Abstract

Background: Marek’s disease (MD) is a highly contagious disease of chickens caused by Marek’s disease virus (MDV). It causes economic losses in poultry industry estimated to be more than 1 billion per year. The aim of this study was to design a peptide vaccine against Marek’s disease virus serotype 1 (MDV-1) by targeting the Glycoproteins H and B as an immunogens to stimulate protective immune response. A total of 43 Glycoprotein H and 33 glycoprotein B of Gallid alphaherpesvirus 2 (MDV-1) were retrieved from the National Center for Biotechnology Information database (NCBI) in the 13th of October 2017. Several tests at Immune Epitope Database (IEDB) were used to detect the highly conserved immunogenic epitopes that elicit B and T cells and could be used as efficient vaccine candidates. In our results three epitopes from glycoprotein H namely; _91-FYKRPVSKLL_-100, _255-LKPYEPVDKF_-264, and _684-PRPL_-687 and three epitopes of glycoprotein B; _162- EKQV_-165, _234-YGLSPPE_-240, and _363-YNDSHVK_-369 were fulfilled the criteria of surface accessibility, antigenicity for becoming the most probable B cell epitope. While Four epitopes of glycoprotein H; _425-YVLRSAYAF_-433, _175-LTSELTGTY-₁₈₃, _476-LYYAFASIF_-484, and _367-MITETLSTF_-375 were addressed as potentially promising epitopes as they bound the highest number of both MHC-I and MHC-II alleles with a high binding affinity to chickens MHC-I molecule (BF2*2101) haplotype in the structural level. Also two epitopes of glycoprotein B; _598-FLFGSGYAL_-606, _727-FMSNPFGAL_-735 were bound with the highest number of both MHC-I and MHC-II with high binding affinity. Taken together Marek’s disease is a significant disease of poultry. We addressed epitopes from glycoprotein H and B that could act as candidates’ vaccine. To our knowledge there is no in silico epitope based vaccine for Marek’s disease virus serotype 1 (MDV-1). An in vitro and in vivo application is required to prove the efficacy of the predicted epitopes as peptide vaccine.

1. Introduction

Marek’s disease (MD) is a highly contagious disease of chickens caused by Marek’s disease virus (MDV) leads to the formation of T-cell tumors in various body tissues and neurological manifestations ^{1, 2, 3, 4, 5}. The disease transmitted by inhalation ⁶ and classified as a B-list infectious disease in Office International des Epizooties (OIE) with important economic losses in poultry industry estimated at more than 1 billion per year ^{7, 8, 9}. The morbidity and mortality rate depending on host genetic susceptibility and virulence of the MDV strain. The Mortality rates were commonly 10% to 30% and on incident could reach 60% to 80% ^{10, 11, 12}. Marek’s disease (MD) is widespread in the worldwide. Recently, OIE estimates that about half of the world countries have reported cases of MDV infection such as the United States, the central Ethiopia, Saudi Arabia, Egypt, India ^{13, 14, 15, 16, 17, 18, 19}, and China is an endemic area with outbreaks reported ^{20, 21}.

Marek’s disease virus (MDV) is double-standard DNA that belongs to the Herpesviridae family, subfamily Alphaherpesvirinae in the genus Mardivirus. It has three serotypes different in their virulence in chicken or their susceptibility to induce T-cell lymphomas. These serotypes are Marek’s disease virus serotype 1 (MDV-1) (Gallid herpesvirus 2), serotype 2 (Gallid herpesvirus 3), and serotype 3 (herpesvirus of turkeys) (HVT or MDV3) ^{22, 23}. Among these serotypes, serotype 1 MDVs are oncogenic ^{24, 25, 26, 27}, while other serotypes are non-oncogenic chicken viruses ^{28, 29, 30}. MDV-1 genome is 175 to 180 kb, depending on the strain and is predicted to encode 103 glycoproteins ^{31, 32}. Glycoproteins are virion surface components and represent potent immunogens. Among them are glycoprotein B, C, D and H ³³. Glycoproteins H (gH) and B (gB) were the main target of immune response to the virus ^{4, 34, 35, 36}.

The MD is still good controlled by vaccinations as the first successful live vaccine to control MD is herpesvirus of turkey (HVT) since the 1970s ³⁷. HVT is a highly cell-associated virus but vaccine preparations require special handling and storage ³⁸. MDV strain required the introduction of new generations of MD vaccine such as CVI988 (Rispens) vaccine, a naturally attenuated MDV-1 to induce protection against very virulent plus MDV strains ^{39, 40}. But these vaccines prevented the tumor development but not the MDV infection ⁴¹. In spite of that, the recombinant DNA vaccines overcome these problems associated with MD vaccine ^{42, 43} but it provided partial protection against MDV ^{38, 44}. Therefore, it is important to consider alternative approaches for (MDV-1) vaccine development.

Peptide –based vaccine is a one of immunoinformatics applications. It is based on identification and chemical synthesis of B-cell epitopes which mainly induce antibody production and the T-cell epitopes that induce cellular response and cytokine secretion as cytotoxic T-cells ^{45, 46, 47, 48, 49}. Peptides have become more desirable vaccine candidates owing to their relatively easy production and construction, chemical stability, and absence of infectious potential, which lessens the time and reduce cost ^{45, 46, 47, 48, 49}. Moreover these approaches serve as therapeutic and vaccine candidates for many infectious diseases ^{50, 51, 52, 53, 54}.

Therefore aim of this study was to design a peptide vaccine against Marek’s disease virus serotype 1 (MDV-1) using immunoinformatics approach. Glycoprotein H and B were used as an immunogens to stimulate protective immune response.

2. Materials and Methods

To identify the best likely B- and T-cell peptides which could be used to design an effective vaccine, different methods were taken into attention in this study and an outline of the methodology was shown in Figure 1.

A total of 43 Protein sequences of glycoprotein H and 33 of glycoprotein B of Gallid alphaherpesvirus (MDV-1) were retrieved from the National Center for Biotechnology Information database (NCBI) in the 13^th of October 2017. The retrieved sequences and their accession numbers as well as the collection area were listed in Table 1.

Retrieval sequences of Glycoprotein H and Glycoprotein B of MDV-1 were aligned to obtain the conserved regions using Multiple Sequence Alignment (MSA) by Clustal-W as applied in the Bio-Edit program (version 7.2.5.0) ⁵⁵. Which were considered as candidate epitopes were further analyzed by different prediction tools from Immune Epitope Database IEDB analysis resource (http: //www.iedb.org/)

B-cell epitope is defined as part of antigen that is recognized by B-cell receptor to elicit antibody in humeral response ⁵⁶. The epitopes of a native protein must be accessible at the antigen surface and possess antigenic reactivity with antibodies. To predict these physicochemical and structural parameters are usually calculated based on propensity scales for each of the 20 amino acids ^{57, 58}. The most epitope prediction tools available in immune epitope database IEDB. The reference sequences of glycoprotein H and glycoprotein B of MDV-1 were subjected to different B cell tools from immune epitope database (https://tools.iedb.org/bcell/) to predict linear B-cell epitopes using BepiPred linear epitope prediction, surface accessibility by Emini surface accessibility prediction tool, and antigenic reactivity using Kolaskar and Tongaonkar antigenicity tool.

A functional T-cell response requires MHC–peptide binding and a suitable interaction of the MHC–peptide ligand with a specific T-cell receptor (TR) ⁴⁵. Therefore this requires the prediction of peptides bind to MHC class I &II.

For prediction of peptides bind to MHC class I; the reference sequence of Glycoprotein H and Glycoprotein B of MDV-1 were submitted in MHC-I Binding prediction tool (http: //tools.iedb.org/mhci/) in IEDB. Prediction method of Artificial Neural Network (ANN) ⁵⁹ was used to calculate IC50 values of peptide binding to MHC1 molecules. Analysis was done using human leucocyte antigen (HLA) alleles ⁶⁰. For all the alleles, peptide length was set to 9 amino acids prior to the prediction ⁶¹. The alleles having binding affinity IC₅₀ less than 100 nm were suggested to be promising candidate epitopes.

For prediction of peptides bind to MHC class II; the reference sequence of Glycoprotein H Glycoprotein B of MDV-1 were assessed by the IEDB MHC-II prediction tool at (http: //tools.iedb.org/mhcii/). For the screening of promising epitopes, human allele reference set (HLA DR, DQ, DP) were used ^{62, 63}. NN-align prediction method ⁶⁴ in IEDB was used with (IC₅₀) 0f 1000 nm. Peptides less or equal to the (IC₅₀) value were chosen for further consideration.

For creation of the 3D structure of the reference sequence of Glycoprotein H (YP_001033950.1) and glycoprotein B (YP_001033956) using Raptor X protein structure prediction server available at (http: // raptorx.uchicago.edu/Structure Prediction/predict/). Chimera 1.8 ⁶⁵ was used to visualize the selected epitopes belonging to the B cell, MHC-I, and MHC-II.

The MHC class I of Chickens BF2*2101 haplotype (CAK54661.1 ) was retrieved in FASTA format in the 15^th of October 2017 from the National Center for Biotechnology Information (NCBI) and uploaded to the Raptor X (http: //raptorx.uchicago.edu/StructurePrediction/predict/) to obtain the 3D structure. Four peptides of glycoprotein H and two peptides of glycoprotein B are bound the largest number of alleles were selected to predict 3D peptide structure using Pep fold available at the mobyle web server ⁶⁶. The proposed models for each of the selected MHC class I binding peptides were docked with the MHC class I protein (BF2*2101) using Patch dock server ⁶⁷ and visualized using Chimera 1.8.

3. Results

Figure 2 showed the conserved regions in the retrieval sequences of Glycoprotein H and Glycoprotein B of MDV-1. Moreover the thresholds for B- Cells for the linear epitopes, surface accessibility and antigenicity were shown in Figure 3.

The reference sequence of glycoprotein H and glycoprotein B of MDV-1 were subjected to different tools in B-cell epitope prediction at IEDB. Several epitopes were predicted to interact against B cells for both glycoproteins H and B. Glycoprotein H epitopes were shown in Table 2 while the best three epitopes of glycoprotein H were shown in Table 3 with their scores. Table 4 showed the total epitopes that obtained from glycoprotein B and Table 5 demonstrated the best three epitopes from Glycoprotein B with their scores. These selected epitopes fulfilled the criteria of surface accessibility, antigenicity. Moreover the 3D structures of the selected epitopes from glycoprotein H and B were shown in Figure 4.

MHC class-I of various HLA alleles with interaction of T-cell epitopes of glycoprotein H and glycoprotein B were predicted. Successful candidate’s epitopes had a half maximal inhibitory concentration (IC50) < 100 nM, were listed in Table 6 and Table 7. The 3D structure of these epitopes was demonstrated in Figure 5.

T- Cell epitopes and interaction with MHC Class II were predicted based on the NN-align method with half maximal inhibitory concentration (IC50) ≤ 1000 nm. The most promising four epitopes of glycoprotein H and two of glycoprotein B that had binding affinity with human MHC class II alleles were shown in Table 8 and Table 9 repectively. The 3D structure of these epitopes was depicted in Figure 5.

Four docked epitopes; ₄₂₅-YVLRSAYAF_-433, _175-LTSELTGTY_-183, _476-LYYAFASIF_-484 and _367-MITETLSTF_-375 of glycoprotein H and two epitopes; _598-FLFGSGYAL_-606 and _727-FMSNPFGAL_-735 of glycoprotein B were found to have binding affinity to chickens MHC I molecule (BF2*2101) haplotype which the binding energy score for four epitopes as shown in Table 10 as obtained from patch dock. Figure 6 visualize the binding interactions between MHC I receptor and epitopes in the structural level.

4. Discussion

Vaccination is usually considered to be the most effective method of preventing infectious diseases. Inactivated vaccines live attenuated vaccines, Subunit vaccines, and DNA vaccines were shown with drawbacks. They were characterized by time-consuming process and active infectious particles can be administered together with the vaccine ⁴⁶. The increase of incidence of viral infections in animals and human provided the need of new available technologies. For instance the peptides based vaccines approach enables achieving effective, cost-efficient vaccines development. The process is based on identification and chemical synthesis of B-cell and T-cell epitopes that can induce specific immune responses ⁴⁵.

Therefore, in this study, we aimed to design epitopes based vaccine for Marek’s disease virus serotype 1 (MDV-1). Marek’s disease (MD) caused by Marek’s disease virus (MDV) serotype 1 (MDV-1) is oncogenic which causes economic losses in poultry industry estimated to be more than 1$ billion per year ^{10, 26, 68}. In this report both glycoprotein H and B of Marek’s disease were targeted as an immunogens and tested for their efficacy in eliciting immunity against B-cell and T-cell. In our results three epitopes of glycoprotein H (_91-FYKRPVSKLL-₁₀₀, _255-LKPYEPVDKF_-264, and _684-PRPL_-687) and three epitopes of glycoprotein B (_162-EKQV_-165, _234-YGLSPPE_-240, and _363-YNDSHVK_-369) were shown to elicit the B cells. They fulfilled the criteria of surface accessibility, antigenicity. Therefore they were proposed as B cell epitopes since they got scores above thresholds in Bepipred, Emini surface accessibility and Kolaskar and Tongaonkar antigenicity prediction methods and showed 100% conservancy.

During virus infection, the importance of MHC I and II in fighting the infection is crucial ⁶⁹. Most importantly since there were no data available in the IEDB concerning chicken alleles, the human alleles were used to predict the allelic interaction with MHC1 and MHC11. Several studies concluded the highly similarities between human and chickens MHC molecules ^{60, 62, 63, 70}. Interestingly four epitopes namely (₄₂₅-YVLRSAYAF_-433, _175-LTSELTGTY_-183, _476-LYYAFASIF_-484 and _367-MITETLSTF_-375) from glycoprotein H and two epitopes namely (_598-FLFGSGYAL_-606 and _727-FMSNPFGAL_-735) from glycoprotein B were found to interact with high binding affinity to both MHC1 and MHC11 alleles. Therefore these epitopes were selected as promising peptides vaccine against Marek’s disease. Furthermore for critical binding the predicted epitopes were further docked against MHC1 molecule. Strikingly the predicted epitopes from the glycoprotein H and B demonstrated low binding energy score to chickens MHC class I molecule (BF2*2101) haplotype in the structural level. This result could further solidify the ability of the predicted epitopes to act as strong vaccine epitopes

To our knowledge there is no epitope based vaccine for the Marek’s disease virus serotype 1 (MDV-1) using in silico approach. The advantages of this approach is focusing in immune response, enhancing immunity and reducing costs ^{47, 71}. It is noteworthy the peptide based vaccine approach have successfully used in therapeutic and designing peptide-based vaccine in a considerable number of human and animal viruses and diseases such as Influenza virus, Paratuberculosis, HIV, Ebola virus, cancer and others ^{72, 73, 74, 75, 76, 77, 78}.

This study proposed an interesting epitopes of glycoprotein H and glycoprotein B that have very strong binding affinity to both B and T cells (MHC1 and MHC11 alleles) which indicates strong potential to formulate peptide vaccine for Marek’s disease virus serotype (MDV-1). An in vitro and in vivo application is required to prove the efficacy of the predicted epitopes as peptide vaccine.

5. Conclusion

In conclusion, this study indicated that immunoinformatics wide screening of vaccine targets of emerging highly pathogenic pathogens is a promising strategy to accelerate their vaccine development, which lessens the time and cost required for laboratory analysis of pathogen gene products.

Acknowledgments

Authors would like to thank the staff members of College of Veterinary Medicine, University of Bahri, Khartoum/ Sudan for their cooperation and support.

Competing Interest

The authors declare that they have no competing interests.

References