The protective effect of the Mycobacterium bovis BCG vaccine is increased by coadministration with the Mycobacterium tuberculosis 72-kilodalton fusion polyprotein Mtb72F in M. tuberculosis-infected guinea pigs

Abstract

A tuberculosis vaccine candidate consisting of a 72-kDa polyprotein or fusion protein based upon the Mtb32 and Mtb39 antigens of Mycobacterium tuberculosis and designated Mtb72F was tested for its protective capacity as a potential adjunct to the Mycobacterium bovis BCG vaccine in the mouse and guinea pig models of this disease. Formulation of recombinant Mtb72F (rMtb72F) in an AS02A adjuvant enhanced the Th1 response to BCG in mice but did not further reduce the bacterial load in the lungs after aerosol challenge infection. In the more stringent guinea pig disease model, rMtb72F delivered by coadministration with BCG vaccination significantly improved the survival of these animals compared to BCG alone, with some animals still alive and healthy in their appearance at >100 weeks post-aerosol challenge. A similar trend was observed with guinea pigs in which BCG vaccination was boosted by DNA vaccination, although this increase was not statistically significant due to excellent protection conferred by BCG alone. Histological examination of the lungs of test animals indicated that while BCG controls eventually died from overwhelming lung consolidation, the majority of guinea pigs receiving BCG mixed with rMtb72F or boosted twice with Mtb72F DNA had mostly clear lungs with minimal granulomatous lesions. Lesions were still prominent in guinea pigs receiving BCG and the Mtb72F DNA boost, but there was considerable evidence of lesion healing and airway remodeling and reestablishment. These data support the hypothesis that the coadministration or boosting of BCG vaccination with Mtb72F may limit the lung consolidation seen with BCG alone and may promote lesion resolution and healing. Collectively, these data suggest that enhancing BCG is a valid vaccination strategy for tuberculosis that is worthy of clinical evaluation.

FIG. 1.

(A) Spleen cell secretion of IFN-γ following stimulation with Mtb72F. Mice were immunized with BCG or BCG boosted with Mtb72F in an AS02A formulation. Data shown are from pooled cells from three mice. PPD, purified protein derivative. (B) Elispot analysis of immunized mice, vaccinated as described above. (C) CTL responses to Mtb32c. Harvested spleen cells were tested for cytolytic activity against EL4 cells expressing Mtb32c. Data shown are the individual responses of three mice against EL4 expressing Mtb32c (filled symbols) or EL4 cell controls (open symbols). E:T ratio, effector-to-target cell ratio. (D) Serum antibody responses to Mtb72F. Open symbols show immunoglobulin G1 antibody titers, and closed symbols show immunoglobulin G2a responses. O.D., optical density.

FIG. 2.

Expression of protective immunity in the lung conferred by vaccination. Both BCG and BCG boosted with Mtb72F-AS02A significantly reduced bacterial loads in the lung (P < 0.01), but the vaccine groups were not themselves significantly different. Data shown are means ± standard errors of the means (n = 5 mice) for animals sacrificed 30 days after aerosol challenge.

FIG. 3.

Kaplan-Meier survival curves (n = 5) showing that covaccination of guinea pigs with BCG mixed with Mtb72F protein substantially increased survival after low-dose aerosol challenge with M. tuberculosis.

FIG. 4.

Histologic appearance of lung tissues harvested from guinea pigs vaccinated with either BCG alone or with BCG plus Mtb72F. (A) Saline control animal showing extensive tissue destruction and necrosis (week 20). (B) Typical appearance of BCG control animals showing extensive consolidation of the lung by lymphocytic granulomatous tissue formation (week 42). (C to F) Representative sections from surviving animals vaccinated with BCG plus Mtb72F. There are occasional small areas of granulomatous tissue and fibrosis; most of the lung fields are clear.

FIG. 5.

Representative light photomicrographs of lungs from animals dying despite Mtb72F boosting. Extensive multifocal to coalescing granulomatous inflammation effaces the majority of pulmonary parenchyma (A). A higher magnification of these affected areas (rectangle in panel A) shows marked thickening of the interalveolar septa with infiltrates of lymphocytes and macrophages, fewer neutrophils, and cellular debris (arrow) that fill remnant alveoli (B). The sections were stained with hematoxylin and eosin.

FIG. 6.

Kaplan-Meier survival curves (n = 5) comparing the survival of BCG controls versus guinea pigs vaccinated with BCG and boosted twice with DNA encoding Mtb72F.

FIG. 7.

Histologic appearance of lung tissues harvested from guinea pigs vaccinated with BCG alone or vaccinated with BCG and boosted twice with Mtb72F DNA. (A) Saline control guinea pig euthanized at week 17 showing extensive coalescing granulomatous inflammation with a central focus of necrosis that is undergoing mineralization (arrow). (B) DNA vector control animal (week 30) with region (arrow) of central coagulative necrosis. (C) BCG-vaccinated guinea pig euthanized at week 79. A large granulomatous structure has degenerated with central necrosis (arrow) but with no apparent mineralization. (D) Covaccinated guinea pig surviving to curtailment of the study. Lesions in animals from this group had less-extensive mixed inflammation than animals from other groups, with extensive fibrous connective tissue deposition and small airway reestablishment (arrow), indicating lesion resolution. Bars, 100 μm.

FIG. 8.

Further illustration of airway reestablishment. Light photomicrographs from guinea pigs surviving to the end of the experiment are shown. There are extensive foci of resolving granulomatous inflammation in which highly branched and regenerating bronchioles appear (thick arrow) (A). These airways are supported by fibrous connective tissue (thin arrow) that effaces the pulmonary parenchyma (B). Higher magnifications of the affected area (C and D) show regenerating bronchioles and alveoli lined by a hyperplastic epithelium (arrowheads) and increased fibrous connective tissue (D), with minimal residual inflammation. The sections shown in panels A and C were stained with hematoxylin and eosin, and the sections shown in panels B and D were stained with Masson's trichrome.