Immunization with a DNA vaccine cocktail protects mice lacking CD4 cells against an aerogenic infection with Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is the most common opportunistic disease and a potentially fatal complication among immunocompromised individuals infected with human immunodeficiency virus (HIV). Effective vaccination against TB in persons with HIV has been considered unlikely because of the central role that CD4 cells play in controlling tuberculous infections. Here we show that the vaccination of CD8(-/-) mice with a TB DNA vaccine cocktail did not significantly enhance protective responses to a Mycobacterium tuberculosis infection. In contrast, immunization with a DNA vaccine cocktail or with the current TB vaccine, Mycobacterium bovis BCG, induced considerable antituberculosis protective immunity in immune-deficient mice lacking CD4 cells. In vaccinated CD4(-/-) animals, substantially reduced bacterial burdens in organs and much improved lung pathology were seen 1 month after an aerogenic M. tuberculosis challenge. Importantly, the postchallenge mean times to death of vaccinated CD4(-/-) mice were significantly extended (mean with DNA cocktail, 172 +/- 7 days; mean with BCG, 156 +/- 22 days) compared to that of naïve CD4(-/-) mice (33 +/- 6 days). Furthermore, the treatment of DNA-vaccinated CD4(-/-) mice with an anti-CD8 or anti-gamma interferon (IFN-gamma) antibody significantly reduced the effect of immunization, and neither IFN-gamma(-/-) nor tumor necrosis factor receptor-deficient mice were protected by DNA immunization; therefore, the primary vaccine-induced protective mechanism in these immune-deficient mice likely involves the secretion of cytokines from activated CD8 cells. The substantial CD8-mediated protective immunity that was generated in the absence of CD4 cells suggests that it may be possible to develop effective TB vaccines for use in HIV-infected populations.

FIG. 1.

CD8⁺ and CD4⁺ T cells from vaccinated mice produced significantly more IFN-γ than cells from naïve controls. Fourteen days after a low-dose aerosol infection with M. tuberculosis Erdman, splenic CD8 (left) and CD4 (right) cells from TB DNA cocktail-vaccinated or naïve WT C57BL/6 mice were isolated by magnetic cell sorting and then were restimulated in vitro with M. tuberculosis-infected bone marrow-derived macrophages. The amounts of IFN-γ that were secreted in these ex vivo cultures were determined by enzyme-linked immunosorbent assays.

FIG. 2.

Vaccination with either BCG or the DNA cocktail resulted in a significant decline in lung and spleen bacterial burdens in CD4^−/− mice (left) and WT mice (right). Organ bacterial CFU values were assessed 28 days after a low-dose aerosol challenge with M. tuberculosis Erdman. The vaccine-induced reduction in bacterial burden is represented as the difference between the CFU values (log₁₀) of naïve controls and immunized mice. For this representative experiment, the CFU values (log₁₀) for the CD4^−/− mice were 8.6 (lung) and 6.6 (spleen) 28 days after the aerogenic infection. For the WT mice, the lung and spleen CFU values were 6.8 and 5.6, respectively.

FIG. 3.

Vaccination of CD4^−/− mice with either BCG or the TB DNA cocktail resulted in a substantial improvement in lung histopathology and highly reduced numbers of acid-fast bacilli compared to naïve CD4^−/− controls. Lung sections were fixed and stained with either hematoxylin and eosin (a, b, and c) or Ziehl-Neelsen reagent (d, e, and f) 28 days postchallenge. (a) Numerous necrotic, suppurative lesions containing many neutrophils and overall severe and acute coalescing inflammation with little functional tissue were seen in nonvaccinated CD4^−/− mice. (d) An overwhelming number of acid-fast bacilli were also detected in the naïve controls. In contrast, lung sections from TB DNA cocktail (b and e)- and BCG (c and f)-vaccinated mice exhibited subacute lymphocytic granulomatous bronchopneumonia with no necrosis, and substantial amounts of intact functional tissue with few acid-fast bacilli were observed.

FIG. 4.

Kaplan-Meier plot of survival data. The plot shows that vaccination with BCG (▴) or the TB DNA cocktail (•) dramatically extended the survival time of CD4^−/− mice after a low-dose aerogenic challenge with M. tuberculosis Erdman relative to that of naïve controls (▪).

FIG. 5.

Treatment of DNA-vaccinated CD4^−/− mice with anti-CD8 or anti-Thy-1.2 antibody resulted in a significant reduction in lung CFU compared to nontreated CD4^−/− vaccinated mice. CD4^−/− mice vaccinated with the TB DNA cocktail were treated with 0.2 mg of anti-CD8 or anti-Thy-1.2 monoclonal antibody i.p. 2 days before, the day of, and twice per week after an aerogenic infection with M. tuberculosis Erdman. The protective response is represented as the reduction in CFU values (log₁₀) for antibody-treated relative to untreated CD4^−/− mice.