I )Bordetella pertussis VIRULENCE DETERMINANTS
Does adenylate cyclase-hemolysin toxin interact with a receptor on eucaryotic cells ? [N. Khelef, in collaboration with P. Guermonprez and C. Leclerc]
We previously shown that adenylate cyclase-hemolysin (AC-Hly) toxin, secreted by B. pertussis, is responsible in vivo and in vitro of alveolar macrophages death via apoptosis but is not cytotoxic for epithelial cells. For this reason we made the hypothesis that a specific receptor for AC-Hly exists on the macrophage surface. In fact, using different specific antibodies we recently showed that the integrin Mac-1 is the receptor of AC-Hly. Since epithelial cells do not express Mac-1, they are resistant to AC-Hly toxin.
What is the localisation of adenylate cyclase-hemolysin inside macrophages or epithelial cells ? [N. Khelef in collaboration with P. Gounon]
Our first results show, using confocal microscopy and immunofluorescence labelling, that AC-Hly binds to the plasma membrane of macrophages very rapidly and is localised in small vesicules probably derived from the Golgi. The localisation seems to be different in epithelial cells.
Which are the adenylate cyclase-hemolysin epitopes important for the induction of a protective immunity ? [A. Le Flèche in collaboration with P. Sebo]
We previously shown that the post translationally modification and the C-terminal part of B. pertussis AC-Hly are important for the induction of a protective immunity. B. pertussis and B. bronchiseptica harbor the same activities and more than 95 % sequence identity. The differences are located at the C-terminal domain. We purified both enzymes as well as recombinant enzymes expressed by E. coli K-12. We showed that i) there is no cross reactive protective immunity between both enzymes ii) both recombinant enzymes are unable to induce a protective immunity. All these results confirm that the C-terminal part of AC-Hly is important for the induction of a protective immunity and that recombinant enzymes are different from the enzymes produced by B. pertussis and B. bronchiseptica.
II )PHYSIOPATHOLOGY OF BORDETELLA INFECTIONS
Interactions of Bordetella pertussis with human tracheal epithelial cells [L. Bassinet, P. Gueirard in collaboration with P. Gounon]
Epithelial cell plays a role in the recrutment of inflammary cells via cytokines secretion. For this reason our aim is to analyse which cytokines are secreted by human tracheal epithelial cells infected by B. pertussis or in contact with purified toxins or adhesins. Our preliminary results show that B. pertussis strains induce IL-6 secretion while there is no induction or inhibition of secretion of IL-8.
Do Bordetella pertussis or Bordetella bronchiseptica survive intracellularly. In which cell ? [P. Gueirard, S. Thiberge]
The study is performed in vivo using our murine respiratory model and in vitro. Is the target cell a dendritic leukocyte ?
III )Whooping cough prevention
Does immunity induced by acellular vaccines last as long as the immunity induced by whole-cell vaccine ? [E. Njamkepo in collaboration with GlaxoSmithKline laboratory]
In order to answer this question we compared immunity induced by both vaccine in children, in France. Both vaccines induced similar type of immune response although the intensity of the immune response is higher after vaccination with acellular vaccine, the duration of immunity is similar.
What is the prevalence of whooping cough in adults in France ? [E. Njamkepo, A. Le Flèche, P. Gueirard in collaboration with I. Parent (AMP), S. Gilberg (SFTG) and Aventis-Pasteur and Aventis Pasteur-MSD]
In France, since several years a resurgence of whooping cough is observed with infants contamined by adolescents and adults. In order to confirm this epidemiology we undertook with 80 general practitioners, a study to determine the prevalence of whooping cough in adults, describe the clinical symptoms during the disease and evaluate the ability of biological
diagnosis such as culture, PCR and serology to diagnose the disease in adult. 70 cases were confirmed over 217 which represent a prevalence of 32 %. All confirmed case were characterized by : a higher frequency of work stops, whoops cough.
Is there a genetical drift in Bordetella pertussis population over vaccinal pressure ? [C. Boursaux-Eude, F. Rimlinger, C. Weber]
Analysis, by typing technique or sequencing of toxin and adhesin structural genes, of isolates circulating in France, a vaccinated country since more than thirty five years with a whole-cell vaccine, confirmed that B. pertussis isolates circulating before and after introduction of generalized vaccination are different. Our analysis suggests that B. pertussis population varies every 3-5 years.
We previously shown that PRN expressed by B. pertussis, B. parapertussis and B. bronchiseptica are very similar but do not induce a cross reactive protective immunity. The differences between the three proteins are located on two regions of the PRN protein (I and II) containing repeated regions. Analysis of several B. pertussis, parapertussis and bronchiseptica PRN indicates that B. pertussis PRN variation is located on region I whereas B. bronchiseptica PRN variation is mainly located on region II. This result shows the importance of PRN region II for the induction of protective immunity. One can suppose that variation of this region could induce vaccine failure.
Analysis of isolates circulating in Japan, a vaccinated country using acellular vaccines since twenty years showed that circulating isolates are similar to the vaccinal strain. Use of acellular vaccines does not seem to induce a strong selection pressure. However, a greater number of isolates has to be analysed before to draw any conclusion.
Is Bordetella pertussis polymorphism affecting vaccine efficacy ? [S. Thiberge in collaboration with GlaxoSmithKline and Aventis-Pasteur Laboratories]
Our murine intranasal model, was used to compare the ability of different whole cell and acellular vaccines. Till now, all vaccines are able to induce a protective immunity against infections due to different variants.
IV )BORDETELLA BRONCHISEPTICA HUMAN AND ANIMAL INFECTIONS
Bordetella bronchiseptica human and animal infections [P. Gueirard, S. Thiberge, L. Guillemot in collaboration with C. Barny]
B. bronchiseptica is a respiratory pathogen for many mamals species, including human. In human, B. bronchiseptica is considered as an opportunist pathogen for immunodepressed patient or patient with respiratory problems. We previously described that this bacterium can induce chronic infections. We confirmed the zoonotic character of this infection with two patients contaminated with either infected rabbit or dog. Furthermore, samples from dogs showed that the bacterium can be isolated on vaccinated dogs.
Studies will continue because of the increase of immunocompromised patients, absence of cross protection of new pertussis vaccine, low efficacy of veterinary vaccines and antibiotic resistances carried by this bacterium.
V )CENTRE NATIONAL DE REFERENCE
CNR [G. Coralie, P. Gueirard, S. Thiberge]
The missions of this Centre is to confirm the identification of Bordetella isolates collected in France, to maintain the collection, to teach French bacteriologists, to analyse isolates by Pulsed-Field-gel electrophoris, as well as the toxins and adhesins they express to set up sensitive and specific biological diagnosis techniques.
VI )QUALITY INSURANCE
Quality Insurance [F. Fouque]
The set up of procedures and quality insurance in the laboratory was pursued.