An unbiased peptide-wide discovery approach to select Mycobacterium tuberculosis antigens that target CD8+ T cell response during infection

Abstract

Accruing data strongly support the possible role of CD8+ T cells in immunity against tuberculosis (TB). Multivalent vaccines against Mycobacterium tuberculosis (Mtb) that incorporate CD8+ T cell antigens with those that elicit CD4+ T cells are therefore highly desirable. To screen for potential CD8+ T cell antigens that are produced by Mtb during infection, we isolated pathogen-derived peptides that bound to MHC Class I molecules expressed in adherent splenocytes obtained from Mtb-infected mice. Mass spectroscopy analysis revealed the following four nonamer peptides that had 100% homology with Mtb proteins: DGYVGAPAH (MT_0401), TTMPLFAD (MT_1164), RSGAATPVR (MT_2160.1) and LAAVVGVVL (MT_0078). The gene MT_0401 codes the protein 5'-phosphoribosylglycinamide transformylase 2 and the other three genes code for hypothetical proteins with unknown function. The NCBI/Blast analysis showed that among the four peptides DGYVGAPAH had the highest maximum alignment score and lowest E value (number of alignments expected by chance). Therefore, we assessed whether MT_0401 expressed in two genetic vaccine formulations was capable of stimulating CD8+ T cell response that is specific to DGYVGAPAH peptide. When mice were immunized with a recombinant plasmid DNA and an E1/E3-deleted Adenovirus 5 expressing MT0401 protein, using both homologous and heterologous prime-boost protocols, they developed strong DGYVGAPAH-specific CD8+ T cell response as well as antibody and CD4+ specific T cell response to the full length MT0401 protein. Equally important was the observation that mice infected with Mtb developed DGYVGAPAH-specific CD8+ T cell responses in both spleen and lungs. These results demonstrate that Mtb antigens that are processed and presented via MHC Class I machinery can be readily identified by the described approach and may be useful candidate antigens to stimulate specific CD8+ T cell responses in vaccine development programs.

Keywords: CD8 associated antigen; Immunogenicity; MT0401; Tuberculosis.

Figure 1. Expression of MT0401 in recombinant DNA and recombinant Ad5 vaccine vectors

293A cells were transfected with 50 μg of DNA [DNA sham (DNA, lane 1) or DNA-MT0401 (DNA, lane 2)] or infected with 10⁹ IFU rAd5 [rAd5 sham (Ad5, lane 1) or rAd5-MT0401 (Ad5, lane 2)]. The lysates of the DNA-transfected or rAd5-infected cell were resolved in 4–20% SDS-PAGE gels and transferred to PVDF membrane. Presence of MT0401 on the membrane was determined using rabbit anti-rMT0401 followed by goat anti-rabbit IgG labeled with horseradish peroxidase. Bound conjugates were detected using ECL enhanced chemiluminescence system and proteins were visualized by autoradiography. Arrow points to the bands of ~45kDa that is recognized by the specific anti-rMT0401 on lanes DNA 2, Ad5 2 as well as in lane R (purified recombinant MT0401 from E. coli). Note that the migration of the purified recombinant protein is slightly higher than the molecular mass of the molecules produced by the 293A cells. This minor difference is expected because of the addition of the His tag sequence and a thrombin site added to the recombinant protein to facilitate its purification. The rMT0401 antigen produced by the 293A cells do not contain the His tag sequence and thrombin site.

Figure 2. Recognition of DGYVGAPAH and rMT0401 by CD8+ and CD4+ T cells from spleens of genetically immunized mice

C57BL/6 mice were either immunized with saline (A) or immunized i.m. with DNA-MT0401 (40μg) three times (4 weeks interval) (B), or immunized once with Ad5-MT0401 (5×10⁸ PFU) (C). In D, mice were primed with DNA-MT_0401 (40ug - 2x, four weeks interval) followed by one boost with Ad5-MT_0401 (5×10⁸ PFU). Four weeks after the last immunization mice were sacrificed and spleens were removed. Mononuclear cell suspensions were prepared and stimulated for six hours with either medium, the peptide DGYVGAPAH, or with rMT0401 protein. Production of IFN-γ by CD8+ T cells (cultures stimulated with DGYVGAPAH) and by CD4+ T cells (cultures stimulated with rMT0401) was assayed by intra-cellular cytokine staining. Results are expressed as the percentage of IFN-γ positive CD8+ or CD4+ T cells. This is a representative experiment of three experiments with the same results.

Figure 3. Recognition of DGYVGAPAH and rMT0401 by CD8+ and CD4+ T cells from lungs of genetically immunized mice

C57BL/6 mice were either immunized with saline (A) or immunized i.m. with DNA-MT0401 (40μg) three times (4 weeks interval) (B), or immunized once with Ad5-MT0401 (5×10⁸ PFU) (C). In D, mice were primed with DNA-MT_0401 (40ug - 2x, four weeks interval) followed by one boost with Ad5-MT_0401 (5×10⁸ PFU). Four weeks after the last immunization mice were sacrificed and lungs were removed. Mononuclear cell suspensions were prepared and stimulated for six hours with either medium, the peptide DGYVGAPAH, or with rMT0401 protein. Production of IFN-γ by CD8+ T cells (cultures stimulated with DGYVGAPAH) and by CD4+ T cells (cultures stimulated with rMT0401) was assayed by intra-cellular cytokine staining. Results are expressed as the percentage of IFN-γ positive CD8+ or CD4+ T cells. This is a representative experiment of three experiments with the same results.

Figure 4. Recognition of DGYVGAPAH and rMT0401 by CD8+ and CD4+ T cells from mice infected with M. tuberculosis

C57BL/6 mice were infected i.v. with 2×10⁶ CFU of M. tuberculosis strain 18b. Six weeks after infection mice were sacrificed and both spleen and lungs were removed. Mononuclear cell suspensions were prepared from these organs and stimulated for six hours with either medium, the peptide DGYVGAPAH, or with rMT0401 protein. Production of IFN-γ by CD8+ T cells (cultures stimulated with DGYVGAPAH) and by CD4+ T cells (cultures stimulated with rMT0401) was assayed by intra-cellular cytokine staining. Results are expressed as the percentage of IFN-γ positive CD8+ or CD4+ T cells. This is a representative experiment of two experiments with the same results.