Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis. More than 98 countries are reported as endemic for leishmaniasis, a vector-borne disease transmitted by sand flies. Drug-resistant forms have emerged and there is an increased need to develop advanced preventive strategies. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The lizard protozoan parasite, L. tarentolae, is nonpathogenic to humans and has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with salivary proteins has been shown to protect mice against cutaneous leishmaniasis. Herein, we used DNA/Live and Live/Live prime-boost vaccination strategies against cutaneous leishmaniasis based on recombinant L. tarentolae stably expressing CPA/CPB genes with and without the sand fly salivary antigen PpSP15 in both resistant and susceptible mice models. Assessment of the immune response and parasite burden in vaccinated mice at different time intervals post-challenge demonstrated that combination of recombinant L. tarentolae CPA/CPB with PpSP15 DNA elicits an enhanced protective immune response against cutaneous leishmaniasis in mice. This parasite- and insect vector-derived antigen combination represents an important step forward in the development of new vaccine strategies against Leishmania infections.

Vaccination with durable immunity is the main goal and fundamental to control leishmaniasis. To stimulate the immune response, small numbers of parasites are necessary to be presented in the mammalian host. Similar to natural course of infection, strategy using live vaccine is more attractive in compare to other approaches. Live vaccines present the whole spectrum of antigens to the host immune system in the absence of any adjuvant. Leishmanization (LZ) was the first effort for live vaccination and currently used in a few countries against cutaneous leishmaniasis, in spite of their obstacle and safety. Then, live attenuated vaccines developed with similar promotion of creating long term immunity in the host with lower side effect. Different example of attenuated strains are generated through long-term in vitro culturing, culturing under drug pressure, temperature sensitivity and chemical mutagenesis, but none are safe enough and their revision to virulent form is possible. Attenuation through genetic manipulation and disruption of virulence factors or essential enzymes for intracellular survival are among other approaches that are intensively under study. Other designs to develop live vaccines for visceral form of leishmaniasis are utilization of live avirulent microorganisms such as Lactococcus lactis, Salmonella enterica and Leishmania tarentolae called as vectored vaccine. Apparently, these vaccines are intrinsically safer and can harbor the candidate antigens in their genome through different genetic manipulation and create more potential to control Leishmania parasite as an intracellular pathogen.