Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Label_Title

Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Modeling and in Silico Analysis for Prediction of Epitopes Vaccine against Norwalk virus from Capsid Protein (VP1) through Reverse Vaccinology

Elsideeq E. M. Eltilib, Yassir A. Almofti*, Khoubieb Ali Abd-elrahman, Mashair A. A. Nouri

Department of Molecular Biology and Bioinformatics, College of Veterinary Medicine, University of Bahri, Khartoum- Sudan

Abstract: Noroviruses are the leading cause of acute gastroenteritis and is responsible for approximately 685 million cases and 200,000 deaths annually worldwide. Currently, there is no vaccine to prevent human norovirus infection, and there is no specific therapy available to treat it. This study aimed to predict epitopes from the capsid VP1 protein that elicited the immune system and acted as safer efficacious vaccine. A total of 21 noroviurse strains were retrieved from the NCBI database. The IEDB analysis resources were used for epitopes prediction against B and T cells. The population coverage was calculated for the proposed epitopes against the whole world. Eight epitopes (₄₈QVNP₅₁, ₁₅₉EVPLE₁₆₃, ₂₂₄VEQK₂₂₇, ₂₄₅RAPLP₂₄₉, ₃₇₆ISPPS₃₈₀, ₄₀₉VYPP₄₁₂, ₄₇₃FKAY₄₇₆ and ₄₉₂PQQLP₄₉₆) successfully passed all B cell prediction tools and were shown to be antigenic, nonallergic and nontoxic. Thus were proposed as B cells epitopes. For cytotoxic T cells, a total of 103 epitopes were found to interact with MHC-I alleles. However, only 22 epitopes were shown to be antigenic, nonallergic and nontoxic. Among them four epitopes namely (₁₄₀₋AQATLFPHV-₁₄₈; ₂₁₆₋FLFLVPPTV₋₂₂₄; ₄₉₉₋GVFVFVSWV₋₅₀₇ and ₄₁₀₋YPPGFGEVL₋₄₁₈) interacted with high number of MHC-I alleles and demonstrated favourable population coverage and thus were proposed as cytotoxic T lymphocytes MHC-1 epitopes. Moreover helper T cells, a total of 421 core epitopes were found to interact with MHC-П alleles. However, only 105 epitopes were shown to be antigenic, nonallergic and nontoxic. Eight epitopes namely (₂₁₆₋FLFLVPPTV₋₂₂₄; ₄₉₉₋GVFVFVSWV₋₅₀₇; ₄₃₃₋LPCLLPQEY₋₄₄₁; ₉₀₋NPFLLHLSQ₋₉₈; ₃₉₄₋NYGSSITEA₋₄₀₂; ₂₄₇₋PLPISSMGI₋₂₅₅; ₂₂₀₋VPPTVEQKT₋₂₂₈; ₄₁₀₋YPPGFGEVL₋₄₁₈) were interacted with most frequent MHC class II alleles, demonstrated higher population coverage and three of them (₂₁₆₋FLFLVPPTV-224; ₄₉₉₋GVFVFVSWV₋₅₀₇ and ₄₁₀₋YPPGFGEVL₋₄₁₈) were shown to interact with both MHC-I and MHC-II alleles. Therefore they were proposed as T helper cells epitopes. The population coverage was 60.35% and 99.96% for MHC-I and MHC-II epitopes, respectively, and 100% for all T cells epitopes. Taken together 17 epitopes successfully proposed as vaccine candidate against noroviruse. In vivo and in vitro clinical trials studies are required to elucidate the effectiveness of these epitopes as vaccine.

Keywords: Noroviruses, NCBI, IEDB, Insilico vaccine, B cells, T cells

Received February 11, 2020; Revised March 14, 2020; Accepted March 24, 2020

Citation: Elsideeq E. M. Eltilib, Yassir A. Almofti, Khoubieb Ali Abd-elrahman, Mashair A. A. Nouri, Modeling and in Silico Analysis for Prediction of Epitopes Vaccine against Norwalk virus from Capsid Protein (VP1) through Reverse Vaccinology. American Journal of Infectious Diseases and Microbiology, 8 (1). 29-44. January 2020. doi:10.12691/ajidm-8-1-5

1. Introduction

Noroviruses are the leading cause of acute gastroenteritis and is responsible for approximately 685 million cases and 200,000 deaths annually worldwide [1,2]. These viruses cause gastrointestinal disease, resulting in recurrent vomiting and diarrhea [1,3,4]. The infection mostly characterized by high excreted viral loads [5], severe infectivity [6] and short-term immunity [7].

Norwalk viruses are the main cause of outbreak and irregular of nonbacterial gastroenteritis [8,9] and connected to almost one over five of all reported cases are acute gastroenteritis internationally [9,10]. Contamination causes outbreaks of water - borne and food - borne illness in the society and generates signs of vomiting and diarrhea [11,12,13,14]. Diarrheal diseases are associated with high mortality rates [15,16,17,18,19] and noroviruses are an important cause of epidemic gastroenteritis together in children and adults worldwide [10,20,21,22,23,24].

Noroviruses are an assemblage of single, positive-stranded RNA viruses classified into the Norovirus genus in the family Caliciviridae [11,25,26,27]. This family composed of five types; Norovirus, Sapovirus, Lagovirus, Vesivirus and Nebo virus. The first two genera are human while the others are animal genera [11,12,17,18,27]. Genetically the human noroviruses are dissimilar [19] and are divided into six genogroups based on the amino acid sequence of the major structural proteins VP1 and VP2 [10,11,12,18].

The norovirus genome has three open reading frames (ORFs) of which ORF2 and ORF3 encode the major capsid protein (VP1) that determines the antigenicity of the virus, as well as the minor capsid protein (VP2). ORF1 encodes a large polyprotein that is cleaved by the viral protease in mature non-structural proteins, including the RNA-dependent RNA polymerase [28]. To date, all norovirus vaccine candidates contain non-infectious recombinant VP1 proteins, either as virus-like particles (VLP), as P-particles, or as recombinant adenoviruses [29].

No vaccine is obtainable to put off norovirus illness or infection [8,21], however there was attempts to make a vaccine from a novel strain of NOVs as plant - based oral vaccine, but unfortunately was invaluable [30]. In this study we attempted to propose an epitopes from the capsid protein (VP1) of the virus that could elicited both B and T lymphocytes and act as safer vaccine.

2. Materials and Methods

2.1. Protein Sequence Retrieval and Alignment Tool

The protein sequences of 21 capsid proteins (VP1) were retrieved from the NCBI database (https: //www.ncbi.nlm.nih.gov/). The retrieved strains, accession numbers, country and date of collection were shown in (Table 1).

Table 1. Retrieved strains of capsid protein (VP1) with their date of collection, accession numbers and geographical regions

Determination of the conserved regions

The retrieved protein sequences were further aligned to obtain conserved regions using multiple sequence alignment (MSA) tools, Clustal W in the BioEdit program, version 7.0.9.0 [31]. Multiple sequence alignment (MSA) analysis was performed to analyze the conserved residue sequence amongst the screened B and T cell epitopes.

2.2. Evolution Analysis

The retrieved capsid protein sequences (VP1) were subjected to evolutionary divergence analysis and a phylogenetic tree was constructed to determine the common ancestor of each retrieved strain using MEGA7.0.26 (7170509) software [32].

2.3. Determination of B Cells Epitopes

The conserved regions of the candidate epitopes were analyzed by different prediction software tools obtained by Immune Epitope Database (IEDB) analysis (https: //www.iedb.org/). The reference sequence of VP1 was used as an input for the IEDB software resource analysis.

2.4. B Lymphocytes Epitopes Prediction

Tools from IEDB were used to identify the B cell epitopes (http: //tools.iedb.org/bcell/). This includes Bepipred linear epitope prediction analysis [33], Emini surface accessibility prediction [34] and Kolaskar and Tongaonkar antigenicity scale [35]. These methods predicted specific epitopes in the capsid protein that were linear, at surface and immunogenic, respectively, and can bind to B cell receptors

2.5. T-lymphocytes Epitopes Prediction

The IEDB tools were used for the identification of the T cell epitopes prediction. The prediction method includes the major histocompatibility complex class I and П (MHC-I, MHC-П)

2.6. MHC-I Binding Predictions

Analysis of epitopes binding to MHC-I molecules was assessed by the software in IEDB MHC-I prediction tools (http: //tools.iedb.org/mhci/). The prediction method was obtained by Artificial Neural Network (ANN), Stabilized Matrix Method (SMM) or Scoring Matrices derived from combinatorial peptide libraries. Before the prediction step, epitopes lengths were set as 9mers. The conserved epitopes that bind to alleles at score equal to or less than 300 was considered as half-maximal inhibitory concentrations. Epitopes equal to or less than the IC50 were selected for further analysis

2.7. MHC-П Binding Predictions

Analysis of epitopes binding to MHC-II molecules was performed by the IEDB MHC-II prediction tools (http: //tools.iedb.org/mhcii/). The neural networks align (NN-align) that allow for simultaneous identification of the MHC-II binding core epitopes and binding affinity was used. All the predicted conserved epitopes that bind to many alleles at score equal to or less than 3000 half-maximal inhibitory concentration (IC50≤3000) was considered. Epitopes that equal to or less than the IC50 were selected for further analysis.

2.8. Antigenicity of the Predicted Epitopes

For determination of the antigenicity of the predicted epitopes, the sequence of the predicted epitopes for B and T lymphocytes were submitted to the VaxiJen v2.0 server (http: //www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) for antigenicity prediction. The threshold of VaxiJen v2.0 server was set to the default threshold (0.4). Epitopes with antigenicity were further investigated for allergenicity and toxicity.

2.9. Allergenicity and Toxicity of the Predicted Epitopes

The following servers; AllergenFP [36], AllerTOP [37], Allermatch [38] and PA³P [39] were used to predict the allergenicity of the proposed epitopes for B and T lymphocytes. Epitopes found to be non-allergenic were further assessed for toxicity level by ToxinPred server [40].

2.10. Population Coverage Analysis of Predicted Epitopes

For the calculation of the population coverage for all potential MHC-I and II epitopes, the IEDB tools was used (http: //tools.iedb.org/population/). The capsid protein was assessed for population coverage against the whole world with selected MHC-I and MHC-II interacted alleles.

2.11. Homology Modeling

Raptor X for 3D structure prediction server (http: //raptorx.uchicago.edu/StructurePrediction/predict/) was used for creation the 3D structure of the reference protein. The reference sequence was used as an input and Chimera 1.8 [41] was used as a tool to visualize the selected epitopes belonging to B cell and T cell (MHC-I and MHC-II). Homology modeling was used for visualization of the surface accessibility of the B lymphocytes predicted candidate epitopes as well as for visualization of all predicted T cell epitopes in the structural level.

3. Results

3.1. Phylogenetic Tree

According to the result of the conserved regions of the retrieved strains of the VP1 proteins using multiple sequence alignment analysis, the protein sequences demonstrated various strains with less mutated regions. This resulted in closed relationship of the retrieved sequences despite the possibilities of mutations in VP1 capsid protein. Generally Figure 1 provided the phylogenetic tree of the retrieved strains and the strains demonstrated molecular evolutionary divergence.

Figure 1. Phylogenetic tree of the capsid protein (VP1) of the retrieved strains. The retrieved strains demonstrated divergence in their common ancestors

Figure 2. Multiple sequence alignment (MSA) of the retrieved strains of the capsid protein (VP1) using Bioedit software and ClustalW. Dots indicated the conservancy of the retrieved strains and letters within the rectangular indicated no conservancy (mutation) in amino acid

3.2. Alignment

The multiple sequence alignment was performed to obtain the regions of conservancy among Norwalk virus variants using multiple sequence alignment (MSA) tools, of Clustal W in the BioEdit program, version 7.0.9.0. As shown in Figure 2 the dots meant that amino acids at that position were conserved while the letters meant that the amino acids at that position were mutated. Generally the retrieved strains demonstrated less mutated regions during the alignment process. Thus multiple epitopes were predicted to be conserved among the retrieved strains. It is noteworthy that only the conserved epitopes (100% conserved epitopes) were selected for further analysis. Thus the proposed epitopes elected when they showed 100% conservancy among the retrieved strains

Figure 3. Prediction of B-cell epitopes by different IEDB scales (a- Bepipred linear epitope prediction, b- Emini surface accessibility, c- Kolaskar and Tongaonkar antigenicity prediction) for the capsid protein. Regions above threshold (red line) were proposed as a part of B cell epitope while regions below the threshold (red line) were not

Table 2. B-cell epitopes prediction from the capsid protein, the position of peptides is according to the position of amino acids in the capsid protein

Figure 4. Position of proposed eight conserved B cell epitopes in structural level of the capsid protein (VP1). The epitopes were shown in dark red ball and sticks shapes. These epitopes showed conservancy, high score in surface accessibility and antigenicity using IEDB software, nonallergic and nontoxic. The position of these epitopes was according to their position in the capsid protein

Table 3. The 15 epitopes of the B-cell that overlapped the Bepipred linear epitope prediction, Emini surface accessibility and Kolaskar and Tongaonkar antigenicity prediction tools and further subjected to antigenicity, allergenicity and toxicity. The position of epitopes is according to the position of amino acids in the capsid protein (VP1)

3.3. Prediction of B Cell Epitope

The capsid protein (VP1) sequence was subjected to Bepipred linear epitope, Emini surface accessibility and Kolaskar and Tongaonkar antigenicity methods in IEDB. Figure 3 demonstrated the thresholds of the methods used to predict the B cell epitopes. For instance the Bepipred linear epitopes prediction method showed a threshold binding score to B cell of 0.350 (minimum -0.005 and maximum 2.401). This method predicted thirty four linear epitopes eliciting the B cell from the conserved regions. In Emini surface accessibility the prediction threshold of the surface accessibility area of the epitopes was 1.000 (minimum of 0.054 and maximum of 7.701). Twenty five epitopes were potentially in the surface by passing the default threshold 1.000. For Kolaskar and Tongaonkar antigenicity the average of antigenicity was 1.038 (minimum of 0.871 and maximum of 1.190). Nineteen epitopes gave score above the default threshold 1.038. All these epitopes and their scores against the B cell were provided in Table 2. However, only fifteen epitopes successfully overlapped the three tools and were shown in Table 3. These fifteen epitopes were further investigated for their antigenicity, allergenicity and toxicity. Eight epitopes namely (₄₈QVNP₅₁, ₁₅₉EVPLE₁₆₃, ₂₂₄VEQK₂₂₇, ₂₄₅RAPLP₂₄₉, ₃₇₆ISPPS₃₈₀, ₄₀₉VYPP₄₁₂, ₄₇₃FKAY₄₇₆ and ₄₉₂PQQLP₄₉₆) were shown to be antigenic, nonallergic and nontoxic and thus were proposed as B cell epitopes. The positions of the proposed epitopes in the 3D structural level of VP1 capsid protein were shown in Figure 4.

Table 4. The 22 epitopes that interacted with MHC-1 from the capsid protein (VP1) of the Norwalk virus that demonstrated antigenicity, nonallergic and nontoxic. The population coverage for each predicted epitope was calculated and election of the proposed epitopes was based on the higher population coverage score. The position of epitopes is according to the position of amino acids in the capsid protein

Table 5. The best four proposed epitopes from the capsid protein (VP1) of the Norwalk virus that interacted with MHC class I alleles. The position of epitopes is according to the position of amino acids in the capsid protein

Figure 5. T cell proposed epitopes that interact with MHC-I alleles. Four epitopes (₁₄₀₋AQATLFPHV-₁₄₈; ₂₁₆₋FLFLVPPTV₋₂₂₄; ₄₉₉₋GVFVFVSWV₋₅₀₇ and ₄₁₀₋YPPGFGEVL₋₄₁₈) were shown in this figure since they interacted with MHC-1 alleles. The positions of these proposed epitopes was according to their position in the capsid protein

3.4. Prediction of Cytotoxic T-lymphocyte Epitopes and Interaction with MHC Class I

The reference sequence of the capsid protein was analyzed using IEDB MHC-1 binding prediction tools to predict T cell epitopes interacting with MHC Class I alleles. Based on Artificial Neural Network (ANN) with half-maximal inhibitory concentration (IC50) ≤300, 103 epitopes were predicted to interact with different MHC-1 alleles. All these epitopes were further investigated for their antigenicity, allergenicity and toxicity. Only 22 epitopes were shown to be antigenic, nonallergic and nontoxic. Furthermore these 22 epitopes were investigated for their population coverage against the whole world. The 22 epitopes and their population coverage were shown in Table 4. Four epitopes namely ₁₄₀₋AQATLFPHV-₁₄₈; ₂₁₆₋FLFLVPPTV₋₂₂₄; ₄₉₉₋GVFVFVSWV₋₅₀₇ and ₄₁₀₋YPPGFGEVL₋₄₁₈ demonstrated favourable population coverage and thus were proposed as cytotoxic T lymphocytes MHC-1 epitopes. The proposed four epitopes with their corresponding MHC-1 alleles were shown in Table 5. The positions of the proposed epitopes in the 3D structural level of capsid protein were shown in Figure 5.

3.5. Prediction of T Helper Cell Epitopes and Their Interaction with MHC Class II

The reference sequence of VP1 capsid protein was analyzed using IEDB MHC-II binding prediction tools. Based on NN-align with half-maximal inhibitory concentration IC50)≤3000 there were 421 predicted epitopes found to interact with MHC-II alleles. When these epitopes subjected to antigenicity, allergenicity and toxicity investigations, only 105 epitopes were shown to be antigenic, non-allergic and nontoxic and the population coverage of each epitope alleles was calculated. The 105 epitopes were provided in Table 6.

Table 6. The 105 epitopes from the capsid protein (VP1) and their interaction with MHC class II. These epitopes demonstrated antigenicity, nonallergic and nontoxic. The population coverage for each predicted epitope was calculated and election of the proposed epitopes was based on the higher population coverage score.

Among the 105 epitopes, eight epitopes namely (₂₁₆₋FLFLVPPTV₋₂₂₄; ₄₉₉₋GVFVFVSWV₋₅₀₇; ₄₃₃₋LPCLLPQEY₋₄₄₁; ₉₀₋NPFLLHLSQ₋₉₈; ₃₉₄₋NYGSSITEA₋₄₀₂; ₂₄₇₋PLPISSMGI₋₂₅₅; ₂₂₀₋VPPTVEQKT₋₂₂₈; ₄₁₀₋YPPGFGEVL₋₄₁₈) were interacted with most frequent MHC class II alleles, demonstrated higher population coverage and three of them were shown to interact with both MHC class I and MHC class II alleles. Therefore they were proposed as T helper cells epitopes. The eight epitopes and their corresponding MHC-II alleles were shown in Table 7. The position of these predicted epitopes in the 3D structural level in the capsid protein was illustrated in Figure 5 and Figure 6.

Table 7. The best eight epitopes that were proposed as a vaccine candidate from the capsid protein (VP1) of the Norwalk virus and interacted with high affinity with MHC class II alleles. The position of epitopes is according to the position of amino acids in the capsid protein

Figure 6. T cell proposed epitopes that interact with MHC-1I alleles. Three epitopes ( ₂₁₆₋FLFLVPPTV₋₂₂₄; ₄₉₉₋GVFVFVSWV₋₅₀₇ and ₄₁₀₋YPPGFGEVL₋₄₁₈) were not shown in this figure since they were shown in figure (5) (interacted with MHC-1 and MHC-1I alleles). The other five epitopes (433-LPCLLPQEY-441; 90-NPFLLHLSQ-98; 394-NYGSSITEA-402; 247-PLPISSMGI-255 and 220-VPPTVEQKT-228) were only shown in this figure since they only interacted with MHC-1I alleles. The positions of these proposed epitopes was according to their position in the capsid protein

3.6. Analysis of the Population Coverage

The predicted epitopes from the capsid protein (VP1) that interacting with MHC class I and II alleles were subjected to population coverage analysis. The proposed vaccine epitopes were elected as peptide vaccine based on their high population coverage score, number of their interacted alleles and/or their interaction with both MHC class I and II alleles. As shown in (Table 4) 22 predicted epitopes interacted with MHC class I alleles with different population coverage scores. Among them four epitopes scored high population coverage % and highly interacted with most frequent MHC class I alleles. This strengthen their potentiality to act as promising vaccine candidate against MHC class I therefore were proposed as peptide vaccine. The epitope set of these four epitopes against MHC-1 alleles was 60.35% (Table 8).

In addition to that, as shown in Table 6 MHC class II, 105 predicted epitopes interacted with MHC class II alleles with different population coverage scores and were shown to be antigenic, nonallergic and nontoxic. Among them eight epitopes highly interacted with most frequent MHC class II alleles therefore they were proposed as peptide vaccine.

Table 8. The population coverage (PC) of MHC-I and MHC-II proposed epitopes. The population coverage of MHC-I was 80.53%, MHC-II was 99.99% and the combined alleles was 100% for all proposed epitopes )PC: Population Coverage)

The epitope set of these eight epitopes against MHC-II alleles was 99.96% (Table 8). Furthermore as shown in Table 8 all proposed epitopes were subjected to population coverage tools to assess population coverage of their MHC-I and MHC-II combined alleles. The population coverage of the proposed epitopes against the combined alleles was 100%.

4. Discussion

Norwalk viruses are considered as a leading etiology of epidemic acute gastroenteritis as well as an important cause of sporadic cases of acute gastroenteritis [42]. Currently, there is no vaccine to prevent human norovirus infection, and there is no specific therapy available to treat it [20]. The norovirus genome has three open reading frames (ORFs) of which ORF2 and ORF3 encode the major capsid protein (VP1) that determines the antigenicity of the virus, as well as the minor capsid protein (VP2). The majority of the studies performed to design vaccine for norovirus used VP1 as a vaccine construct. For instance vaccine using recombinant adenovirus expressing the norovirus major capsid protein VP1was developed. The vaccine measured the cross-reactive neutralizing antibody responses which are required for a successful norovirus vaccine [43]. A study by Tucker et al (2008) [44] developed a currently human clinical trials vaccine that employs a recombinant adenovirus expressing the norovirus GI.1 major capsid protein (VP1) in an oral tablet formulation developed by Vaxart, Inc. Moreover recombinant adenovirus vaccine expressing the norovirus GII.4 major capsid protein VP1 [45] and multiple VLP vaccine developed from VP1 protein [46] were developed by the Chinese center for disease control and prevention. Both studies demonstrated the antigenicity of VP1 as a vaccine candidate. Therefore this study aimed to propose multiple epitopes vaccine candidates from the capsid protein (VP1) to elicit B and T lymphocytes and act as a vaccine candidate using immune-informatics tools.

In the current study the B cell epitopes were predicted from the capsid protein VP1 to ﬁnd the potential epitopes that would interact with B lymphocytes and initiate immune response. For the vaccine to be recognized by the B cell antibodies it must be linear and located on the surface of the antigen protein to be easily accessible. In addition to that the candidate vaccine should demonstrate greater antigenicity to elicit antibodies production. Therefore several tools from IEDB analysis resources were used to identify B cell epitopes such as Bepipred linear epitope prediction analysis [33], Emini surface accessibility prediction [34] and Kolaskar and Tongaonkar antigenicity scale [35]. These tools provided multiple epitopes that were linear, on the protein surface and antigenic. However, for the epitopes to be proposed as a vaccine candidate it should be nonallergic and nontoxic to the host cells [47]. Thus the predicted epitopes further subjected to Vaxigen antigenicity, allergenicity and toxicity investigations. Eight epitopes eight epitopes namely ₄₈QVNP₅₁, ₁₅₉EVPLE₁₆₃, ₂₂₄VEQK₂₂₇, ₂₄₅RAPLP₂₄₉, ₃₇₆ISPPS₃₈₀, ₄₀₉VYPP₄₁₂, ₄₇₃FKAY₄₇₆ and ₄₉₂PQQLP₄₉₆ passed these criteria and proposed as a B cell epitopes.

Since the immune response of T cell is long lasting response compared to B cell, where the antigen can easily escape the antibody memory response. This considered that CD8+T and CD4+T cells response play a major role in antiviral immunity [48]. Cytotoxic CD8+T lymphocytes are considered as an important parameter in recognizing and killing infected cells or producing specific cytokines that prevent the infection in the body [49,50]. Thus, T cell epitope-based vaccination is a unique process of eliciting strong immune response against infectious agents such as viruses [51]. In this study four epitopes were shown to interact with Cytotoxic CD8+T lymphocytes with high number of MHC-1 alleles and they demonstrated antigenicity and were shown to be nonallergenic and nontoxic. Beside that the four proposed epitopes showed favourable population coverage against the whole world population with epitopes set 60.35%. Therefore were proposed as Cytotoxic CD8+T lymphocytes epitopes.

For Helper CD4+T lymphocytes eight epitopes namely ₉₀₋NPFLLHLSQ₋₉₈; ₂₁₆₋FLFLVPPTV₋₂₂₄; ₄₉₉₋GVFVFVSWV₋₅₀₇; ₄₃₃₋LPCLLPQEY₋₄₄₁; ₃₉₄₋NYGSSITEA₋₄₀₂; ₂₄₇₋PLPISSMGI₋₂₅₅; ₂₂₀₋VPPTVEQKT₋₂₂₈; ₄₁₀₋YPPGFGEVL₋₄₁₈ were shown to interact with Helper CD4+T lymphocytes with high number of MHC-11 alleles and they were shown to be antigenic, nonallergenic and nontoxic. Among the eight epitopes three epitopes namely ₂₁₆₋FLFLVPPTV₋₂₂₄; ₄₉₉₋GVFVFVSWV₋₅₀₇ and ₄₁₀₋YPPGFGEVL₋₄₁₈ were shown to interact with both MHC-1 and MHC-11 alleles. This favoured them to be elected as a vaccine candidate. Also the eight epitopes demonstrated favourable interaction with the whole world population coverage with epitopes set 99.96%. Therefore were proposed as Helper CD4+T lymphocytes epitopes. The overall epitope set for the MHC-I and MHC-II combined alleles for the proposed epitopes showed excellent population coverage against whole world population (100%). Accordingly these epitopes were strongly recommended as promising epitopes vaccine candidates against T lymphocyte cells.

Recently a study by Azim et al (2019) [47] used multiple bioinformatics tools to predict epitopes from the VP1 and VP2 proteins of the noroviruse. However none of their predicted epitopes for B and T lymphocytes were corroborated to our proposed epitopes. This might be attributed to the differences in the software used in both studies to predict vaccine candidates.

5. Conclusion

The developing of an effective and safe vaccine is recommended to combat the infection and noroviruses. Vaccine design using reverse vaccinology prediction methods is highly appreciated as it provided specific epitopes in protein to act as a vaccine without using the virus particles as components of the vaccine. In this study eight epitopes were successfully proposed to interact against B cells. Moreover nine epitopes were successfully predicted to interact against T cell with population coverage epitope set of 100%. These epitopes provided excellent results as promising vaccine against noroviruses. However in vitro and in vivo trials are required to achieve the effectiveness of these epitopes as vaccine candidates.

Acknowledgments

Authors would like to thank the staff members of the Department of Molecular Biology and Bioinformatics, College of Veterinary Medicine, University of Bahri, Sudan for their cooperation and support.

Competing Interest

The authors declared that they have no competing interests.

Funding

No funding was received.

References

[1]   | Lopman, B. Global Burden of Norovirus and Prospects for Vaccine Development. CDC Foundation. Available online: https: //www.cdc.gov/norovirus/downloads/global-burden-report.pdf. 2015 (accessed on9 September 2017).
[2]   | Centers for Disease Control and Prevention. Norovirus Worldwide. 2016. Available online: https: //www.cdc.gov/norovirus/worldwide.html (accessed on 9 September 2017).
[3]   | Ahmed, S.M.; Hall, A.J.; Robinson, A.E.; Verhoef, L.; Premkumar, P.; Parashar, U.D.; Koopmans, M.; Lopman, B.A. Global prevalence of norovirus in cases of gastroenteritis: A systematic review and meta-analysis. Lancet Infect. Dis. 2014, 14, 725-730.
[4]   | Bartsch, S.M.; Lopman, B.A.; Ozawa, S.; Hall, A.J.; Lee, B.Y. Global economic burden of norovirus gastroenteritis. PLoS ONE 2016, 11, e0151219.
[5]   | Chan, M.C.; Sung, J.J.; Lam, R.K.; Chan, P.K.; Lee, N.L.; Lai, R.W.; Leung, W.K. Fecal viral load and norovirus-associated gastroenteritis. Emerg. Infect. Dis. 2006, 12, 1278-1280.
[6]   | Teunis, P.F.M.; Moe, C.L.; Liu, P.; Miller, S.E.; Lindesmith, L.; Baric, R.S.; Le Pendu, J.; Calderon, R.L. Norwalk virus: How infectious is it? J. Med. Virol. 2008, 80, 1468-1476.
[7]   | Karst, S.M.; Wobus, C.E.; Lay, M.; Davidson, J.; Virgin, H.W. STAT1-dependent innate immunity to a Norwalk-like virus. Science 2003, 299, 1575-1578.
[8]   | Velasquez LS, Hjelm BE, Arntzen CJ, Herbst-Kralovetz MM. An intranasally delivered Toll-like receptor 7 agonist elicits robust systemic and mucosal responses to Norwalk virus-like particles. Clinical and vaccine immunology: CVI. 2010; 17(12): 1850-8.
[9]   | Ramani S, Neill FH, Opekun AR, Gilger MA, Graham DY, Estes MK, et al. Mucosal and Cellular Immune Responses to Norwalk Virus. The Journal of infectious diseases. 2015; 212(3): 397-405.
[10]   | Kavanagh O, Estes MK, Reeck A, Raju RM, Opekun AR, Gilger MA, et al. Serological responses to experimental Norwalk virus infection measured using a quantitative duplex time-resolved fluorescence immunoassay. Clinical and vaccine immunology : CVI. 2011; 18(7): 1187-90.
[11]   | Czako R, Atmar RL, Opekun AR, Gilger MA, Graham DY, Estes MK. Serum hemagglutination inhibition activity correlates with protection from gastroenteritis in persons infected with Norwalk virus. Clinical and vaccine immunology : CVI. 2012; 19(2): 284-7.
[12]   | Vongpunsawad S, Venkataram Prasad BV, Estes MK. Norwalk Virus Minor Capsid Protein VP2 Associates within the VP1 Shell Domain. Journal of virology. 2013; 87(9): 4818-25.
[13]   | Liu P, Yuen Y, Hsiao HM, Jaykus LA, Moe C. Effectiveness of liquid soap and hand sanitizer against Norwalk virus on contaminated hands. Applied and environmental microbiology. 2010; 76(2): 394-9.
[14]   | McCarthy M, Estes MK, Hyams KC. Norwalk-like virus infection in military forces: epidemic potential, sporadic disease, and the future direction of prevention and control efforts. The Journal of infectious diseases. 2000; 181 Suppl 2: S387-91.
[15]   | Tamura M, Natori K, Kobayashi M, Miyamura T, Takeda N. Interaction of recombinant norwalk virus particles with the 105-kilodalton cellular binding protein, a candidate receptor molecule for virus attachment. Journal of virology. 2000; 74(24): 11589-97.
[16]   | Kobayashi S, Sakae K, Suzuki Y, Shinozaki K, Okada M, Ishiko H, et al. Molecular cloning, expression, and antigenicity of Seto virus belonging to genogroup I Norwalk-like viruses. Journal of clinical microbiology. 2000; 38(9): 3492-4.
[17]   | Sharp TM, Crawford SE, Ajami NJ, Neill FH, Atmar RL, Katayama K, et al. Secretory pathway antagonism by calicivirus homologues of Norwalk virus nonstructural protein p22 is restricted to noroviruses. Virology journal. 2012; 9: 181.
[18]   | Shoemaker GK, van Duijn E, Crawford SE, Uetrecht C, Baclayon M, Roos WH, et al. Norwalk virus assembly and stability monitored by mass spectrometry. Molecular & cellular proteomics : MCP. 2010; 9(8): 1742-51.
[19]   | Czako R, Atmar RL, Opekun AR, Gilger MA, Graham DY, Estes MK. Experimental human infection with Norwalk virus elicits a surrogate neutralizing antibody response with cross-genogroup activity. Clinical and vaccine immunology: CVI. 2015; 22(2): 221-8.
[20]   | Chen Z, Sosnovtsev SV, Bok K, Parra GI, Makiya M, Agulto L, et al. Development of Norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of Norwalk virus gastroenteritis. Journal of virology. 2013; 87(17): 9547-57.
[21]   | Atmar RL, Bernstein DI, Harro CD, Al-Ibrahim MS, Chen WH, Ferreira J, et al. Norovirus vaccine against experimental human Norwalk Virus illness. The New England journal of medicine. 2011; 365(23): 2178-87.
[22]   | Lindesmith LC, Donaldson E, Leon J, Moe CL, Frelinger JA, Johnston RE, et al. Heterotypic humoral and cellular immune responses following Norwalk virus infection. Journal of virology. 2010; 84(4): 1800-15.
[23]   | Ball JM, Hardy ME, Atmar RL, Conner ME, Estes MK. Oral immunization with recombinant Norwalk virus-like particles induces a systemic and mucosal immune response in mice. Journal of virology. 1998; 72(2): 1345-53.
[24]   | Asanaka M, Atmar RL, Ruvolo V, Crawford SE, Neill FH, Estes MK. Replication and packaging of Norwalk virus RNA in cultured mammalian cells. Proceedings of the National Academy of Sciences of the United States of America. 2005; 102(29): 10327-32.
[25]   | Nakata S, Honma S, Numata KK, Kogawa K, Ukae S, Morita Y, et al. Members of the family caliciviridae (Norwalk virus and Sapporo virus) are the most prevalent cause of gastroenteritis outbreaks among infants in Japan. The Journal of infectious diseases. 2000; 181(6): 2029-32.
[26]   | Qu L, Vongpunsawad S, Atmar RL, Prasad BV, Estes MK. Development of a Gaussia luciferase-based human norovirus protease reporter system: cell type-specific profile of Norwalk virus protease precursors and evaluation of inhibitors. Journal of virology. 2014; 88(18): 10312-26.
[27]   | Ajami NJ, Barry MA, Carrillo B, Muhaxhiri Z, Neill FH, Prasad BV, et al. Antibody responses to norovirus genogroup GI.1 and GII.4 proteases in volunteers administered Norwalk virus. Clinical and vaccine immunology : CVI. 2012; 19(12): 1980-3.
[28]   | Thorne LG, Goodfellow IG. Norovirus gene expression and replication. J Gen Virol. 2014; 95: 278-291.
[29]   | Claire P. Mattison, Cristina V. Cardemil & Aron J. Hall: Progress on Norovirus vaccine research: Public health considerations and future directions, Expert Review of Vaccines.
[30]   | Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM, Arntzen CJ. Human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes. The Journal of infectious diseases. 2000; 182(1): 302-5.
[31]   | Hall, T.A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. in Nucleic acids symposium series. 1999. [London]: Information Retrieval Ltd., c1979-c2000.
[32]   | Kumar, S., G. Stecher, and K. Tamura, MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular biology and evolution, 2016. 33(7): p. 1870-1874.
[33]   | Jespersen, M.C., Peters, B., Nielsen, M., Marcatili, P. BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes. Nucleic Acids Res. 2017; 45, W24-W29.
[34]   | Emini, E.A., Hughes, J.V., Perlow, D., Boger, J. Induction of hepatitis A virus-neutralizing antibody by a virus-speciﬁc synthetic peptide. J. Virol. 1985; 55, 836-839.
[35]   | Kolaskar, A., Tongaonkar, P.C. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett. 1990; 276, 172-174.
[36]   | Dimitrov, I., Naneva, L., Doytchinova, I.A., Bangov, I. Allergen FP: allergenicity prediction by descriptor ﬁngerprints. Bioinformatics. 2014; 30, 846-851.)
[37]   | Dimitrov et al., 2013 Dimitrov, I., Bangov, I., Flower, D.R., Doytchinova, I.A. AllerTOP v.2- a server for in silico prediction of allergens. J Mol. Model. 2013; 20, 2278.
[38]   | Fiers, M.W.E.J., Kleter, G.A., Nijland, H., Peijnenburg, A., Nap, J.P., Ham, R. Allermatch™, a webtool for the prediction of potential allergenicity according to current FAO/WHO Codex alimentarius guidelines. BMC Bioinform. 2004; 5, 133.
[39]   | Chrysostomou, C., Seker, H. Prediction of protein allergenicity based on signal-processing bioinformatics approach. In: 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. 2014.
[40]   | Gupta, S., Kapoor, P., Chaudhary, K., Gautam, A., Kumar, R. Open source drug discovery consortium, Raghava GP in silico approach for predicting toxicity of pep-tides and proteins. PLoS One. 2013; 8 (9), e73957.
[41]   | Peng, J. and J. Xu, A multiple‐template approach to protein threading. Proteins: Structure, Function, and Bioinformatics. 2011. 79(6): p. 1930-1939.
[42]   | Glass RI, Parashar UD, Estes MK. Norovirus gastroenteritis. N Engl J Med. 2009; 361: 1776-85.
[43]   | Ettayebi K, Crawford SE, Murakami K, et al. Replication of human noroviruses in stem cell-derived human enteroids. Science. 2016; 353(6306): 1387-1393
[44]   | Tucker SN, Tingley DW, Scallan CD. Oral adenoviral-based vaccines: historical perspective and future opportunity. Exp Rev Vaccines. 2008; 7: 25-31.
[45]   | Guo L, Wang J, Zhou H, et al. Intranasal administration of a recombinant adenovirus expressing the norovirus capsid protein stimulates specific humoral, mucosal, and cellular immune responses in mice. Vaccine. 2008; 26; 460-468.
[46]   | Guo L, Zhou H, Wang M, et al. A recombinant adenovirus prime-virus-like particle boost regimen elicits effective and specific immunities against norovirus in mice. Vaccine. 2009; 27: 5233-5238.
[47]   | Azim KF, Mahmudul H, Hossain Md N, Somana SR et al. Immunoinformatics approaches for designing a novel multi epitope peptide vaccine against human norovirus (Norwalk virus). Infection, Genetics and Evolution. 2019; 74 (103936)
[48]   | Black M, Trent A, Tirrell M, Olive C. Advances in the design and delivery of peptide subunit vaccines with a focus on toll-like receptor agonists. Expert review of vaccines. 2010; 9(2): 157-73.
[49]   | Hasan, M., Azim, K.F., Begum, A., Khan, N.A., Shammi, T.S., Imran, A.S., Chowdhury, I.M., Urme, S.R. Vaccinomics strategy for developing a unique multi-epitope monovalent vaccine against Marburg marburgvirus. Infect. Genet. Evol. 2019; 70, 140-157.
[50]   | Garcia, K.C., Teyton, L., Wilson, I.A. Structural basis of T cell recognition. Annu. Rev. Immunol. 1999; 17, 369-397.
[51]   | Shrestha, M.P., et al., Safety and efficacy of a recombinant hepatitis E vaccine. New England Journal of Medicine, 2007. 356(9): p. 895-903.