CA2466995A1 - Method of evaluating the pathogenic potential of a strain of listeria - Google Patents

Abstract

The present invention relates to Listeria surface protein, namely the internalin and its use in the evaluation of the pathogenic potential of Listeria species such as Listeria monocytogenes.

Description

METHOD OF EVALUATING THE PATHOGENIC POTENTIAL
OF A STRAIN OF LISTERIIA
FIELD OF THE INVENTION
The present invention relates to a Listeria surface protein, namely the internaiin and its use in the evaluation of the pathogenic potential of Listeria species.
BACKGROUND OF THE INVENTION
Listeria monocytogenes is a pathogenic bacteria responsible for deadly human infections originating from food (mortality rate : 20 to 30 %). In most industrialized countries, agencies for dietary surveillance have set forth severe rules to 1- detect the presence of Listeria monocytogenes in the food chain;
and 2-order the withdrawal from sale of contaminated foods, with rapid diffusion of a sanitary alert. Major complications arising during human listeriosis are foetoplacental attack in pregnant women and central nervous system infections (meningitis and encephalitis).
To this day, all strains of Listeria monocytogenes identified in food (by PCR
and/or by biochemical characteristics and the presence of hemolysis on agar in blood) are considered as being equally pathogenic, although numerous animal studies converge to affirm 'that all strains are not identical with regard to their virulence. A relevant prio art on this aspect is WO 89106fi99 which discloses a DNA probe capable of hybridizing to a portion of the genome of pathogenic Lisferia monocyfogenes but which does not hybridize to portions of the genomes of other Listeria species and other humilytic bacteria. This probe is useful to identify food sources infected with Listeria monoc,ytogenes and to distinguish these food services from those infected nearly with non-pathogenic Listeria species.
Up to now, there is no method to evaluate virulence differences in Listeria monocytogenes strains isolated from the environment (food), or in asymptomatic subjects (intestinal portage) while allowing to evaluatE; their dangerousness or their epidemiogenic capacity. Therefore, there is a need for a method for identifying a pathogen strain of Listeria.

The present invention fulfils these needs and also other needs which will be apparent to those skilled in the art upon reading the following specification.
SUMMARY OF THE INVENTION
The present invention relates to a Listeria surface protein, namely the internalin and its use in the evaluation of the pathogenic potential of Listeria species.
More specifically, one object of the present is to provide a method for identifying a pathogen strain of Listeria in a sample. much a method comprises the step of detecting in said sample a Listeria strain comprising a functional internalin, whereby detection of a Listeria strain comprising a functional internalin is indicative of an increased likelihood of pathogenicity of said strain compared to a strain of Listeria having a non-functional internalin.
Another object is to provide a kit for the detection of a pathogen strain of Listeria in a sample, comprising:
- an antibody that binds specifically to a functional internalin located at the surface of a Listeria strain;
- a reagent to detect internalin-antibody immune complex;
- a biological reference sample lacking a functional internalin that immunologicaily bind with said antibody; and - a comparison sample comprising a functional internalin which can specifically bind to said antibody;
wherein said antibody, reagent, biological reference sarnple, and comparison sample are present in an amount sufficient to perform said detection.
A further object is to provide a method for imaging tissue interface where E-cadherin is expressed comprising the following steps a) contacting the tissue where E-cadherin is to be detected with purified internalin; and b) detecting interaction between E-cadherin and internalin.
Yet, another object is to provide a method for targeting placenta, wherein said method comprises the step of administering intemalin to a pregnant mammal.
Furthemore, another object is to provide a kil: for imaging tissue interface where E-cadherin is expressed comprising internalin.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 - Immunohistological study of the location of L,. monocytogenes in the placenta and amnion of women with listeriosis~. Bacteria are labeled with polyclonal antibodies to L, rnonocytogenes and appear red-brown after immuno-enzymatic color development. Sections are counterstained with hematoxylin.
Blood-borne L. monocyto~enes are located in i:he inter-vnous space (H), unassociated with macrophages or polymorphonuclear cells. Some bacteria associated with the syncytiotrophoblast (A, arrow) or cytotrophoblast (B, arrow).
Other bacteria have invaded the syncytiotrophoblast cytoplasm (C) or penetrated through this trophoblast layer to the villous core adjacent to fetal capillaries (D, arrow). Bacteria are seen throughout the cross :aection of some villi, either adhering to or localized within the syncytiotrophoblast (E) or contained within the villous core connective tissue where fetal vessels are in close proximity (E, F).
Isolated villi (G) or villous clusters (H) contain foci of bacteria and microscopic abscesses, with fibrin deposition in the inter-villous space (H). The amnion epithelium (I) is markedly infected, yet there are no bacteria detectable on its chorionic surface (I, J). Bars, 10 pm, except for H, 200 pm.
Fig. 2 - E-cadherin is expressed in placental cytotrophoblasts and syncytiotrophoblasts, and in the amniotic epithelium. Formalin-fixed, paraffinembedded (A) or methanol-fixed (B-J) cryose~:,tions of human placenta and amnion were stained with an E-cadherin mAb (HIoCD-1 ). Immuno-reactive E-cadherin appears red (A) or green (B-J). Sections h<~ve been counterstained with hematoxylin (A) or a blue nuclear marker (Bis-benzimide in B-D and To-Pro-3 iodide blue in E-H) for identification of tissue organization. E-cadherin is apparent in formalin-fixed cytotrophoblast (A, arrow). A lower intensity, discontinuous signal is also seen along the basal plasma membranE; of the syncytiotrophoblast (arrowhead). In the immuno-stained cryosections shown in panels B and C, cytotrophoblasts (e.g., arrows) and the basal plasma membrane of syncytiotrophoblasts (e.g., arrowheads) contain detectable E-cadherin. At higher magnification, a less intense, E-cadherin signal is apparent on the apical microvillous surface of syncytiotrophoblasts (see arrow in panel D). Confocal microscopic images of cryosections with HELD-1 (E, F) confirm the apical surface membrane localization of Ecadherin (e.g., arrows) and the absence of cytoplasmic staining in the syncytiotrophoblast. When placental tissue was formalin fixed to eliminate the possibility of endocytosis and immunostained with HECD-1 before embedding (see Materials and Methods), a clearly detectable apical, microvillous surface signal for E-cadherin remains on the syncytiotrophoblast (G, H). As expected, no cytotrophoblast or basal syncytiotrophoblast plasma membrane signal is evident. The amnion epithelium (I, J) contains immnuoreactive E-cgdherin, in contrast to the associated mesenchyme o~f the chorion, adjacent to the maternal deciduas (positioned to the left of the epithelium). Bar, 10 pm.
Fig. 3 - Listeria binding and invasion assays of BeWo cells. (A-D') Immunohistochemical study of cells cultured in the presence or absence of 1 mM
8-BrcAMP and infected with L. innocua (Li), or L. innocua expressing InIA
(LiA).
Bacteria appear in red, E-cadherin green and nuclei blue in each panel. The enlargement (D') of the boxed region in panel D shov~rs that L. innocua expressing internalin is able to recruit E-cadherin on the surface of syncytiotrophoblasts. (E-H) Histograms displaying the results of invasion assays. Data have been normalized and are expressed as the number of intracellular CFU of bacteria relative to the number in added to the plate, per condition tested. Mean values ~ S.D are presented (each assay/strain/condition was done in triplicate; n = 3 independent experiments). EGD refers to the wild type L. monocytogenes strain, DA to its isogenic InIA-deficient mutant derivative. Results for untreated BeWo cells (mononucleated cytotrophoblasts) are presented in panels E and G, while data obtained from cAMP-treated cultures (syncytiotrophoblasts) are presented in panels F and H. Bars in A-D, 10 pm.
Fig. 4 - E-cadherin localization and Listeria invasion assays of placental explants.
Shown are the results of Gram stains of tissue sections (A, B), immunohistochemical assays of E-cadherin (green) and bacterial (magenta) localization, (C, D, F), and the number of gentamicin-resistant viable bacteria (E) after incubation of explants with isogenic wild type (IEGD) (A-D) or InIA-deficient (~A) (F) strains of L, monocytogenes. Panels A, and C illustrate findings obtained after a 1 h infection and a 2h incubation with gentamicin, while panels B, D
and F

5 contain representative results after a 1 h infection, followed by a 2h incubation with gentamicin, and a 21 h incubation in culture medium without antibiotics. The graph in panel E displays the relative level of infection after 3 and 24 h, with either EGD
or ~A strains (mean values ~ SD are plotted, n = 3 independent experiments, each done in quadruplicate). Bars in A-D and F, 10 pm.
Fig. 5 - Anatomy of the maternofetal interface in humans. (A) A view of the pregnant uterus (adapted from (38)). (B) Expanded view of a small segment of the placental villous tree. A cross section of a villous stained with hematoxylin (C) or with hematoxylin and eosin (D) with an associated diagrammatic outline of the histology (D') reveals a relatively empty inter-villous space surrounding a villous in cross section. This space is normally filled with maternal blood. The multinucleated syncytiotrophoblast, a true syncytium without lateral cell membranes, covers the villi and an underlying discontinuous layer of cytotrophoblasts in the second half of pregnancy. The subjacent cytotrophoblast are mitotically active and through a process of differentiation and fusion give rise to more' syncytium. The two villous trophoblast cell types share a basement membrane that delimits the villous core connective tissue where fetal vessels pass. The placental "barrier" in the villous tree thus includes the apical micro-villous surface membrane of the syncytiotrophoblast, the basal surface membrane of the syncytiotrophoblast, the trophoblastic basement membrane, and the villous core connective tissues. Some fetal vessels are positioned very close to the basal surface of the trophoblast layer, an area termed a "vasculo-syncytial" membrane (arrowhead, panel D). A
brightfield image of a cross section of the amnion (E;) is schematized in panel E'.
The amnion delineates the extra-placental maternofetal interface. The amniotic epithelium is in direct contact with the amniotic fluid that surrounds the developing fetus. The amniotic epithelium rests on a basement membrane that faces the chorion and maternal decidual tissues of the uterine wall. Bars in C-E, 10 pm.

Fig. 6 - Dissection of placental explants. Fetal face (A) (http:Ilwwwmedlib.med.Utah.eduIWebPathIPLACHTMI.IPLAC031.html) and maternal face (B) (http:l/www-medlib.med.utah.eduIWebPath/PLACHTMLIPLAC032.html) of a human placenta.
Placental explants were dissected from the maternal face, and rinsed in saline before being transferred to the culture medium. Panel C shows an explant being rinsed in saline. The scale on the left is in centimeter.
Fig. 7 - Listeria binding and invasion assays of primary trophoblasts.
Immunohistochemical studies and invasion assays of trophoblasts cultured for 8h (mononucleated cytotrophoblasts) are presented in panels A, C, E, and G, while those obtained from cells cultured for 96h (syncytiotrophoblasts) are shown in panels B, D, F, and H. Results from invasion assays are presented as described in the legend to Fig. 4. Bars in A-D, 10 Vim.
Fig. 8 - Invasion assay of amnion explants. The fetal side of the amnion membrane is permissive to InIA-dependant entry but the maternal side is not.
Results are expressed as relative values, as defined iin the legend to Fig. 4 (mean values ~ SD are plotted, n = 3 independent experiments, each done in triplicate).
Fig. 9. shows the nucleic acid sequence encoding the Internalin (InIA) from Lisferia monocyfogenes and identified as SEQ ID NO" 1.
Fig.10. shows the amino acid sequence of the Internalin (InIA) from Listeria monocytogenes and identified as SEQ ID NO 2 Fig.11 The various forms of internalin expressed by L, monocytogenes strains. Black arrows point to the carboxy-terminal end of the respective internalin variants. lnIA1 corresponds to full-length internalin, similar to that produced by the reference EGD strain. InIA2 is a carboxyl-terminal truncated intemalin, similar to that produced by L028 strain. InIA3 is a truncated internalin smaller than that produced by the L028 strain. InIA4 is another truncated version: one that is larger than that expressed by the L028 strain but smaller than full-length internalin. InIA1 is the only version that promotes L. monocytogenes entry into host cells expressing human E-cadherin: in contrast to the truncated version InIA2, InIA3 or InIA4, it retains the carboxy-terminal motif necessary 'for anchoring to the bacterial surface.
DETAILED DESCRIPTION ~F THE INVENTION
The present invention relates to Listeria surface protein, namely the internalin and its use in the evaluation of the pathogenic potential of Listeria species, such as Listeria monocyfogenes. Indeed, the inventors have found that almost all of the strains isolated in patients with human listeriosis (=!3 %) and all of the strains isolated in the context of a maternofoetal infection (100 °/~) express a full-length internalin, functional and anchored to the bacterial surface. However, the inventors have also found that a high proportion of strains of food origin (35 %) express a truncated internalin, secreted and non-functional.
Therefore, an object of the present invention is to provide a method for identifying a pathogen strain of Listeria in a sample. The present method comprises the step of detecting in the sample a Listeria strain comprising a functional internalin, whereby detection of a Listeria strain comprising a functional internalin is indicative of an increased likelihood of pathogenicity of said strain compared to a strain of Listeria having a non-function<~l internalin.
As used herein, the term "functional internalin" refers to an internalin, and more preferably the internalin A (Inl A), that is located on the surface of a Listeria species. Alternatively, a non-functional internalin is an internalin that is secreted by a Listeria species and more preferably, a truncated form of the internalin generally at its C-terminus. In this connection, it will be understood that a functional internalin may also refers to a full-length expressed internalin.
As used herein, the term "sample" refers to~ a variety of sample types obtained from an individual and can be used in a diagnostic or detection assay.
The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof. The term "sample" also encompasses a food sample, either liquid or solid.
According to a first preferred embodiment of the present invention, the step of detecting in said sample a Listeria strain comprising a functional internalin is obtained by the following steps:
a) contacting the sample with an antibody that binds specifically to a functional internalin for a time and under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a).
One of skill in the art will recognize that tllis preferred immunological detection method may take several forms, such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay, essentially to determine whether a functional internalin is present in the tested sample.
The term "antibody°' is used in the broadest sense and specifically covers single monoclonal antibodies. The term "monoclonal antibody'° (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are higlhiy specific, being directed against a single antigenic site. Furthermore, ire contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal"
indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. f=or example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). The "monoclonal antibodies°' also include clones of antigen-recognition and binding-site containing antibody fragments (Fv clones) isolated from phage antibody libraries using the techniques clescribed in Clackson et al., Nature, 352:624-628 (1991 ) and Marks et al., J. Mol. E3iol., 222:581-597 (1991 ), for example.
With respect to antibodies of the invention, the term "specifically binds to"
refers to antibodies that bind with a relatively high affinity to one or more epitopes of a protein of interest, but which do not substantially recognize and bind molecules other than the ones) of interest. As usecl herein, the term "relatively high affinity" means a binding affinity befinreen the .antibody and the protein of interest of at least 106 M-', and preferably of at least aibout 10' M-' and even more preferably 10$ M-' to 10'° M-'. Determination of such affinity is preferably conducted under standard competitive binding immuinoassay conditions which is common knowledge to one skilled in the art.
Preferably, the monoclonal antibodies conltemplated by the present invention are those described in Cossart et al. 199E~ (Infection and Immunity p.
5430-5433) and more specifically those designated 14.4 and L7.7. These antibodies were respectively produced from hybridoma A 4-4 and L 7-7 deposited at CNCM on May 3, 2004 under reference I-3205 and I-3206. It will be understood that the designations 14.4 and A 4-4 refer to the same imonoclonal antibody.
According to a second preferred embodiment, the step of detecting In said sample a Listeria strain comprising a functional internalin is obtained by a step of determining the presence or absence of expression of full length internalin in said sample, whereby identification of expression of full Length internalin is indicative of an increased likelihood of pathogenicity of said strain compared to a strain of Listeria having a non-full length expressed internalin.
In this connection, a full-length expressed internalin as contemplated by the present invention has an amino acid sequence having at least 80% homology, or even preferably 90% homology to the amino acid sequence of Genbank Accession no. AAA25289. More specifically, the full length inlternalin has an amino acid sequence as shown in SEQ ID NO: 2.
The full-length expressed internalin may also be encoded by a nucleic acid sequence which is at least 65% identical, more particularly 80% identical and even more particularly 95% identical to the nucleic ae;id sequence of Genbank Accession no. M67471. More specifically, the full length internalin is encoded by a nucleic acid sequence as shown in SEQ ID N0:1.
Amino acid or nucleic acid (nucleotide) sequence "identity" and "similarity"
are determined from an optimal global alignment between the two sequences 5 being compared. An optimal global alignment is achiE:ved using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.
48:443-453). "Identity" means that an amino acid or nucleotide at a particular position in a first polypeptide or polynucleotide is identical to a corresponding amino acid or nucleotide in a second polypeptide or polynucleotide that is in an 10 optimal global alignment with the first polypeptide or holynucleotide. In contrast to identity, "similarity" encompasses amino acids that are conservative substitutions.
A "conservative°' substitution is any substitution that has a positive score in the blosum62 substitution matrix (Hentikoff and Hentikoff, 1992, Proc. Natl. Acad.
Sci.
USA 89: 10915-10919). By the statement "sequence A is n% similar to sequence B" is meant that n% of the positions of an optimGrl global alignment between sequences A and B consists of identical residues or nucleotides and conservative substitutions. By the statement "sequence A is n% identical to sequence B" is meant that n% of the positions of an optimal global alignment between sequences A and B consists of identical residues or nucleotides.
Preferably, the step of determining the presence or absence of expression of full length internalin in said sample is achieved by any processes known to one skilled in the art, such as PCR, DNA or protein sE;quencing, and/or restriction fragment length polymorphism (RFLP) A further object is to provide a method, preferably conducted in vivo, for imaging tissue interface where E-cadherin is expressed comprising the following steps a) contacting the tissue where E-cadherin is to be detected with purified internalin; and b) detecting interaction between E-cadherin arid internalin.
Preferably, the tissue is placenta or blood brain barrier.
Yet, another object is to provide a method for' targeting placenta, wherein said method comprises the step of administering internalin to a pregnant mammal.

more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, rodents, primates, and pets.
The present invention further provides kits for use within any of the above detection methods. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers andlor equipment. For examplE;, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to an internalin. Such antibodies or fragments may be provided attached to a support material known to one skilled in the art. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay.
In this connection, the present invention provides a kit for the detection of a pathogen strain of Lisferia in a sample, comprising:
- an antibody that binds specifically to a functional internalin located at the surface of a Listeria strain and more specifically an antibody as described above;
- a reagent to detect internalin-antibody immune complex;
- a biological reference sample lacking a functional internalin that immunologically bind with said antibody; and - a comparison sample comprising a functional internalin which can specifically bind to said antibody;
wherein said antibody, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.
The present invention also provide a kit for imaging tissue interface where E-cadherin is expressed comprising internalin.
The present invention will be more readily understood by referring to the following examples. These examples are illustrative of the wide range of applicability of the present invention and is not intended to limit its scope.
Modifications and variations can be made therein without departing from the spirit and scope of the invention: Although any methods and materials similar or equivalent to those described herein can be used in 'the practice for testing of the present invention, the preferred methods and materials are described.

EXAMPLE 1: Targeting and crossing of the human maternofetal barrier by Listeria monocytogenes : Role of internalin interaction with trophoblast E-cadherin Listeria monocytogenes produces severe fetoplacental infections in humans. How it targets and crosses the maternofetal barrier is unknown. The inventors used immunohistochemistry to examine the location of L.
monocyfogenes in placental and amniotic tissue samples obtained from women with fetoplacental listeriosis. The results raised the possibility that L.
monocytogenes crosses the maternofetal barrier through the villous syncytiotrophoblast, with secondary infection occurring via the amniotic epithelium.
Since epidemiological studies indicate that the bacterial surface protein, internalin (InIA), may play a role in human fetoplacental listeriosis, the inventors investigated the cellular patterns of expression of its host receptor, E-cadherin, at the maternofetal interface. E-cadherin was found in villous~ cytotrophoblasts and on the basal and apical plasma membranes of syncylriotrophoblasts. Established trophoblastic cell lines, primary trophoblast cultures, and placental villous explants were each exposed to isogenic InIA+or InIA- strains of L. monocytogenes, and to L. innocua expressing or not InIA. Quantitative <~ssays of cellular invasion demonstrated that bacterial entry into syncytiotrophoblasts occurs via the apical membrane in an InIA-E-cadherin dependent manner. In human placental villous explants, bacterial invasion of the syncytiotrophoblast barrier and underlying villous tissue and subsequent replication produces haistopathological lesions that mimic those seen in placentas of women with listeriosis. Thus, the InIA-E-cadherin interaction that plays a key role in the crossing of the intestinal barrier in humans, is also exploited by L. monocytogenes to target and cross the placental barrier.
This is the first identified ligand-receptor interaction that allows a bacterial pathogen to specifically cross the placental villous trophoblast barrier.

Introduction Listeria monocytogenes is a food-borne pathogen that. causes gastroenteritis, bacteremia, as well as central nervous system (CNS) and maternofetal infections (1-3). Pregnant women constitute 60% of all cases of listeriosis in individuals less than 40 years of age (3). Their ~20-fold higher risk of infection compared to otherwise healthy adults is presumed to be a consequence of the pregnancy-associated immunosuppression that allows tolerance of the fetoplacental allograft (4-7). Nonetheless, the predilection of the fetoplacental unit for infection raises the possibility that a specific mechanism may be responsible for targeting L.
monocytogenes to the maternofetal barrier. L. monoc;ytogenes can enter cultured non-phagocytic human cells through a process mediated by the interaction of internalin (InIA), a bacterial surface protein, with E-cadherin, a cell surface transmembrane protein expressed by various epithelial lineages (8). InIA has a high degree of specificity for human E-cadherin: i.e., a single amino acid polymorphism between the orthologous human and mouse proteins is responsible for the relatively low pathogenicity of L. monocytogenes when administrated orally into mice (9). Studies of transgenic mice that express human E-cadherin in their intestinal epithelium and that have been orally inoculated with isogenic InIA+
or InIA- strains of L. monocytogenes, established that this bacterial protein plays an essential role in crossing the intestinal epithelial barrier (10).
A recent epidemiologic study indicates that InIA may also play a pivotal role in penetration of the maternofetal barrier (11 ). In this study, InIA expression was assessed by immunoblot assays of 300 clinical strains collected from sporadic cases of listeriosis occurring in France in a single year, plus a representative set of 150 strains obtained from food products during the same period. One hundred percent of L. monocytogenes isolates recovered from pregnant women (61/61) expressed the functional protein, whereas only 65% (981150) of food isolates expressed it (p=1x10-7) (11). The human maternofetal barrier contains two anatomically distinct components: the chorioalNantoic placenta and the chorioamnion (Fig. 5A-E). The barrier is formed at the placental level by the villous syncytiotrophoblast. This specialized epithelial lineage is in direct contact with maternal blood circulating through the intervilfous space. In a subjacent layer, mononuclear cytotrophoblasts divide, differentiate and fuse to renew overlying multinucleated syncytiotrophoblasts (Fig. 5D,D'). A basement membrane separates these trophoblastic cells from a connective tissue core that contains fetal capillaries (Fig. SD,D'). The amniotic epithelium forms the maternofetal interface in the chorioamnion. The apical surface of this epithelium is exposed to amniotic fluid while its basal surface sits on a basement membrane that overlies the amniotic mesoderm (Fig.SE,E'). In contrast to the villous tree in the hemochorial placenta of the human (Fig. 5), rodent placentas are hemochorial and labyrinthine, with up to three layers of trophoblasts bathed by maternal blood and overlying the connective tissue of the labyrinthine core where fetal vessels reside (12). This arrangement results in a different maternofetal interface compared to human placental villi, with associated changes in maternal blood flow.
The species-specificity of InIA-E-cadherin interactions (9) and of placental structures between mice and humans (12), the absence of a transgenic mouse model where human E-cadherin is expressed in all epithelial lineages (13), plus our epidemiological findings (11 ), prompted us to examine the role of the InIA-E-cadherin interaction in crossing of the maternofetal barrier in humans by immunohistopathological studies of placentas from women with tisteriosis, and by incubating strains of L. monocyfogenes and L. innoce~a that did or did not express a functional InIA with (y an established human trophoblast cell line (BeWo) that differentiates in vifro into syncytiotrophoblasts when exposed to cAMP, (iii primary cultures of human cytotrophoblasts that spontaneously differentiate into syncytiotrophoblasts and (iii') human placental villous explants. Our results demonstrate that InIA mediates attachment to E-cadherin receptors in syncytiotrophoblasts, and subsequent invasion of the underlying villous tissue.
Therefore, L. monocytogenes ability to target and cross the human maternofetal barrier relies on the interaction between the bacterial protein internalin and its cellular ligand E-cadherin at the villous trophoblast barrier level. This is the first identified ligand-receptor interaction that allows a bacterial pathogen to specifically target and cross the placental villous trophoblast barrier.

Materials and Methods Bacterial strains, cell lines and antibodies The L. monocytogenes type strain EGD (BUG 600), isogenic EGD mutants that 5 lack InIA (DA; BUG 947), InIB (~B; BUG 949), or InIA and InIB (AAB; BUG
949), plus L. innocua transformed with pRB474 harboring the iniA gene (LiA; BUG
1489), or the empty pRB474 expression vector (BUG 1528), have been described in previous publications (14, 15). BeWo cells were obtained from the American Type Culture Collection. Mouse mAbs to human E-cadherin (HECD-1 ), and Bcl-2 10 (Bcl-2-100) were purchased from Zymed Laboratories. (South San Francisco, CA).
Previously characterized rabbit polyclonal antibodies that recognize L.
monocytogenes or L. innocua (R11 and R6, respectively) are described in ref.
(16). Alexa Fluor 488 conjugated monkey antibodies to mouse Ig, and Alexa Fluor 594 conjugated monkey antibodies to rabbit Ig werE: purchased from Molecular 15 Probes (Eugene, OR).
Placental sections from women with pregnancy-associated listeriosis Formalin-fixed, paraffin-embedded blocks of placent<~I tissues and chorioamnion from seven women with maternofetal listeriosis were retrieved from the archived tissue collection of the Histopathology Department: of the Cochin-Port Royal Hospital (Assistance Publique-H~pitaux de Paris, France). Five-micron thick sections of tissues were stained with hematoxylin and eosin. Adjacent sections were stained with R11 primary antibodies [final dilution = 1:500 in blocking buffer (1 % BSA10.3% Triton X-100 in PBS)], followed by HRP-conjugated monkey anti-rabbit Ig (1: 400). Antigenantibody complexes werE: visualized using reagents supplied in the Envision kit (Dakocytomation, Carpinteria, CA). Sections were counterstained with hematoxylin.
Culture of trophoblastic cell lines BeWo cells were seeded in 24-well polystyrene plates (Nalgenunc, Rochester, NY) at a density of 2x104 cells/cm2 in MEM (InvitrogenlGibco, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT). After a 24hincubation (37oC, under 5% C02 atmosphere), :,ells were exposed to fresh media with either 1 mM 8-Br-cAMP (Sigma, St l-ouis, MO; induces BeWo differentiation to syncytiotrophoblasts (17)), or with vehicle alone (PBS).
Medium was changed every 24h (2 cycles). After 72h, cell;> were exposed to different strains of Lisferia.
Isolation and culture of primary cyfotrophoblastrlsyncytiotrophobasts and placental vinous explants Informed consent for the use of human placentas was obtained using a protocol approved by the Institutional Review Board of Washington University School of Medicine. Placentas were obtained immediately after a repeat cesarean section delivery at the end an uncomplicated full-term pregnancy. None of the patients received antibiotics prior to the placental harvest. Cytotrophoblasts were isolated by trypsin-DNAse gradient centrifugation descrik~ed by Kliman (18) with modifications (19). Cells were subsequently seeded in 24-well plates, at a density of 3 x 105/cm2, in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen/Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone), incubated for 8h or 96h at 37oC in 5% C02, and exposed to various bacaerial strains. A 0.5 cm thick section of decidual basalis was removed from the placenta, and villous tissue dissected from multiple cotyledons, avoiding the ch~orionic plate (see Fig.6).
A
single villous explant (average weight, 100 ~ l0mg; average size, 125 ~ 5 mm3) was then placed in each well of a 24-well plate containing DMEM, and exposed to various bacterial strains.
Infections of cells and tissue explants A 1 ml aliquot of an overnight bacterial culture was inoculated into 10 ml of BHI
liquid medium (Difco, BD, Franklin Lakes, NJ), and incubated at 37oC with shaking until an OD600 of 0.8 was reached. Bacteria were then recovered by centrifugation, re-suspended in an equal volume of DMEM and diluted 50-fold in DMEM. Medium from 24-well plates containing culturE:d cells or placental explants was removed, and cells or tissue fragments rinsed tvwice in DMEM. One ml of the diluted bacterial suspension was then added per well. Following a 1 h-incubation at 37oC in 5% C02, cells or tissues were rinsed three times in DMEM (5minlcycle;
25oC), and then incubated for an additional 2h in DMEM supplemented with 10pglml gentamicin (InvitrogenlGibco) to kill extracellular bacteria.
Gentamicin-containing medium was removed, fresh medium was introduced, and the material incubated for an additional 22h in DMEMl10% FCS. At the end of an infection, cells or placental explants were rinsed three times in DMEM. Cells were lysed by incubation in 0.2% Triton X-1001PBS for 15 min at 4~oC. Placental explants were disrupted by homogenization in 10 ml sterile tubes (Nalgenunc) using a Tekmar homogenizes (Analytical Instruments/LLC, Golden Valley, MN; 1 min; 25oC; 10 rpm). The number of CFU of intracellular or intratissular bacteria was determined by plating serial dilutions of the resulting lysateslhomogenates on BHllagar plates.
The level of invasion was defined by dividing the number of viable bacteria obtained after gentamicin treatment by the number of viable bacteria that had been inoculated into each well of the 24-well plates. Invasion assays were performed using a minimum of three donor placenta:, each assayed in triplicate for each condition tested. Methods used for normalizing data are described in the figure legends.
Cell and tissue immunolabeling BeWo cells (with or without exposure to cAMP), and primary cytotrophoblasts were seeded and cultured at 37oC on Lab-Tek chamber slides (Nalgenunc) under the same conditions used for culture on 24-well plates. After 72h (BeWo) or 8h and 96h (primary trophoblasts), cells were infected for 1 h with the same inoculum used for the invasion assays described above. Cell s were subsequently rinsed three times in DMEM and fixed for 5 min in freshly made 3%
paraformaldehyde/PBS. Placental explants were fixed in 3% paraformaldehyde for 15 min after a 1 h or 24h infection, incubated for 2h at 25oC in 15%
sucroseIPBS, then placed in a cryomold, overlaid with OCT compound (Tissue-Tek, Sakura, Torrance, CA), frozen (Cytocoof I1, Richard-Alfan Scientific, Kalamazoo, Mf), and stored at -80°C until use. Cryosections (5-7 pm thick) were cut and post-fixed in methanol for 5 min at -20 °C, then rinsed for 5 min in water, and transferred in PBS (5 min; 25oC). Fixed cells and placental tissues were pre-incubated for 1 h in blocking buffer (see above), and subsequently stained for 1 h with HECD-1 (final dilution in blocking buffer = 1:500), R11 (1:500), or R5 (1:500) antibodies.
Following PBS washes (3 cycles; 5 min each), Alexa Fluor 488-conjugated anti-mouse Ig, or Alexa Fluor 594-conjugated antirabbit Ig were added for 1 h.
Following another round of PBS washes, nuclei werE: stained with bis-benzimide (Sigma; 1:20 000) for 5 min or with To-Pro-3 iodide (IMolecular Probes; 1:400) for 15 min. To visualize E-cadherin on the surface of placental explants, tissue fragments were fixed for 15 min in 3% paraformaldehydeIPBS, incubated for 1 h with RECD-1 (1:500), post-fixed for 5 min in 3% paral~ormaldehyde/PBS, and then processed as described above, except that tissue sections were directly incubated with Alexa Fluor 488-conjugated anti-mouse Ig (1:400; 45 min at 25oC).
Placental explants harvested after infections were also fixed in formalin, embedded in paraffin and treated with hematoxylin and eosin or Gram's stain. Sections were viewed and photographed with a Zeiss Axiophot microscope equipped with Olympus UPlan F1 objectives, a Spot RT Slider digitall camera and Spot3 software (Diagnostic Instruments, Sterling Heights, MI), or with a Nikon C1 confocal microscope and EZ-C1 software (version 1.~0).
Results Immunohistologic studies of human placentas from patients with listeriosis suggest that bacterial invasion follows a traps-placental rather than a trans-amniotic route.
We analyzed the location of L. monocytogenes in placentas obtained from women with listeriosis (n = 7). Immunohistochemical studies of multiple sections prepared fromeach placenta revealed blood-borne bacteria in intervillous spaces (Fig.
1A).
Bacteria were also detected on the surfaces of syncytiotrophoblasts and cytotrophoblasts (Fig.1A,B), in the cytoplasm of syncytiotrophoblasts (Fig.
1C), and in villous core adjacent to fetal capillaries (Fig. 1 D-F). Isolated villi (G) and villous clusters (H) contained foci of bacteria forming abscesses (Fig. 1G,H).
Bacteria were also detected on and in amniotic epithelial cells (Fig. 11), but were not apparent in the connective tissue of the subjacE~nt chorion that overlies the maternal decidua (Fig. 1J). These findings suggested that L. monocytogenes infection of the human fetoplacental unit follows a traps-placental route.
They also suggested a testable hypothesis: namely, that extracellular bacteria present in the maternal blood that bathes placental villi recognize and bind to a specific surface receptor, leading to penetration of the syncytiotrophoblast layer and subsequent invasion of the fetal vascular compartment in the villous core.
Villous trophoblasts express the InIA receptor, E-cadherln As mentioned in the Introduction, epidemiological evidence suggests a role for internalin (InIA), an E-cadherin ligand, in maternofetal listeriosis (11 ).
Immunohistochemical studies of sections of paraffin-embedded placental tissue, using a well-characterized mouse mAb raised against human E-cadherin (HECD-1 ) revealed the protein in villous cytotrophoblasts and localized areas of the basal plasma membrane of syncytiotrophoblasts (Fig 2A). This finding was consistent with previously reported analyses (20-22), but 'was inconsistent with the hypothesis that blood-borne L.monocytogenes would interact with E-cadherin at the apical surface of syncytiotrophoblasts. However, the irnrentors' previous work on intestinal tissue had established that immunofluoreacent labeling of E-cadherin with HECD-1 and fresh cryosections yielded a specific: and far more intense signal than with immunoenzymatic labeling of formalin fixed, paraffin-embedded tissue (10). Therefore, the inventors combined epifluoresence techniques with scanning confocal microscopy to survey cryosections prepared from placentas delivered by women after normal term pregnancies. The inventors found that RECD-1 labeled the surface of cytotrophoblasts, and produced prominent staining of the basal and, to a lesser extent, the apical surface membranes of syncytiotrophoblasts (Fig.

F). The inventors were also able to label the E-cadherin pool that is accessible from the intervillous space by fixing placental tissue fragments prior to incubating with the HECD-1 mAb, before embedding and sectioning. The results revealed a clear signal on the apical surface membrane of sync~~tiotrophoblasts (Fig.
2G,H).
Control experiments that omitted the primary antibody did not produce any signal (data not shown). Moreover, staining placental fragments with a mAb specific for the intracytoplasmic protein, Bcl-2, produced no surface staining in either cytotrophoblast or syncytiotrophoblasts (data not shown). E-cadherin was detected by HECD-1 in the amniotic epithelium but was absent from the mesenchyme of the chorion positioned adjacent to the maternal decidua (Fig. 21,J). Together, these results demonstrate that E-cadherin is expressed at the human maternofetal interface.

Studies of the human trophoblastic cell lins~ BeWo indicate that L.
monocytogenes InIA mediates attachment to, and entry into E-cadherin-expressing syncytiotrophoblasts 5 Apical membrane-associated E-cadherin is positioned to act as an accessible receptor for InIA produced by virulent strains of L. monocytogenes that may have entered the maternal blood space. The inventors used a well-established model of in vitro syncytiotrophoblastic differentiation to test this hypothesis (17).
The human BeWo cell line reproducibly converts from a cytol:rophoblast phenotype to a 10 syncytiotrophoblast phenotype when cultured in presence of 1 mM 8-Br-cAMP
(17) (Fig. 3A,B). The inventors found that the apical membranes of syncytiotrophoblasts formed after a 72h incubation of BeWo cells with cAMP, contain E-cadherin (Fig. 3A,B). The levels of E-cadheirin on apical membranes are lower than in untreated cytotrophoblast-like BeWo cells (n = 6 comparisons;
each 15 performed in a blinded fashion) (Fig. 3A,B) (23). The role of InIA in attachment and invasion of syncytiotrophoblasts was subsequently tested by incubating 8-Br-cAMP-induced BeWo syncytiotrophoblasts with L innocua harboring an InIA
expression vector or the vector alone, or with isogenic InIA+ or InIA- strains of L.
monocytogenes. Invasion was assessed by gentamicin survival assay (24). As 20 with cultured enterocytes (25), the results confirmed that bacteria attach to syncytiotrophoblasts in an InIA-dependant manner and recruit E-cadherin to their sites of attachment (Fig. 3D,D'). The level of invasion of cAMP-induced syncytiotrophoblasts by L. innocua expressing InIA (LiA) was 436~112 greater than with L. innocua that did not express internalin (Li) (n = 3 independent experiments, each performed in triplicate; p<0.001; Student's t-test) (Fig.
3B,D,F).
The level of invasion of wild type L. monocytogenes (strain EGD) was 27~10 greater than an isogenic InIA- strain (L1A) (Fig.3H).
Similar differences were noted using cytotrophoblast-like BeWo cells that had not been exposed to cAMP_ i.e., strain LiA exhibited 600~57 greater entry compared to Li (p<0.001 ) (Fig. 3A,C,E), while wild type L. m~onocy~ogenes (strain EGD) manifested 18~5 fold greater internalization compared to the isogenic InIA-strain DA (Fig. 3G).

InIA mediates bacterial invasion of primary syncytiotrophoblasts BeWo cells are derived from a malignant choriocarcinoma (26), and thus may have a number of differences when compared to normal cytotrophoblasts and syncytiotrophoblasts. Therefore, the inventors used primary cultures of trophoblasts, obtained from placentas harvested after normal term-pregnancies, to examine E-cadherin expression and the role of InIA in bacterial attachment and entry. Harvested cells are known to differentiate from cytotrophoblasts to syncytiotrophoblasts over the course of a 4d culture in DMEM medium supplemented with 10% FCS (19, 27, 28). We found that E-cadherin expression in the differentiated syncytiotrophoblasts was lower than in cytotrophoblasts (Fig.
7A,B) (23). Attachment and internalization was markedly higher for L. innocua expressing InIA compared to L. innocua (10.4~1.7-fold; p<0.001 ) (Fig.
7B,D,F), and for the wild type EGD strain of L. monocytogenes compared to the InIA-isogenic strain DA (16.7~7-fold; p<0.001 ) (Fig. 7H). Multilabel immunohistochemical studies also established that InIA-directed attachment to the cell surface was accompanied by recruitment of E-cadherin (Fig. 7D,D') InIA mediates crossing of the trophoblastic barrier in human placental explants Having demonstrated that InIA directs E-cadherin recruitment, cell surface attachment, and entry into cultured CAMP-treated BeWo-derived, as well as primary syncytiotrophoblasts, the inventors asked whether the InIA-E-cadherin interaction was critical for bacterial targeting and crossing of the trophoblastic barrier in the normal intact placenta. To address i:his question, the inventors exposed placental villous explants to the EGD strain, and to its InIA-deficient isogenic mutant derivative, DA.
InIA mediates bacterial invasion of primary syncyfiotrophoblasts BeWo cells are derived from a malignant choriocarcinoma (26), and thus may have a number of differences when compared to normal cytotrophoblasts and syncytiotrophoblasts. Therefore, the inventors used primary cultures of trophoblasts, obtained from placentas harvested after normal term-pregnancies, to examine E-cadherin expression and the role of InIA in bacterial attachment and entry. Harvested cells are known to differentiate from cytotrophoblasts to syncytiotrophoblasts over the course of a 4d culture in DMEM medium supplemented with 10% FCS (19, 27, 28). The inventors found that E-cadherin expression in the differentiated syncytiotrophoblasts was lower than in cytotrophoblasts (Fig. 7A,B) (23). Attachment and internalization was markedly higher for L. innocua expressing InIA compared to L. innocua (10.4~1.7-fold;
p<0.001 ) (Fig. 7B,D,F), and for the wild type EGD strain of L. monocytogenes compared to the InIA- isogenic strain DA (16.7~;7-fold; p<0.001 ) (Fig. 7H).
Multilabel immunohistochemical studies also established that InIA-directed attachment to the cell surface was accompanied by recruitment of E-cadherin (Fig.
7D,D').
InIA mediates crossing of fhe trophoblastic b~~rrier in human placental explants Having demonstrated that InIA directs E-cadherin recruitment, cell surface attachment, and entry into cultured cAMP-treated BeWo-derived, as well as primary syncytiotrophoblasts, the inventors asked whether the InIA-E-cadherin interaction was critical for bacterial targeting and crossing of the trophoblastic barrier in the normal intact placenta. To address 'this question, the inventors exposed placental villous explants to the EGD strain, and to its InIA-deficient isogenic mutant derivative, DA. As in primary cell cultures, E-cadherin was detected on the apical surfaces of syncytiotrophoblasts (Fig. 4). After a 1 h infection, InIA-dependent attachment to and invasioin of the syncytiotrophoblast layer was evident and the IniA-expressing EGD strain was prominently represented in vasculo-syncytial areas of the trophoblast layer where the maternal and fetal circulations are in close proximity (Fig. 4 ~A,C), while the InIAdeficient mutant was noticeably absent. Quantitative assessment of bacterial invasion using the gentamicin survival assay disclosed that the level of EGD invasion was more than ten-fold higher than that of iniA mutant at the 1 h and 24h time points, respectively (p<0.001 in each case) (Fig. 4E). At the 24h time point, which corresponds to a 1 h infection with L, monocytogE~nes, a 2h incubation with gentamicin to kill extracellular bacteria, and an overnight cuiture in DMEM
without antibiotics to allow intratissular bacterial growth, bacterial replication produced histological lesions indistinguishable from those observed in the villi of placentas harvested from women with maternofetal listeriosis (compare Figs. 1 C-F and 4B,D). In contrast, placental villitis was not observed in explants infected with the .A mutant: at the 24h time point, DA bacteria were only detected in non-trophoblastic components of the placenta (e.g., in fibrin-containing deposits and in maternal neutrophils and phagocytes in the interviilou;~ space blood; Fig.
4F).
Analogous invasion assays perfiormed with amnion tissue (n = 3 independent assays, each performed in triplicate) revealed that the fetal-facing E-cadherin-positive surface of the amniotic epithelium is susceptible to InIA-dependant invasion, while the connective tissue of the maternal face of the chorio-amnion does not exhibit this dependency (Fig. 8).
Taken together, our results demonstrate that InIA mediates attachment to Ecadherin receptors in human placental syncytiotrophoblasts. The resulting bacterial invasion of the syncytiotrophoblast barrier and underlying villous tissue allows L.monocytogenes to replicate and recapitulate in this ex vivo model the histopathological lesions seen in intact placentas of women with listeriosis.
Discussion The inventors' studies of the location of L. monocytogenes in placental and amniotic tissue samples from women with documented fetoplacental listeriosis suggested that L, monocytogenes crosses the maternofetal barrier at the villous syncytiotrophoblast level, with secondary infection via the amnion epithelium.
Since our recent epidemiological studies indicated that InIA plays a role in human fetoplacental listeriosis (11 ), the inventors explored thE: pattern of expression of the InIA receptor, E-cadherin, at the maternofetal interfacE> and found that it is present in villous cytotrophoblasts and at the basal and apical plasma membranes of the syncytiotrophoblasts. The inventors used established trophoblastic cell lines, primary trophoblast cultures, and placental villous explants to show that L.
monocytogenes invades syncytiotrophoblasts via the apical membrane in an InIAdependent manner.

Pregnancy-associated infections are often silent for the mother but can have devastating effects on the fetus. A wide range of intracellular pathogens, ranging from Toxoplasma gondii to rubella virus and L. monocyfogenes, are able to cross the maternofetal barrier. Host microbial interactions that mediate placental tropism have been described. For example, Plasmodium falciparum-infected erythrocytes express the malarial protein PfEMP1, which interacts with chondroitin sulfate A
expressed on placental villi (29). To the inventors knowledge, the bacterial InIA-trophoblast E-cadherin interaction described in the current study is the first ligand-receptor interaction that has been identified which allows a bacterial pathogen to specifically cross the placental villous trophoblast barrier. The physiological significance of apical E-cadherin in syncytiotrophoblasts is unknown. Its presence may simply reflect the presence of remnant surface membranes left over from the fusion of mononuclear cytotrophoblasts to multinucleated syncytiotrophoblasts during normal villous trophoblast differentiation (30, 3'I ). The syncytiotrophoblast is the only syncytial epithelium in humans. The absence of lateral cell membranes distinguishes this barrier from other epithelial barriers. Differences in the expression of proteins in the apical and basal membranes of the syncytiotrophoblasts, compared to other epithelia, have been described. For example, Na+,K(+)-ATPase is apically and basally expressed in syncytiotrophoblasts, but is restricted to the basolateral surfaces of other epithelia (32). Another unique aspect of syncytiotrophoblasts is their direct exposure to maternal blood in the intervillous space, effectively making the syncytiotrophoblast a specialized form of endothelium. L. monocyfogenes appears to take advantage of this unique feature so that blood-borne bacteria can adhere directly to the trophoblast epithelium, invade the trophoblast layer, and thereby gain access to the core of placental villi. We propose that this particular feature provides a molecular explanation for the striking placental tropism of L. monocytogenes in humans.
Although the inventors found that InIA can bind to the apical surfiace of amniotic epithelium, this effect was only detected when infection was initiated on the fetal side of the amnion. Consequently, InIA-dependant infection of the amnion may amplify a pre-existing infection acquired via a traps-placental route and thereby increase the known risk for premature delivery in women with listeriosis. The listerial surface is decorated with an exceptionally high number of surface proteins (33, 34). Would proteins other than InIA also be implicated in crossing the 5 placental barrier? Another well-documented protein also involved in entry into mammalian cells in vitro is InIB. This protein mediatE>s bacterial entry into a wide variety of non-phagocytic ceil types including epithelial, endothelial and fibroblastic cell lines (8). The three known InIB receptors --c-Met, gC1 q-R and glycosaminoglycans - are expressed in virtually all (human tissues, including the 10 placenta (35-37). Invasion assays with isogenic InIB + or InIB- L.
monocytogenes strains indicated that InIB does not play a role in bacterial entry into cultured BeWo-derived or primary syncytiotrophoblasts (data not shown). Nevertheless, the inventors found in villous explants a lower level of infection of the inlB
mutant, compared to the parental EGD strain: the level of inv<~sion was comparable to that 15 of the inlA mutant and that of an inIAB double mutant (data not shown).
This finding is not surprising since virtually ali cell types tested so far -in the presence or absence of a functional IniA pathway- internalize InIB-expressing bacteria (8).
However, in contrast to the InIA-E-cadherin interaction, the ubiquitous expression 20 of InIB receptors does not make InIB a good candidate for mediating the specific targeting of L. monocytogenes to the human placenta. Accordingly, maternofetal infections do not appear to occur in mice as electiveiy as in humans, an observation that correlates with the fact that the mouse is permissive for the InIB
pathway and non-permissive for InIA-Ecadherin int:eractians (9). In conclusion, 25 our results show that L. monocytogenes deploys a common strategy to target and cross the intestinal and placental barriers. This raises 'the possibility that L.
monocytogenes placental tropism may be a consequence of its evolved mechanism for targeting the intestinal epithelium. Interestingly, the blood-brain barrier is composed of E-cadherin expressing microvascular endothelium and choroid plexus epithelium (13).
Thus, it is tempting to speculate that L. monocytogerres targeting to and invasion of the CNS may also be mediated by the interaction between InIA and E-cadherin.

Other human pathogens such as Toxoplasma gondii or human cytomegalovirus (CMV) exhibit a similar tropism for these three barriers. Future studies focused on deciphering the similarities and specificities of these barriers may extend our understanding of the strategies microbes deploy to breach them.
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EXAMPLE 2: A molecule marker for evaluating the pathogenic potential of foodborne Listeria monocytogenes Abstract Background: Internalin mediates Listeria monocyfogenes entry into same human cultured cell lines and crossing of the intestinal barrier in transgenic mice expressing its receptor, human E-cadherin, in enterocytes. The relevance of these results for humans is challenged by the observation that some L. monocytogenes isolates express a truncated non-functional form of internalin.
Methods: The inventors investigated internalin expression by immunoblot assay of 300 clinical strains collected in France in a single year, and a representative set of 150 strains collected in food products during the same period.
Results: Clinical strains expressed full-length internalin far more frequently [2881300 (96%)] than strains recovered from food products [981150 (65%); Odds ratio (OR) =12.73; 95% CI 6.27-26.34; P<1x10-']. All 61 strains from pregnancy related cases (100%), 55 of 56 strains from central nervous system infections (98%), and 151 of 162 strains from patients with bacteremia (93%) expressed full length internalin. All 110 strains belonging to serovar 4b, the most frequent serovar implicated in human listeriosis, expressed full-length internalin.
Conclusions: This study demonstrates the critical role of internalin in the pathogenesis of human listeriosis. It provides a molecular explanation for the predominance of serovar 4b among clinical isolates and supports the usefulness of studying internalin expression as a marker of virulence for humans.
Introduction There is a great sense of urgency to develop public health policies that effectively protect the population against pathogens that can invade our food supply. An ideal program would deploy a rapid and reliable molecular diagnostic test that would be able to detect the organism in components of the food supply, and predict the pathogenic potential of isolates.
The Grarn-positive foodborne pathogen Listeria monocytogenes can cause large outbreaks [1-7], and produce high case-fatality (20-30%). L.
monocytogenes has the ability to cross the intestinal, blood-brain and fetoplacental barriers:
septicemia, central nervous system infections, miscarriages and stillbirths are risks to those who are immunodeficient or pregnant [8].
The annual cost of acute foodborne illness due to L. monocyfogenes in the USA
is ~$2.3 billion (ht~:I/www.ers.usda.govlbriefinglFoodborneDisease/listerial).
The cost of surveying national food supplies for L. monocytogenes contamination is great [9] as are the challenges of product recalls: e.g., in October 2002, L.
monocytogenes contamination led a U.S. poultry producer to recall 27.4 million pounds of fresh and frozen ready-to-eat poultry products [10].
Current surveillance schemes regard all recovered L, monocytogenes strains as equally pathogenic, since phenotypic or genotypic markers for assessing virulence are lacking. However, studies of bacterial surface immunodeterminants in clinical versus food strains [11, 12], plus studies of infection in animal models [13-16], suggest that L, monocyfogenes virulence is heterogeneous.
Virulence relies on the organism's capacity to survive in phagocytic cells [17, 18]. Recent studies have also shown that L. monocytogene,~ can enter and replicate in cultured non-phagocytic cells [19, 20] through a process mediated by the bacterial cell surface protein internalin [21-23]. The receptor for internalin is E-cadherin, a transmembrane protein expressed by epithelial cells [24).
Intemalin has a high degree of specificity for human E-cadherin [25]. Once L.
monocytogenes enters host cells, a bacterial pore-forming toxin, listeriolysin (LLO) lyses phagocytic vacuoles, releasing the organism into the cytoplasm, where it 5 multiplies. Bacteria move within and between human cells by using its ActA
protein to polymerize actin [20]. Infection of mice, genetically engineered to produce human E-cadherin in their intestinal epithelium, with strains of L.
monocytogenes that have, or that lack internalin, indicate that this bacterial protein plays an essential role in mediating crossing of the intestinal barrier [26].
10 The finding that some L. monocytogenes strains express a truncated non-functional internalin [27] prompted the inventors to underkake an epidemiological study to examine the expression and size of internalin in members of a panel of strains recovered from patients and from food surveillance programs, and to analyze whether internalin polymorphism could be correlated with disease 15 manifestations. The inventors' goal was to decipher the role of internalin in human listeriosis, and determine whether it could be used as a readily assayable biomarker to assess the virulence potential of strains that contaminate the food supply.
20 Materials and Methods Study design - All 450 L, monocytogenes strains studied were recovered within a single year (1995) by the National Reference Center (NRC; Institut Pasteur, Paris, France, http:llwww.pasteur.fr!), the central collection facility for foodborne L. monocytogenes surveillance in France. Three hundred strains were 25 from the patients representing sporadic cases of listeriosis that were referred to the NRC in 1995 [11]: of these, 61 (20%) were pregnancy-related. The remaining 239 non-pregnancy related strains included 162 (68%) from the blood of patients with bacteremia, 56 (23%) from central nervous system infections, and 21 (9%) from focal infections. A total of 150 strains from various types of food were 30 randomly selected from the 4,995 food strains isolated by French laboratories and received by the NRC in 1995, and designed 'food strains'. Sixty of the strains were isolated from milk or dairy products, 50 strains were from meat or meat products, 23 strains were from seafoods, 15 strains were from other foods, and 2 strains were from unknown sources. Foods included raw and ready-to-eat versions. The stage of the food chain at which foods were sampled was unknown. All strains were identified as L. monocytogenes and serotyped using standard methods [28, 29]. Strains EGD (serovar 1/2a from the Trudeau Institute, Saranac Lake, NY) and L028 (serovar 112c originally recovered from the feces of a pregnant woman [30]) were used as reference strains.
Immunoblotting - Internalin was assayed using an immunoblot assay that employed previously characterized monoclonal antibodies specific for the amino-terminal leucine-rich region of the protein required for bacterial entry into host cells [31]. Proteins were extracted from each strain [11], separated by electrophoresis through 8% polyacrylamide gels containing 0.1 % SDS, and transferred to nitrocellulose filters. Filters were treated for 1 h (37°C) with blocking buffer (phosphate buffered saline (PBS) supplemented with 0.2% Tween-20 and 5%
dried skim milk), followed by 1 h in the same buffer containing 1 pg/ml of G6.1 or 14.4 mouse monoclonal antibodies [31]. Filters were washed in PBS/0.2% Tween-20, and treated for 1 h with horseradish peroxidase conjugated goat anti-mouse immunoglobulin. Antigen-antibody complexes were detected with the Phototope-horseradish peroxidase Western blot detection kit (New England Biolabs).
Data analysis - Chi-square or Fisher's exact tests were used for statistical analysis (Epilnfo software version 6.04). All tests of significance were at the 5%
two-sided level.
Results Expression of internalin - Internalin was detected in 448 o~f the 450 strains surveyed. Four variants were identified. The most common (386/448 strains;
86%), co-migrated with the full-length internalin produced by the reference type strain, EGD [31] (Table 1 ). Sixteen strains (4%) expressed a truncated protein that co-migrated with internalin produced by t_028, the strain in which a carboxy-terminal truncated variant was originally described [27]. This mutant lacks the ability to anchor to peptidoglycan on the bacterial surface, and, thus, to direct internalization of L. monocytogenes into human E-cadherin-expressing cells [27, 32] (Fig. 11 ). Forty-one strains (9%) expressed a truncated internalin whose size was less than that of L028, while 5 isolates (1 %) expressed a variant intermediate in size between EGD and L028 internalin (Table 1 ). The specificity of the monoclonal antibodies for the intact amino-terminal leucine-rich repeats of internalin and the known structure of the L028 variant provide evidence that each of the three truncated internalin variants lacks the carboxyl-terminal region of the protein necessary for anchorage to bacterial surface peptidoglycan and internalization [27, 32, 33] (Fig. 11 ).
Full-length infernalin in clinical strains - Expression of full-length internalin was strongly associated with clinical strains: 2881300 (96%) versus 981150 (65%) of food strains (Tables 1 and 2), whereas expression of truncated internalin was strongly associated with food strains (OR=12.73; 95% CI 6.27-26.34; P<1x10-~;
Table 2).
Ninety five percent of the strains recovered from non-pregnancy-related cases (227!239) produced full-length internalin (Tables 2 and 3). All strains responsible for the pregnancy-related cases (61 /61 ) expressed the full-length protein (Table 2).
Statistically significant differences were observed when comparing the proportion of full-length internalin-expressing strains from each clinical category studied (pregnancy, bacteremia, CNS and focal infection) with strains recovered from food (Table 2). In addition, among the clinical categories, there was a statistically significant higher proportion of full-length internalin-expressing strains that are able to cross the intestinal and fetoplacental barriers (i.e. strains responsible for pregnancy-related cases) compared to strains that 'only' cross the intestinal barrier (bacteremia strains) (P=4x10-2) (Table 2). Together, these results provide evidence that support the importance of the interaction between full-length internalin and E-cadherin in allowing translocation through these barriers.
Full-length intemalin expression among L. monocytogenes serovars - The distribution of serovars in the 150 food strains was identical to their distribution among the entire 4,995-member collection (Table 1 and data not shown).
Clinical strains more frequently belonged to serovar 4b (OR=5.58; 95% CI 2.63-9.87;
P<1x10-') and food strains to serogroup 112 (i.e. serovars 112a, 112b and 112c) [11]. All 110 strains belonging to serovar 4b (98 clinical, 12 food), and all 84 strains belonging to serovar 1/2b (58 clinical, 26 food) expressed full-length internalin (Table 1 ). None of the 25 strains from serovar 1/2c, to which strain L028 belongs, expressed full-length internalin (8 clinical, 17 food) (Table 1 ).
Serovar 112a strains expressed full-length internalin (83%) or a truncated internalin (16%) (Table 1 ). In this serovar, full-length internalin expression was more often associated with clinical strains than food strains (127/131 clinical strains versus 56/89 food strains; OR=18.71; 95% CI 5.94-65.58; P<1x10'') (Table 1 ).
Discussion The finding that expression of full-length internalin is highly associated with clinical strains provides epidemiological evidence in support of its critical role in human listeriosis. The inventors' study also reveals that internalin can be used as a biomarker for stratifying the virulence of strains recovered from food surveillance programs.
L. monocytogenes has been considered a foodborne pathogen for two decades [1]. However, its intestinal tropism has only been reported recently [4-7].
The significantly higher proportion of full-length internalin in strains responsible for bacteremia compared to food strains supports an observation first made in a genetically engineered 'humanized' mouse model that expresses the human E-cadherin receptor for internalin in their small intestinal epithelium [26]:
namely, that internalin is required for crossing the intestinal barrier. These results are also consistent with a recent suggestion that expression of truncated internalin might be associated with asymptomatic carriage of L. monocytogenes [33].
The cellular constituents of two important biological barriers in humans express E-cadherin: endothelial and choroid plexus cells in the blood-brain barrier, and cytotrophoblastic cells in the fetoplacental barrier [25, 34]. Evidence supporting a role for internalin in crossing these barriers has not been reported previously. We have found that the proportion of strains responsible for pregnancy-related infections that express full-length internalin is significantly higher than the proportion of strains responsible for bacteremia (100% versus 93%; P=0.04 and p<1 x 10~' when pregnancy-associated strains are compared to food strains).
This finding strongly suggests that internalin is involved in the crossing of the fetoplacental barrier. There was no statistically significant difference between the proportion of strains from central nervous system infections expressing full-length internalin and the proportion of bacteremia strains that produce this form of the protein (98% versus 93%; P=0.3). Therefore, evaluating the role played by internalin in crossing the blood-brain barrier will require further investigation.
In this study, full-length internalin appeared dispensable in strains causing a small number (4%) of cases of listeriosis (11 bacteremia and 1 CNS infection strains). However, all 12 of these patients had severe predisposing conditions for listeriosis (Table 3). The fact that bacteremia is observed in the absence of functional internaiin in certain immunodeficient patients fits with the experimental observation that internalin-independent translocation across the intestinal barrier to the systemic circulation is markedly increased in immunodeficient mouse models [35]. Nonetheless, it is important to emphasize that even in the immunodeficient population in our study that had L. monocytogenes bacteremia, the proportion of strains expressing a truncated internalin was small (11/156, 7%).
Most major foodborne outbreaks of invasive listeriosis in Europe and North America have been caused by L, monocytogenes strains belonging to serovar 4b.
Serovar 4b has also been responsible for numerous sporadic cases [36]. In our study, all serovar 4b strains (clinical and food origin) expressed full-length intemalin. Moreover, serovar 4b was more frequent among clinical compared to food isolates. These findings support the notion that serovar 4b is associated with higher pathogenic potential. Interestingly, none of the serovar 112b strains expressed truncated internalin variants, and serovar 1,/2b is closest phylogenetically to serovar 4b [37-39].
The rarity of serotype 1/2c in our panel of clinical strains can be correlated with their expression of truncated non-functional internalins (100% of isolates).
Internalin expression was heterogeneous in serovar 112a with truncated forms of internalin encountered more frequently in food than in clinical strains.
Together, these results indicate that if attenuated or non-virulent strains for humans exist, they are likely to belong to serovars 1/2a and 1/2c, rather than to serovar 4b or 1/2b [11].
In summary, internalin provides a new tool to assess the risk associated with consumption of food products contaminated with L. monocytogenes. The inventors propose that risk should not only be evaluated based on levels of bacterial contamination, but also on the functionality of internalin (full-length versus truncated) produced by a contaminating strain. This should allow for more rational risk assessment and, thus, more efficient and cost effective control measures for foodborne listeriosis. In addition, from a clinical point of view, determining 5 intemalin functionality in L. monocytogenes strains isolated from stools of pregnant women may help to better assess the risk for fetoplacental listeriosis.
References 10 1. Schlech WF, 3rd, Lavigne PM, Bortolussi RA, et al. Epidemic listeriosis--evidence for transmission by food. N Engl J Med 1983;308:203-6 2. Fleming DW, Cochi SL, MacDonald KL, et al. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N Engl J Med 1985;312:404-7 3. Linnan MJ, Mascola L, Lou XD, et al. Epidemic listeriosis associated with Mexican-style cheese. N Engl J Med 1988;319:823-8 4. Salamina G, Dalle Donne E, Niccolini A, et al. A foodborne outbreak of gastroenteritis involving Listeria monocytogenes. Epidemiol Infect 1996;117:429-5. Dalton CB, Austin CC, Sobel J, et al. An outbreak of gastroenteritis and fever due to Listeria monocytogenes in milk. N Engl J Med 1997;336:100-5 6. Aureli P, Fiorucci GC, Caroli D, et al. An outbreak of febrile gastroenteritis associated with corn contaminated by Listeria monocyfogenes. N Engl J Med 2000; 342:1236-4.1 7. Frye DM, Zweig R, Sturgeon J, et al. An outbreak of febrile gastroenteritis associated with delicatessen meat contaminated with Listeria monocytogenes.
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8. Wing EJ, Gregory SH. Listeria monocytogenes: clinical and experimental update. J Infect Dis 2002;185 Suppl 1:S18-24 9. Buzby JC, Roberts T. Economic costs and trade impacts of microbial foodbome illness. World Health Stat Q 1997;50:57-66 10. From the Centers for Disease Control and Prevention. Outbreak of listeriosis--Northeastern United States, 2002. JAMA 2002;288:2260.
11. Jacquet C, Gouin E, Jeannel D, Cossart P and Rocourt J. Expression of ActA, Ami, InIB, and listeriolysin O in Listeria monocyfogenes of human and food origin.
Appl Environ Microbiol 2002;68:616-22 12. Tabouret M, de Rycke J and Dubray G. Analysis of surface proteins of Listeria in relation to species, serovar and pathogenicity. J Gen Microbiol 1992;138:743-53 13. Barbour AH, Rampling A and Hormaeche CE. Variation in the infectivity of Listeria monocytogenes isolates following intragastric inoculation of mice.
Infect Immun 2001;69:4657-60.
14. Roche SM, Velge P, Bottreau E, Durier C, Marquet-van der Mee N and Pardon P. Assessment of the virulence of Listeria monocytogenes: agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice.
Int J Food Microbiol 2001;68:33-44.
15. Larsen CN, Norrung B, Sommer HM and Jakobsen M. In vitro and in vivo invasiveness of different pulsed-field gel electrophoresis types of L isteria monocytogenes. Appl Environ Microbiol 2002;68:5698-703.
16. Olier M, Pierre F, Lemaitre JP, Divies C, Rousset A and Guzzo J.
Assessment of the pathogenic potential of two Listeria monocytogenes human faecal carriage isolates. Microbiology 2002;148:1855-62.

17. Mackaness GB. Cellular resistance to infection. J Exp Med 1902;118:381-406 18. Mackaness GB, Hill WC. The effect of anti-lymphocyte globulin on cell-mediated resistance to infection. J Exp Med 1969;129:993-1012 19. Southwick FS, Purich DL. Intracellular pathogenesis of listeriosis. N Engl J
Med 1996;334:770-6 20. Cossart P, Lecuit M. Interactions of Listeria monoc~togenes with mammalian cells during entry and actin-based movement: bacterial factors, cellular ligands and signaling. EMBO J 1998;17:3797-806 21. Gaillard JL, Berche P, Frehel C, Gouin E and Cossart P. Entry of L.
monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell 1991;65:1127-41 22. Lecuit M, Ohayon H, Braun L, Mengaud J and Cossart P. Intemalin of Listeria monocytogenes with an intact leucine-rich repeat region is sufficient to promote internalization. Infect Immun 1997;65:5309-19 23. Cossart P, Pizarro-Cerda J and Lecuit M. Invasion of mammalian cells by Listeria monocytogenes: functional mimicry to subvert cellular functions.
Trends Cell Biol 2003;13:23-31.
24. Mengaud J, Ohayon H, Gounon P, Mege RM and Cossart P. E-cadherin is the receptor for internalin, a surface protein required for entry of L.
monocytogenes into epithelial cells. Cell 1996;84:923-32 25. Lecuit M, Dramsi S, Gottardi C, Fedor-Chaiken M, Gumbiner B and Cossart P.
A single amino acid in E-cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes. EMBO J 1999;18:3956-63 26. Lecuit M, Vandormael-Pournin S, Lefort J, et al. A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier. Science 2001;292:1722-5.
27. Jonquieres R, Bierne H, Mengaud J and Cossart P. The inlA gene of Listeria monocytogenes L028 harbors a nonsense mutation resulting in release of internalin. Infect Immun 1998;66:3420-2 28. Seeliger H, Hohne K. Serotyping of Lisferia monocyfogenes and related species. In: Bergan T, Morris J, eds. Methods in microbiology: Academic Press, New York, N.Y., 1979:33-48 29. Bille J, Catimel B, Bannerman E, et al. APB Listeria, a new and promising one-day system to identify Listeria isolates. Appl Environ Microbiol 1992;58:1857-30. Vicente MF, Baquero F and Perez-Diaz JC. Cloning and expression of the Listeria monocytogenes haemolysin in Escherichia coli. FEMS Microbiol Lett 1985;30:77-79 31. Mengaud J, Lecuit M, Lebrun M, Nato F, Mazie JC and Cossart P. Antibodies to the leucine-rich repeat region of internalin block entry of Lisferia monocytogenes into cells expressing E-cadherin. Infect Immun 1996;54:5430-3 32. Lebrun M, Mengaud J, Ohayon H, Nato F and Cossart P. Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells. Mol Microbiol 1996;21:579-92 33. Osier M, Pierre F, Rousseaux S, et al. Expression of truncated internalin A is involved in impaired internalization of some Listeria monocytogenes isolates carried asymptomaticaily by humans. Infect Immun 2003;71:1217-24 34. Lecuit M, Cossart P. Genetically-modified-animal models for human infections:
the Listeria paradigm. Trends Mol Med 2002;8:537-42.

35. Czuprynski CJ, Faith NG and Steinberg H. Ability of the Listeria monocytogenes strain Scott A to cause systemic infection in mice infected by the intragastric route. Appl Environ Microbiol 2002;68:2893-900.

36. Kathariou S. Listeria monocytogenes virulence and pathogenicity, a food safety perspective. J Food Prot 2002;65:1811-29.

37. Brosch R, Chen J and Luchansky JB. Pulsed-field fingerprinting of listeriae:
identification of genomic divisions for Listeria monocyfogenes and their correlation with serovar. Appl Environ Microbiol 1994;60:2584-92 38. Graves LM, Swaminathan B, Reeves MW, et al. Comparison of ribotyping and multilocus enzyme electrophoresis for subtyping of Listeria monocytogenes isolates. J Clin Microbiol 1994;32:2936-43 39. Nadon CA, Woodward DL, Young C, Rodgers FG and Wiedmann M.
Correlations between molecular subtyping and serotyping of Listeria monocytogenes. J Clin Microbiol 2001;39:2704-7.

40. Goulet V, Marchetti P. Listeriosis in 225 non-pregnant patients in 1992:
clinical aspects and outcome in relation to predisposing conditions. Scand J Infect Dis 1996;28:307-74 Table 1- Internalin and serovar distributions among clinical and food strains Full- Truncated internalins b Number length -Serovar ~ Origin of strains internalin Total InlA2 InlA3 InlA4 InlAO
[i.e. InlA1]
t~:, ' " ,~ ~ ~~ o."~~a ~EEt,y t r f.
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~,.u,e ;° .~ '°~ t~', a a a.~ iT~.t~a t~53.~, y~~:t~~°°~.':"' . ., f~"« ~ 1."a. xn:.~'F, ,~".,~~ , ;,x".a"
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Clinical 131 127 (97%) 4 (3%)~ 4 (3%) 0 (0%) 112a Food 89 56 (63%) 33 (37%) 0 (0%) 32 (36%) 0 (0%) 1 (1%) Total 220 183 (83%) 37 (17%) 36 (16%) 1 (<1%) Clinical 58 58 (100%) l/2b Food 26 26 (100%) 0 (0%) - - - -Total 84 84 ( 100%) Clinical 8 8 (100%) 6 (75%) 0 (0%) 2 (25%) 0 (0%) l/2c Food 17 0 (0%) 17 (100%) 10 (59%) 3 {18%) 3 (18%) 1 (5%) Total 25 25 {100%) I6 (64%) 3 {12%) 5 (20%) 1 (4%) Clinical 98 98 (100%) Food 12 12 (100%) 0 (~%) - _ 4b - -Total 110 ( I00%) 5 Legend a Only major serovars are listed.
b See Fig. 11 for definitions of InIA1-A4. InlAO indicates that there was no detectable internalin in the strain. Sequence analysis of the internalin gene of the two bacterial strains in which internalin was not detected revealed the presence of stop codons that would lead to synthesis of 10 polypeptides with truncated leucine-rich repeats (LRR) which would render them undetectable by monoclonal antibodies specific for this region of the protein (22, 31].

Table 2 - Full-length internalin expression in L, monocytogenes strains Comparison with Comaarison with Number Fnll-lengthTruncated of Clinical food strains bacteremia origin strains strains internalininternalin OR (95% CI) and OR (95%
P value a CI) and P value 12.73 (6.27-26.34) All 300 288 (96%)12 (4%) -P<1 10'' x 7.28 (3.47-15.61) Bacteremia 162 151 (93%)11 (7%) -P<1 10'' x Pregnancy- _7 -z 61 61 (100%)0 (0%) P=1x10 P=4x10 OR=8.45 related cases (1.11-177.58) 29.18 (4.17-583.38) CNS infections56 55 (98%)1 (2%) P=3x10-' P=1x10'z P=2x 10'6 Focal infections21 21 (100%)0 (0%) P=1x10'3 P=6x10-' Legend a OR, odds ratio; CI, confidence interval; P values were calculated using Chi-square or Fisher's exact tests.

Table 3 - Characterization of L. monocytogenes strains responsible for non-pregnancy-related cases Internalin Number of Predisposing Clinical Mean age Sex ratio form type strains conditions b Truncated 11 70 2.7 11/11 BacteremiaFull-length 1 S 1 67 1.6 144/ 145 All 162 67 1.6 155/156 Truncated 1 SS - 1/1 CNS

Full-length SS 60 2.2 46/51 infections All S6 60 2.3 47/52 Truncated 0 - - -F
l oca Full-length 21 73 1.2 21/21 infections All 21 73 1.2 21/21 Truncated 12 69 3.0 12/ 12 Total Full-length 227 66 1.7 211/217 All 239 66 1.7 223/229 Legend a Male/female ratio Patients with severe immunosuppression: individuals with AIDS or receiving chronic immunosuppressive therapy for malignancies, organ transplants or other diseases; patients affected by immunosuppressive risk factors such as dialysis, diabetes me:Ilitus, alcoholism, hepatic failure or malignancies without immunosuppressive therapy, patients with cardiovascular, respiratory and neurological diseases and patients >64 years old [40]. Clinical information was not available for 10 patients (6 with bacteremia; 4 with CNS infections).
Focal infections: 9 peritonitis, 3 aortic aneurysm infections, 2 pleurisies, 2 urinary tract infections, 1 endocarditis, 1 vasculitis, 1 pan-ophthalmia, 1 lymphadenitis and 1 cervical abscess.

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.y.= .
IN1EltNATIDNJ1L DG,POSITh(IY 4Vi"IIDAItY 'r.'.; ;' DECLARATIO N ~N VUE D'x~N v~,~oT ~~t~'~c'~A.x. COri~Ol~.'wz~;mtEt~'r .~ rah, zRCGL~ sx .~tc'~tet7?~Ertt ire dae case of an original deposit pursuant ro II~..rtle 61 F.;N V'tJ'E DUNE CfJNVI:.R.StflN cONx~oR,.~~'MC~z~I'Ir A Lh RJ:.GI,tE G.~t.c~
' W the case of a coaver~ion parsaant to Rule e~4.d _ ' ' 1GN V'(TE D'UN CON'L'RA'~ASSOCxIL~ (SO~C~k'tE-1XC3TE O'C.T COlVx~'OSA.N7~ ~~
in tha case of ara assacfated canu~act (host ~tYUn Or corrllnartNJtt) .x~ ~ TYPE.DE MICf~00~iG/~IrIISNtE, ' ~. : '. ~ GEILTUR1""c C~E(..L.(,~~.ALR~
~"yp~ of lnieroarganism' ~ ~ Cell culture ~ ~ ' ~ ~ ~ ~ ~ ~ ' -* MiCROCtRGAivtiSME lSOL.E
,Single microorganism MELAhIGE DE MIGROOI'~GANiSMcS
Mixture of rrticrodrganisms --------- ;,--~------,-'~--- --~-"
Da ceIIuIes 9ttfcctces, cuixti~minEes, ... INpIQUER LE CASi ECIiEANT LE HOMHRE
OE MICROUR6APIISMES ET LEURS TYP(;S
uracfected Cellf, cOJttatttiriated C~tIS, etC GIVE THE NtJtifsEn pf kIICROOticultl,SMS AtiG TNEIft KINDS HIIeRC rPrLICanIE
LE SOLTSSIGNE DIa'OSE I.E I1~A1:'lL:k~I~'.i~ IDI°aN'I'Xxrl~', ~:X-t'i,~'ItES ~7' S'ENG.~GE.As, N1C: h,A,S RLt~TIRFR x.E DEt~U'JC' lf.~ND~.Nx' LA,~PEI~OfDE PRE,f:ISETS ~~ z,n mcL.r ~.x.
the undcrsi~r~ed lxereby deposits the rr,Iaterxat ide>llijad hcr~undcrrmsd.
Iwidt:rlakes r:ot to witlub'a~u Uxe dcp~sit fal' U9e period specified en rT,ete 9.1.
. ~R~FCREPICE 0'6DENTIFICA"fION ~ . Y NUH~RO OU SYMBOLES. ~ 6KEMPLE, DDNNCS
fAR LE DEPQSANT~LIU MATERTEL
lC~8I7~'If%C~tlall rC3fPr2IlCE »nceER. SrMUot.s, ecc, 'clvCx To n~C raTE:~rn4 aY TW oFPOSITUR ' ~ 7-7 .
2. o~POSat~Tts) t~o~.(s) r:~ adccasc(s) SfR'fICE IRS BREYL"TS
laapositar (sJ ~,°I=~ttwE_~,,~°~~~;;;
NCnIIC:~S~ and athlrers(cs') j , ., INSTXT'CIT PAST~.;C.L' ' . 25r28, rvc d a Ooctaur Roux 757?. ~ Pons Cedex 75 _ l ~. ' _., - ",~,~~._~"_,_"~ ""~",.,_ ) CETTE FORMULE DOLT ETRC REhIP~.IF. SllttS RATURES piI Su(tCHARGES .
C.'T'.T.T.961i . . ~atr.
It,E~~I~NCED'IDFN'X'xl~'~CA~FOi~i' ...~L''T-~ ' , .pate G/OG/U4 -l6ieliLlJ6GaliOrt referCrtCe - , ---~ . '--"-----.~-~--~---' .....,..».....~,..,.. ~, 3 .,, D)"SCRiPTION SCtENT)FIQLfE Fusion callulaire alttre une c~:l3.u~.E de my~lome SCi~l7tli'iC description - de sourf.s et ~un 3.amnuzzocyte de saur~,s issu de la ' rate. de 7l t au~mn,l ' COt.'HER St t)ES IKft)RMATrONS CAMPLEMEHT~4IRES SaGT F'AUR?LIES StIR tlNE
FFUILLE JUIttTE
fVUtK >dI'nl A CPO55 If AOOII7oHAC INFtitrlATloN TS GIVER off A!P ATTACHED
SHEET
Tyi)~ Gt' Or?,~tl7c (Or~;:krrC~tiSSti, PS~CCC ~nitn.~lC~ ...~ , Tjrpe arid arigiri (or~anltissur, ccrurreal species, ctc) Rate dE sours.;:' . . ~ , ~ ,' . . .. ~ ~ . : .. -Cac~cteristiqnes et prodetctions des ecttrltes C~:araetcristirs arsd prtpdteeGS of the calls ~ .
GOCHt:A LES CASES QUI ~OftVIERNCNT ET DONtt'ER DES IHFQRMATIOKS
Ct7htPLt;MENTAiEtES ' ' wuac wlnl A caves ul,.eae AaI~.I~,aIF aHa sIY~ ~onltroNnwNS'aarwrloN
Hybridnme lVlyetot~ir utiiis~. - X 63~Ag8--G . S . 3 ,FlyBridorna MyalonTac daxigr:atian .
Sp~c~cit~ untit;~ttxiquc ' .~inti~r~ie,slrecifscicy - 't'nt~:ra~:Lin~.: A .
G'lasse I~ T 1;1 / k .Rnti6ody subclars - .
StabltitC do la secrCtxon ~otane etr :Ll3.iutite:~
Stability of sacretiarx - , Ligt~~e ri~ndtiqudment taxodifa~e Yccteur s) tJersetically modifccd cell line - ~ Yeccor(sS ----'°."-' .
. . ~ . ~ . . y . . , . : . . . ' ~: y ~ -A.c.uurlciqne(s) i><sser~(s) ~ .~ ' ~ . . ~ .. , _, .
. . - . . . , , . , ': Nucleic acid irlsa't(s) . , . . _ -Cetlules inPectecs (W rue sauwcgc) Calls infected by a wiltt-type viru~r 'fa~nce celluiaire ntouvc)lcrxrra't ~ttbtie . .
Newly established cell lira Autrr-x particutarites Fccrrhcr or otlserpurticularities Dcscriytioni . .. . . . . , - . . . - , .
Details ' - , ' ' .. . ' ~ . . , . , . ' Iiybx3,dame ~Lssu de 1.a tus~ands'~o.l~.ules spleniques cue sourta immuxtis~os cunt:rc' Tnte'rrta:Llne A ell.? Li:;t~F~rl.a ntoxzocyt.ogett'i~s .wN.~.r. 7.c~
ril;l~;mucj.romc A~;F3 ~X x.5.3 l2~fcriextePS biblionr:e~hieyees . ' ~flE?'drllTe Teferdr~lCc:S
L'~NOICATICN OE CC5 z'IVFOitM11TI4HS EST FACUt.iATIVE, tfAlS SIIVFMENT
RECOiIMANI7Et: AUX 'fEfitMES OE t,A REGL,E 6.I:b I 'iil$ 511PPLY1HG OF S(iCH TNFURI'ATION IS IiPTIdNAL, BUT S'fRONt:LY
It~COMftEHOEQ tN ASCOROAriCf StI311 atll~6.Z_b CE'1°iC FURtIUt,E tIOIT ETRE REMPLtE SAttS MTUR~5 KI SBRCNARGES
Cl~LL96/Z
TH:c Cenft'SfInUCff t5E 011tY CrriPLFYEU WITHOtif OEL~TIOlIS IlA.
ALTF1TR'YtnNc Rrlr~cr,~N~ii p'z~r.~TII~ z~a.~ Iorr ~ . ~ ~, ~ - nR~ ~; I c~ 4 l o ~~
Idmtti~rQtion refercrtca ---- -~-,-..,-._~._.. Z7ate ,_..
4 . '1'ROPRtE'1'~S DANGEREUSES PC?UR L.A SANTE oU t; ENV'lftCl~tI~IEMENT
ProyErties dangerous to f>ealth or environment T~.-ucultux'e-ou le zya6lsza~e identifir sdus Ie chiffre x a les yrnpri~t~s stltwantes qui pz'~seatemt ou g~cuvent prc'sentex des datxgcrs ians Ia santE otzlet 1'czxvirontnezzaczxt Tls~' ticlttu a or trt8 f7tLYttlT4 iGlElItd !Pd undcu Z above has the following ,~rnpertics wliich arc .nr»tdy be dart$erdscs to > ~alth orland titc~ enviroltr7aenf COCEf(:R SI 0~5 INFOft~tATIONS COMpLEMCNTAI~R~5 SORT FOITANIES SUFI U!t~
FfUIt,IC JOINTS
WIBK HiTtl A CR455 IE AI~ITiONAL t~F~f~NTIUH IS GIVrM ON A11 A'1'Tr7CHE0 SIiE$T
Le sotissi;ts~ zz'a p:zs coazxaissance de tetles ~rop0.~ivtes.
g 27te ttndarsi~~~,d is not aware of such proprrties.
. TOUfk: ACTION DIREC'fE t?U INOIRGCTE. CONNUE t1U pREVISIBt.E, SUft Qll~LQU'ORGA
NISMC QUE CE SOTT (AlIIMAL. VEGE'i'AP. OU AUYRE) OOIT E1RC 5I6HALGE.
COCFI~R lA CASE QUI CONVIENT ARY OItLCT QN INGIftECi' KluWlt Oit LIKELY' TO
flc E%Ff~C~"aD EFFEG'("ON ANY AtIIIWL, Y6GErAL c7R a1'hitt MaNC SIxn1 A clio55 THE APP~LG1HL1 6oX Op~ISM ltvsT gE iHOIGitTEn 5. CCINL7tTlOt~tS DE SECUt~ITE PQUR t.A MAriIIPUl..ATtQN DE LA CULTURE
Biosafety rn$asures required t't? manipulate the culture ~X l.1 J P't ~ L.~ / P2, ~ L.3 I P3 ~ A.utxcs G~dier~
(IFS: Safa Btotachnoio9Y ! FEaERAI REGISTER: sut~a7Snea for R~soa~ch Involvtpp Rccan6tpent DtYA Naleculr_c) CC1ND(TtOhIS DE CUI.~TUHE ~ - partie i ~ . . . , . , - ~. . . . - , , ._. -. Canditiorts far cultivation .:- part i ' ~ ~ ~ , ,. . _. , ~ . , : ~, _ _ , -. ~ ' ; ~ . . ' .. , . . . .
COCHfR SI DES,xNFORhIATIONS (OMPLEtI~NTATR~5 SON"f FOURNIES SUR UNE FEUIILE

HARK IdiTfi A CItOSS IF AGt)ItioNAi. INPORMA'YIpN IS 19iY1;N GN AN AfTAdII;D
51(EE,1 Milieu de cuhure (xr~ec Ies r6f~rences prcciscs des cozra osxxats) Cullicne nxediunx (give f ell details i, f .rp~cial folrnettation~
- MEM de 'E:~I;I.e -- Euz:obia ~- Pyruvat:e de sad~.xua - Gibco BRx, - Glutsmi.ne - Gibco I3IZI.
- Amtib3~at,3.que_s : pe>.ii-s't.rept:auzycine ~- Gibca Bll~;t, .- S~ruat de cherra:f. 10~ _ ~3i~:trUanate de soditrea (m~) 7 , 2 g,Z Sarau~ ~~'~ ' °r) Clie°val 10~a Irft -Sodistfrx bicarbonate , , ,p~~ 7 a 4 '~'~mp~ratnre optilnalc .~ 7 o C ,~ltrnospb~rc 7/ C4~
pptirsuatl tcrnpcf-axttrY! CYascalcsphasc Prccautinns particulii.res a tsi decortgdIation Dt:=coisgaltnr z',~.gidemeL~.t 3 37 ° C, . liver 3.z;~uieediatetu~ut PrucatttiDrwtobetnlcEatforthawirtg dues ~,~, mil,yGU+scrtim. Cua.turEa cur, pLaquc do its pm~.t .. . aE ~. znl, . .
~rLc:xiitions plrticul~i~res ~ 1'iucubal:ion ..
Furtlser-derails: shalcingsystent, ete.
Saltztions tLisprxsazites Tttili sties Solcttions tcscQ for cell disper:tion CETTE FORMUtE OaIT ETRE REhIPLIE SANS RAl'UR~S NI SUItrHAR6f-.S ~ C;1CLLJGt3 rnls ~oa~.s sipwcfl ce ewY asHr~erEO t~_nrour os~rtatts oR At.TfnArtons R.ia~'1:.RENCE ta'IDI:N7.'(P'~~A,~I(,7N L. I-7 ' l7atc y/C14/(74 Idertfificctlion refarcrtce , .~....-- ,L~afc -------,-,~-_ .......
6h . CONDITLQNS DE Ct~f_TtJRI= - partie 2 , Conditions for cultiv~~tiar7 - Purr, COGlIER SI DES rNFURMArXONS COMFLEMGNl'AIRC5 SONT t='OURIti~5 SUR UN~ FEUILi,~
.tfltltT'k MAAK tlt?I! A Cp9SS iF AODtTtONAt. IHFORW~TIUR I5 6IYEN OH, lW4 ATTAGHCD SHEET
'Type do cuTtura Lcetlntes en susgectsiocc, ectlcltes mdlterentcs, ...) et morpitolo~ie cettulairt: attcndsze Type of culaue s sttsperuiort, monolaycr, eLt:, arcd capcctEd cell rrempltoIa~r Call.u~.~S ~n 5u~pctzs~.ts'ci Temps de doubtetnent de poputalion Z 5 li . ~ Temgs optim:ci ent.~e les ~tassz~es ~ j ouxt;
~'opttlalian doublucg uinte t?ptirrtal split rYxtia pen,sitG ecltulaure tcttextda ~ S~t 0~ T3urec de vie titoaitCe x~,x3.m3,t~~
E.tpected cell derssity Z,irnited lifealaarx DetaiLS pour Ie trnssagc eTes cellutes p~.r d~daublattcent Teclutiqtce formcttircc sub-eultur~e ~''t"'"~~"~ n~azit Carttments 7 . ~ ACTTVtTES A V~EiIFtEt~ PL1UR CONFiRMER L0. ViABtLtTE AU DEPOT ~ . ~ , ~
. . . , .
.4ct'ivities to bo-.c~fiecke, d ;confir~ttinc~.~,the viabilit y of,.ttle deposlt~. . ~ y . ~. , . ~ - . ;
COCHER 5I D~5 tHFORMATLOlIS COt~PLEI~IEHTAIRES SaNT FOUitNLES SUR UNE F~tIILLE
JOLNT~
W1RK NtTH A CROSS IF ADDITIONAL 1NFDN14ATIoH IS GIYEIt CN AH ATTACHED SHEET
product~.ou d'anticorps )~.orsquG le depot ports sur un m~lan~c ~C ~att%'OOr~3niSntrso In d~clnt~o.tion dnii cotcteuit~ en, otxtxe 1;~ dlescriptioct das cctmposants øu tabtange et d'uu taotng une dts >u~~tl~odcs permettant de ve:xitier lcur pre<sCnce (Rc~;te 6.~..a.i~ii).
~Ylterc: tt r~stxiure v, f rrticrnur,~ani.rrr~x is depvsifed, dcsscrx~.
tipr~.r of the cam,~artertts of the mixizcre and at least orce pf dt~:
mcdcods perncittirz~ dce checking of thr:ir presence should be divers crc accordance wilt ,kttlE fr.l.a.iii.
8. CUNDtTIC?NS.DIr CC~NSERVAT'ION ' . ' .' . ' . ~, . , Conditions for-storecde - ' ~ ~ . ,~ . ' . ~ ~ . ~ , . ..
COCIiFR SI D~S IIiFOAMATiO~tS GOMpLEMFMTAFR)_5 SONT FOURNIF"5 SUR UNF_ FEULLCE
JOtRTE
MIIic!tt de SttSpCngioil H~~'~ITit A CRO55 IF ADOIIIoNiIL 1HFORs~ATIOH IS
GIYEH OH AN ATTACttED SHi:G1' Suspendir~g fluid 9s~ sE.rum dE cheva,l - 5%' DMSO
Ntndalit~s utelz rGcoite ties ccllu'l~:;s ~ pr~le~,o~ent de 1t>. pus .c~nsiora cel~.u~.afire, .cdn.tr~fti o.tioz?.
Techniclrte forcetl liarvestlrtg P g et: repr3.se du culot ds.tzs le mt3.a,et>, de c;ongelct:Lon ,tvripdalices dels Cori~el~,tiou _ 5~~p6 Go,~~,ulQ$ dads Lm~. de mi.lxeu dC
congclat~.on.
Tecl:rtigurforfrcczing , 2E3,~~a --z0°C, 3 a 4 ,ours a -F3(7°C, pu:i~ za.zot:e l~.quado.
Aatrca ini'orntations Furt)tw coat»tents C~TfE FOR.HUI.E DOIT ~TiIE RC~IP4IE SANS RATUR~S NI SDRGHARG~S .
C1~'.~.1rL96~~4 THIS FOAM 5110ULD 0E DULY CCftPLETED NITHOUT DEI.CT(ON5 OH ALTEftATIOHS .

R7~gERI:N'C'.~ ~'LDEIVTII;ICATIOl'I ~L 7-7' ~ . Date (~ /. ()tv /.04 Ielrnti,~CCation refarence ----..--r----. Date: ._..~,..~,.,.~..~
xx: ' ~N~o~i~nATiarcs suA trA cuI.TUFit~ TRAr~snnisE
n~cossalres ~. des fins d°impottatiosl et/ou,d'exportatior~
Details on the transr»itt~d culture reqmred for expart/ir~tporf forrrralities .~.,..
COCIiGR SI DES INFORCSAT IONS t:OMPLF.MEtiTAiRES SORT FOURHI~S SUR UNE
FI;UILL1" JOIN.TE
. ~ PWiK 41ITH A CROSS I~ AODTI'to~tAA(, IIIFOIUMTIDiV Is fiiVRX OK AN
ATTALIiiiD SHEET i RLt4rences d~s sui~strats Uxg:inic~ttes (~aar~xilssc~ur, r~f~reaxcG, lut, datr,, pays d'oriGine) Rcjerences of ace o aortic subsecttcs (sup,~lier, rcfe:rcm; c, batch, date, country a,/vragir>;,1 ' -. MMirM 0111 - ' Eurobia ~ ~ . ' . ' . .' ~ , . - Sei:uTri de cVeval~ -- .N° X73 39 37G~ - Eaycic D1~~.~n.ost:~.c~:
France . .
-- pyxuvate de sod3lLaa - Gibco ~RL France .
- L-Glutamin.e - G3bco I;R1~ T'raz~ccc ' - Antib~,ot~.ques -- Gibco BRL Fxa.nce 9 a. AUTRES 1NFORMATIONS SUR LA CULTURE TRAhlSMtSE
Further details on the transmitted culture COCHfR SL DES INFpRt'fAI'IOtfS COMPLEMEN'i'AYRES SONT FOtIRNI~S SUR tJLtF
1=~UILL~ JOIN't<:
!SARK ifLTN A CNOSS L~ AOOITIDNAL INFD(W0.TIOH x5 f:LV~N ON A,i ATTACHED
Stlfri' ~Live:au do passage de L, ee>ttt'rrre tra>ntsuxL9e ,l~assal,.e tcvea of the tmrrsrrcitted adttrre Date de ~r~~aratiou 6./ ~ 1! / ~ fA
~.L7ate of prc,~rrrir~Q . ~ . . _ , - ~ . : ~ . . , ~ . ' ~ . . . . , , . ~ .
~ , .' . ' ' ~Coi<.eentratiun rn, celtates ~ ygy06 ~Gell/n~~,' ~ . ~ . ' ' ~ ' ' ' ~ ~ ' .
~Cett cvncenlration '~ ' ' ' Date drx dernier cunirGte de viaiyllit~ ~ ~ n c! / ~~
Lexst viability dacck (date) Date du contr3le d'~tnncFalte des ancapoutes x.ast airtiglluiess clsaek'of the vials (date).
CONTRt7L$S D~ ~UI~'I'~; c~cGUts Date / Px~sa,~e t~~.suttats LustPLI~1?YGH~CIC:S . Taatc I Passage Icvet licsults ~BCLCCia - ~tt1(Lgt ,.
CO j~15t9111 virats~
EN CRS DE CONTAMINATION. LF DEPOT' OOIT ETRE DECLARE COMMt: 't9Gt.AttG~
DF MICROORGANiShII:S' (VOIR LES xHSTRUCTIDNS DONNF.ES SOUS LE CpIFFtrt 7) saHElt~ A. pEPOSIT IS CGNTJWINATED. IT Sft0UI3J 9~ 17FJ10'IEO A5 A 'HI%TURE OF
HICROOGGANISHf:S' (SEE IHSTftUCTIGNS CIYCN UNDER 7 0.f10YE) .~ fllltrC'S i1'I~OlClnat:~QtIIS . ' .
~''tlrtlsG'r GOr7IrJ2BrIt,C
CES INFOnMATiONS NE SOPtT I.I~ES, NL AUX DISP05LTIONS DFS ttEGLES 6.1.A, 6.2.a.iii, 7.6 CT
8, N3 AUX DXSP05LTIOMS OGS RE6t_ES 6.l.a.iii, ET il.A.~f_ LEUR INL1ICATLON EST
FACULTATIVE
SUCH htI'oRMVITOtI IS Nt:ri'tlEtc LINK>':I! TO THE PaOVIStONS OV tIYLE s.L.e, ftDLE &.t.a.ill. R>n.s r.s.
_~ _ AND Ncr~' a, NOxTO TftE pRa'rssTON9 OF aUt.r 6.La.if1 AND RUtY 11.s.~.
ITS FUANLSilING IS oPfLONnE,.
CET'('E FQRMiILI: DOIT E'fRE KEMPLI(: SANS RATURES N!; SIiRCSiARGES ~ ~ ~
C:T'i.l,x. Qrm TH15 FOttM SttOtAD a& nLItY C~cFTrn VITNnnT nW trrYnne nn e~rr...~......

.Ir,~FIr~tGNCl; n~xbr..N7rIr~ It..ATIGN ' 1;, ~-, . Date ti / 04 / OG
fdentifccatiorl ref~~encp _ . . Data ..."".."..._ g~ " '.IN~Ot'~MAT'IOiVS SUPPt_~MEf~ITAli~ir5 Additional infot->7»fion COCHER St PIES INFORMATIQNS CI7MPLI:MENTdITfiES SONY FOU!'tNI~5 SUR UIVE
FGUILL~ JOINTE
HaftK NI'iil A Gttas5 TF ADaI'CIOttAt, INiWRIVITION IS GIY>=g ON (itt ATTACIiLD StIEET
Qrt~tllc de to culture t:elltclairc ou dtt IxICiambe ( = cornplculent;
Eventual a~ ind.icatious rionraEes s~atxs le rtlifilc; 3) Source of the cell cu111crt or tl~te rmixxttrc (ar far crs not g'iuerl urtder 3 above) ' . Hybri.danae : - c~7.Xulc ~ spleniduer de souri,s , - myi:lonte rie soux3.. ~Bnlb/.c.P3 A~8 X 63, d.5.3. . , .
. ~:
Lt~nee Et~niatie~r>or In8ezlierZ.e dea ~hnt~corp~ , to 2010/91!
Cell Zirce establrshed by (date j , C~e3txli<; cton~. pax Z~~~Yi~.orle des Anticorps 1G 1. J. / 3.0/4 Cell clawed by ~ . (date) )uxire~istrcmcat Bans d'urrlrcs tostitutior~s de deptrt (Noa:3, dates, unmEros attribellcs, ...~~
ltegiscratinn ire any od:erdepositary frlSIttICtYOrt (Nanr.es, dates, refrrerscer, ete) Attires ii~Corna~dans Fttrthcr cornnlcrlts Cf.S INFORMATIONS HF. SONT lI~~S, Hz AUX tIISPOSITI0N5 DES Rt~Gt.~S ~.l.b, 6.2.a.itl. 7.G I;T
4, NX AUX DISPOSxTIUHS 17E5 !iFGLES fi.x.a.iii, Efi 11.4.fi. LEUR INl7iCATiON
EST FACUI.I'A'fiVE
SUCIt INF4RHATION I5 ltEITlit;4 LINKED Ya~TNE VKOYISIANS D4 PULE GY1.6, ftUt~
G.;~a.tv nuLC r_s nND RULE e, NoR To Ttte PImYISIDHS of RULE s.t.a.iit. AKD RULE 1l.s.t. ITS
EUaIIISNIhG ~s opTIONAL.
'~ Norm, :~dresse e:C nusr>«ro de tei~phoae (ou/et de tElGtopieuc~) dd scicxltiGqoc ru~oxssvzblc de lu caIture truusmise Narn~, rtddrerr, pleane arcdlor fctx tzranber of the scientist resporscr'F~le for tJ'ze culture transrr:i fled t4 1 * ~ .)E;u vue du ddptrt la cultccrG ceIItllairc sera. trnrlslYCisC, co=rditio>'nEe ez< c,artao~l,xce , ou t~n axt~te lLy~tltcte The ceh culcctre released fear tJ:e purpose of deZrositiarl will ba trunsnzitted irt dry ics ~ or in Iiquid nitrogen soul for>;>:ce dn; 12 eclt<xatiIloris, d'un mane loft, en tubes ~tt:unelxCS, marque~.~ contortnec>rceut aux ~rigQrACes iuedic~fies_ as y.2 aanlptes frvnl the xa:r:e batelt, ire airtight vials, rrtarlsrd as rec,~tcerferl * DATE SiCPtATURE(S) S'.(~HATIIRE(S) d~(~es~ scz~rrrz~z~u~(s~
D~~t~e~~ ~''~~ ~ "~, . Signs hure(sJ t~F~POf~~r~~~E~,sa.-~~---_,_..~.
G~~:~:~: : .. _ Chef ~'..~ e;.!~li'.~.-'.'. ';?-'>' rc: cuy ' ' ~ t~ !r~ys~.:or,s LE5 N0~1S t)RCTYLpGhAPHiES DES, PfiRSONNES PHYSIQUES I~UI SIGti~:pl'C AU NOM
~OE LA PEtZ$UfINE M01~LE
OULVCNT ACCOMPAGN~:R L6S SiGNArURFS. .
THE TYP6HRITtEN tuitaES OI~ TItE HATUIiAL PERSGHS SIGttTMC ON dEHAI.F of THE
LCWtL ~ItTItY 5ltnUw ACCOrPmtY THE SIGttnruRFt, C~TTE FOWiUL(: UOIT ETRF. ftEFIPLI1; SANS RATURES NI SURCHI~RGES C~l,r, gG/G
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A I ~ r: . ~ D E D E P 0 T ~ o t 111tST1TUT 1~~1,STEIL~f~ ~4DDENDU6f 1'0 TX~ dPPLICaTIOX fOF~d ~:.
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fCaRGANiSIItiE GENE'nC~iIEMEuT MQaiFiE OUi ~ non Genefically rrrodi~ed arga»ism yes ~~ na ~ °
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(*) AISQUE PDUIL L' EHVIROHNCiltntlT '" 1 v ut "''~~.',~ E11't~ONREIfIRt, R~.SK
v .r 1 .,a ~~.. ..~........... ~, ...~ ._.... u1 1~. w ~ is... ~ ~ a v r Le soussigna declare a~roir procede a toutes ies notifications rt:quises par ies regiementatians _ ~r-'~..a nationaies en vigueur la cancernant quant a !'utiiis~tian et ~ !a dissemination du m'icco-s=H W
w~" r~ organisms cite ci-dessus et de totttes les compvsantes assvciees et auair te~u dps autorit~s w w o_ i:a r.. , .
cotripetentes les autorisatioris sty rapportant.
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;,.1.. w~~e r" The undersigned declares thathe fias prnceeded.to the notifcatiorrs required by /iis own o t~ V
'~'. ~ ~ ~ ~ ~ ~' ~natianal rer~ulati4ns in ford rAaticemtng the use and ~tfie ~ release of ~ the abot~o mentJOned H-u 4 ; -~1 microorganism and all tfie associated components, and .that he has got fmin tfia competent rir_. ~,w autfioritres the relavant pe~~its. ~ , ~s 47 H ~t uN .
Le!s~ d~po~ant(s) Le(5~ scietitiii~ue(s~ cesponsablc~(sj de~t~~~~b~ata~~.,u~~~r:~
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~"~1 TRI~I'4'E DE EUiIAPEST SUR lA RF~NlUtISSRNC"4 tllTEtINATIO;tALi. P1U pEPOT GeS
HIC.~OUNIiANI9lcs nUX FINS OE tA PACIt:pU!2E EN
t411't21(& of OAEYETS ! ACCOf~tO UU Z5 4f11f1' 19713' ENi~ 1.'attGANISATSC1H
FUApP~rItNE CCS ItaElfEiS Gr U1 C:1(7i, IHUTIsU't PAS'ftUR. P0.RIS. N.aniFCE LE 4 ot;OB2E 1902 QSRCi'~NfViT LE DEPO; tiH
hICCROORrntiiSixS EN AYPt,tCnTIOA OES t7EGt,1<5 2~ ~7 Znbts ~>: US tomfENTllJtt 5UR lE BREVcY tatAOP~6H.
CONTF~AT ratahli aux termes ~ ' de tea r~gfe 6.3.a.{v) du Trai2~ de Budapest du poust ~t3.a.(v) de i'Accard bitatt en ~ppiic~tiar~ den ri':gles ~8 et 2t3bia .
de ~a Corzvet~tian sur 1e brevet europe:en , . . . , entre 1es parties d~sign~es ci-Bessou5 Collection N~tia~ale ~ ~T'TI1VYFH'I'L4I~TS

de Cultures de lutiGroorganisrrtcs ! NSTITUT PASTEUR , 25, Rue du Docteur Roux , F-TST~4 PARIS-C~O~7C 't5 ' .
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DireCtcurAdlnfnistrpti, j . ,AL!"COFttTb, DE ll:»f'OT . .. . . ' . ; GEl'CISAhIT(S) 1'd'oxx~;(s) Ct a.dresso(;;)~ . ' , relatif au microorganistne (*) desigrlis ci-aprias ~ ~ ~ ' ~ ' ' ~Iybxa.dome:. do sauz'~.s - 't, ~_t ' ... .. ...r..~, ~1 ~IEFEREI~ECE 1~'IDEN"i't~ICATIUN lIUMERO t1U SYM80L6S, pAR EX>~MPI,~, DflNN~S PAR l.~ D'c°OSANT AU MICROOt~GANISMF
x.-Lc d~posant recanrla~,it avoir eonualss:~ncc dcs cxigences et dca rec:nzu:zlandatiorls rclatavcs au d~,l~c~t do mi,croorr~,n.ismes attx LCrIxIES du Trait6 de Budapest vu au.~. tera~xes do 1'Accord bilst~r:~I cIx application dca roles 28 et 28bi~ de is Caavention sur le brcvct europ6en. ' ' ?: La CNCM :ICCepte lc xr~icrobr~aniJ.,suo idct>.ti.fle~ ci-dsrssu<,, ~us~.e'folS' quc sotlt a;5ur ~;es toulcs LCS COIIdttlprlS do va>.idztc du d4pot cu vcri:u dG Ia z'eglc ~,1_.(:y ou 62.(~<a) ct do la r~~lt 63.(:1) du ~'raitc do Eudapcst oa do pouxt 12.(a) ou 32_(h) et du poizlt 7.~.(a) do 1'Accord bilatcrc~l, ucccssittant, cratrc autre, la r~ceptiom par la CNC:ivI de, . dntl-r_ ~Gchantialon~, du mi.croor~auz;inc 'idamkif:~ cz-do;;sus, prcparc: 3 partir d'uuc mbznc subeuliurc, en'vue dune lt5nr~.,tae cous~r~raeinn, conformctamnt au7e W dications Cournics (~f~), portent do faros: 'lisibte ct~i>ad~lcbih 3~1 r~C~ror,ICC d'idcntii't.catinr, et la date de prGpazatiolx, et ~ .
l'caameu prr~lisnin.~zir~: d'uzz dCS c~CbantillanS r~~us rar.Ia CNCM cnwilc do la cutiaatation. do la validitb dc:
rcns~i~~tteumnts ~ourzsi.s en vCrlll de Ia rc~~(e f~~.~~.~ii'x) du ~:'raat~
d;: Btldap~st ou du point 12.a.(zii) de 1'A,ecocd bilatcrax ct an vrlG da 1:1 canstatatit~tt do $'acceptubilitE du m:~t,~zacl tr~an.,rxLis pour d~pGt aII vcrtu do la r~gle 6.'t~.(a) rlu Trai;te de Budal)esc ou du pauxt ~.~.(a) de 1"Accord bilateral.
' ('~) On elttend par 'MIGROtIl2GACldSidC' tc>'.rt matc:ricl bioSog~iqcto quc 1a Ct6C;'s cst ~:ausc~ptif~l4 d'ac~E!t7ter en vut' d'ttn di..>.lWt aux 'hormes r!u Trai~t~t de f3~ad:Ipest au dc~ 1'~lccord hilatEral. .
(~) LPs SAtpouie~ datvent titre Cotup:itiEl4!S aveC 1~S dispasivifs cte eoaxervation de; to CrlCl4, ~tar.ci»s,zt sans risquc d~s iissuratian, de !-lJpturt: au d'ezplosion pr~nd::nt.la p~riade da tanserva'Cion pr4vum ~
to r~gta 9.~. du Tra,tl: dc: 8udappst ou au paint 31. de 1'Accnrd bita~t~ral.

3: ~ Ux~ num~rv d'enregistsemeat peuc etrc connir~,uzziqui: a,u deposant~d~s rc~copCion du nnicroorgani.;me. La r~coi,tioa~
et I'es~rc~istrement d'un miczaorganismc n'implidue pas son acceptation.
4.- Un rcfus d'scccpCation du rrZiervar auismc pent en's natiFsc daxas Ies conditions prcvucs ~ la r~gl~ 6.4.(a) du Traite da; Budapest au au paint I4~a) de !'Accord billt6ral.
5.- Si lea conditions de validite tin dvpT~t ne sant pas toutcs rc:mplies, unc ~roc~dura de report d'accoptatioa c.~t appti,duEe: T.a CriCM ~ ztot~o 1es raisons et it.YC lc delai de uxi moil au d6pnsant pear qu'~I satisfaase a toutes Ies e.~n~uuce.~. Si Ias ddposant rre satisfait pas aux axi~enccs daps le.
ddiai 1'Lre, la CNCM proccdc a1 1'annulation . da.la.dcmaudc"dc; depot et a !a dcstructiva,du mat~~scl Iriolatiquc traexsrnis. , , .
Ccttc procEdure impliqua un d~pof ~ de re>ztplaceme>«t (~) si unc ir~rd~ularitc de ~ocma, de quantity ou do prEsentatxor>, cat cox>atatce par Ia CNCM s;:r Ie materiel bzoto~iquc transm~
par le dc:posant arx vac d'un dEp~t mina! ou d un ttou~ean d~,pot. Un dcpi~t do xemplaccmcnt eat con .id8rc cornxne un autre depot initial tact due la viarZitc du depot cn attcnte d'acccptataon nest pas ~tablic.
s 6: Chaquc fois quc du n~atEriGl bivla~iquo eat transmis par la dEpos~tstt ~ 1n CNCM. la CNCM pex~ ~oft la tu,~co do conservatzan pravuc ~ la regic 12.1.a.(~) du Tra.itc do Budapest ou apt point 26.a.(i) de 1'Acayox b~t~ral.
'7.- Si lc dEpc~t eat accoptE, Ie nuaWro d'ordrc attribuc au dEpof par Ia CNCM
cat idwntique au num~ro d'enten-i.,-Gccznent, ct la d,3tc do d~pat eat la dato de rcccptaort par la CNCM du microorgatxismc ~dcntif~~ ei-dessus, 8.- La notific~tior< dG 1'acccgtation , du rc~us ou do rannulatioa do to deuiaxide de dEpot cat 6tablic daas un dvltei do six rzaois apr~s r6ccption du microorganisme. L'ncccptation est aCtcste:a par lc rcc,rp'~,,s~.
9.- I,a CN'CM nc proc~du 1u prcnucr coniri;lc do viabilite ttu'uxtc foil assuree qua le d~pasatzt a sutisfait '~ tonics Ies eri~ences ofz ruati~rc de depot.
' ~.~ lf) - La pretnic:TC dee3aratioti soar to viabilile atteste la vilidativ'n ou 'facxnalntiofz~dit dGpt)t : lc dd~pbt eat valitie si ie . _ . :. . uticroor~aniSmu est~viatsls,, i1 rse annuls: si Le trxicroflrganisme n'rst..pas Yiable. ; . . . ., , . , , .
lI: En cas de rm~us, d annulation de demznde de d,rp8t au d'a~ulatioat do dEpot , lee Echarttilloct,, de materiel biota-' gialuc transrais soot dctruitx ; cn cas do d6saccord sur Ics n~,otiPs du rcfus oc: do fannulation , its ~euvt:nt ~tre ooc~.ervtrs v i3 fwNCM ; its ne sent pax ra~titu~s uu dc: osarst , san.F
accord particulier is<tervcnu aprc.s r6ccption clc la nvti~toativn du reins ou dr.1'annulacian pat le d~pasaut.; an aueun cas , lea Eehantillacas ae pcuvent dovnr~r Iitu ii un dcp8t 3 la. C1~ICM ~ d'autres fin:.
22: La taxc dc, conserYatian prc:vue ~ la. r~s;le 32.La.(i) dtt 'Z;'raitd de I3udapcat ou <ut poiat 2G.a.(i) de 1'Accard bilateral eat dua daps tou.5 ies cas, que lc c~icpot snit acccptE, rcfusaott annu~~.
~3.- Lc dEpasant s'rn~arc it d~postr soils eontrat assoc's~ tout mat6iiel vivaxtt , non ou daflwxcilemcnt aece,. Bible:
aeccesslirc att.~c eonk~~oles edou iZ Lx co>tx:~erva,riocz du x<azcr ootpanisrrac idexxiit'a.c ei~tYcssus.
1~-: Le d osant s'cn~a~e a~ fouruir tons Lc.~ 'substrata noa ~nu ,dif~cilEextent aceessihles, nccessaires au.~c'eontr"vlcs atloula conservation du ncxicraor~wi -~-mt ident~c ci-desstts, en quaxttitc 'suti~Cisante pour dauze ~prcuves ou passa~:.s. ' 15: Le ddyo.~t cErtitic avoir fourni toute iudicatioa dont il a eoa~uaissa~aco Bur lr:a pcopri4tes du mice oor~an'ssmc identili~ ci-dessvs qui prEscatent on pcuvent priseuter oat; action dircctc ou indirccia , connuc ou prdvisx~le, Bur 1'hocnatc ou sur quoIqu'or~llsuic'que cc soft, animal, v~a~tal t~u autre. - , II s'tnGage a porter izamtcliatcrxaent 'a 1 a cacaaaiaancr, de la CNG'lYi totttc nouvc;llc information y :elxtiv;:. . .
x6: Apes accc~tation du microoraanismc , !a ~IYCM Ic coaccrvc , assure lcs cvntrolcs de vialalite y,~ ctablst iC5 ClcCIMT'2tlOn.i, attC$Catlpn5 tit tl~ti~ic."RLiUnS , rGtllct 1~5 i;C~~tritt~llCltlS atL'C ~~~irtlCS c'ttEf;ClriSGCS , Ceri.;ItC:CS , UU
tequEr:tntcs, couforlxri~racnt ~. la reglctxxentatiott applicable.
(S) oat entend pnr 'DEPOT DE I;EhfPt,.~VCEttf:RT' one .serfs CocnplLmenfiaire d'ec:;ant;itlons du'rtticroorganisme ayat~k fail. 1"ot~~ct d'un depot initial vu d'un nouveau d~pi5t en atc~nte d'ac;te(n:~tion, prtparEs cortform~inf:nt a.ux t'exiqencu: de la' CltCtf zwt transm~s en ane seut.r. fois par Ic deposant $ is dernande et ~ 1'adresse c1e la CNCM dans IC d~~~1ai ffxn ~u poiW 5 du present tontrai. accompagne'~s d°p~(:e di:cl~~tr.~tion Sem6labiN
ta:Ile defistic & to rt~te 8.2.(a) cu lrrdST-! de Budapest.

17: Chaquc ~ois qne I.r CNCM ~us_te ~ oppc~rtvn d'adressor as ~ de~no:;ara.t ('~~') uu 'dclaantillon d'urzc subcuttuxe du rzricrdor=,~anism~ idraatif'~c ci~c;,sias crzvuc d'urx Cbzttx'Sle do conCarmit~, Ic dd_~osaii4 (~'~) vGrifio 1'cmressioa dcs proprictcs dudit micsoor~r~isxuE daa~rs la dice subcultur;: ct rczsvoit a la GNCM, daxxs 1G dclai de trois s~aai5 aprcs reception de 1'Eehantillon, la Foruznlc joi~ta ~ 1'cxxvoi apr~s 1'avair dumcni rex-.xplic at si~aee.
L:, deposant ~~*) reconaait qu'cn css do zznn~rbpons.~ a unc dcmandc; d'accord do coniornnitti tcllc dy'cllr est pr~seut~G ci- cssus , lc., proprict~s de Ice. subculture cu q,~,sastioz~ sons :c coztai~3~ccr identiquc:, aux proprictEs de Irt subculfurc trausmisc ~ la CNCM 31a date du ddpot.
~8.- Lc ddprasant xxe peat pas retircr , annulcr ou .modifier lc dcc cat pendanit la pcriodc do caixscrvatioxz prcvuc 11a r~~lc 9.1 du TraztG cG°. k3udap :st ou au poi~ut 1,1 Rio 1'~Caord bi~tCral. . . . ' Coctc p~x~iadc do coxrservatiQix est do trcntc-cinq :urs daps tons lcs cas a 1a CNCM.
' 19.- A i'~cpiratic~a de 1<~ pcriode de ~ccrnsGr~natirazx , tout lc rsxa.tv~ricl biolon,~que co;aservG est ~dctrrait , sauF daces It cas d'nnc demands pUrticuli~r~ ~vrmul~;, pH.-.. le dEgouaat uu courant do la trexita'.cinqxtiexue wade de c0li5Crvab,4n. a .
20.. L.ors~ue , pour quclque raison quo ~ soit , la, CNC>u( ne pcnt pas rcmattxa d'er~tilloas du microocgunisma ideuGfic ca-dcssus, uae foil aeccptd et dcclard viably , Ie d4~osa~e~.t praccdc c4ai'rsrnn~Ennent aux dispcr:.itiozxs doT.a,rdglGmerxtatiou, appfteable ~ txxz upcxvcau dBpSt dcxdit micxtaor~tai~ue d-m, arc delai do trrnis .mail ~' comptcr do to data de r~aaption do la noti,ficatioa corrcsponda~atG.
X,.a. d~.C~uihiora dcs rai::ozt~ do boa usage cz1 anati~'re dautoriCc de depot , intcrdisaat la remise d'L~chxatiltorz;, est soumisc 3. la saulc aptian do to CTiCIVF.
2x.- T.a Ct~lCivi cst d~~gde do touts respoxssabilit6 ca ca.$ de variation da caract~rcs du a~,teriel biologique dcpagc. dl an c5t de rarznc si aces pcrtc de viabilitc , ume contanoination ou' aces destt'vtaior~ aCC.id.CntClle ~I;ait con.~tat~~ uxalE~ ~ 1'applic~atavn d~,s pr~cau~tion:: pr8vues pour st coase,xvatiog:. ' . , .~ ~'?.2 .. ~SI ;, p~.r 15,'Iaiitd ou lai n~ ,li~ancc dri deposaxxt , iui Fait : domuzaneahlrr~ ~ la GNCVI auzvient a Ice . r~ae~titrn',.a.1a:
. . '-. manipulation uu'pcrxdant ~a dui~e de ,conscrvatioa du ddp~t;~~.
d6gosatit .ixuiermaiae;;la-CNC1.VI'~uii,'~c~fud'x~c-subi. ' - ~ ' ' 23: ~ !l ~aius que le fit doxnma~uble be soit impniab'le i11~~t FautG ott 1 13 n~~li g,case do Ls. CNCM , lc depasaxtt antit cclh~ci corttre tease action a sore caacoxxtrc cn reparation d'un prcyudics >~ie ~ 1a rcmase d'ua ~cl~xtil-' do znicxoor~;azusmc xdentiF'z~ ci-dossus. .
24.- frn cas do coratcstation la lei .fraarraae est applicable: ct Fe txx'l~unal couxpdtout tst cclxzi dG Paris.
~(s) nF'~~c~~ I?ate.ct si~.atrzrt(s) ~ ~~~~...pTJ ~r ~~~~~' _ .. , . I,I~tS~T.3TII~"d' ~p~iS'1'~CIR .~ ' - . ~ , . .
' . _ ' ' . . ~ ~ Danie(i~ ~~'EF~NEiVl~r~i ' ' -~ .
2~-28. nic du DocteurRor~r chef du Service de~ 8ctsve(s 78?'24PIQrr, Cedcx 95 & InY~Ri;ens ' - - _ . _ X.E(B) SG~NTdI~ I~U1~(S17It~..S1'~'1N5A8LE(S) Datc ct si~atnzG(sj _.~~~. r . ~~ .
. ~-~~-("'') l.es enc~ar~khnents indiquds au paint I7 peuveni titre assurC~ indf ffinre~nt par 'ie tiepasant ou 1C re~pocrsablc $cientvf~qua indique par le dpposant. ' .
('~ri ) C.e.h noau dactyl ographi Cs dH s iersonnes pl~y~i ques qui s S grant au nom d' une peracnne ma#-al a dog vent acca~n-p;ryner Tes s;gnaturc:;. t,orsqu'une dcmanQr: d~ dcpc3t e,~C prdswt~~ p~~tr plus'icars pr~-sanry~;:, t~ formulaire et to central correspandants itaivent potter to slctnature do chvRCUn d~s d~posants. Cans Ca c~.s, une des per--sonnte: .dolt i~tre designee aEP~t~SEti1'AN1' IfHxQt~E, hahi l i t~~ a raceva i r , 1 as documcnrs or'i g; nwux Lrmi s par ~ a CttCM pnndan'r l x p~ri ode ds corts~:rvati on 'prev,ie duns T a rtgl ementaCi on .:ppl i cabt e. , FO!v~SUiAIRE OE DEPU't 'TiZAII'E DE tiUDAP~ST SUft Lfl R~CONNAISSAhCE
IP1't'ERIdATIaNALG nU bEPOT
APPL1G1T(pH fnapi ~ OES M1CR00RGANISME;S AUX FINS DE LA PRDCEDUP.E EN
MA'CI~RF. (TE BREVETS
~ ~ H!tOAPEST TREATY D!t T'NC TMTERN,ITttNllil. 0.ECOGNFTIUtI t1f' Ti~G
OEPDaIT
UP 1'dIGRCORD,iNIStlS fDt? T~IE p~Rl'OSES OF i'ATENT PAOCEDUiIE
CNCM
Colteotion flatiosTSle de Gulturos de Microorganismes INSTlTUT f~ASTF_UR.
25, Rue dfx Dactettr Roux -7a~24 PARIS CEDi=.X 15 Yvanne CERT.SIER
DirccteccrAdnainistratif AU'tORiTE t)E t)>_POT IN'ff=RNATI~ltyALE
INTEiINATtt7NAt. OEPOSITAHY AUTItOtIITY
* t3 ~GLARATIC7 N lrrl VrJL D'UN I7LQUT Ll'T11C1A,X. COl~'OItMEML;~iT.A. LA
REGl~I~ G.I
Std t~Il7 Bn t us fhe case of an original deposit pcusaiatta to ,Mule: G..T -H, L» '~ D~~~E Ca~EI~xQ~ ~o~.a~E~~:~T ~ ~. ~G>,:>F ~ ~t.a ~1 in the case of a corzver$son pursuant to Rule f.4.r~
. EN V~L.TC D'TIN C~I~tTFL~t.T ASSOCdE (SO'UCIE~T~.-ItC!'ZE Ot,1 COt~)E'OS
fr1 tltc Case of art dssaaiated contract (ItostsWaire orepn:~pftrrtt) * ' TYPE.L7E lVtlCHC7t~RGi4NIStIItE ~ . ~ ~ '. . GULTUFi~ CEE.I:.tILArRI~ .~ ~
. ~ .. . ' . , . . . .
7"'Y;o~ of ii~i~ri~orgarrisrri ~ ' ~ ~ Cell culture ~ . ~ ~ ~ . . . . ~ . . ~~
* MIGROORGANISME (SOLE ~ ' Single microorganism . ' M~LANGC ClE M1CROOFtGAt~ItSMES
Mixture of microdrganisms 0o cellutes i;nfCCteus CUIttalnia~es,... ~ INptQU>~R LE CAS LCMEANT LE HOME(R~
D~ MICROt7RGARISMES ET LEURS TYPES
arinfccted celtr, contarraFrtated cells, ttc GtY~ ThE rUMD~R OT
HZclIOOKWtNISHS AdB Ttl~IR KINDS Nti~RE APP4IcAtil.E
z.lG; SOUSSxGI~TE DrPflS7~ L.I'a M,h.TERIEL liv~;l~2TIh'IE", CJt-t~?E'~'5 E'I' S'I:,NGA.Cx;~.
NE PAS Yt~7CJC.FtEIL JLl<. DEli'O'JL= YL.',i~f I~A~t"~' x~A'PEItxO'IaIC
I'Kh:C:ISF~ A LA 'l<th,Gl:.F~ ~.1..
Tytc undcrsi~rmd l'tereby deposits tire rreaterial edcr:lifted icercetanden arid undertakes riot to witlTdraw the deposit far the period spcdfed in rule 9.Z. .
1. , "FiEFER!~NCE D'tDENTIf'lCA"f 1UN ~ . NtIMERO bu SYMOOLES, PNi EXEMPt,E, UaNNES PAR C.E '~EPp'SANt' ~ AU MATERIEL. ~ .
jC~t;'ntification reft=rence NUhIOER. SYNGo4S, ntc. 'GZv~N TO THE tinT>:RUI OY
THE df:PmsTToR ' ' ' A tt ..., t' 2. DEPQSANT(S) riv>;n(s) et :i~essc(s) ~EfV~CE h~~:S BR'~'!'S
Depositvr(s) ..-,.~-~ ; .
Nanse(.s) and addresses) ~~f~"~"''''~"'~ . :.
INS">'ITLI"C ~1PA'ST~.L'i~.
25-28, rue du f~octcur Roux 7724 f'ar i~ Ccdcx 15 C~TTE FORMUL~ l7flIT ETRE REtipLIE SANS RATURES HI SuRCf~lARGES ' Ck!%IrIL.'~6/:
TIiIS fOti(i $NU~ILU QG OULY COMPLETED 4I'fHOUT GELFT'jDNS DIl ALTENA~IOn'a D'al'tL
RE~~RL:NCE D'IDENTIFICiiTI Oi't ~ - ~~ _ L4 ~ Date Iderttif tatiott reference , , ~ -------~~ ---. ... . .
3 .~ DESGRtPTIGN SCII~NTiFiQUE Fusion ccllsx?~zire centre unc cell_a~,~ de my'cloute '"
SCIG'l7tl~IC description ~ cic souxis et txn i.atnux~.ocyrF de svuric; 3.sstt d~ la ~~ ' . rate de ~. ° alximal CDCHE~ sI ttES IttFDRMnIIONS CaMPL>=rlEN1'AIRES SUNT FOURN'CES 5UR ttNG
FEUILLE ,loiHTE
Wt(iK METH ~l C0.p5S IR pDpI7IpHA~, teFOftMATtON x5 6IYEK ON AH ATTACNtp SHCLT
Type et ori~itle (organa,/tissu, csr4ce nailnaslc, ...) Type rind Dri~fu (or~un/tissue, arztncal species, etc) Rate~de so~xxs~ . ... ~ ' . .~ ' . _ . ~ ' ' .
Caraetcristiqtyes et ~rrodtxctiolns des eellnlcs Cltaradcristics curd puodttcts of tltf Cells COCHER LLS CASES QUI~,CON11IENNENT
ET OONNER DCS IHF'DRMAI'IONS COhIPLEMEN"CAIRGS
' w~xK urru w caass WtreRS Ars~,.zcaaLe nno rays ~orn~nl. irsFOarv~rxon Hybridoute Myelome utilisC . X f 3-Ag$-6 . S . 3 .
' ' ~ .
ybridoma Myclonta desi$rtatiorc Sp ~Ci~cite nfttxg~itique .
.drttigcnic speCe~city - :1'.Tt 1. c. r. n ~ :L itt a t1 .
.

Claese X~ :C. ~ I, / k ,~rtttbody subclass -StaLIIit~ do la shcri:fiat>< ' Stabilityofsccrctivn ~ ~oitne et. ilXim;Ltdc lfi;~nGe gLn6tiqttettacnt naodii'a6c ~ V'eckcuc(s) C-re~tet~Cally rnodifred cell Tine Yector(s) ' ' ~ ~ ~ ' ~ . ~ ~~~le.x~,uclcique(s) irtst~rd(s) ~ ~ ' . . . ' ~ ' .
.. ' . . ' . . . ' : ' . , ~. ~ . : . . . v NucdeiG acid irssert(s,) - . - , .
, , . .
_".~......._,.. ~.~,., ".
Cellulcs infcctecs ('~'II'LtS 53idVa.~,TC) Colds infeCtctt by d zarild-type virus xi~W a cclluluirc nouvcllclncnt cttacblic , , Newly establisltcd Cell tine .~.rxires parfjculrxrites I"uriJter or otJterpartictdtxriticS
Description ' . ' _ . ' : ' . .
Dctaiis ~ . . ' .' . .. . : ~ . . . ' ~ . ~ . . . . .
~lybri.dome ~.ssu do Ia fusiandc'celluLes slalen~.q~,t,s rte ~ourxs a.tizmun~secs~ coatcre LtiCNrn<<Line A dt: Ia~isl.cri~~. inc»lc:~t.:ytof;enc.._~, weC lr~=
y~:lra:;tzioryGc~mE~ A~t3 X E~.5.3 I~Cfi.CCitCP,S ~illltph,CRpl1!(~(IiCS
~.itcratcu~e refcrertc:es L'INUICATIOH DE CGS IttFOtIMEITI0N5 EST FACUt,TATIVE, lIAIS UTVEMEP1T
REC014MAHDEE NJX TEFttIES DG LA RrGLE 6.I,.h TStE SUapLYiHG of TUG11 INFpRHATioH IS ovTxoN~IL, gUT' S~HOHGLY RECOt~tF_NpEb IN ACCOtttuthcf NITfI RuL~ a. LA
CETT'E rORMUI>= DOLT ETRE (iEMPLIG SANS ItATURr;S NI StlRCfiARGES CEL.L.~~/.', TItIS FOIiH'SHpC'p 8E oULY CONPLFTEp uiTliOUT oCtlTIOtlS OR ALT~a~tT70NS

JCLIa.(t ~ RF..NCG D'IDL1~I'I I7~ICr~,,TIOl~f ,A 4-r~ _~",~"~"._. bate ,.",~"~
/ C '~y / C t~
Iderttificatiarl refurnec ~---~.~-----4; 'Pf~OPRIE'EES DANGEREUSES POUR LASANTI; OU L.'1::N'SIIROi~iN>rMEN'1C
Properties dong~rr~us to health or erlviranment La-culture.ou le tWlange idc~xti~6 soul Ie chiffrc 1 a 1es proprlEti~
sxilvuzztes tlui, I11"E:SCi1te51t ou peuvent prcacnter dos d~xn~ers roar is sxn~t~ ou~ct 1'enrirun~nennant Tlie culttcre ar elre niFxttcre identified ttndrrr.t above laas the ,~bhowiu~
prapenEes wrhicli arc orntcry be dan~crous tv health orland the envirnrtrr:ent Ct7CHER SI DES IMFt)IZHJITItINS CL)MPLEMF:NTAIRES 50N'C FOURNIES SUR UNE
FEUIi.I.!= JOINT'E
pHAK HLTN A CROSS IF AOOI'fIONAI INFOiZi41TI0H IS GIVEN off AN ATTAUiGO SHEET
1Lc sdvcssign,v n'a pas coannissuncc de tellcs prnl'xit:,t~s~
The uncter~i4ne,~t is not aware tlfstteh prapenies, s TaUTE ncTION DIR~CTE oU INDIRECTE. CONNUfi OU pR~rISiOLE, salt qUEt.Qu'ORG~-NI&ME QUE CE SOIL' (dU~tIMAt,, vE.6ETAt. OU AUTRE} DaI'C ETKE SIGHALEE.
COCH~R LA CASE QUI CONUIENT ANY OIRLLT OR INDIRECT 1410NH OR LIKELY TO of EXPEClEO EFp~CT Otl ANY ANIM~I1.. VEGETAL OR O7HER
W1RK lrlTh A ASS THE APPLICABLE flOX ~~tISM HNST BC IItDtCPvItO -5.. CONDtTlONS DE SECURlTE POUR LA MANIPULA'T'ION DE LA CUl.'11TRE
Biosizfel'y m~~sures required to m~nipuf3te tfie culture L1 l 1~' 1 ~ ~ 12 / P2 Q i-3 / P3 ~ Autres , Cl tJ:ers (EFB: Safa 9loteehno5opy / FEOLiLN, AECIStt:R: GuideltH~a for Rsaoareh .InV°tvinp ReeaenirianC ONA HoLeeulas) his. CONOlTIONS OE GU,LTURE: - patiie ~ ~ ~ . . ~ . . ' . . ~ ~ ~ .
. Gorlditioris for cuJ~ivation.- Pare ~ ~ ~. ~ ~: . - ~ . . . . : ~ .
COCNt:R SI D~S INFDIL'MTIONS CCN~l1'LNTAIR~S SONT FOURNIES SUR IfNE FEUILLI;=.
JOINT~
HARK HLTiI A CROSS IF AOOIfIONhL INFOtiIIATLOH IS GIVGH 0h AH ATTALIt(0 SHEET
Milieu de culture (aver Ies rt:fc~xeutces preeises dca roctiposnu~s}
trulture median: (givo ficlt details if special fornmlrxtian) - MFl~t d~ I:~tglc - Euxobio - Pyruvat:e de sodium - G~,bao ERT, - G7.utamfz~u -- Gibuo BRL
~- A~nt3,biot~.quee : peni-stxept;omyr.l.x~e -- Gtbe.o BRL
- Se>cum de chcval 1Q~ _ Bicarbonate dC sodiuru Serum SodiurnGltarbonatE f~) 2~2 g/~. S~t,it (tie - °lo) Chcval rp~ . ~~
7.4 Temptrrlture optirattle ~ ~ o C A.taaxosph<xe pptirrral terrtperrtttwe Gaseottsplxase ~ ~ GO~ , ~'XGC~utioa5 parlicutie-res it la d~CQIln~a<ltton Dtcott.~eler x~2p~.dern~nt ~. 37°C, ~ lavcr ivrimad3.atemcnc !~'recautions to bs taJccrs fortltawlrlg drta;, le milie~.rt-serum. Gult:ure sue plague de 24 pub.
- ' d C 1 ~m~. . .
PrecS.utions particuiirrsc5 :~ I'izxCtibatiotv .
l''ttrtltertletails:.rhakin~sysrern, etc , $oTaztioas cllsgrKSRhtes uiili..5t~es Solcuions uscd)''or cell dispersion CE1'TE FORMUI.t: oOIT ~TtiC isEMPLTE SANS RATU>;ES RI SUftCtiARGtS ~
.CII,.'L'~G!=
THIS FORM SIRiUIA 6E POLY COHI'L~'rF,O NITNaOT OEtEfiaNS OR ALTCwITIONS

R1GFERTNCIr; D'IDENTfFxCA'~XUN ~ late ~~ / C1l' / 04 ILI~'IZtl~CflllOlt J'~~~J'IJICB -.--,-~ A ~'"t~ .--.---,...~-~-----.---r-~,-,..r ~atr, fib . COPIDtTI,ONS DE CULTURE: - pttrRie 2 .
CCondi('ions for cultivation - P~rt2 . ', COCI4Ett ST nFS IKi~ORMArrOtas COMt~LEMtNTRIRt:S SONT FOURSIES SUR UNE
Ft»UIt_L~E ~OINrE
tlaatc tIiTH A Cwn55 IF AOOtTI0wlil INFOaHATIOft IS 6tYEJl ON. AH ATTAGflEb SpEET .
Typo de cultaarc (cellules cax sus7tcarsian, ccllultas atilatrentes, ...) et rmarplxolo~;ie celluIaire attetaduc i Tjpe of ctclrune .~'susperaxioaa, rrJVraalaycr, etc, and eyECted Cell.
morpltdn~y II Ce~rul.es en suspar~s3on , . . , ' Temps <1e doublcment de popuiatiue 1~ ~ , '1'cnaps opti~uaZ eutre ley passaccs g ~ ouxs Yvpulation doublir:g tome t?prirnal ,rplit ralirr Dcasite ctllullire uttcixctu! 5~1 QG Dr>;rec. de vic: tir~ait~~ x~,lymia: ~~
Ezpcctcd cell density Limited lifesparc.
lJetaiis pour le passage dcs cellutcs pax d,~dauhlament Tcchniqccs fvr routirte s~eb-cldtztrc>
Rc;aauarques u~ aRt CfjrlJlTtBJi~s , 7 . ~ AC'TtldiT~S A V~Rt~tCR RDUFt CCINE»IRNtEI~ ~A VIAE3Il.ITE bU Di;PC~T ~ .
. . .
Activities to:be ychi'cke, d ~ccinfrming ;?fie viability .of the clep. osir:
~: .. . : ; ., COCt(E? SI OCS INFORMATIOttS COMPt.FMEN't'AIRES SONT FOU(tNILS SUR UNE
FFUII,IE JOINTF
NAhY ItlTit A G0.05S IF AGUITICNAI. IHfOR.WITIbN IS GIY~H od pH ATTACI~p SHEET
prodtxation d'szxzticoxps Larsquo 1e dc=Alit porte sur un aa~t~lan~e de: microax~anisrnes, la $i:clarntian do,it contcreir cn autxa; la desGt'i~~Lioax des cam~JUSants du m~lan~e ct d'aa mains one des mdthodcs pex><ucttant do r~~riticr icu~ pr~sGnce (ttrgle 6~..u.iii).
1%VJaere a ntixtruc of raticraorxrxrcin7~ts is drpvsited, descnp Lions of llse cont~JVrae~ats of the r~tixtstre arid' at lc,ast arse of tTtc nrathocrs pErmitting flee checking of Uteirpresence s7zhuld ba ~;iverx tn accordance wFtl:.ltzcta d~l.a.iif.
8. CC1NUITIONS.DE Ct~NStrRVI~TIt'~N . ~ . ' . ~ . ~ . ~ . ~.
Conditions far st'arage ' ' ~ ~ , . . ~ , ' COCi(ER Si EI~S IHFOtirif~TIOpIS COh(PI.EMENTAIRt=S SONT FOURtiIf"S 5UR UNE
FEUILLE JOINTf blilieu dC StiS~~JlSipcl INK kit'S A CROSS IF A40I120NAt. INFbhNATI9H IS GIVEN
OH AN ATTnCNEb SIIECT
Secspendirag flccad _ 95% serum de chev~I - 5% DMSO .
LV(a:itlif~s ale la ti;cr~lte des cciiul~s ~ gx~xeva.Jrient de ~.a aus 'ension Ce1.1u1~i,.re, ,centxi~u ation TechrxicTece fvr Cell laarvrstirig p cat repra.se du cu~.ot rlaus 1~ tn~.l:Leu de coza~c~latio'Ix )vxadalltCs do Ia con~t:Iatio>n . 5g~~6 o~l_~u~es daua Im7, cie mi,Iicu de caxt.g~~at~.aa.
Tecluaic,~uueforfigezrn~ , ~ 2~:a --z0°C, 3 z ~ jouxs a --~10°C, pu:i.s a,zote ~,x,clui,sta.
.tlLlt2'CS itllormal;ionS
~'iLItJIEJ' C~l7tlPIErItS
CETtE FORMUCF UOIT t:'CRE REMPLtt; 5dP15 RATURES NI SURCHAizcES . . C~Li.9G/4 TUiS F0/1M SIUIULO LIE OUCY CbJIPlEI'k0 WIT1IQUT 0&t~TIOtlS ON ALTEIUSTEulL

X'~I~:L~I:ItGNC~ 17'IDENJC~XC:A.TIDN ~ A 4-.tc . . Date ~u/I>~~J~ts Id~ntz~cation referertce _.~.------ Date .
xx: ~ lNFORMA'('IPNS SUFI LJ~t CULTURE T~tANSMISI=
nbGessalres ~ dea frls d'import~ition c!/au-d'expartation Datails on tha t'ransmit~'cd culture required for eapor-t/i,mport forrnaliries COCHEa Si, 0~5 INFORMA1'IONS COtit'Li~I~HTnIRFS SORT FOURNIES SUR uN~ FEVILLE

hAItK NITiI A CltOr~5 IF ADDITIONAL IHFOfEIIATION IS GIV(:Y ON AN A1'TAfiIIED
SkCCIr References de5 sntjstrats urg~~niques (fournisseur, refexanee, lot, dote, pays d'ori= inc) Refererttex ojt)te orb ani~ st~bscraees (supplier, Jwfsrence, butch, date, country of ori~r~) ' ~ .-- MEM 0111 -- ~ Euroba.v -. ' . ' . , .
- Seicnm de chcval -- .I~° 173 39 374 - Aayer Diagwcsat~.ce 1r'ranCe ~
' ~~rruvat.e de sodium -- Gibco IiRL trance , - L-Glutami>~e -- G~.bco BItT, France -- An.t3.blotiques -- G~.'bco BRF~ I:'ran.ce 9a. AUTR~S tNf~C7tiMA1'tCNS SUR LA CUL.TUR>~ TRAhISMISE _ further details on, the ,transrlTitted culture COCHER SI p~5 INFOR14ATIaHS COMPI,EMEHTAtRES SOKT FOURP(Y6S SUIt URE t=~UILLE
.70ItV'TE
MARK tiI711 A CROSS If AOnITIaNAI. INFONHdtTIDti ZS 6tYCN ON AN ATTACHEa Stlfs~' NivrE:aa de Itassa~a de It caltnre txal~smiat: . .
Passage level of t!=e trallsntitted ~ctchure lDnte rlcprL~poratiomt ' ~1i74/04 ~Dat~ of,~mpsuing ' . , _ ' , . . , . , ' _ . ,'. . . . . ' ~ , . , : ', .
. . Concentration tan celltilcs ~ ~ . ~ ~. . . . , . . _ . : . , . .. '. . , .
~ '. .' . .
Celt cortcentralivn ° ~SX106 ~cell/ml' ' ~ .
Datfc du delrnier rontrole de vi~tbsl~te: Z J t)~~ /04 .
LCXSt viability checlc (date) DatC dtt contrSlc d'~lz~nclxtiitv des a~m~ou'teS
Last airttgJwters cl leek ~f trse vials .(daee)~ ' CpNTRCILE,,S ln~ PC3><t~'TF ri:cettts Daar / 1f'a5s.'t~e ItEstIlGxtx Last FUT.t~"xCl~'F..CIGS . l.7ate /Passage level ,Results Iiactcria - ~ mt~i _ Myeoplnstzxa r.
Yirus~ ~ . ~ . , ' . . . , . . . ' EN CAS IYc" CONTAMINa'fIOR, LE" DEPOT pOIT ETTtE QECLAR~ COMME 'DIEtJiNGC
Dt: MiCR00RGAHISMES' (VOIR LE5 INSTRUCTIONS pONNEES SOUS t.E CHIFrIiF 7) WHEIt~ A. pIsFOSIT IS CcYTAKINAIEb. tT s~l0utn dE pENOT~O n5 ~( 'htXYVRE Of !t'LC(t90ft~lutISMES' XS~k IHSTAUCTXONS CXYEH UttDEA 7 NBOYE) ALxtrCS iJlfD1'IIIilttp4S
Further corilrnertex CivS ItiFOtZMATLOt~S NC SONT LItES, ttI AU7( pISFOSITIORS OET RI;GI_ES 6.Lb, f>.~.a. i i i , 7.6 ET
e, NI AUx DL~SPDSITIONS pES REGt_ES fi.l.a,i~ii, EC Il.~.f. LOUR INDICATION
E5T FlICULTA't'.fUE
5uC11 IIIFUf:l4\TlOk I5 NGItHER L>;NKEO To 'niE PROYIsION& OF RtItE G.t.b, kULE 6.x.a,fil. kULE 7.6, ;~~"". ~ AND RtttE a, t~R To THE PkrNISIONS OP aul.E s.i.o.tst ~Nu Rat.E
xi,a.;. res~FUanzshzxG is ~TIOHAL, , CETTE FORtiULE DOLT ET(tE REMPLIE SANS RATUR~S NI SURCHARGES . ~~~~~ ~
° ~ 'C_L:LL~C~I
lkl5 FONtt SHOULD 6( DULY cptPLETfb uZINOUT OEtETIOfI: 00. ALT'ttnTIOHS

x~;r~cItINCr n'xn~;r~~Cira.c~rxorr ~ l~_<< . hac~ f / U4 / olt l'dantificatiorr reference . ~ .w---.~~ ~,~.----.~---.. l'.~rxte .,«. .
9~.. ',1NFCJRMATIONS SUPPLSMEI~tTAIRES ~ . ~
~ldditiarra! infor~matfan COCH(:R St Df:S INFDItMATI'ONS CDMPLEMCNTJIIltES SDNT FDURNIES SUft UNF
FEUILt.E .IOtNTF:
~tK Nmi A cKOSS 'tP ApDITIaNAt, iNF~ITIat~t IS GIYEK oN AK Ar'tnalEO SHEET
C7ri~ine: de to cultrsre. ceiluhitre au du rurnxxrbe ( --- comIyIZrsxerxt e:vt:ntucI nutx indie.~tions darra~es sots Ic chitirc 3}
Source of tlse cell cetltzu~ or tFlc ntz.~'ture (as far at not ~ivrn rattler 3 a6ovc~ - ' ' . liybr~.dom~ : -- cu~,Lul~s spl.en3.ques de. souxxu , .-- olyelom.e de spur3.,s 'Bal.~/.c ~'3 A~8 ~C 6:1 6.5.3. .
Ll~a6e~ta6iic~lar ~n~~rii.crie dues An~tcorps . to ?0/4'3/~3G
Cell liree estabkshcn by (date) Cetiulocloii~epar Tnp~niexie de. Aat:icorps !e :1.1/1()/94 Geli cloned by - . (dare) T~:ssret,~istrenierrt daps d'autr,e.s irtstitut~oms de dEtr"ut (Noays, dates, uum~ros a~.ttri~l~~uGs, «.) lte~isuntinn in any otl:er d~yositary uutitc~tion (Names, dates, r~fcrer:ecs, ctc) Autres iatormations Further eorrvrlerlts CE5 IHFORliATIONS N~ S~NT CIEES, NI AUX DISt'OSIT10NS DES R:GLES 6.I.b.
G.Z.a.fii, 7.6 ET
8, NI AU% QISPOSI1'IONS DES REGI.ES 6.l.a.iti, FT I'i.4.f, LEUR INOtCATI0l1 C51' FACULTRTIVC
SUCH INv~oArv~TipH 15 h'~iTN~K itrttcrn TO YNr pltaYl3IONs OF Aut~ 6.i.b, AULE
r,.2.a.1rl, RULE T.6, ANa RULE 8, NOA TG THE PlinYiSroNS CF AUIL G.l.a.tri, AKD AUS,K 11.A.P. ITS
FUfINI511IxG IS ONtIOrbtl._ '~ Norsr, adrease et rxusncra d.~: t8ldpho:4c (uu/et do t~l6caliieur) do ;rrientifique 1c opal~sabtc de la caIture transnnise Name, address, plxnrtc anrlJar fax nrcmber of the scientist resportscTxlC,~'ar the culture Warlsrriiiied -~~-~ ~.Zc ~_: ~ ~ ~'~fl ~.. f ~ '~' ~ 9 'g ~'' ~ Fn vut rltt digiit la culture cr;Iluiaire sera frartstnise, colulit'.torende en clrll~o~L~ce. ~ ors car axate Ii~uxrie 'l'7tr, cell ectlucre released fur the ptcrpose of de~rorition will be transmitted irt dry ice ot~ ir. liqltid rsitragrtt saes forlue do i2 Eeh:ratillarss, d'exn m~rao lot, en trstses GtnnchPSr snarqsrcis eorsfurxxremeat >tux cx~eu~cs lndiciu~es_ as 9sy samples frr~r~a the sc:rne batch, in airtl~ltr virrTs, rraarlcc,~d crs requested.
* Dh,TE ~ SlGf~iATUt~E(S) SIGtZhTURC(.~~ dU(d~S) SCxEiI~TIFIQUE(S) L?~t'e '~. ~a ~.~.~ "t . Si~'n~turersJ IZF.~PON~~iBI~( S) 1.. ~'--~=-~~J~I~ _~~
~r ~ ~._,......'''._ ....r '.' .eq"
na;,,s~.: ~.", ,::~.:.Ahf CIIPrI ~II1. Can.' v s~G ~ ~ j ~ I
~~ivrar~7 ~~~
' ~ a ~nv~ry~ons ~
LE:S NoPiS DACI'YtOGItAPNIES OCS rERSONf'(E5 pIiYSIQUES QtII SI6NEplT AU NOM
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DaIVLHT ACCOMPAGttLtt L~5 SIGNIiI~UAt;S. ..
THE TYPt'WftrTILe NAMES or. TI;E NarnttnE FE2.>ons SIIrNIN. oN rrtT~pLF Ot' TFI'c LEC~nt. $lfTI7Y SHOULD /tCCpMf°AKT Ttl~ SIGNrII'UxFS.
Cf7TG FORI4Ul.F DOFT R'PRE REHPLIE SAYS RA'TURES tlI SURCHAR~S c~>G,~9rrG
T11I5 FOtiM swOUiD aE OULY caa~ayEl'EO utTNauT DEtF1'IOrts oR AETE~nYioNS

~ti ~ DtREGTIV 9S/BB/CEi: NATIVE: A lai.PROTECTION LYES 'fRAV,'1ILL~ItRS f~--1=
.....w CONTTtE 1.ES Ir:St~tll;S LIES A tINE EXt~OSITIOe;

' r A OE5 AGENTS 9TDLOGIQUES A11 TRA'IAIL - 1~IRInL'TiV'c 90/2r9/CEE
~~=t . ;;, ilEt.,ATIVE A P,.'UTILISA"CTDN COtIFIN'E,.t' DE

::i'..r HxCR~RGANISHFS G~ttETIQUaI~tiT Mc.'DiFIES -. aIRrCTiVE 90/7.?0%'CEE ~
ttLAYxVE A to t7iSSEMItanTxON VOL.Dt~rl'ATftF_ r p~ORGANiSMES 6EHt;TIQU~T H~IFIES pAttS L'E11VIROtINEric,"NT

DECRE"f n 94.352 flU 4 MAI 1994 ittATir A 1J1 PROTEC'fI4N nES
TRAVni~i~EtIRS~Ct)N'iT'eL
tES EtISQUES RES11I,TAHT Ot -.r ~- LGtlr; EXFOSITIpH A aE5 AGENTS BLOLDC:TQiIES - I.DI n 9z-~a nu x3 c.~
w 3UILI,E:T !~2 RLATiVt: AU CON'ttttll,F t7E

t, t1'fTlxS~tTION ET Q~E t.A OISSc?~tlf~tATxDtt pE5 ORG-~ftI5Mt:5 f:':~ GFNEfIQUF~tEttT MdOIFTES

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Le saussigne declare avoir plocede a tocttes ies nvtrFications requises par les reglemertfiat~or~s .

~;~ ~r na~anales en vigueur le concemantuant ~ 1'utilisatiori q et a la ~dassemination du rnicro~

organisrne city ci-dessus ee composarttes associees et de toates l et avc~ir re~u des autarit~s oe ~~ campetentes lee autarisatians w s'y rapporiant.
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~~ ~ W ~'he undersigned declares ~ that he his pmceeded to the nof~cations required by hrs dwn national Ft=gulations in t'orcr' t:onreming the use and tfie -release of~ the above mentioned microorganisrrr and all tfie associated components, and that he has gof from f~he campelent ~a'authon~tns the re%tiant permits.

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lIICMORGAlIISnES Ctt /~pPLICJ17I0t1 0~5 REf~t$ 28 i'Y
28519 OC lA COriYE!lTlat SUR lE IIRRY&'f EvROPECH. , CO1~I'f~x'/.'~T et~mi au : 3errr~es de to t'~gie s.~.ae.(v) du Traitd do t3udape~t du point ~i3.a.(v) de 1"IAta:ord bilat~raf en appiicatioa d~ r~gles 28 et 2t3bis . I _ de to Cvnventivn sur !~ Brevet ~utoQ~en .
entry les parties dEsign~es ci-dessous _ . _ .. , Cattectiort Nationals de Cultutres de Microorganisrtm, ' iNSTCTUT PasTEUt~

2S, Rue du Docteur Raux ~ 7ST~4PARtS~CED~C 15 . ~ tI~tSTXTtI'r PAS't'EIIZ~.

T4lcphone. 33-1 45 68 8; 50 ~

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x''vanrre CF..,i~.f'SIF...R r57~4 Paris Ccdrrx 75 ,birectetu'.fldrni~:istmtif y - aUTORiTE.DE DIwYOT . ,. ~ ' , ~ , ..DEPOSI~T(S) Nom(s) et adresse(s). , , ~ . , relaiif xu microarganisme~ (~~ designs ci-apr~s ' ~ ' . , ...~....._._..~,......,_.r.._.-n..., ; ' ,.....~....._..~. , , Hybr3.dome. de souris - A 4~4 tREF~RHNCE D'1DENTt~=ICAT10N~ NUMEttO OU SYMtl4lE5, PAR EXEMPLE;, DONNi~S PAR
LE (7E1'tISANT AU MICROOttCAliISMF
1~.~ Lc dcposant reconnait avoir conntti~sanct d:~ txz~tlccs tt dc,, xecomrnandations rclativt., sa dttp$t de microor~a~sxncs a.ttx ternle, du ~'rail:c de x3ud.~pcst otI au.~c tCrs~Ies de 1'A.ecord lSiIatcxul eu ahplicat'sotr dcs r~o,;lc::, 28 ct 28c la Coavcntiosx sur lc brevet GuxopBcn.
2.-La Gl'ICM acccpte Ie microargaui:;mo.identi~~ ci dGa.,Shs; une.~ois'quc scant xssxlr~~,s toutcs ~;: etanditiozzJ de v-aliditG du dEpBt en vrxtu de to r~~Ie b_i.(a) ou 6.2(a) et rie Ia r~~le 63,(a) du Trait do Srxdapest au du poznt 7.2.(a) ou 12.(b) ct do point 1~.(a,) de I'Accord bilat6ral, ndccssitant, cntrc autrc, 13 rEc:.ptiom par L~ CN'C'1Ir do dotlze cciyasitillores dax zuicrooar~is:ne idcmt;~r ci-dcs; u5, prirparc;. a partir ' d'itxte alerue subculture, esa'vue d'unc lon~ua conservation, cot~for:nctns».t aux indications fournies (~), gortant dt facori Iisible ct~iuddlcbile la rdfv.-cnce ~idcnttfic~tioa et lay date de prcparatiae, eL ' .
1'e.,calncm prcliminaire d'ua des Gel:~Ciilons rebus ar da CNCM to vue de 11 ra~nstatatiocl. do IsI valxdit~ dcs rea5eiguculGnts ~ourni:, cax Yerlu do J~a rcrlc 6.~.a iii) du Traiii; dw TiudaptsE flu dtI ~Uint XZa.(iii) do f~S,CCprd bilatlral et en vtle da la coestatatioxc de i'acGCptabxlit~ du materiel Cr'arssmis pour depot etz vcrtu de la rtr'tlr G.°l~.(a) clu ~'raitc de filldapirst on du hc~iat ~~.(a) de:1' ~,cc:ard bilat~rai. ' (') On rn-tencl p:ar 'MICRppti6N'IISdih:' tout; mat~t-itzl bioTogique qae la CttCN~ t~s4 susc~ptible d'accep~Ls:,r en vuc; d ut1 t!i-p8t aux termes du Tra~i~C~ de Budc:pesfi ou de 1'Accard bila2~ral.~
(r) Les ampouit~s.doivent ~tre compatibles avee les dl::pnsiti~s dp aorrserva~cion de la C~tC'rl, ~tanches_et sans risquc= d.°. fissura2ion. Cc. rupture ou d'oxplosion pcndant.la pc:rlcscte~, de cnnservatinn prevut~ e1 7aI regle 9.1. du 'franc dc, Budapest ou au point lI. cie 1 °nccard bi tat~ral .

3: ~ Un uumarr~ d'eztre~istrGxncrat pctii litre commtuxicluc au de:posanf:dc,;
reception du tnicroorganisme. L.:t ri,ception et I'e~n.registremcnC d'un micrnax~;a,txismc n'ianplique pas svn acccptaCiou.
4.- 'Un refus d'accepfaCzoax, du microor nnismc pent cerc noti~'x6 duns toc, conditions prcvur..s ~ la rcgtc 6.~:(:x) du TraitG de ?iudapeSC ou au point I4.1'(a,> do 1'A.CGocd bilateral.
5: 5i Ics co~tditions de validit6 du d~pat zte sons pas tautos reznplies, .unc proc6durc cio report d'acceptation ct appliguec; La CN'Ct~ e:a noUFo Ics razso~zs ~t i'LVC le ddl3i rlc un mocs au dGprisaat ~o~~r qu it sati:,faac a tatxtes !es cxt . ences. Si le dGpc~sant nc satiNfaiC pas aux cxiffcaxces dazts le dulai fier:, !a C.N ..ivi proci:dx; ~. 1"annulation de Iz ~c:mandc 'dc di:p3t et a Ia destruction du matGrxel binlcx~-iquc traaa.smi; . . .
Ce~tte pcocEdtirc impl'iqtte uu depot ~ du remptacecxyent,($) Ni uexe irrG~uIarit~: de forzne, de quzntiti: ~ou do orb: catatlon cst constatEc par Ia CNCM s:zr to zxtatcricl bsoto~ique trsnsxttas par lc ddposant en vue d'un d~p5t uiltaal ou d'un nouvc;au d~pc3t. Un dbpot de r~mptacc:zn~ent ca cpnsiderG
coanmc un autx~r~ depot initial tart qae to viabilitG du d6pot en attento d'act~~ptsaiozt zt'cst pas Etabtie.
6: Chaquc ~ois quc du r iat~ricl biolo~;iquc; o st trazasruis par 1e d~posant ~ fa CNCM. la CNCM pcr~oit la, taxe de consezvetioci 'prdvue ~ la rc"~.glc:12.1.a.(i) du'lCraiCc de Budapc;;t ou au point 2G.a.(i) de I'Aeeor~b~at~cal.
'7.- Si Ic depot est acccptG, Ie nuzndrn d'ordrc attritbu~ xcx dx:p6t par la CNCM cst idcntiaue au ntzmGro ci'enxt~x~-is.
Crcmcixt, et la dxtc de dEpot esL la date de r~eeption par la CNCM du microorganzsme identiFid ci-de ,svs.
8: L.a notificztion de I'acca~ptation , du rc~'us au do 1'attimlatioa do Ia domxzadc do dEpdt cst Etablic darts urx dGlai do si~e znois apr8s r6ccptina du znicroor~azaisnnc. L'aiccGptaeion cst attestce par !c xvcGpissG.
9: La CNCM nc proci:dc au pre~xoer controls do viabilai~ qu'unc faiN assu.rLC
quo lc; dGposaext a satis~ait '~, tatetes Lcs axi~euces en zxsatic'.re dsr d~pirt. ' .
~.Q; I;a ~rcmi~ie.dEcLaration sur la vrabilitd a.ttGsta to validation øax, I'annulaiion du~d~~pt5t ':~ lc dc:p3t est valid si to :. ..~z~ oorgaziisntc est viable, al. est'atmulc. si to mie~roor~anismc n.'c.". pas via'bic. : , . , . - . , , , . , 7.1: ~n car, do refa5, d'annulation do dem~ande do depot ou d'aztrylation de dbp8t , Iea Gchantitlons do materiel biolo~,' ~iquc trap .mi, sont dGtxuits ; c», c;as de desaccord sur les xnotiFa du rufus nu drx . D'axx~.tulation , iL, ~euvcnt ecz~c cnn.,orvfs 3 la Cl'~CM ; i1s ne sont pas rc:,tituc.~.s au cx..G~posant , ;
nut' a.car~rd partiCUlier intcrvcnu aprrbs recept'sort de la ztotiFication dti refus ou de 1'annutatiocx pa,r lc clcposan.t; sex aixc;un cas , les Grhantillans nc peuvcnt dcatatx4r lira ~ tux dcp6t a Ia CNCM ~ d'autras fins.
12: L.a ta,~c de conservation nrwue ~ Ia rc~.r~ic 1?..l,,a.(i) du Traiti: de Luda~c,;,t ou au ~eoznt 2G.a.(i) de 1'Accord bilateral est dun da.n , i:ous 1es cas, que Ie c~C.pnC sQSt scccpt~, re~us~ ou annuls:. ' .
1.3: r o d::pusant s'cneage 's dcpasor sotxs coiztrat associG tout mat~ricl vivant , rnon ou di~ciloruextt ncccs.,ibte, u~cussaixe ax>;c coutralcs r:h/au ~ I:a canservatinn din mzcroor~anisrtte identifi,i: ei-dessu :.
.A~: LC dC~.pa:;~n,t S'Cawll~~ ~, loi,tril.ir tDit;, I.eS Suhar~ttS ripn: OU.
, CIs~xCtlCIriCxit' "siGCC:r,,SlblCS , n~CCSSairC5 Ctli.,C . COGtrpla5 ' et/bu ~ 1a cozzscrvatioa do m~,croor~anisnxe i.d:eatitic: Gi-dc.ssus, eu quantitc, siaflvsantc pour douze Gprcuvcs ~ou pa,ssagcs. ' 15... T..c d~po ,grit ccrtittc avair fourni touts indication dota i7 a comxaissa.ucc ,ur Icy; pcourict~s du microor~ani .rnc idcntxtu ci-dcssixs qui pr~setttent ou Pcuvent pr~.sentGr one action directe ou indirccte , con:oue ou pr~vi,sxole, sur 1'horauxc "ou sur cltxelqu'vr~anisaie'quc: cc soft, anim~il, vdrrrtal ott a.utre. - , Il s'cra~age a port:.~,r zmmr~diateznent .'~' la conuaissaace do Ia CNCTvi tO~ae uouvel:lt inrn~~xat'tolt y relative:.. , 7,G.- Apria acceptation du uxiexoorc anisxne , 'la CNCM le conserve ., assure les eontxUlec do viablite , ellbI~t 1C._a ~IcClarltlOn; , ;tCCC; t~ttl~7a5 'Nt nOt~C~.trQXxS , reIllCt 1~5 CCllI~rpti~FOriS ~T. itX pslrtlG:; ~IItOCISt,'4S ~ Cf~,~tiflCCS , Uu requcrantcs, con~o,rruc~meuC ~ t;i rrr~lcmatxt:2tion apptic.-iblc. , (SJ On antcnif par 'OEF~OT 0~ REMfaI.ACEMNT° unc si:ric Campl~metitxire d'cc~Ryt911ons du nicraarg~crlisme ayant fait 7''ob,jr~t d'un d~p5t initial ou d'un nouveau den~it en ant~ntc d'acCLpt~tiaO, prH:pot~us cranformement aux exigenee~: de la' CMCH et transmis en one seuTr.; f~is per te: dcposant 4 ls't tiemttndr et i"r 1'adrt~asr~ de 1a CNC~I daps 1a d~lai fiat au point 5 du pre>ent eonLrat, aorptup.riynG:, d'ut~.~; dEctarZtiun sernlii~thle s cetlc dGfinie d la r~egte b.?,.(a) du TraSt~ <ta~ Budapc~st_ I7: Glxaque foi . que la CNCM ~u~o . olyortum~ d'adre:~scr au . dr.:pr~s:ant (~' '") mz vcI"antillon dune su'~culture du znicroor~unisrne identiii.c ci-cst:asus cn vac d usx cantr8le de ConLUrmit~, le Cieposarit (x'~') vGri<ic ft ;arcssiorc des propriEtEa dudik microoraani_~.rae den.., la, dite subculture ct remvoiL ~t la C'NGM, deal Ic dela.i de troi" moil apn;
rEception do 1'eehautiJlan , Ia formnle jointe .d ~'onvai apr~s favair diaazerxt rcrxpl.ic ct si~nEe.
La dGrosarxt ~*"') rscos>n3a du'cn cal de noo,-r'dposzse ~ une- dcmaxxdc d'a,ccord de cr~niorsz:itE tclle qu'nlle t,~t prE~..;eutcc ci- essus , Ics pro~~~rvctEs de Ia sutrculturc ctt question soot ~ cansidcxcr idcrxtiques aux px~orx:xetes da is SubCxlli'xirC tT~mL;C ~ 11 G~CM ~.1~. datC du dGpaC. ' ~8.- Le d~pos~nt nc peat prs rctircr , ln~auler ou .modifier Ic d~pcat peradarit la lacriode do conservation p,vvuc :z 11 ~rcglc ~.1 du Traitd d- I3udapcst au au poizzt L1 do (Accord i~i~tcraL . ' . .
Cctxc pdzzodc do cor>serv3Eioiz cst de txentc-eirxq aa~s dazes foul Ies cal a la GhTCZvf.
. , ~.9: !1 1'e.~iration do h1 periode do coaservatitsn. , tout lc mat6riel bialo~iqne ooz~~.~rvc cst ~ d6tx-uit , sata~ cans lc r.,a:, d'tmc de~cmaudc purticulic;r~ formulE4 pear. lc ddposant ttu cour;~ul: de la ts~cnte~citiquzemc :zxznde de COfJISCt'~I~tlOn. a ~'Ø.. Lorsc~xit , dour quclduc raisoxx C;uG CO SOiC , la C:NC.rlrf sue pout pas remGttxc d'cchantilloxxs du microorganisms idcut~nb c~ dessu., , nnc foi~ aces tE et dEclarc vxablc , C dEpasant proccde conform~zuent aux divpositions de la 'rE~lemcntatioa lpplicable ~ vn nouveau depot dudiL microott,~nis~mc duns tux dGlai do trail ,zezois fit' coznpccr de la daxc de r~ccptivn do la notification c~orrc.,rhond~tc.
La dE ,finition dcs raison, do bon nsago en nzxti'~rc d'autoritc do dcp3t , ~tGrdi,:u~.t la rezxxise d'cchantillozt5, est souuaisc ~ la seals spprdciatian do is GNCM.
'~.,- La CNC.N~ cst dE~as;Ec de touts rc:,pans~xbilitE csi ca . do °variatiocx de ca:r'acicrcs du rnatC;,rict biologiquc depor,f . ~1 cn cst do memc si vxxG pcrtc de via6ilite , ur~t contamination ou~ unc dcstructioxy aGCideutellc btait constatc:c malcpt~E fapplicariou dcs prEcautiorxs prcvues gear sa consc;rvatian.
'2Z.- ~Si ,.; psr li f<cute ocx taw~~li~encc du dE.posant, i~zi fait: doajzxw ~;.abIc ~ fa. CNCivt . survieait a I1 tccCptqO~n~~~, :~. )a, msxupxtiatiaa ow pendant ~wuu.z~e dc: cocsse: vation. du dEp~c;~C . de:nosanC
Sndcmr7l.S~ ~:la ' CNC11~I~ : y'i , pt~judiae - ' su.U~. ° . ~ .
23..- A mains qw lc fait dommaGcablc ne soit imptxtablc a Ia flute ou ix la nylr~cncc do la C:NCIYI , lc d~pos~t gasantit crlic-sit centre toute action 3 sou exzcontxc en reparation d'u,n pscudice li4 -''a la remiss d'un Echantil ' loo du miczoor~aniszxse idenc%fi~ ci-dcssu.~. .
2~.- ~n ca: de contcstadon la loi franr~aise cst ap~alieatilc et Ie tn'bunal eozxxp~teat est eclu; de ~'aris.
'lx'(''~ D'F.~.'f~le~~~,'~~ T.~ate~et -°sz~aturc(r,) ~ ~'~~-dI7 ~il~iSTITLI"~ ~~ASTLcLII~ ' . . . . , . . . . ~Gw . . . ' . . . °-,---.~._._.
' - . ~ . ' . ~ . . ~ Daniell~'BEI~NEItit~~,t~ ' . . ~ ' - 25 28, nic du Docteur Roux Chef du Service de ~ ~rev~fs i,57Z4fari~. Ccdex'15 & lny~n60~1s x.~cs) SC~NTI:(~'a(~UE(SI RIESPC)i~IS~;(~) T3nte ct si~,~:z~aarc(s~
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(~'*) Lea engtrgetnanC.> inct-iques au paint 17 pruvrnt ~cre~ assure~, inditf~rerr~nent par 3e dcposanr au ie rcsPonsabic scientifiiqu~t indiqu~ par le dsrpasant. ° .
("'""~) Le. Yri~m~ 4aictylognaptriia des, pe;r>onnes pivy~~iqut:;; qui sigttettt au nary dune pcrscnno morale doivent acccm- , pagner ies s;c3rvaturcs_ l.or~~qu'une demandu dr: d~pbt est Em4~:oni:c:e par plu.;i~rtrs peranran~s, le fiorrru~laivw e.t lE~ con~krat correspondancs doivenx porter 7a siqnxLurc do chaccrr des d~paa:.irlt~3. Cans tr ezs, uroe des per-gonnes dolt trtrr? design~~ RGRRESENTRMT UNTQftE, h2biliee':; 3 r~Gevoir, lc, daeummts vriqinauX ~tttis par is Ct!C14 pendant la periods de conseruaLion 'prewe dart;, la rigicmrentation appli~ak~le. , CNCM
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Claims (18)

1. A method for identifying a pathogen strain of Listeria in a sample, comprising the step of detecting in said sample a Listeria strain comprising a functional internalin, whereby detection of a Listeria strain comprising a functional internalin is indicative of an increased likelihood of pathogenicity of said strain compared to a strain of Listeria having a non-functional internalin.

2. The method of claim 1, wherein the step of detecting in said sample a Listeria strain comprising a functional internalin is obtained by the following steps:
a) contacting the sample with an antibody that binds specifically to a functional internalin for a time and under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a).

3. The method of claim 1, wherein the step of detecting in said sample a Listeria strain comprising a functional internalin is obtained by a step of determining the presence or absence of expression of full length internalin in said sample, whereby identification of expression of full length internalin is indicative of an increased likelihood of pathogenicity of said strain compared to a strain of Listeria having a non-full length expressed internalin.

4. The method of claim 3, wherein the step of determining the presence or absence of expression of full length internalin in said sample is achieved by PCR and/or sequencing.

5. The method of claim 1, wherein the step of detecting in said sample a Listeria strain comprising a functional internalin is obtained by a step of determining the presence of internalin at the surface of said strain and/or the absence of secreted internalin

6. The method of claim 5, wherein the presence of internalin at the surface of the strain and/or the absence of secreted internalin is achieved by means of antibodies that binds specifically to internalin.

7. The method of claim 3, wherein the full length internalin has a nucleic acid sequence as shown in SEQ ID NO:1.

8. The method of claim 3, wherein the full length internalin has an amino acid sequence as shown in SEQ ID NO:2.

9. The method according to any one of claims 1 to 8, wherein the strain of Listeria is Listeria monocytogenes.

10. A method for imaging tissue interface where E-cadherin is expressed comprising the following steps:
a) contacting the tissue where E-cadherin is to be detected with purified internalin; and b) detecting interaction between E-cadherin and internalin.

11. The method of claim 10, wherein said tissue is placenta or blood brain barrier.

12. The method of claim 10, wherein said method is conducted in vivo.

13. A method for targeting placenta, wherein said method comprises the step of administering internalin to a pregnant mammal.

14. The method of claim 12 for targeting a molecule of interest to placenta, wherein said method comprises administering said molecule of interest linked to internalin to a pregnant mammal.

15. A kit for imaging tissue interface where E-cadherin is expressed comprising internalin.

16. A diagnostic kit for the detection of a pathogen strain of Listeria in a sample, comprising:
- an antibody that binds specifically to a functional internalin located at the surface of a Listeria strain;
- a reagent to detect internalin-antibody immune complex;
- optionnaly a biological reference sample lacking a functional internalin that immunologically bind with said antibody; and - optionnaly a comparison sample comprising a functional internalin which can specifically bind to said antibody;
wherein said antibody, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.

17. The diagnostic kit according to claim 16, wherein said antobidy is produced from hybridoma A 4-4 or L 7-7 deposited at CNCM on May 3, 2004 under reference I-3205 and I-3206, respectively.

18. The method according to claim 2, wherein the antibody is produced from hybridoma A 4-4 or L 7-7 deposited at CNCM on May 3, 2004 under reference I-3205 and I-3206, respectively.