This project deals with both laboratory and clinical aspects of infection caused by the intestinal nematode, Strongyloides stercoralis. The laboratory work is now focused upon preparation and purification of recombinant antigens from infective (L3) larvae of the parasite. The clinical studies involve assay and evaluation of immune responses in individuals infected with S. stercoralis. The diagnosis of chronic Strongyloides infection is often difficiult because larval excretion is scanty and intermittent. Therefore, a serologic test and other immunologic methods are useful in identifying infected individuals. Assay of antibodies of different isotypes, such as IgG, IgG4 and IgE, has been found useful in immunodiagnosis. An immediate hypersensitivity skin test mediated by parasite-specific IgE using crude parasite antigens continues to be studied. The effect of infection with the retrovirus, HTLV-1, upon individuals also infected with S. stercoralis was found to affect the likelihood of severe disease due to the parasite and efficacy of treatment. The immunologic mechanism for this was found to involve in-vitro activation of T cells with production of interferon-gamma (INF-gamma). This was associated with down-regulation of Th2 cytokines, as well as low serum IgE levels. This stimulus to INF-gamma production has been considered to be due to TAX protein, a product of HTLV-1 infected CD4 positive T cells. However, there may be other sources of INF-gamma since Mitre, et al. found that the majority of INF-gamma producing CD4 positive cells in patients with HTLV-1 infection do not express TAX protein. Since helminth infections are associated with Th2 cytokine responses we examined whether such infections would influence the pro-viral load in patients also infected with HTLV-1. No consistent pattern of either increase or decrease of pro-viral load was found in cells from 20 patients infected with S. mansoni and S. stercoralis. The homology of recombinant NiE antigen with allergen 5 of insect venoms did not interfere with immuno-diagnostic properties of NiE antigen. Methods: 1. Screening of recombinant clones from cDNA library with patients' immune serum. 2. Testing the antigenic activity of recombinant antigens by ELISA and Western blots to various IgG subclasses and for histamine-releasing properties. 3. Culture of lymphocytes from blood of HTLV-1-infected patients after antigen and mitogen stimulation and analysis for intracellular cytokines by FACS scanning of different classes of T cells. 4. Skin testing of patients for immediate hypersensitivity reactions to S. stercoralis antigens. 5. DNA sequencing of recombinant antigens. Goals and Objectives: 1. Select a recombinant antigen for the parasite S. stercoralis that has optimum reactivity with sera from infected patients. 2. Characterize one or more recombinant antigens and develop optimum purification methods so they could be used as skin test antigens in patients. 3. Elucidate the mechanism(s) by which HTLV-1-infected cells stimulate production of gamma-interferon and downregulate Th2 cytokine production and expression. 4. Select one or more candidate recombinant S. stercoralis antigens that stimulate the release of histamine from the blood of patients infected with S. stercoralis (basophils with parasite-specific IgE on surface receptors). Research Significance: 1. A recombinant antigen from S. stercoralis that would be equally or more specific and sensitive than the currently available somatic antigen in immunodiagnosis would be very useful and obviate the need to maintain the parasite in immunosuppressed animals. If such an antigen were also suitable as an immediate skin test it would facilitate and simplify diagnosis of infection. 2. From the recent observations of Mitre, et al (personal communication) it appears that PBMCs from HTLV-1-infected individuals can produce IFN-gamma without the presence of TAX protein. This observation needs to be confirmed and expanded to include more HTLV-1 carriers and more patients co-infected with S. stercoralis. Chronic TH1 cytokine activation could have an important influence upon manifestations of disease in individuals with various bacterial or other parasitic infections. Future Plans: 1. Characterization of a full length clone of a metallo-protease gene from larvae (L3o)of S. Stercoralis. 2. Characterization of several additional recombinant clones which showed homology to the recombinant NiE antigen. 3. Analysis of proviral loads in cells from HTLV-1 patients also infected with intestinal helminths (S mansoni and S. stercoralis) to see if the helminth infection influences the proviral load.
