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Stable integration and expression of the Plasmodium falciparum circumsporozoite protein coding sequence in
F Haeseleer1, J F Pollet, M Haumont
1Laboratory of Applied Genetics, Faculty of Sciences, Free University of Brussels, Nivelles, Belgium.
Abstract:
The DNA coding for the circumsporozoite protein of Plasmodium falciparum (CSP; aa 1-412) has been placed under the control of the mycobacterial promoter derived from the gene encoding the 64-kDa antigen of Mycobacterium bovis-BCG. This expression cassette was cloned into pJRD184, an Escherichia coli multicloning site vector, together with the kanamycin resistance gene from Tn903 and the attachment site and integrase gene from the temperate mycobacteriophage FRAT1. One of the resulting plasmids, pNIV2173, introduced by electroporation into both Mycobacterium smegmatis and M. bovis-BCG, integrated at a specific site in the genome of each recipient. Recombinants expressed immunoreactive polypeptides, ranging in size from 62 to 43 kDa, at a level of about 1% of total soluble proteins. Part of this material was present in the culture medium indicating that mycobacterial recombinants were able to secrete the CSP. The M. smegmatis and M. bovis-BCG recombinants, transformed with pNIV2173 and grown in absence of antibiotic, were followed for more than 400 and 50 generations respectively. Over this time span, neither DNA rearrangement nor loss of expression was observed. Inoculation of the recombinant BCG to mice did not induce humoral response to CSP nor proliferative response to CSP Th2R CD4+ T lymphocyte epitope.
Insights
Researchers engineered Mycobacterium bovis-BCG to express Plasmodium falciparum circumsporozoite protein (CSP). The recombinant BCG showed stable expression and secretion of CSP without inducing an immune response in mice.
Area of Science:
- Molecular biology
- Immunology
- Microbiology
Background:
- The circumsporozoite protein (CSP) of Plasmodium falciparum is a key target for malaria vaccines.
- Developing effective vaccine delivery systems is crucial for malaria control.
Purpose of the Study:
- To create a recombinant Mycobacterium bovis-BCG (BCG) strain expressing Plasmodium falciparum CSP.
- To assess the stability and secretion of CSP by the recombinant BCG.
- To evaluate the immunogenicity of the recombinant BCG in a mouse model.
Main Methods:
- Cloning the DNA for Plasmodium falciparum CSP (aa 1-412) into a mycobacterial expression vector.
- Transforming Mycobacterium smegmatis and M. bovis-BCG with the expression plasmid.
- Assessing CSP expression levels and secretion.
- Monitoring genetic stability and expression over multiple generations.
- Inoculating recombinant BCG into mice to assess immune responses.
Main Results:
- Recombinant BCG strains stably expressed and secreted immunoreactive CSP polypeptides.
- CSP expression was maintained for over 400 generations in M. smegmatis and 50 generations in M. bovis-BCG.
- Recombinant BCG did not induce a humoral or cellular immune response to CSP in mice.
Conclusions:
- Mycobacterium bovis-BCG can be engineered as a stable expression system for Plasmodium falciparum CSP.
- The recombinant BCG secreted CSP but did not elicit a detectable immune response in mice, suggesting potential for further development as a vaccine vector.