Intramuscular immunization with genetically inactivated (ghosts) Actinobacillus pleuropneumoniae serotype 9 protects pigs against homologous aerosol challenge and prevents carrier state.

Bacterial ghosts are empty cell envelopes achieved by the expression of a cloned bacteriophage lysis gene and, unlike classical bacterins, suffer no denaturing steps during their production. These properties may lead to a superior presentation of surface antigens to the immune system. Currently available porcine Actinobacillus pleuropneumoniae vaccines afford only minimal protection by decreasing mortality but not morbidity. Pigs which survive infection can still be carriers of the pathogen, so a herd once infected remains infected. Carrier pigs harbour A. pleuropneumoniae in their nasal cavities, in their tonsils, or within lung lesions. A dose-defined nose-only aerosol infection model for pigs was used to study the immunogenic and protective potential of systemic immunization with ghosts made from A. pleuropneumoniae serotype 9 reference strain CVI 13261 against an homologous aerogenous challenge. Pigs were vaccinated twice intramuscularly with a dose of 5x10(9) CFU ghosts (GVPs) or formalin-inactivated A. pleuropneumoniae bacterins (BVPs). After 2 weeks vaccinated pigs and non-vaccinated placebo controls (PCs) were challenged with a dose of 10(9) CFU by aerosol. The protective efficacy of immunization was evaluated by clinical, bacteriological, serological and post-mortem examinations. Bronchoalveolar lavage in pigs was performed during the experiment to obtain lavage samples (BALF) for assessment of local antibodies. Isotype-specific antibody responses in serum and BALF were determined by ELISAs based on whole-cell antigen. Immunization with ghosts did not cause clinical side-effects. After aerosol challenge PCs developed fever and pleuropneumonia. GVPs or BVPs were found to be fully protected against clinical disease or lung lesions in both vaccination groups, whereas colonization of the respiratory tract with A. pleuropneumoniae was only prevented in GVPs. Specific immunoglobins against A. pleuropneumoniae were not detectable in BALF after immunization. A significant systemic increase of IgM, IgA, IgG(Fc'), or IgG(H+L) antibodies reactive with A. pleuropneumoniae was measured in GVPs and BVPs when compared to the non-exposed controls. BVPs reached higher titers of IgG(Fc') and IgG(H+L) than GVPs. However, prevention of carrier state in GVPs coincided with a significant increase of serum IgA when compared to BVPs. These results suggest that immunization with ghosts, that bias antibody populations specific to non-denaturated surface antigens, may be more efficacious in protecting pigs against colonization and infection than bacterins.

Pigs aerogenously immunized with genetically inactivated (ghosts) or irradiated Actinobacillus pleuropneumoniae are protected against a homologous aerosol challenge despite differing in pulmonary cellular and antibody responses.

Aerosol immunization is a safe way to induce complete protection against pleuropneumonia in pigs caused by the lung pathogenic bacterium Actinobacillus pleuropneumoniae. In order to determine the local immune responses of vaccinees in concomitant with protection, lung lining fluid before and 3 weeks after immunization from pigs immunized three times with aerosols of either genetically inactivated ghosts which represent whole cell envelope preparations, or irradiated bacteria were examined following an homologous aerosol challenge. Specific antibody isotypes in the bronchoalveolar lavage were assayed by whole cell ELISAs. Total and relative numbers of cells including lymphocyte subsets were determined. In both vaccinated groups a net influx of plasma cells and lymphocytes, as well as a significant increase of specific IgG occurred. Concurrently, the CD4+/CD8+ ratio was found to increase after aerosol immunization. The lymphocyte subsets of IgG+ and IgA+ cells were found significantly higher in the group immunized with irradiated bacteria when compared to pigs immunized with bacterial ghosts. The latter group showed a significant increase of IgA, IgM, and a net influx of lymphoid blasts and granulocytes in the bronchoalveolar lining fluid. Although differences between the local immune responses of both immunized groups occurred, a significant increase of specific IgG and a net influx of plasma cells and lymphocytes were found to be associated with complete protection against a homologous aerosol challenge infection.

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.

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.

Induction of protective immunity by aerosol or oral application of candidate vaccines in a dose-controlled pig aerosol infection model.

In order to outline basic concepts for the design of a bacterial aerosol infection model, the development of a pig model with Actinobacillus pleuropneumoniae is described. First, reproducibility of aerosol parameters should be maintained by optimizing generating and sampling conditions. Survival rates of the chosen strain must be predictable. Secondly, inhalation conditions for the recipients have to be standardized to enable the determination of deposition sites and the dose administered. Subsequently, dose-response relationship should be evaluated to find a suitable challenge dose. Furthermore, it seems necessary to establish methods to obtain local specimens for determination of the local immune responses. The present study demonstrates that after aerosol challenge pigs were completely protected after inhalation and partially protected after oral application of A. pleuropneumoniae vaccines and describes techniques to administer bacteria in a dose-dependent, viable way. Using the infection model several stages of the disease from acute pleuropneumonia to chronic infection can be induced for research purposes.