key: cord-0303056-rjacm17z
authors: Elhag, Mustafa; Abdelmoneim, Abdelrahman Hamza; Sati, Anfal Osama; Saadaldin, Moaaz Mohammed; Ahmad, Nagla Mohammad; Hassan, Mohammed A.
title: Design of Epitope Based Peptide Vaccine against Brucella Abortus OmpW Family Protein using Immunoinformatics
date: 2021-12-30
journal: bioRxiv
DOI: 10.1101/2021.12.27.474190
sha: 2c590c536fa6ab85cf557acf20342dd1eacc38d9
doc_id: 303056
cord_uid: rjacm17z

Brucella abortus is a small aerobic, non-spore-forming, non-motile intracellular coccobacilli localized in the reproductive organs of host animals and causes acute or chronic disorders. It infects approximately 200 cases per 100,000 of the population and has become endemic in many countries. OmpW family protein is an outer membrane protein involved in the initial interaction between the pathogen and it’s host. This study predicts an effective epitope-based vaccine against OmpW family protein of Brucella abortus using immunoinformatics tools. Sequences were obtained from NCBI and prediction tests were accomplished to analyze possible epitopes for B and T cells. Seven B cell epitopes passed the antigenicity, accessibility and hydrophilicity tests. Forty-three MHC I epitopes were the most promising, while 438 from MHC II. For the population coverage, the epitopes covered 99.97% of the alleles worldwide excluding certain MHC II alleles. We recommend invivo and invitro studies to prove it’s effectiveness.

Brucellosis is a zoonotic infection caused by the bacterial genus Brucella. The disease is an old one and has been known by various names, including Mediterranean fever, Malta fever, gastric remittent fever, and undulant fever. Humans are the accidental hosts. In humans, they are characterized by a variable incubation period (ranging from several days up to several months), and clinical signs and symptoms of continued, intermittent or irregular fever of variable duration with headaches, weakness, profuse sweating, chills, depression and weight loss. Localized suppurative infections may also occur. The course of the disease can be variable, especially in persons either not or inadequately treated. Diagnosis of clinical brucellosis in humans and animals is initially made by use of appropriate serological or other immunological tests, and confirmed by bacteriological isolation and identification of the agent, but brucellosis continues to be a major public health concern worldwide and is the most common zoonotic infection [1] [2] [3] . Brucella organisms, which are small aerobic intracellular coccobacilli, localize in the reproductive organs of host animals, causing acute or chronic disorder. They are spread widely in the animal's urine, milk, placental fluid and other fluids. Till date, eight species have been identified, named primarily for the source animal or features of infection. Of these, four moderate-to-significant human pathogenicity species are Brucella melitensis (from sheep; highest pathogenicity), Brucella suis (from pigs; high pathogenicity), Brucella abortus (from cattle; moderate pathogenicity), and Brucella canis (from dogs; moderate pathogenicity) [3] . B. abortus is a gram-negative alpha-proteobacterium in the family Brucellaceae and is one of the causative agents of brucellosis. The rod-shaped pathogen, classified under the domain prokaryotic bacteria, is non-spore-forming, non-motile and aerobic. The mortality in recognized symptomatic acute or chronic cases of brucellosis is very low, certainly less than 5% and probably less than 2%. B. abortus primarily affects cattle, other biovidae and cervidae. It is usually the result of the rare instance of Brucella endocarditis or is the result of severe CNS involvement, often as a complication of endocarditis. Post-mortem analysis confirms that the burden of acute brucellotic infection is borne by tissues of the lymphoreticular system. Members of the genus can grow on enriched media like blood agar or chocolate agar. Identification in species level can be done by agglutination with monospecific serum, cultivating the strains in the presence of dyes and/or with PCR methods. They are catalase, oxidase and urea positive bacteria [1] [2] [3] [4] . It is an intracellular coccobacilli pathogen that infects genitourinary tract of cattle and humans who are infected after exposure to B. abortus or was previously identified to be immunogenic in animals infected with Brucella species to infected animals or contaminated meat or dairy products. The response against B. abortus involves the whole gamut of the immune system, from innate to adaptive immunity resulting from stimulation of antigen-presenting cells, NK cells, CD4+ and CD8+ T cells, and B cells [5] . Brucellosis is an important human disease in many parts of the world, as much as 200 cases per 100,000 of the population in some regions of the world; besides, the infection has become endemic in many countries [6] . It is a bacterial infection which is known as undulant fever, Mediterranean fever, or Malta fever. It is a zoonosis and the infection is almost invariably transmitted to people by direct or indirect contact with the infected animal (eg eating raw or unpasteurized dairy products or air ) [7] . To treat or cure this infection, we need to identify its outer membrane proteins such as OmpW family protein. However, most of the gram negative bacteria have evolved many mechanisms of attaching and invading host epithelial and immune cells. In particular, many outer membrane proteins (OMPs) are involved in this initial interaction between the pathogen and it's host. This can make a useful use of it in the designing vaccine. A number of small pore-forming OMPs are all composed of eight stranded βbarrel proteins and include members of the OmpA, OmpW with a beta-barrel structure consisting of eight non-parallel beta strands. OmpW family are widely distributed among gram-negative bacteria. These proteins, together with the related OmpA-like peptidoglycan associated lipoproteins, are involved in interactions with host cells and are mediators of virulence. In many cases, these proteins interact with host immune cells and can be considered as pathogen associated molecular patterns (PAMPS) due to their ability to signal through toll like receptor molecules and other pattern recognition receptors [8] . In some studies, they discovered that the 14-kDa protein possessed immunoglobulin binding and hemagglutination properties that appeared to be based on the protein's lectin-like properties. Hemagglutination inhibition experiments suggested that the 14-kDa protein has affinity towards mannose. Disruption of the gene encoding the 14-kDa protein in virulent B. abortus strain 2308 induced a rough-like phenotype with an altered smooth lipopolysaccharide (LPS) immunoblot profile and a significant reduction in the bacterium's ability to replicate in mouse spleens. However, the mutant strain was stably maintained in mouse spleens at 2.0 to 2.6 log(10) CFU/spleen from day 1 to week 6 after intra-peritoneal inoculation with 4.65 log(10) CFU. In contrast to the case for the smooth virulent strain 2308, in the rough attenuated strain RB51 disruption of the 14-kDa protein's gene had no effect on the mouse clearance pattern. These findings indicate that the 14-kDa protein of B. abortus possesses lectin-like properties and is essential for the virulence of the species, probably because of its direct or indirect role in the synthesis of smooth LPS [9] . The vaccine strains are the most commonly used to protect livestock against infection and abortion. However, due to some disadvantages of these vaccines, numerous studies have been conducted for the development of effective vaccines that could also be used in other susceptible animals. In this article, we compared different aspects of immunogenic antigens that have been a candidate for the brucellosis vaccine to get the most effective, efficient, active and harmless one [10] .

The sequences of the B. abortus strains were retrieved from the National Center for Biotechnology Information (NCBI) database in May 2019 in FASTA format. These strains were collected from different parts of the world for immunoinformatics analysis. The retrieved protein strains had a length of 227 with the name OmpW family protein.

BioEdit Sequence Alignment Editor Software (version 7.0.5.3) was used to determine the conserved regions of the retrieved sequences of B. abortus OmpW family using Clustal-W multiple sequence alignment (MSA). Amino acid composition and Molecular weight of the protein were also obtained.

The prediction of Linear B cell epitopes is done by objected reference sequence of B. abortus to Bepipred Linear Epitope Prediction tool 2.0 at Immune Epitope Database and Analysis Resource IEDB (http://tools.iedb.org/bcell/) [11] . Bioedit sequence alignment editor was used to identify the epitope Conservancy. Only epitopes with 100% conservancy were selected and analysed for surface antigenicity through Kolaskar & Tongaonkar Antigenicity tool, Emini Surface Accessibility Prediction tool for surface accessibility and Parker Hydrophilicity Prediction tool for hydrophilic, accessible, or mobile regions with thresholds of 1.033, 1.000 and 0.779 respectively [12] [13] [14] . Epitopes that pass all tests were predicted as B cell epitope.

Analysis of peptide binding to the MHC (Major Histocompatibility complex) class I molecule was calculated by the IEDB MHC I prediction tool (http://tools.iedb.org/mhci/) to predict cytotoxic T cell epitopes. To predict the binding affinity, we used Artificial Neural Network (ANN) 4.0 prediction method [15, 16] . Before the binding analysis, all the alleles were selected, and the length was set to 9 amino acids before the prediction was done. Then, the conserved epitopes were classified according to their half-maximal inhibitory concentration (IC50) into high affinity (IC50<100), moderate affinity (IC50<500) and lowaffinity epitopes (IC50 <500). Only high-affinity epitopes with their corresponding alleles were subjected to population coverage analysis.

Prediction of T cell epitopes was identified by using the IEDB MHC II prediction tool (http://tools.iedb.org/mhcii/) consuming the NN-align method [17] . To determine the interaction potentials of T cell epitopes and their respective MHC class II alleles, all human allele references were set. All conserved epitopes that bind to alleles at a score less than 500 half-maximal inhibitory concentration (IC50<500) were selected for further analysis.

AllerTop 2.0 (http://www.ddg-pharmfac.net/AllerTOP/), an online server, was used to analyze the predicted allergenicity of the selected epitope based on the main physiochemical properties of proteins. The predicted epitopes were classified as either "Probable Allergen" or "Probable Non-allergen" [18] .

Population coverage analysis tool of IEDB was used for predicting the population coverage of the whole world (http://tools.iedb.org/tools/population/iedb_input) [19] . Given a set of epitopes with their corresponding HLA alleles, this tool calculates the fraction of individuals predicted to cover. Epitopes with the highest frequency were selected for modelling.

RaptorX (https://www.raptor.uchicago.edu) was used to predict the 3D structure of B. abortus OmpW family protein by comparing it to a set of homologs. For visualization and analysis of the molecular structure of the obtained 3D protein structure, UCSF Chimera (version1.8) (http://www.cgl.ucsf.edu/chimera) was used.

Sixty-one sequences of the OmpW family protein of B. abortus were subjected to multiple sequence alignment against the reference protein to look for the conserved regions. 

Forty-seven epitopes passed the four B-cell prediction tools (Bepipred linear epitope 2, Emini surface accessibility (threshold of 1), Kolaskar & Tongaonkar antigenicity (threshold of 1.033), Parker hydrophilicity prediction (threshold of 0.779)) and the Allertop test. Below is the result of the top seven B-cell epitopes that had passed all four tests and have the largest length (Table 1) . 

The reference sequence was analyzed using (IEDB) MHC class I binding prediction tool to predict for possible T cell epitopes interacting with different MHC class I alleles with IC50 <100. Forty-three peptides were predicted to interact with different MHC class I alleles. All conserved epitopes with their corresponding MHC class I alleles and IC50 scores are shown in (Table 2 ). 

The reference sequence was analyzed using (IEDB) MHC class II binding prediction tool. There were 438 predicted epitopes found to interact with MHC class II alleles. The most promising epitopes with the number of HLA hits and their IC50 scores were shown in (Table  3) . 

All MHC class I and II epitopes were assessed for global population coverage using IEDB population coverage tool. For MHC I, the epitope with the highest global population coverage was LLAATALAL with coverage percentage of 45.62% (Table 4 and Figure 2 ).

MHC II epitope with the highest global population coverage was ITYYFTKNIAAELIL with a 99.97% coverage (Table 5 and Figure 3 ). Raptor X homology modeler software used for prediction of the most identical OmpW protein structure of B. abortus, and the PDB ID obtained was 2f1tA. The most promising peptides 3D structure was afterwards, edited and visualized using the Chimera software (version 1.13.1rc). 

In the current study, we proposed different peptides that can be recognized by B and T cells to produce vaccine against OmpW family protein of Brucella abortus. Immunoinformatics based peptide vaccines overcome the side effects of conventional vaccines. Peptide vaccines require less time and cost to be produced, can stimulate effective immune response and can target rapidly mutating pathogens [20, 21] . The reference sequence of OmpW family protein of B. abortus was subjected to Bepipred linear epitope prediction 2 test, Emini surface accessibility test, Kolaskar and Tongaonkar antigenicity test, Parker hydrophilicity test and Allertop to determine the binding ability to the B cell and to test the surface accessibility, immunogenicity, hydrophilicity and allergenicity respectively. Furthermore, multiple sequence alignment was used to study the conservation of the protein against sixty similar protein sequences which showed a high degree of conservation except at three sites (29, 51 and 192) , which signify the necessity of this protein for the survival of B. abortus species ( Figure 1 ). Forty-seven peptides passed four B-cell prediction tools (Table 1) . Bepipred linear epitope 2, Emini surface accessibility, Kolaskar & Tongaonkar antigenicity, Parker hydrophilicity prediction, and Allertop methods. Of these peptide seven were promising as they passed all prediction tools. These peptide has the ability to produce effective antibody response. The reference sequence was further analyzed using IEDB MHC I and II binding prediction tools to predict T cell epitopes. Forty-three peptides were predicted to interact with MHC I alleles with IC50 <=100 ( Table 2) . Seven of them were most promising and had the ability to bind to the highest number of MHC I alleles (HFTNFGAFK, YHFTNFGAF, LALTAPAAF, AELILGTTY, WLIGTGITY, FTKNIAAEL and LLAATALAL). Forty three predicted epitopes interacted with MHC II alleles with IC50 <= 500. Twenty four of them were most promising and had the ability to bind to the highest number of MHC II alleles ( Table 3) .

The best epitope with the highest population coverage for MHC class I was LLAATALAL with 45.62% in two HLA hits, one of them is HLA-A*02:01 -the worldwide predominant MHC I allele which is capable of inducing powerful CTL responses and the coverage of population set was 97.4% for the whole MHC I epitopes (Table 4 ). Regarding population coverage for MHC class II, the best epitope was ITYYFTKNIAAELIL scoring 99.97% with thirty HLA hits and the coverage of population set was 99.99% for the whole MHC II epitopes (Table 5 ). This high coverage percentage makes these peptides excellent targets for the vaccine design. The combined coverage for both MHC I and MHC II is 100%, which further solidify the significance of these epitopes (Figure 4) . Many studies had predicted peptide vaccines for different microorganisms such as madurella mycetomatis, HIV, pulmonary tuberculosis, treponema pallidum, pseudomona aeruginosa, COVID19 and malaria using immunoinformatics tools [22] [23] [24] [25] [26] [27] [28] . Many different approaches to reduce brucellosis risk were tested over the years like killed vaccines, subunit vaccines using recombinant proteins, or vector vaccines, with variable success [29] . Currently, vaccines for Brucella are made with live cells, so the use of OmpW family protein of B. abortus would be much safer [30] . In the present study, we identified several B and T cell epitopes that could be used as targets for vaccine design against B. abortus. However, several in vivo and in vitro studies will be needed in the future to confirm these results.

Vaccination is one of the most successful public health measures to reduce the burden of disease. Moreover, using insilico prediction methods will reduce the cost, time and effort needed to design these vaccines. In this work, we presented different peptides that can produce effective immunity in human against OmpW family protein of B. abortus for the first time. Seven B cell epitopes passed the antigenicity, accessibility, hydrophilicity and allergenicity tests. Seven promising MHC I epitopes were chosen with highest population coverage, while twenty four peptides for MHC II were chosen as the best targets for the vaccine. An astonishing and surprising combined population coverage for both MHC I and MHC II of 100% was obtained through this vaccine design.

The data which support our findings in this study are available from the corresponding author upon reasonable request.

The authors declare that there is no conflict of interest.

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Many thanks go to the National Center for Biotechnology Information (NCBI), BioEdit, Immune Epitope Database (IEDB) Analysis Resource, RaptorX server, and UCSF Chimera team.