Cell envelope protein PPE68 contributes to Mycobacterium tuberculosis RD1 immunogenicity independently of a 10-kilodalton culture filtrate protein and ESAT-6

Abstract

The protective efficacy of Mycobacterium bovis BCG can be markedly augmented by stable integration of Mycobacterium tuberculosis genomic region RD1. BCG complemented with RD1 (BCG::RD1) encodes nine additional proteins. Among them, 10-kDa culture filtrate protein (CFP-10) and ESAT-6 (6-kDa early secreted antigenic target) are low-molecular-weight proteins that induce potent Th1 responses. Using pools of synthetic peptides, we have examined the potential immunogenicity of four other RD1 products (PE35, PPE68, Rv3878, and Rv3879c). PPE68, the protein encoded by rv3873, was the only one to elicit gamma interferon (IFN-gamma)-producing cells in C57BL/6 mice infected with M. tuberculosis. Anti-PPE68 T cells were predominantly raised against an epitope mapped in the N-terminal end of the protein. Importantly, inactivation of rv3873 in BCG::RD1 did not modify CFP-10 and ESAT-6 secretion. Moreover, the generation of IFN-gamma responses to these antigens following immunization with BCG::RD1 was independent of PPE68 expression. Taken together, these results show that PPE68 is an immunogenic product of the RD1 region, which does not interfere with the secretion and immunogenicity of CFP-10 and ESAT-6.

FIG. 1.

(A) Frequency of IFN-γ-secreting cells in the splenocytes of H37Rv-infected mice upon stimulation with peptide pools deriving from PE35, PPE68, Rv3878, and Rv3879c, or in the absence of peptides (Ctrl), as measured by ELISPOT assay. (B) Frequency of IFN-γ-secreting cells in the splenocytes of mice infected with BCG::RD1 or BCG after stimulation with individual peptides deriving from five peptide pools. The activity of peptides from pool Rv3873-2 was also tested on splenocytes from H37Rv-infected mice. Data are representative of three independent ELISPOT experiments.

FIG. 2.

(A) Position of Pep26, Pep27, and Pep3873 in the PPE68 amino acid sequence. The human T-cell epitope in PPE68, as predicted by ProPred, is underlined in Pep3873. Results from sequence alignment of Pep3873 with the PPE proteins of M. tuberculosis using the BLAST program are shown. (B) Diagram comparing the genetic organization of the esx gene clusters containing PPE68, PPE47, PPE46, PPE4, PPE65, PPE27, and PPE25.

FIG. 3.

Frequency of Pep3873-specific IFN-γ-secreting cells in the splenocytes of mice infected with BCG::pYUB412 vector control (BCG), BCG::RD1, Rv3873 ko, or H37Rv, as measured by ELISPOT assay. Cells were prepared 3 weeks postimmunization and restimulated in vitro in presence of Pep3873. Controls include cells cultured in the absence of stimulatory agent (medium) and cells stimulated with PPD or ConA. Error bars, standard deviation.

FIG. 4.

Western blot analysis of the subcellular localization of PPE68 (A) and ESAT-6 (B) in M. tuberculosis H37Rv, BCG::pYUB412 vector control (BCG), BCG::RD1, and Rv3873 ko cultures.

FIG. 5.

(A) Lymphoproliferative response to ESAT-6 in mice infected with BCG::RD1 or Rv3873 ko, compared to BCG::pYUB412-vaccinated animals (BCG). Splenocytes were prepared 3 weeks postimmunization and stimulated in vitro with an ESAT-6 immunodominant peptide (PepESAT), an immunodominant peptide from the Ag85A mycobacterial antigen (PepAg85A), or PPD. Control cells incubated with an immunodominant peptide from an irrelevant recombinant protein (PepMalE) were included. (B) IFN-γ production, as measured by enzyme-linked immunosorbent assay, by splenocytes stimulated with PepESAT, PepAg85A, and PepMalE or with recombinant ESAT-6, CFP-10, and MalE proteins. Control cells stimulated with ConA or PPD or in the absence of stimulatory agent (medium) were included. Data are representative of three independent experiments (error bars, standard deviations).