Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's Disease in ruminants such as cattle, sheep, and goats [[1], [2], [3], [4]]. The disease symptoms usually appear 2–5 years after infection, and the clinical presentations include diarrhea, progressive weight loss, and decreased milk production in cattle [2]. The organism is a slow-growing intracellular bacterium that causes general deterioration of health and productivity, leading to tremendous economic losses in the dairy industry worldwide [5]. MAP is typically transmitted through the fecal-oral route by contacting contaminated materials or surfaces, but studies show MAP might also be transferred by body fluid or aerosols [6,7]. The major problem in the control of Johne's disease is the limited reliability of detection methods of infected animals without clinical signs, resulting in difficulty in eliminating MAP from the environment. Moreover, several studies point out a possible correlation between MAP and Crohn's disease in humans, increasing public health concerns about this potential zoonotic threat [8,9].

Johne's disease is not limited to the conditions that exist within a single animal but can adapt to changing conditions among different hosts. In order to facilitate transmission, MAP has developed an effective infection mechanism for entry into intestinal epithelial cells within 30 min of contact [10]. After penetrating the intestinal epithelial barrier, MAP invades and persistently resides in macrophage phagosome by inhibiting phagosome acidification and phagolysosome fusion [11]. Mycobacteria also disturb the maturation of endosomes through the MAPK-p38 signaling pathway by Toll-like receptor 2 (TLR2), which is activated by mannosylated lipoarabinomannan (Man-LAM) on the MAP cell wall [12,13]. TLR2 has been identified as one of the principal mediators of macrophage activation in response to mycobacterial cell wall lipoproteins [14]. Membrane vesicles (MVs), which are naturally released from Gram-positive bacteria, carry a variety of proteins from bacterial surfaces, periplasm, and cytosol. Distinct from the well-known outer membrane vesicles (OMVs) from Gram-negative bacteria, MVs are budded from the thick cell wall and are populated with different cell wall components, which vary depending on the surrounding environment. In addition, Rafael Prados-Rosales et al. demonstrated that MVs derived from M. bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis could trigger inflammatory responses in a TLR-2 dependent manner, indicating the roles of MVs in modulating host immune responses and Mycobacterium pathogenesis [15]. Moreover, exosomes produced from M. tuberculosis-infected macrophages inhibited activation of CD4+ T cells as well as reduced production of IL-2, which reduced T cell proliferation, illustrating the complex mechanisms involved in host-pathogen interaction [16].

The most effective way to control this disease is by using a vaccine. Heat-killed vaccines have been used to eradicate MAP from livestock [17,18]. However, heat-killed vaccines do not elicit sterile immunity [19]. Efforts have been made to develop a subunit vaccine against MAP [19,20]. Modern immunoinformatics has revolutionized the study of immunology and the design of vaccines. A multi-epitope-based vaccine development strategy is a progressive approach for targeting multiple diseases associated with bacteria, viruses, and tumors [[21], [22], [23]]. Some of the multi-epitope vaccines have entered clinical trials [21]. The subunit/multi-epitope-based vaccine is generally considered safe as compared to live attenuated vaccine [24]. Also, the cost of developing the MEV is relatively low compared to traditional vaccines. However, a multi-epitope-based vaccine against MAP designed using an in-silico approach has not been reported.

In this study, we successfully collected MVs from MAP and analyzed their protein composition. Secretory proteins with high antigenicity and adhesion capability with no homology with bovine proteome are selected for epitope prediction. Screened B-cell and T-cell epitopes from 6 vaccine candidates are stitched together using a linker to form a multi-epitope vaccine. The MEV was docked with bovine TLR2 to validate its efficacy in stimulating the innate immune response. In addition, we also demonstrated that OMV could increase pro-inflammatory cytokine expression in bovine-derived macrophages, contributing to the virulence and pathogenesis of MAP infection.