A chimeric HLA-A2:β2M:Ig fusion protein for the study of virus-specific CD8+ T-cells

Ribeiro, Ágata Lopes; Araújo, Franklin Pereira; Martins, Júlia Pereira; Lourenço, Alice Aparecida; Huang, Jing; Pereira, Felipe Valença; Andrade, Luis Adan Flores; Paim, Adriana Alves Oliveira; Fonseca, Flávio Guimarães da; Barbosa-Stancioli, Edel Figueiredo; Martins‐Filho, Olindo Assis; Peruhype-Magalhães, Vanessa; Tsuji, Moriya; Coelho-dos-Reis, Jordana Grazziela Alves

doi:10.1016/j.jim.2021.112997

Cited by 2 publications

(3 citation statements)

References 24 publications

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“…After the background staining, the microarray slide was incubated with standard buffer at room temperature for 15 minutes. After complete removal of the buffer, 1µg/mL, 10µg/mL and 30µg/mL of HLA-A*02:01 dimer (HLA-A2:β2M:Ig Protein) ( 23 ) diluted in staining buffer were added to the microarray glass slide and incubated for 16 hours at 2-8°C. After complete removal of the HLA-A*02:01 dimer, 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) previously diluted (1:5,000) in staining buffer.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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“…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).…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2023

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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.

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A chimeric HLA-A2:β2M:Ig fusion protein for the study of virus-specific CD8+ T-cells

Cited by 2 publications

References 24 publications

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

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

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

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