Polytope DNA Vaccine Development Against Hepatitis C Virus: A Streamlined Approach from In Silico Design to In Vitro and Primary In Vivo Analyses in BALB/c Mice

Abstract

For chronic viral infections like Hepatitis C, CD8-CTLs have emerged as important protective tools. Hence, isolated dominant epitopes arranged as polytope DNA or peptide vaccines represent a promising approach. However, because of controversial rules governing the polytope construction and epitope processing, proper design and primary analysis of such vaccines are prior to the costly transgenic animal studies. In this study, based on in silico epitope selection, four HLA-A2 (C132, E614 and N1406) and H-2d (E405 and C132) immunodominant CD8-epitopes of HCV were selected. The codon optimized nucleotide sequences of the epitopes were assembled by overlap extension PCR in three different sequential tandems for the best proteasomal cleavage predictions and cloned into pcDNA3.1 vector. In addition, to enhance particulate formation, three other plasmids containing the fusion of polytopes with hepatitis B surface antigen gene (HBsAg) were also constructed. Proper expression of all constructs in transfected Cos-7 cells was verified by RT-PCR, immunofluorescence, Western-blot, ELISA and dot blot techniques. Moreover, particle formation of HBsAg-fused polytopes was manifested by their secretion to the culture media albeit in lesser amounts compared to sole HBsAg protein. Finally, the positive delayed-type hypersensitivity (DTH) response of vaccinated mice indicated the in vivo expression of all constructs and efficient stimulation of immune response, which was stronger for HBsAg fusion constructs. In addition, proper processing of the epitopes was evidenced by the DTH response towards H-2d epitopic peptides. These data provide enough support and merit for the further evaluation of the designed constructs in HLA-A2 transgenic mice.

Fusion of HBsAg and prime/boosting augment Th1 and CTL responses to HCV polytope DNA vaccine

“…3A). Absence of detectable responses against irrelevant CTL peptides (H-2 d ; core [16][17][18][19][20][21][22][23][24][25] and HLA-A2; E 6 and N) in both CTL and ELISpot assays indicated the specificity of the responses against HCVspecific C, E 4 and CE 4 E 6 N (IFN--ELISpot only) peptides. Results of IFN--ELISpot assay ( Fig.…”

“…In silico design and detailed construction procedure of polytope DNA vaccines exploited in this study, as well as their expression analyses through RT-PCR, dot-blot, Western-blot and immunofluorescence techniques was already described (20). In brief, a polytope DNA sequence encoding the H2-D d -restricted epitopes of C (core 132-142 : DLMGYIPLVGA) [21] and E 4 (E2 405-414 : SGPSQKIQLV) [22], besides HLA-A2-restricted epitopes of E 6 (E2 614-622 : RLWHYPCTI) [23] and N (NS3 1406-1415 : KLSGLGLNAV) [24] in the linear CE 4 E 6 N order was codon optimized to support preferred human codon usage (www.kazusa.org.jp/codon/), synthesized by SOEing PCR method and inserted into pcDNA3.1 plasmid (Invitrogen, CA) to provide the pcPOL construct (Fig.…”

“…Of note, the CE 4 E 6 N epitope order was selected as the optimized sequence for both proteasome-mediated epitope processing and reduced junctional epitopes (which can be created by the juxtaposition of two epitopes), based on the results of PAProc (http://paproc.de) and RankPep (http://bio.dfci.harvard.edu/RANKPEP/) modelings, respectively. The potency of particle formation for pcHPOL plasmid was also indirectly assessed through the measurement of HBsAg-based chimeric particles in the supernatant of transfected cells using a commercial HBsAg-ELISA assay (Hepanostika HBsAg Uni-Form II, Biomérieux, France) [20].…”

“…Peptides corresponding to CD8 + -epitopic peptides (C, E 4, E 6 , N) and an irrelevant H-2 drestricted epitope (core [16][17][18][19][20][21][22][23][24][25] ; NRRPQDVKFP), besides the synthetic CE 4 E 6 N long polytopic peptide were synthesized by solid phase Fmoc chemistry with 97% purity (Institute de Biochimie, Universite de Lausanne, Swiss). In this study, HLA-A2-restricted CD8 + -epitopic peptides (E 6 and N) were also used as irrelevant (negative control) peptides for all assays in BALB/c mice.…”

“…Recently, we reported the engineering and construction of polytope DNA vaccine plasmids encoding four immunodominant CD8 + CTL epitopes derived from both structural and NS antigens of HCV and showed their eligibility by in vitro and primary in vivo analyses [20].…”