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As severe acute respiratory coronavirus 2 (SARS-CoV-2) variants continue to emerge, it is important to characterize immune responses against variants which can inform on protection efficacies following booster vaccination. In this study, neutralizing breadth and antigen-specific CD8+ T cell responses were analyzed in both infection-naïve and infection-experienced individuals following administration of a booster bivalent Wuhan-Hu-1+BA.4/5 Comirnaty® mRNA vaccine. Significantly higher neutralizing titers were found after this vaccination compared to the pre-third booster vaccination time point. Further, neutralizing breadth to omicron variants, including BA.1, BA.2, BA.5, BQ.1 and XBB.1, was found to be boosted following bivalent vaccination. SARS-CoV-2-specific CD8+ T cells were identified, but with no evidence that frequencies were increased following booster vaccinations. Spike protein-specific CD8+ T cells were the only responses detected after vaccination and non-spike-specific CD8+ T cells were only detected after infection. Both spikespecific and non-spike-specificCD8+ T cells were found at much lower frequencies than CD8+ T cells specific to cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza (Flu). Taken together, these results show that the bivalent Wuhan-Hu-1 +BA.4/5 Comirnaty® mRNA vaccine boosted the breadth of neutralization to newer SARS-CoV-2 variants and that vaccination is able to induce spike protein-specific CD8+ T cell responses, which are maintained longitudinally. [ABSTRACT FROM AUTHOR]

MHC-multimers are reagents used for the detection and enumeration of antigen-specific T cells (ASTs). These reagents exploit the mechanism by which T cell receptors (TCR) on cytotoxic CD8 T cells recognize specific antigens in the context of a major histocompatibility complex (MHC) molecule during antigen presentation. MHC-multimers are fluorescently-labeled dextran polymers that carry MHC Class I molecules and peptide sequences that can be modified to represent specific cognate sequences of the antigen of interest with dextramers having a 10-fold multiplicity of the MHC/peptide combination within a single multimer. Since the binding of antigen-specific dextramers mimics antigen presentation to the TCR, the present study sought to determine whether this TCR engagement on the AST was sufficient to elicit a functional T cell response. The effect of binding of CMV specific dextramers on the activation of the NFAT signal transduction cascade was assessed in peripheral blood from bone marrow transplant recipients previously determined to be positive for CMV-ASTs (CASTs). NFAT activation was quantified by measuring nuclear translocation of NFAT1 in CD8+ CASTs and CD8+ non-CASTs by imaging flow cytometry. Our results demonstrate that an increase in the nuclear localization of NFAT1 was detectable in the CASTs following the CMV-dextramer binding and could be observed as early as 10 min post-exposure. The NFAT1 activation correlated with a downstream functional response in the form of interferon gamma production. Sample preparation, temperature, and duration of dextramer exposure were important parameters affecting the dextramer-induced NFAT activation with 2 h exposure in whole blood at room temperature being the optimal of the conditions tested. Intra- and inter-individual heterogeneity was observed with regards to the NFAT activation in the CASTs. Importantly, no effect of the dextramers was observed in the CD8+ non-CASTs, and therefore dextramer negative cell populations. Exposure to PMA/ionomycin following dextramer exposure resulted in a homogeneous NFAT activation in both the dextramer-positive but NFAT1 nonresponsive CAST and non-CAST cells. Thus, the data demonstrate that binding of antigen-specific dextramers to ASTs specifically results in activation of NFAT, that the NFAT activation correlates with a downstream functional response and that the response can be heterogeneous. This functional parameter may provide insight to the issue whether enumeration alone of ASTs is a sufficient parameter to assess an individual’s immune status against a specific antigen. [ABSTRACT FROM AUTHOR]

As severe acute respiratory coronavirus 2 (SARS-CoV-2) variants continue to emerge, it is important to characterize immune responses against variants which can inform on protection efficacies following booster vaccination. In this study, neutralizing breadth and antigen-specific CD8 + T cell responses were analyzed in both infection-naïve and infection-experienced individuals following administration of a booster bivalent Wuhan-Hu-1+BA.4/5 Comirnaty ® mRNA vaccine. Significantly higher neutralizing titers were found after this vaccination compared to the pre-third booster vaccination time point. Further, neutralizing breadth to omicron variants, including BA.1, BA.2, BA.5, BQ.1 and XBB.1, was found to be boosted following bivalent vaccination. SARS-CoV-2-specific CD8 + T cells were identified, but with no evidence that frequencies were increased following booster vaccinations. Spike protein-specific CD8 + T cells were the only responses detected after vaccination and non-spike-specific CD8 + T cells were only detected after infection. Both spike-specific and non-spike-specific CD8 + T cells were found at much lower frequencies than CD8 + T cells specific to cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza (Flu). Taken together, these results show that the bivalent Wuhan-Hu-1+BA.4/5 Comirnaty ® mRNA vaccine boosted the breadth of neutralization to newer SARS-CoV-2 variants and that vaccination is able to induce spike protein-specific CD8 + T cell responses, which are maintained longitudinally., Competing Interests: Authors KJ, DI, and LB are employed by the company Immudex ApS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Underwood, Sølund, Jacobsen, Binderup, Fernandez-Antunez, Mikkelsen, Inekci, Villadsen, Castruita, Pinholt, Fahnøe, Ramirez, Brix, Weis and Bukh.)

After allogeneic hematopoietic stem cell transplantation (HSCT), the emergence of circulating cytomegalovirus (CMV)- specific T cells correlates with protection from CMV reactivation, an important risk factor for non-relapse mortality. However, functional assays measuring CMV-specific cells are time-consuming and often inaccurate at early time-points. We report the results of a prospective single-center, non-interventional study that identified the enumeration of Dextramerpositive CMV-specific lymphocytes as a reliable and early predictor of viral reactivation. We longitudinally monitored 75 consecutive patients for 1 year after allogeneic HSCT (n=630 samples). The presence of ≥0.5 CMV-specific CD8+ cells/mL at day +45 was an independent protective factor from subsequent clinically relevant reactivation in univariate (P<0.01) and multivariate (P<0.05) analyses. Dextramer quantification correlated with functional assays measuring interferon-γ production, and allowed earlier identification of high-risk patients. In mismatched transplants, the comparative analysis of lymphocytes restricted by shared, donor- and host-specific HLA revealed the dominant role of thymic-independent CMV-specific reconstitution. Shared and donor-restricted CMV-specific T cells reconstituted with similar kinetics in recipients of CMV-seropositive donors, while donor-restricted T-cell reconstitution from CMV-seronegative grafts was impaired, indicating that in primary immunological responses the emergence of viral-specific T cells is largely sustained by antigen encounter on host infected cells rather than by cross-priming/presentation by non-infected donor-derived antigen-presenting cells. Multiparametric flow cytometry and high-dimensional analysis showed that shared-restricted CMV-specific lymphocytes display a more differentiated phenotype and increased persistence than donor-restricted counterparts. In this study, monitoring CMV-specific cells by Dextramer assay after allogeneic HSCT shed light on mechanisms of immune reconstitution and enabled risk stratification of patients, which could improve the clinical management of post-transplant CMV reactivations.