Synthesis, characterization and in vitro and in vivo investigation of effectiveness as vaccine candidate against visceral leishmaniasis of poly(lactic-co-glycolic acid) nanoparticles containing combination of different antigens of leishmania infantum

Leishmaniasis, one of the serious public health problems of Turkey and the world, is a tropical disease which is obligatory intracellular Leishmania parasites. Through the world, nearly 350 million people are under the risk of this disease. The disease is also in Turkey, where endemic in 98 countries, the number of people infected is 12 million. The number of incidents in the Southeastern Anatolia region has increased considerably due to the civil wars especially in Syria. Climate change, the development of resistance to the drugs and also many mammals have a disease reservoir play a major role in this widespread disease. In addition, the problems in the diagnosis of the disease, the difficulties in treatment and the development of resistance clearly reveal the necessity of an effective and safe vaccine the against the disease. Even though vaccine candidates have been developed in three generations to date, still there is no an effective and safe vaccine because of their different disadvantages. Vaccines from the first generation group consist of killed parasite antigens or attenuated Leishmania parasites. The attenuated vaccines were risky as the parasite was likely to be reactivated. Therefore, its use is limited. Due to the contain many target proteins in their structure, soluble leishmania (SLA) and autoclaved leishmania antigens (ALA) can interact with many MHC molecules and produce a high immune response. Besides this, it is easy to obtain and low cost. However, these antigens alone and/with the use of various adjuvants to date showed a low degree of protection. So, in order to increase the immune response of these antigens, it is very important to investigate more appropriate adjuvants. Second generation vaccines based on mostly surface molecules, intracellular molecules, stress sensitive molecules and many other biomolecules in metabolic pathways as a antigen. Among these, lipophosphoglycan (LPG) is a molecule that most immunogenic and abundant in parasite surface. However, subunit antigens alone cannot produce adequate immune responses. DNA vaccines in the third generation are advantageous because they are relatively easy to produce, easy to direct, and have the ability to combine many genes in their transport. However, reasons such as low efficiency and not being able to provide adequate protection and also cost factors are DNA vaccines' disadvantages. Recent advances in nanotechnology have brought a new dimension to vaccine studies. Nanoparticular allows controlled and effective release of antigens without degredation in the biological system and their targeted transport. In particular, the combination of multiple antigens in the form of the same antigen presenting cell is one of the most important features of the nanoparticular antigen-carrying system. FDA-approved PLGA (poly lactic co-glycolic acid) nanoparticles are commonly used as carrier systems due to the biocompatible, biodegradable and also adjuvant properties. When we review the literature, there is no study about PLGA nanoparticles and immunogenic properties of soluble and autoclaved leishmania antigen combination with lipofosfoglycan. Accordingly, the aim of this thesis is to encapsulate the lipofosfoglycan molecule isolated from L.infantum promastigotes in combination with soluble leishmania antigen or autoclaved leishmania antigen to FDA-approved PLGA nanoparticles, to investigate the effectiveness of the nanoformulations in vitro and in vivo against the visceral leishmaniasis. In this thesis, the parasite cultured in a bioreactor in order to obtain biomass for isolation of SLA, ALA and LPG antigens. The obtained antigens used as a both single and binary combination to encapsulated into PLGA. The characterization of the nanoformulations was then performed using different devices. The amount of antigen and the release profile of the nanoformulations were determined using the UV-spectrophotometer. Both LPG, SLA and ALA antigens alone (F1, F2 and F3) and SLA-LPG and ALA-LPG binary antigens (F4 and F5) encapsulated in PLGA was investigated toxicity by MTT method in J774 macrophage and L929 cell culture. The potential for the production of cytokine and nitric oxide (NO) was investigated in macrophage cell culture. Immunization was carried out at two week intervals to investigate protective activities of these nanoformulations as a vaccine candidate in vivo, and the amount of antibody level was determined by ELISA from the serum samples of the experimental animals. After 8 immunizations, mice in each group were infected with L.infantum parasites and control of infection was monitored by Giemsa staining and micro-culture method. One month later after the detection of infection in the control group, the mice were sacrificed; The parasite load (LDU) in the spleen liver and bone marrow were determined. In addition, IL-4, IL-10, IL-12 and IFN-γ cytokines secreted from lymphocytes were determined by a commercial kit. According to the results, it was found that the different concentrations of nanoformulations in J774 macrophage and L929 cell culture did not show a significant toxic effect up to 500 μg/ml compared to the control. When the effectiveness of the formulations on the NO production of macrophages was examined, it was found that the highest effect on NO production in macrophage cells was F4 nanoformulation which increased NO production by 15-fold and F5 nanoformulation which increased 20-fold compared to control. Similarly, vaccine formulations increased the levels of IL-12 and IFN-γ cytokines produced by macrophage cells to 4 to 6 fold compared to the control group, while no significant difference was observed with antigen combinations alone. The reason for this may be the release of only a small amount of antigens into the medium after 72 h of short time in time the antigens within the PLGA nanoparticles. There was no significant change in IL-4 and IL-10 levels. IL-12 and IFN-γ cytokines are known to induce Th1 response and IL-4 and IL-10 cytokines induce Th2 response. High levels of IL-12 and IFN-γ levels suggest that the immune response may develop in the Th1-type. The all groups were then examined on Balb/c mice. The highest antibody level was observed in F5 nanoformulation groups. In addition, it was observed that 75% and 78 % protection against parasitic infection in experimental animals immunized with respectively F4 and F5 formulation following sacrification of mice. Another examined parameter is cytokine levels in spleen cells. It was found that F4 and F5 nanoformulations increased 9 and 15 fold for IL-12 level and 10 and 18 fold for IFN-γ cytokine, respectively. On the other hand, no change was observed for IL-4 levels and 2-fold decrease IL-10 levels shown in F5 nanoformulation compared to control. According to the results obtained, both F4 ve F5 nanoformulations clearly demonstrate the immunogenicity of these vaccine formulations in which both the increase in antibody level and IL-12 and IFN-γ cytokine levels inducing the Th1 immune response and also decrease the parasite load in mice are evident. This result suggests that the administered vaccine formulations significantly inhibit the parasite infection in the organs by inducing cellular and humoral response. Thus, for the first time in this study, nanoformulations containing binary leishmania antigens such as SLA-LPG and ALA-LPG for the first time have been shown to have significant potential as a vaccine candidate against visceral leishmaniasis in vitro and in vivo.