INSTITUT PASTEUR D' IRAN
Presented for partial fulfillment of the degree
of Philosophy Doctorate (Ph.D)
Cloning and Expression of HCV Capsid and CTL- Based Polytopic-Peptide in Plant and
Evaluation of the Immunogenicity in BALB/c Mice
Supervisors:
Dr. Soheila Ajdary
Dr. Parastoo Ehsani
Dr. Ali-Hatef Salmanian
Advisors:
Dr. Farzin Roohvand
Dr. Arash Memarnejadian
By:
Sara Mohammadzadeh Sadegh
March 2015
Abstract
Hepatitis C virus (HCV) is the major cause of chronic hepatitis, which despite its 3% prevalence
of the world population, no effective vaccine has been approved against until now. On the other
hand, world demand for low-cost and safe vaccines has led plants to gain attention as an
alternative system for most recent expression systems.
In the recent study, the possibility of the expression of HCV core protein (HCVcp) and HCV
polytope in fusion with HBsAg (HCVpolytope-HBsAg) as candidate antigens in designing
vaccines against HCV were studied in plant expression system. Furthermore, the possibility of
applying different expression platforms including transgenic expression in canola (Brassica
napus L.) seeds and transient expression in tobacco (Nicotiana tabacum) leaves using classical
binary and virus-based vector to produce HCVcp was evaluated. In the end, the immunogenicity
of expressed HCVcp protein in canola oilseed and the effect of using oil body in immune
responses were examined.
In this respect, HCVcp and HCVpolytope genes were optimized for expression in the plant hosts.
The designed HCVcp gene from N- to C-terminal in tandem included kozak sequences, hexahistidine (6×His)-tag peptide, HCVcp (1-122 residues), and KDEL for retention in ER. The
optimized HCVpolytopic construct encoding the Kozak sequence, 6×His-tag, and HCV
polytope, which was N-terminally fused with HBsAg gene, was cloned into Potato virus X
(PVX-GW) vector. Additionally the optimized HCVcp was inserted into Potato virus-X (PVX)
and classic pBI121-binary vectors in separate cloning reactions. The resulted recombinant
plasmids (pVX-poly-HBs, pVX-core, and pBI121-core) after confirmation by restriction and
sequencing analyses were transferred into Agrobacterium tumefaciens and were vacuum
infiltrated into tobacco leaves. The effect of gene silencing suppressor P19 protein from TBSV
was tested on expression yield of HCVcp and HCVpoly-HBsAg was also evaluated by ELISA.
For transgenic expression of HCVcp in canola (Brassica napus L.) seeds, the optimized HCVcp
subcloned into pBI1400 binary vector under the control of the seed specific promoter FAE1 and
transformed into canola. Transgenic lines were screened and the presence of the transgene in the
T0 plants was confirmed by PCR. The quantity and quality of the HCV core protein (HCVcp) in
the plant were evaluated by ELISA and western blot respectively. Transgenic seeds and HCVcp
purified from E. coli in combination with oil bodies were evaluated in immunogenicity studies in
BALB/c mice.
Results of optimization design demonstrated that the codon adaptation index (CAI) value
increased to more than 85% for the genes. Sequencing results confirmed the correctness of
cloning steps for constructed cassettes and the transformed Agrobacteria with the constructs
were confirmed by colony-PCR. Moreover, sandwich ELISA results indicated the expression of
HCVcp and HCVpoly-HBsAg and the expression level enhanced 3-5 fold in P19 coagroinfiltrated tobacco leave as well. The plant-derived HCVcp (pHCVcp) was identified by
HCV-infected human sera. Western blot analysis using anti-His and specific antibodies
confirmed the presence of a 15 kDa protein in seeds of T1 transgenic lines and tobacco leaves.
The amount of HCV core protein expression in the seeds of transgenic lines and tobacco leaves
was estimated 0.02% to 0.05% of the total soluble protein (TSP). The results of immunogenicity
evaluation indicated the ability of transgenic canola seeds in inducing specific immune response
against HCVcp and the effect of oil bodies on type of immune response. In these studies, mixed
Th1 and Th2 responses were induced as well. The results demonstrated minute amounts of
rHCVcp expressed in Canola oilseed were capable of inducing immune responses comparable to
10 fold of the same purified recombinant protein, expressed in E. coli.
Considering that plant is a useful expression system for the production of vaccine antigens, the
result of the present study has been able to provide useful information on development of plantbased vaccines especially in HCV studies.
Key words: HCV core protein, HCVpolytope, seed specific expression, Brassica napus,
Nicotiana tabacum.Transient expression, Cellular Immunity, Cytokine response, IFN-γ secreting
lymphocytes,