WO2006003518A2

WO2006003518A2 - 6-phosphorylcholine-n-acetyl-d-galactosamine conjugate molecules and their use in vaccinal, therapeutic and diagnostic applications against bacterial infection of the respiratory tract

Info

Publication number: WO2006003518A2
Application number: PCT/IB2005/002316
Authority: WO
Inventors: Sylvie Bay; Pierre Lafaye; Patrick England; Jean-Michel Alonso; Valérie HUTEAU; Mireille Leduc; Farida Nato; Muhamed-Kheir Taha
Original assignee: Institut Pasteur; Centre National De La Recherche Scientifique; Universite De Paris 11-Sud
Priority date: 2004-06-30
Filing date: 2005-06-30
Publication date: 2006-01-12
Also published as: WO2006003518A3; CA2472818A1

Abstract

The present invention relates to carbohydrate-phosphorylcholine conjugate molecule, and more particularly to phosphorylcholine-N-acetyl-D-galactosamine molecules and their use for treating and/or preventing a bacterial infection of the respiratory tract. More specifically, in this present application, the inventors aimed at raising high-affinity antibodies against ChoP preferably in its bacterial context in order to target several pathogens of the respiratory tract. By mimicking the S. pneumoniae model, the inventors preferably synthesized two carbohydrate-ChoP (GalNAc-ChoP) protein conjugates and showed that these immunogens induce hapten-specific antibodies which recognize two major bacterial pathogens of the respiratory tract : such as a Gram-positive bacterium, S. pneumoniae, and a Gram-negative bacterium, N. meningitidis.

Description

CARBOHYDRATE-PHOSPHORYLCHOLINE CONJUGATE MOLECULES AND THEIR USE IN VACCINAL, THERAPEUTIC AND DIAGNOSTIC

APPLICATIONS

FIELD OF THE INVENTION

The present invention relates to carbohydrate-phosphorylcholine conjugate molecule, and more particularly to synthetic phosphorylcholine-N-acetyl-D- galactosamine molecules and their use for treating and/or preventing a bacterial infection of the respiratory tract. Furthermore, the present invention is concerned with compositions, vaccines and methods for providing an immune response and/or a protective immunity to animals against a bacterial infection of the respiratory tract and methods for the diagnosis of bacterial infections of the respiratory tract.

BACKGROUND OF THE INVENTION

Neisseria meningitidis and Streptococcus pneumoniae are major causative bacterial agents of invasive respiratory infections and meningitis. These bacterial species are genetically and antigenically variable, and, therefore, currently available vaccines are far from satisfactory^'' ■ 2. Moreover, an increasing number of S. pneumoniae strains are resistant to various antibiotics and the emergence of N. meningitidis strains with diminished susceptibility to β-lactams becomes a matter of concern^. Antibody-based therapies could therefore gain renewed interest for the prophylaxis and treatment of these respiratory infections 4. Phosphorylcholine (ChoP) is frequently incorporated in the surface antigens of several prokaryotes (Haemophilus, Streptococcus, Neisseria,

Mycoplasma, Salmonella and Pseudomonas) and eukaryotes (pathogenic helminths and nematodes) (for review see ⁵)

In respiratory infections, ChoP is thought to be directly involved in the steps of adhesion and colonization of the respiratory epithelium, as well as in the inflammatory process leading to the invasion of the host. Through ChoP, H. influenzae and S. pneumoniae are able to bind to the platelet activating factor receptor (PAF-receptor), thus mimicking endogenous processes of cellular signaling ⁶> ⁷. Furthermore, several proteins of S. pneumoniae, which are important for its virulence, are attached to the cell wall via ChoP ⁸. ChoP also appears to be responsible for the triggering of innate immune reactions against H. influenzae, mediated by the C-reactive protein (CRP), which is the natural ligand for ChoP in blood and acts as a complement-binding opsonin ⁹.

Therefore, ChoP is an attractive target for the development of immunotherapies directed against these major bacterial infections of the respiratory tract ¹ °. It has been shown previously that ChoP-specific T15-idiotype antibodies, despite their low affinity, are protective when used in passive immunizations 11> 12 Likewise, the injection of ChoP coupled to a protein carrier induced the production of high-affinity specific antibodies in mice ¹³. Moreover, intranasal ¹⁴> ¹⁵ or parenteral ¹⁶ immunization experiments with a similar ChoP- protein conjugate protected mice against a lethal challenge with S. pneumoniae.

However, the epitope recognized by the induced antibodies is not always limited to ChoP but also includes the covalent link used for the coupling with the carrier ¹³. In the first studies, ChoP-protein conjugates used for the immunizations contained a diazophenyl linker between ChoP and the tyrosine and histidine residues of the protein carrier, resulting in immunodominant responses directed against aromatic rings. To minimize this irrelevant immune response, efforts have been made to replace the aromatic rings by an aliphatic linker. The resulting immunogen provided total protection of Xid mice against a lethal challenge with S. pneumoniae, whereas the diazophenyl-containing conjugate did not ¹⁷> ¹⁸.

These results suggest that the nature of the linker between ChoP and the protein carrier can be critical for the induction of high affinity protective antibodies intended for anti-bacterial therapies.

According to the pathogen, ChoP is coupled to a variety of bacterial cell structures. In the case of N. meningitidis, ChoP is linked to the glycoproteins of the pili, but its molecular carrier (saccharide or aminoacid) has not yet been clearly identified ¹⁹> ²⁰. In H. influenzae, ChoP is grafted to the surface lipopolysaccharides 21. In S. pneumoniae, ChoP is part of the C-polysaccharide (teichoic acid) and F-antigen (lipoteichoic acid) ²²> ²³. Despite this diversity in the native macromolecular backbone, it appears that, at least for H. influenzae and S. pneumoniae, ChoP is presented in position 6 of an hexose or an hexosamine, respectively. Whether this carbohydrate part of the epitope is involved in the pathogenic process remains an open question.

Thus, there is a need for new molecules that use ChoP linked to a carbohydrate as a target for the development of immunotherapies against bacterial infections of the respiratory tract.

The present invention fulfils this need 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 carbohydrate-phosphorylcholine conjugate molecule, and more particularly to phosphorylcholine-N-acetyl-D-galactosamine molecules and their use for treating and/or preventing a bacterial infection of the respiratory tract. More specifically, in this present application, the inventors aimed at raising high-affinity antibodies against ChoP preferably in its bacterial context in order to target several pathogens of the respiratory tract. By mimicking the S. pneumoniae model, the inventors preferably synthesized two carbohydrate-ChoP (GalNAc-ChoP) protein conjugates and showed that these immunogens induce hapten-specific antibodies which recognize two major bacterial pathogens of the respiratory tract : such as a Gram-positive bacterium, S. pneumoniae, and a Gram-negative bacterium, N. meningitidis.

In this connection, one object of the present is to provide a hemi-synthetic conjugate molecule having the following formula:

(I)

wherein X and Y are optionally present and wherein:

X is a spacer group;

Y is chosen from biotin or a derivative thereof or a carrier protein; n >1.

Another object of the invention is to provide immunogenic composition against bacterial infection of the respiratory tract comprising a hemi-synthetic conjugate molecule of the invention.

Another object of the invention is to provide a purified polyclonal or monoclonal antibody capable of specifically binding to a synthetic conjugate molecule of the invention.

Another object of the invention concerns a method for treating and/or preventing a bacterial infection of the respiratory tract in an animal, the method comprising the step of administering to the animal an effective amount of a hemi- synthetic conjugate molecule of the invention, an immunogenic composition or an antibody as defined above.

Yet, another object of the invention concerns a method for immunizing an animal against a bacterial infection of the respiratory tract, comprising the step of administering to the animal an effective amount of an immunogenic composition as defined above. Furthermore, another object of the invention is to provide a method for preparing a hemi-synthetic conjugate molecule of the invention. The present invention is also directed to a method for detecting the presence or absence of a bacterial strain bearing a phosphorylcholine molecule in a sample, comprising the steps of: a) contacting the sample with an antibody as defined above 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).

Another object is to provide a diagnostic kit for the detection of a bacterial strain bearing a phosphorylcholine molecule in a sample, comprising: an antibody that binds specifically to a phosphorylcholine molecule (ChoP) located at the surface of said bacterial strain; a reagent to detect ChoP-antibody immune complex; optionally a biological reference sample lacking a ChoP molecule that immunologically bind with said antibody; and optionally a comparison sample comprising a ChoP molecule 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. Yet, another object of the invention concerns a process for screening of an active molecule interacting with a phosphorylcholine molecule having the following steps :

1) contacting a phosphorylcholine molecule or a product containing a phosphorylcholine molecule with an active molecule to be tested; 2) adding a sample containing labelled antibodies according to the invention

3) revealing the presence or absence of forming complex between the product obtained in step 1 and the antibodies of step 2. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the ELISA measurement of heat-inactivated S. pneumoniae cells reactivity for murine anti-TT-GalNAc-ChoP (♦) and ASA-GalNAc-ChoP (■) immune sera. (0) and (o): corresponding sera of non immunized animals.

Figure 2 shows an lmmunoblot of anti-ASA-GalNAc-ChoP immune serum with N. meningitidis extracts. A wild-type pilin-positive (PiIi+), ChoP-positive serogroup C strain, was tested in parallel with its isogenic pilE mutant (devoid of pilin, PiIi-). The specific monoclonal anti-ChoP antibody TEPC-15 was used as positive control, and a polyclonal rabbit anti-pilin IgG was used to co-localize associated ChoP in N. meningitidis.

Figure 3 shows the synthesis of GalNAc-ChoP according to a preferred embodiment of the present invention. Reagents and conditions : (a) 30% HBr in CH₃COOH, RT, 1h30 (99%) ; (b) Zn, NMI, dry AcOEt, reflux, 2h ; (c) (NH₄)₂Ce(NO₃)6, NaN₃, CH₃CN, -25⁰C, 3h (2 steps, 44%) ; (d) LiBr, dry CH₃CN, RT, 3h (99%); (e) AgOTf, collidine, dry CH₂CI₂, HO-(CH₂)₂-NHZ -40⁰C, 15 h ; (f) NaBH₄, H₃BO₃, NiCI₂, EtOH, RT, 1h30 ; (g) Ac₂O, EtOH, RT, 1h (3 steps, 54%) ; (h) MeONa, MeOH, RT, 10min ; (i) DMTrCI, Pyr, RT, 2h ; 0) Ac₂O, Pyr, RT, 3h ; (k) 2% ABS in CH₂CI₂/MeOH:7/3, 0⁰C, 2min (4 steps, 60%) ; (I) chloro 2- cyanoethyl (Λ/,Λ/-diisopropy!)phosphoramidite, DIEA, dry CH₃CN, RT, 20min ; (m) Choline* Tos^", tetrazole, CH₃CN, RT, 24h ; (n) l₂/Pyr/THF/H₂O, CH₃CN, RT, 15min (3 steps, 31%) ; (o) MeONa, MeOH, RT, 15min ; (p) H₂, Pd/C, EtOH, RT, 1h (2 steps, 55%).

Figure 4 shows the synthesis of GalNAc-ChoP-based synthetic glycopeptides. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to carbohydrate-phosphorylcholine conjugate molecules, and more particularly to synthetic phosphorylcholine-N-acetyl-D- galactosamine (ChoP-GalNAc) molecules and their use for treating and/or preventing a bacterial infection of the respiratory tract. More specifically, the present invention is concerned with compositions, vaccines and methods for providing an immune response and/or a protective immunity to animals against a bacterial infection of the respiratory tract and methods for the diagnosis of bacterial infections of the respiratory tract.

As used herein, the term "immune response" refers to the T cell response or the increased serum levels of antibodies to an antigen, or presence of neutralizing antibodies to an antigen, such as a carbohydrate-phosphorylcholine protein conjugate of the invention, or to a hapten, such as a Chop-GalNAc of the present invention. The term "immune response" is to be understood as including a humoral response and/or a cellular response and/or an inflammatory response.

The term "protection" or "protective immunity" refers herein to the ability of the serum antibodies and cellular response induced during immunization to protect (partially or totally) against a bacterial infection of the respiratory tract which may be caused by, but not limited to, Neisseria meningitidis, Steptococcus pneumoniae, Pseudomonas aeruginosa or Heamophilus influenzae. Thus, an animal immunized by the compositions or vaccines of the invention will experience limited growth and spread of such bacteria.

As used herein, the term "animal" refers to any animal that is susceptible to a bacterial infection of the respiratory tract. Among the animals which are known to be potentially infected by these bacteria, there are, but not limited to, humans, farm animals, sport animals, zoological garden animals, and wild animals. 1. Carbohydrate-phosphorylcholine conjugate molecules

An aspect of the present invention is concerned with a hemi-synthetic conjugate molecule having the following formula:

(I)

wherein X and Y are optionally present and wherein:

X is a spacer group;

Y is chosen from biotin or a derivative thereof or a carrier protein; n ≥1.

Preferably, the hemi-synthetic conjugate contains a pharmaceutically acceptable salt.

As used herein, the expression "derivatives of biotin" refers to any synthetic derivatives of Biotin known in the art, which can be covalently attached to form a molecule as generally defined by formula (I).

The present invention also includes pharmaceutically acceptable salts of the synthetic conjugate molecule of formula (I).

Preferably, when Y is a carrier protein in the hemi-synthetic conjugate molecule of formula (I), it is preferably a protein or a peptide, or fluorescent derivatives thereof. More preferably, the carrier protein is a bacterial protein, such as tetanus toxoid or alpaga serum albumin.

As used herein, the term "spacer group" means a molecular spacer between polymerizable group and reactive group in a functional molecule, such as an aminoacid or a carbohydrate.

As used herein, the term "carbohydrate" refers to a class of molecules including, but not limited to, sugars, starches, cellulose, chitin, glycogen, and similar structures. Carbohydrates can also exist as components of glycolipids and glycoproteins.

In a particular embodiment of the synthetic conjugate molecule of formula (I), the spacer group X is comprised between -(CH₂)₂NH to -(CH₂)4oNH-. More particularly, the spacer group X is -(CH₂)₆NH-.

In a specific embodiment of the synthetic conjugate molecule of formula(l), when Y is a carrier protein, it preferentially forms a molecule with formula (II):

It will be understood that according to a preferred embodiment of the invention, when the carrier protein is preferably tetanus toxoid, n is 17 and when the carrier protein is preferably alpaga serum albumin, n is 29. As shown in the

Example Section, such molecules are respectively designated TT-GalNAc-ChoP and ASA-GalNAc-Chop.

In another specific embodiment of the synthetic conjugate molecule of formula (I), when Y is chosen from biotin or a derivative thereof, it preferentially forms a molecule with formula (III)

In a related aspect, the present invention is concerned with a method for producing a hemi-synthetic conjugate molecule as defined above. More specifically, the method comprises the steps of a) reacting a molecule of formula(IV)

(IV) wherein R1 is a leaving group and PGi, PG₂ and PG₃ are primary or secondary alcohol protecting groups, with a molecule of formula(V)

V-XPG₄ (V) wherein PG₄ is a protecting group and V is a functional group capable of reacting with R1 ; b) reducing the 2-azido group to a 2-amino group; c) protecting the amino group obtained in step b; d) removing PGi from the primary oxygen; e) reacting the product obtained in step d) with a phosphorylating agent and choline; f) removing PG₂ ,PG₃ and PG₄ ; g) reacting the product obtained in step f) with one or more equivalents of a group Y or a suitable derivative thereof, and optionally separating the product obtained from the reacting medium. In a preferred aspect of the method of the invention, the molecule of formula (IV) is 3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-gaIactopyranosyl bromide, whereas the molecule of formula(V) is 6-(benzyloxycarbonylamino)-1-hexanol.

In yet another preferred aspect of the method of the invention, the phosphorylating agent is 2-cyanoethyl (Λ/,Λ/-diisopropyl)phosphoramidite.

As one in the art may appreciate, Figure 3 shows a synthesis scheme for a specific example of a carbohydrate intermediate (11) which can react with group Y or a suitable derivative thereof, to form the conjugate molecule of formula(l).

Furthermore, the reaction conditions for the synthesis of two protein conjugates of carbohydrate (11), namely tetanus toxoid and Alpaga serum

Albumin, are given in example 1 , and further detailed in Annex A, whereas the reaction conditions for the synthesis of a prefered biotinylated derivative of carbohydrate (11) are given in Annex B.

2. Antibodies

In another embodiment, the invention features purified antibodies that specifically bind to the carbohydrate-phosphorylcholine conjugate molecules of the invention. More specifically, the antibody is a purified polyclonal or monoclonal antibody that specifically binds to a hemi-synthetic conjugate molecule as defined above and even more specifically to the ChoP part of such a molecule. Preferably, the antibody is a monoclonal antibody that is secreted by a hybridoma, such as the one designated 13-1 deposited under No. I-3248 at the CNCM, 25 rue du Docteur Roux, 75724 Paris Cedex 15, on June 28^th, 2004 or the one designated D18-3 deposited under No. I-3286 at the CNCM₁ 25 rue du Docteur Roux, 75724 Paris Cedex 15, on August 30^th, 2004.

The antibodies of the invention may be prepared by a variety of methods using carbohydrate-phosphorylcholine conjugate molecules described above. For example, the protein conjugates TT-GalNAc-ChoP and ASA-GalNAc-Chop of the invention, may be administered to an animal in order to induce the production of polyclonal antibodies. Alternatively, antibodies used as described herein may be monoclonal antibodies, which are prepared using known hybridoma technologies (see, e.g., Hammerling et a/., In Monoclonal Antibodies and T-CeII Hybridomas, Elsevier, NY₁ 1981).

As mentioned above, the present invention is preferably directed to antibodies that specifically bind to carbohydrate-phosphorylcholine conjugate molecules of the invention. In particular, the invention features "neutralizing" antibodies. By "neutralizing" antibodies is meant antibodies that interfere with the biological activities of any of the phosphorylcholine macromolecules of the bacterium that can cause a respiratory tract infection. Any standard assay known to one skilled in the art may be used to assess potentially neutralizing antibodies. Once produced, monoclonal and polyclonal antibodies are preferably tested for specific ChoP molecule recognition by Western blot, immunoprecipitation analysis or any other suitable method.

Antibodies that recognize ChoP expressing bacteria and antibodies that specifically recognize carbohydrate-phosphorylcholine conjugate molecules, such as those described herein, are considered useful to the invention. Such an antibody may be used in any standard immunodetection method for the detection, quantification, and purification of ChoP molecules. The antibody may be a monoclonal or a polyclonal antibody and may be modified for diagnostic purposes. The antibodies of the invention may, for example, be used in an immunoassay to determine the amount of ChoP molecules in a biological sample and evaluate the presence or not of a bacteria that can cause a respiratory tract infection. In addition, the antibodies may be coupled to compounds for diagnostic and/or therapeutic uses such as gold particles, alkaline phosphatase, peroxidase for therapy. The antibodies may also be labeled (e.g. immunofluorescence) for easier detection.

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 hapten of interest, but which do not substantially recognize and bind molecules other than the one(s) of interest. As used herein, the term "relatively high affinity" means a binding affinity between the antibody and the hapten of interest of at least 10⁶ M^"1, and preferably of at least about 10⁷ M^"1 and even more preferably 10⁸ M^"1 to 10¹⁰ M^"1. Determination of such affinity is preferably conducted under standard competitive binding immunoassay conditions which is common knowledge to one skilled in the art. As used herein, "antibody" and "antibodies" include all of the possibilities mentioned hereinafter, antibodies or fragments thereof obtained by purification, proteolytic treatment or by genetic engineering, artificial constructs comprising antibodies or fragments thereof and artificial constructs designed to mimic the binding of antibodies or fragments thereof. Such antibodies are discussed in Colcher et a/. (Q J Nucl Med 1998; 42: 225-241). They include complete antibodies, F(ab')₂ fragments, Fab fragments, Fv fragments, scFv fragments, other fragments, CDR peptides and mimetics. These can easily be obtained and prepared by those skilled in the art. For example, enzyme digestion can be used to obtain F(ab')2 and Fab fragments by subjecting an IgG molecule to pepsin or papain cleavage respectively. Recombinant antibodies are also covered by the present invention. Alternatively, the antibody of the invention may be an antibody derivative.

Such an antibody may comprise an antigen-binding region linked or not to a non- immunoglobulin region. The antigen binding region is an antibody light chain variable domain or heavy chain variable domain. Typically, the antibody comprises both light and heavy chain variable domains, that can be inserted in constructs such as single chain Fv (scFv) fragments, disulfide-stabilized Fv (dsFv) fragments, multimeric scFv fragments, diabodies, minibodies or other related forms (Colcher et a/. Q J Nucl Med 1998; 42: 225-241). Such a derivatized antibody may sometimes be preferable since it is devoid of the Fc portion of the natural antibody that can bind to several effectors of the immune system and elicit an immune response when administered to a human or an animal. Indeed, derivatized antibody normally do not lead to immuno-complex disease and complement activation (type III hypersensitivity reaction).

Alternatively, a non-immunoglobulin region is fused to the antigen-binding region of the antibody of the invention. The non-immunoglobulin region is typically a non-immunoglobulin moiety and may be an enzyme, a region derived from a protein having known binding specificity, a region derived from a protein toxin or indeed from any protein expressed by a gene, or a chemical entity showing inhibitory or blocking activity(ies) against WNV or Dengue virus proteins. The two regions of that modified antibody may be connected via a cleavable or a permanent linker sequence.

Preferably, the antibody of the invention is a human or animal immunoglobulin such as IgGI, lgG2, lgG3, lgG4, IgM, IgA, IgE or IgD carrying rat or mouse variable regions (chimeric) or CDRs (humanized or "animalized"). Furthermore, the antibody of the invention may also be conjugated to any suitable carrier known to one skilled in the art in order to provide, for instance, a specific delivery and prolonged retention of the antibody, either in a targeted local area or for a systemic application.

The term "humanized antibody" refers to an antibody derived from a non- human antibody, typically murine, that retains or substantially retains the antigen- binding properties of the parent antibody but which is less immunogenic in humans. This may be achieved by various methods including (a) grafting only the non-human CDRs onto human framework and constant regions with or without retention of critical framework residues, or (b) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues. Such methods are well known to one skilled in the art.

3. Compositions and vaccines

The carbohydrate-phosphorylcholine conjugate molecules of the present invention and the polyclonal or monoclonal antibodies may be used in many ways for the diagnosis, the treatment or the prevention of bacterial infection of the respiratory tract. In another embodiment, the present invention relates to an immunogenic composition for eliciting an immune response or a protective immunity against a bacterial infection of the respiratory tract. According to a related aspect, the present invention relates to a vaccine for preventing and/or treating a bacterial infection of the respiratory tract. As used herein, the term "treating" refers to a process by which the symptoms of a bacterial infection of the respiratory tract are alleviated or completely eliminated. As used herein, the term "preventing" refers to a process by which a bacterial infection of the respiratory tract is obstructed or delayed. The composition or the vaccine of the invention comprises a carbohydrate-phosphorylcholine conjugate molecule associated with a carrier protein, wherein said carbohydrate- phosphorylcholine conjugate molecule consists of phosphorylcholine-N-acetyl-D-galactosamine. Such a composition may further comprise a pharmaceutically acceptable carrier.

As used herein, the expression "a pharmaceutically acceptable carrier" means a vehicle for containing a carbohydrate-phosphorylcholine protein conjugate of the invention that can be injected into an animal host without adverse effects. Suitable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i. e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like. Further agents can be added to the composition and vaccine of the invention. For instance, the composition of the invention may also comprise agents such as drugs, immunostimulants (such as α-interferon, β-interferon, γ- interferon, granulocyte macrophage colony stimulator factor (GM-CSF), macrophage colony stimulator factor (M-CSF), interleukin 2 (IL2), interleukin 12 (IL12), and CpG oligonucleotides), antioxidants, surfactants, flavoring agents, volatile oils, buffering agents, dispersants, propellants, and preservatives. For preparing such compositions, methods well known in the art may be used.

The amount of carbohydrate-phosphorylcholine conjugate molecules of the invention present in the compositions or in the vaccines of the present invention is preferably a therapeutically effective amount. A therapeutically effective amount of the carbohydrate-phosphorylcholine conjugate molecule of the invention is that amount necessary to allow the same to perform their immunological role without causing, overly negative effects in the host to which the composition is administered. The exact amount of carbohydrate- phosphorylcholine conjugate molecules to be used and the composition/vaccine to be administered will vary according to factors such as the type of condition being treated, the mode of administration, as well as the other ingredients in the composition.

4. Methods of use In another embodiment, the present invention relates to methods for immunizing an animal against a bacterial infection of the respiratory tract or for treating and/or preventing a bacterial infection of the respiratory tract in an animal are provided. The method comprises the step of administering to the animal an effective amount of an immunogenic composition as defined above and/or an antibody of the invention.

The vaccine, antibody and composition of the invention may be given to an animal through various routes of administration. For instance, the composition may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally- acceptable diluents or solvents. They may be given parenterally, for example intravenously, intramuscularly or sub-cutaneously by injection, by infusion or per os. The vaccine and the composition of the invention may also be formulated as creams, ointments, lotions, gels, drops, suppositories, sprays, liquids or powders for topical administration. They may also be administered into the airways of a subject by way of a pressurized aerosol dispenser, a nasal sprayer, a nebulizer, a metered dose inhaler, a dry powder inhaler, or a capsule. Suitable dosages will vary, depending upon factors such as the amount of each of the components in the composition, the desired effect (short or long term), the route of administration, the age and the weight of the animal to be treated. Any other methods well known in the art may be used for administering the vaccine, antibody and the composition of the invention. The present invention is also directed to a method for detecting the presence or absence of a bacterial strain bearing a phosphorylcholine molecule in a sample, comprising the steps of: a) contacting the sample with an antibody characterized by the properties of the monoclonal antibodies as defined above 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).

It will be understood that a bacterial strain bearing a phosphorylcholine molecule is preferably one that can cause a respiratory tract infection such as Neisseria meningitidis, Steptococcus pneumoniae, Pseudomonas aeruginosa and Haemophilus influenzae.

It will be further understood that the term "sample" as used herein 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 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 and/or equipment. For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a ChoP molecule. 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 bacterial strain bearing a phosphorylcholine molecule in a sample, comprising: an antibody that binds specifically to a phosphorylcholine molecule (ChoP) located at the surface of said bacterial strain; a reagent to detect ChoP-antibody immune complex; - optionally a biological reference sample lacking a ChoP molecule that immunologically bind with said antibody; and optionally a comparison sample comprising a ChoP molecule 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 concerns a process for screening of an active molecule interacting with a phosphorylcholine molecule having the following steps:

1- contacting a phosphorylcholine molecule or a product containing a phosphorylcholine molecule with an active molecule to be tested;

2- adding a sample containing labelled antibodies according to the invention; and

3- revealing the presence or absence of forming complex between the product obtained in step 1 and the antibodies of step 2.

EXAMPLES

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:

Phosphorylcholine-carbohydrate protein conjugates efficiently induce hapten-specific antibodies which recognize both Streptococcus pneumoniae and Neisseria meningitidis: A potential multi-target vaccine against respiratory infections

GalNAc-ChoP specific antibodies which recognized S. pneumoniae and N. meningitidis

Phosphorylcholine (ChoP) is commonly expressed at the surface of pathogens of the respiratory tract, including Streptococcus pneumoniae and Neisseria meningitidis. We designed a synthetic hapten comprising ChoP and part of its native carrier structure in S. pneumoniae, i.e. Λ/-Acetyl-D- galactosamine (GaINAc). Protein conjugates of this hapten induced GaINAc- ChoP specific antibodies which recognized ChoP on both S. pneumoniae and N. meningitidis. GalNAc-ChoP could therefore lead to the rational design of a novel multipurpose vaccine against respiratory infections.

Synthesis of the bacterial hapten GalNAc-ChoP In S. pneumoniae, one or two ChoP molecules are linked at the position 6 of the /V-Acetyl-D-galactosamine residues within the repeating unit of the C- polysaccharide [-6)-β-D-Glcp-(1-3)-α-AATp-(1-4)-α-D-GalpNAc-(1-3)-β-D-

GaIpNAc-(I -1)-D-ribitol-5-P-(O-] (AAT = 2-acetamido-4-amino-2,4,6-trideoxy-D- galactose) ²²> 23 Fragments of this repeating unit have been synthesized for structural 24 and immunological ²⁵ studies. However none of them bear a ChoP residue.

To mimic the bacterial environment of the ChoP, the inventors designed a synthetic antigen comprising both ChoP and part of its native carrier structure i.e. the 6-substituted Λ/-Acetyl-D-galactosamine residue. The synthesis is summarized in Figure 3. Starting from 1 ,2,3,4,6-penta-O- acetyl-β-D-galactopyranoside ±, a succession of bromination, reductive dehalogenation, azidonitration, bromination, Koenigs-Knorr reaction with 6- (benzyloxycarbonyl)hexanol linker, reduction-acetylation of the azido group, and then selective deprotections/protections gave the 6-OH key intermediate 8 (9 steps, overall yield 14%).

The ChoP was then coupled at the position 6 of the GaINAc residue with the phosphoramidite method. By using ³¹P NMR, we could follow the completion of the different reactions, and the three following steps were sequentially performed one-pot. 8 was reacted with chloro 2-cyanoethyi [N₁N- diisopropyl)phosphoramidite in the presence of DIEA. After disappearance of the ³¹P signal assigned to the starting material (δ 182.41 ppm) and concomitant appearance of the new phosphoramidite signals (δ 150.63 and 150.21 ppm), phosphitylation was performed by adding choline tosylate to the reaction mixture together with tetrazole. Reaction was completed after 24h as shown by ³¹P NMR. The resulting new ³¹P signals account for the two phosphite triester diastereoisomers (δ 141.62 and 141.41ppm). After oxidation, the formation of the phosphotriester diastereoisomers 9 was indicated by new ³¹P signals in the spectrum (δ -1.96 and -2.03ppm) (3 steps, overall yield 31%). The carbohydrate moiety, the cyanoethyl group, and the linker were deprotected to afford the hapten ΛΛ_ (2 steps, overall yield 55%).

Synthesis of the GalNAc-ChoP protein conjugates

The conjugation of ϋ to the tetanus toxoid protein (TT) or the Alpaga serum Albumin (ASA) through activation with the EDC/SulfoNHS method yielded the expected GalNAc-ChoP protein conjugates. The ChoP:protein ratio of the conjugates TT-GalNAc-ChoP and ASA-GalNAc-ChoP were estimated at, respectively, 17:1 and 29:1 by a microphosphate assay ²⁶. Induction of GalNAc-ChoP specific antibodies which recognize S. pneumoniae and N. meningitidis

Biozzi mice were immunized either with TT-GalNAc-ChoP or with ASA- GalNAc-ChoP and the immune sera were tested for reactivity with the parental immunogen after boost injections (for further details see Annex C). Each animal developed either a strong anti-TT-GalNAc-ChoP response or a strong ASA- GalNAc-ChoP response (data not shown). For each immunogen, further experiments were performed with the serum showing the highest reactivity. The specificity of the serum for ChoP was assessed in inhibition assays using TT, ASA, GalNAc-ChoP, GaINAc alone or p-nitro-phenyl-ChoP. The 50% inhibition concentrations (IC₅₀) are shown in Table 1. The results suggest that immunization with both antigens generates antibodies specific for ChoP. Moreover these antibodies have a stronger avidity towards GalNAc-ChoP (IC₅O = 1x10^"6 M for TT-GalNAc-ChoP and IC₅₀ = 0.15x10^'6 M for ASA-GalNAc-ChoP) than towards ChoP alone (IC₅₀ = 20x10^"6 M for TT-GalNAc-ChoP and IC₅₀ = 7x10-⁶ M for ASA-GalNAc-ChoP).

To further characterize the quality of the murine anti-ChoP antibody response, immune sera were tested against heat-inactivated S. pneumoniae (Figure 1). The results suggest that both sera contain antibodies that are able to recognize S. pneumoniae. The specificity of these responses was assessed in inhibition assays with S. pneumoniae cell-wall polysaccharide (C-Ps), because this structure contains specifically ChoP linked to GaINAc. The IC₅₀ was about 35 ng/ml and 20 ng/ml for the TT-GalNAc-ChoP and for the ASA-GalNAc-ChoP sera, respectively (data not shown). Binding of the immune sera to N. meningitidis pilin-associated ChoP was tested by immunoblotting on whole bacterial cell extracts 27. As shown in figure 2, sera obtained by immunization with synthetic ASA-GalNAc-ChoP specifically recognized pilin-associated ChoP in the wild-type N. meningitidis strain but not in the pilin-defective mutant. Cross-reactive recognition of the S. pneumoniae uncapsulated ChoP-positive strain R6 (ATCC 39937) was also confirmed by these assays (data not shown). Conclusion

This study demonstrates the ability of ChoP-carbohydrate protein conjugates to raise a strong hapten-specific antibody response against two phylogenetically unrelated bacteria, S. pneumoniae and N. meningitidis, that express ChoP at their surface. The efficacy of the immune response is currently investigated in animal models.

Taken together, these results described herein highlight the potential of the ChoP-carbohydrate epitope as a model antigen for the rational design of a wide spectrum vaccine which could confer cross-protection against several pathogens of the respiratory tract. An additional advantage of such ChoP-carbohydrate conjugates vaccine is their safety. Indeed, since they closely mimic the native bacterial antigen, they should induce more specific immune response and prevent potential cross-reactivities with phosphatidylcholine-bearing macromolecules of the host.

EXAMPLE 2:

Preparation of a ChoP-GalNAc based MAG synthetic vaccine

Although the proteinic conjugates have appeared to be efficient in numerous cases, they present some disadvantages such as low density of the epitope, protein immunogeneity, among others. An objective of the present invention is to prepare a MAG (Multiple Antigenic Glycopeptide)-type synthetic immunogen in order to overcome such disadvantages. Previous research results on tumorous markers have revealed the efficiency of such vaccines ²⁹. On the MAG, ChoP-GalNAc epitope is tetravalently presented on the non-immunogenic skeleton (tri-lysine) in association with a CD4+ peptidic epitope stemming from the polio virus (Figure 4, R = Lys₂-Lys-βAla, n = 4). Subsequently to the glycopeptide dendrimer preparation, the present invention also proposes i) studying the immune response in mice, ii) preparing anti-ChoP Mab and iii) analyzing the "protection" efficiency of the antibodies.

EXAMPLE 3:

Analysis of the "protection" efficiency of monoclonal antibodies (Mab) anti- ChoP against S. pneumonia

The first phase of the infectious process comprises the adhesion of the pathogenic agent on the target cells. During this process, a molecule from the bacteria is specifically linked to a cellular receptor. Once the adhesion process has occured, the bacteria begin to in situ multiply, leading to mucous colonization. This crucial phase determines the next invasive process (epithelium and then endothelium crossing) leading to the bacteremia, such bacteremia being a prerequisite of meningitis. In this way, it has been demonstrated that for S. pneumonia, ChoP acts as an adhesine, by linking to the PAF (Platelet-Activating Factor) receptor of the respiratory mucous cells. It is known that ChoP is also present on the surface of some others pathogenic bacterium such as N. meningitis, H. influenzae and P. aeruginosa. Thanks to the two protective Mab (D18-3 and 13-1) of the present invention used against N. meningitis, the present invention also concerns the study of the Mab of the invention efficiency to prevent infections caused by S. pneumonia.

EXAMPLE 4:

Synthesis of linear glycopeptid based on ChoP-GalNAc (Figure 4, R = OH, n= 1)

Dendrimere-type structures (MAG) are relatively complex, and may lead to numerous secondary reactions. Furthermore, monitoring the reactions of such molecules is difficult. The inventors have set up the coupling reaction on a linear peptidic structure that is simpler, in order to further link it to the MAG dendrimere. The inventors also functionalized the ChoP-GalNAc using a thioacetyl group. At the same time, the peptide modified on its N-termina! position by a maleimide group was prepared. After deprotection in situ, the coupling reaction between the two fragments was monitored and optimized using HPLC chromatography. The coupling reaction leads to the linear glycopeptide ChoP-GalNAc (Figure 4, R = OH, n = 1). After purification, the product is characterized by amino-acids analysis and mass spectrometry.

EXAMPLE 5:

Analysis of "protection" efficiency of anti-ChoP Mab against S. pneumonia

A series of experiments on the passive protection were performed against a challenge of S. pneumonia Pn40 (serotype 14) ChoP+.

This strain has the advantage of being representative of the dominant sero- group in the invasive pneumococcal diseases. From an experimental point of view, this strain has the disadvantage of not infecting immunocompetent mice by the respiratory tracts. The present experimental protocol consists in first inducing a leucopeny in mice by injecting an infra-toxic maximum dose of cyclophosphamide (200 mg/kg), four days before the challenge, this period corresponding to a maximum leucocitary depletion phase (until D7). In contrast to meningococcal diseases, against which protective antibodies act essentially by inducing a complement-dependent bactericidy and thus justifying the interest in an IgM, pneumococcy diseases involve dependent- antibodies mechanisms which are essentially dominated by the immunophagocytosis. IgG intervention, linking Fc-gamma receptors to the phagocytes surface, is at this point essential for bacterial clearance. An IgM recognizing an antigen on the pneumococcus surface, such as anti-ChoP IgM of the present invention (Monoclonal antibody D18-3), is assumed to inhibit bacterial attachment. Therefore, intranasal administration of the antibodies is performed in order to inhibit interactions between the pneumococcus and the receptors on the respiratory epithelium.

In these experimental conditions, two independent experiments indicate that D18-3 (anti-ChoP IgM) antibody fixing is able to reduce the pulmonary infectious load of a log™ relative to those of control mice treated by a control IgM.

These first results clearly demonstrate the importance of ChoP-based vaccines for targeting two different bacteria, such as S. pneumoniae and N. meningetidis.

The present invention represents a starting point for the rational conception of broad-range vaccines, leading to a crossed protection against several pathogens of the respiratory tract. Particularly, one of these research goals is to combat nosocomial pneumopathy.

IC₅₀ (M) Competitor

Immunogen TT ASA GaINAc GalNAc-ChoP p-nitro-phenyl-ChoP

TT-GalNAc-ChoP >10^"4 ND >10^"4 HO^"6 20.10-⁶

ASA-GalNAc-ChoP ND > κr⁴ >10^"4 0.15 .10^"6 7.10^"6

Table 1: ELISA binding profile of immune sera to coated S. pneumoniae. The specificity of the serum for ChoP was assessed by measuring the 50% inhibition concentration (IC₅₀) using different antigens carrying or not ChoP. ND : not determined. References

1. Lipsitch, M. Bacterial vaccines and serotype replacement: lessons from Haemophilus influenzae and prospects for Streptococcus pneumoniae. Emerg. Infect. Dis. 1999, 5, 336-345.

2. Swartley, J. S.; Marfin, A. A.; Edupuganti, S.; Liu, L. J.; Cieslak, P.; Perkins, B.; Wenger, J. D.; Stephens, D. S. Capsule switching of Neisseria meningitidis. Proc. Natl. Acad. ScL USA 1997, 94, 271-276.

3. Neu, H. C. The crisis in antibiotic resistance. Science 1992, 257, 1064-1073. 4. Casadevall, A.; Scharff, M. Return to the past: the case for antibody-based therapies in infectious diseases. CHn. Infect. Dis. 1995, 21, 150-161. 5. Trolle, S.; Andremont, A.; Fattal, E. Towards a multipurpose vaccination using phosphorylcholine as a unique antigen. STP Pharma. Sci. 1998, 8, 19- 30. 6. Cundell, D. R.; Gerard, N. P.; Gerard, C; Idanpaan-Heikkila, I.; Tuomanen, E. I. Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor. Nature 1995, 377, 435-438.

7. Swords, W. E.; Buscher, B. A.; Ver Steeg Ii, K.; Preston, A.; Nichols, W. A.; Weiser, J. N.; Gibson, B. W.; Apicella, M. A. Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor. MoI. Microbiol. 2000, 37, 13-27.

8. Rosenow, C; Ryan, P.; Weiser, J. N.; Johnson, S.; Fontan, P.; Ortqvist, A.; Masure, H. R. Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae. MoI.

Microbiol. 1997, 25, 819-829.

9. Lysenko, E.; Richards, J. C; Cox, A. D.; Stewart, A.; Martin, A.; Kapoor, M.; Weiser, J. N. The position of phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae affects binding and sensitivity to C-reactive protein- mediated killing. MoI. Microbiol. 2000, 35, 234-245.

10. Musher, D. M. How contagious are common respiratory tract infections? New Engl. J. Med. 2003, 348, 1256-1266. H. Briles, D. E.; Forman, C; Grain, M. Mouse antibody to phosphocholine can protect mice from infection with mouse-virulent human isolates of

Streptococcus pneumoniae. Infect. Immun. 1992, 60, 1957-1962. 12.Yother, J.; Forman, C; Gray, B. M.; Briles, D. E. Protection of mice from infection with Streptococcus pneumoniae by anti-phosphocholine antibody.

Infect. Immun. 1982, 36, 184-188. 13. Brown, M.; Schumacher, M. A.; Wiens, G. D.; Brennan, R. G.; Rittenberg, M.

B. The structural basis of repertoire shift in an immune response to phosphocholine. J. Exp. Med. 2000, 191, 2101-2112. 14.Trolle, S.; Chachaty, E.; Kassis-Chikhani, N.; Wang, C; Fattal, E.; Couvreur,

P.; Diamond, B.; Alonso, J.-M.; Andremont, A. Intranasal immunization with protein-linked phosphorylcholine protects mice against a lethal intranasal challenge with Streptococcus pneumoniae. Vaccine 2000, 18, 2991-2998. 15. Fattal, E.; Pecquet, S.; Couvreur, P.; Andremont, A. Biodegradable microparticles for the mucosal delivery of antibacterial and dietary antigens.

Int. J. Pharm. 2002, 242, 15-24. 16.Wallick, S.; Claflin, J. L; Briles, D. E. Resistance to Streptococcus pneumoniae is induced by a phosphocholine-protein conjugate. J. Immunol.

1983, 130, 2871-2875. 17. Kenny, J. J.; Guelde, G.; Fischer, R. T.; Longo, D. L Induction of phosphocholine-specific antibodies in X-linked immune deficient mice: in vivo protection against a Streptococcus pneumoniae challenge. Int. Immunol.

1993, 6, 561-568.

18. Fischer, R. T.; Longo, D. L; Kenny, J. J. A novel phosphocholine antigen protects both normal and X-linked immune deficient mice against

Streptococcus pneumoniae. Comparison of the 6-O-phosphocholine hydroxyhexanoate-conjugate with other phosphocholine-containing vaccines.

J. Immunol. 1995, 154, 3373-3382.

19.Serino, L.; Virji, M. Phosphorylcholine decoration of lipopolysaccharide differentiates commensal Neisseriae from pathogenic strains: identification of licA-type genes in commensal Neisseriae. MoI. Microbiol. 2000, 35, 1550-

1559. 20. Warren, M. J.; Jennings, M. P. Identification and characterization of pptA : a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis. Infect. Immun. 2003, 71, 6892-6898.

21.Schweda, E. K.; Brisson, J. R.; Alvelius, G.; Martin, A.; Weiser, J. N.; Hood, D. W.; Moxon, E. R.; Richards, J. C. Characterization of the phosphocholine- substituted oligosaccharide in lipopolysaccharides of type b Haemophilus influenzae. Eur. J. Biochem. 2000, 267, 3902-3913.

22. Fischer, W.; Behr, T.; Hartmann, R.; Peter-Katalini, J.; Egge, H. Teichoic acid and lipoteichoic acid of Streptococcus pneumoniae possess identical chain structures. A reinvestigation of teichoid acid (C polysaccharide). Eur. J.

Biochem. 1993, 215, 851-857.

23. Karlsson, C; Jansson, P. E.; Skov Sorensen, U. B. The pneumococcal common antigen C-polysaccharide occurs in different forms. Mono-substituted or di-substituted with phosphocholine. Eur. J. Biochem. 1999, 265, 1091- 1097.

24.Qin, H.; Grindley, T. B. Determination of the configuration of ribitol in the C- polysaccharide of Streptococcus pneumoniae using a synthetic approach. Can. J. Chem. 1999, 77, 481-494.

25. Smid, P.; de Zwart, M.; Jδrning, W. P. A.; van der Marel, G. A.; van Boom, J. H. Stereoselective synthesis of a tetrameric fragment of Streptococcus pneumoniae type 1 containing an α-linked 2-acetamido-4-amino-2,4,6- trideoxy-D-galactopyranose (SUGp) unit. J. Carbohydr. Chem. 1993, 12,

1073-1090.

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27. Kolberg, J.; Hoiby, E. A.; Jantzen, E. Detection of the phosphorylcholine epitope in streptococci, Haemophilus and pathogenic Neisseriae by immunoblotting. Microb. Pathog. 1997, 22, 321-329.

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A N N E X A

Cold water was added to the reaction mixture and the watα phase extracted vήfh CHjCfe. "Che organic phase was veasfaed with, aq 5% NaHCOj, HzO, and evaporated. Jhe residue was purified βo silic* gel using solvent A as eluent (from 100/0 to 9SΛt). The β aπoπαer % wis fiist elmed(i.52g, 59%) followed by the o. anoπief (0.52& 20%).

¹H NMR (CDCJb) i 87.37 (m, 5HE, Ph), 5.72 (d, IH, NH Ac, Λw#=S.^ Hz), 5-27 (4 IH. H- A), 5.20 (da, IH, H-3, JJy=I 1.2 Sz, ,/_3<_=2.83 Hz), 5.04- C-H, CBJM), 4.78 (m, UEI, Ni-CH₂), 4.58 (d, lH H-I, J_\Λ~4.$S Hz), 4.12-4.02 (m, 2H, H-6, H-SO, 3.89 (Jn,^' IH, H-2), 3.89-3.74 (m, 2B, ICHaO, K-S), 3.41 (m, IH, iCEfeO), 3.12 (m, 2H, CfiϊNH), 2ΛXS, 1.96, 1.91, 1.87 (4s, 12H, CEC₃ Ao), 1.55-1.20 (m, SH, CH₂ spacer) i ^UC NMR (CDCl₃) : 5 170.82, 170,69 (CO Ao), 156.95 (OCONH), 137.08 (C pha), 128.92-128.29 (CH Bus), 101.13 (C-I), 70.96 (C-5). 70.36 (C-3), 69.77 (CH₃O). 67-22 (C-4), 66.96 (CHtaPhe), 61.85 (C-6% 52.06 (C-2), 40,97 (CHiNH), 30.13, 29.3S₃ 2631, 25.49 (4C CH₁ qiac«), 23.81, 21.10, 21.08 (CHj Ac). FABMS for C₂₈H_WNIOII : (caiβA 580.26) τaJz 58)1.22 CM+HJ*.

(6-amlnohϊχyt) 2_HicεtaiΛida*2-dC4>^6Η-phθφhoctιotinβ~β-J>-gaLact(}pyrιinό$iώ>) or

[GalNAc-ChaPJU.

To a solution of § (2.28g, 4.24πnnol) in CBtCN (57ml) were added dusβpropyteUjylaniiαe (4.43ad, 6 βqulv.) and chloro 2-cyaαoethyl (Λy^-diieopropyl^liosphDraπJidite (1.14-oL, 1.2 equiv.) (³¹I¹ : 6 182.4 ippm) at room tenπpeiatαie imder argon. The reaction was complete in

A N N E X B

°C), ppm. aa d

Claims

1. A hemi-synthetic conjugate molecule having the following formula:

wherein X and Y are optionally present and wherein:

X is a spacer group;

Y is chosen from biotin or a derivative thereof or a carrier protein; n >1.

2. A hemi-synthetic conjugate as defined in claim 1 containing a pharmaceutically acceptable salt.

3. The hemi-synthetic conjugate molecule according to claim 1 , wherein said carrier protein is a protein or a peptide and fluorescent derivatives thereof.

4. The hemi-synthetic conjugate molecule according to claim 1 , wherein said carrier protein is a bacterial protein.

5. The hemi-synthetic conjugate molecule according to claim 4, wherein said bacterial protein is chosen from tetanus toxoid or alpaga serum albumin.

6. The hemi-synthetic conjugate according to claim 1, wherein said spacer group is chosen from an aminoacid or a carbohydrate.

7. The hemi-synthetic conjugate molecule according to claim 1 , wherein X is comprised between -(CH₂) 2NH- to -(CH₂)₄₀NH-.

8. The hemi-synthetic conjugate molecule according to claim 6, wherein X is - 5 -(CHz)₆NH-.

9. The hemi-synthetic conjugate molecule according to claim 1 having the following formula:

0

10. The hemi-synthetic conjugate molecule according to claim 9 wherein the carrier protein is tetanus toxoid and n is 17.

11. The hemi-synthetic conjugate molecule according to claim 10 wherein the 5 carrier protein is alpaga serum albumin and n is 29.

12. The hemi-synthetic conjugate molecule according to claim 1 having the following formula:

>.O

13. An immunogenic composition against bacterial infection of the respiratory tract comprising a hemi-synthetic conjugate molecule according to any one of claims 1 to 12.

14. A purified polyclonal or monoclonal antibody capable of specifically binding to a hemi-synthetic conjugate molecule according to any one of claims 1 to 12.

15. A purified monoclonal antibody as defined in claim 14, secreted by a hybridoma designated 13-1 and deposited at the CNCM, 25 rue du Docteur

Roux, 75724 Paris Cedex 15, under accession no. I-3248.

16. A purified monoclonal antibody as defined in claim 14, secreted by a hybridoma designated D18-3 and deposited at the CNCM, 25 rue du Docteur Roux, 75724 Paris Cedex 15, under accession no. I-3286.

17. Use of a synthetic conjugate molecule according to any one of claims 1 to 12, an immunogenic composition of claim 13, an antibody as defined in any one of claims 14 to 16 for treating or preventing bacterial infection of the respiratory tract.

18. A method for treating and/or preventing a bacterial infection of the respiratory tract in an animal, the method comprising the step of administering to the animal an effective amount of a hemi-synthetic conjugate molecule according to any one of claims 1 to 12, an immunogenic composition of claim 13, an antibody as defined in any one of claims 14 to 16.

19. The method of claim 18, wherein said bacterial infection is caused by a bacterium selected from the group consisting of Neisseria meningitidis,

Steptococcus pneumoniae, Pseudomonas aeruginosa and Haemophilus influenzae.

20. A method for immunizing an animal against a bacterial infection of the respiratory tract, comprising the step of administering to the animal an effective amount of an immunogenic composition as defined in claim 13.

21. A method for preparing a hemi-synthetic conjugate molecule having the following formula:

wherein X and Y are optionally present and wherein:

X is a spacer group;

Y is chosen from biotin or a derivative thereof or a carrier protein; n >1 ; comprising the steps of: a) reacting a molecule of structure (IV)

(IV) wherein R1 is a leaving group and PGi, PG2 and PG₃ are primary or secondary alcohol protecting groups; with a molecule of structure 5

V-XPG₄ (V) wherein PG₄ is a protecting group and V is a functional group capable of reacting with R1; b) reducing the 2-azido group to a 2-amino group; c) protecting the amino group obtained in step b; d) removing PG₁ from the primary oxygen; e) reacting the product obtained in step d) with a phosphorylating agent and choline f) removing PG₂ ,PG₃ and PG₄ ; g) reacting the product obtained in step f) with one or more equivalents of a Y or a derivative thereof, and optionally separating the product obtained from the reacting medium.

22. A method for preparing the hemi-synthetic conjugate molecule according to claim 21 wherein said molecule of structure (IV) is 3,4,6-tri-O-acetyl-2- azido-2-deoxy-β-D-galactopyranosyl bromide.

23. A method for preparing the hemi-synthetic conjugate molecule according to claim 21 wherein said molecule of structure (V) is 6- (benzyloxycarbonylamino)-i-hexanol.

24. A method for preparing the hemi-synthetic conjugate molecule according to claim 22 wherein the phosphorylating agent is 2-cyanoethyl [N, N- diisopropyl)phosphoramidite.

25. A method for detecting the presence or absence of a bacterial strain bearing a phosphorylcholine molecule in a sample, comprising the steps of: a) contacting the sample with an antibody characterized by the properties of the monoclonal antibodies according to any one of claims 14 to 16 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).

26. The method of claim 24, wherein the bacterial strain is selected from the group consisting of Neisseria meningitidis, Steptococcus pneumoniae,

Pseudomonas aeruginosa and Haemophilus influenzae.

27. A diagnostic kit for the detection of a bacterial strain bearing a phosphorylcholine molecule in a sample, comprising: an antibody that binds specifically to a phosphorylcholine molecule

(ChoP) located at the surface of said bacterial strain; a reagent to detect ChoP-antibody immune complex; optionally a biological reference sample lacking a ChoP molecule that immunologically bind with said antibody; and - optionally a comparison sample comprising a ChoP molecule 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.

28. The diagnostic kit of claim 27, wherein the antibody is a monoclonal antibody secreted by a hybridoma deposited under No. I-3248 or I-3286 at the CNCM, 25 rue du Docteur Roux, 75724 Paris Cedex 15.

29. A process for screening of an active molecule interacting with a phosphorylcholine molecule having the following steps :

1) contacting a phosphorylcholine molecule or a product containing a phosphorylcholine molecule with an active molecule to be tested;

2) adding a sample containing labelled antibodies according to any one of claims 14 to 16; and

3) revealing the presence or absence of forming complex between the product obtained in step 1 and the antibodies of step 2.