In silico analysis and expression of a new chimeric antigen as a vaccine candidate against cutaneous leishmaniasis

Abstract

Objectives: Since leishmaniasis is one of the health problems in many countries, the development of preventive vaccines against it is a top priority. Peptide vaccines may be a new way to fight the Leishmania infection. In this study, a silicon method was used to predict and analyze B and T cells to produce a vaccine against cutaneous leishmaniasis.

Materials and methods: Immunodominant epitope of Leishmania were selected from four TSA, LPG3, GP63, and Lmsti1 antigens and linked together using a flexible linker (SAPGTP). The antigenic and allergenic features, 2D and 3D structures, and physicochemical features of a chimeric protein were predicted. Finally, through bioinformatics methods, the mRNA structure was predicted and was produced chemically and cloned into the pLEXY-neo2 vector.

Results: Results indicated, polytope had no allergenic properties, but its antigenicity was estimated to be 0.92%. The amino acids numbers, molecular weight as well as negative and positive charge residuals were estimated 390, ~41KDa, 41, and 30, respectively. The results showed that the designed polytope has 50 post-translationally modified sites. Also, the secondary structure of the protein is composed of 25.38% alpha-helix, 12.31% extended strand, and 62.31% random coil. The results of SDS-PAGE and Western blotting revealed the recombinant protein with ~ 41 kDa. The results of Ramachandran plot showed that 96%, 2.7%, and 1.3% of amino acid residues were located in the preferred, permitted, and outlier areas, respectively.

Conclusion: It is expected that the TLGL polytope will produce a cellular immune response. Therefore, the polytope could be a good candidate for an anti-leishmanial vaccine.

Keywords: Bioinformatics; Leishmania major; Polytope; TLGL; Vaccine.

Figure 1

Schematic scheme of a polytope construct with restriction sites

Figure 2

Tendency scale plot from polytope construct (TLGL). A) Surface accessibility; (B) Antigenicity; (C) Beta-turn; (D) Flexibility; (E) Hydrophilicity. The threshold/mean rank is shown by a horizontal red line. Desirable areas of interest properties are shown in yellow (over the threshold). Green (below the threshold) demonstrates undesirable areas of interest features

Figure 3

(A) Prediction of the secondary structure of TLGL with GOR4 online service (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html). H, helix, e, extended strand, and c, coil; (B) The graph indicating TLGL secondary structure predicting with GOR 4

Figure 4

The SWISS-MODEL server product (https://swissmodel.expasy.org/). (A) SWISS-model template alignment; (B) global model quality estimation; (C) Sequence similarity as well as coverage; (D) local model quality estimation; (E) Comparing with non-waste PDB structure; (F) produced a 3D model for polytope construct (TLGL)

Figure 5.

Validating the polytope construct tertiary structure by Ramachandran plot. Ramachandran plot analysis, the overall preferred and Pre – Pro preferred areas are in Dark blue. Pale blue color demonstrates the overall allowed as well as Pre – Pro allowed areas. Glycine favored and allowed areas are represented by dark and pale orange, respectively. White color illustrates the disallowed areas. (A) Analysis of statistics Ramachandran plot for the primary model. (B) The RAMPAGE outcome after refinement

Figure 6

Bioinformatics assessment related to the phosphorylation and acylation areas of polytope construct (TLGL). (A) Prediction of phosphorylation sites in protein construct; (B) If the remnant is not phosphorylated, either due to lower score than the threshold, or owing to no Ser, Thr, or Tyr remnant, such area is denoted using (‘.’). The remnants characterized by predicting scores more than the threshold indicated as ‘S’, ‘T’ or ‘Y’, respectively

Figure 7

The predicted mRNA construct without hairpin and pseudoknot at the 5′ end

Figure 8

SDS-PAGE analysis of the level of expression of chimeric sequence that successfully subcloned to pLEXY-neo2 and expressed in Lishmania tarentolae. Lane 1, Protein molecular weight marker (10–140 kDa); Lanes 2: Logarithmic phase secretory sample, Lanes 3: Stationary phase secretory sample, Lanes 4: Logarithmic phase cytosolic sample, Lanes 5: Stationary phase cytosolic sample and Lanes 5: Control (L. tarentolae secretory sample) (A). Western blot analysis ( B)