key: cord-0334876-39onotmd authors: Herzberg, J.; Fischer, B.; Lindenkamp, C.; Becher, H.; Becker, A.-K.; Honarpisheh, H.; Guraya, S. Y.; Strate, T.; Knabbe, C. title: Persistence of immune response in health care workers after two doses BNT162b2 in a longitudinal observational study date: 2021-12-17 journal: nan DOI: 10.1101/2021.12.17.21267926 sha: 8b2844eb2531f7327581c826a314d7a6be3ea27d doc_id: 334876 cord_uid: 39onotmd Background The mRNA-based vaccine BNT162b2 of BioNTech/Pfizer has shown high efficacy against SARS-CoV-2 infection and a severe course of the COVID-19 disease. However, little is known about the long-term durability of the induced immune response resulting from the vaccination. Methods In a longitudinal observational study in employees at a German hospital we compared the humoral and cellular immune response in 184 participants after two doses of the BioNTech/Pfizer vaccine (BNT162b2) with a mid-term follow-up after 9 months. Anti-SARS-CoV-2 binding antibodies were determined using both a quantitative and a semi-quantitative assay. For a qualitative assessment of the humoral immune response, we additionally measured neutralizing antibodies. Cellular immune response was evaluated by measuring Interferon-gamma release after stimulating blood-cells with SARS-CoV-2 specific peptides using a commercial assay. Results In the first analysis, a 100% humoral response rate was described after two doses of BNT162b2 vaccine with a mean antibody ratio of 8.01 {+/-} 1.00. 9 months after the second dose of BNT162b2, serological testing showed a significant decreased mean antibody ratio of 3.84 {+/-} 1.69 (p < 0.001). Neutralizing antibodies were still detectable in 96% of all participants, showing an average binding inhibition value of 68.20% {+/-} 18.87%. Older age, (p < 0.001) and obesity (p = 0.01) had a negative effect on the antibody persistence. SARS-CoV-2 specific cellular immune response was proven in 75% of individuals (mean IFN-gamma release: 579.68 mlU/ml {+/-} 705.56 mlU/ml). Conclusion Our data shows a declining immune response 9 months after the second dose of BNT162b2, supporting the potentially beneficial effect of booster vaccinations, the negative effect of obesity and age stresses the need of booster doses especially in these groups. The mRNA-based vaccine BNT162b2 of BioNTech/Pfizer has shown high efficacy against SARS-CoV-2 infection and a severe course of the COVID-19 disease. However, little is known about the long-term durability of the induced immune response resulting from the vaccination. In a longitudinal observational study in employees at a German hospital we compared the humoral and cellular immune response in 184 participants after two doses of the BioNTech/Pfizer vaccine (BNT162b2) with a mid-term follow-up after 9 months. Anti-SARS-CoV-2 binding antibodies were determined using both a quantitative and a semi-quantitative assay. For a qualitative assessment of the humoral immune response, we additionally measured neutralizing antibodies. Cellular immune response was evaluated by measuring Interferon-gamma release after stimulating blood-cells with SARS-CoV-2 specific peptides using a commercial assay. In the first analysis, a 100% humoral response rate was described after two doses of BNT162b2 vaccine with a mean antibody ratio of 8.01 ± 1.00. 9 months after the second dose of BNT162b2, serological testing showed a significant decreased mean antibody ratio of 3.84 ± 1.69 (p < 0.001). Neutralizing antibodies were still detectable in 96% of all participants, showing an average binding inhibition value of 68.20% ± 18.87%. Older age, (p < 0.001) and obesity (p = 0.01) had a negative effect on the antibody persistence. SARS-CoV-2 specific cellular immune response was proven in 75% of individuals (mean IFN-gamma release: 579.68 mlU/ml ± 705.56 mlU/ml). Our data shows a declining immune response 9 months after the second dose of BNT162b2, supporting the potentially beneficial effect of booster vaccinations, the negative effect of obesity and age stresses the need of booster doses especially in these groups. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint In order to fight the global coronavirus disease 2019 (COVID-19) pandemic, a variety of vaccines were applied worldwide, beginning in December 2020, and were felt to be the pivoting point in this pandemic situation. The mRNA vaccines, provided by BioNTech/Pfizer (BNT162b2) and Moderna (Spikevax) have shown high efficacy in clinical trials and real-worlddata [1, 2] . Until now, real-world-data regarding the persistence of the induced humoral and especially cellular immune response post-vaccination are rare. Even in short-term studies, elderly people were reported to have a less intense immune response after two doses of the BioNTech/Pfizer vaccine BNT162b2 [3, 4] . This was also seen in patients under immunosuppression, such as after organ transplantation [5] . Initial studies reported a decrease in antibodies after 6 months [4, 6] or a correlating increased number of infections after complete vaccination [7] ; but data regarding the longitudinal serological dynamics of immunization after BNT162b2 vaccine is limited. In this trial we report the mid-term dynamics of immune response, 9 months after second dose of BNT162b2, by determining anti-SARS-CoV-2-immunglobulin G (IgG) antibodies, T-cellresponse and neutralizing antibodies. The study cohort consisted of a well-defined group of health care employees, known to be under a higher risk for COVID-19. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint In April 2020 we initiated a longitudinal study in health care workers measuring seroepidemiological data during the COVID-19 pandemic [8] . All employees of the secondary care hospital located in the province of Schleswig-Holstein near the border of the city of Hamburg in Northern Germany were invited to participate. All employees were offered vaccination against SARS-CoV-2 starting in December 2020. In April 2021, all participants were invited to provide a blood specimen to evaluate the antibody prevalence after vaccination or For this study, an additional analysis of all participants around 9 months after second dose of BioNTech/Pfizer vaccine was made to evaluate the longitudinal persistence of the vaccineinduced immune response. All blood samples were collected on November 13 th -14 th 2021 and all participants were asked to complete an additional questionnaire, regarding post-vaccination reactions and previous SARS-CoV-2 infections. All participants provided written and informed consent prior to enrolment. This study was prospectively registered at the German Clinical Trial Register (DRKS00021270) after approval by the Ethics Committee of the Medical Association Schleswig-Holstein. All study activities were conducted in accordance with the Declaration of Helsinki. The fully automated semiquantitative anti-SARS-CoV-2-ELISA (IgG) from Euroimmun (Lübeck, Germany) was used to detect the S1 domain of the SARS-CoV-2 spike-protein. In accordance to the manufacturer this test shows a specificity of 99.0% and sensitivity of 93.8% [10] . As this was the same test used within the previous analyses, a longitudinal comparability was ensured. A ratio below 0.8 was considered negative, a ratio ≥ 0.8 to < 1.1 was considered equivocal, and a ratio ≥ 1.1 was considered positive as defined by the manufacturer. In addition, a fully automated quantitative anti-SARS-CoV-2-assay (IgG) from Abbott (Chicago, USA) was performed. 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. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 17, 2021. ; https://doi.org/10.1101/2021.12.17.21267926 doi: medRxiv preprint All samples were analyzed for neutralizing anti-SARS-CoV-2 antibodies using the NeutraLISA™ SARS-CoV-2 Neutralization Antibody Detection KIT (Euroimmun, Lübeck, Germany) in accordance to the manufacturer's instructions. Binding inhibition values above 35% were considered positive, whereas values between 20% and 35% were considered equivocal. 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). The assay was performed according to manufacturer´s instructions. In brief, 500 µl of heparinized blood was stimulated with SARS-CoV-2 specific peptides covering regions of the viral S1-domain. After incubating the tubes (37°C, 22 h), plasma was collected and tested for Interferon-gamma release using an ELISA-assay (Quan T-cell ELISA, Euroimmun). Background IFN-gamma values were assessed by incubating blood without prior peptide-stimulation. As a positive control, blood cells were stimulated with mitogen, resulting in a broad and unspecific IFN-gamma secretion. IFN-gamma concentration was expressed as mIU/ml. IBM SPSS Statistics Version 25 (IBM Co., Armonk, NY, USA) was used for statistical analysis. Graphics were elaborated using IBM SPSS Statistics Version 25 (IBM Co., Armonk, NY, USA) and GraphPad Prism 9. All variables are presented as means with standard deviation. Categorical variables are shown as numbers with percentages. Fisher's exact test or chi-square test was used to determine relationships between categorical variables depending on size of groups. Exact 95% confidence intervals were provided where appropriate. Differences between groups were analyzed using Wilcoxon test. Inter-group differences were analyzed using Mann-Whitney-U test or Kruskal-Wallis-test. A linear regression analysis was done to investigate the joint effect of age, sex, body mass index and current smoking on antibody and t-cell response using the backward selection method. The t-cell response had a skewed distribution and was logarithmized for the regression analysis. A p-value < 0.05 was considered statistically significant. . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint A total of 184 participants provided a blood sample 9 (range 7-9 months) months after receiving their second dose of BioNTech/Pfizer. This meant a follow-up rate of 58.41% compared to the analysis after the second dose of vaccine (315 participants in April 2021) [9] . In this follow-up, the study characteristics did not differ significant to the previous follow-ups with 73.4% female and 26.6% male and a mean age of 46.32 ± 10.91 years. 3 participants reported a previous SARS-CoV-2 infection. Of these, 1 was reported prior to vaccination, and 2 cases occurred between the second dose of vaccine and this follow-up ( Table 1) . These participants are included in the following analysis. In the previous analysis, all participants showed a positive antibody-ratio after two doses of BioNTech/Pfizer, whereas two participants seroconverted to an equivocal result in this followup after 9 months. The antibody-ratio was significantly lower in the follow-up analysis after 9 months (8.01 ± 1.00. vs. 3.84 ± 1.69; p < 0.001). The mean reduction of the IgG antibody-ratio was 53.11% ± 17.95%. In order to further improve the assessment of the humoral immune response, quantification of anti-SARS-CoV-2 antibodies was performed according to WHO standards (BAU/ml). All participants showed antibody levels above the manufacturer's cutoff (mean: 124.67 ± 104.41 BAU/ml) (Figure 1) . . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Overall, 96% of study-participants showed neutralizing antibodies against SARS-CoV-2. Our data show a mean binding inhibition capability of 68.20% ± 18.87% 9 months after the second vaccination using BioNTech/Pfizer vaccine (Figure 2 ). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 17, 2021. ; https://doi.org/10.1101/2021.12.17.21267926 doi: medRxiv preprint We identified several factors associated with a lower T-cell response and a lower level of neutralizing antibodies ( Table 2) . A BMI above 30 (p = 0.004) and smoking (p = 0.034) was associated with a reduced level of neutralizing antibodies. Male participants showed a lower T-cell response at this timepoint, whereas this difference remained insignificant following the Mann-Whitney-U-test. During the 9 months follow-up period, the antibody-ratio showed a significant decrease in older participants (p = 0.003 following the Kruskal-Wallis-test) (Figure 4) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The additionally performed linear regression analysis confirmed the univariate analyses regarding the differences in persisting immune response after 9 months. A significant negative effect on antibody persistence was observed for older (p<0.001) and obese (p=0.01) participants. Smoking had also a negative effect, with p-value slightly above the limit (p=0.08) Table 3 gives the regression estimates and the corresponding p-values and 95% confidence intervals. Sex had no effect. The same result was also found for neutralizing antibodies as dependent variable. Table 4 gives the corresponding results. Thus, the estimated neutralizing antibodies value for a nonsmoker, age 50 and BMI 25 is 68.705%. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 17, 2021. ; https://doi.org/10.1101/2021.12.17.21267926 doi: medRxiv preprint This study analyzes the persistence of the humoral and cellular response, including neutralizing antibodies, to BioNTech/Pfizer vaccine in a well-defined group of hospital employees as part of a longitudinal evaluation. Up until now, little is known about the persistence of the immune response after vaccination with BioNTech/Pfizer. This study reveals a significant antibody decrease in a mid-term-followup in all subgroups, affecting especially older people and people with a high BMI. Currently there is minimal data about the long-lasting effect following active immunization with mRNA vaccines. In our cohort, the antibody ratio decreased in the 9-month follow-up to about the half of the initial value. This was not surprising, as first half-year follow-ups showed a decrease during that timeframe [4] , and it is well known, that not all plasmablasts commit as memory plasma cells [11, 12] . The decreased antibody ratio is also detected in the decreased neutralization capacity. Several studies found this decrease to begin as early as 3 months after vaccination [4, 13] . Discussing the long-lasting effect of mRNA vaccines (such as the one from BioNTech/Pfizer) in protecting against COVID-19 disease, the role of cellular immunity in addition to humoral response might be important but is often disregarded due to elaborate assays, which are not feasible in standard-laboratories. We determined cellular immunity in a simplified approach by IFN-gamma release after stimulation of blood-cells with specific SARS-CoV-2 peptides. In our cohort, 73.40% showed a detectable T-cell response even 9 months after vaccination. This is in line with data presented by Naaber et al. for their 6-months follow-up [4] , and in keeping with the initial phase I/II trial showing an activation of T-cells after using mRNA vaccines [14] . Compared to our data, Tober-Lau et al. determined a higher median IFN-gamma release six months after BNT162b2-vaccination within their HCW-cohort using the same IGRA-assay (1198.0 mIU/ml vs. 412.0 mIU/ml) [15] . In addition, binding inhibition capability of neutralizing antibodies was considerably lower within our cohort (88.1 % vs. 68.2 %). Reasons for this may be the different time points of examination after second vaccination and the higher average age of our cohort. In our follow-up study, we found a correlation between age, obesity and smoking with respect to almost all considered immune responses. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint As shown in previous studies, we confirmed the negative correlation between antibody responses and the age of vaccinated individuals [3, 4] . In addition to the reduced antibody response after vaccination older people are known to have a faster decrease after vaccination [4] . Interestingly, obese participants with a BMI higher than 30 showed a significantly higher decrease in the antibody ratio and also significantly lower neutralizing antibodies not only in the univariate analysis but also following the regression analysis. This effect was first described by Watanabe et al. [16] and seen by Malavazos et al. [17] in the first months after vaccination. The effect of smoking immediately after vaccination was already discussed [9, 16] and is reported to influence the effectiveness of other vaccines due to a general immunosuppression caused by smoking [16, 18] . In this 9-month evaluation, for the only significant difference was with respect to neutralizing antibodies. These findings are important as obese people and elderly are known to have a higher risk not only for SARS-CoV-2 infection, but especially for a severe course of 20] . Further studies are needed to evaluate the longitudinal course of cellular and humoral immune response not only after two doses of BioNTech/Pfizer but also after a third dose. The major limitation of this trial is its single-center design. Due to the inclusion of hospital employees, women are relatively overrepresented and other groups with a higher risk are underrepresented. Especially elderly participants, with an age over 70 years, are not included in this study. It cannot be excluded that there were asymptomatic, undetected SARS-CoV-2 infections among the participants during the 9 months after second vaccination, which may lead to a slight bias in the results. This study showed an age-dependent decrease of vaccine-induced anti-SARS-CoV-2-IgG antibodies in a midterm-follow-up after 9 months. People with a higher risk for a severe COVID-19-disease course, such as elderly or obese people, showed a reduced immune response at this timepoint after vaccination. These results encourage the use, and need, of is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 17, 2021. ; https://doi.org/10.1101/2021.12.17.21267926 doi: medRxiv preprint so-called booster doses 6 months after the second dose of the BioNTech/Pfizer vaccine BNT162b2, especially in vulnerable individuals such as elderly or obese people. 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 (linguistic enrichment) for her intense and rapid help. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 17, 2021. ; https://doi.org/10.1101/2021.12.17.21267926 doi: medRxiv preprint FDA-authorized mRNA COVID-19 vaccines are effective per real-world evidence synthesized across a multi-state health system Interim Estimates of Vaccine Effectiveness of BNT162b2 and mRNA-1273 COVID-19 Vaccines in Preventing SARS-CoV-2 Infection Among Health Care Personnel, First Responders, and Other Essential and Frontline Workers -Eight Age-dependent immune response to the Biontech/Pfizer BNT162b2 COVID-19 vaccination Dynamics of antibody response to BNT162b2 vaccine after six months: a longitudinal prospective study Poor humoral and T-cell response to two-dose SARS-CoV-2 messenger RNA vaccine BNT162b2 in cardiothoracic transplant recipients Effectiveness of mRNA BNT162b2 COVID-19 vaccine up to 6 months in a large integrated health system in the USA: a retrospective cohort study Resurgence of SARS-CoV-2 Infection in a Highly Vaccinated Health System Workforce A Prospective Sero-epidemiological Evaluation of SARS-CoV-2 among Health Care Workers in a German Secondary Care Hospital. 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