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  Director : GICQUEL Brigitte (bgicquel@pasteur.fr)



Caused by mycobacteria of the Tuberculosis complex, tuberculosis still represents a major problem of public health: it is responsible for about three million deaths each year. BCG, the only vaccine available today is of a relative efficacy. As for the use of antibiotics, it faces the emergence of multiresistant strains of bacteria. This unit is involved in the characterisation of M. tuberculosis virulence factors, and of the immune responses induced in the host by this bacterium and BCG. This work could lead to the identification of targets for new drugs against tuberculosis, of bacterial components to include in a new vaccine, or even to the isolation of a new attenuated strain, more efficient than BCG. The Unit is also studying the possibility to use recombinant strains of BCG to protect against other diseases, such as AIDS.



Genetic study of virulence determinants (M. Jackson, O. Neyrolles, B. Gicquel)

A thermo-sensitive vector derived from pAL5000 of Mycobacterium fortuitum and containing the counterselective sacB (ts/sacB vector) marker was used to construct allelic exchange and transposon mutants in species belonging to the M. tuberculosis complex. This vector was used to identify allelic exchange events leading to the inactivation of certain genes, chosen for study based on their similarity to genes known to be involved in virulence or intracellular survival in other pathogens. In collaboration with Carlos Martin and his team (Zaragoza Medical School, Spain), we recently isolated a mutant in which phoP, which encodes a putative regulator, is inactivated by an insertional mutation. This gene, which is close to the putative phoR gene, is essential for the multiplication of M. tuberculosis in cultured mouse macrophages in vitro and in mice, particularly in the lungs and, to a lesser extent, in the spleen and kidneys. The phoP/phoR gene pair is similar to the two-component system phoP/phoQ, which regulates several virulence determinants in Salmonella. In the M. bovis stain with multiple antibiotic resistance that caused an epidemic in Spain, the IS6110 insertion sequence is located several base pairs upstream from phoP. This suggests that this gene is regulated differently in this clinical isolate, possibly rendering this strain more virulent and therefore more transmissible than most multiple antibiotic-resistant M. tuberculosis strains.

The ts/sacB vector has also been used to build transposition mutant libraries for M. bovis BCG and M. tuberculosis. One library, containing 7000 mutants derived from M. tuberculosis was ordered and used for a PCR search for mutations in candidate genes. A second library containing 4000 mutants was constructed using a tagged transposon. This library was used for STM (signature-tagged transposon mutagenesis) to search directly for mutants with attenuated virulence in mice. About 2000 mutants were screened for their ability to multiply in the lungs of mice. Sixteen attenuated mutants were selected and the corresponding mutations were characterized. Four mutations were identified in a 50 kb region of the genome containing 13 genes involved in the synthesis and transport of phthiocerol dimycoserosates. The role of this lipid in pathogenesis is being studied in the laboratory. We are also interested in characterizing other attenuated mutants isolated by STM that present other deficiencies in their cell envelope. New screening techniques of STM mutants have been developed which should lead to the identification of new virulence factors of the tubercle bacillus.

The protective efficacy of the M. tuberculosis attenuated strains isolated in the laboratory is being studied as part of the EEC "TB vaccine cluster" coordinated by Pr. Brigitte Gicquel: http://www.pasteur.fr/EC_TBvaccine/.

Functional analysis of virulence factors (Jean-Marc Reyrat)

Reporter genes (alkaline phosphatase), tagged insertional mutagenesis and computer tools have been used to identify a number of virulence genes. Several virulence loci are currently being studied, including those encoding Rv1395, a transcriptional activator of the AraC/XylS family and Erp (Rv3810), a protein secreted by M. tuberculosis.

Erp, which is secreted by the bacteria of the tuberculosis complex, was identified by a method involving the use of alkaline phosphatase as a reporter. In M. tuberculosis, inactivation of the erp gene by allelic exchange greatly attenuated virulence both in vitro, in cultured macrophages, and in vivo, in the mouse model of tuberculosis. We recently showed that Erp is ubiquitous in mycobacteria. This secreted protein is composed of three domains. The central domain consists of a tandem repeat of the PGLTS domain. This central domain is the most variable between species: with four repeats in M. leprae and 24 repeats in M. xenopi (Figure 1). Analysis of the genomic region surrounding the erp gene showed that most of the genes in this region are cell wall biosynthesis genes. We are currently studying the role of Erp in development of the structure of the mycobacterial wall and the effect of the number of PGLTS repeats on M. tuberculosis virulence.

Another aspect of the project concerns secretion by myobacteria. A certain number of virulence factors and antigens are present on the surface of the bacillus or in the extracellular environment. We used the Staphylococcus aureus nuclease gene as a reporter gene to show that this protein is secreted into the extracellular environment independently of any potential signal sequence. Cytoplasmic markers showed that this phenomenon was independent of bacterial autolysis. These results suggest the existence of an unidentified secretion pathway independent of the general pathway (Recchi et al., Microbiology, in press).

BCG and new vaccines derived from BCG (Nathalie WINTER)

Mycobacterium bovis BCG, is one of the six vaccines of the Expanded Program for Immunisation of the World Health Organisation. It is the most widely used live attenuated bacterial vaccine in humans and confers some protection against tuberculosis and leprosy. Its ability to persist in professional antigen presenting cells such as macrophages and dendritic cells makes it an attractive vector for the development of recombinant vaccines with long term efficacy. Thanks to the developments of mycobacterial genetic tools, particularly in our laboratory, it is possible to engineer BCG to express heterologous genes of various origins. In order to develop candidate vaccines against AIDS, we have constructed several recombinant BCG (rBCG) strains expressing genes from the Simian Immunodeficiency Virus SIVmac251. Macaques infected with SIVmac251 develop an AIDS like syndrom, making this animal model useful for evaluating vaccine strategies against HIV.

Equal doses of three rBCG strains expressing the SIVmac251 nef gene involved in the regulation of the viral cycle and the structural gag(p26) and env genes were administered together as a mixture (the rBCG-SIV3 vaccine) to cynomolgus macaques. Monkeys received one shot of the rBCG-SIV3 vaccine by the intradermal route, the classical administration route for humans. Over 80% of HIV infections are transmitted sexually meaning that local mucosal immune responses will likely play an important role in protection. Thus, a booster dose of rBCG-SIV3 was administered to the animals either by the oral or the rectal route. Immune responses induced by rBCG-SIV3 vaccination were assessed in Peripheral Blood Mononuclear Cells (PBMC). A cytotoxix T-cell response (CTL) directed against the Env and Gag antigens was observed in most animals inoculated with rBCG-SIV3. Lysis was accompanied by production of IFNg indicating that rBCG-SIV3 vaccination induces a Th1/Tc1 cellular immune response. After the mucosal booster, a high increase in IFNg production was detected. The SIVmac251 challenge was administered by the rectal route 4 months after the mucosal rBCG-SIV booster. Although all animals became infected, one monkey receiving the rBCG-SIV3 booster by the rectal route completely controlled its plasmatic viral load indicating that the rBCG-SIV3 vaccine induces some level of protection against SIV. After the viral challenge, a strong humoral and cellular anamnestic response against SIV Gag(p26) and Env antigens was observed in the rBCG-SIV3 vaccinated animals showing that a memory response is induced. We have also observed that the rBCG strains used in this study were genetically unstable in vivo. These studies were carried out in collaboration with Dr Roger Le Grand (CEA, Fontenay-aux-Roses) and Dr Alain Venet (Faculté de Médecine Paris Sud).

In order to improve genetic stability of rBCG strains, we have recently developed new integrative expression vectors. Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the nef and gag(p26) genes from SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopy replicating vector and the expression levels of both nef and gag in the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of the nef-gag operon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, the (nef-gag) operon was inserted site-specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer-lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes despite the fact that a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude than the replicative rBCG strain containing several copies of the genes (Méderlé et al., 2002, Infection and Immunity, 70, 303-314).

The laboratory is also interested in the mechanisms by which the immune response is induced after administration of the BCG vaccine. Of particular importance is the interaction between BCG and dendritic cells (DC), the most professional antigen presenting cells. In collaboration with Eric Prina and Jean-Claude Antoine (Unité d'Immunophysiologie et Parasitisme Intracellulaire) we are characterising the mycobacterial vacuole after BCG infection in DC. In collaboration with Claude Leclerc's team (Unité de Biologie des Régulations Immunitaires) we have shown that DC play a pivotal role in the very early steps of mycobacterial infection in mice by initiating the immune response and producing IL-12, a key-cytokine in the Th1 orientation of the immune response (Jiao et al., Journal of Immunology, In press).

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  Office staff Researchers Scientific trainees Other personnel

SINNO Helena, executive secretary IP, hsinno@pasteur.fr

GICQUEL Brigitte, Professor I.P., bgicquel@pasteur.fr

REYRAT Jean-Marc, CR 1, INSERM, jmreyrat@pasteur.fr

WINTER Nathalie, CR 2 IP, nwinter@pasteur.fr

CAMACHO Luis Reinaldo, graduate student

EBRAHIMI RAD Mina, post-doctoral fellow

FERNANDEZ BOECHAT Neio, MD, graduante student

FILLACHET Fabrice, graduate student

FILLON-JACKSON Mary, Roux fellow, I.P.

GHAEM-MAGHAMI, post-doctoral fellow

KORDULAKOVA Jana, graduante student

MARTINEZ Valérie, MD, graduante student

MEDERLE-MANGEOT Isabelle, graduate student

RECCHI Chiara, graduate student

ROUSSEAU Cécile, graduate student

SONDEN Berit, post-doctoral fellow

SUPPLY Philip, postd-doctoral fellow

AUBERT-PIVERT Elisabeth, engineer IP, epivert@pasteur.fr

BADELL-OCANDO Edgar, engineer I.P., ebadell@pasteur.fr

BARNAY-VERDIER Stéphanie, engineer IP. CDD, sverdier@pasteur.fr

BORDAT Yann, technician I.P., ybordat@pasteur.fr

CHARLES Patricia, technical agent I.P.

ENSERGUEIX Danielle, technician I.P, densergu@pasteur.fr

RAUZIER Jean, technician I.P., jrauzier@pasteur.fr


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