Stéphane Rioux

Prevention of Pneumococcal Disease in Mice Immunized with Conserved Surface-Accessible Proteins

ABSTRACT The development of a vaccine against Streptococcus pneumoniae has been complicated by the existence of at least 90 antigenically distinct capsular serotypes. Common protein-based vaccines could represent the best strategy to prevent pneumococcal infections, regardless of serotype. In the present study, the immunoscreening of an S. pneumoniae genomic library allowed the identification of a novel immune protein target, BVH-3. We demonstrate that immunization of mice with BVH-3 elicits protective immunity against experimental sepsis and pneumonia. Sequence analysis revealed that the bvh-3 gene is highly conserved within the species. Since the BVH-3 protein shows homology at its amino-terminal end with other pneumococcal proteins, it was of interest to determine if protection was due to the homologous or to the protein-specific regions. Immunoprotection studies using recombinant BVH-3 and BVH-3-related protein fragments as antigens allowed the localization of surface-exposed and protective epitopes at the protein-specific carboxyl termini, thus establishing that BVH-3 is distinct from other previously reported protective protein antigens. Immunization with a chimeric protein comprising the carboxyl-terminal regions of BVH-3 and of a BVH-3-related protein improved the protection by targeting two surface pneumococcal components. Thus, BVH-3 and the chimeric protein hold strong promise as vaccine components to control pneumococcal disease.

Protection from Group B Streptococcal Infection in Neonatal Mice by Maternal Immunization with Recombinant Sip Protein

ABSTRACT The protective potential of antibodies directed against group B streptococcus (GBS) Sip surface protein was determined by using the mouse neonatal infection model. Rabbit Sip-specific antibodies administered passively to pregnant mice protected their pups against a GBS lethal challenge. In addition, active immunization with purified recombinant Sip protein of female CD-1 mice induced the production of specific antibodies that also confer protection to the newborn pups against GBS strains of serotypes Ia/c, Ib, II, III, and V. These data confirm that Sip-specific antibodies can cross the placenta and conferred protective immunity against GBS infections.

Localization of Surface Immunogenic Protein on Group B Streptococcus

ABSTRACT The localization and accessibility of the group B streptococcus (GBS) surface immunogenic protein (Sip) at the surface of intact GBS cells were studied by flow cytometric assay and immunogold electron microscopy. Antibodies present in pooled sera collected from mice after immunization with purified recombinant Sip efficiently recognized native Sip at the surfaces of the different GBS strains tested, which included representatives of all nine serotypes. Examination of GBS cells by immunogold electron microscopy revealed that the Sip-specific antibodies attached preferentially to polar sites and the septal region. This result confirmed that Sip is exposed at the intact-cell surface, but it also suggests that its distribution is restricted to certain regions of the cell.

Isolation and characterization of LPS mutants of Actinobacillus pleuropneumoniae serotype 1

Abstract. The major adhesin of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, has been previously identified as lipopolysaccharide (LPS). The purpose of the present study was to isolate and characterize A. pleuropneumoniae LPS mutants. Screening of LPS mutants was performed with colony dot and sensitivity to novobiocin. One mutant obtained by colony dot (F19) and one mutant selected for its increased sensitivity to novobiocin (33.1) did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 O-antigen compared with the parent strain. Mutants F19 and 33.1 did not express high-molecular-mass LPS bands as determined in silver-stained SDS-PAGE gels. The core-lipid A region of mutant 33.1 and of the parent strain had similar relative mobilities and reacted with serum from a pig experimentally infected with the serotype 1 reference strain of A. pleuropneumoniae, while the same region in mutant F19 showed faster migration and did not react with this serum. Use of piglet tracheal frozen sections indicated that mutant F19 was able to adhere to piglet trachea as well as the parent strain, while mutant 33.1 adhered [half as much as] the parent strain. Finally, both LPS mutants were markedly less virulent in mice than the parent strain. Taken together, our observations support the idea that LPS is an important virulence factor of A. pleuropneumoniae.

Evaluation of the protective efficacy of Actinohacillus pleuropneumoniae serotype 1 detoxified lipopolysaccharides or O-polysaccharide-protein conjugate in pigs

The major adhesin of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, has been previously identified as the lipopolysaccharide (LPS). Experiments in our laboratory have shown that mice immunised with different A pleuropneumoniae serotype 1 LPS preparations were protected against a challenge with a virulent A pleuropneumoniae serotype 1 isolate. The purpose of the present study was to evaluate the protection of pigs against experimental A pleuropneumoniae infection following immunisation with two of these LPS preparations. Groups of five specific pathogen free (SPF) pigs were injected twice with one of the following antigen preparations: detoxified LPS, O-polysaccharide-BSA conjugate, a commercial bacterin, or PBS. Two weeks after the second injection, pigs were challenged intranasally with a virulent A pleuropneumoniae serotype 1 strain. Upon macroscopic examination, fibrino-haemorrhagic pleuropneumonia, compatible with A pleuropneumoniae infection, was observed in one to four pigs in each group. The more extensive lesions were present in control, unimmunised pigs and in animals vaccinated with the O-polysaccharide-BSA conjugate. The highest survival rate was recorded when the pigs had been immunised with detoxified LPS or the commercial bacterin. Taken together, our results suggest that a protection comparable with the one obtained with a commercial bacterin was observed when pigs were immunised with a single class of molecules, detoxified LPS. Most importantly, these results confirm the important role of A pleuropneumoniae LPS in protection against porcine pleuropneumonia. Finally, our results also support the idea that mice are not an appropriate model for the evaluation of porcine pleuropneumonia vaccines.

Evaluation of protective efficacy of an Actinobacillus pleuropneumoniae serotype 1 lipopolysaccharide-protein conjugate in mice

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. The major adhesin of A. pleuropneumoniae has previously been identified as a lipopolysaccharide (LPS), and more recently, we demonstrated that high molecular mass LPS were involved in A. pleuropneumoniae adherence to porcine respiratory tract cells. We postulated that immunization with a LPS-based vaccine may confer a protective immunity. The high molecular mass O-polysaccharides obtained after acid hydrolysis and chromatographic separation were conjugated to bovine serum albumin (BSA) as a protein carrier. Groups of mice were injected twice with the following antigen preparations: whole-cell preparation, outer membrane preparation, O-polysaccharide-BSA conjugate, hydrolyzed LPS and phenol/water extracted LPS. A combination of different adjuvants was also used during these immunization procedures to induce a stronger immunological response to the polysaccharide antigen. Two weeks after the second injection, the mice were challenged intranasally with either homologous A. pleuropneumoniae serotype 1 strain or a serotype 5 strain. The highest survival rate, up to 80%, compared to the control groups (P < 0.05), was recorded when the mice were injected twice with 15 micrograms of carbohydrates of O-polysaccharide-BSA conjugate mixed with the saponin-derived adjuvant Quil A. Survival rates of between 60 and 70%, twice those observed in the control groups immunized with PBS, were recorded in mice injected with the O-polysaccharide-BSA conjugate mixed with other adjuvant preparations such as alhydrogel, peanut oil and Freunds incomplete adjuvant. However, the protection induced by the conjugate antigen preparation was serotype specific, because mice challenged with a serotype 5 strain were killed. Taken together, these results confirm the important role of A. pleuropneumoniae LPS in pathogenesis.

Identification of genes involved in biosynthesis of Actinobacillus pleuropneumoniae serotype 1 O-antigen and biological properties of rough mutants

Actinobacillus pleuropneumoniae is an important pathogen of swine. Lipopolysaccharide (LPS) has been identified as the major adhesin of A. pleuropneumoniae and it is involved in adherence to porcine respiratory tract cells. We previously generated seven rough LPS mutants of A. pleuropneumoniae serotype 1 by using a mini-Tn10 transposon mutagenesis system [Rioux S, Galarneau C, Harel J et al. Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1. Can J Microbiol 1999; 45: 1017—1026]. The purpose of the present study was to characterize these mutants in order to learn more about LPS O-antigen biosynthesis genes and their organization in A. pleuropneumoniae, and to determine the surface properties and virulence in pigs of these isogenic mutants. By mini-Tn10 insertions in rough mutants, four putative genes (ORF12, ORF16, ORF17, and ORF18) involved in O-antigen biosynthesis in A. pleuropneumoniae serotype 1 were found within a region of 18 ORFs. This region is homologous to the gene cluster of serotype-specific O-polysaccharide biosynthesis from A. actinomycetemcomitans strain Y4 (serotype b). Two mutants showed homology to a protein with identity to glycosyltransferases (ORF12); two others had the mini-Tn10 insertion localized in genes encoding for two distinct proteins with identity to rhamnosyltransferases (ORF16 and ORF17) and three showed homology to a protein which is known to initiate polysaccharide synthesis (ORF18). These four ORFs were also present in A. pleuropneumoniae serotypes 9 and 11 that express an O-antigen that serologically cross-reacts with serotype 1. Evaluation of some biological properties of rough mutants seems to indicate that the absence of O-chains does not appear to have an influence on the virulence of the bacteria in pigs and on the overall surface hydrophobicity, charge and hemoglobin-binding activity, or on LAL activation. An acapsular mutant was included in the present study in order to compare the influence of O-chains and capsule polysaccharides on different cell surface properties. Our data suggest that capsular polysaccharides and not O-chains polysaccharides have a major influence on surface properties of A. pleuropneumoniae serotype 1 and its virulence in pigs.

Universal Proteins As an Alternative Bacterial Vaccine Strategy

In the last two decades, discoveries in biological sciences have allowed vaccine research to expand rapidly. Progress in the understanding of the regulatory mechanisms of the immune response to infection, molecular biology, genomics, proteomics and bioinformatics have revolutionized the way vaccines are designed. Vaccinology has established its own credibility, and it is no longer only a subject in microbiology and immunology classes but a true complex discipline. Vaccines are no longer just crude and complex preparations of killed or attenuated microorganisms but can be defined as proteins, polysaccharides (Ps), or nucleic acids that are delivered to the immune system as single entities, as part of complex particles, or by live attenuated agents or vectors, thereby inducing specific responses that inactivate, destroy, or suppress pathogens1.