Exploring rotavirus proteome to identify potential B- and T-cell epitope using computational immunoinformatics

Abstract

Rotavirus is the most common cause of acute gastroenteritis in infants and children worldwide. The functional correlation of B- and T-cells to long-lasting immunity against rotavirus infection in the literature is limited. In this work, a series of computational immuno-informatics approaches were applied and identified 28 linear B-cells, 26 conformational B-cell, 44 T_C cell and 40 T_H cell binding epitopes for structural and non-structural proteins of rotavirus. Further selection of putative B and T cell epitopes in the multi-epitope vaccine construct was carried out based on immunogenicity, conservancy, allergenicity and the helical content of predicted epitopes. An in-silico vaccine constructs was developed using an N-terminal adjuvant (RGD motif) followed by T_C and T_H cell epitopes and B-cell epitope with an appropriate linker. Multi-threading models of multi-epitope vaccine construct with B- and T-cell epitopes were generated and molecular dynamics simulation was performed to determine the stability of designed vaccine. Codon optimized multi-epitope vaccine antigens was expressed and affinity purified using the E. coli expression system. Further the T cell epitope presentation assay using the recombinant multi-epitope constructs and the T cell epitope predicted and identified in this study have not been investigated. Multi-epitope vaccine construct encompassing predicted B- and T-cell epitopes may help to generate long-term immune responses against rotavirus. The computational findings reported in this study may provide information in developing epitope-based vaccine and diagnostic assay for rotavirus-led diarrhea in children's.

Keywords: Immune epitope; Non-structural proteins; Rotavirus; Structural proteins.

Figure 1

Schematic representation of an in-silico strategy followed for prediction of B- and T-cell epitope using proteome of rotavirus.

Figure 2

Summary of rotavirus protein-derived B- and T-cell epitopes. Heat map showing the distribution of (A) linear (continuous) B-cell epitopes, (B) HLA-class I and (C) II epitopes across the structural and non-structural protein sequences of rotavirus. Strong binding affinity epitopes with <0.5% rank and 2% rank, to HLA class I and class II, respectively, for each HLA molecule are represented here. Red color represents likely antigenic epitopes that were predicted using the methods described in Figure 1.

Figure 3

Schematic diagram of multi-epitope chimeric constructs. The multi-epitope constructs sequence consisting adjuvant followed by T- and B-cell epitope. Adjuvant and CTL epitope has been joined by EAAAK linker, whereas the AAY, KK and GGGGS linkers were used to join the CTL, HTL and linear/conformational B-cell epitopes, respectively. A. Construct 1 (VP6A/B/C), B. Construct 2 (VP6/4/7), C. Construct 3 (VP2/3/4/6/7), D. Construct 4 (NSP2/3/4/5) and E. Construct 5 (VP2/3/4/6/7-NSP2/3/4/5), F. Construct 6 (VP6A/B/C–B), G. Construct 7 (VP4/6/7-B), H. Construct 8 (VP4/A), I. Construct 9 (VP6/A) and J. Construct 10 (VP7/A); (BL- Linear B-cell epitope, BC- Conformational B-cell epitope). A/B/C: VP6 sequence of group A, B and C rotaviruses; A: group A rotavirus; B: B-cell epitopes (Both linear and conformational).

Figure 4

Graphical representation of secondary structure obtained for the multi-epitope constructs using PSIPRED server. A. Construct 1, 52.2% helix, 3.0% sheet and 44.8% coil, B. Construct 2, 10.43% helix, 9.71% sheet and 79.86% coil, C. Construct 6, 28.5% helix, 4.0% sheet and 67.5% coil and D. Construct 7, 19.2% helix, 4.8% sheet and 76.0% coil.

Figure 5

Molecular dynamics simulation study of final multi-epitope constructs representing root mean square deviation. A simulation was carried out for time duration of 20 ns. Representative graphs for construct 1, 2, 6 and 7 are provided.

Figure 6

Tertiary structure modeling and structure validation of multi-epitope constructs. Cyan color represents CTL epitopes, orange represents HTL epitopes, blue represents linear B- cell epitopes and conformational B-cell epitope is highlighted with green. A. Construct 1; B. Construct 2; C. Construct 3; D. Construct 4; E. Construct 5; F. Construct 6; G. Construct 7; H. Construct 8; I. Construct 9; and J. Construct 10.

Figure 7

Surface accessibility of linkers in the final multi-epitope constructs. A. Construct 1; B. Construct 2; C. Construct 3; D. Construct 4; E. Construct 5; F. Construct 6; G. Construct 7; H. Construct 8; I. Construct 9; and J. Construct 10. Blue color represents AAY linker, cyan represents KK linker and GGGGS is represented by red color.

Figure 8

Conformational B-cell epitopes prediction for the final multi-epitope constructs by Ellipro. A. Construct 1, B. Construct 2, C. Construct 3, D. Construct 4, E. Construct 5, F. Construct 6, G. Construct 7, H. Construct 8, I. Construct 9 and J. Construct 10. The epitopes are represented as colored spheres in the final vaccine model where each color represents one epitope.

Figure 9

Structure prediction and validation of final multi-epitope constructs. Ramachandran plot analysis of the simulated structures. Summary of residues in favored, allowed and in outlier part is provided in Table 4.

Figure 10

Docked complex of multi-subunit vaccine constructs with integrin receptor. A. Construct1 interaction with αIIbβ3 B. Construct 1 with α_Vβ₃ C. Construct 2 with αIIbβ3 D. Construct 2 with α_Vβ₃ E. Construct 6 with αIIbβ3 F. Construct 6 with α_Vβ₃ G. Construct 7 with αIIbβ3 H. Construct 7 with α_Vβ₃. Integrin receptor chain A and B has been shown in cyan and silver color, respectively, whereas magenta color represents the multi-epitope vaccine constructs in the docked complex.

Figure 11

Cloning, expression, and affinity purification of four chimeric constructs in E. coli. A) Confirmation of recombinant clones using PCR. Construct 1 (expected gene size 693 bp); Construct 2 (expected gene size 837 bp with 201 bp from vector sequence due to use of T7 forward primer), Construct 6 (expected gene size 603 bp) and Construct 7 (expected gene size 690 bp) were synthesized and cloned into champion pET directional TOPO expression vector (pET100/D-TOPO). C: Negative control without template DNA; M: GeneRuler 1 kb DNA ladder (SM0311, Thermo Scientific). B) SDS-PAGE analysis showing the expression of recombinant chimeric proteins induced with IPTG (200 μM) at 18 °C, 25 °C and 37 °C induction temperature. Construct 1 (expected size 28.9 kDa including tag); Construct 2 (expected size 34 kDa including tag); Construct 6 (expected size 28.9 kDa including tag); Construct 7 (expected size 34 kDa including tag); UI: uninduced E. coli whole cell lysates C) Silver stained-SDS-PAGE gel electrophoresis showing the purity of multi-epitope antigens. Different concentrations of BSA were loaded to determine the approximate concentration of purified proteins estimated using Bradford assay. M: Prestained protein ladder (Cat. 26616, ThermoScientific). See fig. S5 for full, uncropped image.