Cryptosporidium parvum sporozoite pellicle antigen recognized by a neutralizing monoclonal antibody is a beta-mannosylated glycolipid.
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The protozoan parasite Cryptosporidium parvum is an important cause of diarrhea in humans, calves, and other mammals worldwide. No approved vaccines or parasite-specific drugs are currently available for the control of cryptosporidiosis. To effectively immunize against C. parvum, identification and characterization of protective antigens are required. We previously identified CPS-500, a conserved, neutralization-sensitive antigen of C. parvum sporozoites and merozoites defined by monoclonal antibody 18.44. In the present study, the biochemical characteristics and subcellular location of CPS-500 were determined. CPS-500 was chloroform extractable and eluted with acetone and methanol in silicic acid chromatography, consistent with being a polar glycolipid. Following chloroform extraction and silicic acid chromatography, CPS-500 was isolated by high-pressure liquid chromatography for glycosyl analysis, which indicated the presence of mannose and inositol. To identify which component of CPS-500 comprised the neutralization-sensitive epitope recognized by 18.44, the ability of the monoclonal antibody to bind CPS-500 treated with proteases, or with alpha- or beta-glycosidases, was determined. Monoclonal antibody 18.44 did not bind antigen treated with beta-D-mannosidase but did bind antigen treated with alpha-D-mannosidase, other alpha- or beta-glycosidases, or a panel of proteases. These data indicated that the target epitope was dependent on terminal beta-D-mannopyranosyl residues. By immunoelectron microscopy, 18.44 binding was localized to the pellicle and an intracytoplasmic tubulovesicular network in sporozoites. Monoclonal antibody 18.44 also bound to antigen deposited and released onto substrate over the course travelled by gliding sporozoites and merozoites. Surface localization, adhesion and release during locomotion, and neutralization sensitivity suggest that CPS-500 may be involved in motility and invasion processes of the infective zoite stages. (+info)
Immunoglobulin subclass distribution and diagnostic value of Leishmania donovani antigen-specific immunoglobulin G3 in Indian kala-azar patients.
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Visceral leishmaniasis, or kala-azar, a fatal tropical disease, remains problematic, as early diagnosis is difficult and treatment often results in drug resistance and relapse. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA), using leishmanial membrane antigenic extracts (LAg) to detect specific antibody responses in 25 untreated Indian visceral leishmaniasis patients. To investigate the pathogenetic significance of isotype markers in kala-azar, relative levels of specific immunoglobulin G (IgG), IgM, IgA, IgE, and IgG subclasses were analyzed under clinically established diseased conditions. Since LAg showed higher sensitivity for specific IgG than lysate, the immunoglobulin isotype responses were evaluated, with LAg as antigen. Compared to 60 controls, which included patients with malaria, tuberculosis, leprosy, and typhoid and healthy subjects, visceral leishmaniasis patients showed significantly higher IgG (100% sensitivity, 85% specificity), IgM (48% sensitivity, 100% specificity), and IgE (44% sensitivity, 98.3% specificity) responses. Low levels of IgA in visceral leishmaniasis patients contrasted with a 13-fold-higher reactivity in sera from patients with leprosy. Among IgG subclasses, IgG1, -3, and -4 responses were significantly higher in visceral leishmaniasis patients than in the controls. IgG2 response, however, was significantly higher (twofold) in leprosy than even visceral leishmaniasis patients. The rank orders for sensitivity (IgG = IgG1 = IgG3 = IgG4 > IgG2 > IgM > IgE > IgA) and specificity (IgM = IgG3 > IgE > IgG4 > IgG2 > IgG > IgG1 > IgA) for LAg-specific antibody responses suggest the potentiality of IgG3 as a diagnostic marker for visceral leishmaniasis. (+info)
Cytokine profile induced by Cryptosporidium antigen in peripheral blood mononuclear cells from immunocompetent and immunosuppressed persons with cryptosporidiosis.
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The proliferative response of peripheral blood mononuclear cells (PBMC) to a crude extract from Cryptosporidium parvum (CCE) was studied in persons who acquired cryptosporidiosis in the same outbreak (15 immunocompetent subjects with prior cryptosporidiosis and 22 human immunodeficiency virus [HIV]-positive persons with various levels of immunosuppression and active cryptosporidiosis) and in individual patients (8 HIV-positive patients with active cryptosporidiosis and 15 HIV-positive persons without history of cryptosporidiosis). PBMC from HIV-positive persons showed less proliferation to CCE and mitogens than did PBMC from immunocompetent subjects with prior cryptosporidiosis, independent of CD4 cell count. In immunocompetent subjects, cytokine gene expression was consistent with cytokine production, whereas in HIV-positive subjects it was not. The production of interferon-gamma in CCE-stimulated PBMC from both immunocompetent and HIV-positive subjects with cryptosporidiosis and the lack of interferon-gamma in CCE-stimulated PBMC from HIV-positive subjects without cryptosporidiosis indicate that C. parvum mainly induces a Th1 response. (+info)
HLA class II factors associated with Plasmodium falciparum merozoite surface antigen allele families.
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In Plasmodium falciparum malaria, certain human leukocyte antigens (HLA) and the parasite's merozoite surface antigens 1 and 2 (MSA-1, MSA-2) have been shown to influence the course of the infection. This report is on associations of distinct HLA factors with the occurrence of particular MSA families in a group of patients with either severe or mild P. falciparum malaria in Gabon. Different distributions of HLA-DPB1 alleles were found in the 2 groups. DR *04 alleles were observed more frequently among patients with severe malaria. Several alleles of different loci were associated with distinct MSA allele families. In addition, carriers of the amino acid methionine at position 11 of the DPA1 allele were more often infected by MSA-1 K1 parasites and less frequently by MSA-1 RO33 parasites. Furthermore, associations of HLA factors with polyclonal infections were found. (+info)
Susceptibility to infectious diseases: Leishmania as a paradigm.
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The diverse response of individuals within populations to infectious pathogens remains poorly understood, although genetic determinants undoubtedly contribute in substantial ways to the outcome of infection. In a mouse model of infection with the intramacrophage protozoan Leishmania major, susceptibility correlates both with aberrant helper T cell differentiation biased towards the production of interleukin 4 and with the presence of an endogenous CD4 T cell repertoire that recognizes an immunodominant parasite antigen with high frequency. In the setting of the particular ecological niche occupied by Leishmania, this combination of otherwise unrelated factors synergizes to result in exquisite susceptibility to this single pathogen, without seemingly compromising host defenses against other agents. Similar paradigms could underlie susceptibility to other pathogenic organisms. (+info)
Immunization of mice with DNA-based Pfs25 elicits potent malaria transmission-blocking antibodies.
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Immunological intervention, in addition to vector control and malaria chemotherapy, will be needed to stop the resurgence of malaria, a disease with a devastating impact on the health of 300 to 500 million people annually. We have pursued a vaccination strategy, based on DNA immunization in mice with genes encoding two antigens present on the sexual stages of Plasmodium falciparum, Pfs25 and Pfg27, to induce biologically important antibodies that can block development of the parasite in the Anopheles mosquito and thus transmission of the disease. DNA encoding Pfs25 when administered by the intramuscular route, either alone or with DNA encoding Pfg27, had the most potent transmission-blocking effects, resulting in up to a 97% decrease in oocyst numbers in mosquito midguts and a 75% decrease in rate of infection. Immunization with DNA encoding a Pfg27-Pfs25 fusion protein was less effective and DNA encoding Pfg27 elicited antibodies in sera that had only modest effects on the infectivity of the parasite. These results show for the first time that DNA vaccination can result in potent transmission-blocking antibodies in mice and suggest that the Pfs25 gene should be included as part of a multicomponent DNA vaccine. (+info)
Antibodies reactive with the N-terminal domain of Plasmodium falciparum serine repeat antigen inhibit cell proliferation by agglutinating merozoites and schizonts.
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The serine repeat antigen (SERA) is a vaccine candidate antigen of Plasmodium falciparum. Immunization of mice with Escherichia coli-produced recombinant protein of the SERA N-terminal domain (SE47') induced an antiserum that was inhibitory to parasite growth in vitro. Affinity-purified mouse antibodies specific to the recombinant protein inhibited parasite growth between the schizont and ring stages but not between the ring and schizont stages. When Percoll-purified schizonts were cultured with the affinity-purified SE47'-specific antibodies, schizonts and merozoites were agglutinated. Indirect-immunofluorescence assays with unfixed parasite cells showed that SE47'-specific immunoglobulin G (IgG) bound to SERA molecules on rupturing schizonts and merozoites but the IgG did not react with the schizont-infected erythrocytes (RBC). Furthermore, double-fluorescence staining against SE47'-specific IgG and anti-human RBC membrane IgG showed that the RBC membrane disappeared from SE47'-specific-IgG-bound schizonts after cultivation. These observations suggest that the SE47'-specific antibodies inhibit parasite growth by cross-linking SERA molecules that are associated with merozoites in rupturing schizonts with partly broken RBC and parasitophorous vacuole membranes, blocking merozoite release. (+info)
Role of gamma interferon in cellular immune response against murine Encephalitozoon cuniculi infection.
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Microsporidia are obligate intracellular protozoan parasites that cause a wide variety of opportunistic infection in patients with AIDS. Because it is able to grow in vitro, Encephalitozoon cuniculi is currently the best-studied microsporidian. T cells mediate protective immunity against this parasite. Splenocytes obtained from infected mice proliferate in vitro in response to irradiated parasites. A transient state of hyporesponsiveness to parasite antigen and mitogen was observed at day 17 postinfection. This downregulatory response could be partially reversed by addition of nitric oxide (NO) antagonist to the culture. Mice infected with E. cuniculi secrete significant levels of gamma interferon (IFN-gamma). Treatment with antibody to IFN-gamma or interleukin-2 (IL-12) was able to neutralize the resistance to the parasite. Mutant animals lacking the IFN-gamma or IL-12 gene were highly susceptible to infection. However, mice unable to secrete NO withstood high doses of parasite challenge, similar to normal wild-type animals. These studies describe an IFN-gamma-mediated protection against E. cuniculi infection that is independent of NO production. (+info)