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Open Access Research

Evaluation of the immune responses induced by four targeted DNA vaccines encoding the juvenile liver fluke antigen, cathepsin B in a mouse model

Rama Jayaraj126*, David Piedrafita3, Terry Spithill4 and Peter Smooker5

Author Affiliations

1 Biotechnology & Environmental Biology, School of Applied Sciences, RMIT University, Bundoora West Campus, PO Box 71, Bundoora, Vic 3083, Australia

2 Allied Health Program, School of Environmental and Life Science, Charles Darwin University, Casuarina, NT 0810, Australia

3 Animal Biotechnology Research Laboratories, Physiology Department, School of Biomedical Sciences, Monash University, Clayton, Vic, 3800, Australia

4 Department of Agricultural Science, Latrobe University, Melbourne, Vic, 3086, Australia

5 Biotechnology & Environmental Biology, School of Applied Sciences, RMIT University, Bundoora West Campus, PO Box 71, Bundoora, Vic, 3083, Australia

6 School of Environmental and Life Sciences, Charles Darwin University, Casuarina, NT, 0909, Australia

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Genetic Vaccines and Therapy 2012, 10:7  doi:10.1186/1479-0556-10-7

Published: 31 August 2012

Abstract

Background

Liver fluke can infect cattle and sheep, and is also emerging as a human pathogen in developing countries. Cathepsin B (Cat B2) is a major cysteine protease secreted by the juvenile flukes. To enhance the immune responses of Cat B2, the cDNA sequence was fused with four different DNA vaccine vectors. The induced cellular and antibody responses were compared in vaccinated mice.

Methods

The following recombinant DNA vaccine constructs were constructed: empty vector VR1012 as negative control, cytoplasmic construct pVR1012 Cat B2, secretory construct pVR1020 Cat B2, chemokine-fused construct pMCP3 Cat B2 and lymph node targeting construct pCTLA-4 Cat B2. Plasmids were constructed using standard procedures, and positive constructs screened and selected using restriction digestion analysis followed by sequence analysis. The constructs were then tested in Cos-7 cells for in vitro expression, which was analysed using immunoblotting. Subsequently, female BALB/c mice were immunised with DNA constructs as vaccines. Elicited antibody responses were measured using ELISA. The ratio between IgG1 and IgG2a antibody responses was estimated among different vaccine groups. IgG antibody avidity assay was performed and the relative avidity index was calculated. The induced cytokine production from splenocytes of vaccinated animals was estimated using ELISPOT.

Results

DNA vaccine constructs carrying Cat B2 were expressed in Cos-7 cell lines and encoded protein was recognised using western blotting using rat anti- cathepsin B antibody. DNA vaccines elicited high Cat B2- specific IgG, IgG1, IgE and also modest IgG2a antibody responses. Cat B2 specific IL-4‚ÄČT cell responses were also observed in Cat B2 vaccinated mice. The comparison of immunogenic potential in each of these constructs was demonstrated as enhanced antibody responses on the lymph-node targeting vector pCTLA-4 Cat B2, the high antibody avidity of chemo-attractant pMCP3 Cat B2 and stronger T cellular responses of non-secretory DNA vaccine pVR1012 Cat B2 in vaccinated animals.

Conclusion

This study showed that the targeting DNA vaccine strategies enhanced specific immune responses to juvenile fluke Cat B2. The results of our current study have demonstrated that a gene-based vaccine as an immunotherapeutic approach to combat Fasciola infection may be feasible.

Keywords:
Cathepsin B2; Fasciola hepatica; DNA vaccination; CTLA-4; MCP3