Background: The development of safe and effective vaccines against SARS-CoV-2 was crucial for controlling COVID-19 and establishing long-lasting immune memory in the population. Methods: This study evaluated cellular immune memory in individuals vaccinated with different regimens in Rio Grande do Sul using flow cytometry. Additionally, the rs2228059 polymorphism in the IL15RA gene was genotyped. A total of 62 participants were randomly recruited. Results: A decrease in memory T cell subsets in response to SARS-CoV-2 stimuli was observed in total CD3⁺, CD4⁺, and CD8⁺ T cells. Regarding the timing of the last vaccine dose, 94.4% of participants had received their final COVID-19 vaccination at least two years prior to recruitment. The rs2228059 polymorphism was genotyped in 443 individuals from the Rio Grande do Sul population. Among participants who received the ChAdOx1/ChAdOx1/BNT162b2 vaccination regimen and carried the TT genotype, an increase in CD8⁺ naive, CD8⁺ effector and CD4⁺ naive subsets was observed in stimulated cells. Although preliminary, the results suggest no major differences between vaccination regimens. Conclusions: The progressive reduction in memory T cell counts supports the need for booster doses, which is essential not only in the context of new emerging variants but also especially to maintain adequate cellular immune protection.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by the presence of pathogenic germline variants in TSC1 or TSC2 genes. However, most TSC1 and TSC2 genetic testing strategies are limited and about 20% of patients have inconclusive or negative results. In this study, we standardized a low-cost technique based on RT-PCR followed by Sanger sequencing, aimed to complement genetic testing strategies. Patients with clinical suspicion of TSC and TSC1 and TSC2 inconclusive (n=3) or negative (n=1) genetic testing results were recruited. Exon 5 skipping in TSC1 gene was identified in one patient with a previous negative genetic testing result. In addition, a 9-base-pair intronic inclusion was found in the TSC1 of an individual with a previously identified variant of uncertain significance, c.664-10A>C. No alterations were found in the other two patients. The present study was successful in identifying errors in mRNA processing in two TSC patients with previously negative or inconclusive genetic testing results. Furthermore, this strategy was useful to reclassify a VUS to pathogenic. Finally, the validated technique could be used to complement coding region genetic tests and to investigate causality of VUS in TSC and other monogenic diseases. Highlights: - A novel cDNA analysis method revealed the presence of exon 5 skipping in the - TSC1 gene - A nine-base-pair retention in TSC1 mRNA is caused by the intronic variant c.664-10A>C - The variant of uncertain significance c.664-10A>C in TSC1 was reclassified as pathogenic - A strategy using cDNA could be a good complement to conventional molecular diagnosis protocols