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Mucosal and systemic immune responses induced by intranasal immunization of recombinant Bacillus subtilis expressing the P97R1, P46 antigens of Mycoplasma hyopneumoniae.

ABSTRACT: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the pathogen of swine enzootic pneumonia, a chronic respiratory disease affecting pigs of all ages. The ciliated epithelial cells of the respiratory tract are the main target invaded and colonized by M. hyopneumoniae. Therefore, the ideal vaccine would be mucosally administered and able to stimulate suitable mucosal immunity and prevent the adherence of pathogens to mucosal cell surfaces. Currently, Bacillus subtilis as a recombinant vaccine carrier has been used for antigen delivery and proved to be effectively enhancing the innate immunity of nasal mucosa. Here, our study attempts to construct recombinant Bacillus subtilis (B.S-P97R1, B.S-P46), which can express the P97R1 or P46 antigen of M. hyopneumoniae, and to evaluate the immune responses in BALB/c mice. Initially, we respectively successfully constructed recombinant B.S-P97R1, B.S-P46 and validated the expression of antigen proteins by Western analysis. Then, recombinant B.S-P97R1 or B.S-P46 were respectively intranasally (i.n.) immunized in mice. Both strong P97R1-specific and P46-specific immunoglobulin G (IgG), secretory immunoglobulin A (SIgA) antibodies were induced in sera, bronchoalveolar lavage fluids (BALs) by ELISA analysis. Moreover, the levels of specific IL-4, IFN-? in the immunized mice were elevated, and the proliferation of lymphocytes was also enhanced. In general, intranasal inoculation of recombinant B.S-P97R1 or B.S-P46 resulted in strong mucosal immunity, cell-mediated and humoral immunity, which was a mixed Th1/Th2-type response. In addition, our results provided a potential novel strategy that may be applied to the development of vaccines against M. hyopneumoniae.

SUBMITTER: Wang Y

PROVIDER: S-EPMC6822509 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Mucosal and systemic immune responses induced by intranasal immunization of recombinant Bacillus subtilis expressing the P97R1, P46 antigens of Mycoplasma hyopneumoniae.

Wang Yongheng Y Wang Jialu J Zhou Mengyun M Liu Peng P Zhang En E Li Yuchen Y Lin Jian J Feng Zhixin Z Yang Qian Q

Bioscience reports 20191001 10

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the pathogen of swine enzootic pneumonia, a chronic respiratory disease affecting pigs of all ages. The ciliated epithelial cells of the respiratory tract are the main target invaded and colonized by M. hyopneumoniae. Therefore, the ideal vaccine would be mucosally administered and able to stimulate suitable mucosal immunity and prevent the adherence of pathogens to mucosal cell surfaces. Currently, Bacillus subtilis as a recombinant vaccine carrier ...[more]

PMID: 31492763

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Project description:Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) is the etiological agent of swine enzootic pneumonia (EP), which has been associated with considerable economic losses due to reduced daily weight gain and feed efficiency. Adhesion to the cilia is important for Mhp to colonize the respiratory epithelium. Therefore, a successful vaccine must induce broad Mhp-specific immune responses at the mucosal surface. Recombinant attenuated Salmonella strains are believed to act as powerful live vaccine vectors that are able to elicit mucosal immune responses against various pathogens. To develop efficacious and inexpensive vaccines against Mhp, the immune responses and protection induced by recombinant attenuated Salmonella vaccines based on the P42 and P97 antigens of Mhp were evaluated. In general, the oral inoculation of recombinant rSC0016(pS-P42) or rSC0016(pS-P97) resulted in strong mucosal immunity, cell-mediated immunity, and humoral immunity, which was a mixed Th1/Th2-type response. In addition, the levels of specific IL-4 and IFN-γ in the immunized mice were increased, and the proliferation of lymphocytes was also enhanced, confirming the production of a good cellular immune response. Finally, both vaccine candidate strains were able to improve the weight loss of mice after a challenge and reduce clinical symptoms, lung pathological damage, and the inflammatory cell infiltration. These results suggest that the delivery of protective antigens with recombinant attenuated Salmonella vectors may be an effective means by which to combat Mhp infection. IMPORTANCE Mhp is the main pathogen of porcine enzootic pneumonia, a highly infectious and economically significant respiratory disease that affects pigs of all ages. As the target tissue of Mhp infections are the mucosal sites of the respiratory tract, the induction of protective immunity at the mucosal tissues is the most efficient strategy by which to block disease transmission. Because the stimulation of mucosal immune responses is efficient, Salmonella-vector oral vaccines are expected to be especially useful against mucosal-invading pathogens. In this study, we expressed the immunogenic proteins of P42 and P97 with the attenuated Salmonella Choleraesuis vector rSC0016, thereby generating a low-cost and more effective vaccine candidate against Mhp by inducing significant mucosal, humoral and cellular immunity. Furthermore, rSC0016(pS-P42) effectively prevents Mhp-induced weight loss and the pulmonary inflammation of mice. Because of the effectiveness of rSC0016(pS-P42) against Mhp infection in mice, this novel vaccine candidate strain shows great potential for its use in the pig breeding industry.

Project description:The P46 and P65 proteins of Mycoplasma hyopneumoniae are two membranous proteins carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the genes encoding entire P46 (1,260 bp) and P65 (1,803 bp) and N-terminally truncated P65(c) (1,200 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species that commonly colonize the porcine respiratory tract. Both amplified genes were then cloned into the pGEX-4T-1 vector to be expressed in Escherichia coli cells as recombinant fusion proteins with glutathione S-transferase (GST). Prior to generation of expression constructs, TGA nonsense codons, exceptionally used for tryptophan residues by M. hyopneumoniae, had been converted to TGG codons by PCR-directed mutagenesis. Following induction by IPTG (isopropyl-beta-D-thiogalactopyranoside), both GST-P46 and GST-P65(c) recombinant fusion proteins were recovered by disrupting transformed cells by sonication, purified by affinity chromatography, and then cut with thrombin to release the P46 and P65(c) moieties. The enriched E. coli-expressed P46 and P65c proteins were used to immunize female BALB/c mice for the generation of anti-P46 and anti-P65(c) monoclonal antibodies (MAbs). The polypeptide specificities of MAbs obtained was confirmed by Western blotting with cell lysates prepared from the homologous strain. Cross-reactivity study of the anti-P46 and anti-P65(c) MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture, suggested that the MAbs obtained against both membranous proteins were directed against highly conserved species-specific epitopes. No reactivity to other mycoplasma species tested was demonstrated. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs that had been inoculated intratracheally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. Both anti-P46 and anti-P65(c) MAbs permitted effective detection by indirect immunofluorescence and indirect immunoperoxidase assay of M. hyopneumoniae in, respectively, frozen and formalin-fixed, paraffin-embedded lung sections from pigs that were killed after the 6- to 7-week observation period.

Dataset Information

Mucosal and systemic immune responses induced by intranasal immunization of recombinant Bacillus subtilis expressing the P97R1, P46 antigens of Mycoplasma hyopneumoniae.

Publications

Mucosal and systemic immune responses induced by intranasal immunization of recombinant Bacillus subtilis expressing the P97R1, P46 antigens of Mycoplasma hyopneumoniae.

Similar Datasets

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