Cellular and humoral immune response to a third dose of BNT162b2 COVID-19 vaccine - a prospective observational study (preprint)

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.

Short-term drop in antibody titer after the third dose of SARS-CoV-2 BNT162b2 vaccine in adults (preprint)

Little is known about the longevity of antibodies after a third dose of BNT162b2 (BioNTech/Pfizer). Therefore, the serum antibody levels were evaluated after the third dose of BNT162b2 which dropped significantly within 11 weeks from 4155.59 {+/-} 2373.65 BAU/ml to 2389.10 {+/-} 1433.90 BAU/ml, p-value <0.001 but remained higher than after the second dose. These data underline the positive effect of third dose of BNT162b2 but shows a rapid and significant drop of antibodies within a short span of time.

SARS-CoV-2 IgG seroprevalence in blood donors located in three different federal states, Germany, July 2020 to June 2021 – a follow-up (preprint)

Seroprevalence studies can contribute to better assess the actual incidence of infection. Since long-term data for Germany are lacking, we determined seroprevalence of IgG-antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 3,759 regular blood donors in three German federal states between July 2020 and June 2021. The IgG seroprevalence was 5.48% (95% confidence interval (CI): 4.77–6.25) overall, ranging from 5.15% (95% CI: 3.73–6.89) in Lower Saxony to 5.62% (95% CI: 4.57–6.84) in North Rhine Westphalia.

Effective high-throughput RT-qPCR screening for SARS-CoV-2 infections in children (preprint)

Systematic SARS-CoV-2 testing is a valuable tool for infection control and surveillance. However, broad application of high sensitive RT-qPCR testing in children is often hampered due to unpleasant sample collection, limited RT-qPCR capacities, and high costs. Here, we developed a high-throughput approach (Lolli-Method) for sensitive SARS-CoV-2 detection in children, combining non-invasive sample collection with an RT-qPCR-pool testing strategy. SARS-CoV-2 infections were diagnosed with sensitivities of 100% and 93.9% when viral loads were >10E6 copies/ml and >10E3 copies/ml in corresponding Naso-/Oropharyngeal-swabs, respectively. For effective application of the Lolli-Method in schools and daycare facilities, SIR-modeling indicated a preferred frequency of two tests per week. The developed test strategy was implemented in 3,700 schools and 698 daycare facilities in Germany, screening over 800,000 individuals twice per week. In a period of 3 months, 6,364 pool-RT-qPCRs tested positive (0.64%) ranging from 0.05% to 2.61% per week. Notably, infections correlated with local SARS-CoV-2 incidences as well as with a school social deprivation index. Moreover, in comparison with the alpha variant, statistical modeling revealed a 31% increase for multiple (>1 child) infections per class following infections with the delta variant. We conclude that the Lolli-Method is a powerful tool for SARS-CoV-2 surveillance and infection control in schools and daycare facilities.

Persistence of immune response in health care workers after two doses BNT162b2 in a longitudinal observational study (preprint)

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.

SARS-CoV-2-antibody response in health care workers after vaccination or natural infection in a longitudinal observational study (preprint)

Background Following a year of development, several vaccines have been approved to contain the global COVID-19 pandemic. Real world comparative data on immune response following vaccination or natural infection are rare. Methods We conducted a longitudinal observational study in employees at a secondary care hospital affected by the COVID-19 pandemic. Comparisons were made about the presence of anti-SARS-CoV-2 immunglobulin G (IgG) antibody ratio after natural infection, or vaccination with one or two doses of BioNTech/Pfizer (BNT162b2), or one dose of AstraZenca (Vaxzevria) vaccine. Results We found a 100% humoral response rate in participants after 2 doses of BNT162b2 vaccine. The antibody ratio in participants with one dose BNT162b2 and Vaxzevria did not differ significantly to those with previous PCR-confirmed infection, whereas this was significantly lower in comparison to two doses of BioNTech/Pfizer. We could not identify a correlation with previous comorbidities, obesity or age within this study. Smoking showed a negative effect on the antibody response (p=0.006) Conclusion Our data provide an overview about humoral immune response after natural SARS-CoV-2 infection or following vaccination, and supports the usage of booster vaccinations, especially in patients after a natural SARS-CoV-2 infection.

Half year longitudinal seroprevalence of SARS-CoV-2-antibodies and rule compliance in German hospital employees (preprint)

Introduction COVID-19, caused by SARS-CoV-2, is an occupational health risk especially for healthcare employees. This study was designed to determine the longitudinal seroprevalence of specific immunglobolin-G (IgG)-antibodies in employees in a hospital setting. Methods All employees including healthcare and non-healthcare workers in a secondary care hospital were invited to participate in this single-center study. After an initial screening, a 6 months follow-up was done which included serological examination for SARS-CoV-2-IgG-antibodies and a questionnaire for self-reported symptoms, self-perception and thoughts about the local and national hygiene and pandemic plans. Results The seroprevalence of SARS-CoV-2-IgG-antibodies was 0.74% among 406 hospital employees (95% confidence interval) (0.75% in healthcare workers, 0.72% in non-healthcare workers), initially recruited in April 2020, in their follow-up blood specimen in October 2020. In this study, 30.54% of the participants reported using the official German corona mobile application and the majority were content with the local and national rules in relation to Coronavirus restrictions. Discussion At the 6 months follow-up, the 0.74% seroprevalence was below the reported seroprevalence of 1.35% in the general German population. The prevalence in healthcare workers in direct patient care compared with those without direct patient contact did not differ significantly.

A combined strategy to detect plasma samples reliably with high anti-SARS-CoV-2 neutralizing antibody titers in routine laboratories (preprint)

The determination of anti-SARS-CoV-2 neutralizing antibodies (NAbs) is of interest in many respects. High NAb titers, for example, are the most important criterion regarding the effectiveness of convalescent plasma therapy. However, common cell culture-based NAb assays are time-consuming and feasible only in special laboratories. Our data reveal the suitability of a novel ELISA-based surrogate virus neutralization test (sVNT) to easily measure the inhibition-capability of NAbs in the plasma of COVID-19 convalescents. We propose a combined strategy to detect plasma samples with high NAb titers (≥ 1:160) reliably and to, simultaneously, reduce the risk of erroneously identifying low-titer specimens. For this approach, results of the sVNT assay are compared to and combined with those acquired from the Euroimmun anti-SARS-CoV-2 IgG assay. Both assays are appropriate for high-throughput screening in standard BSL-2 laboratories. Our measurements further show a long-lasting humoral immunity of at least 11 months after symptom onset.

Evidence of long-lasting humoral and cellular immunity against SARS-CoV-2 even in elderly COVID-19 convalescents showing a mild to moderate disease progression (preprint)

After the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in China in late 2019, a pandemic evolved that has claimed millions of lives so far. While about 80 % of infections cause mild or moderate COVID-19 disease, some individuals show a severe progression or even die. Most countries are far from achieving herd-immunity, however, the first approved vaccines offer hope for containment of the virus. Although much is known about the virus, there is a lack of information on the immunity of convalescent individuals. We here evaluate the humoral and cellular immune response against SARS-CoV-2 in 41 COVID-19 convalescents. As previous studies mostly included younger individuals, one advantage of our study is the comparatively high mean age of the convalescents included in the cohort considered (54 {+/-} 8.4 years). While anti-SARS-CoV-2 antibodies were still detectable in 95 % of convalescents up to 8 months post infection, an antibody-decay over time was generally observed in most donors. Using a multiplex assay, our data additionally reveal that most convalescents exhibit a broad humoral immunity against different viral epitopes. We demonstrate by flow cytometry that convalescent donors show a significantly elevated number of natural killer cells when compared to healthy controls, while no differences were found concerning other leucocyte subpopulations. We detected a specific long-lasting cellular immune response in convalescents by stimulating immune cells with SARS-CoV-2-specific peptides, covering domains of the viral spike, membrane and nucleocapsid protein, and measuring interferon-{gamma} (IFN-{gamma}) release thereafter. We modified a commercially available ELISA assay for IFN-{gamma} determination in whole-blood specimens of COVID-19 convalescents. One advantage of this assay is that it does not require special equipment and can, thus, be performed in any standard laboratory. In conclusion, our study adds knowledge regarding the persistence of immunity of convalescents suffering from mild to moderate COVID-19. Moreover, our study provides a set of simple methods to characterize and confirm experienced COVID-19.

Ultra-fast one-step RT-PCR protocol for the detection of SARS-CoV-2 (preprint)

The COVID-19 pandemic resulted in lockdowns all over the world thus affecting nearly all aspects of social life and also had a huge impact on global economies. Since vaccines and therapies are still not available for the population, prevention becomes desperately needed. One important aspect for prevention is the identification and subsequent isolation of contagious specimens. The currently used methods for diagnostics are time consuming and also hindered by the limited availability of reagents and reaction costs, thus presenting a bottle neck for prevention of COVID-19 spread. Here, we present a new ultra-fast test method which is ten times faster than conventional diagnostic tests using real time quantitative PCR (RT-qPCR). In addition, this ultra-fast method is easy to handle as well as cost effective. We translated published SARS-CoV-2 testing protocols from the Centers of Disease Control and Prevention (Atlanta, Georgia, USA) and the Charite Berlin (Germany) to the NEXTGENPCR (NGPCR) machine and combined it with a fluorescence-based endpoint measurement. Fluorescence was measured with a commercial blue light scanner. We confirmed the NEXTGENPCR results with commercially available positive controls. In addition, we isolated RNA from SARS-CoV-2 infected patients and achieved similar results to clinical RT-qPCR assays. Here, we could show correlation between the results obtained by NEXTGENPCR and conventional RT-qPCR.