| Bacillus anthracis is a Gram-positive spore-forming, rod-shaped bacterium. It can cause fetal anthrax, a zoonotic disease (a disease shared between animals and humans). Untreated anthrax is often fatal, especially the respiratory type of anthrax infection. Anthrax is probably the most widely studied biological warfare and bioterrorism agent. Anthrax vaccine, which is the hotspot of biosecurity research, also is the focus of pathogens and relevant vaccines. The current approved vaccine against anthrax is based on protective antigen (PA) of Bacillus anthracis, requires six injections over an 18-month period and has a known history of side effects. Therefore, there is significant effort towards developing an improved vaccine against B. anthracis.In this study, a 34-kDa cytolysin A hemolysin (ClyA) of Salmonella enterica serovar Typhi was used as a novel export system for the secreted expression of Bacillus anthracis protective antigen PA or PA fusion protein (PA-EFn, PA-CTB, PA-LTB, PA-GFP) in the attenuated Salmonella serovar Typhi live-vector strains.At first, asd gene was cloned into expression vector pACYC184. Then the vector was transformed into S. typhi attenuated strain CVD908-htrA (Ty2 aroC aroD asd htrA) in order to construct a host-plasmid balanced lethal system, and the resulted strain was named SC184. The clyA gene was PCR amplified from S. typhi chromosome and cloned into pACYC184 vector. Afterward, the pag gene which was PCR amplified from Bacillus anthracis vaccine strain A16R was cloned just downstream of clyA gene to constructed vector pSA184P. The efn gene was PCR amplified from A16R genome and added to the 3’end of clyA-pag fusion gene to create vector pSA184Pen which could express ClyA-PA-EFn fusion protein. The ltb gene was also PCR amplified from Escherichia coli K12 genome and cloned just downstream of clyA-pag sequence to constructed vector pSA184P1 which could express ClyA-PA-LTB fusion protein. The ctb gene was PCR amplified from vector pUCT1 and added to the 3’end of clyA-pag sequence, generating vector pSA184Pc which could express ClyA-PA-CTB fusion protein. At last, a reporter gene gfp was PCR amplified from vector pEGFP-Nl and cloned just downstream of clyA-pag sequence, generating vector pSA184Pg which could express ClyA-PA-GFP fusion protein. The vectors were transformed into S. typhi attenuated strain CVD908-htrA, and the resulted clones were named SC184P, SC184PEn, SC184PL, SC184PC and SC184PG respectively. The constructed live vector strains expressed foreign antigens successfully, which were proved by the SDS-PAGE and Western blot assay. The reporter GFP was identified by flow cytometry assay and immunofluorescence microscopy assay. In order to observe whether foreign antigens were secreted or surface display on the outer membrane of the live vaccine strains by ClyA secreted system, some of method including outer membrane protein extraction combining with Western blot assay, whole cell ELISA assay, immunofluorescence microscopy assay, as well as flow cytometry assay were used to exam the subcellular localization of foreign antigens. The experimental results showed that the antigens were partly expressed on the cell surface of live vaccine strains.Mice oral immunized with constructed SC184PEn, SC184PC or SC184P followed by PA-boost resulted in higher levels of PA-specific IgG in sera than those priming with SCI 84 followed by PA-boost. To evaluate the protective quality of the immune response in mice vaccinated with the SC184PEn, SC184PC or SC184P producing PA, we investigated the ability of immune serum to neutralize the cytotoxic activity of anthrax LeTx. At a 1:10 dilution, serum samples from vaccinated mice could protect 40-80% RAW 264.7 cells from killing; even at a 1:160 dilution, serum samples from vaccinated mice could protect 20-40% RAW 264.7 cells from killing. Mice immunized with SC184PEn, SC184PC, SC184P or SC184 followed by PA-boost were challenged i.p. with 1×108cfu of B. anthracis strain A16R spores. Following challenge, mice were monitored for survival for at least 10 days. Mice immunized with SC184 followed by PA-boost were not protected, and all mice died between days 3 to 5 (0/6). Mice immunized with the vaccine SC184P followed by PA-boost were only 17% protected (1/7). In contrast, mice immunized with SC184PEn, SC184PC followed by PA-boost were 60% protected (3/5, 3/5). When mice of each group were challenged i.p. with 8×108cfu of A16R spores, only mice immunized with SC184PEn and SC184PC followed by PA-boost were partially protected (2/6,3/6). The results showed that the live vaccine strain SC184PEn and SC184PC could provide efficient immune response against PA in vaccinated mice and provided protective efficacy in this animal model. |
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