Research

Enhancement of the expression of HCV core gene does not enhance core-specific immune response in DNA immunization: advantages of the heterologous DNA prime, protein boost immunization regimen

Ekaterina Alekseeva¹ , Irina Sominskaya¹ , Dace Skrastina¹ , Irina Egorova^2,3 , Elizaveta Starodubova² , Eriks Kushners¹ , Marija Mihailova¹ , Natalia Petrakova⁴ , Ruta Bruvere¹ , Tatyana Kozlovskaya¹ , Maria Isaguliants^2,4,5 and Paul Pumpens¹

¹  Latvian Biomedical Research and Study Centre, Ratsupites 1, Riga, LV-1067, Latvia

²  Swedish Institute of Infectious Disease Control, SE-17182 Stockholm, Sweden

³  Pasteur Institute, 197101 St Petersburg, Russia

⁴  Microbiology and Tumorbiology Center, Karolinska Institutet, 17177 Stockholm, Sweden

⁵  D.I. Ivanovsky Institute of Virology, 123098 Moscow, Russia

author email corresponding author email

Genetic Vaccines and Therapy 2009, 7:7doi:10.1186/1479-0556-7-7

Published:	8 June 2009

Abstract

Background

Hepatitis C core protein is an attractive target for HCV vaccine aimed to exterminate HCV infected cells. However, although highly immunogenic in natural infection, core appears to have low immunogenicity in experimental settings. We aimed to design an HCV vaccine prototype based on core, and devise immunization regimens that would lead to potent anti-core immune responses which circumvent the immunogenicity limitations earlier observed.

Methods

Plasmids encoding core with no translation initiation signal (pCMVcore); with Kozak sequence (pCMVcoreKozak); and with HCV IRES (pCMVcoreIRES) were designed and expressed in a variety of eukaryotic cells. Polyproteins corresponding to HCV 1b amino acids (aa) 1–98 and 1–173 were expressed in E. coli. C57BL/6 mice were immunized with four 25-μg doses of pCMVcoreKozak, or pCMV (I). BALB/c mice were immunized with 100 μg of either pCMVcore, or pCMVcoreKozak, or pCMVcoreIRES, or empty pCMV (II). Lastly, BALB/c mice were immunized with 20 μg of core aa 1–98 in prime and boost, or with 100 μg of pCMVcoreKozak in prime and 20 μg of core aa 1–98 in boost (III). Antibody response, [³H]-T-incorporation, and cytokine secretion by core/core peptide-stimulated splenocytes were assessed after each immunization.

Results

Plasmids differed in core-expression capacity: mouse fibroblasts transfected with pCMVcore, pCMVcoreIRES and pCMVcoreKozak expressed 0.22 ± 0.18, 0.83 ± 0.5, and 13 ± 5 ng core per cell, respectively. Single immunization with highly expressing pCMVcoreKozak induced specific IFN-γ and IL-2, and weak antibody response. Single immunization with plasmids directing low levels of core expression induced similar levels of cytokines, strong T-cell proliferation (pCMVcoreIRES), and antibodies in titer 10³(pCMVcore). Boosting with pCMVcoreKozak induced low antibody response, core-specific T-cell proliferation and IFN-γ secretion that subsided after the 3rd plasmid injection. The latter also led to a decrease in specific IL-2 secretion. The best was the heterologous pCMVcoreKozak prime/protein boost regimen that generated mixed Th1/Th2-cellular response with core-specific antibodies in titer ≥ 3 × 10³.

Conclusion

Thus, administration of highly expressed HCV core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. Instead, the latter is induced by a heterologous DNA prime/protein boost regimen that circumvents the negative effects of intracellular core expression.