Date of Award
Rodrigo X. Armijos
Background and Significance. The leishmaniases are a diverse group of diseases with four major clinical manifestations: cutaneous, mucocutaneous, visceral, and diffuse. Approximately 21 Leishmania species and 30 sandfly vectors have been implicated in human leishmaniasis. Leishmaniasis is endemic in 88 countries, 72 are developing countries. Currently, 350 million people live in leishmaniasis-endemic areas around the world. The true incidence of the disease is not known. However, the estimated incidence for visceral leishmaniasis is 500,000 cases per year, and for cutaneous and mucocutaneous leishmaniasis 1.5-2 million cases per year. Visceral leishmaniasis causes 70,000 deaths each year. In the Americas region, leishmaniasis extends from the United States to Argentina excepting Canada, Uruguay and Chile. In 2006, 62,000 leishmaniasis cases were registered affecting mostly Brazil, Colombia, Paraguay, Venezuela, Panama, Ecuador, and Peru. In the United States, cutaneous leishmaniasis has been reported in the southern part of Texas and also among military personnel deployed to Iraq and Afghanistan. The first and second line treatments are extremely toxic. Resistant to some of these drugs is very high in some parts of the world.
The only immunizing intervention shown to be effective for preventing human cutaneous leishmaniasis is Leishmanization (LZ), but is not currently recommended due to a small number of complications, such as non-healing lesions, and cases of immunosuppression following LZ. First generation and second generation vaccines are difficult to standardize from cultured parasites. Third generation vaccines using DNA are one of the most promising new developments in vaccination strategies. The introduction of DNA plasmids into the cells of living hosts has the potential to lead to the generation of both humoral and cellular immune responses and protective immunity. DNA vaccination provides specificity, stability, safety, and a cost advantage.
A leishmaniasis vaccine should optimally be composed of several molecules in order to attack the survival mechanism of the parasite. This is because Leishmania parasites have multiple molecules allowing them to enter the host cells and attack the body. For this study, the molecules selected were biopterin transporter (BT), intracellular adhesive molecules (ICAMs), ORFF, and Amastin. Biopterin Transporter is a potential growth promoter of Leishmania parasites. Cell adhesion molecules (ICAMs) allow the survival of the parasite inside the cell. The ORFF gene is reported to induce partial protection against challenge with L. donovani . The Amastin family plays a role in adjusting the cytoplasmic pH allowing the survival of Leishmania parasites inside the cells. For the present study, the BT, ICAM, ORFF, and Amastin antigens were assembled in the pVAX backbone as a bicistronic plasmid in order to examine the poly-antigen vaccination approach. The ability of these molecules to induce immunity and protection was evaluated using a murine model.
Study Objectives. The two major objectives of this experimental phase 0 trial were: (1) to characterize the immune response induced by the DNA vaccine candidate against L. mexicana infection in a murine model, and (2) to determine the efficacy of the DNA vaccine candidate in immunized BALB/c mice challenged with virulent L. mexicana promastigotes.
Hypothesis.. It was hypothesized that the use of a cocktail DNA vaccine would induce protection against L. mexicana infection in BALB/c mice by promoting the activation of protective Th1-related cytokines and decreasing the progression of the disease at infected footpad sites.
Methodology. The experimental design used 29, six-week old BALB/c female mice. The experimental and control groups were housed under identical environmental conditions. The mice were randomized to one of the six experimental or control groups. The three experimental groups were immunized with 100ÂµL of pVAX-BT-ICAM, 100ÂµL of pVAX-ORFF-Amastin, or 100ÂµL of pVAX-BT-ICAM plus pVAX-ORFF-Amastin. The negative control groups were injected with 100ÂµL PBS or 100ÂµL of pVAX. Three weeks after their last immunization, the experimental and control groups were challenged subcutaneously in the left hind footpad with 40ÂµL of 1 x 106 stationary phase virulent L. mexicana promastigotes (LV4 strain). The development of footpad lesions at the infection site was measured weekly for eight weeks. Quantitative Real Time Polymerase Chain Reactions (QRT-PCR) assays were run to quantify gene expression of Th1 indicators (Interleukin-2, Tumor Necrosis Factor-α, and Interferon-γ) and Th2 (Interleukin-4) indicator. The data were analyzed using descriptive and bivariate statistical techniques.
Results. A partial protection and Th1 immune response was suggested in the experimental group receiving the pVAX-BT-ICAM plus pVAX-ORFF-Amastin vaccine candidate. This group had the smallest mean footpad lesion. There was found statistically significant reduction in footpad thickness when this group was compared to the other experimental groups [pVAX-BT-ICAM (P = 0.000) or pVAX-ORFF-Amastin (P = 0.032)] and to the negative controls [PBS (P = 0.032) or pVAX (P = 0.015)]. The group vaccinated with pVAX-BT-ICAM plus pVAX-ORFF-Amastin had a Th1 cytokine profile (high levels of IL-2, TNF-α, and IFN-γ) which is associated with immune protection against L. mexicana. However, there was also an increased IL-4 expression. For this group, no statistically significance differences were found between this group cytokine profiles (P = 0.376).
Conclusion. Only the pVAX-BT-ICAM-I plus pVAX-ORFF-Amastin vaccine formulation provided partial protection in BALB/c mice. This vaccine formulation was able to decrease parasite burden better than single bicistronic plasmids at the end of the eight-week follow-up period. The lymphocytes of the group vaccinated with pVAX-BT-ICAM-I plus pVAX-ORFF-Amastin had a predominant Th1 immune response by showing higher levels of IFN--γ, TNF-α, and IL-2. However, this protective cytokine profile was reduced by the presence of IL-4 which correlates to a partial protection observed after the eight-week follow-up. Hence, in order to successfully attack L. mexicana, the vaccine must be composed of several antigens. Further studies need to be conducted to support these findings as well as to evaluate the efficacy of this vaccine in other Leishmania species causing cutaneous leishmaniasis clinical form.
Received from ProQuest
Rodarte, Rosina, "Evaluation of Protection Induced by DNA Vaccine Candidate Against Leishmania Mexicana in BALB/c Mice Model" (2012). Open Access Theses & Dissertations. 2176.