Identifying salivary antigens of Phlebotomus argentipes by a 2DE approach.

In the Indian subcontinent visceral leishmaniasis, also known as kala-azar, is caused by the protozoa Leishmania donovani and is transmitted to humans by the bite of infected female sand flies Phlebotomus argentipes in an anthroponotic cycle. Sand fly saliva is known to play an important role in host infection outcome after an infective bite. Immunogenicity of P. argentipes saliva has already been described. However, specific antigens that can contribute to these immunogenic properties are unknown. This work focuses on the identification of antigens present in P. argentipes saliva through the combination of two-dimensional electrophoresis (2DE) and Western blot (WB). Analysis of the salivary protein profile showed a gradual increase of the protein content in relation to the age of sand flies, reaching the complete salivary protein pattern at day five, which marked the minimum age for dissections. The 2DE revealed a reproducible protein profile that matched the classic monodimensional SDS-PAGE pattern (1DE). The resulting salivary proteomic map consisted of at least 30 spots located between 10 and 60 kDa. According to their isoelectric points, spots were mostly distributed around pH ranges: 5-6 and 9-10. In the proteomic maps, the presence of isoforms or posttranslational modifications was also highlighted since several spots were identified as the same protein. Analysis by in silico prediction programs located several potential glycosylation and phosphorylation sites in the aminoacidic sequences. On the other hand, pooled sera of immunized hamsters through the bite of uninfected sand flies showed elevated anti-saliva IgG levels. These sera permitted the detection of 4 protein bands and at least 20 protein spots in 1DE and 2DE respectively, followed by WB. The antigens were identified by MALDI-TOF, MALDI-TOF/TOF and de novo sequencing as D7-related proteins, PpSP15-like proteins, antigen 5-related proteins, apyrases, and several proteins without assigned protein family. Absence of cross-reactivity between P. argentipes and Phlebotomus perniciosus saliva antibodies determined by ELISA and WB was highlighted in this study, confirming that specific salivary antigens from different sand fly vectors need to be sought when designing vector-borne vaccines and markers for vector exposure assays.