Immunoregulatory and Antimicrobial Activity of Bovine Neutrophil β-Defensin-5-Loaded PLGA Nanoparticles against Mycobacterium bovis

Abstract

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.

Intranasal bovine β-defensin-5 enhances antituberculosis immunity in a mouse model by a novel protein-based respiratory mucosal vaccine

“…Moreover, antigen-specific IgA, IgM and IgG responses were thought to be related to protection [ 39 , 41 ]. And intranasal B5 could induce IgA response in airway [ 26 ]. Therefore, based on these findings, we chose two Mtb surface antigens to prepare AH-PB with the overarching goal of inducing antigen-specific antibody response in airway.…”

“…B5 was prepared as previously described [ 26 ]. AH was generated through fusion of the genes of Ag85A and HspX with linker (G 4 S) 3 using overlap polymerase chain reaction (PCR).…”

“…Next, the collected protein solution was gradually dialyzed against 20 mM Tris-HCl containing 1 mM EDTA, 10% glycerol, 1 mM GSH, 0.1 mM GSSG, and 0～8 M urea (pH 8.0), and finally dialyzed against PBS at 4℃. Notably, the soluble protein HspX was purified as previously described [ 26 ]. Finally, western blot using the mouse monoclonal His-tag antibody (Proteintech, Wuhan, Hubei, China) and Coomassie blue staining Solution (Solarbio, Beijing, China) were conducted to identify the proteins.…”

“…LL-37, human AMP, administered orally could elicit antigen-specific IgA response [ 25 ]. Our previous study showed that pulmonary B5 (bovine neutrophil β-defensin-5, BNBD5) induced IgA response in airway [ 26 ]. However, the adjuvant activity of B5 in subunit vaccines and the mechanism of B5 regulating antigen-specific immune response have not yet been elucidated.…”

Antimicrobial peptide-based materials: opportunities and challenges

“…B5-PLGA NPs showed a significant increase of TNF-a, IgA in serum and CD4+ T cells in the spleen as compared to B5 AMPs alone. 260 Lung infections caused by other microorganisms have been addressed using AMP-based NPs. The composition of formulations should be made in such a way that they can be easily administered via a nasal route and have a long persistence in the lungs and low systemic toxicity.…”