Display Of Antigens Of Poultry Pathogens On Bacillus Thuringiensis Cell Surface

S-layer protein CTC surface display system of Bacillus thuringiensis was used to testthe possibility of displaying avian influenza virus nucleoprotein(NP), haemagglutininHA1 or protein of Mycoplasma gallisepticum agglutinin (pMGA) on the cell surface ofBacillus thuringiensis for the development of heat-stable, oral, live, veterinary vaccineusing B. thuringiensis as a carrier.1. Safety of the receptor strain and vector plasmid of S-layer protein CTC surfacedisplay system for mice and chickensB. thuringiensis crystal negative strain BMB171 and pHT304 were the receptor strainand the vector plasmid respectively in S-layer protein CTC surface display system. StrainBMB304 was constructed by transferring pHT304 into BMB171. The experiment wasconducted to evaluate the safety of strain BMB304 for mice and chickens and the fate ofstrain BMB304 in the intestinal tracts of chickens in order to determine the feasibility ofB. thuringiensis being used as a live vehicle for the delivery of heterologous antigenprotein for veterinary vaccine purpose.Following injected 10¹⁰ BMB304 vegetative cells by intra-peritoneal route, mice hadno acute toxicity reaction. After mice were given water mixed with 10⁸, 10⁷ and 10⁶BMB304 vegetative cells/mL respectively for 28 days, the appearance, behavior, weights,organ coefficients and the indexes of hematology and biochemistry in the mice have nosignificant difference compared with the control group. Significant pathological changesof inherent organs were not discovered. Following given 10¹⁰ BMB304 spores by oralroute, chickens had no acute toxicity reaction. Feeding the diet added with 10⁸, 10⁷ and10⁶ BMB304 spores/g respectively for 28 days, chickens showed no significant differencecompared with the control group. After 3 days of 10⁶ BMB304 spores/g diet of feeding tochickens, the spore persisted in the intestinal tracts of chickens for more than 5 days butno more than 7 days following spore removal from the diet. These findings demonstratedthat strain BMB304 was safe for mice and chickens and could colonize the avianintestinal tract for several days. It is suggestive that B. thuringiensis may be used fordeveloping novel oral veterinary vaccine that can be delivered with spore.2. Construction of S-layer fusion genes harboring heterologous antigen genesThe heterologous antigen genes np, npp, ha1p and pmga1.2p were amplified by PCR.Eight recombinant plasmids were constructed by replacing 3'-terminal part or the centralpart below the surface anchoring sequence slh of S-layer protein gene ctc with the heterologous antigen genes. The resulting plasmids included pSNP, pCSA-SNP,pCTC-NPP, pCSNPP, pCTC-HA1P, pCSHA1P, pCTC-PMGA1.2P and pCSPMGA1.2P.The pCSA-SNP, pCSNPP, pCSHA1P and pCSPMGA1.2P also harbored csaAB operonwhich was located on the upstream of S-layer fusion genes. The csaAB operon was veryimportant to the anchoring of S-layer protein on cell surface.3. Construction of recombinant strains harboring S-layer fusion genesNine recombinant B. thuringiensis strains were constructed by transferring recombinantplasmids to receptor BMB171. The resulting strains included BN (harboring pSNP), BCN(harboring pSNP and pMIL-CSA), C-S (harboring pCSA-SNP), BCCN (harboringpCTC-NPP and pMIL-CSA), CN (harboring pCSNPP), BCCH (harboring pCTC-HA1Pand pMIL-CSA), CH (harboring pCSHAIP), BCCG (harboring pCTC-PMGA1.2P andpMIL-CSA) and CG (harboring pCSPMGA1.2P). The plasmid pMIL-CSA harboringcsaAB operon was co-transferred into BMB171 with the recombinant plasmids which didnot harboring csaAB operon.4. Assaying the display of heterologous antigens on the recombinant strainsSlide agglutination assay showed all those recombinant NP proteins were displayed onthe cell surface of recombinant strains. Haemagglutination assay showed recombinantHA1 proteins and recombinant pMGA proteins were displayed on the cell surface ofrespective recombinant strains. Haemagglutination inhibition assay showed recombinantHA1 proteins and recombinant pMGA proteins displayed on the cell surface ofrecombinants respectively were specific to standard positive serum of avian influenzavirus and Mycoplasma gallisepticum, respectively.5. Immunogenicity of recombinant strains to mice and chickensAfter immunizing SPF BALB/c mice by intra-peritoneal route with vegetative cells ofrecombinant strains, all recombinant strains elicited humoral respones to respectiveheterologous antigens displayed on the cell surfaces and exhibited immunogenicity asassayed by indirect enzyme-linked immunosorbent assay.The spores of recombinant strains BCCH, CH, BCCG and CG were prepared. The AVcommercial broiler chickens were immunized by oral route with spores. The serumantibody titers of chickens immunized with spores of BCCH and CH were assayed byhaemagglutination inhibition test. The serum antibody titers of chickens immunized withspores of BCCG and CG were assayed by serum plate agglutination test. The results showed that the recombinant strains elicited humoral respones of chickens to respectiveheterologous antigens displayed on the cell surfaces and exhibited immunogenicity.Among all recombinant strains, CN (harboring fusion gene csa-ctc-npp), CH(harboring csa-ctc-halp) and CG (harboring csa-ctc-pmga1.2p) exhibited the highestactivity. That means the best way of constructing S-layer fusion gene is csa-ctc-*(*denotes heterologous antigen gene) which means the central part of S-layer protein genectc replaced by the heterologous antigen gene and csaAB operon located on the upstreamof fusion gene).The strategy developed in this study gives a possibility to generate heat stable, oral,veterinary vaccine with B. thuringiensis S-layer protein surface display system.