Immune memory to SARS-CoV-2 is key for establishing herd immunity and limiting the spread of the virus. The duration and qualities of T-cell-mediated protection in the settings of constantly evolving pathogens remain an open question. We conducted a cross-sectional study of SARS-CoV-2-specific CD4+ and CD8+ T-cell responses at several time points over 18 months (30–750 days) post mild/moderate infection with the aim to identify suitable methods and biomarkers for evaluation of long-term T-cell memory in peripheral blood. Included were 107 samples from 95 donors infected during the periods 03/2020–07/2021 and 09/2021–03/2022, coinciding with the prevalence of B.1.1.7 (alpha) and B.1.617.2 (delta) variants in Bulgaria. SARS-CoV-2-specific IFNγ+ T cells were measured in ELISpot in parallel with flow cytometry detection of AIM+ total and stem cell-like memory (TSCM) CD4+ and CD8+ T cells after in vitro stimulation with peptide pools corresponding to the original and delta variants. We show that, unlike IFNγ+ T cells, AIM+ virus-specific CD4+ and CD8+ TSCM are more adequate markers of T cell memory, even beyond 18 months post-infection. In the settings of circulating and evolving viruses, CD8+ TSCM is remarkably stable, back-differentiated into effectors, and delivers immediate protection, regardless of the initial priming strain.

Background: Reactive oxygen species (ROS) are generated at physiological levels as a result of cellular metabolism and contribute to cellular interaction and immune response. Elevated ROS may cause cell stress, damage, and apoptosis, and have been detected in different pathological states of infectious and non-infectious etiology. Aim: To evaluate the association between intracellular ROS in T-cell subsets and HIV VL in chronic HIV infection. Material and methods: Whole blood samples (Li-heparin, n=33) were analyzed during routine immune monitoring in two groups of HIV+ patients: A (n=21), on continuous cART for at least 2y, with sustained viral suppression (HIV VL<40 copies/ml) and group B (n=12) on cART for less than 2y, average HIV VL 92330 c/ml. Percentage and absolute counts (AC) of CD4+ and CD8+T cells were determined by flow cytometry (Multitest, BD Trucount™ tubes, FACS Canto II). Fluorometric ROS assay kit (Sigma-Aldrich) was adapted for flow cytometry analysis to detect intracellular ROS in CD4+ and CD8+ T-cells (FACSDiva 6.1.2). Results: The average CD4AC did not differ significantly between group A and B (714 vs. 568, p>0.05), unlike the CD4/CD8 ratio (1.2 vs. 0.6, p<0.01). The mean fluorescence intensity (MFI) of CD4+T intracellular ROS was significantly lower in group A (mean MFI 1744 vs. 2492, p<0.05), unlike the CD8+T cell ROS content (1753 vs. 2129, p>0.05). Noteworthy, CD4+T intracellular ROS correlated positively with HIV VL (R=0.5, p<0.05), unlike CD8+T ROS. On the other hand, positive correlations between CD8+T ROS and cART duration, as well as age (R=0.5, p<0.05 for both) were observed in group A. Conclusions: CD4+T ROS production may be an indicator of residual HIV activity in the settings of undetectable HIV VL. The combined effects of ageing and long-term cART affect mostly the CD8+T cell compartment.