Overexpression of O-polysaccharide chain length regulators in Gram-negative bacteria using the Wzx-/Wzy-dependent pathway enhances production of defined modal length O-polysaccharide polymers for use as haptens in glycoconjugate vaccines.

AIMS: O-polysaccharide (OPS) molecules are protective antigens for several bacterial pathogens, and have broad utility as components of glycoconjugate vaccines. Variability in the OPS chain length is one obstacle towards further development of these vaccines. Introduction of sizing steps during purification of OPS molecules of suboptimal or of mixed lengths introduces additional costs and complexity while decreasing the final yield. The overall goal of this study was to demonstrate the utility of engineering Gram-negative bacteria to produce homogenous O-polysaccharide populations that can be used as the basis of carbohydrate vaccines by overexpressing O-polysaccharide chain length regulators of the Wzx-/Wzy-dependent pathway. METHOD AND RESULTS: The O-polysaccharide chain length regulators wzzB and fepE from Salmonella Typhimurium I77 and wzz2 from Pseudomonas aeruginosa PAO1 were cloned and expressed in the homologous organism or in other Gram-negative bacteria. Overexpression of these Wzz proteins in the homologous organism significantly increased the proportion of long or very long chain O-polysaccharides. The same observation was made when wzzB was overexpressed in Salmonella Paratyphi A and Shigella flexneri, and wzz2 was overexpressed in two other strains of P. aeruginosa. CONCLUSIONS: Overexpression of Wzz proteins in Gram-negative bacteria using the Wzx/Wzy-dependant pathway for lipopolysaccharide synthesis provides a genetic method to increase the production of an O-polysaccharide population of a defined size. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods presented herein represent a cost-effective and improved strategy for isolating preferred OPS vaccine haptens, and could facilitate the further use of O-polysaccharides in glycoconjugate vaccine development.

Pertactin deficient Bordetella pertussis present a better fitness in mice immunized with an acellular pertussis vaccine.

Bordetella pertussis is the etiologic agent of whooping cough and has been the target of vaccination for over fifty years. The latest strategies include the use of acellular pertussis vaccines that induce specific immunity against few virulence factors amongst which pertactin is included in three and five component acellular pertussis vaccines. Recently, it has been reported that B. pertussis clinical isolates loose the production of this adhesin in regions reaching high vaccine coverage with vaccines targeting this virulence factor. We here demonstrate that isolates not producing pertactin are capable of sustaining longer infection as compared to pertactin producing isolates in an in vivo model of acellular pertussis immunization. Loosing pertactin production might thus provide a selective advantage to these isolates in this background, which could account for the upraise in prevalence of these pertactin deficient isolates in the population.

Antibody-mediated inhibition of Bordetella pertussis adenylate cyclase-haemolysin-induced macrophage cytotoxicity is influenced by variations in the bacterial population.

Whooping cough is a vaccine-preventable disease presenting with epidemic cycles linked to natural and/or vaccine-driven evolution of the aetiological agent of the disease, Bordetella pertussis. Adenylate cyclase-haemolysin (AC-Hly) is a major toxin produced by this pathogen, which mediates macrophage apoptosis in vitro and in vivo. While current acellular pertussis vaccine (APV) formulations do not include AC-Hly, they all contain pertussis toxin and can comprise filamentous haemagglutinin (FHA), which interacts with AC-Hly, and pertactin (PRN), which has been hypothesized also to interact with AC-Hly. We aimed to study the capacity of specific antibodies to inhibit the in vitro B. pertussis AC-Hly-mediated cytotoxicity of J774A.1 murine macrophages in a background of a changing bacterial population. We demonstrate that: (i) clinical isolates of different types or PRN phenotype are all cytotoxic and lethal in the mouse model of respiratory infection; (ii) lack of PRN production does not impact AC-Hly-related phenotypes; (iii) anti-AC-Hly antibodies inhibit cell lysis whatever the phenotype of the isolate, while anti-PRN antibodies significantly inhibit cell lysis provided the isolate produces this antigen, which might be relevant in vivo for APV-induced immunity; and (iv) anti-FHA antibodies only inhibit lysis induced by isolates collected in 2012, maybe indicating specific characteristics of epidemic lineages of B. pertussis.

Evolution of French Bordetella pertussis and Bordetella parapertussis isolates: increase of Bordetellae not expressing pertactin.

Bordetella pertussis and Bordetella parapertussis are closely related bacterial agents of whooping cough. Whole-cell pertussis (wP) vaccine was introduced in France in 1959. Acellular pertussis (aP) vaccine was introduced in 1998 as an adolescent booster and was rapidly generalized to the whole population, changing herd immunity by specifically targeting the virulence of the bacteria. We performed a temporal analysis of all French B. pertussis and B. parapertussis isolates collected since 2000 under aP vaccine pressure, using pulsed-field gel electrophoresis (PFGE), genotyping and detection of expression of virulence factors. Particular isolates were selected according to their different phenotype and PFGE type and their characteristics were analysed using the murine model of respiratory infection and in vitro cell cytotoxic assay. Since the introduction of the aP vaccines there has been a steady increase in the number of B. pertussis and B. parapertussis isolates collected that are lacking expression of pertactin. These isolates seem to be as virulent as those expressing all virulence factors according to animal and cellular models of infection. Whereas wP vaccine-induced immunity led to a monomorphic population of B. pertussis, aP vaccine-induced immunity enabled the number of circulating B. pertussis and B. parapertussis isolates not expressing virulence factors to increase, sustaining our previous hypothesis.