Inhibitory effect of anti-paraflagellar RODc antibodies on in-vitro growth of trypanosoma brucei brucei

Abstract

The flagellum of a trypanosome possesses a unique compact filamentous structure termed as the Paraflagellar rod (PFR), conserved among trypanosomatids, euglenoids, and dinoflagellates. The PFR is expressed in all life cycle stages of kinetoplastidae except the amastigotes where the flagellum is reduced. In T.b.brucei, the PFR has two major components, PFRc and PFRa proteins of approximately similar molecular masses 73 kDa and 68 kDa respectively. Recent studies in vivo and in-vitro indicate that interfering with the expression of PFR proteins causes cell paralysis and is lethal to the bloodstream forms. Other experimental evidence also seems to suggest that, the PFR protein is highly immunogenic and plays a protective function. However, the role of the PFR protein as a vaccine candidate producing antibodies with protective properties against trypanosomes has not been adequately investigated in vitro. Therefore this study was undertaken to investigate further, the role of this interspecies conserved protein in eliciting specific antibodies which could inhibit the growth of T.brucei parasites in vitro. The PFRc protein was chosen since it is biochemically related to PFRa and hence the similar results are expected. A DNA segment of approximate 672 bp encoding the PFRc protein was successfully amplified using polymerase chain reaction (PCR), cloned and expressed in E.coli (BL21) cells. The expressed and purified recombinant PFR protein (200µg) was used to immunize rabbits. The rabbits produced antibody titres of 2.5x104 reciprocal dilutions following three booster injections with the same immunogen, spaced two weeks apart. The rabbit antibodies specifically bound to a 25kDa protein on western blot analysis of whole parasite lysate, which corresponds to the size of the PFRc protein. Addition of 25% of purified IgG antibodies to T.b.brucei parasite culture inhibited their growth by 70% implying that the antibodies have inhibitory properties in vitro. In conclusion, this study confirms that the PFRc protein is immunogenic and elicits specific antibodies which have a growth inhibitory potential against T.b.brucei parasites in culture. This ability deserves further investigation and probable consideration of the PFRc proteins as a vaccine candidate against trypanosomiasis.