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r-Sm14 - pRSETA efficacy in experimental animals
C R Ramos1, M M Vilar, A L Nascimento
1Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brasil.
Abstract:
Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system.
Insights
New expression systems for Sm14 antigen, a potential vaccine for schistosomiasis, offer improved yield and purification. These systems provide protective activity comparable to previous methods, aiding large-scale production for clinical trials.
Area of Science:
- Parasitology
- Vaccinology
- Biotechnology
Background:
- Recombinant Sm14 (rSm14) produced in Escherichia coli using the pGEMEX system showed high protective activity against Schistosoma mansoni infections.
- This rSm14 was endorsed by the World Health Organization for clinical trials.
- The pGEMEX system has limitations for large-scale production due to time consumption and low yield.
Purpose of the Study:
- To evaluate alternative Escherichia coli expression systems for Sm14 production suitable for scale-up.
- To develop a more efficient method for producing Sm14 for potential large-scale vaccine manufacturing.
Main Methods:
- Expressed two different 6XHis-tagged Sm14 fusion proteins in E. coli using T7 promoter-based plasmids.
- Purified the recombinant proteins using Ni+2-charged resin chromatography.
- Validated protein identity using Western-Blot with anti-Sm14 antibodies and anti-S. mansoni antiserum.
- Assessed protective activity in animal models against S. mansoni cercariae challenge.
Main Results:
- Successfully expressed and purified two 6XHis-tagged Sm14 fusion proteins (18 and 16 kDa).
- The expressed proteins were recognized by specific antibodies, confirming their identity.
- Both new recombinant Sm14 proteins demonstrated protective activity against S. mansoni infection, equivalent to the pGEMEX-derived rSm14.
- The 6XHis-tag facilitated rapid purification, indicating suitability for downstream processing.
Conclusions:
- Alternative E. coli expression systems with 6XHis-tagging are effective for producing Sm14 antigen.
- These systems offer advantages in purification efficiency and scalability compared to the pGEMEX system.
- The findings support the development of Sm14 as a vaccine candidate for schistosomiasis control through improved manufacturing processes.