Computational protocol: Novel Immunoinformatics Approaches to Design Multi epitope Subunit Vaccine for Malaria by Investigating Anopheles Salivary Protein

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Protocol publication

[…] CD8+ cytotoxic T-lymphocyte were shown to inhibit malaria parasitic growth and development, inside the hepatocytes cells. To get an immunogenic CTL epitopes having the ability to elicit cell-mediated immunity and form the memory cells, all 14 salivary protein sequences were subjected to the NetCTL 1.2 server. NetCTL 1.2 is an online web server intended for predicting CTL epitopes among input protein sequences based on the training dataset. NetCTL was selected to predict the CTL epitopes because of its higher prescient execution on all execution parameters as compared to the recently developed servers namely MHC-pathway, MAPPP, and EpiJen. All the salivary protein sequences were submitted in the FASTA format to predict the CTL epitope at the threshold score of 0.75 (default). Those epitopes having a combined score of greater than 0.75 were selected as CTL epitope and further subjected to the Immune Epitope Database (IEDB) MHC class I immunogenicity prediction module. Predicted CTL epitopes for each salivary protein was used as an input sequence and the result was obtained in the form of the score, where higher score determines that greater will be the probability of eliciting an immune response. [...] B lymphocytes, a type of white blood cells, are the key player of humoral immunity by antibody production. The identification of B-cell epitopes is an essential part in vaccine designing. BCPREDS server was used to predict the linear B-cell epitopes of 20 amino acids long. The amino acid sequence of final vaccine construct was used as an input sequence in plain format followed by the selection of fixed length epitope prediction method and length of the epitope. BCPREDS (default method) was selected as the prediction method for the epitope of 20 amino acids long. The specificity threshold was set to be by default at 75% to get the result in a user-friendly format. While conformational epitopes were predicted using ElliPro server for the input of tertiary protein structure of vaccine construct. [...] Antigenicity determines the ability of an antigen to binds with the B- and T-cell receptor that may lead to the immune response and memory cell formation. Therefore, the antigenic nature of predicted vaccine construct was determined to ensure its ability to interact with the immune receptor. ANTIGENpro is a sequence based, pathogen independent and alignment-free prediction method that was used to check the antigenic behavior of vaccine protein. It uses SVM classifier to summarize the probable antigenic or non-antigenic nature of proteins. ANTIGENpro uses the existing protein antigenicity microarray files of eight feature sets for five pathogens to construct two-stage architecture; among them, the first one is multiple representations of the primary protein sequence and the second one is five machine learning algorithms.Allergenicity is the potential of a material to cause sensitization and allergic reactions associated with the IgE antibody response. Therefore, the predicted vaccine construct must be free from the allergenic nature. AllerTOP v. 2.0 was used to check the allergenicity of the vaccine construct based on the method that uses auto cross-covariance (ACC) transformation of protein sequences into uniform equal-length vectors. Input protein sequence of vaccine protein was classified by the k-nearest neighbor algorithm (kNN, k = 1) which is based on the training set of 2427 known allergen from different species and 2427 nonallergen from similar species. [...] The main purpose of vaccination is to induce an immune response after injecting the vaccine into the body. Therefore, it is necessary to define the physical and chemical parameters associated with the vaccine. ProtParam web server, a part of Expert Protein Analysis System (EXPASY), was used to define various physicochemical properties of predicted vaccine construct. The primary protein sequence of the vaccine was used to predict the various parameters including molecular weight (kDa), estimated half-life, theoretical pI, aliphatic index, grand average of hydropathy (GRAVY) and so on. [...] Protein molecule achieves maximum stability in its lowest energy state by proper bending and twisting to form a tertiary structure. It is the interaction between the amino acids side chain residue which is responsible to stabilize the protein structure. The 3-dimensional structure of predicted vaccine construct was obtained by utilizing RaptorX structure prediction server. RaptorX is a pure ab initio method that can be used to build a 3D model in a template-free manner. [...] It is the degree of likeness between the target and available template structure that determines the quality of protein model structure created by contemporary protein structure prediction techniques,. Therefore, it was necessary to improve the template based predicted model beyond the accuracy by utilizing the template information. To fulfill this thought, output model of RaptorX server was subjected to the GalaxyRefine web server, which is based on the CASP10 tested refinement method. GalaxyRefine performs rehashed structure perturbation followed by overall structural relaxation by performing molecular dynamics simulation. [...] Before proceeding to the next step, it was necessary to improve the stability of refined protein model. Disulfide bonds are covalent interactions that emulate the stabilizing molecular interaction and provide a considerable stability to protein model by confirming precise geometric conformations. Disulfide engineering is a novel approach for creating disulfide bonds into the target protein structure. Therefore, the refined model of final vaccine construct was subjected to the Disulfide by Design 2.0 to perform disulfide engineering. Initially, the refined protein model was uploaded and run for the residue pair search that can be used for the disulfide engineering purpose. Total 4 residue pairs were selected to mutate them with cysteine residue using create mutate function of the Disulfide by Design 2.0 server. [...] Molecular docking is a computational method used to predict the preferred orientation of ligand molecule to the receptor molecule in their stable complex form. It can be also used to predict the binding affinity between these two molecules in terms of scoring function. As mentioned in the previous section, TLRs having the capability to recognize the Plasmodium ligands and P. falciparum primes the human TLR-4 response towards high proinflammatory cytokine profile. Therefore, TLR-4 was selected as receptor and its PDB file (PDB id: 4G8A) was obtained from RCSB-Protein Data Bank while the refined model of vaccine protein was used as a ligand. Protein-protein docking was performed using the ClusPro 2.0: protein-protein docking server, to check the binding affinity between them. [...] Molecular dynamics simulation is a widely accepted computational approach which is used to determine the stability of protein-ligand complex at the microscopic level. The protein-protein docked complex output of ClusPro was used as an input to perform the molecular dynamics simulation using Gromacs v5.1.5. Initially, the crystal water of complex was removed followed by the topology generation using a GROMOS9643a1 force field. In the next step, protein complex was centered in a cubic boundary box and filled by water molecule using simple point charge (SPC) water model and chloride ion was used for the charge neutralization of complex. Moreover, energy minimization followed by canonical equilibration (NVT ensemble) and isothermal-isobaric (NPT ensemble) was performed for a time duration of 100 ps. Finally, molecular dynamics simulation was executed for the time duration of 10ns. The root mean square deviation (RMDS) for backbone and root mean square fluctuation (RMSF) for side chain was determined. […]

Pipeline specifications

Software tools ProtParam, RaptorX, GalaxyRefine, DbD
Databases ExPASy CASP
Applications Protein structure analysis, Protein physicochemical analysis
Organisms Homo sapiens, Escherichia coli K-12