Burkholderia is a bacterial genus comprising several pathogenic species, including two species highly pathogenic for humans, B. pseudomallei and B. mallei. B. thailandensis is a weakly pathogenic species closely related to both B. pseudomallei and B. mallei. It is used as a study model. These bacteria are able to exhibit multiple resistance mechanisms towards various families of antibiotics. By sequentially plating B. thailandensis wild type strains on chloramphenicol we obtained several resistant variants. This chloramphenicol-induced resistance was associated with resistance against structurally unrelated antibiotics including quinolones and tetracyclines. We functionally and proteomically demonstrate that this multidrug resistance phenotype, identified in chloramphenicol-resistant variants, is associated with the overexpression of two different efflux pumps. These efflux pumps are able to expel antibiotics from several families, including chloramphenicol, quinolones, tetracyclines, trimethoprim and some β-lactams, and present a partial susceptibility to efflux pump inhibitors. It is thus possible that Burkholderia species can develop such adaptive resistance mechanisms in response to antibiotic pressure resulting in emergence of multidrug resistant strains. Antibiotics known to easily induce overexpression of these efflux pumps should be used with discernment in the treatment of Burkholderia infections.

Recombinant antibodies are a highly successful class of therapeutic molecules, they are well adapted for use against bio-weapons (BW) as they act immediately, are often synergistic with other therapeutic molecules, have a long half-life and are well tolerated. Anthrax is regarded at high risk of being used as BW, and its pathogenic properties depend on toxins, which might be neutralized by antibodies. These toxins are made of three different types of sub-units (PA, LF, EF). Several anti-PA have been developed, including an original approach by our team. We have developed an anti-LF, as recommended by experts. Our anti-PA antibody, and to a lesser extend our anti-LF antibody, will be presented here.

A knockout mutant with a deletion in a quorum sensing system gene qseC was generated from the vaccine strain Francisella tularensis 15 by site-directed mutagenesis. The variant with the inactivated gene qseC differed from the parental strain in growth rate on solid nutrient medium but had the same growth dynamics in liquid nutrient medium. The mutation abolished almost completely the resistance of the vaccine strain to normal rabbit serum and its ability to survive in macrophages; in addition, the strain lost the residual virulence. A significant phenotypic alteration was observed in the lipopolysaccharide of F. tularensis. Particularly, the mutant strain synthesized no noticeable amount of the lipopolysaccharide with the high-molecular-mass O-polysaccharide, presumably as a result of impairing biosynthesis of the repeating unit, namely, a loss of the ability to incorporate a formyl group, an N-acyl substituent of 4-amino-4,6-dideoxy-D-glucose.

The threat posed by bioweapons (BW) could lead to the re-emergence of such deadly diseases as plague or smallpox, now eradicated from industrialized countries. The development of recombinant antibodies allows tackling this risk because these recombinant molecules are generally well tolerated in human medicine, may be utilized for prophylaxis and treatment, and because antibodies neutralize many BW. Recombinant antibodies neutralizing the lethal toxin of anthrax, botulinum toxins and the smallpox virus have in particular been isolated recently, with different technologies. Our approach, which uses phage-displayed immune libraries built from non-human primates (M. fascicularis) to obtain recombinant antibodies, which may later be super-humanized (germlinized), has allowed us to obtain such BWs-neutralizing antibodies.

Burkholderia pseudomallei est l’agent de la mélioïdose. Il s’agit d’une bactérie de l’environnement hydrotellurique, hautement pathogène pour l’homme. En France et en Europe, les cas de mélioïdose sont des cas d’importation, alors qu’en Asie du Sud-est et en Australie, cette maladie sévit sous forme d’endémie. La répartition géographique de cette maladie s’étend et touche maintenant le Brésil, Madagascar et l’Ile de la Réunion. Le diagnostic phénotypique de cette bactérie est aisé, mais il faut savoir penser à ce micro-organisme, qui n’est pas fréquent sous nos contrées. Son antibiogramme spécifique constitue une aide précieuse au diagnostic. Cette maladie est d’autant plus grave que les possibilités de traitement sont très restreintes et reposent sur une lourde antibiothérapie.

Melioidosis is an infection affecting both human and animal health. The causative agent is Burkholderia pseudomallei, a Gram-negative soil bacterium. Melioidosis is endemic in tropical areas of Southeast Asia and Northern Australia, and sporadic in many other countries. Clinical presentation is variable ranging from acute septicemia, isolated pulmonary infection, or chronic granulomatous lesions to asymptomatic forms with positive serology. There is no vaccine and treatment is difficult because B. pseudomallei is resistant to a wide range of antibiotics. Relapses are common. B. pseudomallei is listed as a biological risk class 3 and considered as a potential bioterrorism agent due to its high virulence by inhalation, to the difficulty of treatment, and to the lack of vaccine.

Le risque d’utilisation d’armes biologiques pourrait conduire à la réémergence de graves pathologies, telles que la peste ou la variole, maintenant éradiquées des pays développés. La prise en charge de ce risque, appelé risque biologique provoqué (RBP), comporte notamment le développement d’anticorps recombinants - qui est une voie assez sûre pour obtenir des molécules bien tolérées pour la prévention et le traitement des maladies humaines - parce que les anticorps neutralisent de nombreux agents pathogènes. Des travaux récents ont ainsi permis d’isoler des anticorps recombinants neutralisant la toxine létale du charbon, les toxines botuliques et le virus de la variole en particulier, grâce à différentes technologies. Notre approche, consistant à obtenir des anticorps recombinants en construisant des librairies immunes exposées à la surface de phages à partir de primates non humains (M. fascicularis), puis à humaniser ces anticorps par ingénierie, nous permet de participer efficacement à ces travaux.

Le réseau national des laboratoires Biotox-Piratox est une des composantes des plans nationaux de sauvegarde des populations. Il tire profit des leçons de la crise « charbon » de 2001, afin d’assurer l’utilisation optimale des capacités analytiques des laboratoires nationaux. L’organisation générale du réseau est articulée en trois niveaux de compétence, les laboratoires sentinelles, les laboratoires de zones de défense et les laboratoires à compétence nationale. Il met ainsi à la disposition des autorités locales et nationales une capacité de réponse flexible et adaptée aux actes malveillants ou alertes mettant en cause des agents biologiques ou des toxiques de guerre ou industriels.

La maladie du charbon est une zoo-anthroponose touchant les troupeaux et parfois l’homme en contact avec les produits animaux dans un contexte de maladies professionnelles, industrielles ou de toxi-infections alimentaires. Elle se présente sous formes cutanée, digestive et respiratoire. Bacillus anthracis est l’agent responsable de la maladie du charbon. C’est un bacille à Gram positif, immobile, capsulé, non hémolytique formant des spores. Alors qu’il est facile de traiter le charbon cutané par les antibiotiques, le charbon digestif, le charbon méningé et le charbon pulmonaire (ou d’inhalation) sont redoutables. Le cycle du charbon met en jeu une forme sporulée tellurique et une forme végétative capsulée produisant les toxines charbonneuses chez l’hôte. Toujours présente dans l’environnement hydrotellurique des zones d’enzootie, la maladie réémerge périodiquement en fonction des évolutions climatiques et écologiques ou des activités humaines. La grande résistance des spores dans l’environnement et la virulence du bacille du charbon en font un agent potentiel de guerre bactériologique et de bioterrorisme de premier plan. Il est donc important de savoir faire le diagnostic biologique de cette maladie qui s’appuie sur des techniques de bactériologie classique, de biologie moléculaire et d’immunologie.

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ABSTRACT Protective antigen (PA)-based anthrax vaccines acting on toxins are less effective than live attenuated vaccines, suggesting that additional antigens may contribute to protective immunity. Several reports indicate that capsule or spore-associated antigens may enhance the protection afforded by PA. Addition of formaldehyde-inactivated spores (FIS) to PA (PA-FIS) elicits total protection against cutaneous anthrax. Nevertheless, vaccines that are effective against cutaneous anthrax may not be so against inhalational anthrax. The aim of this work was to optimize immunization with PA-FIS and to assess vaccine efficacy against inhalational anthrax. We assessed the immune response to recombinant anthrax PA from Bacillus anthracis (rPA)-FIS administered by various immunization protocols and the protection provided to mice and guinea pigs infected through the respiratory route with spores of a virulent strain of B. anthracis. Combined subcutaneous plus intranasal immunization of mice yielded a mucosal immunoglobulin G response to rPA that was more than 20 times higher than that in lung mucosal secretions after subcutaneous vaccination. The titers of toxin-neutralizing antibody and antispore antibody were also significantly higher: nine and eight times higher, respectively. The optimized immunization elicited total protection of mice intranasally infected with the virulent B. anthracis strain 17JB. Guinea pigs were fully protected, both against an intranasal challenge with 100 50% lethal doses (LD50) and against an aerosol with 75 LD50 of spores of the highly virulent strain 9602. Conversely, immunization with PA alone did not elicit protection. These results demonstrate that the association of PA and spores is very much more effective than PA alone against experimental inhalational anthrax.

Inhalational anthrax is a life-threatening infectious disease of considerable concern, especially because anthrax is an emerging bioterrorism agent. The exact mechanisms leading to a severe clinical form through the inhalational route are still unclear, particularly how immobile spores are captured in the alveoli and transported to the lymph nodes in the early steps of infection. We investigated the roles of alveolar macrophages and lung dendritic cells (LDC) in spore migration. We demonstrate that alveolar macrophages are the first cells to phagocytose alveolar spores, and do so within 10 min. However, interstitial LDCs capture spores present in the alveoli within 30 min without crossing the epithelial barrier suggesting a specific mechanism for rapid alveolus sampling by transepithelial extension. We show that interstitial LDCs constitute the cell population that transports spores into the thoracic lymph nodes from within 30 min to 72 h after intranasal infection. Our results demonstrate that LDCs are central to spore transport immediately after infection. The rapid kinetics of pathogen transport may contribute to the clinical features of inhalational anthrax.

ABSTRACT The anthrax lethal toxin (LT) consists of two subunits, the protective antigen (PA) and the lethal factor (LF), and is essential for anthrax pathogenesis. Several recombinant antibodies directed against PA and intended for medical use have been obtained, but none against LF, despite the recommendations of anthrax experts. Here we describe an anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque ( Macaca fascicularis ) and was obtained by phage display. Panning of the library of 1.8 × 10 8 clones allowed the isolation of 2LF, a high-affinity (equilibrium dissociation constant, 1.02 nM) scFv, which is highly neutralizing in the standardized in vitro assay (50% inhibitory concentration, 1.20 ± 0.06 nM) and in an in vivo assay. The scFv neutralizes anthrax LT by inhibiting the formation of the LF-PA complex. The genes encoding 2LF are very similar to those of human immunoglobulin germ line genes, sharing substantial (84.2%) identity with their most similar, germinally encoded counterparts; this feature favors medical applications. These results, and others formerly published, demonstrate that our approach can generate antibody fragments suitable for prophylaxis and therapeutics.

Anti-PA recombinant antibodies have been developed for anthrax treatment, but other sub-units have not been targeted despite anthrax experts recommendations. Here, we describe an anti-LF scFv that was obtained from a macaque (Macaca fascicularis) immune antibody gene library (1.8x108 clones) in pHAL14 phagemid vector. One scFv clone (2LF) selected from the library, had a high-affinity (KD = 1.02 nM), was highly neutralizing in the standardized in vitro (IC50 = 1.17 ± 0.06 nM) and in an in vivo assays. The genes encoding 2LF are similar to human immunoglobulin germline genes, and assigned to subgroups of human V, (D) or J genes by IMGT/V-QUEST. 2LF framework regions have a 84% identity with their most similar, germinally encoded human counterparts. This scFv neutralizes the anthrax lethal toxin by inhibiting the formation of the LF-PA complex, as shown in a competition assay. This inhibition suggests that 2LF interacts with domain 1 of LF, which is partially shared with EF and 2LF also reacted with EF, in ELISA and SPR. A 2LF-derived IgG, targeting LF and maybe EF, would be suitable for medical use. Antibody engineering. December, 10-14, 2006. San Diego, CA

Inhalational anthrax is a life-threatening infectious disease of considerable concern, especially as a potential bioterrorism agent. Progress is gradually being made towards understanding the mechanisms used by Bacillus anthracis to escape the immune system and to induce severe septicaemia associated with toxaemia and leading to death. Recent advances in fundamental research have revealed previously unsuspected roles for toxins in various cell types. We summarize here pathological data for animal models and macroscopic histological examination data from recent clinical records, which we link to the effects of toxins. We describe three major steps in infection: (i) an invasion phase in the lung, during which toxins have short-distance effects on lung phagocytes; (ii) a phase of bacillus proliferation in the mediastinal lymph nodes, with local effects of toxins; and (iii) a terminal, diffusion phase, characterized by a high blood bacterial load and by long-distance effects of toxins, leading to host death. The pathophysiology of inhalational anthrax thus involves interactions between toxins and various cell partners, throughout the course of infection.

The recent bioterrorist attacks have stressed the need of a better knowledge of Bacillus anthracis infection pathophysiology. We present here the increasing interests of B. anthracis studies in term of bio-defense, the main pathogen characteristics, the main clinical features of inhalational anthrax (the pulmonary form of the disease), and recent aspects of its physiopathology. Next, we address the main results concerning the toxin effects on immune system through impairing the dendritic cell functions, and we analyze the singular role of anthrax toxins in immune evasion.

Bacillus anthracis secretes 2 toxins: lethal toxin (LT) and edema toxin (ET). We investigated their role in the physiopathologic mechanisms of inhalational anthrax by evaluating murine lung dendritic cell (LDC) functions after infection with B. anthracis strains secreting LT, ET, or both or with a nontoxinogenic strain. Three lung cell populations gated on CD11c/CD11b expression were obtained after lung digestion: (1) CD11c(high)/CD11b(low) (alveolar macrophages), (2) CD11c(intermediate (int))/CD11b(int) (LDCs), and (3) CD11c(low)/CD11b(high) (interstitial macrophages or monocytes). After infection with LT-secreting strains, a decrease in costimulatory molecule expression on LDCs was observed. All CD11c+ cells infected with a nontoxinogenic strain secreted tumor necrosis factor (TNF)- alpha , interleukin (IL)-10, and IL-6. LT-secreting strains inhibited overall cytokine secretion, whereas the ET-secreting strain inhibited only TNF- alpha secretion and increased IL-6 secretion. Similar results were obtained after preincubation with purified toxins. Our results suggest that anthrax toxins secreted during infection impair LDC function and suppress the innate immune response.

The recent bioterrorist attack's have stressed the need of a better knowledge of Bacillus anthracis infection pathophysiology. We present here the increasing interests of B. anthracis studies in term of bio-defense, the main pathogen characteristics, the main clinical features of inhalational anthrax (the pulmonary form of the disease), and recent aspects of its physiopathology. Next, we address the main results concerning the toxin effects on immune system through impairing the dendritic cell functions and we analyze the singular role of anthrax toxins in immune evasion.