- Beckham, Simone, Piedrafita, David, Phillips, Carolyn, Samarawickrema, Nirma, Law, Ruby, Smooker, Peter, Quinsey, Noelene, Irving, James, Greenwood, Deanne, Verhelst, Steven, Bogyo, Matthew, Turk, Boris, Coetzer, Theresa, Wijeyewickrema, Lakshmi, Spithill, Terry, Pike, Robert
- Authors: Beckham, Simone , Piedrafita, David , Phillips, Carolyn , Samarawickrema, Nirma , Law, Ruby , Smooker, Peter , Quinsey, Noelene , Irving, James , Greenwood, Deanne , Verhelst, Steven , Bogyo, Matthew , Turk, Boris , Coetzer, Theresa , Wijeyewickrema, Lakshmi , Spithill, Terry , Pike, Robert
- Date: 2009
- Type: Text , Journal article
- Relation: International Journal of Biochemistry and Cell Biology Vol. 41, no. 7 (2009), p. 1601-1612
- Full Text: false
- Reviewed:
- Description: The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P2 substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5–7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P2 position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P2 Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination.
Evaluation of the immune responses induced by four targeted DNA vaccines encoding the juvenile liver fluke antigen, cathepsin B in a mouse model
- Jayaraj, Ramamoorthi, Piedrafita, David, Spithill, Terry, Smooker, Peter
- Authors: Jayaraj, Ramamoorthi , Piedrafita, David , Spithill, Terry , Smooker, Peter
- Date: 2012
- Type: Text , Journal article
- Relation: Genetic Vaccines and Therapy Vol. 10, no. 1 (Art. No:7) (2012), p. 1-9
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- Reviewed:
- Description: 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 T 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.
- Authors: Jayaraj, Ramamoorthi , Piedrafita, David , Spithill, Terry , Smooker, Peter
- Date: 2012
- Type: Text , Journal article
- Relation: Genetic Vaccines and Therapy Vol. 10, no. 1 (Art. No:7) (2012), p. 1-9
- Full Text:
- Reviewed:
- Description: 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 T 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.
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