Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis complex imposing a high zoonotic threat to human health. The limited efficacy of BCG (Bacillus Calmette–Guérin) and upsurges of drug-resistant tuberculosis require new effective vaccination approaches and anti-TB drugs. Poly (lactic-co-glycolic acid) (PLGA) is a preferential drug delivery system candidate. In this study, we formulated PLGA nanoparticles (NPs) encapsulating the recombinant protein bovine neutrophil β-defensin-5 (B5), and investigated its role in immunomodulation and antimicrobial activity against M. bovis challenge. Using the classical water–oil–water solvent-evaporation method, B5-NPs were prepared, with encapsulation efficiency of 85.5% ± 2.5%. These spherical NPs were 206.6 ± 26.6 nm in diameter, with a negatively charged surface (ζ-potential −27.1 ± 1.5 mV). The encapsulated B5 protein from B5-NPs was released slowly under physiological conditions. B5 or B5-NPs efficiently enhanced the secretion of tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-10 in J774A.1 macrophages. B5-NPs-immunized mice showed significant increases in the production of TNF-α and immunoglobulin A (IgA) in serum, and the proportion of CD4+ T cells in spleen compared with B5 alone. In immunoprotection studies, B5-NPs-immunized mice displayed significant reductions in pulmonary inflammatory area, bacterial burden in the lungs and spleen at 4-week after M. bovis challenge. In treatment studies, B5, but not B5-NPs, assisted rifampicin (RIF) with inhibition of bacterial replication in the lungs and spleen. Moreover, B5 alone also significantly reduced the bacterial load in the lungs and spleen. Altogether, our findings highlight the significance of the B5-PLGA NPs in terms of promoting the immune effect of BCG and the B5 in enhancing the therapeutic effect of RIF against M. bovis.

Respiratory mucosal immunization is an effective immunization strategy against tuberculosis (TB), and effective mucosal vaccines require adjuvants that can promote protective immunity without deleterious inflammation. Mucosal BCG (Bacille Calmette-Guerin) is effective, but it causes a severe inflammatory response in the lung. A novel less cytotoxic mucosal vaccine AH-PB containing Mycobacterium tuberculosis (Mtb) cell surface antigens Ag85A and HspX (AH), as well as polyinosinic-polycytidylic acid (Poly IC) and bovine neutrophil β-defensin-5 (B5) adjuvants were prepared, with the overarching goal of protecting against TB. Then, the immunogenicity and protective efficacy of these vaccines via the intranasal route were evaluated in a mouse model. Results showed that intranasal AH-PB promoted tissue-resident memory T cells (T RMs ) development in the lung, induced antigen-specific antibody response in airway, provided protection against Mycobacterium bovis ( M. bovis ), conferred better protection than parenteral BCG in the later stage of infection, and boosted the protective immunity generated by BCG in mice. Moreover, both B5 and Poly IC were indispensable for the protection generated by AH-PB. Furthermore, intranasal immunization with AH-B5 fusion vaccines also provided similar protection against M. bovis compared to AH-PB. Collectively, B5-based TB vaccine via the intranasal route is a promising immunization strategy against bovine TB, and this kind of immunization strategy may be applied to human TB vaccine development. These findings highlight the potential importance of B5 as a mucosal adjuvant used in TB vaccines or other respiratory disease vaccines.

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M. bovis is the causative agent of animal tuberculosis (TB) and human TB. Research on M. bovis is of great public health significance.

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Mycobacterium bovis is the causative agent of bovine tuberculosis and also responsible for serious threat to public health. Koumiss is a fermented mare's milk product, used as traditional drink. Here, we explored the effect of koumiss on gut microbiota and the host immune response against M bovis infection. Therefore, mice were treated with koumiss and fresh mare milk for 14 days before M bovis infection and continue for 5 weeks after infection. The results showed a clear change in the intestinal flora of mice treated with koumiss, and the lungs of mice treated with koumiss showed severe edema, inflammatory infiltration, and pulmonary nodules in M bovis‐infected mice. Notably, we found that the content of short‐chain fatty acids was significantly lower in the koumiss‐treated group compared with the control group. However, the expression of endoplasmic reticulum stress and apoptosis‐related proteins in the lungs of koumiss‐treated mice were significantly decreased. Collectively, these findings suggest that koumiss treatment disturb the intestinal flora of, which is associated with disease severity and the possible mechanism that induces lungs pathology. Our current findings can be exploited further to establish the “gut‐lung” axis which might be a novel strategy for the control of tuberculosis.

Introduction: The limited efficacy of BCG (bacillus Calmette–Guérin) urgently requires new effective vaccination approaches for the control of tuberculosis. Poly lactic-co-glycolic acid (PLGA) is a prevalent drug delivery system. However, the effect of PLGA-based nanoparticles (NPs) against tuberculosis for the induction of mucosal immune response is no fully elucidated. In this study, we hypothesized that intranasal immunization with culture filtrate protein-10 (CFP10)-loaded PLGA NPs (CFP10-NPs) could boost the protective immunity of BCG against Mycobacterium bovis in mice. Methods: The recombinant protein CFP10 was encapsulated with PLGA NPs to prepare CFP10-NPs by the classical water–oil-water solvent-evaporation method. Then, the immunoregulatory effects of CFP10-NPs on macrophages in vitro and on BCG-immunized mice in vivo were investigated. Results: We used spherical CFP10-NPs with a negatively charged surface (zeta-potential −28.5 ± 1.7 mV) having a particle size of 281.7 ± 28.5 nm in diameter. Notably, CFP10-NPs significantly enhanced the secretion of tumor necrosis factor α (TNF-α) and interleukin (IL)-1β in J774A.1 macrophages. Moreover, mucosal immunization with CFP10-NPs significantly increased TNF-α and IL-1β production in serum, and immunoglobulin A (IgA) secretion in bronchoalveolar lavage fluid (BALF), and promoted the secretion of CFP10-specific interferon-γ (IFN-γ) in splenocytes of mice. Furthermore, CFP10-NPs immunization significantly reduced the inflammatory area and bacterial load in lung tissues at 3-week post-M. bovis challenge. Conclusion: CFP10-NPs markedly improve the immunogenicity and protective efficacy of BCG. Our findings explore the potential of the airway mucosal vaccine based on PLGA NPs as a vehicle for targeted lung delivery.

Paratuberculosis is a chronic wasting disease of granulomatous enteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis, MAP) resulting in heavy economic losses to dairy industries worldwide. Currently, commercial vaccines were not effective in preventing pathogen shedding and were reported with serious side effects. To develop a novel and smarter paratuberculosis vaccine, we utilized PLGA nanoparticles encapsulating the Bfra antigen (Bfra-PLGA NPs). We observed that mice vaccinated with Bfra-PLGA NPs exhibited an enhanced secretory IFN-γ, CD4+ T cells response and antibody IgG against MAP infection. In addition, secretions of the inflammatory cytokine TNF-α and IL-10 were increased following treatment with Bfra-PLGA NPs. A significant reduction in bacterial load was observed in the livers and spleens of animals vaccinated with Bfra-PLGA NPs. Furthermore, Bfra-PLGA NPs were effective to alleviate the pathological lesions of livers in mice. Overall, our approach provides a rational basis for employing PLGA nanoparticles to develop improved vaccines that induced protective immunity against paratuberculosis. Graphical Abstract