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

Abstract

IntroductionThe present work sought to identify MHC-I-restricted peptide signatures for arbovirus using in silico and in vitro peptide microarray tools.MethodsFirst, 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.ResultsThe 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).DiscussionTo 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.

In Silico Physicochemical Characterization of Fusion Proteins from Emerging Amazonian Arboviruses

“…In addition to concentrating regions of high antigenic propensities, the N-terminus half of MAYV, SLEV, and OROV fusion proteins contained spatially equivalent amino acid positions where such a property was at its maximum (i.e., around residues Pro74, Arg85, and Asp131, respectively). However, a poor overlapping of immunogenic peptides amongst these emerging arboviruses was observed in the C-terminus half of their fusion proteins, which is in line with previous findings on distinct members of the Togaviridae, Flaviviridae, and Peribunyaviridae families [19].…”

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

“…Such results were observed not only for all proteins of SARS-CoV-2 Wuhan original reference strain and in its five VOCs (except for ORF8 in the Alpha variant), but also for other HCoVs such as HCoV-NL63, MERS-CoV, and SARS-CoV-1. This is a clear contrast regarding other viruses such as arboviruses, for which the induction of a proinflammatory antiviral profile is predominant (20). High levels of IL-4 have been correlated to a delay in viral clearance of SARS-CoV-2 (62), however, low levels of this cytokine were also associated with long COVID-19 (63), accentuating the importance of a balance between Th1/Th2 axis for the establishment of an effective immune response against SARS-CoV-2.…”

“…To assess the HLA-A*02-restricted peptide signature, a customized microarray was performed by PEPperPRINT © (Heidelberg, Germany) as described previously (20) with modifications. Briefly, 2,555 peptides from HCoV-NL63, MERS-CoV, SARS-CoV-1, and SARS-CoV-2 were printed on a PEPperCHIP © microarray slide and incubated with a HLA-A*02:01 dimeric protein (Ig : DimerX -BD Biosciences, California, USA) at 1µg/mL or 10µg/mL diluted in staining buffer (PBS + 10% bovine serum albumin).…”

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

“…To identify which peptides bind to HLA-A*02:01 and the intensity of binding, we used a secondary reagent, an anti-mouse IgG1 conjugated to the fluorochrome Cy3. The secondary reagent binds to the mouse IgG1 (Fc portion) and is then read out by the microarray reader [ 27 , 28 ]. For the microarray, 345 (9-mer) peptides covering all the extensions of Tax protein were synthesized and adsorbed at 1 nM concentration on spots in duplicates located on a glass slide (75.4 mm × 25.0 mm × 1 mm).…”

“…For the second stage of the assay, the microarray slide was re-equilibrated by a new incubation with standard buffer for 15 min at room temperature. After complete removal of the buffer, concentrations of 1 µg/mL and 10 µg/mL of HLA-A*02:01:Ig dimeric protein produced in-house [ 27 , 28 ] were diluted in staining buffer and added to the microarray glass slide and incubated for 16 h at 2–8 °C. The microarray slide was washed three times using standard buffer and incubated with anti-murine IgG1/Cy3 secondary antibody (BD Biosciences, San Jose, CA, USA) diluted in a 1:5000 ratio for 45 min at room temperature in the dark.…”