Prediction of Epitopes in the Proteome of Helicobacter pylori

Helicobacter pylori (H. pylori) is classified by the World Health Organization (WHO) as a group I carcinogen and is one of the most efficient human pathogens with over half of the world's population colonized by this gram-negative spiral bacterium. H. pylori can cause a chronic infection in the stomach during early childhood that persists throughout life due to diverse mechanisms of immune response evasion. H. pylori has several factors strongly associated with increased risk of disease such as toxins, adhesins, and chemoattractants, some of which are highly polymorphic, phase variable, and have different functions. Conventional treatments involve the use of antibiotics. However, treatment frequently fails due to the resistance H. pylori has progressively developed to antibiotics. This creates the need for different treatments made possible by identifying new therapeutic targets in the pathogen’s genome. The purpose of this study was an in silico prediction of Tand Bepitopes in H. pylori proteins. Twenty-two external membrane proteins from H. pylori Strain 26695 (accession number NC_000915) were identified using the web tool Vaxign (http://www.violinet.org/vaxign/). A total of one-hundred epitopes (60 class I epitopes and 40 class II epitopes) that could be used to develop novel non-antibiotics drugs for an H. pylori infection were predicted.

H. pylori is an important pathogen in Public Health worldwide as one of the main causes of gastric diseases (Hooi et al., 2017).Several studies have shown that H. pylori is present in about 50% of the world population (Pilotto & Franceschi, 2014).In developing countries, the prevalence of infection can exceed 90% in adulthood (Hooi et al., 2017).
The host immune response induced by H. pylori is characterized by neutrophil recruitment followed by macrophages, mast cells, eosinophils, and T and B lymphocytes (Ayraud, Janvier, & Fauchère, 2002).Additionally, there is an increase of IL-1, IL-8, and IL-6, and a reduction in the production of IL-17 (Kabir, 2011).
The current treatment of an H. pylori infection uses a combination of at least three compounds which include a proton pump inhibitor and two antibiotics.This treatment's decline in efficiency has been documented mainly due to its increased resistance to antibiotics (Fallone et al., 2016;Mégraud, 2012).Therefore, it is imperative to develop new therapeutic alternatives.
The rational design of a vaccine based on In silico prediction epitopes is a promising way to approach this problem (Moss et al., 2011).It has been demonstrated that surface proteins of some microorganisms have immunogenic epitopes, which could be used to design and develop novel, safe and efficient vaccines against pathogens (Ni, Wang, Liu, & Lu, 2010).However, the greatest requirement for the candidate epitopes is their ability to bind to molecules of Major Histocompatibility Complex (MHC) class I and / or class II.These molecules cause the epitopes to come to the cell surface to be subsequently recognition by the lymphocytes (Liao et al., 2009).
Vaxign is an online design system that predicts target proteins for vaccine design based on genome sequences using reverse vaccinology strategy (He, Xiang, & Mobley, 2010).The preestablished characteristics in Vaxign include the subcellular localization of the white protein and identifying epitopes that bind to MHC class I and class II (He et al., 2010;Xiang & He, 2009).
This study aimed to predict T-and B-epitopes from outer membrane proteins of Helicobacter pylori that can be used in later work to develop a vaccine against this microorganism or for alternative therapies such as passive immunization.

Identification of Conserved Sequences
The OrthoMCL algorithm was used to find conserved proteins (Yu et al., 2010) and SPAAN software (Sachdeva, Kumar, Jain, & Ramachandran, 2005).The transmembrane helix topology analysis was carried out using optimized HMMTOP (Xiang & He, 2009) is freely available to non-commercial users at http://www.enzim.hu/hmmtop.Source code is also available upon request to academic users.

Identification of Conserved Sequences
The OrthoMCL algorithm was used to find conserved proteins (Chen, Mackey, Stoeckert, & Roos, 2006), OrthoMCL provides a scalable method for constructing orthologous groups across multiple eukaryotic taxa, using a Markov Cluster algorithm to group (putative) orthologs and paralogs.This method performs similarly to the INPARANOID algorithm when applied to two genomes, but can be extended to cluster orthologs from multiple species.A cut off of E-105 was used as the default value for all proteins that could cross-react with human but mouse and/or pig proteins were discarded.

Prediction of Epitopes
For H. pylori protein, the Vaxitop pipeline was used for the prediction of epitopes (http://www.violinet.org/vaxign/vaxitop),following the methodology described above by (Xiang & He, 2013).Calculations were done using a cut-off point of p ≤ 0.05, which provides high sensibility, specificity and balance (He et al., 2010).

H. pylori Genomes
A total of 683 H. pylori genomes were found, of which only 61 sequences corresponded to genomes strains of this microorganism, with an average of 1,571 and 1,454 protein genes (Table 1).

H. pylori Vaccine Candidate Protein Predition
H. pylori strain 26695 was arbitrarily selected for the prediction of protein candidates.For this strain, a total of 1,445 proteins have been described.In this study, 42 outer membrane proteins were identified, with a sticky probability ≥ 0.51.These proteins had no significant homology with pig, mouse or human proteins (Table 2).The outer membrane proteins with a greater likelihood adhesin (0.737) were: NP_207714.1 (a protein with fusion functions) and NP_207405.1 (a toxin-like protein).

Conclusions
In the present study, bioinformatic analysis was made to the complete genome of H. pylori (Strain 26695 with Gen Bank access number of NC_000915).We identified 22 outer membrane proteins using the Vaxign web program.A total of 60 class I epitopes and 40 class II epitopes were predicted.These epitopes could be used in the improvement of active vaccines, for which we consider it important to combine diverse antigens of H. pylori, thus guiding the immune system to defend the host against this bacterium.Accordingly, this study was developed to predict antigenic determinants / epitopes of various outer membrane proteins of H. pylori.We propose probable epitopes of B and T cells that can trigger a desired immune response to these proteins.Additionally, they could be used in the design of therapeutic strategies without the use of antibiotics such as passive immunization, in which antibodies are administered to patients to help control infection by this microorganism, which we propose as the first line of action, before use of antibiotics.

Table 1 .
Genomes of H. pylori strains

Table 2 .
Selection of conserved outer membrane proteins of Helicobacter pylori, with probability of adherence ≥0.51

Table 3 .
Predicted epitopes for MHC I in proteins of Helicobacter pylori.

Table 4 .
Epitopes for MHC class II predicted by Vaxign for Helicobacter pylori