Omicsonline | Full text | HLA Class I and II Binding Promiscuity of the T-cell Epitopes in Putative Proteins of Hepatitis B Virus

Abstract
Hepatitis B virus is a human infectious disease universally caused by the hepatitis B virus. Its genome size is 3.215 kb. Immunoinformatics tools have been used to predict the epitopes from seven putative protein viz. polymerase, large-S- and middle –S- Protein, S and X- protein, Precore/Core Protein, Core and E- antigen. Total 50 epitopes were predicted for MHC class I and 55 epitopes for class II MHC molecules. These epitopes showed highest binding score at optimum threshold. Epitopes may use as an antigen for diagnosis and also might be helpful for designing peptide based subunit vaccine against Hepatitis B virus.

Keywords: Hepatitis B virus; epitopes; diagnosis; vaccine

Introduction
Hepatitis B virus (HBV) is a dsDNA, enveloped virus that replicates in hepatocytes and belongs to family of the Hepadnavirus. The disease, originally known as “serum hepatitis (Barker et al 1996 ) has caused epidemics in various parts of Asia and Africa, and is endemic in China. (Williams 2006 ). The primary routes of transmission are through vertical transmission, blood and sexual exposure. Hepatitis B is potentially a life-threatening liver infection caused by hepatitis B virus. It can cause chronic liver disease and lay people at high risk of death from liver cirrhosis and cancer (Chang 2007 ). It is significantly more transmissible than HIV via blood-borne exposure, and some fluids that do not normally transmit HIV viz. saliva and sweat they contain infectious HBV but at lower levels when compared with blood. In many instances, patient route of infection is not identified. About one- third of world’s population has beeninfected with the hepatitis B virus. This includes 350 million
chronic carriers of the virus. The acute illness causes liver inflammation, vomiting, jaundice and death (WHO report).

Highly pathogenic and communicable virus cannot be cultured easily as it always requires high level of biosafety containment facilities. The culture of hepatitis B virus also requires the cell line for growth and all these steps are time consuming and laborious. Therefore, antigen preparation of
hepatitis B virus is not easily processed. Several immunoinformatics tools are available for prediction and mapping of antigenic epitopes in protein sequence. It assist in designing subunit vaccines that starts from prediction of antigenic epitope through in silico techniques from protein sequence of pathogens independent of their abundance and without need to grow the microorganism in vitro (de Groot and Rappuoli, 2004 ; Rappuoli, 2001 ).

Putative proteins of M.tuberculosis were predicted for cytoplasmic, integral membrane, secretory protein attached to membrane by Lipid anchor in the subcellular localization. The predictions provide a method to annotate Mycobacterium proteomes with subcellular localization information rapidly (Somvanshi a, et al 2008 ). Several proteins are involved in the pathogenesis and regulation of bacterial cell activity. The prediction of protein subcellular localization was used to distinguish the location in cells in V. cholerae. These proteins reside in cytoplasm, periplasm, inner membrane,
outer membrane and extracellular space (Somvanshi b, et al 2008 ).

Prediction of epitopes in putative protein of hepatitis B virus with Propred and Propred1 are the new strategies to produce antigens for diagnosis. Synthetic peptides can use as vaccines to induce either humoral or cell-mediated immunity requires an understanding of the nature of T-cell and B-cell epitopes has been reported (Singh and Raghava, 2001 ; Bhasin et al., 2003 ; Singh and Raghava, 2003 ). The present study was aimed to predict and map the antigenic epitopes in hepatitis B virus.

Materials and Methods
Bioinformatics tools were used for the analysis of genome of hepatitis B virus. The complete sequences of hepatitis B virus (NC_003977) were retrieved from www.ncbi.nlm.nih.gov. The open reading frames were identified from the whole genome using softwares viz. Generunner, ORF finder and DNAstar. The expected molecular weight, and isoelectric point (pI) value were also verified using Generunner and ExPaSy (http:// www.expasy.org/). Propred (http://www.imtech.res.in/raghava/propred/) and Propred1
(http://www.imtech.res.in/ raghava/propred1/) immunoinformatics tools are available to predict the antigenic epitopes in the complete protein primary sequences. Significant efforts have been made in the last few years as several groups devoted their research toward the development of procedures and algorithms that allow more effective and accurate prediction of MHC binding affinity.

The tools cover maximum number of human leukocyte antigen (HLA) in comparison to other epitope prediction tools. All these epitopes were predicted on the 4% threshold level with the highest binding score with the MHC molecules. T-cells interact through their polymorphic T cell receptor
with HLA class I molecules containing endogenously synthesized peptides of 9-11 on the surface of infected cells. The presence of allele-specific amino acid motifs has been demonstrated by sequencing of peptides eluted from MHC molecules. Prediction of T-cell epitopes from protein sequence and have been widely used by experimental researchers without expert knowledge of bioinformatics (Lund et al, 2002 ).

Results and Discussion
In the present study, seven putative proteins of hepatitis B virus were used for the physicochemical analysis such as molecular weight, isoelectric point (pI value) and antigenic nature. The polymerase protein has the highest molecular weight 94.60 kDa and the lowest molecular weight 16.51 kDa was present in X- protein. Isoelectric points of these proteins were ranged between 7.87 to 9.94. The physicochemical propertievs of putative proteins were given (Table 1 ). The pI value of any protein indicates the stability of protein in that particular isoelectric point.

Table 1: Physicochemical properties of different putative proteins of Hepatitis B virus.

Table 2: The predicted epitopes in the putative proteins of Hepatitis B virus.

The prediction of epitopes in Polymerase, large-s- protein, middle –s- Protein, s- protein, x- protein, precore/core protein, core and e- antigen, proteins of hepatitis B virus were investigated. Total 50 epitopes were predicted for class I MHC and 55 epitopes for class II MHC molecules in these proteins. The predicted epitopes having the MHC alleles for putative proteins from hepatitis B virus were performed (Table 2 ). Evaluation of synthetic peptides as potential vaccine candidate for flavivirus has been investigated. Using the computational tools for prediction of epitopes and
synthetic peptides from E glycoprotein of Murray Valley encephalitis (MVE) and DEN 2 viruses were prepared and their immunogenicity was evaluated in mice (Gao et al, 1990). The identification of significant T-cells epitopes from secretory and cell surface proteins virulent proteins of M.tuberculosis H37Rv strain was done. The promiscuous nanomer candidate epitopes from HTL and CTL were recognized (Somvanshi a et al, 2008 ). T-cell analyses of synthetic peptides to other viruses have correlated the association between T- and B-cell responses (Hu et al, 1999 ). A new approach for vaccine design in immunology and the development of bioinformatics tools for T cell epitope prediction from primary protein sequences is essential. Bioinformatics tools have the potential to accelerate research into the design of vaccines and diagnostic tests by exploiting genome sequences. In silico analysis could be combined with in vitro screening methods to identify the peptides that are immunogenic. Chemically synthesized domains of FMDV (Food-mouth disease virus) VP1 were tested as potential peptide vaccine. The peptide corresponding to amino acid 141-160, 151-160 and 200-213 which are located near the C- terminal end of VP1 and 9-24, 17-32 and
25-41, and the N-terminal end of the VP1, were each bound to a separate insert carrier protein (Bittle et al, 1982).

The prediction of T-cells epitopes in highly virulence surface proteins i.e. hemagglutinin and neuraminidase from Influenza A virus H5N1 was performed. The highly conserved and specific epitopes were predicted the Influenza host and strain specific. These epitopes were used for the
immunodiagnostic of Influenza and also as a novel epitope based vaccine candidate (Somvanshi c et al, 2008 ). Immunoinformatics serves as a valuable tool to screen and select antigenic peptide sequences as potential T cell epitopes for binding affinity with HLA alleles. The dengue variants structural protein was studied and promiscuous nanomer candidate epitope for MHC class I and II has been recognized (Somvanshi and Seth, 2008 ).

Conclusion
New theoretical and immunoinformatic approaches are currently used for prediction of epitopes in the proteins sequence of hepatitis B virus without using their cultures. The prediction of hepatitis B virus nanomer epitope for T-cells is recognized against MHC Class II and MHC class I. It may use as an easy and effective way to diagnose the suspected individuals during a future hepatitis B virus epidemic, thereby reducing and containing the transmission. These epitopes may also use for the vaccination against hepatitis B virus.

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