Th1 immune response is essential in the protection against mycobacterial intracellular pathogens. Lipoproteins trigger both humoral and cellular immune responses and may be candidate protective antigens. We studied in BALB/c mice the immunogenicity and the protection offered by the recombinant 27-kDa Mycobacterium tuberculosis lipoprotein and the corresponding DNA vaccine. Immunization with the 27-kDa antigen resulted in high titers of immunoglobulin G1 (IgG1) and IgG2a with a typical Th1 profile and a strong delayed hypersensitivity response. A strong proliferation response was observed in splenocytes, and significant nitric oxide production and gamma interferon secretion but not interleukin 10 secretion were measured. Based on these criteria, the 27-kDa antigen induced a typical Th1-type immune response thought to be necessary for protection. Surprisingly, in 27-kDa-vaccinated mice (protein or DNA vaccines) challenged by M. tuberculosis H37Rv or BCG strains, there was a significant increase in the numbers of CFU in the spleen compared to that for control groups. Furthermore, the protection provided by BCG or other mycobacterial antigens was completely abolished once the 27-kDa antigen was added to the vaccine preparations. This study indicates that the 27-kDa antigen has an adverse effect on the protection afforded by recognized vaccines. We are currently studying how the 27-kDa antigen modulates the mouse immune response.

Mycobacterium paratuberculosis (MPT) is the etiological agent of paratuberculosis. The disease is prevalent throughout the world, and exacts a heavy financial toll. At present, the only means of controlling this disease are culling or vaccination. The existing vaccines are not very efficient and produce a long-lasting local reaction at the point of injection and induce anti-bodies/delayed-type hypersensitivity (DTH) reaction that cannot be differentiated from those of naturally infected animals. New potent acellular vaccines that allow discrimination between infected and vaccinated animals are necessary to improve the control of this disease. We have isolated, overexpressed and purified the 85B antigen of MPT, and characterized the immune response induced by this antigen in mice. Our results showed that the recombinant MPT 85B (rMPT 85B) antigen induced a high production of interferon (IFN)gamma, interleukin (IL)-6, IL-10 and nitric oxide (NO). Spleen cells from mice immunized with rMPT 85B in Ribi adjuvant produced a higher level of IL-10 and NO than spleen cells of mice immunized with rMPT 85B only. In contrast, the addition of Ribi to the immunization protocol resulted in a lower amount of IFNgamma released by spleen cells. The levels of spleen cells proliferation in mice vaccinated with the rMPT 85B protein alone or with rMPT 85B with Ribi adjuvant were, respectively, four times or five times greater than in the control mice. The Ribi adjuvant induced significantly higher anti-85B antibody production of all classes tested and increased the IgG1/IgG2a ratio. DTH responses in mice footpads were observed only in mice immunized with rMPT 85B emulsified in Ribi. rMPT 85B induced both a Th1 and Th2 type of immune response with the later slightly more pronounced when the vaccination protocol comprised Ribi as an adjuvant. The rMPT 85B antigen elicited a strong immune response and can be considered as a potential candidate for a future acellular vaccine.

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The immunotherapeutic effects of interleukin-1 alpha (IL-1 alpha) encapsulated within 1-5 microns-diameter poly (D, L-lactide) microspheres and delivered intratumorally into fibrosarcoma-bearing mice were investigated. Such microspheres are avidly taken up by macrophages, and directing IL-1 alpha into these cells may activate them to participate in antitumor responses in vivo. Treating of tumor-bearing mice with IL-1 alpha microspheres has increased their survival rate, as compared with control mice, untreated or treated with microspheres containing bovine serum albumin (BSA). In 20% of the IL-1 alpha-treated mice, a complete tumor regression was observed. The timing of treatment with IL-1 alpha microspheres was crucial; optimal survival and regression rates were observed in mice treated 24 hr postinjection of the tumor cells. Administration of three doses of IL-1 alpha microspheres on days 1, 8, and 15 postinjection of tumor cells resulted in longer survival rates. Histopathology studies on regressed tumors revealed extensive areas of tumor cell degeneration and necrotic tissue surrounded by a large number of inflammatory cells. A similar picture was observed when IL-1 alpha microspheres were administered into the footpad of control mice, whereas the tissue reaction to BSA microspheres was much milder. Thus, it appears that tumor regression is mainly due to the antitumor effects of IL-1 alpha. Further studies are being aimed at increasing the immunotherapeutic efficiency of microspheric IL-1 alpha, used as a single treatment or in combination with other treatment modalities.

We reported previously that even though immunization with the recombinant mycobacterial 27-kDa lipoprotein (r27) induced a Th1-type response in mice, the vaccinated mice became more susceptible to challenge with Mycobacterium tuberculosis. In this study we show that r27 stimulates naive splenocytes to proliferate. Acylation of r27 was crucial for this effect, since a nonacylated mutant of r27, termed r27⌬SP, failed to stimulate splenocytes either in vitro or in vivo. Depletion experiments indicated that only B cells were proliferating in a T-cell-independent manner. We also found that r27 is recognized by TLR2, which is involved in mitogenic stimulation. Interestingly, r27 but not r27⌬SP induced high gamma interferon levels in splenocyte supernatants, whereas no significant interleukin-2 levels were detected. Since B-cell polyclonal activation might aggravate pathogen infection, we asked whether the antiprotective effect of the r27 lipoprotein is associated with its mitogenicity. We showed that, as in the case of r27, immunization of mice with the nonmitogenic r27⌬SP lipoprotein resulted in increased M. tuberculosis multiplication. We conclude that the antiprotective effect of the r27 lipoprotein must be linked to properties of the polypeptide portion of the lipoprotein rather than to its lipid moiety and its mitogenicity.The 27-kDa antigen, the product of the Mycobacterium tuberculosis lprG gene, is a putative lipoprotein that is present exclusively in the membrane of the M. tuberculosis species complex (5). Previously, it was reported that mice immunized with r27 were more susceptible to M. tuberculosis challenge than nonimmunized mice (15). Splenic CFU counts in mice immunized with the recombinant mycobacterial 27-kDa lipoprotein (r27) were significantly higher, by about 0.5 to 0.8 log unit, than those in nonimmunized mice. Furthermore, the protection afforded following immunization with the Mycobacterium bovis BCG vaccine was completely abolished when r27 was added to the BCG vaccine (15). These results were unexpected, because r27 induces a typical Th1-type immune response thought to be crucial for protection against intracellular pathogens such as M. tuberculosis. It was found recently that M. tuberculosis with a knockout of the 27-kDa (lprG) gene has an attenuated virulence phenotype in mice (A. Cataldi, personal communication). This finding supports our previous results and indicates that the native 27-kDa lipoprotein might also have a role in enhancing M. tuberculosis infection.Lipoproteins have been reported to be powerful antigens that induce strong antibody-and cell-mediated immune responses (7, 10). The lipid moiety on mature acylated lipoproteins or synthetic acylation of peptides increased their immunogenicity and induced their adjuvant-like properties (4). Bacterial lipoproteins are recognized by the innate immune system through toll-like receptor 2 (TLR2) together with TLR1 or TLR6, all of which induce antibacterial activity in macrophages (27-29). These findings indicate that lipoproteins migh...

Mycobacterium paratuberculosis (MPT) is the etiologic agent of paratuberculosis. The disease is prevalent in cattle worldwide, and exacts a heavy financial toll. Effective control requires the development of acellular vaccines offering a better protection than the current available vaccines without side effects and allowing the discrimination between infected and vaccinated animals. We studied the immune response of mice to the MPT superoxide dismutase (SOD) alone or adjuvanted by Ribi. We cloned, overexpressed and purified this antigen in Escherichia coli. Spleen cells from immunized mice, after exposure to recombinant MPT SOD (MPT rSOD), produced significant levels of IFNgamma, TNFalpha and IL-6. IFNgamma and TNFalpha production was increased by the addition of Ribi. In contrast, low levels of NO, IL-4 and IL-10 were secreted by spleen cells culture from immunized mice. The immunoglobulin isotype distribution analysis showed that Ribi adjuvant clearly induced a significantly higher anti-MPT rSOD antibody production of all classes tested and decreased the IgG1/IgG2a ratio thus improving the Th1 response. Delayed-type hypersensitivity responses in mice footpads were observed only in mice immunized with MPT rSOD emulsified in Ribi. Vaccination of MPT rSOD emulsified with Ribi induced both a Th2 and Th1 type of immune response with the later slightly more pronounced. The results presented here on the immunogenicity of MPT SOD suggest that this antigen should be further tested as a candidate antigen for a future acellular vaccine against paratuberculosis.