Immunoinformatics Prediction of Epitope Based Peptide Vaccine Against Listeria Monocytogenes Fructose Bisphosphate Aldolase Protein

Listeria Monocytogenes represents an important food-borne pathogen worldwide that can cause life-threatening listeriosis disease especially in pregnant women, fetuses, elderly people, and immuno-compromised individuals with high mortality rates. Moreover, no vaccine against it exists. This study predicts an effective epitope-based vaccine against Fructose 1,6 Bisphosphate Aldolase (FBA) enzyme of Listeria Monocytogenes using immunoinformatics approaches. The sequences were retrieved from NCBI and several prediction tests were conducted to analyze possible epitopes for B-cell, T-cell MHC class I and II. 3D structure of the promising epitopes was obtained. Two epitopes showed high binding affinity for B-cells, while four epitopes showed high binding affinity for MHCI and MHCII. The results were promising to formulate a vaccine with more than 98% population coverage. We hope that these promising epitopes serves as a preventive measure for the disease in the future and recommend invivo and invitro studies.


INTRODUCTION
Bacteria of the genus Listeria that are widely distributed in the environment comprise a group of grampositive, facultative anaerobe, non-sporulating rods [1]. It consists of ten different species with Listeria Monocytogenes commonly found in humans [2]. Listeria Monocytogenes represents an important food-borne pathogen worldwide, that can cause life-threatening listeriosis disease in the susceptible groups including pregnant women, fetuses, elderly people, and immuno-compromised individuals, with a a considerable mortality rate (20-30%) [3]. The disease includes, but not limited to sepsis, meningitis, encephalitis, spontaneous abortion, or fever and self-limiting gastroenteritis in a healthy adult [2]. Food-borne listeriosis has a global economic and health burden due to the wide spread of Listeria Monocytogenes in food and food processing environments [4]. It capable of proliferating in different stressful environmental conditions, including high salinity, low temperature (refrigerator), and a wide range of pH values [4]. Although the incidence of listeriosis is relatively rare compared to other food-borne illnesses, listeriosis accounts for approximately 19% of deaths among all food-borne illness [5]. Studies have shown that Listeria Monocytogenes is the third leading cause of death from food-borne illness in the United States, with approximately 260 deaths annually. Mortality rates with confirmed Listeria Monocytogenes infections are around 15% but can be higher depending on patient status and comorbidities [6]. Listeria Monocytogenes has 13 different serotypes based on a variety of flagella and surface antigens. However, there are only three serotypes (1/2a, 1/2b, 4a) that can cause disease in humans [2]. Mendonça et al., (2016) mentioned that the surface exposed antigens of fructose-1,6-bisphosphate aldolase (FBA) class II in Listeria species is the antigen target of the previously described a hybridoma-derived antibody (mAb-3F8). They reported FBA as a novel immunogenic surface target useful for the detection of the genus Listeria [7]. 30-kDa protein is a fructose-1,6-bisphosphate aldolase (FBA), an enzyme of the glycolytic pathway that catalyzes the cleavage of its substrate fructose-1,6-bisphosphate (FBP) into glyceraldehyde 3phosphate (G3P) and dihydroxyacetone phosphate (DHAP). There are two main classes of FBA: class 1 is known to form tetramers, and is present mainly in higher eukaryotes, such as animals, plants, and algae; while class II can form many different multimers, and is present mainly in bacteria [8]. Class II FBA has been studied as a potential target of new antibiotics [9,10] and as vaccine antigen [11]. Besides, many studies have shown that FBA may play a role in pathogenesis by interacting with host's plasminogen [12,13] or promoting adhesion of T cell epitopes and MHC Class II allele (HLA DR, DP and DQ). NN-align method was used to predict the binding affinity. IC50 values at score less than 500 were selected. [34][35][36][37]

Population Coverage
In IEDB, the population coverage link was selected to analyse the epitopes. This tool calculates the fraction of individuals predicted to respond to a given set of epitopes with known MHC restrictions (http://tools.iedb.org/population/iedbinput). The appropriate checkbox for calculation was checked based on MHC I, MHC II separately and combination of both. [38]

3D Structures
3D structure was obtained using raptorX (http://raptorx.uchicago.edu) i.e. a protein structure prediction server developed by Xu group, excelling at predicting 3D structures for protein sequences without close homologs in the Protein Data Bank (PDB). USCF chimera (version 1.8) was the program used for visualization and analysis of molecular structure (http://www.cgl.uscf.edu/chimera). [39,40]

Multiple Sequence Alignment
The conserved regions and amino acid composition for the reference sequence of Listeria Monocytogenes FBA is illustrated in figure 1 and 2 respectively.

B-cell epitope prediction
The reference sequence of Listeria Monocytogenes FBA was subjected to Bepipred linear epitope, Emini surface accessibility, Kolaskar & Tongaonkar antigenicity and Parker hydrophilicity Prediction methods to test for various immunogenicity parameters (Table 1 and

Prediction of cytotoxic T-lymphocyte epitopes and interaction with MHC class I
The reference sequence was analyzed using (IEDB) MHC-1 binding prediction tool to predict T cell epitopes interacting with different types of MHC Class I alleles, based on Artificial Neural Network (ANN) with halfmaximal inhibitory concentration (IC50) <100 nm. 61 peptides were predicted to interact with different MHC-1alleles. The most promising epitopes and their corresponding MHC-1 alleles are shown in (Table 2) followed by the 3D structure of the proposed T cell epitope ( Figure 9).

Prediction of the T cell epitopes and interaction with MHC class II
Reference sequence was analyzed using (IEDB) MHC-II binding prediction tool based on NN-align with halfmaximal inhibitory concentration (IC50) <500 nm; there were 388 predicted epitopes found to interact with MHC-II alleles. The most promising epitopes and their corresponding alleles are shown in (Table 3) along with the 3D structure of the proposed epitope ( Figure 10).   In population coverage analysis of MHC II; 12 alleles were not included in the calculation , therefore the above (*) percentages are for epitope sets excluding these alleles:

DISCUSSION
Different proposed peptides that can be recognized by B cell and T cell to produce antibodies against FBA of Listeria Monocytogenes were presented for the first time. Peptide vaccines overcome the side effects of conventional vaccines. The reference sequence of Listeria Monocytogenes FBA was subjected to Bepipred linear epitope prediction 2 test, Emini surface accessibility test and Kolaskar and Tongaonkar antigenicity test and Parker hydrophilicity test in IEDB, to determine the binding to B cell and to test the immunogenicity and hydrophilicity respectively. Out of the five predicted epitopes using Bepipred 2 test, only two epitopes passed the other three tests (VDYAHAKG, VREKLATDDKVY). 26 epitopes were predicted to interact with MHCI alleles with IC50 < 100. Four of them were most promising (ITVPVAIHL, MPIVNMTDM, MLKKALAGK, YMGGFKTVV). 134 predicted epitopes were interacted with MHCII alleles with IC50 < 500. Four of them were most promising (VVDYAHAKGVSVEAE, MTDMLKKALAGKYAV, NMTDMLKKALAGKYA, TDMLKKALAGKYAVG). Eleven epitopes (YAHAKGVSV, ALAAALGSV, TVVKMTEGL, MLKKALAGK, ITVPVAIHL, HAKGVSVEA, IVNMTDMLK, ALGSVHGPY, KALAGKYAV, NIDALAAAL, LAAALGSVH) were shared between MHC I and II. Excluding certain alleles for MHCII, population coverage for the most promising epitopes covered 98.94% of the whole world (MHCI & MHCII combined). Many studies predicted peptide vaccines for different microorganisms such as, Rubella, Ebola, Dengue, Zika, HPV, Lagos rabies virus, and mycetoma using immunoinformatics tools. [41][42][43][44][45][46][47][48][49][50] Limitations include the exclusion of certain HLA alleles for the MHC II. We hope that the whole world will benefit from this epitopebased vaccine upon its successful development following invivo and invitro studies to prove it's effectiveness.

CONCLUSION:
Peptide vaccines overcome the side effects of conventional vaccines. We presented different peptides that can produce antibodies against FBA of Listeria Monocytogenes for the first time. Two B cell epitopes passed the antigenicity, accessibility and hydrophilicity tests. Four MHCI epitopes were the most promising ones, while four for MHC II. Eleven epitopes were shared between MHC I and II. For the population coverage, the epitopes covered 98.94% of the alleles worldwide excluding certain ones.

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