Mycobacterium tuberculosis-secreted protein antigens: immunogenicity in baboons.

The effective control of tuberculosis (TB) requires the development of improved vaccines. It is now well established that Mycobacterium tuberculosis-secreted antigens represent promising candidates to be included in subunit vaccine preparations. It also is accepted that studies in nonhuman primate models will be required to further develop these vaccine preparations. As a necessary step in this direction, we have assessed the immunogenicity of M. tuberculosis-secreted antigens in baboons. Animals received a total of three intramuscular injections consisting of M. tuberculosis culture filtrate proteins resuspended in an adjuvant formulation (MPL-SE) and were tested for development of specific antibody and cellular responses. All animals produced antibody and cellular proliferative responses in the absence of detectable delayed-type hypersensitivity reactions. Production of gamma-interferon following stimulation of peripheral blood lymphocytes with culture filtrate proteins was either absent or present at low levels. Results from this study show that, although M. tuberculosis-secreted protein antigens are relatively safe and immunogenic in baboons, alternative immunization approaches must be identified for the induction of T-helper type 1 responses.

Immunogenicity and safety of Mycobacterium tuberculosis culture filtrate proteins in non-human primates.

The development of improved vaccines is considered a high priority in the effort to control tuberculosis (TB) world wide. Results from several studies performed in relevant animal models have demonstrated that Mycobacterium tuberculosis secreted antigens may represent major components of improved TB vaccines. To characterize further the M. tuberculosis secreted antigens as they relate to specific features important for vaccine development, rhesus macaques were immunized with either one of two different preparations containing M. tuberculosis culture filtrate (CF) proteins. These preparations differed in relative protein content and in the presence or absence of lipoarabinomannan. Animals received a total of three monthly intramuscular injections consisting of CF proteins resuspended in RIBI adjuvant and were tested for development of specific antibody and cellular proliferative responses. In addition, all animals were constantly monitored for local and systemic reactions as well as for the development of DTH reactions to intradermal tuberculin injection. Results from this study show that the two CF preparations are relatively safe and immunogenic in non-human primates. These two CF preparations differed in their ability to induce specific antibody responses, but were comparable in their ability to induce specific cellular proliferative responses. Induction of both humoral and cellular responses occurred even in presence of pre-existing antibodies directed against M. tuberculosis antigens. However, these responses appeared to be short-lived. Only one of the four animals produced interferon-gamma (IFN-gamma) in response to immunization with CF proteins. No DTH reaction to intradermal tuberculin injection was observed in any immunized animal. Although it is clear that additional studies are required to design strategies for the improvement of the immunogenicity of CF proteins, our observations support the currently accepted view that secreted protein-based preparations may represent promising vaccine candidates for TB.

DNA-based immunization induces anti-CD4 antibodies directed primarily to native epitopes.

DNA-based immunization is one of the most promising strategies to induce protective immunity against a variety of pathogens, presenting clear advantages as compared to the use of recombinant antigens. One of these advantages might be the ability to induce antibodies directed primarily against conformational determinants, as compared to immunization with recombinant proteins. To test this possibility, we have analyzed the antibody responses induced in mice by immunization with either recombinant soluble CD4 (rCD4) or by immunization with plasmid DNA-encoding CD4 (CD4-DNA). Mice immunized with CD4-DNA had lower titers of antibodies able to recognize rCD4 than mice immunized with rCD4. However, immunization with CD4-DNA induced antibodies reactive with the native cell surface CD4 molecule in all mice, whereas only two out of five mice immunized with rCD4 produced antibodies reactive with cell surface CD4, thus demonstrating that the genetic immunization approach may lead to an antibody response more consistent and superior at a qualitative level as compared to immunization with the corresponding recombinant protein. In addition, differences in the kinetics of appearance of antibodies directed against the native CD4 molecule were observed between mice immunized with CD4-DNA or rCD4. In the first case, antibodies reacting with cell surface CD4 were present 28 days after the first immunization, whereas mice immunized with rCD4 produced antibodies directed against the native molecule only following a booster injection. Finally, the two groups of mice produced antibodies with a different isotype distribution. No clear predominance of a specific IgG subclass was detected in the antibody population produced in response to DNA immunization. Conversely, mice immunized with rCD4 produced predominantly antibodies of the IgG1 isotype, indicating generation of a TH2 response. Together, results from this study indicate that the CD4 molecule endogenously produced following DNA immunization is expressed, at least partially, in a native conformation. This feature confers a major advantage to the DNA immunization approach as compared to immunization with the corresponding recombinant protein, which seems to elicit antibodies predominantly directed to epitopes uniquely expressed on the recombinant molecule.