Pulmonary necrosis resulting from DNA vaccination against tuberculosis

Abstract

The use of DNA constructs encoding mycobacterial proteins is a promising new approach to vaccination against tuberculosis. A DNA vaccine encoding the hsp60 molecule of Mycobacterium leprae has previously been shown to protect against intravenous infection of mice with Mycobacterium tuberculosis in both the prophylactic and immunotherapeutic modes. It is shown here, however, that this vaccine was not effective in a more realistic aerosol infection model or in a model of latent tuberculosis in the lungs. Moreover, when given in an immunotherapeutic model the immunized mice developed classical Koch reactions characterized by multifocal discrete regions of cellular necrosis throughout the lung granulomas. Similar and equally severe reactions were seen in mice given a vaccine with DNA coding for the Ag85 antigen of M. tuberculosis. This previously unanticipated safety problem indicates that DNA vaccines should be used with caution in individuals who may have already been exposed to tuberculosis.

FIG. 1.

Prophylactic vaccination in C57BL/6 mice. Animals were given DNA or control vector DNA three times and then rested for 1 month prior to low-dose aerosol challenge with M. tuberculosis H37Rv. Controls received injections of saline or a single dose of BCG. Data shown are the mean numbers of bacteria recovered from the lungs 30 days after aerosol exposure (n = 5) (error bars, standard errors of the means). Only mice receiving BCG were protected against the challenge infection (P > 0.01).

FIG. 2.

Photomicrographs of lungs of C57BL/6 mice harvested 30 days (A and B) or 80 days (C and D) after aerosol exposure to M. tuberculosis. Mice received prophylactic vaccination with hsp60/lep (A and C) or saline (B and D). Alveoli contain large numbers of macrophages and lower numbers of lymphocytes, but there is no appreciable neutrophil infiltration or necrosis. At day 80 perivascular accumulations of lymphocytes are seen; these were particularly prominent in the hsp60/lep vaccinated animals.

FIG. 3.

Effect of hsp60/lep vaccine in the modified Cornell model. Four weeks after exposure to low-dose aerosol challenge with M. tuberculosis H37Rv, the bacterial load was assessed (day 30 counts), and then the remaining mice were treated with isoniazid for 60 days, after which the reduction in bacterial load was assessed (Post-INH). Mice were then given three vaccinations with the hsp60/lep DNA or control injections. Thirty days after the final vaccination the bacterial load was determined, and it was then determined again after 8 weeks of treatment with dexamethasone. In each case the mean value is shown (n = 5) (error bars, standard errors of the means).

FIG. 4.

Representative histologic appearance of lungs from BALB/c mice given immunotherapeutic vaccination with hsp60/lep. (A) In mice given saline only small granulomatous foci were seen in the lungs of these animals 130 days after low-dose aerosol challenge with M. tuberculosis H37Rv. Perivascular aggregates of lymphocytes can be seen extending into the adjacent parenchyma, which is variably surrounded by macrophages. Size bar = 100 μm. (B) In mice given hsp60/lep DNA the lesions are very large (size bar = 100 μm) and show evidence of extensive tissue destruction. There is a sharp line of demarcation with normal lung parenchyma, and alveoli are filled with infiltrates of mononuclear cells and some neutrophils. There is loss of alveolar wall detail, with multifocal necrosis. (C) At a higher power of magnification (size bar = 10 μm), these multiple necrotic foci are characterized by fibrillar acidophilic cellular debris with clear clefts that represent extracellular cytoplasmic lipid deposition (arrows). These foci contain a mixture of normal to completely degenerate macrophages. (D) Acid-fast staining revealed multiple bacilli (example indicated with an arrow) visible within the cytoplasm of foamy macrophages as well as in the extracellular space of necrotic foci. Such bacilli were only rarely seen in lesions of control mice at this stage of the infection.

FIG. 5.

Immunotherapeutic vaccination in C57BL/6 mice. Animals were given three injections of DNA, vector DNA control, or BCG on days 60, 81, and 102 after exposure to low-dose aerosol challenge with M. tuberculosis H37Rv. The data shown are the mean numbers of bacteria recovered from the lungs after a further 30 days (n = 5) (error bars, standard errors of the means). No protection was seen in any groups at this time, or after a further seventy days (data not shown).

FIG. 6.

Histologic appearance of lungs of C57BL/6 mice 30 days after the third immunotherapeutic vaccination with hsp60/lep. (A) Alveoli and interalveolar septae are effaced by infiltrates of macrophages, some of which have abundant foamy cytoplasm. There are random foci of necrosis containing karyorhectic and cellular debris with intralesional aggregates of neutrophils (indicated by an arrow). Mice given saline (B) or DNA vector control (C) have prominent perivascular accumulations of lymphocytes and intra-alveolar macrophages but no necrosis or neutrophilic infiltration.

FIG. 7.

Histologic appearance of lungs one hundred days after immunotherapeutic vaccination with hsp60/lep DNA. Foci of necrosis contain debris (small arrows) and clefts of accumulated cholesterol (large arrows). Alveoli contain macrophages with abundant foamy cytoplasm, and aggregates of lymphocytes are prominent. Overall, the appearances of lungs from vaccinated (A) and saline controls (B) were similar.