Here we evaluated responses to the Gamma, Mu, and Delta variants as they contributed to a significant disease burden during their active circulation in Colombia. third dose of the vaccine caused a rise in antibody titers. The dynamics of the antibody response upon vaccination depended on the previous SARS-CoV-2 exposure. Lower levels of vaccine-induced antibodies were associated with the development of breakthrough infections. Vaccination resulted in central memory spike-specific CD4+ T cell responses that cross-recognized peptides from the Gamma and Mu variants, and their duration also depended on previous SARS-CoV-2 exposure. In (+)-α-Tocopherol addition, we found cross-reactive CD4+ T cell responses in unexposed and unvaccinated individuals. These results have important implications for vaccine design for new SARS-CoV-2 variants of interest and concern. Keywords: SARS-CoV-2, variants, natural infection, vaccination, antibody, CD4+ T cell, hybrid immunity, breakthrough infections 1.?Introduction The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has been present for more than three years, resulting in widespread morbidity and mortality throughout the world (1). One of the main measures to mitigate the impact of COVID-19 has been the generation of vaccines targeting conserved viral epitopes. Most of the current vaccine platforms that target the (+)-α-Tocopherol Spike (S) protein of SARS-CoV-2, are based (+)-α-Tocopherol on the ancestral Wuhan variant, and aim to PYST1 induce virus-specific antibodies and T cells (1C9). However, new viral variants may be differentially recognized by the immune system, leading to decreased protection after natural infection and/or vaccination (10C12). While current COVID-19 vaccines are highly immunogenic and effective at preventing severe disease, several questions remain unanswered (1): a) How do COVID-19 vaccines induce protection and how can we asses this protection (i.e. what are the correlates of protection); b) How effective are COVID-19 vaccines against viral variants of concern (VOCs) or variants of interest (VOIs), and at preventing breakthrough infections (BTI); c) Are vaccine-mediated protection and its durability determined by the pre-vaccination history of natural infection and/or the populations geographic region? Although several studies have evaluated the presence of neutralizing antibodies to viral variants (11C15), there are no studies evaluating both the humoral and cellular responses against the Mu variant, which circulated widely in Colombia in 2021 and has been shown to evade the humoral response (12). In this regard, previous studies in US, British and Chinese populations have observed that the T cell response is conserved for several variants after natural infection and vaccination (3, 10, 16), but this is not clear for the Mu variant. Moreover, it is incompletely known whether immunity generated by vaccination and/or natural infection with the ancestral variant persists over time and whether it is effective in preventing BTI with new viral variants (4, 11, 16). An important aspect to consider regarding vaccine-induced immunity against SARS-CoV-2 is preexisting immunity (both previous natural exposure to SARS-CoV-2, and cross-reactive responses to human common cold coronaviruses [HCoVs]). It has been observed that multiple SARS-CoV-2 exposures enhance the magnitude of IgG responses but cause a low impact on the frequency of S-specific T cells (1). However, these T cells resulting from hybrid immunity may exhibit a wider polyfunctional profile than those from vaccination alone (17). Thus, the impact of previous antigen exposure on the levels and durability of humoral and cellular responses and the interplay of viral variants.