Biomarkers of Attenuation in the Leishmania donovani Centrin Gene Deleted Cell Line—Requirements for Safety in a Live Vaccine Candidate

Biomarkers of the attenuated phenotype are needed to develop live attenuated parasites into safe Leishmania vaccines. The centrin-1 gene deleted strain of Leishmania donovani (LdCEN1 -/- ) shows promise as a vaccine candidate. To identify genes whose expression patterns are indicators of attenuation, the LdCEN1 -/- line was compared to wild type by gene expression microarray. Two genes, one coding for a 27kDa protein (p27) and another coding for putative Argini- nosuccinate Synthase (AS) have such expression patterns. Both genes express a higher RNA level in the amastigote stage than in the promastigote stage of wild type cells; however they are down-regulated in the LdCEN1 -/- amastigote cells. Western blots indicated that the AS protein level is also reduced in the LdCEN1 -/- amastigotes, while the p27 protein level is not reduced even when its mRNA level has diminished. Northern and Western blot analysis with these two biomarkers showed that LdCEN1 -/- parasites recovered after five weeks of infection in mice had the same expression pattern as they had prior to infection and episomal expression of centrin in the LdCEN1 -/- cells restored normal expression of both genes. Measurement of the expression of these two genes in infected macrophages by RT-PCR indicated the same pattern as in cultured cells. Therefore, both the mRNA and/or the protein levels of these two genes could be used as biomarkers of at- tenuation to monitor the safety of the LdCEN1 -/- cell line as it is developed as a potential vaccine.