The hepatitis C virus (HCV) is a plus stranded RNA virus belonged to the Flaviviridae family. HCV causes acute and chronic hepatitis, and more than half of HCV patients develop liver cirrhosis or hepatocellular carcinoma (1). Globally, 130–150 million people are living with HCV (2).

Hepatitis C has six major genotypes, which demonstrate many variations in geographic distribution, response to therapy and disease progression (3). The most prevalent genotypes of HCV are 1, 2 and 3, which occur around the globe. HCV genotype 4 is predominant in Africa and the Middle East, while in South Africa and Hong Kong genotypes 5 and 6, respectively are predominant (4). The most prevalent genotype in Pakistan is 3 (3).

There is currently no vaccine available for protection from HCV. Between 2001 and 2011, interferon and ribavirin were administered for HCV treatment. These types of medication resulted in limited responses with many adverse effects. Numerous interferon-free therapeutic strategies are at various stages of development and few of these produce a high response rate with minimum adverse effects (5,6). The prevalence of HCV is particularly high in the multitransfused patient population and in individuals who inject drugs (7,8).

The genome of HCV is comprised of 9,600 nucleotides and encoded as a single polyprotein. The genome is composed of four structural proteins and six non-structural proteins. HCV nonstructural protein 3 (NS3) protein is a 631-amino acid long dual-function protein, with a serine protease domain in the N terminal one-third, while the two-thirds region of the C terminus is RNA helicase domain (9,10). The NS4A region is a cofactor for the activation of the protease subunit of HCV (11). HCV protease has proven to be crucial for viral replication and is considered to be the best target for the development of anti-HCV therapeutic strategies (12). Thus, the aim of the present study was to develop a global consensus sequence of the HCV serine protease and predict conserved B- and T-cell binding epitopes.

The NS3-4A gene of HCV has a highly conserved catalytic triad comprised of His57, Asp81 and Ser139 residues. The catalytic triad is essentially required for the proteolysis of the HCV polyprotein (9). Replacing any of the catalytic triad amino acids, histidine, aspartate or serine with any other amino acid eliminated the proteolytic cleavage by NS3 (14). The consensus sequence alignment demonstrates that the residues His57, Asp81, and Ser139 remained conserved across all HCV genotypes.

Previous X-ray observations and computational modeling analysis have revealed that a zinc-binding site is present opposite to the catalytic triad of HCV protease (11). In the present study, results from the consensus sequence analysis indicated that the zinc-binding site amino acids are well conserved among all HCV genotypes.

NS4A is a 54-amino acid long protein forming a non-covalent heterodimer with protease domain of NS3 (11). Mutation analysis reveals that the N-terminus 22 amino acids of NS3 are involved in the interaction with the central region 21–34 residues of the NS4A protein. Mutations affecting the non-covalent bonding between these two proteins cause a significant decline or inhibition of protease activity, confirming that the configuration of the bonded complex is vital for protease function (15). Numerous amino acids located in the middle section of the NS4A protein develop elaborate hydrophobic interactions with various hydrophobic side-chains in two β strands of NS3 serine protease, forming a sandwich-like configuration between the β-barrels of NS3 and the NS4A cofactor. These hydrophobic amino acid residues of NS4A primarily include Val23, Ile25, Ile29 and Leu31. The consensus sequence analysis reveals that the residues, Val23 and Ile25 are conserved among all HCV genotypes. The residue Ile29 has been replaced by Leu and Val in genotype 2 and 4, respectively, which are similar branched-chain amino acids. However, the residue, Leu31 has been mutated to Thr in genotype 6, which is a significant mutation.

Highly conserved B- and T-cell binding epitopes were predicted from the consensus sequence of HCV. Among B-cell epitopes, certain epitopes demonstrated 8%-100% conservation among all six genotypes of HCV. Various MHC class I and II predicted epitopes exhibited maximum allele-binding affinity confirming them as potential T-cell epitopes. The epitopes B1, B8 and B9 are considered to be the best targets for B cell-based vaccine development and are >95% conserved across six major HCV genotypes. Similarly, M4, M5, M7 and M10 are the best MHC class I epitopes to be adopted as synthetic vaccines against multi-isotypes of HCV. Additionally, epitopes T5, T7 and T10 are ideal MHC class II specific epitopes with high antigenicity scores and high conservancy across major genotypes. In comparison to the epitopes derived from highly variable genome regions, the use of conserved epitopes from the consensus sequence may provide broader protection against HCV. Therefore, these predicted epitopes may be invoked as effective vaccine candidates against major genotypes of HCV.

In conclusion, regardless of numerous variations in the NS3-4A gene sequences from different genotypes of HCV, the functionally important residues of the serine protease and helicase are highly conserved. These regions of the NS3-4A sequence may be useful in developing antiviral agents or peptide vaccines against HCV. Prediction of epitope immunogenicity and characterization on the basis of peptide sequences is important in developing a potent peptide vaccine for HCV. Though as the antigens predicted in the present study were based on computer software analysis, the antigenic potential of the peptides should be further characterized in animal models.