二次接种后 9 个月）的基线水平相比，观察到抗 SRBDs 大幅上升 804%。最近的一项研究还描述了 97 名 65 岁以上的以色列人的高抗 S IgG 抗体^。5

此外，还检查了各种附加参数对 NAb 和抗 SRBD 滴度的影响。检查了几个因素以确定它们是否会影响每天的抗体水平或由于加强剂量而报告的免疫反应增加，包括年龄、性别、病史（即合并症）和 BMI。对于 NAbs 或抗 SRBDs，男性和女性之间没有发现差异；对于 NAb 和抗 SRBD，NAb 和抗 SRBD的 Mann-Whitney p值分别为 0.803 和 0.670。此外，当参与者分为三个年龄组时，NAbs（Kruskal-Wallis p值 = .230）或抗 SRBDs（p  = .779）抗体均未发现统计学上的显着差异。

这项研究的一个局限性是样本量相对较小，这可能导致无法调查特定的病理生理情况。此外，患有严重伴随疾病（如癌症）的患者被排除在当前研究之外。已证明使用活性免疫抑制药物会显着导致 COVID-19 疫苗接种后体液反应不佳，但此类患者未包括在本研究中。关于性别的作用，应该强调的是，男性和女性的样本量不同，应该考虑到这一点。女性在劳动力中的比例几乎是男性的两倍。虽然不等的样本量通常会导致结果有偏差，但这在我们的情况下不是问题，因为不平衡不是极端的，而是正常的（33.8% 对 66.2%）。

我们得出结论，第三剂疫苗，即 BNT162b2 疫苗完全接种后 9 个月，会产生非常高的 NAb 和抗 SRBD IgG 滴度，无论年龄和性别如何。这可能是导致接受第三剂疫苗的人的 COVID-19 发病率较低的原因，⁶或感染 omicron 变种的人的 COVID-19 症状较轻的原因。