Comparison of the Immunogenic Properties of Lactiplantibacillus plantarum Carrying the Mycobacterial Ag85B-ESAT-6 Antigen at Various Cellular Localizations

Abstract

The bacille Calmette-Guèrin (BCG) vaccine has been used for a century; nonetheless, tuberculosis (TB) remains one of the deadliest diseases in the world. Thus, new approaches to developing a new, more efficient vaccine are desirable. Mucosal vaccines are of particular interest, considering that Mycobacterium tuberculosis first enters the body through the mucosal membranes. We have previously demonstrated the immunogenicity of a recombinant Lactiplantibacillus plantarum delivery vector with TB hybrid antigen Ag85B-ESAT-6 anchored to the cell membrane. The goal of the present study was to analyze the impact of antigen localization in the immune response. Thus, we assessed two novel vaccine candidates, with the TB antigen either non-covalently anchored to the cell wall (LysMAgE6) or located intracellularly (CytAgE6). In addition, we compared two expression systems, using an inducible (LipoAgE6) or a constitutive promoter (cLipoAgE6) for expression of covalently anchored antigen to the cell membrane. Following administration to mice, antigen-specific CD4+ T-cell proliferation and IFN-γ and IL-17A secretion were analyzed for lung cell and splenocyte populations. Generally, the immune response in lung cells was stronger compared to splenocytes. The analyses showed that the type of expression system did not significantly affect the immunogenicity, while various antigen localizations resulted in markedly different responses. The immune response was considerably stronger for the surface-displaying candidate strains compared to the candidate with an intracellular antigen. These findings emphasize the significance of antigen exposure and further support the potential of L. plantarum as a mucosal vaccine delivery vehicle in the fight against TB.

Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark

“…Western blot analysis was performed to investigate protein production levels at the various expression conditions. E. coli and L. plantarum harboring expression plasmids were lysed and applied to a SDS–PAGE as previously described 31 , with minor adjustments. Briefly, harvested cells were resuspended in 250 µl NP-40 buffer (150 mM NaCl; 1.0% Triton X-100; 50 mM Tris, pH 8.0) containing 1 mM phenylmethylsulfonyl fluoride (PMFS) and lysed using a FastPrep FP120 Cell Disrupter (MP Biomedicals, Santa Ana, CA).…”

“…Other lactic acid bacteria, such as Lactiplantibacillus plantarum , also show significant potential for the overproduction of heterologous proteins 25 , 26 . Several inducible and constitutive expression systems are compatible with lactobacillal s pecies, including the pheromone inducible pSIP vectors, which have been extensively used for the expression, secretion, and surface anchoring of a range of heterologous proteins 25 , 27 – 31 . The pSIP system is based on the promotors and regulatory genes involved in the production of the class-II bacteriocin sakacin P in Latilactobacillus sakei.…”

The extracellular domain of site-2-metalloprotease RseP is important for sensitivity to bacteriocin EntK1

“…pEV, EfmRseP and the N359A mutant under inducing (+) and non-inducing (-) conditions. Cells were harvested, lysed and subjected to SDS-PAGE as previously described ( 50 ), with minor modifications. The harvested cells were resuspended in 250 μl NP-40 lysis buffer containing 1 mM PMSF (phenylmethylsulfonyl fluoride).…”

The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System

“…Surface display vaccines can increase the immunogenicity of foreign antigens by facilitating their recognition by the immune system. For example, the surface display of antigens derived from Mycobacterium tuberculosis on Salmonella typhimurium [1] or Lactiplantibacillus plantarum [2] evokes a stronger immune response compared to that with cytoplasmic delivery. Surface display vaccines also have advantages such as their ease of use and low manufacturing costs.…”