An immunoinformatics study reveals a new BoLA-DR-restricted CD4+ T cell epitopes on the Gag protein of bovine leukemia virus

Abstract

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), which has been reported worldwide. The expression of viral structural proteins: surface glycoprotein (gp51) and three core proteins - p15 (matrix), p24 (capsid), and p12 (nucleocapsid) induce a strong humoral and cellular immune response at first step of infection. CD4+ T-cell activation is generally induced by bovine leukocyte antigen (BoLA) region- positive antigen-presenting cells (APC) after processing of an exogenous viral antigen. Limited data are available on the BLV epitopes from the core proteins recognized by CD4+ T-cells. Thus, immunoinformatic analysis of Gag sequences obtained from 125 BLV isolates from Poland, Canada, Pakistan, Kazakhstan, Moldova and United States was performed to identify the presence of BoLA-DRB3 restricted CD4+ T-cell epitopes. The 379 15-mer overlapping peptides spanning the entire Gag sequence were run in BoLA-DRB3 allele-binding regions using a BoLA-DRB- peptide binding affinity prediction algorithm. The analysis identified 22 CD4+ T-cell peptide epitopes of variable length ranging from 17 to 22 amino acids. The predicted epitopes interacted with 73 different BoLA-DRB3 alleles found in BLV-infected cattle. Importantly, two epitopes were found to be linked with high proviral load in PBMC. A majority of dominant and subdominant epitopes showed high conservation across different viral strains, and therefore could be attractive targets for vaccine development.

Figure 1

Distribution of the BoLA-DRB3-restricted CD4+ T-cell epitopes along the Gag polyprotein. The labeled blue bars in the upper part of figure refer to the identified 22 epitopes 1A–15B. The figure shows the localization of the epitopes for the most commonly detected BoLA-DRB3 (on the left side of the figure). The distribution of the epitopes for the all analysed alleles is shown in Supplementary Fig. S3.

Figure 2

(a–d) Representation of the dominant and subdominant CD4+ T cell epitopes on 3D protein structure model of BLV Gag. The model is shown as space-filled images of opposite sides arbitrarily named side A and side B. (A–B) Gag structure contain 11 dominant epitopes: 1A (red), 1B (forest green), 2 (blue), 3 (yellow), 4A (cyan), 4B (orange), 4C (green), 5 (cornflower blue), 6 (magenta), 7 (goldenrod), 8 (sky blue), 1B + 3 [255–267] (brown), 4A + 6 [42–55] (purple), 4B + 4C (163–164): silver. (C–D) Gag structure contain 11 subdominant epitopes: 9 (red), 10 (forest green), 11A (blue), 11B (yellow), 12A (cyan), 12B (orange), 13A (green), 13B (cornflower blue), 14 (magenta), 15A (goldenrod), 15B (all residues overlap with one or more other sequences), 9 + 15B [192–194] (brown), 12B + 15B [180–181] (purple), 10 + 13A [141–142] (silver), 11B + 14 [116–119] (navy blue), 12A + 13B [237–250] (coral), 10 + 14 [125–131] (dark red), 9 + 12B + 15B [182–191] (black).

Figure 3

Association between the number of BoLA-DRB3-restricted CD4 + T-cells epitopes on the Gag and BLV proviral load. A polynomial trend line of the fourth degree is plotted in the graph.

Figure 4

Association between 73 BoLA-DRB3 alleles and two BoLA-DRB3- restricted CD4 + T cell epitopes (1A and 2). Log2 Rank predicted binding score for Gag peptides observed for the BoLA-DRB3 alleles distinguished in the two groups: Group A (n = 22 alleles) marked in red line on the graph; Group B (n = 51 alleles) marked in grey line.

Figure 5

Comparison of BLV copy number between cattle carrying BoLA-DRB3 alleles with no affinity to the Gag protein CD4+ T-cells 1A and 2 epitopes on and alleles with strong affinity to the epitopes using the student t-test for 2 independent means.

Figure 6

Gag protein sequence alignment for selected BLV isolates, containing amino acid changes within CD4+ T cell epitopes that change the degree of binding affinity of BoLA-DRB3. The names of the isolates and their corresponding BoLA-DRB3 are listed on the left side of the alignment. Amino acid changes, which generate new BoLA-DRB3 binding affinity site are marked with red arrows; amino acid changes that impair BoLA-DRB3 binding affinity sites are marked with green arrows; changes that enhance BoLA-DRB3 affinity are marked with orange arrows, the changes that generate lack of peptides interactions with BoLA-DRB3 are marked with grey arrows. BoLA-DRB3-restricted CD4 + T-cell epitopes along the Gag polyprotein are labeled in the upper part of the figure as blue bars.