Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag(p26) genes from the simian immunodeficiency virus (SIV) SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopyreplicating vector, and the expression levels of both nef and gag in the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of the nef-gag operon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, the nef-gag operon was inserted site specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes even though a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude as that of the replicative rBCG strain containing several copies of the genes.Mycobacterium bovis BCG, an attenuated mycobacterial strain administered to protect against tuberculosis and leprosy, is a particularly attractive vector for the delivery of heterologous antigens. This live and persistent vaccine, which can be administered at birth, has been given to over three billion people with remarkably low levels of major side effects in immunocompetent individuals. Its excellent immunomodulating properties and the fact that it can be administered by mucosal routes make BCG one of the most attractive live vectors for the development of vaccines against other diseases.
Toxoplasma gondii-specific antibody responses in serum, intestinal secretions, and milk were identified with an enzyme-linked immunosorbent assay following a single oral infection of mice with strain 76K cysts of T. gondii. Immunoglobulin A (IgA) production began during week 2 of infection in serum and milk and during week 3 of infection in intestinal secretions and persisted in all three throughout the experiment (17 weeks). IgG but not IgM antibodies were detected in intestinal secretions later in the infection. Serum and milk IgG and IgM production began at the same time after infection as did the IgA response. In Western blotting (immunoblotting), intestinal IgA antibodies were shown to react with antigens comigrating with the T. gondii proteins p22, p23, p30, and p43, the 28-kilodalton antigen, and the 55-and 60-kilodalton rhoptry proteins, as recognized by specific monoclonal antibodies. Milk IgA antibodies reacted with antigens comigrating with p30 and p43. Most of the antigens recognized by IgA antibodies were also detected by IgG antibodies. IgA antibodies from all three biological samples detected the same major T. gondii antigens; thus, there was apparently no specific antibody production unique to one locality.
Following oral immunization of C57BLU6 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.
Our study demonstrates that cholera toxin (CT) markedly enhances the intestinal anti-T. gondii antibody response following oral immunisation of mice with a T. gondii sonicate (TSo) and CT. The antibodies induced were mostly IgA and secretory IgA but a small quantity of IgG was also produced. In contrast, no intestinal anti-T. gondii IgM antibodies were detected. Anti-CT IgA antibodies were also present in intestinal secretions but in much lower quantities than the T. gondii-specific IgA. No anti-CT IgG nor IgM antibodies were detected. Western blot analysis showed that CT induced not only an increase of the intensity of the intestinal IgA antibody response to the 30-kDa band but also induced intestinal IgA antibodies against other major T. gondii proteins (p22, and the 28-kDa antigen) as recognised by specific monoclonal antibodies. The amplification of the anti-T. gondii secretory IgA response by means of an appropriate adjuvant may be one major step leading towards an orally induced immune protection against toxoplasmosis.
The activation of a predominant T-helper-cell subset plays a critical role in disease resolution. In the case of Toxoplasma gondii, the available evidence indicates that CD4+protective cells belong to the Th1 subset. The aim of this study was to determine whether T. gondii antigens (in T. gondii sonicate [TSo]) presented by splenic dendritic cells (DC) were able to induce a specific immune response in vivo and to protect CBA/J mice orally challenged with T. gondiicysts. CBA/J mice immunized with TSo-pulsed DC exhibited significantly fewer cysts in their brains after oral infection with T. gondii 76K than control mice did. Protected mice developed a strong humoral response in vivo, with especially high levels of anti-TSo immunoglobulin G2a antibodies in serum. T. gondii antigens such as SAG1 (surface protein), SAG2 (surface protein), MIC1 (microneme protein), ROP2 through ROP4 (rhoptry proteins), and MIC2 (microneme protein) were recognized predominantly. Furthermore, DC loaded with TSo, which synthesized high levels of interleukin-12 (IL-12), triggered a strong cellular response in vivo, as assessed by the proliferation of lymph node cells in response to TSo restimulation in vitro. Cellular proliferation was associated with gamma interferon and IL-2 production. Taken together, these results indicate that immunization of CBA/J mice with TSo-pulsed DC can induce both humoral and Th1-like cellular immune responses and affords partial resistance against the establishment of chronic toxoplasmosis.
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.
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