Online monitoring of Escherichia coli ghost production.

Controlled expression of cloned phi X174 gene E in gram-negative bacteria results in lysis of the bacteria by the formation of a transmembrane tunnel structure built through the cell envelope complex. Production of bacterial ghosts is routinely monitored by classical microbiological procedures. These include determination of the turbidity of the culture and the total number of cells and the number of reproductive cells present during the time course of growth and lysis. Although conceptually simple, these methods are labor intensive and time consuming, providing a complete set of results after the determination of viable cell counts. To avoid culturing methods for bacterial growth, an alternative flow cytometric procedure is presented for the quantification of ghosts and polarized, as well as depolarized, nonlysed cells within a culture. For this method, which is based on the discriminatory power of the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol, a staining protocol was developed and optimized for the maximum discrepancy in fluorescence between bacterial ghosts and viable cells. The total quantitative analysis procedure takes less than 2 min. The results derived from classical or cytometric analyses correlate with respect to the total cell numbers and the viability of the culture.

Extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli.

Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174. Gene E expression is tightly controlled by the rightward lambda pR promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12. The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines. Expression of gene E is stringently repressed at temperatures up to 30 degrees C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30 degrees C due to thermal inactivation of the cI857 repressor. As a consequence, the production of ghosts requires that bacteria have to be grown at 28 degrees C before the lysis process is induced. In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the lambda pR promoter/cI857 repressor system, allowing pathogens to grow at 37 degrees C before induction of lysis. In this study we describe a mutation in the lambda pR promoter, which allows stringent repression of gene E expression at temperatures up to 36 degrees C, but still permits induction of cell lysis at 42 degrees C.

New strategies for combination vaccines based on the extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a great variety of bacteria and are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts do not need the addition of adjuvants to induce immunity in experimental animals they can also be used as carriers or targeting vehicles or as adjuvants in combination with subunit vaccines. Matrixes like dextran which can be used to fill the internal lumen of ghosts can be substituted with various ligands to bind the subunit or other materials of interest. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. As carriers of foreign antigens there is no limitation in the size of foreign antigens to be inserted and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. Using the different building blocks and combining them into the recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Cold-sensitive E-lysis systems.

The release of recombinant bacteria into the environment is undesirable because of possible risks associated with the genetically modified organisms. The aim of this study was to establish a cold-sensitive killing system with a lethal gene, activated when bacteria encounter lower environmental temperatures. To obtain cold-sensitive lysis vectors, the lambdacI857 repressor/pR promoter expression system was combined with either the lacI/lacZpo or the phage 434 cI/pR system that control the expression of the lysis gene E of bacteriophage phiX174. Escherichia coli strains harbouring such suicide vectors are able to grow at 37 degrees C, but cell lysis takes place at temperatures below 30 degrees C. By replacing gene E with a beta-galactosidase reporter gene we also showed that the onset of beta-galactosidase activity corresponds with the onset of lysis at 28 degrees C. Results indicate that these newly combined promoter/repressor systems can also be used to confer cold-sensitive expression to any gene of interest.

Bacterial ghosts as multifunctional vaccine particles.

Expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a variety of bacteria including Escherichia coli. Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Klebsiella pneumoniae, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Pasteurella haemolytica, Pasteurella multocida, and Helicobacter pylori. Such ghosts are used as non-living candidate vaccines and represent an alternative to heat or chemically inactivated bacteria. In recombinant ghosts, foreign proteins can be inserted into the inner membrane prior to E-mediated lysis via specific N-, or C-, or N- and C-terminal anchor sequences. The export of proteins into the periplasmic space or the expression of recombinant S-layer proteins vastly extents the capacity of ghosts or recombinant ghosts as carriers of foreign epitopes or proteins. Oral, aerogenic or parenteral applications of (recombinant) ghosts in experimental animals induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts used as vaccines or as carriers of relevant antigens. The inserted target antigens into the inner membrane or into S-layer proteins are not limited in size.