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D Bout

Publications and source records attributed to D Bout.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mapping of B epitopes in GRA4, a dense granule antigen of Toxoplasma gondii and protection studies using recombinant proteins administered by the oral route.

GRA4, a dense granule protein of Toxoplasma gondii elicits both mucosal and systemic immune responses following oral infection of mice with cysts. We studied the antigenicity and immunogenicity of truncated and soluble forms of GRA4 expressed as glutathione S-transferase fusion proteins in Escherichia coli. Protein C (amino-acids 297-345) was particularly well recognized by serum IgG antibodies, milk IgA antibodies and intestinal IgA antibodies from T. gondii infected mice and by serum IgG antibodies from T. gondii infected humans and T. gondii infected sheep. One major B epitope was localized within the last 11 C-terminal residues of GRA4. A second epitope, recognized with lower frequency, was mapped within the region 318-334. In contrast, the N domain of GRA4 (amino acids 25-276) was poorly recognized. Oral immunization of C57BL/6 mice with N, C or NC (amino acids 25-276 fused to 297-345) in association with cholera toxin induced a significant production of serum anti-GRA4 IgG antibodies but a weak and inconsistent intestinal anti-GRA4 IgG antibody response and afforded partial resistance to oral infection with T. gondii. These results provide new molecular and immunological understanding of GRA4 and indicate that it is a potential candidate for oral vaccination against T. gondii.

Administration, Oral↗
PubMed1997

Cell mediated immunity induced in mice by HPV 16 L1 virus-like particles.

Recombinant human papillomavirus (HPV) type 16 L1 virus-like particles (VLPs) expressed in the baculovirus system were used to investigate the cellular immune response to human papillomavirus type 16. The cell-mediated immune response was evaluated through immunization of mice with HPV 16 L1 virus-like particles using a lymphoproliferation assay and cytokine production and cytometric analysis of lymphocyte subsets. A significant proliferative response was observed which was associated with secretion of both interferon-gamma and interleukin-2. FACS analysis of splenic lymphocytes revealed that CD8+ T-cells were increased in the immunized mice. These results demonstrate that HPV 16 L1 VLPs induce a T-cell response characterized by a Th1 profile and confirm that the HPV 16 VLP is a reasonable candidate for vaccine development.

Animals↗
PubMed1997

Differences in immunological response to a T. gondii protein (SAG1) derived peptide between two strains of mice: effect on protection in T. gondii infection.

This study presents the analysis of the immunogenicity, antigenicity and protective effects of a peptide derived from the major surface antigen of Toxoplasma gondii, SAG1. This synthetic peptide carrying three predicted H-2k restricted T cell epitopes was used to immunize mice. The protective effect of the peptide was evaluated in CBA/J and C57BL/6 mice using the decrease in brain cyst load as evidence of protection. Immunization of C57BL/6 mice yielded high antibody titres but had no protective effect after oral challenge. Immunized CBA/J, mice which responded with a lower titre, showed a 35% reduction in cyst burden after oral challenge. Both strains yielded antibodies which recognized the cognate SAG1 protein on immunoblot assay. Using the BIAcore, system, it was shown that at lower titres the CBA/J mouse sera recognized the native SAG1 protein more effectively than the C57BL/6 mouse sera, yielding much higher anti-peptide titres. Lymphoproliferation assays using the peptide experimentally confirmed the predicted T-cell epitopes and showed that they were also recognized by cells of T. gondii infected mice. The anti-peptide subclass analysis suggested a Th1 orientation in CBA/J mice, whereas a Th2 orientation was observed in C57BL/6 mice. Finally, fine analysis of sequences recognized under MHC class I indicated the existence of a T-cell epitope in the H-2k haplotype (CBA/J mice) but not in the H-2b haplotype (C57BL/6 mice). This study provides a structural basis to the understanding of the vaccination response to one of the T. gondii antigens in different strains of mice.

Animals↗
PubMed1996

Immunochemical characterization of antibody-coated nanoparticles.

A new method using surface plasmon resonance (SPR) through the BIAcore was used to demonstrate the specific interaction between an anti-CD4 monoclonal antibody (IOT4a), adsorbed on poly(methylidene malonate 2.1.2) (PMM 2.1.2) nanoparticles, and the CD4 molecule. The results obtained were compared with the interaction of the same immunonanoparticles with rabbit anti-mouse Fc antibodies. The molar ratio (Fc)/(Fab) was 1, suggesting that the same number of epitopes on the Fc and the Fab fragments were accessible after IOT4a adsorption onto nanoparticles. Comparing the observed association rates of free antibody and antibody adsorbed on nanoparticles, the number of molecules of IOT4a antibody on PMM 2.1.2 nanoparticles was estimated as between 2.6 and 3 per nanoparticle. The properties of the antibody-coated nanoparticles are compatible with their use as antibody-targeted pharmacophores.

Adsorption↗
PubMed1996

Intranasal immunization with SAG1 protein of Toxoplasma gondii in association with cholera toxin dramatically reduces development of cerebral cysts after oral infection.

SAG1 protein of Toxoplasma gondii was evaluated as a protective antigen in mucosal immunization with cholera toxin as an adjuvant. CBA/J mice intranasally immunized with a combination of SAG1 and cholera toxin exhibited significantly fewer cysts in the brain after oral infection with the 76K strain of T. gondii than control mice. This acquired protection lasted at least 5 months. Protected mice developed high levels of serum anti-SAG1 immunoglobulin G antibodies as well as an enhanced systemic cellular response, as assessed by the proliferation of splenocytes in response to SAG1 restimulation in vitro. This cellular proliferation was associated with an increase of interleukin-2 and interleukin-5 synthesis and with barely detectable gamma interferon production. Splenic immune T cells were shown to convey modest protection to recipients against development of brain cysts following oral infection with T. gondii. Significant production of anti-SAG1 immunoglobulin A was induced in intestinal secretions of protected mice. These results indicate that intranasal immunization with SAG1 and cholera toxin can induce mucosal and systemic immune responses and affords partial and long-lasting resistance against the establishment of chronic toxoplasmosis.

Administration, Intranasal↗
PubMed1995

Analysis of human CD4-antibody interaction using the BIAcore system.

Interaction between CD4 cell surface protein and HIV-bearing gp120 has been described as the initial step for HIV entry into host cells. Some anti-CD4 antibodies were shown to inhibit this interaction. Biosensor studies using the BIAcore were performed to determine kinetic and thermodynamic parameters of the interaction of one of these antibodies (i.e. IOT4a, clone 13B8-2) with immobilized recombinant soluble CD4 (rsCD4). A non-linear regression method was used to analyze the sensorgrams, showing the existence of a double exponential time curve. A KA of 5.2 x 10(7) M-1 was calculated at 25 degrees C. The complex formation was exothermic (-4.5 kcal.mol-1( and entropically positive (+20 cal.mol-1.K-1). The reaction rate (0.234 x 10(5) M-1.s-1 at 25 degrees C) as well as the enthalpy change of the activated complex (+9.7 kcal.mol-1) are not compatible with a diffusion controlled reaction. The thermodynamic values calculated from equilibrium data corresponded to those calculated from kinetic data confirming the validity of the theoretical approach. As for most antigen-antibody interactions, complex formation was enthalpy driven. The overall positive entropy contribution to the stabilization of the complex is in contrast to that observed for the lysozyme-anti-lysozyme model and is probably due to electrostatic interaction between the epitope and the antibody combining site.

Antibodies, Monoclonal↗
PubMed1995

Peritoneal macrophages from C57BL/6 mice orally immunized with Toxoplasma gondii antigens in association with cholera toxin possess an enhanced ability to inhibit parasite multiplication.

Gamma-interferon (IFN-gamma) has been reported to be a major mediator of resistance to toxoplasma infection, mainly through macrophage activation. Cholera toxin used as oral adjuvant induces enhanced protection. Following oral immunization of C57BL/6 mice with a Toxoplasma gondii sonicate (TSo), in association with either cholera toxin (CT) or its B subunit (CTB), the ability of primed sensitized peritoneal macrophages (PM phi) to prevent T. gondii intracellular proliferation in vitro was examined both with and without rIFN-gamma activation. Under these conditions, the inhibition of T. gondii multiplication was greatly enhanced in PM phi from mice immunized with a TSo and CT as an oral adjuvant. In contrast, PM phi from mice immunized with a TSo in association with CTB showed a decrease in their microbiostatic activity towards T. gondii. This negative effect on IFN-gamma-treated PM phi was cancelled out by the addition of a small amount of CT in association with TSo and CTB in the immunization regimen. These data suggest that CT could act as an oral adjuvant in vaccination against toxoplasmosis by increasing the microbiostatic activity of M phi activated with IFN-gamma. Further studies, using intestinal effector cells such as enterocytes, are needed to confirm the value of CT for enhancing this major mechanism of protection against T. gondii infection.

Adjuvants, Immunologic↗
PubMed1994

Toxoplasma gondii oral infection induces specific cytotoxic CD8 alpha/beta+ Thy-1+ gut intraepithelial lymphocytes, lytic for parasite-infected enterocytes.

The first line of defense of the host after primary infection by Toxoplasma gondii is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IEL) (mainly of CD8 phenotype), mucus, and secretory Igs. T. gondii cysts were administered orally to CBA/J mice to determine whether parasite-specific cytotoxic IEL can be elicited within the intestine. We found that oral infection led to an increase in the relative percentages of the CD8 beta + and Thy-1+ IEL populations between day 9 and day 13 after infection. At these times, T. gondii-primed, but not control mice, generated parasite-specific cytotoxic effector IEL for toxoplasma-infected macrophages. This cytotoxic activity was genetically restricted. By using magnetically activated cell sorting, the effector IEL were shown to be CD8 alpha/beta + Thy-1+ T lymphocytes and FACScan analysis revealed that they mainly express TCR-alpha beta. A significant level of cytotoxicity was also observed against T. gondii-infected epithelial cells from the MODE-K cell line, suggesting that parasite-specific IEL may ensure epithelial integrity by rapidly killing infected enterocytes. Finally, toxoplasma stimulation in vitro led to IFN-gamma production by T. gondii-primed IEL. Taken together, these data suggest that antigen-specific IEL, bearing the CD8 alpha/beta + Thy-1+, TCR-alpha beta + phenotype, can act directly as effector cells through a specific CTL activity at the intestinal level and may indirectly activate other mucosal effector mechanisms through IFN-gamma secretion.

Administration, Oral↗
PubMed1994

Epitopic analysis of the Toxoplasma gondii major surface antigen SAG1.

T and B cell epitopes of the major Toxoplasma gondii surface antigen SAG1 were studied following CNBr fragmentation. Three fragments, F1, F2 and F3, were obtained, of 19, 16.5 and 14 kDa, respectively. The positions of F1 F2 and F3 within the SAG1 protein were identified by N-terminal sequence determination. The F1 fragment located on residues 125-269 contains the C-terminus, and the fragment F2 (residues 1-124) is located at the N-terminal region. F3 is a C-terminal peptide about 40 amino acids shorter than the F1 fragment (residues 165-269). Polyclonal antibodies obtained from infected animals or humans and a monoclonal anti-SAG1 antibody did not recognize either the reduced protein or the reduced fragments on immunoblotting. The monoclonal antibody 1E5 did not recognize fragment F1. Mouse IgA and IgG antibodies from infected mouse sera and intestinal secretions, as well as human IgG antibodies, only recognized the whole protein and the F1 fragment. These results suggest that the fragment F1 encompasses all B cell epitopes recognized on the SAG1 protein after infection with the parasite and that the sequence 125-165 is essential for the structural integrity of these B cell epitopes. Murine anti-SAG1 T cell proliferation was observed in SAG1 immunized CBA/J mice (H-2k) and BALB/c mice (H-2d), but not in C57BL/6 mice (H-2b). The three fragments F1, F2 and F3 were able to induce specific proliferation of anti-SAG1 T cells from CBA/J mice, while only the F1 and F2 fragments induced specific blastogenesis of anti-SAG1 T cells from BALB/c mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Oral immunization with Toxoplasma gondii antigens in association with cholera toxin induces enhanced protective and cell-mediated immunity in C57BL/6 mice.

Following oral immunization of C57BL/6 mice with a Toxoplasma gondii sonicate (TSo) in association with either cholera toxin (CT) or CT B subunit, the T. gondii-specific in vitro proliferation of splenic T lymphocytes was determined. Cytokines produced by these T cells were then characterized. After oral challenge with T. gondii 76K cysts, the percentage of cumulative survival was assessed, as was the number of brain cysts in the mice which survived. The TSo-specific proliferation of splenic T lymphocytes was greatly enhanced by the use of CT, whereas CT B subunit alone did not lead to amplification of splenic T-cell proliferation. The use of CT was associated with an increase of interleukin-2 (IL-2) and gamma interferon synthesis by TSo-stimulated splenic T cells, whereas no enhancement of IL-5 and IL-6 production was observed. IL-4 was not detected. A significant protection of mice immunized orally with TSo plus CT was observed in comparison with those immunized with TSo alone. This protection was associated with a large decrease in the number of brain cysts compared with the number found in naive mice infected orally with a sublethal dose of T. gondii 76K cysts. Further studies, using well-defined T. gondii proteins which are known to induce both mucosal and systemic immune responses, are needed to confirm the value of CT in the enhancement of protection against oral toxoplasmosis.

Adjuvants, Immunologic↗
PubMed1993

Mucosal and systemic cellular immune responses induced by Toxoplasma gondii antigens in cyst orally infected mice.

This study was performed to determine the T-cellular immune responses following Toxoplasma gondii oral infection and to assess further toxoplasma antigens on their ability to stimulate in vitro mucosal and systemic T-cell immunity. Parasite-specific cellular immune responses in Peyer's patches (PP), in mesenteric lymph nodes (MLN) and in spleen (SPL) were investigated using a lymphoblastic transformation test following oral infection of mice with strain 76K cysts of T. gondii. An early toxoplasma sonicate-induced mucosal T-cell proliferation occurred in MLN and PP with a peak responsiveness on day 6 post-infection (PI) and rapidly reached background levels on day 7 PI in PP and on day 8 PI in mesenteric lymph nodes. A later splenic cellular blastogenesis was observed from day 28 PI and persisted throughout the experiment (day 91). At the time of T-cell proliferation, FACS analyses revealed a decrease in the relative percentages of CD4+ and CD8+ T cells with a predominance of CD8+ lymphocytes which leads to an inversion of the CD4/CD8 ratios. We found that CBA/J is a high responder mouse strain in the induction of mesenteric and splenic T-lymphocyte blastogenesis compared to the intermediate responder BALB/c and low responder C57BL/6. Toxoplasma gondii antigens SAG1 (30,000 MW) and GRA4 (40,000-41,000 MW), which are known to induce locally IgA antibodies, are shown to stimulate primed mucosal T lymphocytes from CBA/J and BALB/c mice whereas no proliferation was demonstrated with C57BL/6 T cells. 229-242 peptide, derived from the deduced amino acid sequence of GRA4, only induces detectable proliferation of primed-CBA/J T lymphocytes. Following oral experimental infection, the in vitro mesenteric response to a toxoplasma sonicate is dominated by a Th2-type cytokine pattern whereas a predominant Th1 cytokine response is observed in the spleen. Finally, in vitro stimulation of mesenteric T cells with the three defined toxoplasma antigens resulted in secretion of interleukin-5 (IL-5) and IL-6 (except for SAG1) and interferon-gamma (IFN-gamma) whereas no detectable IL-2 or IL-4 was observed.

Animals↗
PubMed1992

Molecular cloning of GRA4, a Toxoplasma gondii dense granule protein, recognized by mucosal IgA antibodies.

Clones which were selected from a Toxoplasma gondii expression library with the immune serum from a T. gondii-infected rabbit, were further screened using milk and intestinal secretions from mice which had been orally infected with T. gondii cysts. The gene products of several clones reacted strongly with milk IgA and weakly with intestinal IgA. Three of these clones (5.1, 36.1, 37.4) were shown to encode a dense granule protein of 40 kDa (GRA4). The GRA4 protein co-migrates with one of the T. gondii antigens recognized by mucosal IgA. The complete nucleotide sequence of GRA4 has been obtained by cloning genomic T. gondii BamHI fragments containing the 37.4 DNA insert. The coding sequence contains no intron. The deduced amino acid sequence indicates a proline rich (12%) product with an internal hydrophobic region of 19 amino acids and a potential site of N-glycosylation. The primary translation product with a theoretical size of 36,260 Da contains a putative N-terminal signal sequence of 20 amino acids but no apparent glycolipid anchor sequence. Quantitation of the GRA4 gene and Southern blot analysis suggested that the GRA4 gene is single copy. GRA4 gene is translated in tachyzoites to yield a single mRNA species of about 1900 bases.

Amino Acid Sequence↗
PubMed1992

Immunocapture assay for quantification of human IgA antibodies to parasite antigenic enzymes. Application with the alkaline phosphatase of Schistosoma mansoni.

Conditions are described for using solid phase adsorbed jacalins in an immunocapture assay for IgA antibodies to the alkaline phosphatase of Schistosoma mansoni. Microtiter plates were activated with polylysine and jacalins were covalently adsorbed by means of glutaraldehyde. From three different jacalins, the one purified from seeds of Artocarpus tonkinensis showed the lowest non-specific adsorption and was used for further studies. Comparing solutions of bovine serum albumin, ovalbumin and Tween 20, it was shown that the latter was most successful in blocking non-specific adsorption. Low serum dilutions resulted in a less efficient IgA capture by the adsorbed jacalin than higher dilutions. Under optimal working conditions, a high correlation could be shown between the presence of specific anti-alkaline phosphatase antibodies of IgA isotype and IgG isotype.

Alkaline Phosphatase↗
PubMed1992

Parasite enzymes as a tool to investigate immune responses.

Previous evidences reported by us and by other authors revealed the presence of IgG in sera of Schistosoma mansoni-infected patients to immunodominant antigens which are enzymes. Besides their immunological interest as possible inductors of protection, several of these enzyme antigens might be also interesting markers of infection in antibody-detecting immunocapture assays which use the intrinsic catalytic property of these antigens. It was thus thought important to define some enzymatic and immunological characteristics of these molecules to better exploit their use as antigens. Four different enzymes from adult worms were partially characterized in their biochemical properties and susceptibility to react with antibodies of infected patients, namely alkaline phosphatase (AKP, Mg2+, pH 9.5), type I phosphodiesterase (PDE, pH 9.5), cysteine proteinase (CP, dithiothreitol, pH 5.5) and N-acetyl-beta-D-glucosaminidase (NAG, pH 5.5). The AKP and PDE are distinct tegumental membrane-bound enzymes whereas CP and NAG are soluble acid enzymes. Antibodies in infected human sera differed in their capacity to react with and to inhibit these enzyme antigens. Possibly, the specificity of the antibodies related to the extent of homology between the parasite and the host enzyme might be in part responsible for the above differences. The results are also discussed in view of the possible functional importance of these enzymes.

Africa↗