In vitro and in vivo investigation of effectiveness of attenuated Leishmania parasites by immunostimulant polymers as a vaccine

Leishmaniasis, which is one of the important public health problems for both Turkey and the world, commonly occurs in 98 countries around the world and 350 million people are under the threat of this disease. Furthermore, 12 million people arecurrently infected with Leishmaniasis at worldwide and it is estimated that 2 million new cases are being added each year (1.5 million CL, 500,000 VL). Also in Turkey, approximately 20 million people are at the risk of the infection. At the same time,drugs that are used for the treatmenti, are highly toxic, besides the disease pathogens and vectors have developed resistance to the antileishmanial drugs and insecticides. Furthermore, it is known that the disease is spreading all around the world by the effect of global warming. However, although there are several studies to develop aneffective vaccine against Leishmania, these endeavors have not reached to a success. Up to the present, different approaches have been tried to develop a vaccine against Leishmaniasis. One of these approaches is live attenuated vaccines approach. However, obtained attenuated vaccines could not demonstrate sufficienteffectiveness, just like other vaccine approaches so far. One of the main reasons for this failure is gradually decrease of important immunogenic molecules of the parasites(such as LPG and gp63) during the avirulation process in long term culture or silencing of genes of these molecules. In order to obtain highly effective attenuated live xxivaccines which alter these disadvantages, there is a great need to develop new approaches. Accordingly, it is known that polyelectrolytes increase the immune response, especially polyacrylic acid (PAA) and polyoxidonium (POX) shows high adjuvant effect in vivo. On the other hand, there is no study about the effects of the polyelectrolytes those have adjuvant properties and interacted with live Leishmaniaparasites related to immune response and infectivity in vivo and in vitro (macrophage cell cultures). According to this, the aim of this thesis study, as a first time, is to determineinfluence of immunostimulant negative and positive charged polyelectrolytes (PAA and POX) exposed VL agent L. infantum parasites to infectivity rates, resultant cell response effects in vitro and humoral and cellular immune responses in vivo and according to this todevelop an approach in generating attenue vaccine models against Leishmania. Methods: To reach this target, Infectivity determination with Giemsa staining studies, determination of Nitric Oxide with Griess reagent, MTT method to determine cell viability and scanning electron microscopy for morphological studies were used in this thesis. On the other hand, in the in vivo studies total IgG and cytokine (IL-2, IL-4, IL-10 and IFN-?) amounts in the mouse serum was determined by ELISA method while liver and spleen parasite amount in LDU units, % infectivity decrease in peritoneal macrophages was calculated by Giemsa staining method. In addition, presence of parasites in the mouse peripheral blood was determined with Giemsa staining and micro culture method (MCM) while delayed-type hypersensitivity was determined by the DTH test. The data was performed with SPSS version 19.0 for Windows program and p <0.05 was considered as statistically significant.Results: In the in vitro macrophage cells, it has been shown that parasites exposed to the polymer both lost infectivity and induced the production of NO. In in vivo mouse models PAA subjected mouse groups IgG antibody levels significantly increased compared to the control group, while the percentage of reduction in parasite burden compared to the control for PAA spleen 84.27 ± 2.98, for PAA liver 89.75 ± 2.12, for POX spleen 76.92 ± 1.88 and finally for POX liver 80.02 ± 3.11 has been found (p <0.05). In particular PAA exposed promastigotes induces the immune system to Th1 type of cell proliferation, induce the production of IFN-? cytokines that have protective role in L.infantum infections and 7-fold increase compared to control (p <0.05). In addition, results of tests carried out with POX has shown for the first time that Pox doses that are above 6000 ?g / ml, inhibits the proliferation of intracellular parasites.Thus, for the first time in this study it was shown that by using POX and PAA it is possible to develop a attenuated vaccine against leishmaniasis. The obtained results may constitute a basis for the development of a vaccine against leishmaniasis in the future or immunotherapy methods to be developed.Keywords: POX, PAA, J774, L.infantum , toxcicity, MTT, cell culture, amastigot,Nitric Oxide, vacci