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Development of a heat-stable and orally delivered recombinant M2e-expressing B. subtilis spore-based influenza vaccine.

ABSTRACT: Highly conserved ectodomain of influenza virus M2 protein (M2e) is an important target for the development of universal influenza vaccines. Today, the use of chemical or genetic fusion constructs have been undertaken to overcome the low immunogenicity of M2e in vaccine formulation. However, current M2e vaccines are neither orally delivered nor heat-stable. In this study, we evaluated the immune efficacy of an orally delivered recombinant M2e vaccine containing 3 molcules of M2e consensus sequence of influenza A viruses, termed RSM2e3. To accomplish this, CotB, a spore coat of Bacillus subtilis (B. subtilis), was used as a fusion partner, and heat-stable nonpathogenic B. subtilis spores were used as the carrier. Our results showed that CotB-M2e3 fusion had no effect on spore structure or function in the resultant recombinant RSM2e3 strain and that heterologous influenza virus M2e protein was successfully displayed on the surface of the recombinant RSM2e3 spore. Importantly, recombinant RSM2e3 spores elicited strong and long-term M2e-specific systemic and mucosal immune responses, completely protecting immunized mice from lethal challenge of A/PR/8/34(H1N1) influenza virus. Taken together, our study forms a solid basis for the development of a novel orally delivered and heat-stable influenza vaccine based on B. subtilis spore surface display.

SUBMITTER: Zhao G

PROVIDER: S-EPMC4514050 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Development of a heat-stable and orally delivered recombinant M2e-expressing B. subtilis spore-based influenza vaccine.

Zhao Guangyu G Miao Yu Y Guo Yan Y Qiu Hongjie H Sun Shihui S Kou Zhihua Z Yu Hong H Li Junfeng J Chen Yue Y Jiang Shibo S Du Lanying L Zhou Yusen Y

Human vaccines & immunotherapeutics 20140101 12

Highly conserved ectodomain of influenza virus M2 protein (M2e) is an important target for the development of universal influenza vaccines. Today, the use of chemical or genetic fusion constructs have been undertaken to overcome the low immunogenicity of M2e in vaccine formulation. However, current M2e vaccines are neither orally delivered nor heat-stable. In this study, we evaluated the immune efficacy of an orally delivered recombinant M2e vaccine containing 3 molcules of M2e consensus sequenc ...[more]

PMID: 25483702

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Project description:BackgroundClonorchiasis, a food-borne zoonosis, is caused by Clonorchis sinensis. The intestinal tract and bile ducts are crucial places for C. sinensis metacercariae to develop into adult worms. The endospore of Bacillus subtilis is an ideal oral immunization vehicle for delivery of heterologous antigens to intestine. Cysteine protease of C. sinensis (CsCP) is an endogenous key component in the excystment of metacercariae and other physiological or pathological processes.MethodsWe constructed a fusion gene of CotC (a coat protein)-CsCP and obtained B. subtilis spores with recombinant plasmid of pEB03-CotC-CsCP (B.s-CotC-CsCP). CotC-CsCP expressed on spores' surface was detected by Western blotting and immunofluorescence. Immunological characteristics of recombinant spore coat protein were evaluated in a mouse model. The levels of CsCP-specific antibodies were detected by ELISA. Effects of recombinant spores on mouse intestine were evaluated by histological staining. The activities of biochemical enzymes in serum were assayed by microplate. Liver sections of infected mice were evaluated by Ishak score after Masson's trichrome.ResultsThe B.s-CotC-CsCP spores displayed CsCP on their coat. Specific IgG and isotypes were significantly induced by coat proteins of B.s-CotC-CsCP spores after subcutaneous immunization. IgA levels in intestinal mucus and bile of B.s-CotC-CsCP orally treated mice significantly increased. Additionally, more IgA-secreting cells were observed in enteraden and lamina propria regions of the mouse jejunum, and an increased amount of acidic mucins in intestines were also observed. There were no significant differences in enzyme levels of serum among groups. No inflammatory injury was observed in the intestinal tissues of each group. The degree of liver fibrosis was significantly reduced after oral immunization with B.s-CotC-CsCP spores.ConclusionsBacillus subtilis spores maintained the original excellent immunogenicity of CsCP expressed on their surface. Both local and systemic specific immune responses were elicited by oral administration of B.s-CotC-CsCP spores. The spores effectively promoted intestinal health by inducing secretion of acidic mucins, with no other side effects to the liver or intestine. Oral administration of spores expressing CsCP could provide effective protection against C. sinensis. This study may be a cornerstone for development of antiparasitic agents or vaccines against clonorchiasis based on B. subtilis spore expressing CsCP on the surface.

Project description:BackgroundPorcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome, and is associated with a number of other diseases. PCV2 is widely distributed in most developed swine industries, and is a severe economic burden. With an eye to developing an effective, safe, and convenient vaccine against PCV2-associated diseases, we have constructed a recombinant Bacillus subtilis strain (B. subtilis-Cap) that expresses the PCV2 capsid protein (Cap).MethodsElectroporation of a plasmid shuttle vector encoding the PCV2 Cap sequence was use to transform Bacillus subtilis. Flow cytometry was used to evaluate in vitro bone marrow derived dendritic cell (BM-DC) maturation and T cell proliferation induced by B. subtilis-Cap. Orally inoculated piglets were used for in vivo experiments; ELISA and western blotting were used to evaluate B. subtilis-Cap induced PCV2-specific IgA and IgG levels, as well as the secretion of cytokines and the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 9 (TLR9).ResultsWe evaluated the immune response to B. subtilis-Cap in vitro using mouse BM-DCs and in vivo using neonatal piglets orally inoculated with B. subtilis-Cap. Our results showed that the recombinant B. subtilis-Cap activated BM-DCs, significantly increased co-stimulatory molecules (CD40 and CD80) and major histocompatibility complex II, and induced allogenic T cells proliferation. Piglets immunized with B. subtilis-Cap had elevated levels of PCV2-specific IgA in the mucosal tissues of the digestive and respiratory tract, and PCV2-specific IgG in serum (P < 0.05 or P < 0.01). Ileal immunocompetent cells, such as the IgA-secreting cells (P < 0.01), intestinal intraepithelial lymphocytes (IELs) (P < 0.01), CD3+ T lymphocytes (P < 0.01) and CD4+ T lymphocytes (P < 0.01) increased significantly in the B. subtilis-Cap immunized piglets. Additionally, B. subtilis-Cap inoculation resulted in increased the expression of TLR2 and TLR9 (P < 0.01), and induced the secretion of cytokines IL-1β, IL-6, interferon-γ, and β-defensin 2 (P < 0.01).ConclusionsWe constructed a prototype PCV2 vaccine that can be administered orally and elicits a more robust humoral and cellular immunity than inactivated PCV2. B. subtilis-Cap is a promising vaccine candidate that is safe, convenient, and inexpensive. Further in vivo research is needed to determine its full range of efficacy in pigs.

Project description:We recently reported a stable Bacillus subtilis-carrying chicken NK-lysin peptide (B. subtilis-cNK-2) as an effective oral delivery system of an antimicrobial peptide to the gut with therapeutic effect against Eimeria parasites in broiler chickens. To further investigate the effects of a higher dose of an oral B. subtilis-cNK-2 treatment on coccidiosis, intestinal health, and gut microbiota composition, 100 (14-day-old) broiler chickens were allocated into 4 treatment groups in a randomized design: 1) uninfected control (CON), 2) infected control without B. subtilis (NC), 3) B. subtilis with empty vector (EV), and 4) B. subtilis with cNK-2 (NK). All chickens, except the CON group, were infected with 5,000 sporulated Eimeria acervulina (E. acervulina) oocysts on d 15. Chickens given B. subtilis (EV and NK) were orally gavaged (1 × 1012 cfu/mL) daily from d 14 to 18. Growth performances were measured on d 6, 9, and 13 postinfection (dpi). Spleen and duodenal samples were collected on 6 dpi to assess the gut microbiota, and gene expressions of gut integrity and local inflammation makers. Fecal samples were collected from 6 to 9 dpi to enumerate oocyst shedding. Blood samples were collected on 13 dpi to measure the serum 3-1E antibody levels. Chickens in the NK group showed significantly improved (P < 0.05) growth performance, gut integrity, reduced fecal oocyst shedding and mucosal immunity compared to NC. Interestingly, there was a distinct shift in the gut microbiota profile in the NK group compared to that of NC and EV chickens. Upon challenge with E. acervulina, the percentage of Firmicutes was reduced and that of Cyanobacteria increased. In NK chickens, however, the ratio between Firmicutes and Cyanobacteria was not affected and was similar to that of CON chickens. Taken together, NK treatment restored dysbiosis incurred by E. acervulina infection and showed the general protective effects of orally delivered B. subtilis-cNK-2 on coccidiosis infection. This includes reduction of fecal oocyst shedding, enhancement of local protective immunity, and maintenance of gut microbiota homeostasis in broiler chickens.

Dataset Information

Development of a heat-stable and orally delivered recombinant M2e-expressing B. subtilis spore-based influenza vaccine.

Publications

Development of a heat-stable and orally delivered recombinant M2e-expressing B. subtilis spore-based influenza vaccine.

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