Leishmaniasis is a vector-borne disease that is caused by the infection of protozoan parasites of the genus Leishmania. The extracellular promastigote forms of Leishmania are inoculated into humans (and other mammalian hosts) by sandflies (phlebotomine insects), after which the parasites undergo phagocytosis by macrophages and transform to intracellular amastigotes. Clinical manifestations of leishmaniasis are particularly diverse , ranging from subclinical (unapparent infections) to visceral leishmaniasis (VL), which is usually fatal when untreated. Other common forms of the disease are mucocutaneous (MCL), diffuse cutaneous (DCL) and cutaneous leishmaniasis (CL). The clinical outcomes depends upon a number of factors, including the species (and strain) of the parasite, as well as the host's genetically determined immune responses. Thus, Leishmania major and many other Leishmania species cause CL, Leishmania donovani and Leishmania infantum are mainly associated with VL, whereas MCL results after infection with parasites from the Leishmania braziliensis complex .
Leishmaniasis threatens 350 million people worldwide, mainly in developing countries. Annual incidence is estimated at 2 million cases and the overall prevalence is 12 million people . In developing and under-developed parts of the world, AIDS and other immunosuppressive syndromes add to the higher risk of leishmaniasis . In spite of its incidence, leishmaniasis is a neglected disease. Current control strategies rely on reservoir and vector control and pharmacological drugs, but new treatment strategies are clearly needed . Abundant clinical and experimental evidence indicates that leishmaniasis would be preventable by vaccination, but anti-leishmanial vaccines for human use have yet to be developed [6–10].
Effective vaccination against human CL has been practiced for centuries by deliberate inoculation of living organisms from the exudates of active lesions or, more recently, by the inoculation of cultured Leishmania promastigotes (process known as "leishmanization") . The appearance of complications, i.e. developing of severe disease in some individuals, led to abandoning the use of live Leishmania as a prophylactic vaccine. Nevertheless, leishmanization is still currently practiced in some countries including Uzbekistan, Afghanistan, Iraq, and Iran , and there are recent efforts to standardize it as a live vaccine and also to use it for rapidly assessing the efficacy of new vaccines . On the other hand, first generation vaccines (or killed vaccines), prepared using inactivated whole parasites, have been the subject of many studies over decades and are the only vaccine candidates for leishmaniasis which have undergone phase 3 clinical trials. However, evidence of protective efficacy has not emerged from those clinical trials .
The ineffectiveness of vaccines based on either killed parasites or recombinant proteins seems to be a consequence of the short-term immunity they induce . On the other hand, several studies in mice indicate that persistent parasites are important to maintain durable, anti-Leishmania memory responses [12, 16]. These findings have led to the exploration of the use of live, genetically modified-parasites as an appealing strategy for developing vaccines against leishmaniasis [17, 18]. Defined genetic alterations of the Leishmania genome can be achieved through homologous recombination , allowing disruption of essential genes for virulence and/or host survival. The first Leishmania mutant generated by gene replacement assayed as a potential Leishmania vaccine was an L. major line lacking the gene coding for dihydrofolate reductase-thymidylate synthase (DHFR-TS) . This thymidine-auxotroph mutant was found to persist in BALB/c mice for up to 2 months, but it was incapable of causing disease. Interestingly, this dhfr-ts knockout was able to elicit substantial resistance in mice to a subsequent challenge with virulent L. major. However, immunizations with dhfr-ts knockouts derived from L. chagasi, L. donovani, or L. major did not protect against L. chagasi infection in BALB/c mice . In another report, disruption of BT1 genes, encoding a biopterin transporter, in Leishmania donovani allowed the generation of a mutant line with reduced capacity for inducing infection in mice . Furthermore, it was found that inoculation of BT1 null parasites elicits protective immunity in mice against an L. donovani challenge. Another mutant assayed as attenuated vaccine has been an L. major LPG2-knockout, which cannot synthesize LPG and other phosphoglycans; despite this defect, upon infection of mice, the mutants persist for several months without causing disease . When BALB/c mice infected with lpg2
parasites were challenged with virulent L. major, they were protected from disease . However, the in vivo follow-up of these mutants led to the identification of a compensatory mutant (lpg2-REV) that regained virulence even in the absence of phosphoglycan synthesis . In addition, it was found that L. mexicana lpg2
mutants retained their virulence for macrophages and mice . Another genetically-modified L. mexicana line, lacking cysteine proteinase genes cpa and cpb, was successfully used to protect against homologous infection in mice and hamsters [27, 28]. Likewise, vaccination of BALB/c mice with an L. mexicana null mutant for GDP-mannose pyrophosphorylase (GDP-MP) conferred significant and long lasting protection against infection with virulent parasites . Recently, it has been shown that BALB/c mouse infection with L. infantum mutant lacking one of the two SIR2 (silent information regulatory 2) alleles induced a high degree of protection against a virulent challenge . More recently, it was reported that immunization with a centrin deletion mutant of L. donovani protected mice against infection with either L. donovani or L. braziliensis.
In this study, we analyzed the inmunoprotective ability of an L. infantum deletion mutant, lacking HSP70 type II gene (ΔHSP70-II), as a live vaccine against leishmaniasis in the L. major-BALB/c infection model. Immunization with this mutant line elicited specific immune responses and significant levels of protection against a challenge with virulent L. major promastigotes.