Date of Award


Degree Name

Master of Science


Public Health


Rodrigo X. Armijos



Background and Significance.

The leishmaniases are an important group of neglected tropical parasitic diseases caused by parasitic protozoa of the genus Leishmania. International health authorities estimate that 350 million people around the world are at risk for contracting leishmaniasis. Two million new cases occur each year and 12 million people are presently infected worldwide. Leishmaniasis is endemic throughout the Americas region including all of Central America, most of South America, Mexico and southern Texas. Leishmania is also considered a threat to the military readiness of U.S. troops deployed to the Middle East or Latin America.

First- and second-line treatments for leishmaniasis are highly toxic. Drug resistance is on the increase. Elimination of the sandfly vector and mammalian reservoirs has not proven practical. For this reason, the World Health Organization has designated the development of an effective leishmaniasis vaccine as a major global public health priority.

Leishmanization is the oldest form of vaccination against leishmaniasis which had been in practice for hundreds of years. This practice was replaced later by an attenuated Leishmania major vaccine, but severe side effects and complications had limited its use. Research and technology have advanced into third generation vaccines, DNA vaccines. DNA vaccines are stable at high temperatures and inexpensive to produce. However, they possess lower than desired immunogenicity because of the degradation of DNA by DNAse before they reach the target cells. Biopterin transporter (Bt), Intracellular adhesive molecule (ICAM), open reading frames (ORFF), and Amastin are important molecules found in Leishmania amastigote or promastigote stages. Previous studies have shown that Bt and ORFF can produce partial protection against experimental leishmaniasis. Therefore, the development of a method to protect DNA until its delivery to the target cell, i.e., the Leishmania parasite, would be an important scientific advance. Encapsulating DNA within a degradable delivery system such as in nanoparticles could theoretically help to improve the delivery of leishmaniasis DNA vaccines to target cells since degradation would be reduced.


The two major objectives of the Phase 0 experimental study were to: (1) describe the immune response and side effects induced by a DNA vaccine candidate encapsulated within metallo-lipid microparticles and (2) define protection generated by a DNA vaccine candidate metallo-lipid microparticle against infection caused by Leishmania mexicana in murine models.


It is hypothesized that the use of a DNA delivery system incorporating copper or zinc metallo-lipid microparticles will increase the immunogenicity of the DNA leishmaniasis vaccine candidate by decreasing the amount of degradation that occurs prior to the delivery to the target cells.


The experimental study was conducted using 6-week old female BALB/c mice. The mice were randomized to one of seven groups. Group 1 (n=3) received pVAX-bt-icam-I(5ìg) + pVAX-orff-amastin(5ìg)+ metallo-lipid Zinc 32ng (ML-Zn). Group 2 (n=3) received pVAX-bt-icam-I(5ìg) + pVAX-orff-amastin(5ìg)+ metallo-lipid Copper 32ng (ML-Cu). Group 3 (n=4) received pVAX-bt-icam-I(5ìg) + pVAX-orff-amastin(5ìg)+PBS. Group 4 (n=6) received pVAX-bt-icam-I(50ìg) + pVAX-orff-amastin(50ìg). Group 5 (n=4) received metallo-lipid Copper (ML-Cu). One control group, Group 6 (n=6), received 100ìL of PBS and the other, Group 7 (n=7), received only PVax.

Six weeks after the first vaccination, the mice were challenged with Leishmania mexicana (106) in their right hind footpad. Footpad thickness was measured weekly for eight weeks. The mice also were examined for potential adverse effects. Two aliquots of lymph nodes and spleen cells from the sacrificed mice were used to analyze intracellular cytokine expression by flow cytometry. Gene expression was analyzed using Quantitative Real Time Polymerase Chain Reaction with TaqMan Probes (Interleukin-2, Interleukin-4, Interleukin-10, and Tumor Necrosis Factor-á, Interferon- ã). Tissue samples from hind foot pad lesions were processed and plated to determine the amount of parasites present per gram of tissue.


The study results suggested that a partial protective effect occurred in the pVAX-ORFF-Amastin (5ìg) + pVAX-Bt-ICAM (5ìg) + ML-Cu and pVAX-ORFF-Amastin + pVAX-Bt-ICAM (100ìg) experimental groups. These groups had smaller mean footpad lesions compared to the control group mice. In addition, the pVAX-ORFF-Amastin (5ìg) + pVAX-Bt-ICAM (5ìg) + ML-Cu group had a Th1 cytokine profile (high IFN-ã, and low IL-4 and IL-10 with values of 27.5, 0, and 0.57 ÷-fold more RNA copies than the calibrator sample, respectively. This type of pattern has been reported as associated with immune protection against Leishmania infection.


The results of this study suggest that pVAX-ORFF-Amastin (5ìg)+ pVAX-Bt-ICAM(5ìg)+ML-Cu is immunogenic and provides partial protection in the BALB/c mouse model. Further experimentation is needed to increase the immunogenicity of this candidate vaccine such as increasing the concentration of the plasmid or ML-Cu microparticle.




Received from ProQuest

File Size

57 pages

File Format


Rights Holder

Joanna B. Valencia