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Completed Research Projects
Title: Protective Immunity in Experimental Visceral Leishmaniasis
Synopsis: This animal study evaluated the mouse immune system response after being infected by leishmania.
Overall Summary: See project objective.
Overall Project Objective: Characterization of the protective immune mechanisms in experimental visceral leishmania, and identification of the parasite antigens which elicit such protective responses.
Results to Date: Mice infected with L. donovani amastigotes by the intradermal route develop minimal cutaneous swelling, regional lymphadenopathy, but no detectable visceral (hepatic) parasite burden. In contrast, mice infected by the intravenous route develop progressive hepatosplenomegaly and a visceral parasite burden that increases up to 4-6 weeks after infection. To understand the mechanisms associated with the local control of infection and active visceral disease, we have characterized the in situ expression of cytokines (IFN-y, IL-4, IL-10, and IL-12) in the spleen, draining the lymph node, and cutaneous site of inoculation compared to the spleen following systemic inoculation. IL-10 and TGF-b mRNA and protein expression were prominent in the spleens of systemically infected animals. There was significant expression of both IL-10 and IL-12 at the cutaneous site of inoculation starting 7 days after infection. Thus there seems to be a mixed Th1/Th2 type response to primary infection in this model, but local control (skin and draining LN) appears to be associated with markedly increased levels of IFN-y and IL-12 RNA. These differences may be due to more efficient antigen presentation at the LN level following cutaneous infection (possibly from migration of cutaneous cells), or the induction of immunological tolerance following intravenous infection. Additional studies were performed using a similar methodology to characterize the immunopathogenesis of VL in the highly susceptible hamster model. In order to study the model, multiple hamster cytokine genes were cloned, sequenced and used as molecular probes to determine the levels of mRNA expression in response to active visceral disease. There was a strong Th1 cytokine expression in the spleen in spite of the progressive nature of disease in this model of lethal infection. IL-10, but not IL-4 expression was markedly increased in response to infection and is likely to play a role in the progressive nature of this disease. A number of L. donovani antigens previously identified as a target of immune T cells have been cloned for study of vaccine candidates. The open reading frames have been cloned into a eukaryotic expression vector, expressed in mouse fibroblasts and macrophages, and the nature of the T cell response directed toward it is being characterized. Mice have been immunized with these constructs and studies to determine their protective efficacy are in progress. The IL-12 expression vector we have constructed will be used as an adjuvant in these immunization studies.
Project: VA-16
Agency: Department Of Veterans Affairs
Location: VAMC San Antonio
P.I. Name: Peter Melby
Status: Complete
Study Start Date: October 01, 1994
Estimated Completion Date: September 30, 1997
Specific Aims: Studies of the murine model of visceral leishmaniasis will provide a means to characterize the operative protective immune mechanisms as well as identify parasite antigens which have potential use for human vaccination. Characterization of the protective immune mechanisms in experimental visceral leishmaniasis will be accomplished by studies of the in situ cytokine response to primary infection in susceptible mice, as well as identification of responses associated with the acquisition of immunity. Once the the mechanisms associated with protective immunity have been defined the parasite antigens relevant to immunity can be identified. Purified and recombinant Leishmania donovani antigens which elicit an in vitro cytokine response which correlates with in vivo immunity will be identified and characterized.
Methodology: The murine model of visceral leishmaniasis will be used to characterize the mechanisms involved in protective immunity at the tissue level. These studies were performed using a reverse transcriptase polymerase chain reaction methodology for measuring cytokine (IL 4, 11 6, IL 10, IL12, IFN - y) gene expression in skin, Lymph node, and spleen. Comparisons were made between mice infected locally (skin) and systematically (intravenous). Expression of these cytokines in the spleen was also studied at the protein level using immunohistochemical techniques. Because infection of mice with L. donovani does not result in a progressive lethal infection, we have also begun to study the immunopathogenesis of VL in the Syrian hamster model which very closely mimics the progressive, fatal disease seen in humans. Because there were no reagents available to study this model, we cloned and sequenced a number of the hamster cytokine genes (IL-2, IL-4, IL-10, IL-12, IFN-y, TNF alpha, and TGF-beta). These molecular probes were then used to characterize the expression of cytokine mRNAs in active VL in the hamster model. In our work to identify vaccine candidates for this disease, we have cloned recombinant L. donovani antigens into a eukaryotic vector for use in DNA immunization studies. Because of the prominent role IL-12 plays in the local containment of L. donovani infection through induction of a Th1 response, we have constructed an IL-12 expression plasmid for use as an adjuvant in the DNA immunization studies. Once the in situ mechanisms are better defined, in vitro correlates of immunity (e.g. a certain cytokine profile) can be identified. Semipurified or purified soluble antigens will then be tested in vitro for their capacity to elicit a proliferative response and protective cytokine profile in stimulate splenocytes or lymph nodes. A recombinant library will also be directly screened using spleen cells from immune animals. This will enable the identification of vaccine candidates which can subsequently tested in the animal model.
Publications:
Melby P , Tryon V V, Chandrasekar B , Freeman G L. Infect Immun.1998;66:2135-42.
Melby P , Yang Y , Cheng J , Zhao W . Infect Immun.1998;66:18-27.
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