MSA-2c and GPI-4: two prominent vaccine candidates of Babesia bovis to be expressed in the ciliate Tetrahymena thermophila.

MONTES, GUADALUPE; RODRIGUEZ ANABEL; B.C. NUDEL; FLORIN-CHRISTENSEN M; LEONHARD SCHNITTGER; A. D. NUSBLAT

Queensland

Conferencia; 9th Tick and Tick-borne Pathogen Conference & 1st Asia Pacific Rickettsia Conference.; 2017

Glycosylphosphatidylinositol(GPI)-anchored proteins are abundantly expressed on the surface of parasiticprotozoa and involved in the invasion of host cells, thus constitutingattractive vaccine candidates. Free-living protozoa also express GPI-anchoredproteins as has been shown for the ciliate Tetrahymenathermophila. This protozoan has attractive features that make it an optimaleukaryotic expression system for heterologous proteins, and has already beenused as expression platform for GPI-anchored vaccine candidates for malaria andfish white dot disease. In this work, we have selected two promisingGPI-anchored vaccine candidates of the tick-transmitted cattle hemoparasite Babesia bovis for expression in T. thermophila: MSA-2c and GPI-4. Bothare immunodominant and have surface-exposed neutralization-sensitive B cellepitopes. Sequences were optimized to the codon usage frequency of T. thermophila and cloned downstream ofthe metallothionein promoter 1 (Mtt1) of pICY-gtw plasmid. The 3? ends containingencoded GPI-anchor signals were removed, while the original 5? signalpeptide-encoding regions were conserved. Recombinant plasmids were introducedin T. thermophila ex-conjugants byelectroporation and transformed clones were selected by paromomycin resistance.Episomal transformation with both plasmids was verified by PCR. In the case ofMSA-2c, recombinant protein expression was achieved by induction with cadmiumchloride and verified by immunoblot and HPLC-MS. Importantly, the protein wasdetected in Triton X-114 cell membrane extracts, indicating recognition of the B. bovis signal peptide by the T. thermophila secretory machinery.Application of the developed protocols to GPI-4 expression is under way.Financed by MINCyT (PICT 2013-1708) and INTA (PNBIO 1131034), Argentina.