The research proposed for this Physician Scientist Award will study the host immune response in Chagas' disease, a chronic illness caused by Trypanosoma cruzi that affects millions of South and Central Americans. The long-term objective is to gain insight into the immunology of Chagas' disease that will guide the development of vaccines and better treatments. The proposed experiments, to be conducted in mice, seek to bolster the host immune response and to induce protective immunity. A new approach in infectious diseases will be employed in which the pathogen is genetically engineered to secrete immunomodulators to boost the immune response against it. By having the immunomodulator molecule delivered into the microenvironment of infection, the local effects will be maximized and the systemic effects minimized.
The specific aims are: 1. Characterize and further develop an expression system for secretory proteins in T. cruzi. 2. Introduce immunomodulatory genes into T. cruzi that are predicted to favorably affect the outcome of infection 3. Study the effects of the transfected parasites on acute and chronic disease in mice. 4. Test for the development of protective immunity and study the immunologic basis of the protective immunity. The laboratory, with one of its collaborators, has developed an expression vector incorporating portions of the calmodulin-ubiquitin locus of Trypanosoma cruzi. parasites have been transfected with cDNAs for interleukin-2 and gamma-interferon and secrete bioactive proteins. An inhibitor to transforming growth factor beta, decorin, will next to introduced into T. cruzi. The transfected parasites will be tested for normal growth characteristics and stability of the transfected DNA. To maximize secretion, the signal peptide sequence of interleukin-2 will be cloned upstream of the cDNA for the new constructs. Control parasites will be engineered that contain the expression plasmid without the immunomodulator gene. Susceptible mice will be infected with a virulent strain of transfected T. cruzi to study acute disease. Resistant mice will be infected with a less virulent strain of transfected parasites to study chronic disease. Parasitemia, mortality, and tissue pathology will be the primary endpoints. If attenuated infection is observed, then challenge experiments will be performed to test for protective immunity. The basis of this immunity can be studied in detail, with techniques such as adoptive transfer and cell depletion experiments. The findings from the above research will not only help our knowledge of Chagas' disease, but may provide important insights for the treatment and prevention of diseases caused by other chronic intracellular pathogens.
