CA2475736A1 - Vibrio cholerae lps detoxified derivatives and immunogenic compositions containing them - Google Patents
Vibrio cholerae lps detoxified derivatives and immunogenic compositions containing them Download PDFInfo
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Abstract
The presence of a free amino group on the D-glucosamine residue of the detoxified lipopolysaccharide (LPS) of Vibrio cholerae is exploited for the site-specific derivatization of this polysaccharide. The invention allows the preparation of V.
cholerae LPS derivatives useful for the immuno-detection. The invention also provides a convenient way to prepare detoxified LPS of V. cholerae (and especially V. cholerae O1, serotype Inaba)-immunogenic carrier conjugates. Thanks to the single modification introduced following the adopted strategy, all derivatives of the detoxified LPS can be fully analyzed in term of purity and identity. This leads to an improved characterization of the conjugates and a better control of their preparation processes.
cholerae LPS derivatives useful for the immuno-detection. The invention also provides a convenient way to prepare detoxified LPS of V. cholerae (and especially V. cholerae O1, serotype Inaba)-immunogenic carrier conjugates. Thanks to the single modification introduced following the adopted strategy, all derivatives of the detoxified LPS can be fully analyzed in term of purity and identity. This leads to an improved characterization of the conjugates and a better control of their preparation processes.
Description
VIBRlO CHOLERAE LPS DETOXIFIED DERIVATIVES AND IMMUNOGENIG
COMPOSITIONS CONTAINING TIiEM
FIELD OF THE INVENTION
The invention relates to the field of bioorganic chemistry, more specifically to the field of derivatization and conjugation of surface polysaccharide of Vibrio cholerae species. More particularly, the present invention relates to a derivative compound of the detoxified lipopolysaccharide (LPS) and its use for treating and/or preventing a V. cholerae infection. Furthermore, the present invention is concerned with compositions, vaccines and methods for providing an immune response andlor a protective immunity to animals against a V. cholerae infection and diagnostic methods thereof.
BACKGROUND OF THE INVENTION
Cholera remains a major public health concern in the developing world and will at least persist as long as a good level of hygiene will not be achieved in the endemic areas. Before then, the use of an effective vaccine is highly recommended by the WHO to control this infectious disease (WHO meeting, 10-11 december 2002). Two inactivated and one live attenuated oral vaccines are currently available (JD
Clemens ef al., Lancet, 1990, 335, 270-273; DD Trach et al., Lancet, 1997, 349, 231-235; MM
Levine et al., Vaccine, 1993, 7 7, 207-212): however, the high level of protection initially induced by these vaccines declines rapidly especially in infants, which seems to limit their use in some emergency situations and is incentive to develop alternate vaccine approaches.
Giycoconjugate vaccines composed of a protein carrier bound to surface polysaccharides of Vibrio cholerae, the gram-negafive bacterium responsible for the cholera epidemics, might offer such an option stemming from - The unique property of the glycoconjugate vaccines to induce long-term protection including children for many bacterial diseases (JB Bobbins et al., New generation of Vaccines.Second Edition, Revised and Expanded (Levine, M. M., Woodrow, G. G., Kapper, J. B., and Cobon, G. S., Eds) pp803-815, Chapter 52, Marvel Dekker, New York; JB Bobbins et aL,Pure Appl. Chem., 1999, 79, 745-754).
- The central role apparently played by the LPS of V. c:holerae in immunity against cholera since detection of high level of serum vibriocidal anti-LPS antibodies strongly correlates with an efficient protection of V. cholerae exposed or vaccinated populations (JB Bobbins et al., in New generation of Vaccines. Second Edition, Revised and Expanded (Levine, M. M., Woodrow, G. G., Kapper, J. B., and Cobon, G. S., Eds) pp803-815, Chapter 52, Marvel Dekker, New York).
Not ali strains of V. cholerae but those belonging to serograup 01, itself divided info Inaba or Ogawa serotypes, or to serogroup 0139, according to their LPS
structures, can cause cholera. No cross-protection is conferred from either serogroup 01 or 0139 to the other: this lack of protective immunity has bE~en evidenced as the first V.
cholerae 0139 infection has largely affected adults in V. choierae 01 areas of endemicity (MJ Albert, J. Clin. Ivlicrobiol., 1994, 32, 234.5-2349; F. Qadri et al., Clin.
Diagn. Lab. ImmunoL, 1995, 2, 685-688). If protection essentially originates from the LPS, as surmised above, this observation might derived from the complete absence of similarities between the O-specific polysaccharides (O-SP), of the two serogroups.
Concerning 01, vaccines trials in Asia from 1968 to 1971 showed that killed whole-cell Inaba vaccines protected against cholera caused by both Inaba and Ogawa serotypes, whereas Ogawa vaccines protected only against the homologous serotype (WH Mosley et al., J. infect. Dis., 1970, 727, S'I-S9; WH Mosley et ad., Bull.
UVHO, 9973, 49, 381-387; ML Levine and NF Pierce, in Cholera, D. Barua and WB
greenough III, Eds, pp285-327; Pfenum, New York, 1992). The O-specific saccharide of both serotypes is a linear homopolymer of 5-23 (mainly 14-22), a(1~2)-linked 4,C-dideoxy-4-(3-deoxy-L-glycero-tetronamido)-a-D-mannopyranosyl residues (Figure 1 ) (SN Chatterjee and K Chaudhuri, Siochim. 8iophys. Acta, 2003, 9639, 65-79). The only chemical difference reported to date consists on a partial 2-O-methylation of the nonreducing terminal sugar of the Ogawa O-antigen chains which is absent in the Inaba O-specific oligosaccharide. This single methyl is part of a dominant determinant able to elicit antibodies which could selectively protect against the Ogawa serotype but would fail to recognize the Inaba serotype (J Wang et al., J.
Biol. Chem., 1998, 273, 2777-2783; S. Villeneuve ef al., Proc. Natl. Acad.
Sci. USA, 2000, 97, 8433-8438). Otherwise, Inaba and Ogawa serotypes share several common antigenic determinants. One of them, involving sugars from both the O-antigen chain and the core of the LPS, is able to elicit protective antibodies and might be associated with the protection in absence of the Ogawa-specific antigenic determinant (J Wang et al., J. Biol. Chem., 1998, 273, ;?777-2783; S.
Villeneuve ef al., Microbiology, 1999, 97, 2477-2484). The combination of these data could give account for the specificity of the protection observed in the field.
As a consequence, successful LPS-derived choleira vaccines should probably consist on separate or mixed formulations including components directed against at feast V, cholerae 0139 and V. cholerae O1 Inaba.
Series of conjugates against V. cholerae 0139 have been designed from either detoxified LPS or the capsular polysaccharide which is a polymer produced by the V, cholerae 0139 strains whose repeating unit is identical to the O-specific polysaccharide of the LPS (A. Boutonnier et al., Infect. Immun., 2001, 69, 3488-3493;
JA Johnson, Bull. lest. Pasfer~r, 1995, 93, 285-290; S. Kossacka et al., Infecf.
lmmun., 2000, 68, 5037-5043; S. Kossacka and SC S:zu, Glycoconj. J., 2000, 77, 425-433). Following the encouraging results observed in marine models, at least one of them has now entered clinical trial.
Concerning V. chalerae 01, initial attempts relied on the use of alkali or hydrazine-detoxified LPS from both serotypes. The diffE:rent preparations proved to be irnrnunogenic in mice or rabbits (LB Adams et al., J. Clin. Microbiol., 1984, 19, 181-186; S. Kabir, J. Med. Microbiol., 1987, 23, 9-18;. R. K. Gupta et al., Infecf.
Immun., 1992, 60, 3201-3208). However, Inaba conjugai:es failed to elicit a high level of protection in human during a clinical phase 1 evaluation and need to be optimized (RK Gupta ef al., Infect. lmmun., 1998, 66, 3095-3099). Kovar and Wades groups are now privileging the use of synthetic hexasacharides mimicking the non-reducing end of the Ogawa or lnaba O-specific polysaccharidles to address this lack of efficiency. Unsurprisingly, all Ogawa semi-synthetic conjugates induce protective immunity specific for the Ogawa serotype in marine model (A. Chernyak et al., J.
Infect. Dis., 2002, 985, 950-962)). On the other hand, the antibodies induced in mice immunized with Inaba conjugates recognize both serofirpes but are not protective (MD Meeks ef al., Infect. Immun., 2004, 72, 4090-4101 ). One can note that none of the oligosaccharides belonging to the core are incorporated into the synthetic carbohydrate fragments although some of them seem important to induce protective antibodies (S. Villeneuve et al., Microbiology, 1999, 97, 2477-2484).
Taken together, these data suggest that V. cholerae detoxified LPS forms an attractive starting material towards the preparation of a vaccine formulation against cholera. Until now, the LPS, once detoxified, was d~erivatized using cyanogen bromide which can activate any nucleophilic group of the molecule namely the amino, hydroxy and carboxylic acid functions resulting in an ill-defined intermediate whose antigenic determinants) might be altered.
Thus, there is a need for new molecules that u;se LPS as a target for the development of immunotherapies against V. cholerae infE:ctions.
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
The inventors of the present invention hypothesi:7ed that a careful choice of experimental conditions might lead to a specific rather than a random modification of the detoxified LPS. The derivatives thus obtained might be more easily purified and analyzed. As a consequence, their further conjugation to a protein or a peptide carrier would afford well-characterized conjugates. Overall, it would facilitate the process control towards the production of a vaccine formulation. At the same time, it should be possible to prepare detoxified LPS derivatives useful for the immuno-detection.
For instance, as the LPS contains a unique, unprotected amino group on the D-glucosamine, the present inventors hypothesized tllat its derivatization would provide a single point of attachment for conjugation. 'This strategy is even more interesting as the use of a free amino group for conjugation is common when using synthetic oligosaccharides but remains unexplored from natural polysaccharides since, when present, the amine is generally acylated and consequently, unreactive.
' S
Consequently, one object of the present invention concerns an isolated or purified derivative compound of the detoxified LPS having the formula:
O-SP
v 1a pmLPS 01 Inaba: R = H X,X' = O
1b pmLPS 01 Ogawa: R = Me X,X' = O
1c reduced pmLPS 01 Inaba: = H X,X' = OH,H
wherein the derivatization is carried out onto the NH2 group.
Another object of the invention concerns a glycoconjugate comprising an isolated or purified derivative compound as defined above linked to a carrier.
Yet, the present invention also concerns immur~ogenic and pharmaceutical compositions comprising at least a glycoconjugate as defined above.
Other objects and advantages of the present invention will be apparent upon reading the following non-restrictive detailed description, made with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Structure of the acid-detoxified LPS (pmLPS) of V. cholerae 01, serotype Inaba and Ogawa. The point of attachment (dotted line) between the O-specific polysaccharide and the care has not yet been cle<~rly established, see E.V.
vinogranov et al., Eur. J. Biochem., 1995, 233, 152-158.
Figure 2. MALDI-TOF-MS spectra of (a) pmLPS (1a), {b) biotinylated-pmLPS (2), and (c) maleimide activated pmLPS (3).
Figure 3. (a) ELISA binding profiles of biotinylated-pmLP;S (2) with mAbs F-22-30 (v), I-24-2 (~,), and S-20-3IS-20-4 (~); (b) ELISA inhibition of the interaction between Inaba LPS and mAb F-22-30 with various cancentrations of fnaba LPS (O), Inaba pmLPS (0) or biotinylated-pmLPS (2) (v).
Figure 4. Preparation of the pmLPS-Tetanus toxoid (TT), conjugate using the maleimide chemistry.
Figure 5. EL1SA inhibition of the interaction between Inaba LPS and mAb F-22-with various concentrations of Inaba LPS (O) or maleimide activated-pmLPS (3) (v).
Figure 6. Double immunodiffusion analysis. Wells: 1, rnAb I-24-2, 35 pL; 2, Inaba LPS; 3,pmLPS-Mal TT Batch 1, 0.26 mg.mL'~ ; 4, pmLP~> Inaba (1a).
Figure 7. Preparation of the succinimidyl ester derivatives used as modifying agents of the prnLPS 01 Inaba and the tetanus toxoid (TT).
Figure 8. Structure of pmLPS0139 and of its biotinylateci derivative Figure 9. MALDI-TOF mass spectra of pmLPS 0139 (a) and of the corresponding mono-biotinylated compound {b).
Figure 10: Structures of the derivatized-pmLPS
BRIEF DESCRIPTION OF THE INVENTION
The invention relates to the feld of bioorganic chemistry, more specifically to the field of derivatization and conjugation of surface polysaccharide of Vibria cholerae species. More particularly, the present invention relates to a derivative compound of the detoxified lipopofysaccharide (LPS) and its use for treating and/or preventing a V. cholerae infection. Furthermore, the present invention is concerned with compositions, vaccines and methods for providing an immune response andlor a protective immunity to animals against a V. cholerae infection and diagnostic methods thereof.
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 glycoconjugate of the present invention as described herein below. The term "immune response" is to be understood as including a humoral response andlor a cellular respoinse 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 Vibrio cholerae infection which may be caused by, but not limited to, V.cholera selected among serotypes 0139 or 01. Thus, an animal immunized by the compositions or vaccines of the invention will experience limited growth and spread of such V. cholerae.
As used herein, the term "animal" refers to any animal that is susceptible to a Vibrio cholerae infection. Among the animals which are known to be potentially infected by Vlbrio cholerae, there are, but not limited to, humans, farm animals, sport animals, zoological garden animals, and wild animals.
1. 1_PS detoxified derivative compounds and glycocor~jugates comprising same One object of the present invention concerns an isolated or purified derivative compound of the detoxified I_PS having the formula:
M R
HO y~
OH N HO
O-SP O H
NH~~~OH
O~Me H OH n=3-20 H M
N HO H HO
OH ~ un HEN
1a pmLPS 01 Inaba: R = H X,X' = O
1b pmLPS 01 Ogawa: R = Me X,X' = O
1c reduced pmLPS 01 Inaba: = H X,X' = OH,H
wherein the derivatization is carried out onto the NH2 group.
Advantageously, the derivatization is carried out by means of a ligand which is preferably directly labelled by a dye (visible or UV), a colored particle, a pigment or bears at least a radioactive atom.
Specifically, the ligand is a linker molecule preferably selected among the group consisting of the following R structures:
M H
HO H~
OH N HO
H OH
O NH~./\,~' '~'~Me nH " ..,.
HO~~~ M -c ~ N H~0' OH
H
~O
S~NH
2X,X'=O; R= N N~~
O H
3X,X'=O; R=
N
~~~-''O
O
4 X,X'=OH,H; R=C'l ~OMe 5 X,X' = OH,H ; R = ~~'N _ ~NH2 H O v v H
O
9 X,X' = O ; R =
O
H
10X,X'=O; R= N
O O
17X,X'=O; R= N3 18 X,X'=O; R= N' N
'~ 3 O
According to another object, the present invention provides a glycoconjugate comprising an isolated or purified derivative compound of the invention linked to a carrier, wherein the LPS is preferably derived from V.chofera selected among serotypes 0139 or 01.
4t wilt be understood that the carrier preferably contemplated by the present invention is a polypeptide or a protein, such as but not limited to, those selected among the group consisting of: Tetanus Toxoid (TT), Human albumin (HA) and polypeptide PADRE. More preferably, the carrier bears at least one T helper epitope.
2. Compositions The glycaconjugate of the present invention may be used in many ways for the diagnosis, the treatment or the prevention of a V.choJer~a infection of the respiratory tract. For instance, they may be used in order to produce antibodies that specifically bind to the glycoconjugate molecules of the invention. More specifically, the antibody may be a purified polyclonal or monoclonal antibody that specifically binds to a glycoconjugate molecule as defined above.
Such antibodies may be prepared by a variety of methods using glycoconjugate molecules described above. For example, the glycoconjugates 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 ).
In another embodiment, the present invention relates to an immunogenic composition for eliciting an immune response or a protective immunity against a V. cholera infection. According to a related aspect, the present invention relates to a vaccine for preventing andlor treating a V.cholera infection. As used herein, the term "treating" refers to a process by which the symptoms of a V. cholera infection are alleviated or completely eliminated. As used herein, the term "preventing"
refers to a process by which a V. cholera infection is obstructed or delayed. The composition or the vaccine of the invention comprises at least a glycoconjugate of the invention.
Such a composition may further comprises a pharmaceutically acceptable vehicle.
As used herein, the expression "a pharmaceutically acceptable vehicle"
means a vehicle for containing a glycoconjugate of the invention that can be injected into an animal host without adverse effects. Suitable vehicles known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions. Vehicles 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), interleukin 2 (IL2), interleukin 12 (IL12), and CpG
oligonucleotides), antioxidants, surfiactants, 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 glycoconjugate 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 glycoconjugate 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 glycoconjugate molecules to be used and the compositionlvaccine 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 cornposition.
3. Methods of use In another embodiment, the present invention relates to methods for immunizing an animal against a Vibrio cholerae infection, or for treating and/or preventing against a Vibrio choierae infection, 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.
a i2 The vaccine, antibody and composition of the invention may be given to an animal through various routes of adminisfiration. 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 Vibrio cholerae strain 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).
It will be understood that the detection method of the present invention concerns a V. cholerae strain such as one is selected among serotypes 0139 or 01.
!t 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, feces, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof.
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 are 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.
The following examples describe the synthesis of V, cholerae detoxified LPS
derivatives useful for the immuno-detection and for the preparation of conjugates.
These examples also disclose the synthesis of V. choJerae detoxified LPS-immunogenic carrier conjugates from the above mentioned derivatives. The preparation of these compounds relies on the use of a free amino group on the core moiety of the detoxified LPS of V. cholerae. Unicity and superior nucleophilicity of the amine compare to the other functional groups displayed on the detoxified LPS
is exploited to introduce a single, controlled modification.
General (6-biotinamidocaproylamido)caproic acid N hydroxy-sulfo-succinimide ester (sulfo-NHS-Ic-Ic-Biotin), N (6-maleimidocaproyloxy) sulfosuccinimide ester (sulfo-EMCS), and IV-succinimidyl S-acefiylthioacetate (SATA), were purchased from Uptirna Interchim (MontluCon, France). Other chemical reagents were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Avidin from egg white was purchased from Sigma. Tetanus toxoid (TT) (MW 150 kDa), was purchased from Pasteur Merieux Connaught (Mercy I'Etoile, France), dialyzed against 0.05 M
aqueous NaCI to an actual concentration of 20.6 mg.mL-'. PBS, pH 7.3 is a mixture of 0.05 M potassium phosphate buffer and 0.15 M NaCI.
Thin-layer chromatography was performed using E. Merck plates of silica gel 60 with fluorescent indicator. Visualization was effected by spraying plates with 5°ro phosphomolybdic acid in EtOH followed by heating at 120-140°C. Silica gel used in flash chromatography had 230-400 mesh and 60 A pore size. 'H, '3C and 3'P NMR
spectra were recorded on a Broker DRX 400 spectrometer. Chemical shifts are given in ppm. Assignment of signals were confirmed by 'H homonuciear 2D correlation spectroscopy and 'H-'3C heteronuciear single quantum correlation (HSQC), spectroscopy experiments. Low-resolution mass spectra were obtained by either chemical ionization (C1), using NH3 as the ionizing gas, by fast atom bombardment (FAB) or matrix assisted laser desorption ionization (MALDI) techniques. MALDI-TOF-MS spectra were recorded on a Voyager mass spectrometer. Samples were dissolved in MeOHlH20 50:50 and adsorbed on a 2.5-dihydroxybenzoic acid matrix.
Analytical and semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC), separations were perFormed upon a Gilson Unipoint System using a Kromasil C18 (A. f. T. chromato, France) (100 h, 4.6 x 250 mm), column at a flow rate of 1 mL.min-' (monitoring and analysis), or using a Nucleosil 100-5 C18 (Macherey-Nagel) (100 A, 5 arm, 10 x 250 mrn), column at a flow rate of 3 mL.min-' (semi-preparative), with detection at 215 nm. Gradient: 0% B for 5 min, 0-10% B over 10 min, 10-30% B over 40 min. Solvent system A: 0.05% TFA in water;
solvent system B: 0.05% TFA in 60% CH3CN-40% water.
Dialyses were performed with Slide-A-Lyzer~ Dialysis Cassettes (Pierce) and centrifugal concentration were performed on Vivaspin 15R concentrators (Vivascience, Palaiseau, France).
Preparation of LPS and detoxified pm~PS from inaba serotype Bacteria were grown in Tryptic Soy Agar (Difco~, Detroit, Michigan), in Roux flasks at 37°C for 18 h. Ceiis were resuspended in distilled water and LPS was obtained by hot phenol water extraction (58), followed by enzymatic treatment (DNase, RNase and protease), and ultracentrifugatian (100,000 g for 3 h). The supernatant of ultracentrifugation was stored at -20°C. The pellet, containing the LPS, was dialyzed against distilled water and freeze-dried. This preparation contained 0.5% (wlv) protein and less than 0.2% (w/v) nucleic acids.
LPS was treated with acefiic acid to hydrolyze the lipid A-core linkage: LPS
[10 rng.mL-' in 1% (v/v) aqueous acetic acid) was heated at 100°C for 60 min.
Precipitated lipid A was removed by low-speed centrifugation (350 x g for 15 min) at 4°C. The supernatant was extracted with equal volume of chloroform-ethanol (2:1 ).
The reaction mixture was shaken vigorously and centrifuged at 10,000 x g for 60 min at 4°C. The aqueous phase was dialyzed against distilled water to remove ethanol and then freeze-dried. The resulting product is referred to as pmLPS (1 a) (Figure 1 ).
Alternatively, the supernantant was reacted with NaBH3CN (wlw of initial LPS) overnight prior to extraction. The resulting mixture was freeze-dried and the crude residue purified by RP-HPLC. The collected fractions were diluted with water, frozen and freeze-dried to furnish pure reduced pmLPS 01 Inaba (1c) in 26% yield.
pmLPS 01 Inaba (1a) (negative MALDI-TOF-MS): m/z 7057, 6810, 6562, 6315, 6067, 5820, 5573, 5326, 5079, 4831, 4585, 4337, 4090, 3843, 3596, 3348 (M -H)-, (22-mer to 6-mer) (Small amount of pmLPS with loss of one tetronic acid residue are also detected) (see Figure 2a).
reduced pmLPS 01 lnaba (1c) (negative MALDI-TOF-MS): m/z 7305, 7057, 6810, 6563, 6316, 6069, 5822, 5575, 5327, 5080, 4833 (M - H)-, (23-mer to 13-mer).
ELISA
Flat-bottom microplates (immuno Microwell, Nunc), were coated with avidin (Sigma) (100 pL at 1 pg.mL-') in PBS for 1 h at 37°C and washed with PBS-Tween.
Wells were blocked by incubation with 0.5% gelatin (proiabo) in PBS for 30 min at 37°C and washed with PBS containing 0.1 % Tween-20. Biotinylated-pmLPS
(2) dilutions (100 irL) in PBS-Tween-gelatin were added to the wells. The plates were incubated for 1 h at 37°C, washed with PBS-Tween and 100 pLlwell of mAbs were added and the plates further incubated for 1 h at 37°C and washed with PBS-Tween.
An anti-mouse peroxidase-conjugated IgG (heavy- and light-chain specific) (Biorad) diluted 1 in 1000 in PBS-Tween containing 0.5% gelatin was added to the wells. The plate was incubated at 37°C for 45 min and washed with PBS-Tween. The enzyme substrate, o-phenyienediamine dihydrochloride (Sigma) (100 NL at 0.4 mg.mL-') in 0.1 M sodium citrate (pH 5.2), containing 0.02% hydrogen peroxide, was added to each well and the plate incubated. for 10 min at RT. The reaction was stopped by adding 3 M HCI (50 pL per well) and the A,49o was read in an EL 800 spectrophotometer (Bio-Tek Instruments).
ELISA inhibition These assays were carried out in a two-steps ELISA. In step 1, mAb was incubated with solutions of polysaccharides and in step 2, the resulting mixfiures of free and bound antibody were added to microtitre plates coated with lnaba LPS
or pmLPS. Briefly, flat-bottom microplates were blocked by incubation with 0.5°!0 gelatin (prolabo) in PBS for 1 h at 37°C and rinsed with PBS containing 0.1 %
Tween-20.
Polysaccharide dilutions (100 ~rL) and mAb (100 NL) in PBS-Tween-gelatin were added to the wells, at a dilution, determined by direct ELISA titration, giving an A4~ of 0.5. The mixture were incubated for 1 h at 37°C and 100 pL of samples from each well were transferred to a second plate, that had been previously coated by incubation for 2 h at 37°C with Inaba LPS or pmLPS (5 pg.mL'') in Na2CO~/NaHC03 buffer (0.1 M, pH 9.5), blocked with gelatin and washed with PBS-Tween. This plate was incubated at 37°C for 1 h and washed with PBS-Tween. The detection of the inhibition and the reading of the plates were performed as described for direct ELISA.
Double immunodiffusion analysis Double immunodiffusion was performed in 1 % agarose in 0.5 M NaCI for 48 h at 4°C (lmmunochemistry in Practice, A. Johnstone and R. Thorpe, Eds, pp122-125, Chapter 6, 1982, Blackwell Scientific Publications, Oxford).
Example 't: Mono-biotinyfation of the pmLPS 01 lnaba according to the invention; preparation of derivatives useful for the imrnuno-detection The biotin moiety is used to favour the adsorption of the pmLPS derivatives through the formation of a complex with avidin, precoated on micrtiter-plates.
The biotinylation of oligosaccharides has been shown to considerably improve the sensitivity and the reproducibility of ELISA'assay quantitations (K.D.
McReynolds et al., Bioconjugate Chem., 1999, 10, 1021-1031; PJ Meikle et al., J. Immunnol.
Methods, 1990, 932, 255-261 and references cited therein).
Biofinylafion of pmLPS 01 Inaba A solution of pmLPS 01 Inaba (1a) (4.~7 mg, 0.81 pmole, 1 equiv.) in 0.1 M
potassium phosphate buffer, pH 7.3 (500 pL), was added to an eppendorf containing solid sulfo-NHS-lc-Ic-biotin (1.53 mg, 3 equiv.). The reaction mixture was transferred to another eppendorf containing solid sulfo-NHS-Ic-Ic-biotin (1.53 mg, 3 equiv.) after two hours. This operation was repeated once two hours later. After an additional two hours time, the crude reaction mixture was purified by RP-HPLC. The collected fractions were diluted with H2O, frozen and freeze-dried to give mono-biotinylated pmLPS 01 Inaba (2) (Figure 10) (1.60 mg, 31 % yield), together with starting material (1.13 mg, 23%) as white powders.
Negative MALDI-TOF-MS: m/z 7508, 7261, 7014, 6767, 6519, 6273, 6025, 5778, 5531, 5283, 5036, 4788, 4542, 4295, 4047, 3800 (M - H)-, (22-mer to 6-mer) (Small amount of monobiotinylated-pmLPS with loss of one tetronic acid residue is also detected) (see Figure 2b).
Direct ELISA assays using compound (2).
Compound 2 was plated on avidin-coated microtiter plates and further incubated with a series of dilution of monoclonal antibodies. A nice binding was observed with monoclonal IgG3 I-24-2 and monoclonal IgG1 F-22-30, obtained following immunization of mice with a lysate from V, cholerae 01 serotype Inaba.
These two mAbs recognize an antigenic determinant associated with the LPS from both Inaba and Ogawa serotypes and protect mice from mortal challenge (32, 33).
On the other hand, no recognition was observed with monoclonal lgG1 S-20-3/S-4, directed against the 2-O-methylated terminal perosamine of the Ogawa O-SP
and, thus, specific for this serotype (93, 94) (Figure 3a). The specificity of the recognition of compound (2) with the two former mAbs was further characterized by ELISA
inhibition. Biotinylated-pmLPS (2) is as effective as Inaba LPS or Inaba pmLPS
to inhibit the interaction of mAb F-22-30 with Inaba LPS (Figure 3b).
Example 2: Synthesis of maleimide-functionafized pmLPS 01 lnaba according to the invention; use of thereof for its conjugation to a protein carrier Synthesis of maleimide-activated pmLPS 07 Inaba (3J (,Figure 10) A solution of 1a (22.33 mg, 3.7 pmole, 1 equiv.), in 0.1 M potassium phosphate buffer, pH 7.3 (2230 pL), was added to an eppendorf containing solid N
(0-maleimidocaproyloxy)sulfosuccinimide ester, (6.10 mg, 4 equiv). The reaction mixture was transferred to another eppendorf containing anather 4 equiv of solid sulfo-EMCS after 1.5 hours. This operation was repeated once 1.5 hours later.
After an additional one hour, the crude reaction mixture was purified by RP-HPLC.
The collected fractions were diluted with H20, frozen and freeze-dried to give maleimide activated-pmLPS 01 (nabs (3) (9.53 mg, 23% yield), together with starting material (3.36 mg, 15%) as white foams.
Maleimide activated-pmLPS 01 inaba (3): negative MALDI-TOF-MS: m/z 7001, 6752, 6506, 6260, 6011, 5764, 5518, 5270, 502.4, 4777, 4529, 4283, 4035, 3789, 3541 (M - H)-, (21-mer to 7-mer) (see Figure 2c).
Derivatization of the tetanus toxoid (Figure 4) Protocol A
To a solution of tetanus toxoid (29.12 mg, 1400 pL, 1.90 pmole) was added succinimidyl S-acetylthioacetate (SATA) (3 x 2.19 mg, 3 x 36 pL of an 60 mg.mL-' solution in CH3CN, 3 x 50 equiv), in three portions every 45 minutes.
Following an additional reaction period of 45 minutes, the crude reaction mixture was dialyzed against 3 x 2 L of 0.1 M potassium phosphate buffer, pH 6.0 at 4°C to eliminate excess reagent.
Protocol B
To a solution of tetanus toxoid (7.2 mg, 350 pL, 0.047 pmole, 1 equiv.)) was added succinimidyl S acetyithioacetate (SATA) (2 x 0.11 mg, 2 x 36 pL of an 30 mg.mL-' solution in CH3CN, 2 x 10 equiv), in two portions at 45 minutes interval.
Following an additional reaction period of 45 minutes, the crude reaction mixture was dialyzed against 3 x 2 L of 0.1 M potassium phosphate buffer, pH
COMPOSITIONS CONTAINING TIiEM
FIELD OF THE INVENTION
The invention relates to the field of bioorganic chemistry, more specifically to the field of derivatization and conjugation of surface polysaccharide of Vibrio cholerae species. More particularly, the present invention relates to a derivative compound of the detoxified lipopolysaccharide (LPS) and its use for treating and/or preventing a V. cholerae infection. Furthermore, the present invention is concerned with compositions, vaccines and methods for providing an immune response andlor a protective immunity to animals against a V. cholerae infection and diagnostic methods thereof.
BACKGROUND OF THE INVENTION
Cholera remains a major public health concern in the developing world and will at least persist as long as a good level of hygiene will not be achieved in the endemic areas. Before then, the use of an effective vaccine is highly recommended by the WHO to control this infectious disease (WHO meeting, 10-11 december 2002). Two inactivated and one live attenuated oral vaccines are currently available (JD
Clemens ef al., Lancet, 1990, 335, 270-273; DD Trach et al., Lancet, 1997, 349, 231-235; MM
Levine et al., Vaccine, 1993, 7 7, 207-212): however, the high level of protection initially induced by these vaccines declines rapidly especially in infants, which seems to limit their use in some emergency situations and is incentive to develop alternate vaccine approaches.
Giycoconjugate vaccines composed of a protein carrier bound to surface polysaccharides of Vibrio cholerae, the gram-negafive bacterium responsible for the cholera epidemics, might offer such an option stemming from - The unique property of the glycoconjugate vaccines to induce long-term protection including children for many bacterial diseases (JB Bobbins et al., New generation of Vaccines.Second Edition, Revised and Expanded (Levine, M. M., Woodrow, G. G., Kapper, J. B., and Cobon, G. S., Eds) pp803-815, Chapter 52, Marvel Dekker, New York; JB Bobbins et aL,Pure Appl. Chem., 1999, 79, 745-754).
- The central role apparently played by the LPS of V. c:holerae in immunity against cholera since detection of high level of serum vibriocidal anti-LPS antibodies strongly correlates with an efficient protection of V. cholerae exposed or vaccinated populations (JB Bobbins et al., in New generation of Vaccines. Second Edition, Revised and Expanded (Levine, M. M., Woodrow, G. G., Kapper, J. B., and Cobon, G. S., Eds) pp803-815, Chapter 52, Marvel Dekker, New York).
Not ali strains of V. cholerae but those belonging to serograup 01, itself divided info Inaba or Ogawa serotypes, or to serogroup 0139, according to their LPS
structures, can cause cholera. No cross-protection is conferred from either serogroup 01 or 0139 to the other: this lack of protective immunity has bE~en evidenced as the first V.
cholerae 0139 infection has largely affected adults in V. choierae 01 areas of endemicity (MJ Albert, J. Clin. Ivlicrobiol., 1994, 32, 234.5-2349; F. Qadri et al., Clin.
Diagn. Lab. ImmunoL, 1995, 2, 685-688). If protection essentially originates from the LPS, as surmised above, this observation might derived from the complete absence of similarities between the O-specific polysaccharides (O-SP), of the two serogroups.
Concerning 01, vaccines trials in Asia from 1968 to 1971 showed that killed whole-cell Inaba vaccines protected against cholera caused by both Inaba and Ogawa serotypes, whereas Ogawa vaccines protected only against the homologous serotype (WH Mosley et al., J. infect. Dis., 1970, 727, S'I-S9; WH Mosley et ad., Bull.
UVHO, 9973, 49, 381-387; ML Levine and NF Pierce, in Cholera, D. Barua and WB
greenough III, Eds, pp285-327; Pfenum, New York, 1992). The O-specific saccharide of both serotypes is a linear homopolymer of 5-23 (mainly 14-22), a(1~2)-linked 4,C-dideoxy-4-(3-deoxy-L-glycero-tetronamido)-a-D-mannopyranosyl residues (Figure 1 ) (SN Chatterjee and K Chaudhuri, Siochim. 8iophys. Acta, 2003, 9639, 65-79). The only chemical difference reported to date consists on a partial 2-O-methylation of the nonreducing terminal sugar of the Ogawa O-antigen chains which is absent in the Inaba O-specific oligosaccharide. This single methyl is part of a dominant determinant able to elicit antibodies which could selectively protect against the Ogawa serotype but would fail to recognize the Inaba serotype (J Wang et al., J.
Biol. Chem., 1998, 273, 2777-2783; S. Villeneuve ef al., Proc. Natl. Acad.
Sci. USA, 2000, 97, 8433-8438). Otherwise, Inaba and Ogawa serotypes share several common antigenic determinants. One of them, involving sugars from both the O-antigen chain and the core of the LPS, is able to elicit protective antibodies and might be associated with the protection in absence of the Ogawa-specific antigenic determinant (J Wang et al., J. Biol. Chem., 1998, 273, ;?777-2783; S.
Villeneuve ef al., Microbiology, 1999, 97, 2477-2484). The combination of these data could give account for the specificity of the protection observed in the field.
As a consequence, successful LPS-derived choleira vaccines should probably consist on separate or mixed formulations including components directed against at feast V, cholerae 0139 and V. cholerae O1 Inaba.
Series of conjugates against V. cholerae 0139 have been designed from either detoxified LPS or the capsular polysaccharide which is a polymer produced by the V, cholerae 0139 strains whose repeating unit is identical to the O-specific polysaccharide of the LPS (A. Boutonnier et al., Infect. Immun., 2001, 69, 3488-3493;
JA Johnson, Bull. lest. Pasfer~r, 1995, 93, 285-290; S. Kossacka et al., Infecf.
lmmun., 2000, 68, 5037-5043; S. Kossacka and SC S:zu, Glycoconj. J., 2000, 77, 425-433). Following the encouraging results observed in marine models, at least one of them has now entered clinical trial.
Concerning V. chalerae 01, initial attempts relied on the use of alkali or hydrazine-detoxified LPS from both serotypes. The diffE:rent preparations proved to be irnrnunogenic in mice or rabbits (LB Adams et al., J. Clin. Microbiol., 1984, 19, 181-186; S. Kabir, J. Med. Microbiol., 1987, 23, 9-18;. R. K. Gupta et al., Infecf.
Immun., 1992, 60, 3201-3208). However, Inaba conjugai:es failed to elicit a high level of protection in human during a clinical phase 1 evaluation and need to be optimized (RK Gupta ef al., Infect. lmmun., 1998, 66, 3095-3099). Kovar and Wades groups are now privileging the use of synthetic hexasacharides mimicking the non-reducing end of the Ogawa or lnaba O-specific polysaccharidles to address this lack of efficiency. Unsurprisingly, all Ogawa semi-synthetic conjugates induce protective immunity specific for the Ogawa serotype in marine model (A. Chernyak et al., J.
Infect. Dis., 2002, 985, 950-962)). On the other hand, the antibodies induced in mice immunized with Inaba conjugates recognize both serofirpes but are not protective (MD Meeks ef al., Infect. Immun., 2004, 72, 4090-4101 ). One can note that none of the oligosaccharides belonging to the core are incorporated into the synthetic carbohydrate fragments although some of them seem important to induce protective antibodies (S. Villeneuve et al., Microbiology, 1999, 97, 2477-2484).
Taken together, these data suggest that V. cholerae detoxified LPS forms an attractive starting material towards the preparation of a vaccine formulation against cholera. Until now, the LPS, once detoxified, was d~erivatized using cyanogen bromide which can activate any nucleophilic group of the molecule namely the amino, hydroxy and carboxylic acid functions resulting in an ill-defined intermediate whose antigenic determinants) might be altered.
Thus, there is a need for new molecules that u;se LPS as a target for the development of immunotherapies against V. cholerae infE:ctions.
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
The inventors of the present invention hypothesi:7ed that a careful choice of experimental conditions might lead to a specific rather than a random modification of the detoxified LPS. The derivatives thus obtained might be more easily purified and analyzed. As a consequence, their further conjugation to a protein or a peptide carrier would afford well-characterized conjugates. Overall, it would facilitate the process control towards the production of a vaccine formulation. At the same time, it should be possible to prepare detoxified LPS derivatives useful for the immuno-detection.
For instance, as the LPS contains a unique, unprotected amino group on the D-glucosamine, the present inventors hypothesized tllat its derivatization would provide a single point of attachment for conjugation. 'This strategy is even more interesting as the use of a free amino group for conjugation is common when using synthetic oligosaccharides but remains unexplored from natural polysaccharides since, when present, the amine is generally acylated and consequently, unreactive.
' S
Consequently, one object of the present invention concerns an isolated or purified derivative compound of the detoxified LPS having the formula:
O-SP
v 1a pmLPS 01 Inaba: R = H X,X' = O
1b pmLPS 01 Ogawa: R = Me X,X' = O
1c reduced pmLPS 01 Inaba: = H X,X' = OH,H
wherein the derivatization is carried out onto the NH2 group.
Another object of the invention concerns a glycoconjugate comprising an isolated or purified derivative compound as defined above linked to a carrier.
Yet, the present invention also concerns immur~ogenic and pharmaceutical compositions comprising at least a glycoconjugate as defined above.
Other objects and advantages of the present invention will be apparent upon reading the following non-restrictive detailed description, made with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Structure of the acid-detoxified LPS (pmLPS) of V. cholerae 01, serotype Inaba and Ogawa. The point of attachment (dotted line) between the O-specific polysaccharide and the care has not yet been cle<~rly established, see E.V.
vinogranov et al., Eur. J. Biochem., 1995, 233, 152-158.
Figure 2. MALDI-TOF-MS spectra of (a) pmLPS (1a), {b) biotinylated-pmLPS (2), and (c) maleimide activated pmLPS (3).
Figure 3. (a) ELISA binding profiles of biotinylated-pmLP;S (2) with mAbs F-22-30 (v), I-24-2 (~,), and S-20-3IS-20-4 (~); (b) ELISA inhibition of the interaction between Inaba LPS and mAb F-22-30 with various cancentrations of fnaba LPS (O), Inaba pmLPS (0) or biotinylated-pmLPS (2) (v).
Figure 4. Preparation of the pmLPS-Tetanus toxoid (TT), conjugate using the maleimide chemistry.
Figure 5. EL1SA inhibition of the interaction between Inaba LPS and mAb F-22-with various concentrations of Inaba LPS (O) or maleimide activated-pmLPS (3) (v).
Figure 6. Double immunodiffusion analysis. Wells: 1, rnAb I-24-2, 35 pL; 2, Inaba LPS; 3,pmLPS-Mal TT Batch 1, 0.26 mg.mL'~ ; 4, pmLP~> Inaba (1a).
Figure 7. Preparation of the succinimidyl ester derivatives used as modifying agents of the prnLPS 01 Inaba and the tetanus toxoid (TT).
Figure 8. Structure of pmLPS0139 and of its biotinylateci derivative Figure 9. MALDI-TOF mass spectra of pmLPS 0139 (a) and of the corresponding mono-biotinylated compound {b).
Figure 10: Structures of the derivatized-pmLPS
BRIEF DESCRIPTION OF THE INVENTION
The invention relates to the feld of bioorganic chemistry, more specifically to the field of derivatization and conjugation of surface polysaccharide of Vibria cholerae species. More particularly, the present invention relates to a derivative compound of the detoxified lipopofysaccharide (LPS) and its use for treating and/or preventing a V. cholerae infection. Furthermore, the present invention is concerned with compositions, vaccines and methods for providing an immune response andlor a protective immunity to animals against a V. cholerae infection and diagnostic methods thereof.
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 glycoconjugate of the present invention as described herein below. The term "immune response" is to be understood as including a humoral response andlor a cellular respoinse 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 Vibrio cholerae infection which may be caused by, but not limited to, V.cholera selected among serotypes 0139 or 01. Thus, an animal immunized by the compositions or vaccines of the invention will experience limited growth and spread of such V. cholerae.
As used herein, the term "animal" refers to any animal that is susceptible to a Vibrio cholerae infection. Among the animals which are known to be potentially infected by Vlbrio cholerae, there are, but not limited to, humans, farm animals, sport animals, zoological garden animals, and wild animals.
1. 1_PS detoxified derivative compounds and glycocor~jugates comprising same One object of the present invention concerns an isolated or purified derivative compound of the detoxified I_PS having the formula:
M R
HO y~
OH N HO
O-SP O H
NH~~~OH
O~Me H OH n=3-20 H M
N HO H HO
OH ~ un HEN
1a pmLPS 01 Inaba: R = H X,X' = O
1b pmLPS 01 Ogawa: R = Me X,X' = O
1c reduced pmLPS 01 Inaba: = H X,X' = OH,H
wherein the derivatization is carried out onto the NH2 group.
Advantageously, the derivatization is carried out by means of a ligand which is preferably directly labelled by a dye (visible or UV), a colored particle, a pigment or bears at least a radioactive atom.
Specifically, the ligand is a linker molecule preferably selected among the group consisting of the following R structures:
M H
HO H~
OH N HO
H OH
O NH~./\,~' '~'~Me nH " ..,.
HO~~~ M -c ~ N H~0' OH
H
~O
S~NH
2X,X'=O; R= N N~~
O H
3X,X'=O; R=
N
~~~-''O
O
4 X,X'=OH,H; R=C'l ~OMe 5 X,X' = OH,H ; R = ~~'N _ ~NH2 H O v v H
O
9 X,X' = O ; R =
O
H
10X,X'=O; R= N
O O
17X,X'=O; R= N3 18 X,X'=O; R= N' N
'~ 3 O
According to another object, the present invention provides a glycoconjugate comprising an isolated or purified derivative compound of the invention linked to a carrier, wherein the LPS is preferably derived from V.chofera selected among serotypes 0139 or 01.
4t wilt be understood that the carrier preferably contemplated by the present invention is a polypeptide or a protein, such as but not limited to, those selected among the group consisting of: Tetanus Toxoid (TT), Human albumin (HA) and polypeptide PADRE. More preferably, the carrier bears at least one T helper epitope.
2. Compositions The glycaconjugate of the present invention may be used in many ways for the diagnosis, the treatment or the prevention of a V.choJer~a infection of the respiratory tract. For instance, they may be used in order to produce antibodies that specifically bind to the glycoconjugate molecules of the invention. More specifically, the antibody may be a purified polyclonal or monoclonal antibody that specifically binds to a glycoconjugate molecule as defined above.
Such antibodies may be prepared by a variety of methods using glycoconjugate molecules described above. For example, the glycoconjugates 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 ).
In another embodiment, the present invention relates to an immunogenic composition for eliciting an immune response or a protective immunity against a V. cholera infection. According to a related aspect, the present invention relates to a vaccine for preventing andlor treating a V.cholera infection. As used herein, the term "treating" refers to a process by which the symptoms of a V. cholera infection are alleviated or completely eliminated. As used herein, the term "preventing"
refers to a process by which a V. cholera infection is obstructed or delayed. The composition or the vaccine of the invention comprises at least a glycoconjugate of the invention.
Such a composition may further comprises a pharmaceutically acceptable vehicle.
As used herein, the expression "a pharmaceutically acceptable vehicle"
means a vehicle for containing a glycoconjugate of the invention that can be injected into an animal host without adverse effects. Suitable vehicles known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions. Vehicles 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), interleukin 2 (IL2), interleukin 12 (IL12), and CpG
oligonucleotides), antioxidants, surfiactants, 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 glycoconjugate 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 glycoconjugate 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 glycoconjugate molecules to be used and the compositionlvaccine 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 cornposition.
3. Methods of use In another embodiment, the present invention relates to methods for immunizing an animal against a Vibrio cholerae infection, or for treating and/or preventing against a Vibrio choierae infection, 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.
a i2 The vaccine, antibody and composition of the invention may be given to an animal through various routes of adminisfiration. 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 Vibrio cholerae strain 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).
It will be understood that the detection method of the present invention concerns a V. cholerae strain such as one is selected among serotypes 0139 or 01.
!t 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, feces, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof.
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 are 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.
The following examples describe the synthesis of V, cholerae detoxified LPS
derivatives useful for the immuno-detection and for the preparation of conjugates.
These examples also disclose the synthesis of V. choJerae detoxified LPS-immunogenic carrier conjugates from the above mentioned derivatives. The preparation of these compounds relies on the use of a free amino group on the core moiety of the detoxified LPS of V. cholerae. Unicity and superior nucleophilicity of the amine compare to the other functional groups displayed on the detoxified LPS
is exploited to introduce a single, controlled modification.
General (6-biotinamidocaproylamido)caproic acid N hydroxy-sulfo-succinimide ester (sulfo-NHS-Ic-Ic-Biotin), N (6-maleimidocaproyloxy) sulfosuccinimide ester (sulfo-EMCS), and IV-succinimidyl S-acefiylthioacetate (SATA), were purchased from Uptirna Interchim (MontluCon, France). Other chemical reagents were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Avidin from egg white was purchased from Sigma. Tetanus toxoid (TT) (MW 150 kDa), was purchased from Pasteur Merieux Connaught (Mercy I'Etoile, France), dialyzed against 0.05 M
aqueous NaCI to an actual concentration of 20.6 mg.mL-'. PBS, pH 7.3 is a mixture of 0.05 M potassium phosphate buffer and 0.15 M NaCI.
Thin-layer chromatography was performed using E. Merck plates of silica gel 60 with fluorescent indicator. Visualization was effected by spraying plates with 5°ro phosphomolybdic acid in EtOH followed by heating at 120-140°C. Silica gel used in flash chromatography had 230-400 mesh and 60 A pore size. 'H, '3C and 3'P NMR
spectra were recorded on a Broker DRX 400 spectrometer. Chemical shifts are given in ppm. Assignment of signals were confirmed by 'H homonuciear 2D correlation spectroscopy and 'H-'3C heteronuciear single quantum correlation (HSQC), spectroscopy experiments. Low-resolution mass spectra were obtained by either chemical ionization (C1), using NH3 as the ionizing gas, by fast atom bombardment (FAB) or matrix assisted laser desorption ionization (MALDI) techniques. MALDI-TOF-MS spectra were recorded on a Voyager mass spectrometer. Samples were dissolved in MeOHlH20 50:50 and adsorbed on a 2.5-dihydroxybenzoic acid matrix.
Analytical and semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC), separations were perFormed upon a Gilson Unipoint System using a Kromasil C18 (A. f. T. chromato, France) (100 h, 4.6 x 250 mm), column at a flow rate of 1 mL.min-' (monitoring and analysis), or using a Nucleosil 100-5 C18 (Macherey-Nagel) (100 A, 5 arm, 10 x 250 mrn), column at a flow rate of 3 mL.min-' (semi-preparative), with detection at 215 nm. Gradient: 0% B for 5 min, 0-10% B over 10 min, 10-30% B over 40 min. Solvent system A: 0.05% TFA in water;
solvent system B: 0.05% TFA in 60% CH3CN-40% water.
Dialyses were performed with Slide-A-Lyzer~ Dialysis Cassettes (Pierce) and centrifugal concentration were performed on Vivaspin 15R concentrators (Vivascience, Palaiseau, France).
Preparation of LPS and detoxified pm~PS from inaba serotype Bacteria were grown in Tryptic Soy Agar (Difco~, Detroit, Michigan), in Roux flasks at 37°C for 18 h. Ceiis were resuspended in distilled water and LPS was obtained by hot phenol water extraction (58), followed by enzymatic treatment (DNase, RNase and protease), and ultracentrifugatian (100,000 g for 3 h). The supernatant of ultracentrifugation was stored at -20°C. The pellet, containing the LPS, was dialyzed against distilled water and freeze-dried. This preparation contained 0.5% (wlv) protein and less than 0.2% (w/v) nucleic acids.
LPS was treated with acefiic acid to hydrolyze the lipid A-core linkage: LPS
[10 rng.mL-' in 1% (v/v) aqueous acetic acid) was heated at 100°C for 60 min.
Precipitated lipid A was removed by low-speed centrifugation (350 x g for 15 min) at 4°C. The supernatant was extracted with equal volume of chloroform-ethanol (2:1 ).
The reaction mixture was shaken vigorously and centrifuged at 10,000 x g for 60 min at 4°C. The aqueous phase was dialyzed against distilled water to remove ethanol and then freeze-dried. The resulting product is referred to as pmLPS (1 a) (Figure 1 ).
Alternatively, the supernantant was reacted with NaBH3CN (wlw of initial LPS) overnight prior to extraction. The resulting mixture was freeze-dried and the crude residue purified by RP-HPLC. The collected fractions were diluted with water, frozen and freeze-dried to furnish pure reduced pmLPS 01 Inaba (1c) in 26% yield.
pmLPS 01 Inaba (1a) (negative MALDI-TOF-MS): m/z 7057, 6810, 6562, 6315, 6067, 5820, 5573, 5326, 5079, 4831, 4585, 4337, 4090, 3843, 3596, 3348 (M -H)-, (22-mer to 6-mer) (Small amount of pmLPS with loss of one tetronic acid residue are also detected) (see Figure 2a).
reduced pmLPS 01 lnaba (1c) (negative MALDI-TOF-MS): m/z 7305, 7057, 6810, 6563, 6316, 6069, 5822, 5575, 5327, 5080, 4833 (M - H)-, (23-mer to 13-mer).
ELISA
Flat-bottom microplates (immuno Microwell, Nunc), were coated with avidin (Sigma) (100 pL at 1 pg.mL-') in PBS for 1 h at 37°C and washed with PBS-Tween.
Wells were blocked by incubation with 0.5% gelatin (proiabo) in PBS for 30 min at 37°C and washed with PBS containing 0.1 % Tween-20. Biotinylated-pmLPS
(2) dilutions (100 irL) in PBS-Tween-gelatin were added to the wells. The plates were incubated for 1 h at 37°C, washed with PBS-Tween and 100 pLlwell of mAbs were added and the plates further incubated for 1 h at 37°C and washed with PBS-Tween.
An anti-mouse peroxidase-conjugated IgG (heavy- and light-chain specific) (Biorad) diluted 1 in 1000 in PBS-Tween containing 0.5% gelatin was added to the wells. The plate was incubated at 37°C for 45 min and washed with PBS-Tween. The enzyme substrate, o-phenyienediamine dihydrochloride (Sigma) (100 NL at 0.4 mg.mL-') in 0.1 M sodium citrate (pH 5.2), containing 0.02% hydrogen peroxide, was added to each well and the plate incubated. for 10 min at RT. The reaction was stopped by adding 3 M HCI (50 pL per well) and the A,49o was read in an EL 800 spectrophotometer (Bio-Tek Instruments).
ELISA inhibition These assays were carried out in a two-steps ELISA. In step 1, mAb was incubated with solutions of polysaccharides and in step 2, the resulting mixfiures of free and bound antibody were added to microtitre plates coated with lnaba LPS
or pmLPS. Briefly, flat-bottom microplates were blocked by incubation with 0.5°!0 gelatin (prolabo) in PBS for 1 h at 37°C and rinsed with PBS containing 0.1 %
Tween-20.
Polysaccharide dilutions (100 ~rL) and mAb (100 NL) in PBS-Tween-gelatin were added to the wells, at a dilution, determined by direct ELISA titration, giving an A4~ of 0.5. The mixture were incubated for 1 h at 37°C and 100 pL of samples from each well were transferred to a second plate, that had been previously coated by incubation for 2 h at 37°C with Inaba LPS or pmLPS (5 pg.mL'') in Na2CO~/NaHC03 buffer (0.1 M, pH 9.5), blocked with gelatin and washed with PBS-Tween. This plate was incubated at 37°C for 1 h and washed with PBS-Tween. The detection of the inhibition and the reading of the plates were performed as described for direct ELISA.
Double immunodiffusion analysis Double immunodiffusion was performed in 1 % agarose in 0.5 M NaCI for 48 h at 4°C (lmmunochemistry in Practice, A. Johnstone and R. Thorpe, Eds, pp122-125, Chapter 6, 1982, Blackwell Scientific Publications, Oxford).
Example 't: Mono-biotinyfation of the pmLPS 01 lnaba according to the invention; preparation of derivatives useful for the imrnuno-detection The biotin moiety is used to favour the adsorption of the pmLPS derivatives through the formation of a complex with avidin, precoated on micrtiter-plates.
The biotinylation of oligosaccharides has been shown to considerably improve the sensitivity and the reproducibility of ELISA'assay quantitations (K.D.
McReynolds et al., Bioconjugate Chem., 1999, 10, 1021-1031; PJ Meikle et al., J. Immunnol.
Methods, 1990, 932, 255-261 and references cited therein).
Biofinylafion of pmLPS 01 Inaba A solution of pmLPS 01 Inaba (1a) (4.~7 mg, 0.81 pmole, 1 equiv.) in 0.1 M
potassium phosphate buffer, pH 7.3 (500 pL), was added to an eppendorf containing solid sulfo-NHS-lc-Ic-biotin (1.53 mg, 3 equiv.). The reaction mixture was transferred to another eppendorf containing solid sulfo-NHS-Ic-Ic-biotin (1.53 mg, 3 equiv.) after two hours. This operation was repeated once two hours later. After an additional two hours time, the crude reaction mixture was purified by RP-HPLC. The collected fractions were diluted with H2O, frozen and freeze-dried to give mono-biotinylated pmLPS 01 Inaba (2) (Figure 10) (1.60 mg, 31 % yield), together with starting material (1.13 mg, 23%) as white powders.
Negative MALDI-TOF-MS: m/z 7508, 7261, 7014, 6767, 6519, 6273, 6025, 5778, 5531, 5283, 5036, 4788, 4542, 4295, 4047, 3800 (M - H)-, (22-mer to 6-mer) (Small amount of monobiotinylated-pmLPS with loss of one tetronic acid residue is also detected) (see Figure 2b).
Direct ELISA assays using compound (2).
Compound 2 was plated on avidin-coated microtiter plates and further incubated with a series of dilution of monoclonal antibodies. A nice binding was observed with monoclonal IgG3 I-24-2 and monoclonal IgG1 F-22-30, obtained following immunization of mice with a lysate from V, cholerae 01 serotype Inaba.
These two mAbs recognize an antigenic determinant associated with the LPS from both Inaba and Ogawa serotypes and protect mice from mortal challenge (32, 33).
On the other hand, no recognition was observed with monoclonal lgG1 S-20-3/S-4, directed against the 2-O-methylated terminal perosamine of the Ogawa O-SP
and, thus, specific for this serotype (93, 94) (Figure 3a). The specificity of the recognition of compound (2) with the two former mAbs was further characterized by ELISA
inhibition. Biotinylated-pmLPS (2) is as effective as Inaba LPS or Inaba pmLPS
to inhibit the interaction of mAb F-22-30 with Inaba LPS (Figure 3b).
Example 2: Synthesis of maleimide-functionafized pmLPS 01 lnaba according to the invention; use of thereof for its conjugation to a protein carrier Synthesis of maleimide-activated pmLPS 07 Inaba (3J (,Figure 10) A solution of 1a (22.33 mg, 3.7 pmole, 1 equiv.), in 0.1 M potassium phosphate buffer, pH 7.3 (2230 pL), was added to an eppendorf containing solid N
(0-maleimidocaproyloxy)sulfosuccinimide ester, (6.10 mg, 4 equiv). The reaction mixture was transferred to another eppendorf containing anather 4 equiv of solid sulfo-EMCS after 1.5 hours. This operation was repeated once 1.5 hours later.
After an additional one hour, the crude reaction mixture was purified by RP-HPLC.
The collected fractions were diluted with H20, frozen and freeze-dried to give maleimide activated-pmLPS 01 (nabs (3) (9.53 mg, 23% yield), together with starting material (3.36 mg, 15%) as white foams.
Maleimide activated-pmLPS 01 inaba (3): negative MALDI-TOF-MS: m/z 7001, 6752, 6506, 6260, 6011, 5764, 5518, 5270, 502.4, 4777, 4529, 4283, 4035, 3789, 3541 (M - H)-, (21-mer to 7-mer) (see Figure 2c).
Derivatization of the tetanus toxoid (Figure 4) Protocol A
To a solution of tetanus toxoid (29.12 mg, 1400 pL, 1.90 pmole) was added succinimidyl S-acetylthioacetate (SATA) (3 x 2.19 mg, 3 x 36 pL of an 60 mg.mL-' solution in CH3CN, 3 x 50 equiv), in three portions every 45 minutes.
Following an additional reaction period of 45 minutes, the crude reaction mixture was dialyzed against 3 x 2 L of 0.1 M potassium phosphate buffer, pH 6.0 at 4°C to eliminate excess reagent.
Protocol B
To a solution of tetanus toxoid (7.2 mg, 350 pL, 0.047 pmole, 1 equiv.)) was added succinimidyl S acetyithioacetate (SATA) (2 x 0.11 mg, 2 x 36 pL of an 30 mg.mL-' solution in CH3CN, 2 x 10 equiv), in two portions at 45 minutes interval.
Following an additional reaction period of 45 minutes, the crude reaction mixture was dialyzed against 3 x 2 L of 0.1 M potassium phosphate buffer, pH
6.0 at 4°C to eliminate excess reagent.
Preparation and characterization of fhe conjugates (Figure 4) Maleimide activated-pmLPS was added to the appropriate modified TT in 0.1 M potassium phosphate buffer solution at a 16:1 molar ratio. Reaction mixtures were buffered at a 0.5M concentration by addition of 1 M potaissium phosphate buffer, pH
6Ø Then, NH20H, HCl (10 pL of a 2 M solution in 1 M potassium phosphate buffer, pH 6), was added to the different mixtures and the couplings were carried out for 2 h at room temperature. The conjugated products were dialyzed against 3 x 2 L of 0.05 M PBS, pH 7.4 at 4°C, and further purified by gel permeation chromatography on a sepharose CL-6B column (1 m x 160 mm) (Pharmacia Biotech), using 0.05 M PBS, pH 7.4 as eluent at a flow rate of 0.2 mL.min-', with detection by measuring the optical density at 280 nm and the refractive index. The conjugates referred as pmLPS-Mal-TT batch 1 and batch 2 were stored at 4°C in the presence of thimerosal (0.1 mg.mL-'), and assessed for their total carbohydrate and protein content.
Hexose concentration was measured by a colorimetric method based on the enthrone reaction {D. Herbert et al., Methods Microbioi., 1979 , 5B, 209-344), using pmLPS as a standard.
Protein concentration was measured by the Lowry's spectrophotometric method, using BSA as a standard and total acidic hydrolysis (6 N HCl at 110°C for 20 h), using norleucine as an internal standard.
Referenceprotein isolated Glucides Ul/eight Total Glucidelprotein conjugatecontent yieldlproteindeterminationGlucideslweightGlucide(mmolelmmole) (enthrone protein content (mglmg) reagent) /pmLPS D1 lnaba CG0203-673.30 46% 1.69 mg 0.51 34% 13.1 mg Batch CG0203-843.54 48% 2.00 mg 0.56 36% 14.4 mg Batch CG0203-873.25 45% 0.47 mg 0.15 13% 3.7 mg Batch Evaluation of the antigenkity of compound (3) and of pmLPS-Mal-TT batch 7 ELISA inhibition experiments conducted with Inaba LPS were in full agreement with the conservation of an antigenic determinant common to both Inaba and Ogawa serotype (Figure 5).
Antigenicity of pmLPS-Mal-TT was analyzed by double immunodiffusion (Figure 6). A single precipitate, characteristic of the reaction between a glycoconjugate and an antibody, was detected when pmLPS-Mal-TT was tested against mAb I-24-2 (Figure 6, wells 1 and 3). The immuno-precipitation line is fused with those observed when LPS Jnaba or pmLPS lnaba (1a) are tested against the same mAb (Figure 6, wells 1 and 2 and wells 1 and 4, respectively), indicating immunochemical identity for the three oligosaccharides.
Example 3: Synthesis of squarate-functionalized p~nLPSs 01 Inaba according to the invention; use of thereof for their conjugation to a protein carrier Synthesis of squarate-functionalized pmLPS 01 Inaba (4) (Figure 10}
Reduced pmLPS 01 Inaba (1c), (4 mg, 0.6 pmoJe), was reacted with ' 21 dimethylsquarate (1.90 mg, 12 Nmoles, 20 equiv), in potassium phosphate buffer saline, pH 7.3 (400 pL) containing CH3CN (50 pL) for 8 hours at room temperature.
The crude mixture was then dialysed against H2O (3 x 2 L), diluted with water, freezed and freeze-dried to give squarate functionalized-pmLPS 01 Inaba (4) as a white powder which was used without further purification.
Negative MALD1-TOF-MS: m/z 7172, 6925, 6677, 6430, 6182, 5687, 5439, 5192, (M - H)-, (22-mer to 18-mer and 16-mer to 14-mer).
Derivafizafion of reduced pmLPS 01 with dimefhylsquarafe and adipic acid di-hydrazide: Synthesis of derivative 5 (Figure 10) Reduced pmLPS 01 lnaba (7c), (7.6 mg, 1.2 Nmole), was reacted with dimethyisquarate (2.7 mg, 18 Nmol, 15 equiv), in potassium phosphate buffer saline 0.1 M, pH 7.3 (800 pL) containing CH3CN (100 pL) for 8 hours at room temperature.
To the crude mixture was then added adipic acid di-hydrazide (16.4 mg, 94 pmol, 75 equiv) and the reaction was carried out for 24 hours at room temperature.
Hydrazido squarate activated-pmLPS 01 Inaba (5) (8.60 mg), was obtained as a white powder following purification by RP-HPLC and freeze-drying of the collected fractions.
Negative MALDI-TOF-MS: m/z 7314, 7067, 6819, 6572, 6324 (M - H)', (22-mer to mer).
Preparation and characterization of the conjugates (Figure 4) Tetanus toxoid (5 mg, 242 pL of the stock solution) and squarate-modified pmLPS 01 Inaba (4), (2.34 mg, 11 equiv), were dissolved in 0.1 M sodium borate buffer, pH 9 (160 pL) and the reaction mixture was stirred at room temperature for 48 h. The conjugated product was dialyzed against 3 x 2 L of 0.05 M PBS, pH 7.4 at 4°C, and further purified by gel permeation chromatography on a sepharose CL-6B
column (1 m x 160 mm) (Pharmacia Biotech), using 0.05 M PBS, pH 7.4 as eluent at a flow rate of 0.2 mL.min-~, with detection by measuring the optical density at 280 nm and the refractive index. The conjugate referred as pmLPS-sq-TT was stored at 4°C
in the presence of thimerosal (0.1 mg.mL-'), and assessed for its tots( carbohydrate and protein content.
Tetanus toxoid (3.7 mg, 180 pL of the stock solution) and hydrazidosquarafie-modified pmLPS 01 inaba (5), (2.55 mg, 15 equiv), were dissolved in 0.2 M
potassium phosphate buffer, pH 6 ( 120 pL). 1-ethyl-3-(3-dimethylaminopropyi)carbodiimide (EDAC) (0.48 mg, 100 equiv) was added to the solution and the reaction mixture was allowed to stir at room temperature for 8 h. The conjugated product was dialyzed against 3 x 2 L of 0.05 M PBS, pH 7.4 at 4°C, and further purified by gel permeation chromatagraphy on a sepharose CL-6B column (1 m x 160 mm) (Pharmacia Biotech), using 0.05 M PBS, pH 7.4 as eluent at a flow rate of 0.2 mL.min~', with detection by measuring the optical density at 280 nm and the refractive index. The conjugate referred as pmLPS-sqhdz-TT was stored at 4°C in the presence of thimerosai (0.1 mg.mL-'), and assessed for its total carbohydrate and protein content.
Reference proteinisolated Glucides Weight Total Glucidelprotein conjugate contentyieldlproteindeterminationGlucideslweightGlucide(mmolelmmole) (anthrone protein content (mg/mg) reagent) IpmLPS 01 Inaba CG0703-'! 2.40 50% 0.80 mg 0.33 25% 2.7 30 mg pm LPS-Sq-TT
CG0703-131 2.77 75% 2.00 mg 0.72 22% 5.50 mg pmLPS-Sqhdz-Example 4: Synthesis of S-acetylthioacetyl-functionalized pmLPSs 01 Inaba according to the invention; use of thereof for their conjugation to a protein carrier 6-acetylthiohexanoic acid succinimidyl esfer (6) (Figure 7) To a stirred solution of 5-acetylthiohexanoic acid (500 mg, 2.63 mmol, 1 equiv) and N-hydroxysuccinimide (302 mg, 2.63 mmol, 1 equiv) in CH2C12 (3 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide {EDAC) (504 mg, 2.63 mmol, 1 equiv}, and the mixture was allowed to stir at room temperature averr~ight.
The crude reaction mixture was diluted with CHzCl2, and this solution washed with 1 N aq HCI
and brine. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish a crude residue which was purified by rapid chromatography on silica gel (eluent: CH2CI2lAcOEt 96:4).
Succinimidyl ester (fi} (642 mg, 85%) was finally obtained as an pale yellow oil.
Rf = 0.75 (AcOEtlCH2CI2 4:96): ~H NMR (400 MHz, CDCI3) D 2.86 (t, 2 H, J = 7.1 Hz), 2.78 (br s, 4 H), 2.60 (t, 2 H, J = 7.4 Hz), 2.31 (s, 3 H), 1.75 (q, 2 H, J = 7.4 Hz), 1.63-1.56 and 1.52-1.43 (m, 2 H}; '3C NMR (100 MHz, CDCl3) 0 196.2, 169.6 (2 C), 168.8, 31.1, 31.0, 29.4, 29.1, 28.2, 25.9 (2 C), 24.5; CI-MS: m/z 305 [M +
NH4]+, 288 [M + H]+; Anal. Calcd for C,2H~7NO5S: C, 50.16; H, 5.96; N, 4.87; O, 27.84; S, 11.16.
Found: C, 50.14; H, 5.96; N, 4.89.
fi-j(6-acetylfhioohexanoyl)aminoJcaproic acid {7} (Figure 7) To a stirred solution of 6-acetylthiohexanoic acid sucinimidyl ester (6) (150 mg, 0.52 mmol, 1 equiv) in DMF {1 mL) was successively added 6-aminocaproic acid (100 mg, 0.76 mmol, 1.5 equiv) and Et3N (291 pL, 2.08 mmol, 4 equiv), and the mixture was allowed to stir at room temperature for 4 h. The crude reaction mixture was diluted with CH2CIz, and this solution washed 1 N aq HCI. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish the acid 7 in a quantitative yield as a film which was used without further purification.
Rf= 0.70 (AcOEt):'H NMR (400 MHz, CDCI3) p3.18 (t, 2 H, J = 7.0 Hz), 2.89 (t, 2 H, J = 7.3 Hz), 2.33-2.27 (m, 2 H), 2.32 (s, 3 H), 2.19 (t, 2 H, J = 7.4 Hz), 1.68-1.56 (m, 6 H), 1.54 (q, 2 H, J = 7.3 Hz), 1.46-1.34 (m, 4 H}; ~3C NMR {100 MHz, CDCI3) ~ 196.8, 177.5, 173.7, 39.5, 36.9, 34.0, 31.0, 29.6, 29.2, 28.6, 26.6, 25.5, 24.6; CI-MS: m/z 321 [M + NHS.]~', 304 [M + H]+.
6-j(6-acetylfhiohexanoyl)amino]caproic acid succinimidyf ester (8) (Figure 7) To a stirred solution of 6-[{6-acetyithiohexanoyl)amino]caproic acid {7) (88 z4 mg, 0.29 mmol, 1 equiv) and N-hydroxysuccinimide (33 mg, 0.38 mmol, 1 equiv) in CHCl2 (500 pL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (56 mg, 0.38 mmol, 1 equiv), and the mixture was allowed to stir at room temperature for 3 h. The crude reaction mixture was diluted with CH2Cl2, and this solution washed with, 1 N aq HCl and brine. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish the succinimidyl ester (8), (116 mg, quantitative} as an oil which was used without further purification.
Rf = 0.63 (AcOEtICHzCi2 4:96}: ~ H NMR (400 MHz, CDCI3) 0 f~ CJ p ~.y J = 6.1 Hz), 3.28 (dt, 2 H, J = 6.1 and 6.7 Hz), 2.92-2.85 {m, 2 H), 2.88 (br s, 4 H), 2.65 (t, 2 H, J = 7.1 Hz ), 2.34 (s, 3 H), 2.19 (t, 2 H, J = 7.4 Hz), 1.70 (q, 2 H, J =
Preparation and characterization of fhe conjugates (Figure 4) Maleimide activated-pmLPS was added to the appropriate modified TT in 0.1 M potassium phosphate buffer solution at a 16:1 molar ratio. Reaction mixtures were buffered at a 0.5M concentration by addition of 1 M potaissium phosphate buffer, pH
6Ø Then, NH20H, HCl (10 pL of a 2 M solution in 1 M potassium phosphate buffer, pH 6), was added to the different mixtures and the couplings were carried out for 2 h at room temperature. The conjugated products were dialyzed against 3 x 2 L of 0.05 M PBS, pH 7.4 at 4°C, and further purified by gel permeation chromatography on a sepharose CL-6B column (1 m x 160 mm) (Pharmacia Biotech), using 0.05 M PBS, pH 7.4 as eluent at a flow rate of 0.2 mL.min-', with detection by measuring the optical density at 280 nm and the refractive index. The conjugates referred as pmLPS-Mal-TT batch 1 and batch 2 were stored at 4°C in the presence of thimerosal (0.1 mg.mL-'), and assessed for their total carbohydrate and protein content.
Hexose concentration was measured by a colorimetric method based on the enthrone reaction {D. Herbert et al., Methods Microbioi., 1979 , 5B, 209-344), using pmLPS as a standard.
Protein concentration was measured by the Lowry's spectrophotometric method, using BSA as a standard and total acidic hydrolysis (6 N HCl at 110°C for 20 h), using norleucine as an internal standard.
Referenceprotein isolated Glucides Ul/eight Total Glucidelprotein conjugatecontent yieldlproteindeterminationGlucideslweightGlucide(mmolelmmole) (enthrone protein content (mglmg) reagent) /pmLPS D1 lnaba CG0203-673.30 46% 1.69 mg 0.51 34% 13.1 mg Batch CG0203-843.54 48% 2.00 mg 0.56 36% 14.4 mg Batch CG0203-873.25 45% 0.47 mg 0.15 13% 3.7 mg Batch Evaluation of the antigenkity of compound (3) and of pmLPS-Mal-TT batch 7 ELISA inhibition experiments conducted with Inaba LPS were in full agreement with the conservation of an antigenic determinant common to both Inaba and Ogawa serotype (Figure 5).
Antigenicity of pmLPS-Mal-TT was analyzed by double immunodiffusion (Figure 6). A single precipitate, characteristic of the reaction between a glycoconjugate and an antibody, was detected when pmLPS-Mal-TT was tested against mAb I-24-2 (Figure 6, wells 1 and 3). The immuno-precipitation line is fused with those observed when LPS Jnaba or pmLPS lnaba (1a) are tested against the same mAb (Figure 6, wells 1 and 2 and wells 1 and 4, respectively), indicating immunochemical identity for the three oligosaccharides.
Example 3: Synthesis of squarate-functionalized p~nLPSs 01 Inaba according to the invention; use of thereof for their conjugation to a protein carrier Synthesis of squarate-functionalized pmLPS 01 Inaba (4) (Figure 10}
Reduced pmLPS 01 Inaba (1c), (4 mg, 0.6 pmoJe), was reacted with ' 21 dimethylsquarate (1.90 mg, 12 Nmoles, 20 equiv), in potassium phosphate buffer saline, pH 7.3 (400 pL) containing CH3CN (50 pL) for 8 hours at room temperature.
The crude mixture was then dialysed against H2O (3 x 2 L), diluted with water, freezed and freeze-dried to give squarate functionalized-pmLPS 01 Inaba (4) as a white powder which was used without further purification.
Negative MALD1-TOF-MS: m/z 7172, 6925, 6677, 6430, 6182, 5687, 5439, 5192, (M - H)-, (22-mer to 18-mer and 16-mer to 14-mer).
Derivafizafion of reduced pmLPS 01 with dimefhylsquarafe and adipic acid di-hydrazide: Synthesis of derivative 5 (Figure 10) Reduced pmLPS 01 lnaba (7c), (7.6 mg, 1.2 Nmole), was reacted with dimethyisquarate (2.7 mg, 18 Nmol, 15 equiv), in potassium phosphate buffer saline 0.1 M, pH 7.3 (800 pL) containing CH3CN (100 pL) for 8 hours at room temperature.
To the crude mixture was then added adipic acid di-hydrazide (16.4 mg, 94 pmol, 75 equiv) and the reaction was carried out for 24 hours at room temperature.
Hydrazido squarate activated-pmLPS 01 Inaba (5) (8.60 mg), was obtained as a white powder following purification by RP-HPLC and freeze-drying of the collected fractions.
Negative MALDI-TOF-MS: m/z 7314, 7067, 6819, 6572, 6324 (M - H)', (22-mer to mer).
Preparation and characterization of the conjugates (Figure 4) Tetanus toxoid (5 mg, 242 pL of the stock solution) and squarate-modified pmLPS 01 Inaba (4), (2.34 mg, 11 equiv), were dissolved in 0.1 M sodium borate buffer, pH 9 (160 pL) and the reaction mixture was stirred at room temperature for 48 h. The conjugated product was dialyzed against 3 x 2 L of 0.05 M PBS, pH 7.4 at 4°C, and further purified by gel permeation chromatography on a sepharose CL-6B
column (1 m x 160 mm) (Pharmacia Biotech), using 0.05 M PBS, pH 7.4 as eluent at a flow rate of 0.2 mL.min-~, with detection by measuring the optical density at 280 nm and the refractive index. The conjugate referred as pmLPS-sq-TT was stored at 4°C
in the presence of thimerosal (0.1 mg.mL-'), and assessed for its tots( carbohydrate and protein content.
Tetanus toxoid (3.7 mg, 180 pL of the stock solution) and hydrazidosquarafie-modified pmLPS 01 inaba (5), (2.55 mg, 15 equiv), were dissolved in 0.2 M
potassium phosphate buffer, pH 6 ( 120 pL). 1-ethyl-3-(3-dimethylaminopropyi)carbodiimide (EDAC) (0.48 mg, 100 equiv) was added to the solution and the reaction mixture was allowed to stir at room temperature for 8 h. The conjugated product was dialyzed against 3 x 2 L of 0.05 M PBS, pH 7.4 at 4°C, and further purified by gel permeation chromatagraphy on a sepharose CL-6B column (1 m x 160 mm) (Pharmacia Biotech), using 0.05 M PBS, pH 7.4 as eluent at a flow rate of 0.2 mL.min~', with detection by measuring the optical density at 280 nm and the refractive index. The conjugate referred as pmLPS-sqhdz-TT was stored at 4°C in the presence of thimerosai (0.1 mg.mL-'), and assessed for its total carbohydrate and protein content.
Reference proteinisolated Glucides Weight Total Glucidelprotein conjugate contentyieldlproteindeterminationGlucideslweightGlucide(mmolelmmole) (anthrone protein content (mg/mg) reagent) IpmLPS 01 Inaba CG0703-'! 2.40 50% 0.80 mg 0.33 25% 2.7 30 mg pm LPS-Sq-TT
CG0703-131 2.77 75% 2.00 mg 0.72 22% 5.50 mg pmLPS-Sqhdz-Example 4: Synthesis of S-acetylthioacetyl-functionalized pmLPSs 01 Inaba according to the invention; use of thereof for their conjugation to a protein carrier 6-acetylthiohexanoic acid succinimidyl esfer (6) (Figure 7) To a stirred solution of 5-acetylthiohexanoic acid (500 mg, 2.63 mmol, 1 equiv) and N-hydroxysuccinimide (302 mg, 2.63 mmol, 1 equiv) in CH2C12 (3 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide {EDAC) (504 mg, 2.63 mmol, 1 equiv}, and the mixture was allowed to stir at room temperature averr~ight.
The crude reaction mixture was diluted with CHzCl2, and this solution washed with 1 N aq HCI
and brine. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish a crude residue which was purified by rapid chromatography on silica gel (eluent: CH2CI2lAcOEt 96:4).
Succinimidyl ester (fi} (642 mg, 85%) was finally obtained as an pale yellow oil.
Rf = 0.75 (AcOEtlCH2CI2 4:96): ~H NMR (400 MHz, CDCI3) D 2.86 (t, 2 H, J = 7.1 Hz), 2.78 (br s, 4 H), 2.60 (t, 2 H, J = 7.4 Hz), 2.31 (s, 3 H), 1.75 (q, 2 H, J = 7.4 Hz), 1.63-1.56 and 1.52-1.43 (m, 2 H}; '3C NMR (100 MHz, CDCl3) 0 196.2, 169.6 (2 C), 168.8, 31.1, 31.0, 29.4, 29.1, 28.2, 25.9 (2 C), 24.5; CI-MS: m/z 305 [M +
NH4]+, 288 [M + H]+; Anal. Calcd for C,2H~7NO5S: C, 50.16; H, 5.96; N, 4.87; O, 27.84; S, 11.16.
Found: C, 50.14; H, 5.96; N, 4.89.
fi-j(6-acetylfhioohexanoyl)aminoJcaproic acid {7} (Figure 7) To a stirred solution of 6-acetylthiohexanoic acid sucinimidyl ester (6) (150 mg, 0.52 mmol, 1 equiv) in DMF {1 mL) was successively added 6-aminocaproic acid (100 mg, 0.76 mmol, 1.5 equiv) and Et3N (291 pL, 2.08 mmol, 4 equiv), and the mixture was allowed to stir at room temperature for 4 h. The crude reaction mixture was diluted with CH2CIz, and this solution washed 1 N aq HCI. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish the acid 7 in a quantitative yield as a film which was used without further purification.
Rf= 0.70 (AcOEt):'H NMR (400 MHz, CDCI3) p3.18 (t, 2 H, J = 7.0 Hz), 2.89 (t, 2 H, J = 7.3 Hz), 2.33-2.27 (m, 2 H), 2.32 (s, 3 H), 2.19 (t, 2 H, J = 7.4 Hz), 1.68-1.56 (m, 6 H), 1.54 (q, 2 H, J = 7.3 Hz), 1.46-1.34 (m, 4 H}; ~3C NMR {100 MHz, CDCI3) ~ 196.8, 177.5, 173.7, 39.5, 36.9, 34.0, 31.0, 29.6, 29.2, 28.6, 26.6, 25.5, 24.6; CI-MS: m/z 321 [M + NHS.]~', 304 [M + H]+.
6-j(6-acetylfhiohexanoyl)amino]caproic acid succinimidyf ester (8) (Figure 7) To a stirred solution of 6-[{6-acetyithiohexanoyl)amino]caproic acid {7) (88 z4 mg, 0.29 mmol, 1 equiv) and N-hydroxysuccinimide (33 mg, 0.38 mmol, 1 equiv) in CHCl2 (500 pL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (56 mg, 0.38 mmol, 1 equiv), and the mixture was allowed to stir at room temperature for 3 h. The crude reaction mixture was diluted with CH2Cl2, and this solution washed with, 1 N aq HCl and brine. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish the succinimidyl ester (8), (116 mg, quantitative} as an oil which was used without further purification.
Rf = 0.63 (AcOEtICHzCi2 4:96}: ~ H NMR (400 MHz, CDCI3) 0 f~ CJ p ~.y J = 6.1 Hz), 3.28 (dt, 2 H, J = 6.1 and 6.7 Hz), 2.92-2.85 {m, 2 H), 2.88 (br s, 4 H), 2.65 (t, 2 H, J = 7.1 Hz ), 2.34 (s, 3 H), 2.19 (t, 2 H, J = 7.4 Hz), 1.70 (q, 2 H, J =
7.4 Hz), 1.85-1.36 (m, 10 H);'3C NMR (100 MHz, CDCI3) D 196.4, 173.4, 169.7 (2 C}, 168.8, 43.4, 36.7, 31.2, 31.0, 29.7, 29.3, 29.2, 28.7, 26.1; 26.0 (2 C), 25.5, 24.6; CI-MS:
m/z 418 [M + NH4]+, 401 [M + H]~.
Derivatization of the pmLPS 09 lnaba (Ta) wifh succinimidyl esters 6 and 8 (Figure 10) To a solution of pmLPS 01 Inaba (1.5 to 2.5 pmole), in potasium phosphate buffer saline 0.2M, pH 7.3, (900-1500 pL) was added the succinimidyl esters 6 or 8 [3 x 5 equiv dissolved in CH3CN {50 pL}], in three portions every 2 hours.
Following an additional reaction period of 2 hours, the crude reaction mixture was purified by RP-HPLC. The collected fractions were diluted with H20, frozen and freeze-dried to give corresponding activated-pmLPS 01 Inaba as white foams.
prnLPS 01 Inaba-C6SAc (9) (3.7 mg, 28%) was obtained together with starting material (5.5 mg, 42%); (negative MALDI-TOF-MS) m/z 7227, 6978, 6731, 6483, 6238, 5990, 5744, 5497, 5250, 5002 (M - H)', (22-mer to 12-mer).
pmLPS 01 Inaba-Cl2SAc ('t0) (5.2 mg, 33%) was obtained together with starting material (4.3 mg, 29%); (negative MALDI-TOF-MS) m/z 7089, 8843, 6593, 6347, 6100 (M - H)- {21-mer to 17-mer).
Derivafization of the tetanus toxoid Stock solution of tetanus toxoid (24 rng, 1165 pL, 0.42 pmole) was diluted with 0.2 M potassium phosphate bufFer saline, pH 7.3 (840 pL). To this solution was added succinimidyl bromoacetate (3 x 1.90 mg dissolved in 50 irL of CH3CN, 3 x equiv), in three portions every 2 h. Following an additional reaction period of 4 h, the crude reaction mixture was dialyzed against 0.1 M potassium phosphate buffer, pH
6.5 (3 x 2 L} at 4°C to eliminate the excess of reagent.
Preparation and characterization of the conjugates Protocol A
IV-bromoacetylated TT (720 pL), was diluted with 0.1 M phosphate buffer, pH
7.3 (280 pL). pH of the soltion was adjusted to 7 by addition of 2 M NaOH. The mixture was degassed and added under Ag(g) to S-acetylthio-activated-oligosaccharides 9 or 10 at a 1:12 molar ratio. Then 2 M hydroxylamine in 0.1 M
potassium phosphate buffer, pH 7 (7.5 pL), and the reaction mixtures were stirred at room temperature for 30 h. The conjugated products were dialyzed against 3 x 2 L of 0.05 M PBS, pH 7.4 at 4°C, and further purified by gel permeation chromatography on a sepharose CL-6B column (1 m x 160 mm) (Pharmacia Biotech), using 0.05 M
PBS, pH 7.4 as eluent at a flow rate of 0.2 mL.min-', with detection by measuring the optical density at 280 nm and the refractive index. The conjugate referred as pmLPS-sqhdz-TT was stored at 4°C in the presence of thimerosal (0.1 mg.mL-~), and assessed for its total carbohydrate and protein content.
Protocol B
N bromoacetylated TT (270 pL), was diluted with 0.1 M NaHC03 buffer, pH
m/z 418 [M + NH4]+, 401 [M + H]~.
Derivatization of the pmLPS 09 lnaba (Ta) wifh succinimidyl esters 6 and 8 (Figure 10) To a solution of pmLPS 01 Inaba (1.5 to 2.5 pmole), in potasium phosphate buffer saline 0.2M, pH 7.3, (900-1500 pL) was added the succinimidyl esters 6 or 8 [3 x 5 equiv dissolved in CH3CN {50 pL}], in three portions every 2 hours.
Following an additional reaction period of 2 hours, the crude reaction mixture was purified by RP-HPLC. The collected fractions were diluted with H20, frozen and freeze-dried to give corresponding activated-pmLPS 01 Inaba as white foams.
prnLPS 01 Inaba-C6SAc (9) (3.7 mg, 28%) was obtained together with starting material (5.5 mg, 42%); (negative MALDI-TOF-MS) m/z 7227, 6978, 6731, 6483, 6238, 5990, 5744, 5497, 5250, 5002 (M - H)', (22-mer to 12-mer).
pmLPS 01 Inaba-Cl2SAc ('t0) (5.2 mg, 33%) was obtained together with starting material (4.3 mg, 29%); (negative MALDI-TOF-MS) m/z 7089, 8843, 6593, 6347, 6100 (M - H)- {21-mer to 17-mer).
Derivafization of the tetanus toxoid Stock solution of tetanus toxoid (24 rng, 1165 pL, 0.42 pmole) was diluted with 0.2 M potassium phosphate bufFer saline, pH 7.3 (840 pL). To this solution was added succinimidyl bromoacetate (3 x 1.90 mg dissolved in 50 irL of CH3CN, 3 x equiv), in three portions every 2 h. Following an additional reaction period of 4 h, the crude reaction mixture was dialyzed against 0.1 M potassium phosphate buffer, pH
6.5 (3 x 2 L} at 4°C to eliminate the excess of reagent.
Preparation and characterization of the conjugates Protocol A
IV-bromoacetylated TT (720 pL), was diluted with 0.1 M phosphate buffer, pH
7.3 (280 pL). pH of the soltion was adjusted to 7 by addition of 2 M NaOH. The mixture was degassed and added under Ag(g) to S-acetylthio-activated-oligosaccharides 9 or 10 at a 1:12 molar ratio. Then 2 M hydroxylamine in 0.1 M
potassium phosphate buffer, pH 7 (7.5 pL), and the reaction mixtures were stirred at room temperature for 30 h. The conjugated products were dialyzed against 3 x 2 L of 0.05 M PBS, pH 7.4 at 4°C, and further purified by gel permeation chromatography on a sepharose CL-6B column (1 m x 160 mm) (Pharmacia Biotech), using 0.05 M
PBS, pH 7.4 as eluent at a flow rate of 0.2 mL.min-', with detection by measuring the optical density at 280 nm and the refractive index. The conjugate referred as pmLPS-sqhdz-TT was stored at 4°C in the presence of thimerosal (0.1 mg.mL-~), and assessed for its total carbohydrate and protein content.
Protocol B
N bromoacetylated TT (270 pL), was diluted with 0.1 M NaHC03 buffer, pH
8.3 (400 pL) containing pmLPS 01 Inaba-C6SAc (9) (4.66 mg, 0.70 pmol, 12 equiv.). To this solution were added successively NH20H, HCI (15 pL of 2 M
solution in NaHC03, pH 8.3) and nBu3P (10 NL of 1 M solution in iPrOH). The reaction mixtures was allowed to stir at room temperature for 30 h. The conjugated product was dialyzed against 3 x 2 L of 0.05 M PBS, pH 7.4 at 4°C, and further purified by gel permeation chromatography on a sepharose CL-6B column (1 m x 160 mm) (Pharmacia Biotech), using 0.05 M PBS, pH 7.4 as eluent at a flow rate of 0.2 mL.min~~, with detection by measuring the optical density at 280 nm and the refractive index. The conjugate referred as pmLPS-sqhdz-TT was stored at 4°C in the presence of thimerosal (0.1 mg.mL-~), and assessed for its total carbohydrate and protein content.
Reference proteinisolated Glucides Weight Totat Glucidelprotein conjugate contentyield/proteindeterminationGlucideslweightGlucide(mmolelmmole) (anthrone protein content (mglmg) reagent}
lpmLPS 01 Inaba CG0703-149 2,06 52% 0.33 mg 0.16 14% 4 mg protocol A
pmLPS-SC6-TT
CG0203-150 1.80 45% 0.26 mg 0.14 13% 2.8 mg protocol A
pmLPS-SC12-TT
CG0204-176 1.71 57% 0.70 mg 0.41 29% 10 mg protocol B
pmLPS-SC6-TT
Example 5: Synthesis of azido-functionaliaed pmLPSs a1 Inaba according to the invention; use of thereof for their conjugation to a protein carrier 6-azidohexanoic acid (11 ) (Figure 7) To a stirred solution of 6-bromohexanoic acid (1.5 g, 7.7 rnmol, 1 equiv) in DMF (5 mL) was added sodium azide (1 g, 15.4 mmol, 2 equiv), and the mixture was heated at 85°C for 3 h. The crude reaction mixture was diluted in CH2CI2, and this solution washed with 0.1 N aq HCI. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish 6-azidohexanoic acid (11 ) (1 g, 82%) as an oil which was used without further purification.
Rf= 0.63 (AcOEtICH2Cl2 3:7):'H NMR (400 MHz, CDCI3) ~ 11.00 (br s, 1 H), 3.29 (t, 2 H, J = 6.9 Hz), 2.39 (t, 2 H, J = 7.4 Hz), 1.73-1.58 (m, 4 H), 1.49-1.40 (m, 2 H); ~3C NMR (100 MHz, CDCl3) 0 180.3, 51.6, 34.2, 28.9, 26.5, 24.5; Cl-MS: m/z [M + N H4] fi' .
6-azidohexanoic acid succinimidyl esfer (12) (Figure 7) To a stirred solution of 6-azidohexanoic acid (11 ) (590 mg, 3.76 mmol, 1 equiv) and N hydroxysuccinimide (432 mg, 3.76 mmol, 1 equiv) in CHC131DMF 9:1 (1 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (720 mg, 3.76 mmol, 1 equiv), and the mixture was allowed to stir at room temperature overnight. The crude reaction mixture was diluted with CH2C12, and this solution washed with 1 N aq HCI, 5% aq NaHC03, and brine. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish the succinimidyl ester (12) (760 mg, 80%) as an oil which was used without further purification.
Rf = 0.76 (AcOEt/CH2Cl2 3:7): 'H NMR (400 MHz, CDCl3) D 3.29 (t, 2 H, J =
6.8 Hz), 2.82 (br s, 4 H), 2.62 (t, 2 H, J = 7.4 Hz), 1.77 (q, 2 H, J = 7.4 Hz), 1.68-1.56 (m, 2 H), 1.54-1.44 {m, 2 H); '~C NMR {100 MHz, CDC13) 0 169.6 (2 C), 168.8, 51.5, 31.1, 28.8, 26.2, 26.0 (2 C), 24.5; Cl-MS: m/z 272 [M + NH4]+; Anal. Calcd for C~pH~4N4O4: C, 47.24; H, 5.55; N, 22.04; O, 25.17. Found: C, 47.11; H, 5.55;
N, 22.05.
6-((6-azidohexanoyl)aminoJcaproic acid (13) (Figure 7) To a stirred solution of 6-azidohexanoic acid sucinimidyl ester (12) (215 mg, 0.85 mmol, 1 equiv) in DMF (1 mL) was successively added 6-arninocaproic acid (162 mg, 0.85 mmol, 1 equiv) and Et3N (235 pL, 1.69 rnmol, 2 equiv), and the mixture was allowed to stir at room temperature far 4 h. The crude reaction mixture was diluted with CH2Ci2, and this solution washed 1 N aq HCI. The organic layer was dried over anhydrous Na2S04, filtered and concentrafied under reduced pressure to furnish the acid (13) in a quantitative yield as an oil which was used without further purification.
Rf= 0.49 (AcOEt): ~H NMR (400 MHz, CDCI~) D 7.80 (br s, 1 H), 5.87 (br s, 1 H), 3.29-3.18 (m, 4 H), 2.31 (t, 2 H, J = 7.3 Hz), 2.18 (t, 2 H, J = 7.4 Hz}, 1.67-1.55 (m, 6 H), 1.50 (q, 2 H, J = 7.3 Hz), 1.41-1.31 (m, 4 H); '3C NMR (100 MHz, CDCI3) D
177.5, 173.7, 51.6, 39.7, 36.5, 34.2, 29.5, 28.9, 26.6, 25.6, 24.7; CI-MS: m/z 271 [M
+ NH4]+.
6-((fi-azidohexanoyl~amino]caproic acid succinimidyl esfer ('!4) (Figure 7) To a stirred solution of 6-[(6-azidohexanoyl)amino]caproic acid (13} (104 mg, 0.38 mmol, 1 equiv) and N hydroxysuccinimide (42 mg, 0.38 mmol, 1 equiv) in CHCl2 (1 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (71 mg, 0.38 mmol, 1 equiv), and the mixture was allowed to stir at room temperature overnight. The crude reaction mixture was diluted with CH2CI2, and this solution washed with, 1 N aq HCI, 5°!o aq NaHC03 and brine. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish the succinimidyi ester (14) (123 mg, 87%) as an oil which was used without further purification.
Rf = 0.30 (AcOEtICH2C12 3:97): ~H NMR (400 MHz, CDCl3) 0 5.75 (br s, 1 H), 3.31-3.22 (m, 4 H), 2.84 (br s, 4 H), 2.62 (t, 2 H, J = 7.3 Hz), 2.18 (t, 2 H, J = 7.4 Hz), 1.77 (q, 2 H, J = 7.4 Hz), 1.69-1.35 (m, 10 H); '3C NMR (100 MHz, CDCI3) 0 173.2, 169.7 (2 C), 168.9, 51.6, 39.3, 36.8, 31.2, 29.2, 29.0, 26.7, 26.1, 26.0 (2 C), 25.6, 24.6; C(-MS: m/z 385 [M + NH4]+, 368 [M + H]+; Anal. Calcd for C~rH25N505,113H20:
C, 51.46; H, 6.93; N, 18.76; O, 22.85. Found: C, 51.68; H, 6.62; N, 18.58.
2-(Diphenyiphosphany!)phenyl glufarafe (15) (Figure 7) Reference for the first step: O. Herd, A. Hessler, P. Machnitzki, M. Tepper, O.
Steizer, Cafal. Today, 1998, 42, 413-420.
2-lodophenol (810 mg, 3.68 mmol, 1 equiv) and potassium acetate (722 mg, 7.36 mmol, 2 equiv) were dissolved in DMF (10 mL). The mixture was degassed under vacuum and, Ag(g) was bubbled through the solution for 30 min. After addition of palladium(II) acetate (1 mg) and diphenylphosphine (0.637 mL, 3.68 mmol, 1 equiv), ° 29 the reaction mixture was heated at 110 °C for 16 h. The solvent was removed under reduced pressure and the orange residue was diluted in CH2CI2 and washed with HCI. The organic layer was dried over Na2S04, filtered and concentrated under reduced pressure.
Part of the crude residue (758 mg) was dissolved in dry DMF (3 mL) under Ag(g) at 0 °C and sodium hydride {60% dispersion in oil, 120 mg, 3 mmol, 1:1 equiv) was added in several portions. The reaction mixture was stirred at 0 °C
for 20 min and, solid glutaric anhydride (391 mg, 2.73 mmol, 1 equiv) was added. After 30 min at 0 °C, the reaction mixture was poured over 0.1 N HCI and extracted with CH2CI2.
The organic layer was dried over Na2S04, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (eluent: cyclohexane/AcOEt 8:2) to give the acid {15) (786 mg, 74% for the two steps) as an oil.
Rf = 0.46 (AcOEtlcyclohexane 3:7): 'H NMR (400 MHz, CDCI~) 0 10.81 (br s, 1 H), 7.83-7.74, 7.68-7.58, 7.55-7.49, 7.48-7.36, 7.27-7.13 and 7.94-7.90 (m, 14 H), 2.48-2.40 (m, 4 H), 1.92 (q, 2 H, J = 7.2 Hz); '3C NMR (100 MHz, CDCI3) Cl 179.4, 171.3, 153:1 (d, J= 17.1 Hz), 136.0-129.1 (12 C), 126.7, 123.1, 33.3 (2 C), 19.8; 31P
NMR (162 MHz, CDCI3) 0 -14.8; positive Cl-MS: m/z 393 [M + NHa1*
Succinimidyl 2-(diphenylphosphanyl)phenyl glutarate (1G) (Figure 7) To a stirred solution of 2-{diphenylphosphanyl)phenyl glutarate (15) {418 mg, 1.07 mmol, 1 equiv) and IV-hydroxysuccinimide (122 mg, 1.07 mmol, 1 equiv) in CHCI2 {4 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (205 mg, 1.07 mmol, 1 equiv), and the mixture was allowed to stir at room temperature overnight. The crude reaction mixture was diluted with CH2C12, and this solution washed with 0.1 N aq HCI and brine. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude residue was purified by chromatography on silica gel (eiuent: CH2C12IAcOEt 96:4) to furnish the succinimidyl ester (1S) (470 mg, 90%) as an oil.
Rf = 0.71 (AcOEt/CH2Cl2 4:96): 'H NMR (400 MHz, CDC13) ~ 7.4.1-7.32 {m, 11 H), 7.17 (t, 1 H, J = 3.8 Hz), 7.15 (t, 1 H, J = 7.1 Hz), 6.88-6.85 (m, 1 H), 2.74 (br s, 4 H), 2.59 (t, 2 H, J = 7.3 Hz), 2.43 (t, 2 H, J = 7.2 Hz), 1.93 (q, 2 H, J = 7.2 Hz); ~3C NMR
' 30 (100 MHz, CDCIs) ~ 170.8, 169.6 (2 C), 168.5, 153.0 (d, 1 C, J = 17.3 Hz), 135.8 (d, 2 C, J = 10.0 Hz ), 134.4 (d, 4 C, J = 20.6 Hz), 134.1 (d, 1 C, J = 0.9 Hz), 130.7 (d, 1 C, J = 14.7 Hz), 130.4, 129.6 (2 C), 129.1 (d, 4 C, J = 7.3 Hz), 126.7, 123.0, 32.8, 30.3, 26.0 (2 C), 19.8; 3~P NMR (162 MHz, CDCl3) 0 -15.0; positive FAB-MS: m/z 522.2 [M + Na]~, 490.2 jM + H]+; Anal. Calcd for C~7H24N06P,1I3H20: C, 65.45;
H, 5.02; N, 2.83; O, 20.45; P, 6.25. Found: C, 65.54; H, 4.87; N, 2.73; O, 20.19.
Derivatization of the pmLPS 09 Inaba (9a) with succinirnidyl esters 92 and94 (Figure 10) To a solution of pmLPS 01 Inaba (1a) (1.5 to 3.6 pmole), in 0.2 M potassium phosphate buffer saline, pH 7.3, (900-1500 pL) was added the succinimidyl ester 12 or 14 [3 x 10 equiv dissolved in CH3CN (50 pL)], in fihree portions every 2 hours.
Following an additional reaction period of 2 hours, the crude reaction mixture was purified by RP-HPLC (gradient: 0% B for 5 min then 0-30% B over 60 min). The collected fractions were diluted with H20, frozen and freeze-dried to give corresponding activated-pmLPS 01 lnaba 17 and 18 as white foams.
pmLPS 01 Inaba-C6N3 (17) {8.5 mg, 38%) was obtained together with starting material (5.2 mg, 24%); (negative MALDI-TOF-MS) m/z 7197, 6951, 6702, 6455, 6207, 5960, 5713, 5466, 5219, 4970 (M - H)', 7169, 6922, 6675, 6429, 6181, 5933, 5686, 5438, 5192 (M - N2 - H)' (22-mer to 14-mer).
pmLPS 01 Inaba-C12N3 (18) {7.3 mg, 58%) was obtained together with starting material (2.0 mg, 20%); {negative MALDI-TOF-MS) m/z 7312, 7066, 6814, 6572, 6321, 6070 (M - H)' (22-mer to 17-mer).
Derivatization of the tetanus toxoid with succinimidyl ester 9B
Stock solution of tetanus toxoid (8 mg, 390 NL, 0.16 pmole) was diluted with 0.2 M potassium phosphate buffer saline, pH 7.3 (1 mL). To this solution was added succinimidyl 2-(diphenyfphosphanyl)phenyl glutarate (~I6) (3 x 3.84 mg dissolved in 50 pL of CH3CN, 3 x 50 equiv), in three portions every 2 h. Following an additional reaction period of 4 h, the crude reaction mixture was dialyzed against 0.2 M
potassium phosphate buffer saline, pH 7.3 (3 x 2 L) at 4°C to eliminate excess reagent.
Preparation and characterization of fhe conjugates Azido-activated-oligosaccharides (17) or (18) were added in one portion to the phospine-functionalized TT in solution in 0.1 M phosphate buffer, phi 7.3 at a 12:1 molar ratio. the reaction mixture were heated at 47°C for 6 h and then, dialyzed against 0.1 M potassium phosphate buffer saline, pH 7.4 (3 x 2 L) at 4°C, and further purified by gel permeation chromatography on a sepharose CL-6B column (1 m x 160 mm) (Pharmacia Biotech), using 0.1 M potassium phosphate buffer saline, pH
7.4 as eluent at a flow rate of 0.2 mL.min-', with detection by measuring the optical density at 280 nm and the refractive index. The fractions containing the conjugates were pooled and concentrated by using Vivaspin 15R centrifuge! concentrators (Vivascience France, Palaiseau), displaying a membrane cut-off of 10000 Da, at a centrifugal force of 5000 x g. The conjugate was stored at 4°C in the presence of thimerosal (0.1 mg.mL-~) and assessed for its total carbohydrafie and protein content.
Reference conjugateproteinisolated Glucides Weight Total Glucidelprotein contentyield/proteindeterminatioGlucides/weightGlucide(mmolelmmole) n protein content (mglmg) (enthrone reagent) /pmLPS
Inaba CG0703-151 2.35 59% 0.26 mg 0.11 9% 2.8 pmLPS-C6NHC0-TTmg CG0203-152 3.31 83% 0.41 mg 0.12 11 3 %
pmLPS-C12NHC0-mg TT
Exarnpie 6: Mono-biotinylation of the pmLPS 01, serotype Ogawa according to the invention; preparation of derivatives useful for the immuno-detection A solution of pm~.PS 01 Ogawa (1 b) (5.74 mg, 0.94 pmole) in PBS 0.1 M, pH
7.3 (600 pL), was added to an eppendorf containing solid sulfo-NHS-Ic-Ic-biotin (2.61 mg, 4 equiv.). The reaction mixture was transferred to another eppendorf containing solid sulfo-NHS-Ic-Ic-biotin (2.61 mg, 4 equiv.) after two hours. This operation was repeated once two hours later. After an additional two hours time, the crude reaction mixture was purified by RP-HPLC. The collected fractions were diluted with H20, frozen and freeze-dried to give mono-biotinylated pmLPS 01Ogawa (1.70 mg, 28%
yield), as a white powder.
pmLPS 01 Ogawa: (negative MALDI-TOF-MS) m/z 7522, 7276, 7028, 6781, 6533, 6286, 6040, 5792, 5546 (M - H)', 7494, 7248, 7000, 6753, 6506, 6258, 6011, 5764, 5517 (M - H-CO)', (22-mer to 15-mer).
Example 7: Mono-biotinylation of the pmLPS 01, serotype Ogaviia according to the invention; preparation of derivatives useful for the immuno-detection A solution of pmLPS 0139 (4 mg, 9 .70 pmole) in PBS 0.1 M, pH 7.3 (800 NL) and CH3CN (200 pL), was added to an eppendorf containing solid NHS-lc-Ic-biotin (4.83 mg, 5 equiv.). The reaction mixture was transferred to another eppendorf containing solid NHS-Ic-Ic-biotin (4.83 mg, 5 equiv.) after 1 h30min. This operation was repeated once 1 h30min later. After an additional th30min time, the crude reaction mixture was purified by RP-HPLC. The collected fractions were diluted with H20, frozen and freeze-dried to give mono-biotinylated pmLPS 0139 (0.60 mg, 13%
yield), as a white powder.
pmLPS 0139: (negative MALDI-TOF-MS) m/z 2888 [M-Fru+Ac]', 2758 [M-Fru-Col+Ac]- , 2696 [M-Fru-2Col+Ac]'. (See Figures 9 and 8)
solution in NaHC03, pH 8.3) and nBu3P (10 NL of 1 M solution in iPrOH). The reaction mixtures was allowed to stir at room temperature for 30 h. The conjugated product was dialyzed against 3 x 2 L of 0.05 M PBS, pH 7.4 at 4°C, and further purified by gel permeation chromatography on a sepharose CL-6B column (1 m x 160 mm) (Pharmacia Biotech), using 0.05 M PBS, pH 7.4 as eluent at a flow rate of 0.2 mL.min~~, with detection by measuring the optical density at 280 nm and the refractive index. The conjugate referred as pmLPS-sqhdz-TT was stored at 4°C in the presence of thimerosal (0.1 mg.mL-~), and assessed for its total carbohydrate and protein content.
Reference proteinisolated Glucides Weight Totat Glucidelprotein conjugate contentyield/proteindeterminationGlucideslweightGlucide(mmolelmmole) (anthrone protein content (mglmg) reagent}
lpmLPS 01 Inaba CG0703-149 2,06 52% 0.33 mg 0.16 14% 4 mg protocol A
pmLPS-SC6-TT
CG0203-150 1.80 45% 0.26 mg 0.14 13% 2.8 mg protocol A
pmLPS-SC12-TT
CG0204-176 1.71 57% 0.70 mg 0.41 29% 10 mg protocol B
pmLPS-SC6-TT
Example 5: Synthesis of azido-functionaliaed pmLPSs a1 Inaba according to the invention; use of thereof for their conjugation to a protein carrier 6-azidohexanoic acid (11 ) (Figure 7) To a stirred solution of 6-bromohexanoic acid (1.5 g, 7.7 rnmol, 1 equiv) in DMF (5 mL) was added sodium azide (1 g, 15.4 mmol, 2 equiv), and the mixture was heated at 85°C for 3 h. The crude reaction mixture was diluted in CH2CI2, and this solution washed with 0.1 N aq HCI. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish 6-azidohexanoic acid (11 ) (1 g, 82%) as an oil which was used without further purification.
Rf= 0.63 (AcOEtICH2Cl2 3:7):'H NMR (400 MHz, CDCI3) ~ 11.00 (br s, 1 H), 3.29 (t, 2 H, J = 6.9 Hz), 2.39 (t, 2 H, J = 7.4 Hz), 1.73-1.58 (m, 4 H), 1.49-1.40 (m, 2 H); ~3C NMR (100 MHz, CDCl3) 0 180.3, 51.6, 34.2, 28.9, 26.5, 24.5; Cl-MS: m/z [M + N H4] fi' .
6-azidohexanoic acid succinimidyl esfer (12) (Figure 7) To a stirred solution of 6-azidohexanoic acid (11 ) (590 mg, 3.76 mmol, 1 equiv) and N hydroxysuccinimide (432 mg, 3.76 mmol, 1 equiv) in CHC131DMF 9:1 (1 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (720 mg, 3.76 mmol, 1 equiv), and the mixture was allowed to stir at room temperature overnight. The crude reaction mixture was diluted with CH2C12, and this solution washed with 1 N aq HCI, 5% aq NaHC03, and brine. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish the succinimidyl ester (12) (760 mg, 80%) as an oil which was used without further purification.
Rf = 0.76 (AcOEt/CH2Cl2 3:7): 'H NMR (400 MHz, CDCl3) D 3.29 (t, 2 H, J =
6.8 Hz), 2.82 (br s, 4 H), 2.62 (t, 2 H, J = 7.4 Hz), 1.77 (q, 2 H, J = 7.4 Hz), 1.68-1.56 (m, 2 H), 1.54-1.44 {m, 2 H); '~C NMR {100 MHz, CDC13) 0 169.6 (2 C), 168.8, 51.5, 31.1, 28.8, 26.2, 26.0 (2 C), 24.5; Cl-MS: m/z 272 [M + NH4]+; Anal. Calcd for C~pH~4N4O4: C, 47.24; H, 5.55; N, 22.04; O, 25.17. Found: C, 47.11; H, 5.55;
N, 22.05.
6-((6-azidohexanoyl)aminoJcaproic acid (13) (Figure 7) To a stirred solution of 6-azidohexanoic acid sucinimidyl ester (12) (215 mg, 0.85 mmol, 1 equiv) in DMF (1 mL) was successively added 6-arninocaproic acid (162 mg, 0.85 mmol, 1 equiv) and Et3N (235 pL, 1.69 rnmol, 2 equiv), and the mixture was allowed to stir at room temperature far 4 h. The crude reaction mixture was diluted with CH2Ci2, and this solution washed 1 N aq HCI. The organic layer was dried over anhydrous Na2S04, filtered and concentrafied under reduced pressure to furnish the acid (13) in a quantitative yield as an oil which was used without further purification.
Rf= 0.49 (AcOEt): ~H NMR (400 MHz, CDCI~) D 7.80 (br s, 1 H), 5.87 (br s, 1 H), 3.29-3.18 (m, 4 H), 2.31 (t, 2 H, J = 7.3 Hz), 2.18 (t, 2 H, J = 7.4 Hz}, 1.67-1.55 (m, 6 H), 1.50 (q, 2 H, J = 7.3 Hz), 1.41-1.31 (m, 4 H); '3C NMR (100 MHz, CDCI3) D
177.5, 173.7, 51.6, 39.7, 36.5, 34.2, 29.5, 28.9, 26.6, 25.6, 24.7; CI-MS: m/z 271 [M
+ NH4]+.
6-((fi-azidohexanoyl~amino]caproic acid succinimidyl esfer ('!4) (Figure 7) To a stirred solution of 6-[(6-azidohexanoyl)amino]caproic acid (13} (104 mg, 0.38 mmol, 1 equiv) and N hydroxysuccinimide (42 mg, 0.38 mmol, 1 equiv) in CHCl2 (1 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (71 mg, 0.38 mmol, 1 equiv), and the mixture was allowed to stir at room temperature overnight. The crude reaction mixture was diluted with CH2CI2, and this solution washed with, 1 N aq HCI, 5°!o aq NaHC03 and brine. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to furnish the succinimidyi ester (14) (123 mg, 87%) as an oil which was used without further purification.
Rf = 0.30 (AcOEtICH2C12 3:97): ~H NMR (400 MHz, CDCl3) 0 5.75 (br s, 1 H), 3.31-3.22 (m, 4 H), 2.84 (br s, 4 H), 2.62 (t, 2 H, J = 7.3 Hz), 2.18 (t, 2 H, J = 7.4 Hz), 1.77 (q, 2 H, J = 7.4 Hz), 1.69-1.35 (m, 10 H); '3C NMR (100 MHz, CDCI3) 0 173.2, 169.7 (2 C), 168.9, 51.6, 39.3, 36.8, 31.2, 29.2, 29.0, 26.7, 26.1, 26.0 (2 C), 25.6, 24.6; C(-MS: m/z 385 [M + NH4]+, 368 [M + H]+; Anal. Calcd for C~rH25N505,113H20:
C, 51.46; H, 6.93; N, 18.76; O, 22.85. Found: C, 51.68; H, 6.62; N, 18.58.
2-(Diphenyiphosphany!)phenyl glufarafe (15) (Figure 7) Reference for the first step: O. Herd, A. Hessler, P. Machnitzki, M. Tepper, O.
Steizer, Cafal. Today, 1998, 42, 413-420.
2-lodophenol (810 mg, 3.68 mmol, 1 equiv) and potassium acetate (722 mg, 7.36 mmol, 2 equiv) were dissolved in DMF (10 mL). The mixture was degassed under vacuum and, Ag(g) was bubbled through the solution for 30 min. After addition of palladium(II) acetate (1 mg) and diphenylphosphine (0.637 mL, 3.68 mmol, 1 equiv), ° 29 the reaction mixture was heated at 110 °C for 16 h. The solvent was removed under reduced pressure and the orange residue was diluted in CH2CI2 and washed with HCI. The organic layer was dried over Na2S04, filtered and concentrated under reduced pressure.
Part of the crude residue (758 mg) was dissolved in dry DMF (3 mL) under Ag(g) at 0 °C and sodium hydride {60% dispersion in oil, 120 mg, 3 mmol, 1:1 equiv) was added in several portions. The reaction mixture was stirred at 0 °C
for 20 min and, solid glutaric anhydride (391 mg, 2.73 mmol, 1 equiv) was added. After 30 min at 0 °C, the reaction mixture was poured over 0.1 N HCI and extracted with CH2CI2.
The organic layer was dried over Na2S04, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (eluent: cyclohexane/AcOEt 8:2) to give the acid {15) (786 mg, 74% for the two steps) as an oil.
Rf = 0.46 (AcOEtlcyclohexane 3:7): 'H NMR (400 MHz, CDCI~) 0 10.81 (br s, 1 H), 7.83-7.74, 7.68-7.58, 7.55-7.49, 7.48-7.36, 7.27-7.13 and 7.94-7.90 (m, 14 H), 2.48-2.40 (m, 4 H), 1.92 (q, 2 H, J = 7.2 Hz); '3C NMR (100 MHz, CDCI3) Cl 179.4, 171.3, 153:1 (d, J= 17.1 Hz), 136.0-129.1 (12 C), 126.7, 123.1, 33.3 (2 C), 19.8; 31P
NMR (162 MHz, CDCI3) 0 -14.8; positive Cl-MS: m/z 393 [M + NHa1*
Succinimidyl 2-(diphenylphosphanyl)phenyl glutarate (1G) (Figure 7) To a stirred solution of 2-{diphenylphosphanyl)phenyl glutarate (15) {418 mg, 1.07 mmol, 1 equiv) and IV-hydroxysuccinimide (122 mg, 1.07 mmol, 1 equiv) in CHCI2 {4 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (205 mg, 1.07 mmol, 1 equiv), and the mixture was allowed to stir at room temperature overnight. The crude reaction mixture was diluted with CH2C12, and this solution washed with 0.1 N aq HCI and brine. The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude residue was purified by chromatography on silica gel (eiuent: CH2C12IAcOEt 96:4) to furnish the succinimidyl ester (1S) (470 mg, 90%) as an oil.
Rf = 0.71 (AcOEt/CH2Cl2 4:96): 'H NMR (400 MHz, CDC13) ~ 7.4.1-7.32 {m, 11 H), 7.17 (t, 1 H, J = 3.8 Hz), 7.15 (t, 1 H, J = 7.1 Hz), 6.88-6.85 (m, 1 H), 2.74 (br s, 4 H), 2.59 (t, 2 H, J = 7.3 Hz), 2.43 (t, 2 H, J = 7.2 Hz), 1.93 (q, 2 H, J = 7.2 Hz); ~3C NMR
' 30 (100 MHz, CDCIs) ~ 170.8, 169.6 (2 C), 168.5, 153.0 (d, 1 C, J = 17.3 Hz), 135.8 (d, 2 C, J = 10.0 Hz ), 134.4 (d, 4 C, J = 20.6 Hz), 134.1 (d, 1 C, J = 0.9 Hz), 130.7 (d, 1 C, J = 14.7 Hz), 130.4, 129.6 (2 C), 129.1 (d, 4 C, J = 7.3 Hz), 126.7, 123.0, 32.8, 30.3, 26.0 (2 C), 19.8; 3~P NMR (162 MHz, CDCl3) 0 -15.0; positive FAB-MS: m/z 522.2 [M + Na]~, 490.2 jM + H]+; Anal. Calcd for C~7H24N06P,1I3H20: C, 65.45;
H, 5.02; N, 2.83; O, 20.45; P, 6.25. Found: C, 65.54; H, 4.87; N, 2.73; O, 20.19.
Derivatization of the pmLPS 09 Inaba (9a) with succinirnidyl esters 92 and94 (Figure 10) To a solution of pmLPS 01 Inaba (1a) (1.5 to 3.6 pmole), in 0.2 M potassium phosphate buffer saline, pH 7.3, (900-1500 pL) was added the succinimidyl ester 12 or 14 [3 x 10 equiv dissolved in CH3CN (50 pL)], in fihree portions every 2 hours.
Following an additional reaction period of 2 hours, the crude reaction mixture was purified by RP-HPLC (gradient: 0% B for 5 min then 0-30% B over 60 min). The collected fractions were diluted with H20, frozen and freeze-dried to give corresponding activated-pmLPS 01 lnaba 17 and 18 as white foams.
pmLPS 01 Inaba-C6N3 (17) {8.5 mg, 38%) was obtained together with starting material (5.2 mg, 24%); (negative MALDI-TOF-MS) m/z 7197, 6951, 6702, 6455, 6207, 5960, 5713, 5466, 5219, 4970 (M - H)', 7169, 6922, 6675, 6429, 6181, 5933, 5686, 5438, 5192 (M - N2 - H)' (22-mer to 14-mer).
pmLPS 01 Inaba-C12N3 (18) {7.3 mg, 58%) was obtained together with starting material (2.0 mg, 20%); {negative MALDI-TOF-MS) m/z 7312, 7066, 6814, 6572, 6321, 6070 (M - H)' (22-mer to 17-mer).
Derivatization of the tetanus toxoid with succinimidyl ester 9B
Stock solution of tetanus toxoid (8 mg, 390 NL, 0.16 pmole) was diluted with 0.2 M potassium phosphate buffer saline, pH 7.3 (1 mL). To this solution was added succinimidyl 2-(diphenyfphosphanyl)phenyl glutarate (~I6) (3 x 3.84 mg dissolved in 50 pL of CH3CN, 3 x 50 equiv), in three portions every 2 h. Following an additional reaction period of 4 h, the crude reaction mixture was dialyzed against 0.2 M
potassium phosphate buffer saline, pH 7.3 (3 x 2 L) at 4°C to eliminate excess reagent.
Preparation and characterization of fhe conjugates Azido-activated-oligosaccharides (17) or (18) were added in one portion to the phospine-functionalized TT in solution in 0.1 M phosphate buffer, phi 7.3 at a 12:1 molar ratio. the reaction mixture were heated at 47°C for 6 h and then, dialyzed against 0.1 M potassium phosphate buffer saline, pH 7.4 (3 x 2 L) at 4°C, and further purified by gel permeation chromatography on a sepharose CL-6B column (1 m x 160 mm) (Pharmacia Biotech), using 0.1 M potassium phosphate buffer saline, pH
7.4 as eluent at a flow rate of 0.2 mL.min-', with detection by measuring the optical density at 280 nm and the refractive index. The fractions containing the conjugates were pooled and concentrated by using Vivaspin 15R centrifuge! concentrators (Vivascience France, Palaiseau), displaying a membrane cut-off of 10000 Da, at a centrifugal force of 5000 x g. The conjugate was stored at 4°C in the presence of thimerosal (0.1 mg.mL-~) and assessed for its total carbohydrafie and protein content.
Reference conjugateproteinisolated Glucides Weight Total Glucidelprotein contentyield/proteindeterminatioGlucides/weightGlucide(mmolelmmole) n protein content (mglmg) (enthrone reagent) /pmLPS
Inaba CG0703-151 2.35 59% 0.26 mg 0.11 9% 2.8 pmLPS-C6NHC0-TTmg CG0203-152 3.31 83% 0.41 mg 0.12 11 3 %
pmLPS-C12NHC0-mg TT
Exarnpie 6: Mono-biotinylation of the pmLPS 01, serotype Ogawa according to the invention; preparation of derivatives useful for the immuno-detection A solution of pm~.PS 01 Ogawa (1 b) (5.74 mg, 0.94 pmole) in PBS 0.1 M, pH
7.3 (600 pL), was added to an eppendorf containing solid sulfo-NHS-Ic-Ic-biotin (2.61 mg, 4 equiv.). The reaction mixture was transferred to another eppendorf containing solid sulfo-NHS-Ic-Ic-biotin (2.61 mg, 4 equiv.) after two hours. This operation was repeated once two hours later. After an additional two hours time, the crude reaction mixture was purified by RP-HPLC. The collected fractions were diluted with H20, frozen and freeze-dried to give mono-biotinylated pmLPS 01Ogawa (1.70 mg, 28%
yield), as a white powder.
pmLPS 01 Ogawa: (negative MALDI-TOF-MS) m/z 7522, 7276, 7028, 6781, 6533, 6286, 6040, 5792, 5546 (M - H)', 7494, 7248, 7000, 6753, 6506, 6258, 6011, 5764, 5517 (M - H-CO)', (22-mer to 15-mer).
Example 7: Mono-biotinylation of the pmLPS 01, serotype Ogaviia according to the invention; preparation of derivatives useful for the immuno-detection A solution of pmLPS 0139 (4 mg, 9 .70 pmole) in PBS 0.1 M, pH 7.3 (800 NL) and CH3CN (200 pL), was added to an eppendorf containing solid NHS-lc-Ic-biotin (4.83 mg, 5 equiv.). The reaction mixture was transferred to another eppendorf containing solid NHS-Ic-Ic-biotin (4.83 mg, 5 equiv.) after 1 h30min. This operation was repeated once 1 h30min later. After an additional th30min time, the crude reaction mixture was purified by RP-HPLC. The collected fractions were diluted with H20, frozen and freeze-dried to give mono-biotinylated pmLPS 0139 (0.60 mg, 13%
yield), as a white powder.
pmLPS 0139: (negative MALDI-TOF-MS) m/z 2888 [M-Fru+Ac]', 2758 [M-Fru-Col+Ac]- , 2696 [M-Fru-2Col+Ac]'. (See Figures 9 and 8)
Claims (16)
1) An isolated or purified derivative compound of the detoxified LPS having the formula:
1a pmLPS O1 Inaba: R = H X,X' = O
1b pmLPS O1 Ogawa: R = Me X,X' = O
1c reduced pmLPS O1 Inaba: = H X,X' = OH,H
wherein the derivatization is carried out onto the NH2 group.
1a pmLPS O1 Inaba: R = H X,X' = O
1b pmLPS O1 Ogawa: R = Me X,X' = O
1c reduced pmLPS O1 Inaba: = H X,X' = OH,H
wherein the derivatization is carried out onto the NH2 group.
2) The isolated or purified derivative compound of the detoxified LPS
according to claim 1, wherein the derivatization is carried out by means of a ligand.
according to claim 1, wherein the derivatization is carried out by means of a ligand.
3) The isolated or purified derivative compound of the detoxified LPS
according to claim 2, wherein said ligand is directly labelled by a dye (visible or UV), a colored particle, a pigment or bears at least a radioactive atom.
according to claim 2, wherein said ligand is directly labelled by a dye (visible or UV), a colored particle, a pigment or bears at least a radioactive atom.
4) The isolated or purified derivative compound of the detoxified LPS
according to claim 2, wherein the ligand is a linker molecule.
according to claim 2, wherein the ligand is a linker molecule.
5) The isolated or purified derivative compound of the detoxified BPS
according to claim 4, wherein said linker is selected among the group consisting of the following R structures:
according to claim 4, wherein said linker is selected among the group consisting of the following R structures:
6) A glycoconjugate comprising an isolated or purified derivative compound according to claim 1 linked to a carrier.
7) The glycoconjugate of claim 6, wherein the carrier is a polypeptide or a protein.
8) The glycoconjugate of claim 6 or 7, wherein the carrier bears at least one T (helper epitope.
9) The glycoconjugate of claim 7 or 8, wherein the carrier is selected among the group consisting of : Tetanus Toxoid (TT), Human albumin (HA) and polypeptide PADRE.
10) The glycoconjugate according to any one of claims 1 to 6, wherein the LPS
is derived from V.cholera selected among serotypes O139 or O1.
is derived from V.cholera selected among serotypes O139 or O1.
11) An immunogenic composition for eliciting an immune response or a protective immunity against a Vibrio cholerae infection comprising at least a glycoconjugate according to any one of claims 6 to 10.
12) A purified polyclonal or monoclonal antibody capable of specifically binding to a glycoconjugate according to any one of claims 6 to 10.
13) A method for treating and/or preventing a Vibrio cholerae infection in an animal, the method comprising the step of administering to the animal an effective amount of a glycoconjugate according to any one of claims 6 to 10, an immunogenic composition of claim 11 or an antibody as defined in claim 12.
14) A method for immunizing an animal against a Vibrio cholerae infection, comprising the step of administering to the animal an effective amount of an immunogenic composition as defined in claim 11.
15) A method for detecting the presence or absence of a Vibrio cholerae strain in a sample, comprising the steps of:
a) contacting the sample with an antibody as defined in claim 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).
a) contacting the sample with an antibody as defined in claim 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).
16) The method of claim 15, wherein the V. cholera strain is selected among serotypes O139 or O1.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002475736A CA2475736A1 (en) | 2004-07-23 | 2004-07-23 | Vibrio cholerae lps detoxified derivatives and immunogenic compositions containing them |
| CA002513024A CA2513024A1 (en) | 2004-07-23 | 2005-07-22 | Vibrio cholerae lps detoxified derivatives and immunogenic compositions containing them |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002475736A CA2475736A1 (en) | 2004-07-23 | 2004-07-23 | Vibrio cholerae lps detoxified derivatives and immunogenic compositions containing them |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2475736A1 true CA2475736A1 (en) | 2006-01-23 |
Family
ID=35696144
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002475736A Abandoned CA2475736A1 (en) | 2004-07-23 | 2004-07-23 | Vibrio cholerae lps detoxified derivatives and immunogenic compositions containing them |
| CA002513024A Abandoned CA2513024A1 (en) | 2004-07-23 | 2005-07-22 | Vibrio cholerae lps detoxified derivatives and immunogenic compositions containing them |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002513024A Abandoned CA2513024A1 (en) | 2004-07-23 | 2005-07-22 | Vibrio cholerae lps detoxified derivatives and immunogenic compositions containing them |
Country Status (1)
| Country | Link |
|---|---|
| CA (2) | CA2475736A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011528710A (en) * | 2008-07-21 | 2011-11-24 | ザ ブライハム アンド ウイメンズ ホスピタル， インコーポレイテッド | Methods and compositions for synthesizing β-1,6-glucosamine oligosaccharides |
| US10919956B2 (en) | 2002-11-12 | 2021-02-16 | The Brigham And Women's Hospital, Inc. | Polysaccharide vaccine for staphylococcal infections |
| CN116249720A (en) * | 2020-03-13 | 2023-06-09 | 赫菲斯托斯制药公司 | Detoxified lipopolysaccharide (LPS), natural non-toxic LPS and uses thereof |
-
2004
- 2004-07-23 CA CA002475736A patent/CA2475736A1/en not_active Abandoned
-
2005
- 2005-07-22 CA CA002513024A patent/CA2513024A1/en not_active Abandoned
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10919956B2 (en) | 2002-11-12 | 2021-02-16 | The Brigham And Women's Hospital, Inc. | Polysaccharide vaccine for staphylococcal infections |
| JP2011528710A (en) * | 2008-07-21 | 2011-11-24 | ザ ブライハム アンド ウイメンズ ホスピタル， インコーポレイテッド | Methods and compositions for synthesizing β-1,6-glucosamine oligosaccharides |
| CN105085349A (en) * | 2008-07-21 | 2015-11-25 | 布赖汉姆妇女医院 | Methods and compositions relating to synthetic beta-1,6 glucosamine oligosaccharides |
| US9474806B2 (en) | 2008-07-21 | 2016-10-25 | The Brigham And Women's Hospital, Inc. | Methods and compositions relating to synthetic beta-1,6 glucosamine oligosaccharides |
| CN105085349B (en) * | 2008-07-21 | 2018-02-09 | 布赖汉姆妇女医院 | The method and composition related to the glucosamine oligosaccharides of β 1,6 of synthesis |
| US10034927B2 (en) | 2008-07-21 | 2018-07-31 | The Brigham And Women's Hospital, Inc. | Methods and compositions relating to synthetic beta-1,6 glucosamine oligosaccharides |
| US11123416B2 (en) | 2008-07-21 | 2021-09-21 | The Brigham And Women's Hospital, Inc. | Methods and compositions relating to synthetic beta-1,6 glucosamine oligosaccharides |
| CN116249720A (en) * | 2020-03-13 | 2023-06-09 | 赫菲斯托斯制药公司 | Detoxified lipopolysaccharide (LPS), natural non-toxic LPS and uses thereof |
| CN116249720B (en) * | 2020-03-13 | 2024-12-03 | 赫菲斯托斯制药公司 | Detoxified lipopolysaccharide (LPS), natural non-toxic LPS and its uses |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2513024A1 (en) | 2006-01-23 |
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