key: cord-0894465-8b8iyytj
authors: Herzberg, J.; Fischer, B.; Becher, H.; Becker, A.-K.; Honarpisheh, H.; Guraya, S. Y.; Strate, T.; Knabbe, C.
title: Cellular and humoral immune response to a third dose of BNT162b2 COVID-19 vaccine - a prospective observational study
date: 2022-03-13
journal: nan
DOI: 10.1101/2022.03.10.22272204
sha: aa9953beb8f72982bb2e7737259a7ce99d2567d0
doc_id: 894465
cord_uid: 8b8iyytj

Background Since the introduction of various vaccines against SARS-CoV-2 at the end of 2020, rates of infection have continued to climb worldwide. This led to the establishment of a third dose vaccination in several countries, known as a booster. To date, there has been little real-world data about the immunological effect of this strategy. Methods We compared the humoral- and cellular immune response before and after the third dose of BioNTech/Pfizer vaccine BNT162b2, following different prime-boost regimes. Humoral immunity was assessed by determining anti-SARS-CoV-2 binding antibodies using a standardized quantitative assay. In addition, neutralizing antibodies were measured using a commercial surrogate ELISA-assay. Interferon-gamma release was measured after stimulating blood-cells with SARS-CoV-2 specific peptides using a commercial assay to evaluate the cellular immune response. Results The median antibody level increased significantly after the third dose to 2663.1 BAU/ml vs. 101.4 BAU/ml (p < 0.001) before administration of the boosting dose. This was also detected for neutralizing antibodies with a binding inhibition of 99.68% {+/-} 0.36% vs. 69.06% {+/-} 19.88% after the second dose (p < 0.001). 96.3% of the participants showed a detectable T-cell-response after the third dose with a mean interferon-gamma level of 2207.07 mIU/ml {+/-} 1905 mIU/ml. Conclusion This study detected a BMI-dependent increase after the third dose of BNT162b2 following different vaccination protocols, whereas all participants showed a significant increase of their immune response. This, in combination with the limited post-vaccination-symptoms underlines the potential beneficial effect of a BNT162b2-boosting dose.

Following an initially flattened curve of COVID-19 infections, due to vaccination campaigns all over the world, there came a subsequent resurgence of COVID-19 infections worldwide [1] . New variants of SARS-CoV-2 and decreasing immunity after vaccination over time [2, 3] , have caused an increased rate of infection and hospitalization also in vaccinated people [1] .

A well-established approach to handle the so-called secondary vaccine failure, is the use of a booster dose -an additional vaccine dose after the initial round of immunization [1, 4] . The effectiveness of a third dose of BioNTech/Pfizer vaccine was suggested by different studies [5] [6] [7] . Therefore, after approval by the regulatory authorities in US [8] and in the European Union [9] , several countries such as Israel, USA, UK and Germany, initiated a vaccination program for administration of a third dose in vulnerable groups at least 5 months after complete vaccination [1, 10] .

The effect of this third dose on the immune response and the resulting protective effects have been analyzed in register or clinical studies [1, 7, 11, 12] .

As the impact of different vaccines on the immune system varies, considering both cellular and humoral immunity is crucial. The detected levels of neutralizing antibodies have been shown to be higher after administration of the BioNTech/Pfizer vaccine BNT162b2 than those after receiving the AstraZeneca vaccine ChAdOx1, although this could not be shown

for T-cell responses [13] . These differences have led to the discussion regarding potential benefit from a heterologous vaccination strategy [13] [14] [15] [16] .

We aimed to evaluate the humoral-and cellular immune response after a third booster-dose of BNT162b2 following an initial administration of either two doses of BNT162b2 (Group 1), two doses of ChAdOx1 (Group 2) or cross-vaccination of BNT162b2+ChAdOx1 (Group 3) in a real-world setting analyzing one of the most important groups in this global pandemichealth care workers.

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In this study we evaluated the effect of administration of a third dose of BioNTech/Pfizer mRNA vaccine BNT162b2, within a longitudinal study in health care workers initiated in April 2020 [17] . All employees of the Hospital Reinbek St. Adolf-Stift, a secondary care hospital located in Northern Germany, were eligible to participate in the study. The vaccination program for employees was established in December 2020. At the end of October 2021, the Robert-Koch-Institute recommended administration of a third dose in specific groups [18] .

Following this recommendation, beginning in November 2021 all employees who had received their second vaccination more than 6 months prior, were invited for a third boosterdose. In order to have a reference value for the determination of the booster-effect, blood samples were initially collected and analyzed before the administration of the third vaccine dose on November 13 th -14 th 2021 [2] .

To evaluate the efficiency of the third dose all participants were asked to provide an additional blood sample and complete a questionnaire 4 weeks after the booster-vaccination (December 13 th -14 th 2021).

The anti-SARS-CoV-2-igG antibody-titer was expressed in Binding Antibody Units per ml (BAU/ml) to stay in accordance with the WHO standard. Antibodies were determined by using a fully automated quantitative anti-SARS-CoV-2-assay (IgG) from Abbott (Chicago, USA). As recommended by the manufacturer, a value below 7.1 BAU/ml was determined to be negative whereas values ≥ 7.1 BAU/ml were determined to be positive.

To evaluate the neutralizing anti-SARS-CoV-2 antibodies, all samples were additionally tested using the NeutraLISA™ SARS-CoV-2 Neutralization Antibody Detection KIT (Euroimmun, Lübeck, Germany). Results were presented in binding inhibition, where values ≥ 35 % were considered positive.

In addition to the humoral immune response, the cellular immunity to SARS-CoV-2 was also assessed, using the Quan-T-cell SARS-CoV-2 kit (Euroimmung, Lübeck, Germany) working as an Interferon-gamma release assay (IGRA). The analysis for this Quan-T-cell test was performed as previously reported [2] . IFN-gamma concentration was expressed as mIU/ml. Values ≥ 200 mIU/ml were considered positive.

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Before and after administration of the third dose of vaccine, all participants were asked to complete a questionnaire regarding their prior vaccinations, smoking behaviors and especially after the third dose, possible side effects of the third dose. Specifically, participants were questioned about localized symptoms (local pain, lymphadenopathy) and systemic symptoms, such as fever or fatigue.

This study was conducted in accordance with the Declaration of Helsinki. All participants provided written and informed consent before inclusion. This study was prospectively Relationships between categorial variables were tested using the Chi-square test or the Fisher's exact test, depending on the size of the groups. Inter-group differences were analyzed using Mann-Whitney-U test or Kruskal-Wallis-test reporting the mean with standard deviation. Pre-and post-third dose antibody and INF-gamma levels were compared using Wilcoxon sign rank test. A p-value < 0.05 was considered statistically significant.

To investigate the joint effect of age, sex, body mass index and current smoking on the relative increase of antibody-level, a linear regression analysis was done to take the antibody level before booster into account. The IgG-levels had a skewed distribution and was logarithmized for the regression analysis yielding an approximately normal distribution.

As the dependent variable, the difference of the logarithmized IgG-levels after and before booster was used. The backward selection method was used to determine the final model. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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Before administration of the third vaccine-dose, 310 participants provided a blood specimen, of which 243 resubmitted a blood sample and completed questionnaire exactly 4 weeks after the booster dose (follow-up-rate 78.39%).

The participants included 59 (24.3%) male and 184 (75.7%) female participants with a mean age of 46.33 ± 11.44 years. The characteristics of the study cohort are described in Table 1 , grouped according to their initial vaccination protocol. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 13, 2022 Figure 1A ). The median relative increase was 25.9 (IQR 15.5-48.7).

The administration of a third dose of BNT162b2 vaccine also caused a significant increase in neutralizing antibodies, throughout all study groups (99.68 % ± 0.36 % vs. 69.06 % ± 19.88 %; p < 0.001) ( Figure 1B) . No participants showed a binding inhibition capability below 96 % following the booster-vaccination. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 13, 2022. ; https://doi.org/10.1101/2022.03.10.22272204 doi: medRxiv preprint A: Anti-SARS-CoV-2-IgG binding antibodies were determined using a quantitative assay from Abbott. In keeping with the WHO-standard, data were expressed in Binding Antibody Units per ml (BAU/ml). Samples were marked seronegative below 7.1 BAU/ml whereas values above 7.1 BAU/ml were determined to be positive, as mentioned by the manufacturer. B: Binding inhibition capability of Neutralizing anti-SARS-CoV-2 antibodies was determined using the NeutraLISA™ SARS-CoV-2 Neutralization Antibody Detection KIT from Euroimmun. According to the manufacturer, binding inhibition values above 35% were considered positive (horizontal black dotted line), whereas values between 20% and 35% were considered equivocal (horizontal gray dotted line). C: Cellular immunity to SARS-CoV-2 was assessed by using an Interferon (IFN)-gamma release assay (IGRA) from Euroimmun (Quan-T-cell SARS-CoV-2 kit). Values > 200 mIU/ml were considered positive (horizontal black dotted line), whereas values between 100-200 mIU/ml were considered equivocal (horizontal gray dotted line). ****p < 0.0001 (Mann-Whitney U-test).

Anti-SARS-CoV-2 binding antibody levels differed significantly between the subgroups (representing different prior vaccination protocols), prior to booster-administration. In brief, pre-boost antibody levels were highest in Group 2 (183.1 BAU/ml), followed by Group 1 (124.9 BAU/ml) and Group 3 (52.75 BAU/ml). The administration of a third BNT162b2 dose led to a highly significantly increased antibody-level, across all analyzed subgroups.

Inductive effects were highest within Group 3 participants (mean 2358 vs. 52.75 (4,370%), p = 0.002), followed by Group 1 (mean 3661 vs. 124.9 (2,831 %), p < 0.001) and Group 2 (mean 2122 vs. 183.1 (1,058%), p < 0.001) participants (Figure 2 and Table 2 ).

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. booster-dosage. Anti-SARS-CoV-2-IgG binding antibodies were determined before-and 4 weeks after third booster-dosage using a quantitative assay from Abbott. In keeping with the WHO-standard, data were expressed in Binding Antibody Units per ml (BAU/ml). Samples were marked seronegative below 7.1 BAU/ml whereas values above 7.1 BAU/ml were determined to be positive, as mentioned by the manufacturer. *p < 0.05; ***p < 0.001; ****p < 0.0001 (Mann-Whitney U-test).

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Neutralizing antibody levels also differed significantly between the groups prior to boosteradministration (Figure 3 and Table 2 ). Specifically, individuals within Group 3 showed the lowest percentage binding inhibition (49.55%), followed by Group 1 (67.66%) and Group 2 (77.57%) participants. The administration of a third BNT162b2 dose caused a significant induction of binding antibody capability within all three groups, whereby the strongest effect was detected for Group 3 (99.70% vs. 49.55% (induction: 101.21%)), followed by Group 1 (99.71% vs. 67.66% (induction: 47.37%)) and Group 2 (99.59% vs. 77.57% (induction:

28.39%)) participants. Post-booster binding inhibition capabilities did not differ significantly between the different groups. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Prior to the administration of a booster BNT162b2 dosage, SARS-CoV-2 specific IFNgamma release of stimulated blood-cells significantly differed between individuals of each considered group (Figure 4, Table 2 ). Briefly, Group 1 participants showed the lowest IFNgamma mean-value (560.4 mIU/ml), followed by individuals belonging to Group 3 (654.7 mIU/ml) and Group 2 (828.7 mIU/ml). SARS-CoV-2 specific t-cell response significantly 

In the linear regression analysis, we identified the body mass index as a significant predictor for the antibody-level increase. The increase was also dependent on the antibody-level . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 13, 2022. ; https://doi.org/10.1101/2022.03.10.22272204 doi: medRxiv preprint before booster. Neither sex, age or smoking had a significant effect. The estimated effect of a previous COVID-19 infection is negative, however since very few individuals (N=2) had an infection, no conclusion can be draws from this result. Table 3 gives the result of the full regression model. The fit of the model was very good with an R 2 value of 0.43. The regression coefficients remained virtually unchanged when only the significant variables, BMI and antibody-level before booster, remained in the final model ( Table 3) . From the regression model, the estimated antibody-level showed both an absolute and relative increase given the BMI and the antibody-level before booster. Table 4 is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The majority of the study cohort reported some minor side-effect following the booster dose ( . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. [19] . In our study, we considered a much longer time period of 6 months.

Our study shows the highest increase in INF-gamma release, binding-antibody expression, as well as neutralizing antibody capability for participants initially vaccinated two times with ChAdOx1. However, these participants showed the weakest humoral immune response before administration of the booster dose. A regression analysis detected a high BMI to be associated with an increased immune response. Besides the determined overall binding antibody titers, the neutralizing antibodies play a main role in the humoral immune response and are correlated with protection against severe COVID-19 outcome [24] .

Using the commercial ELISA-based NeutraLISA assay from Euroimmun, our data show a substantial increase in neutralizing antibody capability in all participants after boosteradministration. This is in line with the data presented from Atmar et al. [23] . Data from US and Israel underline the protective effect of a third dose of BNT162b2 against a severe COVID-19-course [25] [26] [27] .

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Multiple studies have tried, and are still attempting, to evaluate possible factors associated with a reduced immune response or with a complete non-response to anti-SARS-CoV-2-vaccination.

Within the study cohort presented here, smoking, increased age and BMI have previously been shown to have a negative impact in studies involving two doses of BNT162b2 [28] or 9 months after vaccination, respectively [2] . This could not be seen in the results presented here (Table 3) .

In contrast, a higher BMI was associated with a stronger increase of the antibody titer after the third dose. This may be due to a lower antibody titer before administration of the third dose as reported previously [2] . Soffer et al. also found a positive correlation after COVID-19 infection for antibody titers and BMI, whereas Frasca et al. found the opposite correlation [29] .

Other factors potentially influencing the immune response after a third dose of BNT162b2 are immunosuppression, for instance in patients after solid-organ transplantation [20] .

In our cohort, we could not detect any negative impact of older age, although elderly participants are relatively underrepresented in this trial. Data published by Eliakim-Raz et. al. also described no correlation to higher age in a group older than 60 years [21] .

Further studies are required to provide more accurate information on risk factors associated with reduced immunity to SARS-CoV-2.

Despite the fact this study benefits from its real-life data in an important group of health-care workers, and a relatively large number of participants, it comes with some limitations. This study is limited by its single-center design. The inclusion of health-care workers led to the overrepresentation of women and younger people, whereas groups with a higher risk for low immune response to vaccination, such as elderly and participants with an immunomodulatory treatment, are underrepresented. Unfortunately, the number of individuals in each group of vaccination protocols were unequally distributed, as there was no individual choice regarding the vaccine received.

This study showed a BMI-dependent increase after the third dose of BNT162b2 following different vaccination protocols, although all participants showed a significant increase in their immune response. This effect, in combination with mild-to-none post-vaccination symptoms, underlines the potential beneficial effect of a BNT162b2-booster dose following 2 doses anti-. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted March 13, 2022. ; https://doi.org/10.1101/2022.03.10.22272204 doi: medRxiv preprint SARS-CoV-2-vaccine. Further studies are needed to evaluate thresholds for the immune response -both humoral and cellular -and to detect the longevity of the booster-induced immunity.

We would like to thank all team members at the Krankenhaus Reinbek St. Adolf-Stift, and all participating laboratories that analyzed these additional samples in a phase of high workload.

Thanks also to Rebecca Zimmer for her intense and rapid assistance with linguistic enrichment.

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