The present work aimed at 1) characterization of the E1 and E2
proteins (HCV-E) from an Egyptian hepatitis C virus genotype 4a (HCV-4a) isolate at the molecular and immunological level, 2) in silico identification of the B- and
T-cell epitopes responsible for the immunogenicity of HCV-E, and 3) evaluation of the diagnostic potential of both the recombinant HCV-E and
antibodies raised using mammalian expression constructs encoding the
protein. The region encoding the E1 and E2
proteins was amplified by RT-PCR from
RNA isolated from blood of a human infected with HCV-4 and cloned into the pSC-TA plasmid, and the sequence was verified and used to construct a neighbor-joining phylogenetic tree. The translated nucleotide sequence was used to predict the HCV-E secondary structure using the PREDICT-
PROTEIN server and PSI-
PRED. A 3D model of HCV-E was generated using the online tool 3Dpro. B- and
T-cell epitopes were predicted using the online tools BCPred and Epijen v1.0, respectively. The HCV-E-encoding sequence was later subcloned into the mammalian expression plasmid pQE, and the constructs that were generated were used to immunize mice in the absence and presence of adjuvants of plant origin. The maximum sequence identity obtained by
nucleotide and
protein BLAST analysis with previously published HCV-E sequences was 85 and 77 %, respectively. The
B-cell epitope CFTPSPVVV at position 203 and the
T-cell epitope ALSTGLIHL at position 380 were found to be highly conserved among all HCV genotypes. Both ELISA and Western blotting experiments on crude and purified recombinant HCV envelope
proteins using mouse
antisera raised using the HCV-E mammalian expression construct confirmed the specific antigenicity of the expressed
protein. The
antibodies raised in mice using the HCV-E-encoding construct could efficiently capture circulating
antigens in patients' sera with good sensitivity that correlated with liver
enzyme levels (r = 0.4052, P < 0.0001 for ALT; r = -0.5439, P = 0.0019 for AST). Moreover, combining the HCV-E-encoding construct with extracts prepared from Echinacea purpurea and Nigella sativa prior to immunizing mice significantly (P < 0.05) increased both the humoral (14.9- to 20-fold increase in
antibodies) and the cellular (CD4(+) and cytotoxic CD8(+)- T lymphocytes) responses compared to mice that received the
DNA construct alone or PBS-treated mice. Both recombinant HCV-E
protein preparations and
antibodies raised using the HCV-E-encoding mammalian expression construct represent useful diagnostic tools that can report on active HCV
infection. Also, the immunostimulatory effects induced by the two
plant extracts used at the cellular and humoral level highlight the potential of natural products for inducing protection against HCV
infection. The neutralizing capacity of the induced
antibodies is a subject of future investigations. Furthermore, the predicted B- and
T-cell epitopes may be useful for tailoring future diagnostics and candidate
vaccines against various HCV genotypes.