Researches On Genetically Inactivated Non-denaturated Bacterial Ghost Vaccine

Bacterial ghosts are prepared by the controlled exprssion of the phiX174 lysis gene E in Gram-negative bacteria that retain the antigenic determinants of the envelope with their living counterparts and thus represent ideal vaccine candidates. The objective of this study was to explore the potential utility of the phiX174 gene E driven by theλpLpR-cI8557 regulatory system for the generation of Avian Pathogenic Escherichia coli ghosts (APECGs) and investigate its potential as vaccine candidate.Lyis gene E was obtained by PCR from phiX174 using specific primers, cloned into pBS-T vector, and modified by the introduction of a single base exchange mutation (A-G). The 300bp DNA fragment containing mutated gene E cut off from pBS-T was inserted into the corresponding sites of pBV220, which is designated as pElys1. The lysis cassette (the operator expression lysis gene E) was amplified from pElysl and inserted into the Broad-host-range plasmid pBBR1MCS, which is designated as pBBRlys. pBBRlys was electroporated into Avian Pathogenic Escherichia coli (APEC) to prepare APECGs.Ghosts of Escherichia coli (E. coli) strain XL-1blue carrying pElys1 and APEC carrying pBBRlys were generated successfully by increasing the incubation temperature from 28℃up to 42℃to induce the expression of lysis gene E. Compared to XL-lblue harboring pElys1, in which lysis was observed to begin at about 15 min. and complete within 90 min. after gene E expression, the onset of lysis of APEC harboring pBBRlys occurred 45 and 60 min. after temperature elevation and the lysis process completed in about two hours. At the end of the lysis process, the inactivation efficiency of APECGs and XL-lblue ghosts was determined as 99.9916% and 99.9997% respectively by plating and CFU counting. However, no living cell was detected in lyophilized APECGs and XL-1blue ghosts.Scanning electromicrograph showed that ghost cells retain the basic morphology of E. coli cells, except that the surface of the ghost cells shrunk evidently compared with normal cells. The lysis tunnel through which the cytoplasmic content is expelled is not randomly distributed over the cell envelope but is predominantly in the middle of the cell or at polar sites. The diameter of the transmembrane tunnel ranges between 40 and 200 nm. Scanning electromicrograph also showed merged inner and outer-membrane at the transmembrane tunnel, which means that closed periplasmic space is remained after E-mediated cell lysis.Ducks were intramuscularly vaccinated with 10⁹ CFU APECGs and formalin killed APEC vaccine (FKV) without adjuvant. A booster of the same dose was intramuscularly administered two weeks after the primary vaccination. Four weeks after vaccination, ducks were intramuscularly challenged with homologous APEC strain with a dose of two fold of LD₅₀. Ducks immunized with APECGs once and twice showed RPS of 56.25% and 75% respectively, whereas ducks immunized with FKV showed RPS of 50% and 68.75%. Serum agglutination titers of ducks immunized with APECGs were also higher than those of ducks immunized with FKV.The result of our study suggests that bacterial ghosts is a new kind of vaccine candidate, which potentially has better protective efficiency than traditional formalin killed bacterial vaccines through more intensive and extensive researches.