Immunisation of mice with HCV core protein formulated in montanide-ISA720 and CpG primes CD+8 CTLs and elicits Th1-Th2 responses
Abstract number: 1733_1376
Aghasadeghi M., Sadat M., Khabiri A., Rouhvand F.
Objectives: Hepatitis C (HCV) core protein (HCVcp) is a prime candidate for a HCV vaccine. HCVcp is the most conserved gene in HCV genotypes with several well characterised B cell, T cell and CTL determinants and target cells expressing HCVcp can be identified and destroyed by Core-specific CTLs. Immunisation with recombinant proteins however, requires formulation in proper adjuvants. Herein we describe results of evaluating adjuvant effect of M720 (Montanide ISA 720), CPG ODNs and F127 (Pluronic acid) in different formulations on induction of HCVcp-specific Th1/Th2 responses and CD+8 CTLs to HCVcp in BALB/C mice.
Methods: The HCVcp (amino acids 2122) was expressed in a T7/arabinose induction system in E. coli and purified in native condition on Ni-NTA agarose. Mice were immunised with HCVcp as antigen (Ag) in following formulations; Ag, Ag-Freund (IFA), Ag-CpG, Ag-M720, Ag-F127, Ag-CpG-F127, Ag-CpG-M720. Total IgG, IgG-isotypes and cytokines were quantified by ELISA. Re-stimulated spleenocytes of immunised mice by CD8-specific-HCVcp peptides were analysed for CTLs by LDH release cytotoxic assay.
Results: expressed HCVcp was identified as a 21 kd band by SDS-PAGE and western blotting using anti-penta His and HCV-core mAb and sera of HCV infected patients. All Immunised mice developed anti HCV-core antibodies of IgG class but the mean value for M720 and IM720-CpG groups was significantly higher than for other groups. Cytokine measurement indicated that although CpG and F127-CpG groups showed the highest IFN-g:IL-4 ratio, the levels of each cytokine separately was highest for M720 and M720-CpG adjuvated groups. The CLTs of mice immunised with HCVcp formulated in M720-CpG, M720 and CpG shpwed the highest percent of specific lysis at the effector: target ratio of 20:1, respectively. In contrast, in other HCVcp formulated groups (F127, F127-CpG, IFA, free HCVcp and control adjuvats) no detectable HCVcp-specific CTL responses could be mounted.
Conclusion: HCVcp purified in native from is capable to elicit different level of Th1 and Th2 immune responses when administered in a combination with selected adjuvants. HCVcp administered with M720 or M720-CpG as adjuvants seem to elicit strong and balanced immune responses and HCVcp-specific CD+8 CTL.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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