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Infection and Immunity, December 2007, p. 5609-5614, Vol. 75, No. 12
0019-9567/07/$08.00+0     doi:10.1128/IAI.00781-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transgenic Mice Expressing Human Transferrin as a Model for Meningococcal Infection

Maria-Leticia Zarantonelli,¹ Marek Szatanik,¹ Dario Giorgini,¹ Eva Hong,¹ Michel Huerre,² Florian Guillou,³ Jean-Michel Alonso,¹ and Muhamed-Kheir Taha^1*

Neisseria Unit, National Reference Centre for the Meningococci,¹ Histopathology Unit, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris cedex 15, France,² UMR 6175, Institut National de la Recherche Agronomique, Centre National de Recherche Scientifique, Université de Tours, Haras Nationaux, Physiologie de la Reproduction et des Comportements, 37380 Nouzilly, France³

Received 8 June 2007/ Returned for modification 4 July 2007/ Accepted 13 September 2007

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The pathogenesis of meningococcal disease is poorly understood due to the lack of a relevant animal model. Moreover, the use of animal models is not optimal as most meningococcal virulence determinants recognize receptors that are specifically expressed in human tissues. One major element of the host specificity is the system of meningococcal iron uptake by transferrin-binding proteins that bind specifically human transferrin but not murine transferrin. We developed a new mouse model for experimental meningococcal infection using transgenic mice expressing human transferrin. Intraperitoneal challenge of transgenic mice induced bacteremia for at least 48 h with an early stage of multiplication, whereas the initial inoculum was rapidly cleared from blood in wild-type mice. Inflammation in the subarachnoidal space with a high influx of polymorphonuclear cells was observed only in transgenic mice. Meningococcal mutants that were unable to use transferrin as a source of iron were rapidly cleared from both wild-type and transgenic mice. Thus, transgenic mice expressing human transferrin may represent an important advance as a new mouse model for in vivo studies of meningococcal virulence and immunogenicity factors.

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Neisseria meningitidis is found exclusively in humans, and although it is frequently found in the upper respiratory tract of asymptomatic carriers, it can be the causative agent of life-threatening invasive infections, such as septicemia and meningitis. As it is for several other bacterial pathogens, iron is essential for meningococcal growth and is needed at a micromolar concentration. However, free iron is extremely rare in body fluids as it sequestered in iron-binding proteins, such as transferrin and lactoferrin, that limit the amount of ionic iron available in body fluids to 10^–18 M (4). Therefore, N. meningitidis grows under iron-restricted conditions during infection and expresses several iron-regulated proteins that allow the bacterium to acquire iron by using transferrin and lactoferrin. N. meningitidis can also use hemoglobin and haptoglobin-hemoglobin as iron sources (for a review, see reference 19). N. meningitidis produces outer membrane receptors, called transferrin-binding proteins (TbpA and TbpB), that are highly specific for human transferrin (hTf) (25). TbpA is a 93- to 98-kDa outer membrane porin-like protein that interacts with TbpB to optimally remove iron from transferrin. TbpB varies in size from 68 to 85 kDa (6, 23).

Meningococcal mutants that are devoid of both proteins are unable to bind or utilize iron from transferrin (12). Moreover, Tbp proteins require energy that is transduced through the inner membrane TonB complex (composed of the TonB, ExbB, and ExbD proteins) (20, 26).

Iron acquisition through Tbp is essential to the virulence of pathogenic Neisseria strains. Indeed, a mutant of Neisseria gonorrhoeae lacking a transferrin receptor is avirulent, while the parental strain is virulent in humans (5). A hypoferremic state is a defense mechanism that helps reduce the amount of free transferrin by as much as 70%, allowing control of meningococcal infection (30).

Relevant animal models are key factors for deciphering microbial virulence (2). N. meningitidis is an extracellular pathogen, and its ability to grow under in vivo conditions is crucial for expression of its virulence determinants. Limitation of iron for efficient in vivo growth of meningococci in the mouse could be compensated for in various models by supplementation with exogenous iron or hTf (17, 31). These models may be improved by using transgenic mice expressing hTf. Such mice were developed to study the regulation of gene expression in brain cells (21) and Sertoli cells in testes (15). We aimed in this work to use these transgenic mice to develop an animal model of meningococcal infection.

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Bacterial strains and growth media. N. meningitidis strain Clone 12 (a serogroup C wild-type strain derived from clinical isolate LNP8013) was used to construct the meningococcal mutants used in this study (16, 27). Escherichia coli strain DH5 was used to construct recombinant plasmids (9). N. meningitidis was grown on GCB medium supplemented with Kellogg supplements (14). When needed, kanamycin (Km) was added at a final concentration of 100 µg/ml and deferoxamine (Desferal; Noavrtis) was added at a final concentration of 15 µM.

Construction and analysis of N. meningitidis mutants. A 1,116-bp fragment corresponding to the 3' half of the tbpB gene (codons 337 to 708) was amplified using oligonucleotides tbp1F and tbp100R (Table 1). The amplified fragment was then subcloned into the vector pGEM-T Easy (Promega) to obtain recombinant plasmid pDG26. The tbpB gene was inactivated by insertion, at the AvaI restriction site, of the aph3' cassette that confers resistance to Km. The resulting recombinant plasmid, pDG27, was then used to transform strain Clone 12, and transformants were selected on GCB medium in the presence of 100 µg/ml Km. Integration by homologous recombination into the tbpB gene on the chromosome was controlled by PCR analysis, using oligonucleotides tbp1F and Km6 (in the aph3' gene) (Table 1). One transformant, designated NM05-03, was selected as a tbpB mutant of N. meningitidis for further study.

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TABLE 1. Oligonucleotides used in this study

A 699-bp fragment of the tonB gene corresponding to codons 28 to 260 was amplified using oligonucleotides tonB1 and tonB2 (Table 1). The amplified fragment was then subcloned into the vector pGEM-T Easy (Promega) to obtain recombinant plasmid pEH05-06. The tonB gene was inactivated by insertion, at the Bsu36I restriction site, of the aph3' cassette that confers resistance to Km. The resulting recombinant plasmid, pEH05-03, was then linearized and used to transform strain Clone 12, and transformants were selected on GCB medium in the presence of 100 µg/ml Km. Integration by homologous recombination into the tonB gene on the chromosome was controlled by PCR analysis, using oligonucleotides tonB1 and Km6 (Table 1). One transformant, designated EH05-05, was selected as a tonB mutant of N. meningitidis for further study. The ability of meningococcal wild-type strains, as well as tbpB and tonB mutants, to bind hTf in vitro was tested using peroxidase-labeled hTf as previously described (23).

A 1,134-bp fragment of the ctrA gene (involved in capsule transport) corresponding to codons 9 to 386 was amplified using oligonucleotides ctrA01 and ctrA100 (Table 1). The amplified fragment was then subcloned into the vector pGEM-T Easy (Promega) to obtain recombinant plasmid pSF1. The ctrA gene was inactivated by insertion, at the PmlI restriction site, of the aph3' cassette that confers resistance to Km. The resulting recombinant plasmid, pSF2, was then linearized and used to transform strain Clone 12, and transformants were selected on GCB medium in the presence of 100 µg/ml Km. Integration by homologous recombination into the ctrA gene on the chromosome was controlled by PCR analysis, using oligonucleotides ctrA01 and Km6 (Table 1). One transformant, designated SFCT7, was selected as a ctrA mutant of N. meningitidis for further study. This mutant did not show any agglutination with serogroup C-specific antibody.

Transgenic mice. A strain of transgenic mice expressing hTf bred under specific-pathogen-free conditions was established from line 803 C57B6/SJLJ mice (referred to below as B6/SJL mice) (21). Transgenic mice were designated hTf⁺, and wild-type B6/SJL^+/+ mice were designated hTf^–. Mice were screened for the presence of the transgene in DNA extracted from the tail by PCR using primers In15-16 (located in the junction between intron 15 and exon 16 of the hTf gene) and Ex16 (located at the end of exon 16 of the hTf gene), which amplified a 230-bp fragment (Table 1). Expression of the hTf gene in the organs of mice was analyzed by reverse transcriptase PCR (RT-PCR) assays. Mice were first perfused with a 155 mM NaCl solution, and then total RNAs from different organs were prepared using Trizol extraction (22). Reverse transcription was performed by random priming using random hexanucleotides, and then PCR was performed with cDNA using primers Ex7 (located within exon 7 of the hTf gene) and Ex8 (located within exon 8 of the hTF gene), as previously described (22). These primers amplified a 178-bp fragment from cDNA and 938 bp from genomic DNA. These oligonucleotides are specific for the hTf gene, and no amplification of the murine transferrin (mTf)-encoding gene was detected. mTf and hTf in transgenic mice were titrated in each experiment by a specific enzyme-linked immunosorbent assay (ELISA) (15). The results were expressed in nanograms per milligram of total weight of organ tested (liver or brain) or in nanograms per milliliter of total volume of blood (see Table 2).

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TABLE 2. Levels of expression of of mTF and hTF in transgenic mice

Experimental infections. Mice were kept in a biosafety containment facility in cages with sterile litter, water, and food according to institutional guidelines. The experimental design was approved by the Institut Pasteur Review Board. Mice were infected at 6 weeks of age by intraperitoneal challenge with standardized inocula (5 x 10⁶ CFU). Bacterial counts in the blood were determined 2, 6, 24, and 48 h after meningococcal challenge by plating serial dilutions of blood samples on GCB medium and were expressed in log₁₀ CFU/ml of blood. Experiments with wild-type mice were also conducted by intraperitoneal injection of 5 mg of hTf (Sigma-Aldrich) per mouse. The interleukin-6 (IL-6) in blood of infected mice was quantified by an ELISA (Quantikine; R&D Systems Europe, Abingdon, Oxon, United Kingdom) using blood samples after 2 and 6 h of bacterial infection. Student's t test and analysis of variance were used to examine the data. A P value of 0.05 was considered statistically significant.

Mice were challenged intranasally using our previously described model of respiratory meningococcal superinfection in mice convalescing from a previous infection with an influenza A/H3N2 virus, which permits evaluation of both the colonization of the respiratory tract and the bacteremia (1).

Histological analysis. Groups of four mice were killed by intraperitoneal injection of 300 mg/kg sodium pentobarbital 24 h after intraperitoneal challenge and were exsanguinated by retroorbital puncture prior to removal of the brain, which was immediately immersed in 4% formaldehyde in phosphate-buffered saline for 8 days. The specimens were further processed for embedding in paraffin and stained with hematein and Giemsa stain. To detect the bacteria, we carried out an immunohistochemical analysis with a specific anti-serogroup C antibody. In brief, deparaffinized lung sections were immersed in citrate buffer and treated with proteinase K. Endogenous peroxidase activity was blocked with H₂O₂, and a rabbit anti-N. meningitidis serogroup C antibody (specific for the capsular polysaccharide) was added at a dilution of 1:50. The sections were incubated for 1 h at room temperature and washed with phosphate-buffered saline, and a biotinylated goat anti-rabbit immunoglobulin G antibody (Dako EO432), diluted 1:500, was added for 45 min. The sections were then treated with horseradish peroxidase-conjugated streptavidin (Dako PO397), and the reaction was revealed by using aminoethyl carbazole-H₂O₂. The preparations were then counterstained with hematoxylin.

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Expression of hTf in transgenic mice. The hTf gene is located on a 32-kbp DNA fragment and is composed of 17 exons. The size of the transcript is 2,318 nucleotides. The transgenic mice that were positive for the presence of the hTf gene as determined by PCR with DNA extracted from the tail were first confirmed to express hTf in blood by an ELISA. The expression profile of the hTf gene in transgenic mice was then analyzed by RT-PCR using total RNA from the brain, lungs, kidneys, spleen, liver, stomach, and spinal cord. RT-PCR analysis showed the expected 178-bp fragment corresponding to amplification between exons 7 and 8 after correct splicing only in transgenic mice. Amplification of cDNA was detected for all the organs tested, suggesting that expression of the hTf gene was ubiquitous in these transgenic mice. A 938-bp fragment was also observed by PCR with genomic DNA from transgenic mice but not with genomic DNA from wild-type mice (Fig. 1). When monitored in transgenic mice, the level of hTf expression was lower than the level of expression of mTF in the liver, brain, and blood; the hTf titers were only 10 to 40% of the mTF titers (Table 2).

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FIG. 1. RT-PCR analysis using total RNA extracted from the organs of mice as indicated. Lanes +, B6/SJL hTf+ transgenic mice; lanes –, wild-type (B6/SJL hTf–) mice. RT-PCR was performed using primers Ex7 (located in exon 7 of the hTf gene) and Ex8 (located in exon 8 of the hTf gene), which amplified a 178-bp fragment. PCR with total DNA (938 bp) was used as a control.

Experimental meningococcal infection in transgenic mice after intraperitoneal or intranasal challenge. Six-week-old female B6/SJL mice, either hTf⁺ or hTf^–, were infected by intraperitoneal injection of strain Clone 12. Both transgenic and wild-type mice survived when the dose was 5 x 10⁶ CFU, and bacteremia could therefore be scored by blood culture at 2, 6, 24, and 48 h. The blood bacterial loads were almost 100 times higher in the hTf⁺ transgenic mice than in the hTf^– controls. Bacteria were rapidly cleared from the blood in the control mice, while bacteremia was still detectable at 48 h after challenge in hTf⁺ transgenic mice (Fig. 2A). These results strongly suggest that the expression of hTf in transgenic mice correlated with a higher load and prolonged survival of N. meningitidis in the blood. To further validate the requirement for hTf in this model, we tested two isogenic mutants of strain Clone 12 that were defective in transferrin-binding protein B (TbpB^–) and were unable to bind transferrin (data not shown) or were defective in the energy transducer protein TonB (TonB^–). The survival of both TbpB^– and TonB^– mutants was drastically affected in hTf⁺ transgenic mice, and these mutants were rapidly cleared from the blood in both hTf⁺ and hTf^– B6/SJL mice (Fig. 2B and data not shown). We also challenged B6/SJL hTf^– mice by intraperitoneal injection after hTf was added to the challenge dose of bacteria. Meningococcemia was detected in these mice at levels similar to those in hTf⁺ transgenic mice, while bacteria were rapidly cleared from the blood in hTf^– mice (Fig. 3A). All these results strongly indicate that the production of hTf is correlated with the establishment of sustained meningococcemia in hTf⁺ transgenic mice.

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FIG. 2. Susceptibility of hTf+ transgenic mice to intraperitoneal meningococcal challenge. (A) Transgenic mice expressing hTf (B6/SJL hTf+) or hTf-negative littermates (B6/SJL hTf–) were challenged intraperitoneally with 5 x 106 CFU of N. meningitidis serogroup C strain Clone 12. The data are the means ± standard deviations from five independent experiments with groups of five mice per time point in each experiment. IL-6 levels in the blood were determined after bacterial challenge with the meningococcal wild-type strain (Clone 12). IL-6 levels were measured 2 and 6 h (bars) after intraperitoneal bacterial infection. A significant increase in IL-6 production was induced after 6 h of infection in hTf+ mice. IL-6 concentrations (in pg/ml) are indicated on the right x axis, and the data are means ± standard deviations from two independent experiments with groups of five mice per time point in each experiment. (B) Transgenic mice expressing hTf (B6/SJL hTf+) were challenged intraperitoneally with 5 x 106 CFU of N. meningitidis serogroup C strain Clone 12 or isogenic mutants deficient in TbpA protein (NM05-03) or TonB protein (EH05-05). The data are the means ± standard deviations from three independent experiments with groups of five mice per time point in each experiment. (C) Transgenic mice expressing hTf (B6/SJL hTf+) or hTf-negative littermates (B6/SJL hTf–) were challenged intraperitoneally with 5 x 106 CFU of N. meningitidis serogroup C strain Clone 12 or isogenic mutants deficient in capsule (SFTC7). The data are the means ± standard deviations for groups of five mice per time point.

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FIG. 3. (A) Role of hTf in experimental meningococcal infection in mice. The meningococcal wild-type strain, Clone 12 (5 x 106 CFU per mouse), was used to infect transgenic mice expressing hTf (B6/SJL hTf+), hTf-negative littermates (B6/SJL hTf–), or B6/SJL hTf– mice supplemented with 5 mg of hTf (Sigma-Aldrich) simultaneously with the challenge dose of bacteria. The data are the means ± standard deviations for groups of five mice per time point in each experiment. (B) Susceptibility of hTf+ transgenic, influenza A virus-infected mice to meningococcal respiratory challenge. A mild influenza A virus infection was induced in transgenic mice expressing hTf (B6/SJL hTf+), as well as in control littermates (B6/SJL hTf–), by intranasal administration of 250 PFU. Seven days later, both types of mice were infected by intranasal challenge with a standardized bacterial suspension (1.7 x 109 CFU) of N. meningitidis serogroup C strain Clone 12. Bacterial counting (CFU) was performed using samples of blood and lung homogenates obtained from three mice at 3, 24, and 48 h after challenge. The data are the means ± standard deviations from two independent experiments with groups of five mice per time point.

The capsule of N. meningitidis is a major virulence factor that allows serum resistance of meningococci and survival in the blood (29). To further explore the use of the transgenic mice as a model for experimental meningococcal infections, we compared the kinetics of infection of wild-type strain Clone 12 and an isogenic capsule-deficient mutant of N. meningitidis. The capsule-deficient mutant (SFCT7) was also rapidly cleared from the bloodstream (Fig. 2C). In addition to the intraperitoneal challenge model, we also used a model consisting of respiratory meningococcal superinfection in mice convalescing from a previous infection with an influenza A/H3N2 virus that permits evaluation of both the colonization of the respiratory tract and the bacteremia (1). Figure 3B shows the kinetics of bacterial counts in the lungs and blood of hTf⁺ transgenic mice and of hTf^– mice. Bacteria persisted better in the lungs of hTf⁺ mice. Expression of hTf rendered the mice more susceptible to invasive infection, as revealed by the higher amplitude of meningococcemia in hTf⁺ transgenic mice.

Meningeal lesions during experimental meningococcal infection in hTf⁺ transgenic mice. We quantitatively studied production of the proinflammatory cytokine IL-6 in blood 2 and 6 h after bacterial challenge of both hTf⁺ and hTf^– B6/SJL mice to assess the role of meningococcal growth in inducing the inflammatory response during experimental infection. Similar levels (P = 0.791) of IL-6 were observed 2 h after intraperitoneal challenge due to the equal initial infective doses. However, significantly higher levels of IL-6 (P < 0.001) were detected in hTf⁺ transgenic after 6 h of infection, corresponding to the peak of bacterial growth (Fig. 2A). Since bacteremia is a correlate of invasiveness of the strain and is the first stage before invasion of the meningeal space to provoke meningitis, we looked for the presence of histological lesions of meningitis. Brain samples from all hTf⁺ transgenic mice tested (n = 9) had inflammatory lesions of the choroid plexus and the meninges with important infiltrates of polymorphonuclear cells (Fig. 4A). In some instances, intracytoplasmic diplococci were also observed after immunohistochemical analysis using anti-serogroup C antibodies (Fig. 4B). Neither histological lesions nor cellular influx was seen in samples from control mice.

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FIG. 4. Histological examination of brain sections 24 h after intraperitoneal meningococcal challenge. (A) Hematoxylin and eosin staining (magnification, x400). Note the important infiltrates of polymorphonuclear cells in the meningeal space. (B) Bacteria (red) were detected by immunohistochemical analysis with a specific rabbit anti-serogroup C antibody and a biotinylated goat anti-rabbit immunoglobulin G antibody and were revealed with peroxidase-conjugated streptavidin after treatment with aminoethyl carbazole-H2O2 (see Materials and Methods). The preparation was then counterstained with Giemsa stain (magnification, x1,000). The results are representative of the results for a set of nine hTf+ mice from two independent experiments.

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The development of animal models is a crucial factor in the field of infectious disease as such models permit workers to address the fundamental issue of pathogen-host interaction. The optimal animal model should mimic the natural infection in the route of contamination, as well as in the development of the experimental infection. N. meningitidis is a strict human pathogen, which hinders development of reliable animal models. Hence, transgenic mice should provide an opportunity to engineer new animal models of meningococcal infection. The first model reported consisted of transgenic mice expressing human CD46 (13). This model represents a substantial improvement as bacteria can cross the blood-brain barrier (BBB) after both intraperitoneal and intranasal infection without addition of iron resources (13, 24). However, this model still requires large infectious inocula (10⁸ to 10⁹ CFU) in order to detect bacteria in cerebrospinal fluid after they cross the BBB, and considerable bacterial growth in blood does not occur (13, 24). Meningococcal growth during invasive infection is a primordial aspect for analyzing the host-bacterium interaction. Indeed, massive meningococcal outgrowth may be rapidly fatal in humans (for a review, see reference 28). Hence, the transgenic mice expressing hTf are a further improvement in development of an animal model for N. meningitidis as they provide invasive bacteria with an iron source, which is necessary for meningococcal growth during infection. The hTf gene fragment in the hTf⁺ transgenic mice comprised the 5' and 3' regulatory sequences, and it expressed hTf ubiquitously, although the level was less than the level of mTf. The lower level may be due to tissular differences between mice and humans in recognition of the regulatory sequences of the hTf gene (15). Our data clearly showed that bacterial growth occurred in blood as bacterial counts increased by at least 1 log in blood after 6 h of intraperitoneal infection (Fig. 2). This bacterial growth seemed to be dependent on hTf expression in transgenic mice as it was not observed in wild-type mice. Such growth was not observed in meningococcal mutants (TbpB^– and TonB^–) that are unable to use hTf. Intraperitoneal injection of 8 mg of hTf was reported to allow a concentration of hTf in mice similar to that encountered in humans, hence permitting bacterial growth in blood (18). Our data support these findings and confirm the requirement for transferrin for bacterial growth in vivo. Moreover, our model has the advantage of expressing hTf in several organs, which should permit bacterial growth in other compartments, such as the meningeal space. It is noteworthy that infection in this model can be achieved with a rather low dose when the intraperitoneal route is used (about 10⁶ CFU per mouse).

This mouse model allows analysis of virulence factors, such as the capsule. A capsule-deficient mutant was rapidly cleared from the blood in both hTf⁺ and wild-type mice after intraperitoneal challenge (Fig. 2). However, this mutant survived better in hTf⁺ mice than in wild-type mice due to bacterial growth in hTf⁺ mice (Fig. 2C). Indeed, invasive meningococcal infection caused by capsule null locus strains has been reported by several authors even in immunocompetent patients (7, 10).

Meningococcal growth in hTf⁺ transgenic mice may be responsible for greater release of bacterial mediators of inflammation that may be responsible for crossing of the BBB. Findings that support this explanation are the higher levels of IL-6 in hTf⁺ transgenic mice (Fig. 2A) and the important infiltrates of polymorphonuclear cells in the meningeal space (Fig. 4). Indeed, the levels of several cytokines at admission to the hospital have been reported to be correlated with fatality (3, 11) and were proportional to the bacterial loads in the blood (8).

The hTf⁺ transgenic mouse model of meningococcal infection offers the possibility of measuring tissular kinetics of infection with great sensitivity after either intraperitoneal or respiratory challenge and provides an original model of meningococcal meningitis. Moreover, the intraperitoneal challenge model offers the possibility of assessing passive and active protection with new vaccine candidates. Indeed, we have recently shown the feasibility of such a study of protection using intraperitoneal challenge to test the protective activity of anti-penicillin binding protein 2 antibodies against meningococcal bacteremia in mice (32).

 
We thank Mario Zakin and Bruno Baron for helpful discussions and Sandrine Farci for technical assistance.

This work was supported by the Institut Pasteur.

 
* Corresponding author. Mailing address: Neisseria Unit, National Reference Centre for the Meningococci, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris cedex 15, France. Phone: 33 1 44 38 95 90. Fax: 33 1 45 68 83 38. E-mail: mktaha{at}pasteur.fr

Published ahead of print on 24 September 2007.

Editor: R. P. Morrison

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Infection and Immunity, December 2007, p. 5609-5614, Vol. 75, No. 12
0019-9567/07/$08.00+0     doi:10.1128/IAI.00781-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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