Purification of lypophosphoglican (lpg) from leishmania parasites and investigation of antileishmanial efficiency of lpg-polymer conjugate as vaccine model in vitro and in vivo

Leishmaniasis is a group disease transmitted by the bite of the infected Phlebotomus while obtaining blood from mammalian host. According to the World Health Organization, leishmaniasis is one of the six major tropical diseases. 350 million people in the world are under the risk of the disease. It is endemic in 98 country and 21 million people is infected. Leishmaniasis is also important public health problem in Turkey and 20 million people is under the risk of disease. In our country, visceral and especially cutaneous form is endemic. According to Ministry of Health statistics, 5000 new cases are occurring every year. It is expected that this number will be on increase due to migration from endemic neighboring countries. The important reasons for spread of the disease are resistance to the drugs, global climate changes, difficulty of diagnosis. Also there is still no effective vaccine against to leishmaniasis.Different studies for vaccine development have been conducted since 1940 as first, second and third generation vaccine studies. These studies are based on killed parasites, live-attenuated parasites, purified and recombinant antigen based vaccines, DNA vaccines and dendritic cell based vaccines. Although there have been studies on vaccine development for long years, there is still no effective vaccine against to leishmaniasis. Recent studies give much more importance to LPG, one of the most important surface antigens. LPG, with glycolipid structure, is dominant molecule in the surface of parasites. LPG is responsible for pathogenesis and survival of parasites in mammalian host and vector.LPG is being investigated as a vaccine candidate thanks to its important properties. In a study, purified LPG from L. major and L. mexicana alone or with adjuvant injected to the mice and this application provided a partial protection against the disease. These studies showed that LPG is important immunogenic molecule, however, LPG, alone or with the present adjuvant does not have enough efficiency. Recently, it was revealed that synthetic polyelectrolytes, when introduced in mixtures with typical antigens (proteins, natural microbial polysaccharides, and their synthetic analogs), serve as immunostimulants by enhancing immune response as several times. Also it is theoretically possible to conjugate LPG to PAA by using NH2 groups of LPG and COOH groups of PAA.Therefore, for the first time in this study, the aim is to prepare the conjugate of LPG with poliacrylic acid (PAA) which has adjuvant property, and to investigate the efficiency of the conjugate as a vaccine on mice models against to leishmaniasis.To reach the goal of this study, firstly large-scale culture methods were developed. Then obtained cultures are used in isolation of LPG. Purified LPG was conjugated to polymer and MTT assay and flow cytometry were performed in order to determine the toxicity of the conjugates on various cell cultures. Different vaccine formulations were determined as non-toxic and injected to the BALB/c mice and immune responses were studied with ELISA, IFAT, cytokine measurement, microcultures method, in vitro infectivity of peritoneal macrophages, measurement of the spleen and liver weights, Leishman-Donovan Units (LDU) for determininig the parasite burden.Efficiency of the conjugate has been researched by ELISA, IFAT, micro culture method, additionally in vitro infectivity of peritonal macrophages, spleen and liver mass and parasite burden calculation by Leishman-Donovan Units (LDU) had been investigated by several methods. According to the obtained results, LPG-PAA conjugate possessed the most effective vaccine-Induced protective immune response comparing to the control group bound to all of studied parameters. Especially according to LDU, 35 ?g LPG including LPG-PAA conjugate has a decrease of 81,17% for parasite burden but 53,37% decrease for 10 ?g LPG including group. Especially, the highest decrease of parasite burden exists for 35 ?g LPG including LPG-PAA conjugate with a rate of 81,17%. This rate is 44,93% for immunized of LPG control group. Other groups, 10 ?g LPG including LPG-PAA conjugate has a decrease of 53,37%, 35 ?g LPG including LPG-PAA physical mixture has a decrease of 55,2% , the immunization by just LPG group has a decrease of 44,93% and LPG-Freund?s adjuvant group has a decrease of %65,8. Thereby, for the first time in the world and in our country, a new vaccine oriented to LPG-polymer has been developed against leishmaniasis.