The concept of trained immunity defines the long-term functional reprogramming of innate immune cells, which is evoked by exogenous or endogenous insults and leads to an altered response towards a second challenge after return to a non-activated state and is characterized by several markers, such as specific cytokines, activation markers of innate immune cells and epigenetic modifications, e.g. H3K4me3. Vaccinations have been shown to induce trained immunity and to have heterologous effects on other infections or vaccinations. A recent article showed, that individuals who had received recombinant adjuvanted zoster vaccine (RZV) before the pandemic had a 16% lower risk of COVID-19 diagnosis and a 32% lower risk of hospitalization suggesting a protective heterologous effect of RVZ on COVID19 infections. So far, the mechanisms behind these add-on benefits of RZV vaccination are on the hypothetical level and need further experimental evidence.

Therefore, we aim to investigate the specific humoral and cellular immune response towards COVID-19 vaccine in healthy individuals who were exposed to RZV 1 to 12 months before COVID-19 vaccination compared to individuals who did not receive RZV before. Particular emphasis is layed on COVID-19 vaccine non-responders and individuals with breakthrough infections indicating lower vaccine efficacy compared to those who had no breakthrough infection.

The primary objective is the cytokine profile of spike protein-stimulated T, NK and NKT cells. Spike protein stimulated T, NK and NKT cells are characterized by cell surface markers, transcription factor expression, chemokine receptor expression, activation and proliferation markers and by their lineage-specific cytokine pattern. CD14+ monocytes are magnetically isolated and further characterized by cell-culture experiments imitating a training and resting period after stimulation. Epigenetic modifications by methylation of CpG regions are assessed at promoter, enhancer and regulatory gene regions of immune cell characteristic transcription factors by bisulfite conversion and pyrosequencing. Chromatin immunoprecipitation and ChIP-seq will be performed for analysis H3K4me3 associated with trained immunity. Humoral and cellular reactivity to spike protein is analyzed by adapted ELISA and neutralisation assays and by ELISpot and flow cytometry, respectively, and correlated.

From our findings we expect to learn about the role of previous RZV on immunogenicity and efficacy of COVID-19 vaccination and whether mechanisms of trained immunity play a role for better responses towards COVID-19 vaccination.

Inclusion Criteria:

Group 1, retrospective (samples already stored, CoVaKo study cohort): healthy (n=420) Group 2, prospective (samples to be recruited, CoVaKo study cohort): healthy (n=180)

• Age 18-90 years, stratified into <60 and ≥60 years
• at least 2 COVID-19-mRNA vaccinations as equivalent to primary immunization and the third and/or fourth dose considered as booster, as recommended for all individuals >12 years (third dose) and for risk groups, medical staff, nursing home residents (fourth dose) in Germany Only individuals naive for SARS-CoV-2 (negative N-specific and S-specific-IgG antibody status, no history of PCR+ swab) before COVID-19 vaccination will be included.

Exclusion Criteria:

• High-dose glucocorticoids >10 mg/day
• Immunosuppressive therapy
• Biologics or immunomodulators
• Transplantation
• Cancer
• Autoimmunity
• RZV after COVID-19 vaccination
• COVID-19 PCR+ before COVID-19 vaccination
• Other types of COVID-19 vaccines than mRNA vaccines

Immunologically healthy individuals who were vaccinated at least twice with COVID-19 mRNA vaccine (either Comirnaty or Moderna) are taken from CoVaKo study cohort (Group A). Immunologically healthy is defined at inclusion by normal leucocyte and lymphocyte counts, normal CRP and ESR according to age range, no severe allergies requiring daily therapy, no congenital or acquired immunodeficiency or immunosuppression.

A control group (Group B) consists of individuals who did not receive RZV 1-12 months before COVID-19 vaccination.