Surveillance of SARS-CoV-2 immunogenicity: loss of immunodominant HLA-A*02-restricted epitopes that activate CD8+ T cells.

Introduction and methods: In this present work, coronavirus subfamilies and SARS-CoV-2 Variants of Concern (VOCs) were investigated for the presence of MHC-I immunodominant viral peptides using in silico and in vitro tools. Results: In our results, HLA-A*02 haplotype showed the highest number of immunodominant epitopes but with the lowest combined prediction score. Furthermore, a decrease in combined prediction score was observed for HLA-A*02-restricted epitopes when the original strain was compared to the VOCs, indicating that the mutations on the VOCs are promoting escape from HLA-A2-mediated antigen presentation, which characterizes a immune evasion process. Additionally, epitope signature analysis revealed major immunogenic peptide loss for structural (S) and non-structural (ORF8) proteins of VOCs in comparison to the Wuhan sequence. Discussion: These results may indicate that the antiviral CD8+ T-cell responses generated by original strains could not be sufficient for clearance of variants in either newly or reinfection with SARS-CoV-2. In contrast, N epitopes remain the most conserved and reactive peptides across SARS-CoV-2 VOCs. Overall, our data could contribute to the rational design and development of new vaccinal platforms to induce a broad cellular CD8+ T cell antiviral response, aiming at controlling viral transmission of future SARS-CoV-2 variants.

Diversity of HLA-A2-Restricted and Immunodominant Epitope Repertoire of Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Protein: Novel Insights among N-Terminal, Central and C-Terminal Regions.

The present study sought to search for the immunodominance related to the N-terminal, Central and C-terminal regions of HTLV-1 Tax using novel, cutting-edge peptide microarray analysis. In addition, in silico predictions were performed to verify the presence of nine amino acid peptides present along Tax restricted to the human leukocyte antigen (HLA)-A2.02*01 haplotype, as well as to verify the ability to induce pro-inflammatory and regulatory cytokines, such as IFN-γ and IL-4, respectively. Our results indicated abundant dose-dependent reactivity for HLA-A*02:01 in all regions (N-terminal, Central and C-terminal), but with specific hotspots. Furthermore, the results of fold-change over the Tax11-19 reactivity obtained at lower concentrations of HLA-A*02:01 reveal that peptides from the three regions contain sequences that react 100 times more than Tax11-19. On the other hand, Tax11-19 has similar or superior HLA-A*02:01 reactivity at higher concentrations of this haplotype. The in silico analysis showed a higher frequency of IFN-γ-inducing peptides in the N-terminal portion, while the C-terminal portion showed a higher frequency of IL-4 inducers. Taken together, these results shed light on the search for new Tax immunodominant epitopes, in addition to the canonic Tax11-19, for the rational design of immunomodulatory strategies for HTLV-1 chronic diseases.

In silico and in vitro arboviral MHC class I-restricted-epitope signatures reveal immunodominance and poor overlapping patterns.

Introduction: The present work sought to identify MHC-I-restricted peptide signatures for arbovirus using in silico and in vitro peptide microarray tools. Methods: First, an in-silico analysis of immunogenic epitopes restricted to four of the most prevalent human MHC class-I was performed by identification of MHC affinity score. For that, more than 10,000 peptide sequences from 5 Arbovirus and 8 different viral serotypes, namely Zika (ZIKV), Dengue (DENV serotypes 1-4), Chikungunya (CHIKV), Mayaro (MAYV) and Oropouche (OROV) viruses, in addition to YFV were analyzed. Haplotype HLA-A*02.01 was the dominant human MHC for all arboviruses. Over one thousand HLA-A2 immunogenic peptides were employed to build a comprehensive identity matrix. Intending to assess HLAA*02:01 reactivity of peptides in vitro, a peptide microarray was designed and generated using a dimeric protein containing HLA-A*02:01. Results: The comprehensive identity matrix allowed the identification of only three overlapping peptides between two or more flavivirus sequences, suggesting poor overlapping of virus-specific immunogenic peptides amongst arborviruses. Global analysis of the fluorescence intensity for peptide-HLA-A*02:01 binding indicated a dose-dependent effect in the array. Considering all assessed arboviruses, the number of DENV-derived peptides with HLA-A*02:01 reactivity was the highest. Furthermore, a lower number of YFV-17DD overlapping peptides presented reactivity when compared to non-overlapping peptides. In addition, the assessment of HLA-A*02:01-reactive peptides across virus polyproteins highlighted non-structural proteins as "hot-spots". Data analysis supported these findings showing the presence of major hydrophobic sites in the final segment of non-structural protein 1 throughout 2a (Ns2a) and in nonstructural proteins 2b (Ns2b), 4a (Ns4a) and 4b (Ns4b). Discussion: To our knowledge, these results provide the most comprehensive and detailed snapshot of the immunodominant peptide signature for arbovirus with MHC-class I restriction, which may bring insight into the design of future virus-specific vaccines to arboviruses and for vaccination protocols in highly endemic areas.

A New Flow Cytometry-Based Single Platform for Universal and Differential Serodiagnosis of HTLV-1/2 Infection.

In the present work, we developed and evaluated the performance of a new flow cytometry-based single platform, referred to as "FC-Duplex IgG1 (HTLV-1/2)", for universal and differential serodiagnosis of HTLV-1/2 infection. The proposed technology employs a system for detection of IgG1 antibodies in a single competitive immunofluorescence platform by flow cytometry using fluorescently labeled MT-2/MoT cell line mix coupled to a highly sensitive development system (Biotin/Streptavidin/Phycoerythrin). The stability of fluorescent labeling and the antigenicity of MT-2 and MoT cell lines were confirmed upon storage at -20°C for 2, 6, and 12 months. The anti-HTLV-1/2 IgG1 reactivity, expressed as percentage of positive fluorescent cells (PPFC), was evaluated for each target antigen along the titration curve of test serum samples (1:32 to 1:4,096). Upon selection of target cell line and serum dilutions with higher segregation score between groups, the performance of "FIX" and "FIX & PERM" protocols was evaluated. The "FIX" protocol presented excellent performance indices (Se = 92%/Sp = 94%/AUC = 0.96; Se = 96%/Sp = 100%/AUC = 0.99) for the universal (HTLV-1/2 vs. NI) and differential (HTLV-1 vs. HTLV-2) diagnosis of HTLV-1 infection, respectively. Optimization of the "FIX" protocol using the principle of synchronous and asynchronous pairwise analysis further improved the performance of "FC-Duplex IgG1 (HTLV-1/2)", using the "FIX" protocol for differential diagnosis of HTLV-1 and HTLV-2 infections (Se = 100%/Sp = 100%/AUC = 1.00). In conclusion, the "FC-Duplex IgG1 (HTLV-1/2)" method represents an innovation in the biotechnology segment with the potential to compose a serological kit for differential diagnosis of HTLV-1/2 infection for reference laboratories and blood centers.