Comparative protective effects of recombinant DNA and Mycobacterium bovis bacille Calmette-Guérin vaccines against M. avium infection.

A range of strategies are being explored to develop more effective vaccines against mycobacterial infection, including immunization with DNA plasmids encoding single mycobacterial bacterial genes and the use of recombinant live vectors based on the current vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG). We have compared these two approaches using a model of virulent M. avium infection, and the gene for the immunodominant 35 kDa protein which is shared by M. avium and M. leprae, but absent from BCG. Recombinant BCG over-expressing the M. avium 35 kDa protein (BCG-35) induced strong antigen-specific proliferative and interferon-gamma (IFN-gamma)-secreting T cell responses. These were comparable to those induced by a single immunization with a plasmid expressing the same antigen (DNA-35); however, repeat DNA-35 immunization evoked the strongest IFN-gamma release. Immunization with BCG-35 significantly reduced the growth of virulent M. avium, although this effect was similar to that induced by wild-type BCG. Immunization with DNA-35 resulted in significantly greater (2 x log(10)) reduction in the growth of M. avium. Prime-boost strategies combining DNA-35 and BCG-35 increased the protective effect above that achieved by BCG-35, but they were not more protective than DNA-35 alone. Therefore, recombinant BCG-35 and BCG induced similar levels of protection in this model, and maximal protection against M. avium infection was attained by immunization with DNA encoding the 35 kDa protein.

Dendritic cells infected with Mycobacterium bovis bacillus Calmette Guerin activate CD8(+) T cells with specificity for a novel mycobacterial epitope.

Although CD4(+) T cells are essential for protective immunity against Mycobacterium tuberculosis infection, recent reports indicate that CD8(+) T cells may also play a critical role in the control of this infection. However, the epitope specificity and the mechanisms of activation of mycobacteria-reactive CD8(+) T cells are poorly characterized. In order to study the CD8(+) T cell responses to the model mycobacterial antigen, MPT64, we used recombinant vaccinia virus expressing MPT64 (VVWR-64) and a panel of MPT64-derived peptides to establish that the peptide MPT64(190-198) contains an H-2D(b)-restricted CD8(+) T cell epitope. A cytotoxic T lymphocyte response to this peptide could be demonstrated in M. bovis bacillus Calmette Guerin (BCG)-infected mice following repeated in vitro stimulation. When bone marrow-derived dendritic cells (DC) were infected with BCG, the expression of MHC class I molecules by DC was up-regulated in parallel with MHC class II and B7-2, whereas CD1d expression level was not modified. Moreover, BCG-infected DC activated MPT64(190-198)-specific CD8(+) T cells to secrete IFN-gamma, although with a lower efficacy than VVWR-64-infected DC. The production of IFN-gamma by MPT64(190-198)-specific CD8(+) T cells was inhibited by antibodies to MHC class I, but not to CD1d. These data suggest that mycobacteria-specific CD8(+) T cells are primed during infection. Therefore, anti-mycobacterial vaccine strategies targeting the activation of specific CD8(+) T cells by DC may have improved protective efficacy.

The development, maturation, and turnover rate of mouse spleen dendritic cell populations.

Three distinct subtypes of dendritic cells (DC) are present in mouse spleen, separable as CD4(-)8alpha(-), CD4(+)8alpha(-), and CD4(-)8alpha(+) DC. We have tested whether these represent stages of development or activation within one DC lineage, or whether they represent separate DC lineages. All three DC subtypes appear relatively mature by many criteria, but all retain a capacity to phagocytose particulate material in vivo. Although further maturation or activation could be induced by bacterially derived stimuli, phagocytic capacity was retained, and no DC subtype was converted to the other. Continuous elimination of CD4(+)8(-) DC by Ab depletion had no effect on the levels of the other DC subtypes. Bromodeoxyuridine labeling experiments indicated that all three DC subtypes have a rapid turnover (half-life, 1.5-2.9 days) in the spleen, with none being the precursor of another. The three DC subtypes showed different kinetics of development from bone marrow precursors. The CD8alpha(+) spleen DC, apparently the most mature, displayed an extremely rapid turnover based on bromodeoxyuridine uptake and the fastest generation from bone marrow precursors. In conclusion, the three splenic DC subtypes behave as rapidly turning over products of three independent developmental streams.

Protective effect of DNA immunization against mycobacterial infection is associated with the early emergence of interferon-gamma (IFN-gamma)-secreting lymphocytes.

The development of more effective anti-tuberculosis (TB) vaccines would contribute to the global control of TB. Understanding the activated/memory T cell response to mycobacterial infection and identifying immunological correlates of protective immunity will facilitate the design and assessment of new candidate vaccines. Therefore, we investigated the kinetics of the CD4+ T cell response and IFN-gamma production in an intravenous challenge model of Mycobacterium bovis bacille Calmette-Guérin (BCG) before and after DNA immunization. Activated/memory CD4+ T cells, defined as CD44hiCD45RBlo, expanded following infection, peaking at 3-4 weeks, and decreased as the bacterial load fell. Activated/memory CD4+ T cells were the major source of IFN-gamma and the level of antigen-specific IFN-gamma-secreting lymphocytes, detected by ELISPOT, paralleled the changes in bacterial load. To examine the effects of a DNA vaccine, we immunized mice with a plasmid expressing the mycobacterial secreted antigen 85B (Ag85B). This led to a significant reduction in mycobacteria in the liver, spleen and lung. This protective effect was associated with the rapid emergence of antigen-specific IFN-gamma-secreting lymphocytes which were detected earlier, at day 4, and at higher levels than in infected animals immunized with a control vector. This early and increased response of IFN-gamma-secreting T cells may serve as a correlate of protective immunity for anti-TB vaccines.

Protection against virulent Mycobacterium avium infection following DNA vaccination with the 35-kilodalton antigen is accompanied by induction of gamma interferon-secreting CD4(+) T cells.

Mycobacterium avium is an opportunistic pathogen that primarily infects immunocompromised individuals, although the frequency of M. avium infection is also increasing in the immunocompetent population. The antigen repertoire of M. avium varies from that of Mycobacterium tuberculosis, with the immunodominant 35-kDa protein being present in M. avium and Mycobacterium leprae but not in members of the M. tuberculosis complex. Here we show that a DNA vector encoding this M. avium 35-kDa antigen (DNA-35) induces protective immunity against virulent M. avium infection, and this protective effect persists over 14 weeks of infection. In C57BL/6 mice, DNA vaccines expressing the 35-kDa protein as a cytoplasmic or secreted protein, both induced strong T-cell gamma interferon (IFN-gamma) and humoral immune responses. Furthermore, the antibody response was to conformational determinants, confirming that the vector-encoded protein had adopted the native conformation. DNA-35 immunization resulted in an increased activated/memory CD4(+) T-cell response, with an accumulation of CD4(+) CD44(hi) CD45RB(lo) T cells and an increase in antigen-specific IFN-gamma production. The protective effect of the DNA-35 vectors against M. avium infection was comparable to that of vaccination with Mycobacterium bovis BCG and significantly greater than that for previous treated infection with M. avium. These results illustrate the importance of the 35-kDa protein in the protective response to M. avium infection and indicate that DNA vaccination successfully promotes a sustained level of protection during chronic M. avium infection.

Immunoprophylaxis against Mycobacterium leprae infection with subunit vaccines.

We have investigated the effect of subunit vaccines against infection with Mycobacterium leprae, employing DNA plasmids as the vaccine vectors, and the immunodominant 35 kDa protein of M. leprae as the candidate antigen. A DNA vaccine that expresses the M. leprae 35 kDa protein both stimulated interferon-gamma (IFN gamma)-secreting T cells in mice, and demonstrated protection against M. leprae-infection of mice.

Protection against aerosol Mycobacterium tuberculosis infection using Mycobacterium bovis Bacillus Calmette Guérin-infected dendritic cells.

In the lung, dendritic cells (DC) are key antigen-presenting cells capable of triggering specific cellular responses to inhaled pathogens, and thus, they may be important in the initiation of an early response to mycobacterial infections. The ability of DC to enhance antigen presentation to naive T cells within the lungs was characterized with respect to Mycobacterium bovis Bacillus Calmette Guérin (BCG) vaccination against M. tuberculosis infection. In vitro derived DC were infected with BCG, which induced their maturation, as shown by the increased expression of MHC class II antigens, CD80 and CD86 co-stimulatory molecules. The synthesis of mRNA for IL-1, IL-6, IL-12, IL-10 and IL-1 receptor antagonist was also enhanced. When administered intratracheally in mice, infected DC induced a potent T cell response and the production of IFN-gamma to mycobacterial antigens in the mediastinal lymph nodes, leading to a significant protection against aerosol M. tuberculosis infection. Intriguingly, although the vaccination schedule for BCG-infected DC was much shorter than subcutaneous BCG vaccination (7 days as compared to 100 days), both types of vaccination showed similar levels of protection. These data confirm that DC can be potent inducers of a cellular immune response against mycobacteria and support the concept of combining DC strategies with mycobacterial vaccines for protective immunity against tuberculosis.

Co-immunization with DNA vaccines expressing granulocyte-macrophage colony-stimulating factor and mycobacterial secreted proteins enhances T-cell immunity, but not protective efficacy against Mycobacterium tuberculosis.

The development of more effective antituberculosis vaccines would assist in the control of the global problem of infection with Mycobacterium tuberculosis. One recent vaccination strategy is immunization with DNA plasmids encoding individual microbial genes. Using the genes for the M. tuberculosis-secreted proteins, MPT64 (23 000 MW) and Ag85B (30 000 MW) as candidate antigens, we previously prepared DNA vaccines and demonstrated their ability to stimulate T-cell responses and confer protection in a mouse model of aerosol tuberculosis (TB). The protective efficacy of the DNA vaccines was less than that promoted by the current vaccine Mycobacterium bovis bacille Calmette-Guèrin (BCG). To improve the immunogenicity and protective efficacy of these mycobacterial vectors, co-immunization of a plasmid expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) was investigated. Intramuscular immunization with DNA expressing MPT64 or Ag85B and GM-CSF enhanced the antigen-specific cellular immune response, with increased proliferative response and production of interferon-gamma (IFN-gamma). The titre of antimycobacterial protein immunoglobulin G (IgG) antibodies was unchanged. Mice immunized with DNA vaccines showed reduced pulmonary bacterial load following an aerosol challenge of M. tuberculosis, but codelivery of the plasmid expressing GM-CSF did not increase the protective effect. Therefore, despite modifying the cellular immune response to DNA vaccines, GM-CSF does not improve their protective efficacy at the peak of infection after an aerosol challenge with 100 c.f.u. of M. tuberculosis.

Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis.

The development of more-effective antituberculosis vaccines would assist in the control of the global problem of infection with Mycobacterium tuberculosis. One recently devised vaccination strategy is immunization with DNA plasmids encoding individual microbial genes. Using the genes for the M. tuberculosis secreted proteins MPT64 (23 kDa), Ag85B (30 kDa), and ESAT-6 (6 kDa) as candidate antigens, DNA vaccines were prepared and tested for immunogenicity and protective efficacy in a murine model of aerosolized tuberculosis (TB). Intramuscular immunization with DNA-64 or DNA-85B resulted in the activation of CD4(+) T cells, which produce gamma interferon (IFN-gamma), and high titers of specific immunoglobulin G antibodies. Further, DNA-64 induced major histocompatibility complex class I-restricted CD8(+) cytotoxic T cells. The addition of a eukaryotic leader sequence to mpt64 did not significantly increase the T-cell or antibody response. Each of the three DNA vectors stimulated a significant reduction in the level of M. tuberculosis infection in the lungs of mice challenged 4 weeks after immunization, but not to the levels resulting after immunization with Mycobacterium bovis BCG. The vaccines showed a consistent hierarchy of protection, with the most effective being Ag85B, followed by ESAT-6 and then MPT64. Coimmunization with the three vectors resulted in a greater degree of protection than that induced by any single vector. This protective efficacy was associated with the emergence of IFN-gamma-secreting T cells earlier than in infected animals immunized with a control vector. The efficacy of these DNA vaccines suggests that multisubunit vaccination may contribute to future vaccine strategies against TB.