Abstract
No licensed human vaccines are currently available against leishmaniasis. Several anti-leishmanial vaccines are currently undergoing testing, including genetically modified live-attenuated parasite vaccines. Studies with live attenuated Leishmania vaccines such as centrin deleted Leishmania donovani parasites (LdCen−/−) showed protective immunity in animal models. Such studies typically examined the biomarkers of protective immunity however the biomarkers of attenuation in the parasite preparations have not received adequate attention. As several candidate vaccines enter clinical trials, a more complete product characterization to enable maintenance of product quality will help meet regulatory requirements. Towards this goal, we have determined the complete genome sequence of LdCen−/− and its parent strain Ld1S-2D (LdWT) and characterized the LdCen−/− vaccine strain using bioinformatics tools. Results showed that the LdCen−/− parasites, in addition to loss of the centrin gene, have additional deletions ranging from 350 bp to 6900 bp in non-contiguous loci on several chromosomes, most commonly in untranslated regions. We have experimentally verified a subset of these adventitious deletions that had no impact on the attenuation of the LdCen−/− parasites. Our results identified hitherto unknown features of attenuation of virulence that could be used as markers of product quality in production lots and highlight the importance of product characterization in parasitic vaccines.
Highlights
Visceral leishmaniasis (VL) is a serious global public health problem caused by the infection of blood borne protozoan parasites of Leishmania donovani/infantum/chagasi complex
The whole genome sequencing followed by coverage analysis of LdWT(Ld1S-2D) and LdCen−/− parasites revealed that the open reading frame corresponding to the centrin gene is deleted in the LdCen−/− genome as shown in previous studies (Fig. 2)
To confirm that the homologous recombination method used to delete centrin (LinJ.22.1260) did not perturb other centrin genes, we examined the other four centrin genes located on chromosomes 7, 32, 34 and 36 (LinJ.07.0790, LinJ.32.0690, LinJ.34.2160 and LinJ.36.6370)
Summary
Visceral leishmaniasis (VL) is a serious global public health problem caused by the infection of blood borne protozoan parasites of Leishmania donovani/infantum/chagasi complex. The relative ease of manipulation of the Leishmania genome enabled creation of genetically modified parasites by eliminating genes essential for virulence Following these advances, several genetically modified live attenuated Leishmania donovani/infantum parasites have been developed as potential vaccines in recent years[4]. The ability of Leishmania parasites to rearrange their genomes upon experimental disruption of genes considered to be essential has been reported previously[14,15,16,17] These rearrangements often involved duplication of certain fragments and/or altered expression of the genes as a compensatory effect[14, 18]. This raises important implications for characterization of the genetic stability of the null mutants as a function of attenuation of virulence and safety characteristics. Our results demonstrate the utility of the whole genome sequencing method in obtaining reproducible and novel product characteristics that can be routinely used to assess the manufactured lots
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