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

Abstract

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.

Figure 1

The effect of immunization with DNA-64 and pGM-CSF on T-cell proliferation. Proliferative responses of spleen-derived lymphocytes from mice immunized imi with 100 μg DNA-64, co-immunized with pGM-CSF, or the empty vector. Mice were immunized once or three times at three week intervals. The incorporation of ³H-thymidine (Δc.p.m.±SEM) in response to 10 μg/ml MPT64 was measured as described in the materials and methods. The statistical significance of the differences in the proliferative response was determined by Student's t-test, *P = 0·002, **P = 0·007.

Figure 2

The effect of immunization with DNA-85B and pGM-CSF on T-cell proliferation. Proliferative responses of spleen-derived lymphocytes from mice immunized three times imi, with 100 μg DNA-85B alone (♦), co-immunized with pGM-CSF (•), or the empty vector (▴). The incorporation of ³H-thymidine (Δc.p.m.±SEM) in response to Ag85B was measured as described in the materials and methods. The statistical significance of differences in the proliferative responses was determined by Student's t-test, *P = 0·001, **P = 0·01.

Figure 3

The effect of codelivery of a vector expressing GM-CSF on the protective efficacy of DNA vaccines against challenge with M. tuberculosis. Mice were immunized (100 μg imi) with a combination of DNA-64 and DNA-85B, with or without pGM-CSF, three times, every three weeks. As positive control, mice were given a subcutaneous immunization with BCG (5 × 10⁴ c.f.u.), and the empty vector was used as the negative control. Four weeks later, mice were challenged with aerosol-delivered M. tuberculosis H37Rv (100 c.f.u.). Four weeks postinfection, the bacteria in the lung were enumerated (±SEM), as described in the material and methods. The statistical significance of differences compared to empty vector were determined by the Mann–Whitney test (n = 5, *P < 0·009). There was no statistically significant difference between DNA vaccines with or without pGM-CSF.