CA2472818A1 - Carbohydrate-phosphorylcholine conjugate molecules and their use in vaccinal, therapeutic and diagnostic applications - Google Patents

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 andlor preventing a bacterial infection of the respiratory tract. Furthermore, the present invention is concerned with compositions, vaccines and methods for providing an immune response andlor 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 satisfactoryl ~ 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 {3-lactams becomes a matter of concern3. 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 (Haemophitus, 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

2 receptor (PAF-receptor), thus mimicking endogenous processes of cellular signaling 8. 7. Furthermore, several proteins of S. pneumoniae, which are important for its virulence, are attached to the cell wall via Chop $. 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 g.
Therefore, Chop is an attractive target for the development of immunotherapies directed against these major bacterial infections of the respiratory tract 10. It has been shown previously that Chop-specific T15-idiotype antibodies, despite their low affinity, are protective when used in passive immunizations 1 ~ ~ 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 IV. meningitidis, Chop is linked to the glycoproteins of the pill, but its molecular carrier (saccharide or aminoacid) has not yet been clearly identified 19~ 20, In H. influenzae, Chop is grafted to the surface

3 lipopolysaccharides 2~ . In S. pneumoniae, Chop is part of the C-polysaccharide (teichoic acid) and F-antigen (lipoteichoic acid) 22, 2~n 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 imrnunotherapies 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 andlor 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 (GaINAc-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:

O~~ /O-~h OH O~ ~O-CHZCH2N(CH3)s HO
O X Y
N HAc 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 ~.
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 andlor 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 rnolecuie 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 bacteria!
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;
- optionaly a biological reference sample lacking a Chop molecule that imrnunologically bind with said antibody; and optionaly 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 : ELISA measurement of heat-inactivated S. pneumoniae cells reactivity for marine anti-TT-GaINAc-Chop {() and ASA-GaINAc-Chop (() immune sera. ( O) and {o) : con-esponding sera of non immunized animals.
Figure 2 . Immunoblot of anti-ASA-GaINAc-Chop immune serum with N.
meningitidis extracts A wild-type pilin-positive {Pill+), Chop-positive serogroup C strain, was tested in parallel with its isogenic pilE mutant (devoid of pilin, Pili-}. 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 3a : Synthesis of GaINAc-Chop aReagents and conditions : (a} 30% HBr in CH3COOH, RT, 1 h30 (99%) ; (b) Zn, NM1, dry AcOEt, reflux, 2h ; (c) (NH4}2Ce(NO3)6, NaN3, CH3CN, -25°C, 3h (2 steps, 44%) ; (d) Liar, dry CH3CN, RT, 3h {99%); (e) AgOTf, collidine, dry CH2CI2, HO-(CH2)2-NHZ -40°C, 15 h ; (f) NaBH~, H3B03, NiClz, EtOH, RT, 1 h30 ; (g) Ac20, EtOH, RT, 1 h (3 steps, 54%) ; (h) MeONa, MeOH, RT, 1 Omin ; {i) DMTrCI, Pyr, RT, 2h ; (j) Ac20, Pyr, RT, 3h ; (k) 2% ABS in CH2CIZ/MeOH:713, 0°C, 2min (4 steps, 60%) ; (I} chloro 2-cyanoethyl (N,N diisopropyl)phosphoramidite, DIEA, dry CH3CN, RT, 20min ; (m) Choline+ Tos , tetrazole, CH3CN, RT, 24h ; (n) l2IPyr/THF/H20, CH3CN, RT, l5min (3 steps, 31 %) ; (o) MeONa, MeOH, RT, 15min ; (p) H2, PdIC, EtOH, RT, 1 h (2 steps, 55%).
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-GaINAc} 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-GaINAc of the present invention. The term "immune response" is to be understood as including a humoral response andlor 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 meningifidis, Steptococcus pneumoniae, Pseudomonas aeruginosa or Heamophilus influenzae. Thus, an anima! 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 is concerned with a hemi-synthetic conjugate molecule having the following formula:

o\\
p +
OH O~ ~O-CH2CH2N(CH3y3 HO
O X Y
NHAc 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 ~.
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 bacterial protein, such as tetanus toxoid or alpaga serum albumin.
In a particular embodiment of the synthetic conjugate molecule of formula (I), the spacer group X is -(CH2)6NH-.

In a specific embodiment of the synthetic conjugate molecule of formula(1), when Y is a carrier protein, it preferentially forms a molecule with formula (ll):
O,, i O _ rp, +
OH O O-CH2CH2N(CH3)a HO
O (CH2)B NH carrier protein NHAc n ft 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-GaINAc-Chop and ASA-GaINAc-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) o,, ~o_ p +
OH O~ ~O-CH2CHZN(CH3)s HO O p--(CH2)6 NHC~S~g~./NHCO-Biotin NHAc 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}
PG20 OPG~

(1V) 1a wherein R1 is a leaving group and PG~, PG2 and PG3 are primary or secondary alcohol protecting groups, with a molecule of formula(V) (V) wherein PGa 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 PG~ 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-~i-D-galactopyranosyl bromide, whereas the molecule of formula(V) is 6-(benxyloxycarbonylamino)-1-hexanol.
In yet another preferred aspect of the method of the invention, the phosphorylating agent is 2-cyanoethyl (N,N diisopropyl)phosphoramidite.
As one in the art may appreciate, Figure 3 shows a synthesis scheme far 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(I).
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 specifcally 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.
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-GaINAc-Chop and ASA-GaINAc-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 al., In Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, 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'', and preferably of at least about 10' M-' and even more preferably 10$ M'' to 10'° M-'. 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, proteo(ytic 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 ef al. (Q J Nuc! Med 9998;
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 ef al. 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 Ill 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 IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE or IgD carrying rat or mouse variable regions (chimeric) or CDRs (humanized or "animaiized").
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-phosphoryEcholine 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 comprises an 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 a-interferon, ~i-interferon, y-interferon, granulocyte macrophage colony stimulator factor (GM-CSF), macrophage colony stimulator factor (M-CSF), interfeukin 2 (IL2), interleukin 12 (1L12), 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 andlor preventing a bacteria( 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 andlor 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 meningifidis, Steptocaccus pneumoniae, Pseudomonas aeruginosa and Naernophilus 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 andlor 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 phosphorylchaline molecule in a sample, comprising:
- an antibody that binds spec~cally to a phosphorylcholine molecule (Chop) located at the surface of said bacterial strain;
- a reagent to defect Chop-antibody immune complex;
- optionaly a biological reference sample lacking a Chop molecule that immunologically bind with said antibody; and - optionaly 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 far 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.
EXAMPLE
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:
Phosphorylchotine-carbohydrate protein conjugates efficiently induce hapten-specific antibodies which recognize both Streptococcus pneumoniae and Neisseria meningitides: A potential multi-target vaccine against respiratory infections o~ ~o_ P
Hp O~ ~O-CH2CH2N(CH3~ ~ GaINAc-Chop specific antibodies O which recognized H0~~0-(CH2)s-NH Protein S. pneumoniae and N. meningitides AcHN
Phosphorylcholine (Chop) is commonly expressed at the surface of pathogens of the respiratory tract, including Strepfococcus pneumoniae and Neisseria meningifidis. We designed a synthetic hapten comprising Chop and part of its native carrier structure in S. pneumoniae, i.e. N-Acetyl-D-galactosamine (GaINAc). Protein conjugates of this hapten induced GaINAc-Chop specific antibodies which recognized Chop on both S. pneumoniae and N. meningifidis.
GaINAc-Chop could therefore lead to the rational design of a novel multipurpose vaccine against respiratory infections.
Synthesis of the bacterial hapten GaINAc-Chop In S. pneumoniae, one or two Chop molecules are linked at the position 6 of the N-Acetyl-D-galactosamine residues within the repeating unit of the C-polysaccharide [-6)-~-D-Glcp-(1-3)-a-AATp-(1-4)-a-D-GaIpNAc-(1-3)-[i-D-GaIpNAc-(1-1 )-D-ribitol-5-P-(Cl-] (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 N Acetyl-D-galactosamine residue.
The synthesis is summarized in Figure 3. Starting form 1,2,3,4,6-penta-O-acetyl-a-D-galactopyranoside 1, a succession of bromination, reductive dehalogenation, azidonitration, brornination, Koenigs-Knorr reaction with 6-{benzyloxycarbonyl)hexanol linker, reduction-acetylation of the azido group, and then selective deprotectionslprotections 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 3~P 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-cyanoethyl (N,N-diisopropyl)phosphoramidite in the presence of DIEA. After disappearance of the 3'P signal assigned to the starting material (8 182.41 ppm) and concomitant appearance of the new phosphoramidite signals (b 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 3~P NMR. The resulting new 3~P signals account for the two phosphite triester diastereoisomers (S
141.62 and 141.41 ppm). After oxidation, the formation of the phosphotriester diastereoisomers 9 was indicated by new 3~P signals in the spectrum (8 -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 11 (2 steps, overall yield 55%).
Synthesis of the GaINAc-Chop arotein coniu aq tes The conjugation of 11 to the tetanus toxoid protein (TT) or the Alpaga serum Albumin (ASA) through activation with the EDC/SuIfoNHS method yielded the expected GaINAc-Chop protein conjugates. The ChoP:protein ratio of the conjugates TT-GaINAc-Chop and ASA-GaINAc-Chop were estimated at, respectively, 17:1 and 29:1 by a microphosphate assay 26.

Induction of GaINAc-Chop specific antibodies which recognize S. ~oneumoniae and N. meninaitidis Biozzi mice were immunized either with TT-GaINAc-Chop or with ASA-GaINAc-Chop and the immune sera were tested for reactivity with the parental immunogen after boost injections (for further details see Annex C). Bach animal developed either a strong anti-TT-GaINAc-Chop response or a strong ASA-GaINAc-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, GaINAc-Chop, GaINAc alone or p-nitro-phenyl-Chop. The 50% inhibition concentrations (ICSO) 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 GaINAc-Chop (IC5o = 1x10'6 M for TT-GaINAc-Chop and ICSO = 0.15x10-6 M for ASA-GaINAc-Chop) than towards Chop alone (IC5o = 20x10'6 M for TT-GaINAc-Chop and ICSO = 7x10'6 M for ASA-GaINAc-ChoP).
To further characterize the quality of the marine 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-wal( polysaccharide (C-Ps), because this structure contains specifically Chop linked to GaINAc. The ICSO was about 35 ng/ml and 20 nglml for the TT-GaINAc-Chop and for the ASA-GaINAc-Chop sera, respectively (data not shown).
Binding of the immune sera to N. meningitides pilin-associated Chop was tested by immunoblotting on whole bacterial cell extracts 2~. As shown in figure 2, sera obtained by immunization with synthetic ASA-GaINAc-Chop specifically recognized pilin-associated Chop in the wild-type N. meningifidis strain but not in the pilin-defective mutant. Cross-reactive recognition of the S. pneumoniae encapsulated Chop-positive strain R6 (ATCC 3993T) 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. meningitides, that express Chop at their surtace. 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.
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J. Immunol. 1995, ?54, 3373-3382.

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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.; Jorning, W. P. A.; van der Marel, G. A.; van Boom, J.
H. Stereoselective synthesis of a tetramerie fragment of Streptococcus pneumoniae type 1 containing an a-linked 2-acetamido-4-amino-2,4,6-trideoxy-D-galactopyranose (SUGp) unit. J. Carbohydr. Chem. 1993, 92, 1073-1090.

26. Bartlett, G. R. Phosphorus assay in column chromatography. J. Biol. Chem.
1958, 234, 466-468.

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.

IC~a (M) Competitor Immunogen TT ASA GaINAc GaINAc-Chop p-vitro-phenyl-Chop TT-GaINAc-Chop >10'~ ND >10'4 1.10'6 20.10'6 ASA-GaINAc-Chop ND > 10'~ >10'4 0.15 .10'6 7.10'6 Table 1: ELISA binding profile of immune sera to boated S. pneumoniae. The specificity of the serum for Chop was assessed by measuring the 50% inhibition concentration (ICSo) using different antigens carrying or not Chop. ND : not determined.
Figure 1 2,S

i;
~, I,5 a O

0,5 IIS00 1/1000 112000 114000 1/8000 1/16000 i/32000 Sera Dilutions Figure 2 N. menihgitidis KCB Pih + Pih Y
X ,.~f Y,~~ .~ y, '.
a ~ ~~~ ~g~ ~~~~..
25.0 ~. ~ .. x# i ~h Non-immunised 16.5 ~ ~,~~~
6 5- ~ - mouse serum 25.0 ~y~ ~ ,, ~~~~~ Murine anti-ASA-GaINAc-Chop ' ~~ r ~
16'5- ~~M , ~~ ~s ~ ~ ~a~~ ~'~ immune serum ~~~~~; <3~s:
25.0 -16.5- Rabbit anti-pilin IgG
6.5-25.0 -'r Anti-Chop (TEPC-15) I6.5- ' ~y monoclonal antibod 6.5 - Y

Figure 3a Ac0 OAc Acp OAc Acp OAc O (b) O (~) O
A~ Rt Ac0 ~ A~ N3 Ac0 R~
A R1=a- and p-ONO2 1_ R~=OAc (d) .~y (e) ~ 2_ R~=Br (e) 5_ R1=a-Br -NH2 S Ri=a- and (i-O-(CHz)s (~~(s) R30 p ~ O CH2CHZN(CHs)s AcO ORS
(~)~(m)~tn) O O-(CHz)s-NHZ
R30~~0-(CHz)s-NHRz '~ Ac0 AcHN
AcHN
T R1=AC
9_ R~=O-(CHz)zCN, Rz=Z~ Rs=Ac, Tos (h)I(iNGN(k) 8 R~=H
(c) ~ 10 R~=O , Rz=Z, R3=H
(p) ~ 11 R~=O', Rz=H, Rs=H

Claims (26)

28

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 bacterial protein.

4. The hemi-synthetic conjugate molecule according to claim 3, wherein said bacterial protein is chosen from tetanus toxoid or alpaga serum albumin.

5. The hemi-synthetic conjugate molecule according to claim 1, wherein X is comprised between -(CH2)2NH- to -(CH2)40NH- and preferably is -(CH2)6NH-.

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

7. The hemi-synthetic conjugate molecule according to claim 6 wherein the carrier protein is tetanus toxoid and n is 17.

8. The hemi-synthetic conjugate molecule according to claim 7 wherein the carrier protein is alpaga serum albumin and n is 29.

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

10. An immunogenic composition against bacterial infection of the respiratory tract comprising a hemi-synthetic conjugate molecule according to any one of claims 1 to 9.

11. A purified polyclonal or monoclonal antibody capable of specifically binding to a hemi-synthetic conjugate molecule according to any one of claims 1 to 9.

12. A purified monoclonal antibody as defined in claim 11, secreted by a hybridoma designated 13-1.

13. The hybridoma according to claim 12 deposited at the CNCM under accession no. I-3248.

14. Use of a synthetic conjugate molecule according to any one of claims 1 to 9, an immunogenic composition of claim 10, an antibody as defined in claim 11 or 12, or the hybridoma of claim 13 for treating or preventing bacterial infection of the respiratory tract.

15. 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 9, an immunogenic composition of claim 10, an antibody as defined in claim 11 or 12, or the hybridoma of claim 13.

16. The method of claim 15, wherein said bacterial infection is caused by a bacterium selected from the group consisting of Neisseria meningitidis, Steptococcus pneumoniae, Pseudomonas aeruginosa and Haemophilus influenzae.

17. 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 10.

18. 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) wherein R1 is a leaving group and PG1, PG2 and PG3 are primary or secondary alcohol protecting groups;
with a molecule of structure 5 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.

19. A method for preparing the hemi-synthetic conjugate molecule according to claim 18 wherein said molecule of structure (IV) is 3,4,6-tri-O-acetyl-2-azido-2-deoxy-.beta.-D-galactopyranosyl bromide.

20. A method for preparing the hemi-synthetic conjugate molecule according to claim 18 wherein said molecule of structure (V) is 6-(benzyloxycarbonylamino)-1-hexanol.

21. A method for preparing the hemi-synthetic conjugate molecule according to claim 19 wherein the phosphorylating agent is 2-cyanoethyl (N,N-diisopropyl)phosphoramidite.

22. A method for detecting the presence or absence of a bacterial strain bearing a phospharylcholine 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 claims 11 or 12 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).

23. The method of claim 22, wherein the bacterial strain is selected from the group consisting of Neisseria meningitidis, Steptococcus pneumoniae, Pseudomonas aeruginosa and Haemophilus influenzae.

24. 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.

25. The diagnostic kit of claim 24, wherein the antibody is a monoclonal antibody secreted by a hybridoma deposited under No. I-3248 at the CNCM.

26. A process for screening of an active molecule interacting with a phosphorylcholine molecule having the following steps :
4) contacting a phosphorylcholine molecule or a product containing a phosphorylcholine molecule with an active molecule to be tested;
5) adding a sample containing labelled antibodies according to claims 11 to 12; and 6) revealing the presence or absence of forming complex between the product obtained in step 1 and the antibodies of step 2.