The Apa protein of Mycobacterium tuberculosis stimulates gamma interferon-secreting CD4+ and CD8+ T cells from purified protein derivative-positive individuals and affords protection in a guinea pig model

Abstract

The search to identify Mycobacterium tuberculosis antigens capable of conferring protective immunity against tuberculosis has received a boost owing to the resurgence of tuberculosis over the past two decades. It has long been recognized that lymphoid cells are required for protection against M. tuberculosis. While traditionally the CD4(+) populations of T cells were believed to predominantly serve this protective function, a pivotal role for CD8(+) T cells in this task has been increasingly appreciated. We show that the 50- to 55-kDa Apa protein, specified by the Rv1860 gene of M. tuberculosis, can elicit both lymphoproliferative response and gamma interferon (IFN-gamma) production from peripheral blood mononuclear cells (PBMC) of purified protein derivative (PPD)-positive individuals, with significant differences recorded in the levels of responsiveness between PPD-positive healthy controls and pulmonary tuberculosis patients. Flow cytometric analysis of whole blood stimulated with the recombinant Apa protein revealed a sizeable proportion of CD8(+) T cells in addition to CD4(+) T cells contributing to IFN-gamma secretion. PBMC responding to the Apa protein produced no interleukin-4, revealing a Th1 phenotype. A DNA vaccine and a poxvirus recombinant expressing the Apa protein were constructed and tested for their ability to protect immunized guinea pigs against a challenge dose of virulent M. tuberculosis. Although the DNA vaccine afforded little protection, the poxvirus recombinant boost after DNA vaccine priming conferred a significant level of protective immunity, bringing about a considerable reduction in mycobacterial counts from the challenge bacilli in spleens of immunized guinea pigs, a result comparable to that achieved by BCG vaccination.

FIG. 1.

Purification of recombinant Apa protein. The insert from ARRMTB65 was cloned into pRSETC as described in Materials and Methods. Lanes: 1, IPTG-induced lysate from E. coli BL21(DE3) carrying the pRSETC-65 recombinant after induction with IPTG; 2, Apa protein from lane 1 FPLC-purified by using Ni²⁺-NTA agarose.

FIG. 2.

Human PBMC respond to recombinant Apa protein by lymphoproliferation and IFN-γ production. (A) The SI values plotted for 30 PPD-positive healthy controls and 22 pulmonary TB patients were derived as a ratio of the counts incorporated by PBMC in the presence of Apa antigen to that observed with unstimulated cells in triplicate cultures. (B, C, and D) IFN-γ production by PBMC from the same group of volunteers shown in panel A in response to the Apa protein, total M. tuberculosis sonicate, and the mitogen PHA, respectively. The horizontal lines specify the mean SI or IFN-γ values observed in each group. P values for significance are shown for each data set. P values of <0.05 are significant. Evidence of age-sex interaction with SI or IFN-γ response to the stimulants within the study groups, treated separately or in concert, did not reach statistical significance (P > 0.05).

FIG. 3.

Flow cytometric detection of intracellular IFN-γ in Apa-stimulated (A) and M. tuberculosis sonicate-stimulated (B) T cells. Whole blood was stimulated with recombinant Apa protein of M. tuberculosis or M. tuberculosis sonicate for 6 h, fixed, and stained for CD3 (fluorescein isothiocyanate), CD8 (phycoerythrin), and IFN-γ (Cychrome). Cells were gated sequentially on lymphocytes, followed by CD3, and analyzed for expression of CD8 and IFN-γ. The frequencies in the upper quadrants are IFN-γ-producing cells as percentages of the total CD4⁺ (left quadrants) or CD8⁺ (right quadrants) T cells.

FIG. 4.

Animals immunized with Apa-expressing DNA, followed by a poxvirus boost, are protected against challenge with M. tuberculosis. (A) Animals immunized with APADNA/APAMVA have significantly fewer M. tuberculosis bacilli in the lungs than control animals. Challenged animals were sacrificed at the end of the 6-week observation period, and the CFU of M. tuberculosis in the spleen were enumerated. The data are given as the CFU per spleen for each animal in the groups. P values for significance between the mean CFU values for each group are shown. P values of <0.05 are significant. The lower limit of detection was 50 bacilli per organ (one CFU on a plate seeded with 100 μl of an undiluted 1% sample [5 ml] of a spleen). (B) Weekly weights of animals for 6 weeks after challenge with M. tuberculosis by the intramuscular route in the immunization groups described. Weights are plotted against time in weeks for each animal. Symbols: ▪, DNA/MVA; •, DNAAPA/MVA; ▴, DNAAPA/MVAAPA; ⧫, BCG.