Immunization with large number of plasmids is also the basis for expression library immunization, a powerful but laborintensive strategy for vaccine discovery [46] , which has been used with Leishmania. Immunization of mice with L. major genomic expression library fractions was able to induce significant protection, but these authors did not pursue library fractionation further [47] . In another study, the identification of protective library subsets from an L. donovani amastigote cDNA library and their successive fractionation into smaller protective libraries lead to the identification of novel protective antigens [48] . Interestingly, most of the antigens identified would not have been predicted to be good vaccine candidates. Indeed, they were not surface or secreted proteins, neither stage-specific, but were intracellular and some very conserved such as histones, or ribosomal proteins [48] . Vaccine discovery is also the next logical step following the recent completion of the L. major genome sequencing [49] . In one approach, the random screening of 100 genes upregulated in amastigotes tested as DNA vaccine allowed the identification of 14 novel protective and 7 exacerbating antigens [50, 51] . Again, function and cellular localization would have been poor predictors of the protective efficacy of these antigens, as most were not predicted to be localized on the surface, but shared similarity with ribosomal proteins, cytoskeleton, or metabolic enzymes [51] . It is thus becoming increasingly clear that there is little rationale to limit Leishmania vaccine discovery searches to surface or secreted antigens. Rather, new criteria need to be considered for the rational identification of vaccine candidates as strategies based on such random screening cannot be applied to large genomes such as that of Leishmania, with over 8000 annotated genes.