- Development of a vector for secretion of heterologous proteins in BCG. - Evaluation of BCG as a live carrier for vaccination against intracellular parasites. - Study of Mycobacterium tuberculosis expression signals in BCG.
Research has been carried out with respect to stimulating protective immunity against an intracellular model parasite, using the intracellular live vaccine vector BCG.
An expression cassette using the Mycobacterium tuberculosis Ag 85A promoter and signal sequence was constructed. For comparison, vectors containing the 65 kDa protein heat shock promoter (HSP) in combination with the Ag 85A signal sequence gene or without signal sequence gene were also prepared. The GRA2 protein gene from Toxoplasma gondii was cloned in the vector using the Ag85A promoter and signal sequence. Constructs to express the protein under control of the HSP for comparative reasons are also envisaged. Deletion analysis of the Ag 85A promoter revealed a 50 bp region essential for promoter function.
The T. gondii genes of interest have been cloned in expression vectors and expression vectors and expression in Escherichia coli has been attempted. For both antigens, severe degradation is observed when the complete gene is expressed. Upon expression a carboxyl terminal fragment, good expression results were obtained and the proteins were purified.
Epitope mapping studies using expression constructs or synthetic peptides have shown that B-cell epitopes are localized at the extreme c-terminus of both proteins, although in the case of ROP2 a nonlinear epitope is suggested.
The recombinant ROP2 and GRA2 fragments and T. gondii antigen preparations were used in T-cell stimulation assays with cells from infected and control sheep. Significant proliferative responses to the ROP2 fragment and also to a soluble T. gondii extract were observed in infected but not in control animals. The GRA2 fragment did not elicit T-cell proliferation in these assays, but a strong antibody response to this antigen was detected in sera of infected sheep by indirect enzyme linked immunosorbent assay (ELISA). These results indicate that both antigens are able to stimulate immune responses in sheep infected with T. gondii.
The proposed project aims at the development of a system to stimulate protective immunity against an intracellular parasite (Toxoplasma gondii) by using a non- pathogenic intracellular live vector (BCG, Bacillus Calmette-Guerin). Two well characterized antigens of the parasite, GRA2 and ROP2, were selected since they follow two different pathways of processing and presentation to the immune system. The genes encoding these antigens will be expressed in BCG using the promoter and signal sequence of a gene from the 85A complex, encoding a protein which is secreted by M. tuberculosis. The recombinant BCG strains will be evaluated for their capacity to provoke protective immunity against toxoplasmosis in a mouse and a rat model. The humoral and cellular response towards both model antigens will be studied. Eventually, vaccination experiments with live recombinant BCG/toxo will be performed in sheep. The results obtained with this model system may also have some bearing on protection from infection with other important intracellular pathogens like Plasmodium falciparum.
Funding SchemeCSC - Cost-sharing contracts