Vaccine potential of PE/PPE peptide conjugated-ESAT-6 proteins against Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) is a highly pathogenic bacterium, which has a unique feature of a thick, lipid-rich cell wall. Many mechanisms are associated with this lipid layer, and which is a very important factor in the pathogenicity of Mtb. In addition to the early secreted antigen target 6 (ESAT-6), a representative vaccine candidate and a typical antigen of type VII secretion systems, the Proline-Glutamate and Proline-Proline-Glutamate (PE/PPE) family proteins are virulence factors involved in the formation of the cell wall of Mtb. Although ESAT-6 is a typical T cell antigen, the immune response induced by it against Mtb is limited due to its N-terminal immunodominant epitope. To overcome this disadvantage, I prepared an ESAT-6 fusion protein conjugated with peptides in the PE/PPE family proteins selected using an IFN-γ response assay. PE/PPE peptides were fused to the front part of ESAT-6 and the vaccine efficacy was compared with that of ESAT-6 subunit vaccine only. Mice immunized with the fused proteins exhibited greater secretion of multifunctional T cell cytokines than those immunized with ESAT-6 only, especially the group immunized with multiple fused peptides (ESAT-6: 0.55-0.92% ± 0.17-0.4, PE/PPE+ESAT-6: 0.55-1.35% ± 0.09-0.4 in lung CD4+ T cells). After challenge with the hyper virulent Mtb Beijing strain HN878, increased secretion of double positive IL-2+/IFN-γ+ cytokines, which are known to be associated with the improvement of tuberculosis treatment and latent tuberculosis infection (LTBI), was observed in the group immunized with the fused proteins (ESAT-6: 15.1% ± 1.3, PE/PPE+ESAT-6: 19.87% ± 2.15, in lung CD4+ T cells). Additionally, secretion of triple positive IFN-γ, TNF-α and IL-2 cytokines, suggested to be the main factors that contribute to protection, was maintained after 15 weeks of infection. Ex vivo stimulation with the PE/PPE peptide resulted in higher secretion of the triple positive T cells in the group immunized with the fusion proteins only, confirming the immunogenicity of the fusion proteins and the peptide-induced immune response. The bacterial burden for the group immunized with the protein fused with one peptide was slightly lower than that for the ESAT-6 group, while the two-peptide immunization group showed a more significant decrease in the spleen. Therefore, the effect of one peptide fusion can be expected to be greater than that of ESAT-6 alone and the PE/PPE peptide fusion protein is anticipated to be sufficient as a BCG booster since PE/PPE exists in BCG. Understanding the immunological properties of PE/PPE proteins and utilizing them effectively will lead to the development of improved vaccine candidates.