Bacterial ghosts as an oral vaccine: a single dose of Escherichia coli O157:H7 bacterial ghosts protects mice against lethal challenge.

Enterohemorrhagic Escherichia coli (EHEC) is a bacterial pathogen that is associated with several life-threatening diseases for humans. The combination of protein E-mediated cell lysis to produce EHEC ghosts and staphylococcal nuclease A to degrade DNA was used for the development of an oral EHEC vaccine. The lack of genetic material in the oral EHEC bacterial-ghost vaccine abolished any hazard of horizontal gene transfer of resistance genes or pathogenic islands to resident gut flora. Intragastric immunization of mice with EHEC ghosts without the addition of any adjuvant induced cellular and humoral immunity. Immunized mice challenged at day 55 showed 86% protection against lethal challenge with a heterologous EHEC strain after single-dose oral immunization and 93.3% protection after one booster at day 28, whereas the controls showed 26.7% and 30% survival, respectively. These results indicate that it is possible to develop an efficacious single-dose oral EHEC bacterial-ghost vaccine.

Immobilization of plasmid DNA in bacterial ghosts.

The development of novel delivery vehicles is crucial for the improvement of DNA vaccine efficiency. In this report, we describe a new platform technology, which is based on the immobilization of plasmid DNA in the cytoplasmic membrane of a bacterial carrier. This technology retains plasmid DNA (Self-Immobilizing Plasmid, pSIP) in the host envelope complex due to a specific protein/DNA interaction during and after protein E-mediated lysis. The resulting bacterial ghosts (empty bacterial envelopes) loaded with pDNA were analyzed in detail by real time PCR assays. We could verify that pSIP plasmids were retained in the pellets of lysed Escherichia coli cultures indicating that they are efficiently anchored in the inner membrane of bacterial ghosts. In contrast, a high percentage of control plasmids that lack essential features of the self-immobilization system were expelled in the culture broth during the lysis process. We believe that the combination of this plasmid immobilization procedure and the protein E-mediated lysis technology represents an efficient in vivo technique for the production of non-living DNA carrier vehicles. In conclusion, we present a "self-loading", non-living bacterial DNA delivery vector for vaccination endowed with intrinsic adjuvant properties of the Gram-negative bacterial cell envelope.

Modulation of gene expression by promoter mutants of the lambdacI857/pRM/pR system.

Gene expression driven by the p(R) promoter of the lambdacI857/p(RM)/p(R) system results from inactivation of the temperature-sensitive CI857 repressor. The CI857 repressor, whose gene is transcribed by the divergently orientated p(RM) promoter, is destabilised at temperatures above 30 degrees C. In this study, the lambdacI857/p(RM)/p(R) system was modified by the introduction of a single (A-32G) and a double mutation (A-32G and T-41C). The mutated lambdap(R) expression modules, 32G and 32G/41C, tightly repressed the highly lethal phage PhiX174 lysis gene E at temperatures up to 37 and 39 degrees C, respectively. Expression of protein E and subsequent lysis of Escherichia coli was still induced by a temperature up-shift to 42 degrees C. The impact of the mutations on gene expression levels driven by the lambdap(R) and p(RM) promoters was evaluated at various temperatures using the lacZ reporter gene. Results indicate that the A-32G mutation confers a lambdap(R) promoter-down phenotype. The additional increase in the temperature stability of the 32G/41C expression system is due to the T-41C mutation leading to a higher p(RM) activity. The described lambdap(R) expression modules can be used to obtain a defined expression level at a given temperature and to tightly repress in particular highly lethal genes at different bacterial growth temperatures.