Additionally, individuals who were infected with the ancestral virus strain in 2020 exhibited stronger T cell responses against Omicron BA.1 and BA.2 mutation-specific regions of the spike protein than infection-na?ve individuals. As expected, both T cell and antibody responses significantly increased after booster vaccination. activity significantly increased one month after booster vaccination. Adoprazine (SLV313) Four months after booster vaccination, T cell and antibody responses significantly decreased but levels remained steady thereafter until seven months after booster vaccination. After a similar number of vaccinations, previously infected individuals had significantly higher S1-specific T cell, anti-RBD IgG, and neutralizing IgG responses than infection-na?ve HCWs. Strikingly, we observed overall cross-reactive T cell responses against different SARS-CoV-2 VOC in both previously infected and infection-na?ve HCWs. In summary, COVID-19 booster vaccinations induce strong T cell and neutralizing antibody responses and the presence of T cell responses against SARS-CoV-2 VOC suggest that vaccine-induced T cell immunity offers cross-reactive protection against different VOC. Keywords: COVID-19, SARS-CoV-2, immunity, vaccination, T cell, antibody, variants 1. Introduction More than two years after coronavirus disease 2019 (COVID-19) was declared a pandemic [1], severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still causes a substantial number of infections despite many individuals having been previously vaccinated against COVID-19 or having had a SARS-CoV-2 infection that builds immunity against the virus [2,3,4]. The presence of neutralizing antibodies is generally considered a key correlate of immune protection from SARS-CoV-2 infection [5,6]. These antibodies bind the receptor-binding domain (RBD) of the spike protein, thereby preventing the virus from entering human cells [7]. In addition to the humoral compartment, it is now widely accepted that T cells also play a pivotal role in controlling SARS-CoV-2 infection. For example, lymphopenia is a determinant for worse clinical outcomes after SARS-CoV-2 infection and memory T cell responses are maintained against multiple SARS-CoV-2 epitopes [8]. Accordingly, a considerable number of SARS-CoV-2 immunity studies revealed that SARS-CoV-2 infection and COVID-19 vaccination induce the formation of Adoprazine (SLV313) neutralizing anti-spike antibodies and robust T cell responses against a wide range of viral epitopes [9,10,11,12,13]. Although these immune responses were still detectable up to one year post-immunization, a significant decrease was observed within the first months following immunization [9,14,15,16]. This observation could at least partly explain why a substantial number of immunized individuals are (re)infected with the virus [17,18,19]. Besides the waning of SARS-CoV-2 immunity, the substantial incidence of new SARS-CoV-2 infections after earlier infection or vaccinations can potentially be explained by new emerging SARS-CoV-2 variants of concern (VOC), including the most recent VOC Delta Emr1 (B.1.617.2 lineage) and Omicron (B.1.1.529 lineage) [3]. These SARS-CoV-2 VOC involve mutations in the spike protein and multiple studies reported that spike-specific antibodies partially lost their neutralizing capabilities against new SARS-CoV-2 VOC [20,21,22]. Remarkably, comparable results were observed within the same variant as the Omicron subvariants BA.4 and BA.5 escaped from neutralizing antibodies that were formed after Omicron BA.1 or BA.2 infection [23,24,25,26]. The present study aims to describe the long-term kinetics of SARS-CoV-2 specific humoral and T cell responses after primary and booster vaccinations in previously SARS-CoV-2-infected individuals and compare these to infection-na?ve vaccinated individuals. In addition, we determined whether prior infection and vaccination induce cross-reactive T cell responses against the spike protein of the SARS-CoV-2 Delta and Omicron BA.1 and BA.2 VOC. 2. Materials and Methods 2.1. Study Design This study cohort consisted of previously infected healthcare workers (HCWs) who tested SARS-CoV-2 reverse transcription-quantitative polymerase chain reaction (RT-qPCR) positive between March 2020 and March 2021, recently infected HCWs who tested RT-qPCR positive between December 2021 and May 2022, and infection-na?ve HCWs who never tested SARS-CoV-2 RT-qPCR positive during the study period. All participating HCWs were affiliated with our hospital. For previously Adoprazine (SLV313) infected HCWs, SARS-CoV-2-specific T cell and antibody responses were measured at the following time points: June 2020 (only antibodies) and June 2021 (as part of our previous studies) [9,27], November 2021 (t0), December 2021 (t1), March 2022 (t2), and June 2022 (t3). The recently infected and infection-na?ve HCWs were included in March 2022 (t2) and June 2022 (t3). This study received approval from the Medical Research.