Enhancement of bacteriolysis of shuffled phage PhiX174 gene E.

Bacterial ghosts that are generated using the regulated PhiX174 lysis gene E offer a new avenue for the study of inactivated vaccines. Here, we constructed a library of mutant gene E using a gene-shuffling technique. After screening and recombination with the prokaryotic non-fusion expression vector pBV220, two lysis plasmids were selected. Among which, a novel mutant E gene (named mE), consisting of a 74-bp non-encoding sequence at 5'-end and a 201-bp gene ΔE, significantly increased the lysis effect on prokaryotic Escherichia coli and Salmonella enteritidis. Moreover, lysis efficiency, as measured by the OD600 value, reached 1.0 (109 CFU), avoiding the bottleneck problem observed with other bacterial lysis procedures, which results in a low concentration of bacteria in suspension, and consequent low production of bacterial ghosts. Our results may provide a promising avenue for the development of bacterial ghost vaccines.

Salmonella enteritidis ghost vaccine induces effective protection against lethal challenge in specific-pathogen-free chicks.

Bacterial ghosts (BGs) are empty bacterial envelopes generated by expulsion of the bacterial genome and cytoplasmic contents from bacterial cells, and the process is mediated by lysis protein E encoded on bacteriophage PhiX174. BGs represent a new approach in vaccine development and have been applied to a variety of gram-negative bacterial vaccine candidates. In this study, a BG vaccine generated from Salmonella enteritidis (S. enteritidis) strain DH091 was prepared using the highly efficient plasmid, pBV-mE. The efficacy of the BG vaccine was tested using 75 chicks (Gallus gallus) kept under specific pathogen-free (SPF) conditions. A comprehensive evaluation of the immune response, including humoral and cellular immune responses, interferon-γ (IFN-γ) and interleukin-4 (IL-4) production, and histopathology of various tissues, was performed in BG-vaccinated animals subsequently challenged with S. enteritidis. The results were compared with animals that were immunized with the inactivated vaccine. S. enteritidis ghosts not only promoted the generation of high titer antibodies and IFN-γ and IL-4 production but also stimulated a significant increase in CD8(+) and CD4(+) T lymphocytes. In particular, the dramatic increase in CD8(+) T cells indicated that the vaccine was able to induce clearance of intracellular Salmonella. The protective effects of BG vaccination in SPF chicks against 5×10(9) colony forming units of S. enteritidis were a result of the induction of a more effective immune response than that observed with the inactivated vaccine. These findings demonstrate the potential of S. enteritidis ghosts to be used as effective vaccines.

Positive role for rApxIVN in the immune protection of pigs against infection by Actinobacillus pleuropneumoniae.

The role of in vivo-induced ApxIV toxin of Actinobacillus pleuropneumoniae in protective immunity was evaluated in pigs by administering it alone or added to a multicomponent recombinant subunit vaccine composed of recombinant ApxI, ApxII, ApxIII toxin, and 42-kDa outer membrane protein (OMP). The pigs were immunized with vaccine I (rApxIVN), vaccine II (rApxI+rApxII+rApxIII+rApxIVN+rOMP), vaccine III (rApxI+rApxII+rApxIII+rOMP), or placebo (phosphate-buffered saline+adjuvant). A. pleuropneumoniae serovar 1 field isolate JMS 06 and serovar 2 field strain FX 01 were used as the challenge strains. Pigs that were immunized with vaccine I or vaccine II all developed high antibody titers against rApxIVN. The antibody titers against rApxI, rApxII, rApxIII, and rOMP in pigs immunized with vaccine II were higher than those in pigs vaccinated with vaccine III. Following the challenge, the pigs immunized with rApxIVN alone showed similar results to the pigs in the control group, such as severe respiratory symptoms and severe lung lesions. Pigs that had been immunized with vaccine II or vaccine III were protected against challenge with A. pleuropneumoniae serovar 1 and serovar 2. The pigs immunized with vaccine II had slighter lung lesions and fewer bacterial recovery than those of pigs immunized with vaccine III. These results indicate that rApxIVN contributes to the production of high level of antibodies directed against the vaccination antigens, and thus confers strong protection against challenges with different serovars of A. pleuropneumoniae.

Renaturation and purification of ApxII toxin of Actinobacillus pleuropneumoniae.

ApxII toxin is the only Apx toxin that is produced by Actinobacillus pleuropneumoniae serotype 7. In order to determine whether the recombinant ApxII that derived from Escherichia coli (E. coli) expression is faithful to the natural ApxII so that can be used as additional component in vaccine preparation, the structure gene apxIIA of ApxII toxin was expressed in E. coli with prokaryotic expression vector pGEX-6p-1 (formed pGEX-6p-A). pGZRS-C which is A. pleuropneumoniae-E. coli shuttle vector pGZRS-38 expressing the post-transcriptional activation gene apxII C was co-expressed with pGEX-6p-A. The expression product of rApxII A formed inclusion. The inclusion protein was oxidized, refolded and restored hemolytic activity after denaturation, renaturation and purification. The result indicated that E. coli expressed recombinant ApxII toxin has good fidelity, which makes it possible to produce this valuable antigen for vaccine preparation or diagnosis.