WO2004056851A1 - Peptides mimicking vibrio cholerae o139 capsular polysaccharide and their use to elicit protective immune response - Google Patents

Peptides mimicking vibrio cholerae o139 capsular polysaccharide and their use to elicit protective immune response Download PDF

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WO2004056851A1
WO2004056851A1 PCT/IT2003/000489 IT0300489W WO2004056851A1 WO 2004056851 A1 WO2004056851 A1 WO 2004056851A1 IT 0300489 W IT0300489 W IT 0300489W WO 2004056851 A1 WO2004056851 A1 WO 2004056851A1
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peptide
peptides
cholerae
cooh
capsular polysaccharide
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French (fr)
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Franco Felici
Andrej Weintraub
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Franco Felici
Andrej Weintraub
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Priority to AU2003253301A priority Critical patent/AU2003253301A1/en
Priority to PCT/IT2003/000489 priority patent/WO2004056851A1/en
Publication of WO2004056851A1 publication Critical patent/WO2004056851A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1239—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Vibrionaceae (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to compounds, methods for their preparation, compositions containing them and to their use as medicaments, in particular in the field of infectious diseases, more in particular for the prevention of cholera.
  • Cholera is an acute dehydrating diarrhoeal disease, and epidemics as well as pandemics of cholera are a major health problem in developing countries and cause many deaths each year, especially in children.
  • V. cholerae 0139 has the potential of being responsible for the eighth pandemic of cholera and a vaccine against this serogroup would be of great importance. To date, such a vaccine is not yet available. Prior exposure to V. cholerae 01 does not protect against infection with V. cholerae 0139, thus the 0139 strain is expected to have a selective advantage in regions where 01 infections predominate.
  • V. cholerae 0139 Making available a vaccine against V. cholerae 0139 is very important in endemic areas and also in areas not yet affected by the 0139 to prevent large epidemics in immunologically naive populations.
  • V. cholerae 0139 possesses a capsular pol saccharide as a major cell wall associated antigen. As for majority of encapsulated microorganisms, the immunity against a CPS confers protection against the disease. However, most capsular polysaccharides are T-cell independent type 2 antigens and do not induce immunological memory, neither do they require T cells to induce an immune response. In addition, due to the differences in the structures of cell wall polysaccharides, there is no cross protection from the 01 vaccine.
  • Phage display technology has been used to identify peptides capable of inducing antibodies against a variety of carbohydrate epitopes present on the surface of bacterial pathogens, for example Shigella flexneri, Cryptococcus neoformans, Neisseria meningitidis, Brucella abortus and group B streptococci (G.R. Moe, et al, FEMS Immunology and Medical Microbiology, 26 (1999) 209-226; Charalambous, B.M. & Fea ⁇ ers, I.M., J. Med. Microbiol, 50 937-9, (2001); Partidos, CD., Curr. Opin. Mol.
  • phage display libraries Ther., 2, 74-9 (2000); Meloen, R.H., et al, J. Mol Recognit., 13, 352-9. (2000)).
  • the advantage of phage display libraries is that it allows for rapid screening and identification of reactive peptides.
  • the use of phage display libraries is an efficient and effective way to identify potential vaccine candidates capable of inducing functional, anti- polysaccharide antibodies.
  • WO 99/11660 to Institut Pasteur and INSERM, published on 1999, discloses a purified immunogenic peptide comprising an epitope unit recognized by a protective monoclonal antibody having a high affinity and a high specificity for a surface polysaccharide of a pathogenic microorganism of bacterial, viral or fungal origin.
  • the peptide induces an immune response in vivo against the pathogenic microorganism.
  • Vibrio cholerae serogroups Ol and 0139 are the causative agents of the cholera disease.
  • the major virulence factor in this microorganism is the cholera toxin (CT).
  • CT cholera toxin
  • the V. cholerae 0139 serogroup possesses a capsular polysaccharide (CPS) that has been shown to promote colonization of the microorganism (Nesper, J., et al, Infect. Immun., 70, 5990-6 (2002)). Therefore, it could be surmised that an immune response against the CPS may prevent colonization and therefore the disease.
  • CPS capsular polysaccharide
  • cholerae 0139 has several unique features seldom found in polysaccharides from other micro-organisms (Knirel, Y.A., et al, Eur. J. Biochem., 232, 391-6. (1995)). Since cholera is strictly human pathogen, there are no in vivo animal models for an evaluation of the efficacy of potential vaccine candidates. However, the suckling mouse model, described below is adequate for evaluation of the efficacy of antibodies induced by vaccine candidates. The model is based on the assumption that antibodies prevent the bacteria to colonize the intestine.
  • Object of the present invention are peptide mimics against the capsular polysaccharide of V cholerae 0139.
  • mAbs monoclonal antibodies against the capsular polysaccharide of V. cholerae 0139. Said antibodies are used to screen different phage-displayed random peptide libraries.
  • a further object of the present invention is the use of the above peptide as medicaments, in particular for the preparation of medicaments for the prevention of cholera infections.
  • compositions comprising the above peptides, in particular pharmaceutical compositions.
  • mice We have selected eight different phage clones, characterized them using enzyme immunoassay (EIA) with the mAbs, and then tested the specificity by competition with the V. cholerae 0139 capsular polysaccharide. The peptide sequences were then determined and peptides were synthe- sised and conjugated before immunization of mice.
  • EIA enzyme immunoassay
  • Ncl-1 AEGEFSPGVWKAAFQGDKLPDPAK - SEQ ID No. 1; Vcl-4 : AEGEFYCSGPPDRVCWGPDPAK - SEQ ID No. 2; Vcl-10 : AEGEFPSRMQFHGDQDYGDPAK - SEQ ID No. 3; Vcl-12 : AEGEFMGDICSWCYSSAPDPAK - SEQ ID No. 4; Vcl-22 : AEGEFCSPPDSPGNCGDPAK - SEQ ID No. 5; Vc2-1 : AEGEFSYDMLWMMTPQTGDPAK - SEQ ID No. 6; Vcl2-1 : AEGEFCGFVWPFKCHRIGDPAK - SEQ ID No. 7; Vcl2-2 : AEGEFKVPFRMLNRQVNGDPAK - SEQ ID No. 8.
  • the peptides can also be in cyclic form.
  • the peptides of the present invention are relatively short and can be synthesized and conjugated to different carrier proteins and be used for oral or nasal immunization in order to give rise to a protective mucosal immunity.
  • the application of such a vaccine for the prevention of cholera is novel for the field of enteric vaccines.
  • antigenic portion and equivalent are well understood by the skilled person.
  • antigenic portion means portions which generate an antibody response and the definition of equivalent is given in the above mentioned WO 99/11660.
  • a MAP construct can be an andvantageous embodiment for the purposes of therapeutic application. In any case, the whole technology of vaccines available can be used in the present invention.
  • the peptides of the present invention can be also conjugated with another substance useful in immunization, preferably a protein, such as for example Bovine Serum Albumin or Keyhole Limpet Hemo- cyanin, tetanus toxoid and diphteria toxoids.
  • a protein such as for example Bovine Serum Albumin or Keyhole Limpet Hemo- cyanin, tetanus toxoid and diphteria toxoids.
  • the peptides of the present invention can be prepared by a process comprising the following steps: a) producing a monoclonal antibody against the capsular polysaccharide of V. cholerae 0139; b) using said monoclonal antibody to screen different phage-displayed random peptide libraries; c) isolating the peptides displayed on the surface of the filamentous phages selected in step b).
  • the peptides of the present invention once characterized by their aminoacid sequence, can be scaled up to industrial prepa- ration by classical chemical synthesis, such as disclosed for example Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis by Gregg B. Fields, Sidney P. Colowick, Mel ⁇ in I. Simon (Editors) 1997, Academic Press, Inc., New York.
  • the peptides of the present invention can be also obtained by polynucleotides coding for them.
  • Said polynucleotides can be derived from the amino acid sequence and, once obtained, they can be used for producing the peptide by means of conventional genetic engineering techniques currently used in the field.
  • the peptides of the present invention are used to immunize subjects against Vibrio cholerae 0139.
  • the present invention also provides a diagnostic method for detecting antibodies against Vibrio cholerae 0139 in a subject, for example a subject suspected to be infected or having been infected, the method comprising: a) contacting a biological sample of the subject with at least one peptide of the present invention; b) detecting peptide-antibody complex formation.
  • the peptide can optionally be isolated.
  • the peptides used in the above method will be contained in a suitable diagnostic kit, which is comprised in the scope of this invention.
  • CPS was purified from V. cholerae 0139 AI-1838, a patient isolate from the strain collection at the Laboratory Science Division, International Centre of Diarrhoeal Diseases Research, Bangladesh, Dhaka, Bangladesh, Centre for Health and Population Research, ICDDR,B.
  • LPS from E. coli 0172 CCUG 36540 was used.
  • V. cholerae 0139 strain 1852 also a patient isolate from the strain collection at ICDDR,B.
  • the monoclonal antibodies were prepared basically as described in de St. Groth, S.F. and Scheidegger, D., (1980) J. Immunol Methods 35:1- 21. Briefly, 8-12 weeks old BALB/c female mice were immunized intraperitoneally with 2 x 10 7 heat killed V. cholerae AI-1838 bacteria. After twice booster, fusion of spleen cells from the immunized mice and mouse Sp2/0 cells was carried out three days after last booster dose, using polyethyleneglycol 4000 (PEG 4000, VWR International, Sweden) as the fusing agent. The first screening after 2 weeks was performed by enzyme immunoassay (EIA) against the homologous CPS (V. cholerae AI-1838).
  • EIA enzyme immunoassay
  • the hybridomas secreting antibodies that reacted with the CPS were cloned twice by the limiting dilution method.
  • Antibody classes and subclasses were determined by double radial immunodifusion against rabbit anti-mouse immunoglobulins ⁇ l, ⁇ 2a, ⁇ 2b, ⁇ 3 and ⁇ heavy-chain antisera and rabbit anti-mouse and ⁇ light- chain antiserum (Litton Bionetics, Kensington, MD).
  • the culture supernatants were concentrated 10 times by ammonium sulfate precipitation, followed by intensive dialysis against PBS.
  • V. cholerae strain AI-1838 was grown in a 30 1 fermentor (Belach AB, Sweden) in a rich tryptone-yeast extract (TY-medium) as described in Gustafsson, B., Rosen, A., Holme, T., Infect. Immun. 1982; 38: 449-54 to late logarithmic phase.
  • the culture was checked for purity by Gram-stain and inoculation on blood- and L-agar at the end of the growth cycle and the bacteria killed with formaldehyde (1% wt/vol).
  • the lyophilised aqueous phase after the extraction was subjected to a phenol-chloroform-petroleum ether (PCP) extraction as described earlier (Galanos, C, et al, Eur. J. Biochem., 1969; 9: 245-9; Kasper, D.L., Weintraub, A., Lindberg, A.A., L ⁇ nngren, J., J. BacterioL, 1983; 153: 991-7).
  • PCP phenol-chloroform-petroleum ether
  • the PCP-soluble fraction (containing LPS) was further processed as follows.
  • the chloroform and petroleum ether were removed by evaporation and the LPS was precipitated from the phenol phase by 6 volumes of diethyl ether-acetone (1:5 v/v) (Qureshi, N., et al, J. Biol. Chem. 1982; 257: 11808-15).
  • the CPS was purified from the crude phenol-water extract containing both LPS and CPS.
  • the extract was dissolved in 0.1 M sodium-acetate buffer (pH 4.2), heated for 4 h at 100°C, the precipitate removed by centrifugation, and the CPS isolated by gel-permeation chromatography on Sephadex G-50.
  • the E. coli 0172 was grown as above and the LPS extracted with hot phenol- water extraction (Westphal, O., et al, see above).
  • the LPS was purified from the aqueous phase by treatment with RNase, DNase and Proteinase
  • Peptide mimics were selected by panning six different phage libraries: pVIII9aa, pVIII9aa. Cys, pVIII12aa, pVHI12aa.Cys, pVIIIl ⁇ aa and pVIII-28aa. All libraries have been constructed in the pVIII two- gene/phagemid vector pC89 (Felici, F., et al., J. Mol. Biol,. (1991) 222: 301-310). The libraries are carrying random inserts encoding peptides of various sizes fused into the N-terminal region of the major coat protein (protein VIII) of filamentous phage.
  • protein VIII major coat protein
  • pVIII9aa, pVIII12aa pNIIIl ⁇ aa and pVIII-28aa are four different libraries composed of random 9-mers, 12-mers, 15-mers and 28-mers, respectively, whereas pVIII9aa.Cys and pVHI12aa.Cys are libraries in which the random insert contains two cysteine residues (Luzzago, A., et al, (1993) Gene 128, 51-57). In this latter libraries, cysteines promote the formation of a disulfide bridge that constrains to some extent the conformation of the displayed peptide. All libraries have been generated as described (A. Luzzago and F.
  • phage clones were isolated from the libraries by two rounds of affinity selection as described previously (Felici et al, 1991). In the first round the monoclonal antibody was incubated at 1 ⁇ M concentration overnight at 4°C with 1010 transducing units of library in a total volume of 10 ⁇ l. The mixture was incubated with 0.25 ⁇ g of a biotin-conjugated goat anti-mouse IgG secondary antibody (Fc specific, Sigma, St. Louis, MO), which had been pre-adsorbed overnight at 4°C with 2 x 10 11 phage particles of UV-killed M13K07 to prevent nonspecific binding.
  • Fc specific biotin-conjugated goat anti-mouse IgG secondary antibody
  • the phage-mAb-secondary antibody complexes were bound to streptavidin-coated dishes and bound complexes eluted after a washing step.
  • the second round of biopanning was carried out in the same way, but using 10 nM concentration of mAb and proportionally lower amounts of the secondary antibody. Positive phage clones were identified through plaque immunoscreening (Luzzago, et al (1993)).
  • the phage DNA of the positive clones was sequenced by the dideoxy mediated chain termination method (Sanger, F., et al, (1977) Proc. Natl. Acad. Scl, 74 5463-5467).
  • Bovine Serum Albumin BSA
  • KLH Keyhole Limpet Hemocyanin
  • EIA plates (Costar ® 3590, Corning Inc. Corning, NY) were coated at 4 °C overnight with 100 ⁇ L/well of BSA conjugated peptide, V. cholerae 0139 capsular polysaccharide, and E. coli 0172 lipopolysaccharide (5 ⁇ g/mL in phosphate buffered saline, PBS (10 mM phosphate buffer [pH 7.2], 140 mM NaCl), all in duplicates. After blocking with 0,25% skimmed milk, the plates were washed three times in washing buffer (0,15 M NaCl, 0,05% Tween 20).
  • Plates were incubated at 20 °C and read in a Multiskan Plus EIA reader (Labsystems) at 405 nm after 10, 25, 50 and 100 min. Absorbance values between 0,2 and 1 was chosen and extrapolated to absorbance after 100 min of incubation.
  • mice 2-5 day old Swiss albino mice were separated from the mother two hours before the experiments. The sera were deactivated in 56 °C for 30 minutes. 25 ⁇ l of sera and 10 4 bacteria per mouse were preincubated in room temperature for 30 minutes prior to tube feeding. The mice were then observed for 48 hours.
  • Vc 1, Vc 2 and ICL 12 Three different murine monoclonal antibodies (Vc 1, Vc 2 and ICL 12) have been used in this study. All mAbs, have been tested for specificity using EIA and shown to react only with the homologous antigens, i.e. the CPS and LPS isolated from V. cholerae 0139. Both Vcl and Vc2 are of the IgG ⁇ isotype, whereas the ICL 12 is an IgG 2 b. All three mAbs have been tested in the suckling mouse model described above. The protective efficacy was 100%, 75%, and 83%, respectively.
  • Vcl-1 AEGEFSPGVWKAAFQGDKLPDPAK - SEQ ID No. 1;
  • Vcl-4 AEGEFYCSGPPDRVCWGPDPAK - SEQ ID No. 2;
  • Vcl-10 AEGEFPSRMQFHGDQDYGDPAK - SEQ ID No. 3;
  • Vcl-12 AEGEFMGDICSWCYSSAPDPAK - SEQ ID No. 4;
  • Vcl-22 AEGEFCSPPDSPGVCGDPAK - SEQ ID No. 5;
  • Vc2-1 AEGEFSYDMLWMMTPQTGDPAK - SEQ ID No. 6;
  • Vcl2-1 AEGEFCGFVWPFKCHRIGDPAK - SEQ ID No. 7;Vcl2-2 :
  • the selected peptides have no consensus sequence, they are of different length, some are linear and some have cystein inserts that will form loops.
  • Selected peptides have been sequenced, synthesized and conjugated to Bovine Serum Albumin (BSA) and Keyhole Limpet Hemocyanin (KLH).
  • BSA Bovine Serum Albumin
  • KLH Keyhole Limpet Hemocyanin
  • cholerae 0139 was lower, but still higher than the negative control, in any case sufficient to demonstrate protection, as is shown below. This pattern was the same for all peptides. Although there are no obvious common structural features, the peptides show striking functional similarities, mimic the same antigen and have been identified by using the same procedure, therefore they can be here considered as variants of the same product, and useful for an anti-V. cholera 0139 vaccine.
  • mice The mouse sera against the peptides were further tested for protection in the suckling mouse assay. The results showed that the peptides could induce an antibody response in mice with a protective effect.
  • Vcl-1, Vcl-12 and Vc2-1 showed a protection of 30% or less, Vcl-10, Vcl2-1 and Vcl2-2 a protection of 40-60%, and Vcl-22 and Vcl-4 a protection of 60-70%.
  • Sera against two of the peptides, Vcl-22 and Vcl- 4 were further investigated, and showed a dose dependent decrease in protection when tested in different dilutions in the suckling mouse model.
  • the protective effect could also be neutralised when the sera against the KLH conjugate were adsorbed with the corresponding BSA conjugate.
  • sera from mice immunized with KLH alone or with an S. flexneri peptide conjugate did not show any protective efficacy against V. cholerae 0139 in the suckling mouse model.
  • the antibodies against the peptides react with V. cholerae 0139 CPS and show protective effect against V. cholerae 0139 in the suckling mouse model.
  • the antibody response after immunization is relatively weak against the V. cholerae 0139, explanations for that might be that intrape- ritonally immunization is not ideal or that another adjuvant can be used.
  • mice The results clearly show that the peptides induce a protective immune response in mice. It can therefore, be surmised that these peptides can be used for the preparation of vaccines against the V. cholerae 0139.
  • one possible realisation of the present invention is in the form of diagnostic kits containing the peptides.
  • one possible realisation of the present invention is in the form of vaccines containing the peptides, whose preparation comes within the framework of general knowledge; for further reference purposes the reader is referred to the patent literature cited in the present description.
  • compositions in particular in the form of a vaccine, contain at least one peptide or its derivative as an active ingredient, in an amount such as to produce a significant prophylactic and/or therapeutic effect.
  • the compositions covered by the present invention are entirely conventional and are obtained with methods which are common practice in the pharmaceutical industry, such as, for example, those illustrated in Remington's Pharmaceutical Science Handbook, Mack Pub. N. Y. — latest edition. According to the administration route chosen, the compositions will be in solid or liquid form, suitable for oral, nasal, parenteral or intravenous administration.
  • the compositions according to the present invention contain, along with the active ingredient, at least one pharmaceutically acceptable vehicle or excipient. These may be particularly useful formulation coadjuvants, e.g. solubilising agents, dispersing agents, suspension agents, and emulsifying agents. Particular interest is put on adjuvants used in the formulation of vaccines.

Abstract

The present invention discloses peptide mimics against the capsular polysaccharide of V. cholerae O139, their antigenic portions, their equivalents, MAP construct. These peptides can be conjugated with another substance useful in immunization and used for the preparation of a medicament for the prophylaxis of cholera. Also disclosed are processes for their preparation, pharmaceutical compositions comprising them, in particular a vaccine and a diagnostic kit.

Description

Peptides mimicking Vibrio cholerae 0139 Capsular Polysaccharide and their use to elicit protective immune response
The present invention relates to compounds, methods for their preparation, compositions containing them and to their use as medicaments, in particular in the field of infectious diseases, more in particular for the prevention of cholera.
Background of the invention.
Cholera is an acute dehydrating diarrhoeal disease, and epidemics as well as pandemics of cholera are a major health problem in developing countries and cause many deaths each year, especially in children.
Cholera is caused by Vibrio cholerae 01 and since 1992 also by serogroup 0139 (Albert, M.J., et al, Lancet, 341, 704. (1993); Ramamurthy, T., et al, Lancet, 341, 703-4 (1993)). The 0139 serogroup appeared in 1992 in Southern India and it spread rapidly over the country and to neighbouring countries including Bangladesh. During the epidemic, the normally endemic 01 serogroup was totally displaced, but a new 01 clone emerged in 1993 and again became dominating. The 0139 serogroup disappeared until 1996 when it again became dominating in Calcutta and also reappeared in other parts of India and in Bangladesh. The aetiology of cholera in the future is likely to swing between the two serogroups or a cyclic pattern of predominance of the two serogroups may prevail in cholera endemic areas (Ramamurthy, T., et al., Microbes and Infection, 5, 329-44 (2003)). The V. cholerae 0139 has the potential of being responsible for the eighth pandemic of cholera and a vaccine against this serogroup would be of great importance. To date, such a vaccine is not yet available. Prior exposure to V. cholerae 01 does not protect against infection with V. cholerae 0139, thus the 0139 strain is expected to have a selective advantage in regions where 01 infections predominate.
There is an oral vaccine licensed in many countries of the world for the 01 serogroup, containing inactivated bacteria in combination with the B-subunit of the cholera-toxin. This vaccine gives quite good protection with about 50% protective efficacy after three years of immunization.
Making available a vaccine against V. cholerae 0139 is very important in endemic areas and also in areas not yet affected by the 0139 to prevent large epidemics in immunologically naive populations. There are several vaccine candidates for the V". cholerae 0139 under development. They are based on a glycoconjugate were the CPS has been conjugated to a protein carrier or on inactivated bivalent cholera vaccine comprised of the 01/0139 serogroup and as live oral 0139 vaccine, Bengal 15 (Kossaczka, Z., et al, Glycoconj. J., 17 (2000), 425- 433; Coaster, T.S., et al, Lancet, 345 (1995) 949-952; Jertborn, M., et al, Vaccine, 14 (1996), 1459-65).
None of these vaccine candidates have yet reached the market. One of the reasons could be that the strategies to produce a cholera vaccine against the V. cholerae 0139 are similar to the strategies used previously for the 01 serogroup. Only one vaccine against V. cholerae 01 is at present licensed in Europe and the efficacy of it is rather limited. Another reason for the lack of vaccine against V. cholerae 0139 may be that it was believed that the emergence of this serogroup was a one time event. However, it is now well established that both serogroups do coexist as agents for the cholera disease.
V. cholerae 0139 possesses a capsular pol saccharide as a major cell wall associated antigen. As for majority of encapsulated microorganisms, the immunity against a CPS confers protection against the disease. However, most capsular polysaccharides are T-cell independent type 2 antigens and do not induce immunological memory, neither do they require T cells to induce an immune response. In addition, due to the differences in the structures of cell wall polysaccharides, there is no cross protection from the 01 vaccine.
To circumvent this problem, our approach was to make peptide mimotopes of the V. cholerae 0139 CPS.
Phage display technology has been used to identify peptides capable of inducing antibodies against a variety of carbohydrate epitopes present on the surface of bacterial pathogens, for example Shigella flexneri, Cryptococcus neoformans, Neisseria meningitidis, Brucella abortus and group B streptococci (G.R. Moe, et al, FEMS Immunology and Medical Microbiology, 26 (1999) 209-226; Charalambous, B.M. & Feaυers, I.M., J. Med. Microbiol, 50 937-9, (2001); Partidos, CD., Curr. Opin. Mol. Ther., 2, 74-9 (2000); Meloen, R.H., et al, J. Mol Recognit., 13, 352-9. (2000)). The advantage of phage display libraries is that it allows for rapid screening and identification of reactive peptides. Thus, the use of phage display libraries is an efficient and effective way to identify potential vaccine candidates capable of inducing functional, anti- polysaccharide antibodies.
WO 99/11660, to Institut Pasteur and INSERM, published on 1999, discloses a purified immunogenic peptide comprising an epitope unit recognized by a protective monoclonal antibody having a high affinity and a high specificity for a surface polysaccharide of a pathogenic microorganism of bacterial, viral or fungal origin. The peptide induces an immune response in vivo against the pathogenic microorganism. Although the disclosure of this reference is quite generic, the only enabling embodiment relates to Shigella flexneri.
Vibrio cholerae serogroups Ol and 0139 are the causative agents of the cholera disease. The major virulence factor in this microorganism is the cholera toxin (CT). However, in order to induce the disease, the microorganism needs to colonize the intestine. The V. cholerae 0139 serogroup possesses a capsular polysaccharide (CPS) that has been shown to promote colonization of the microorganism (Nesper, J., et al, Infect. Immun., 70, 5990-6 (2002)). Therefore, it could be surmised that an immune response against the CPS may prevent colonization and therefore the disease. In addition, the CPS of V. cholerae 0139 has several unique features seldom found in polysaccharides from other micro-organisms (Knirel, Y.A., et al, Eur. J. Biochem., 232, 391-6. (1995)). Since cholera is strictly human pathogen, there are no in vivo animal models for an evaluation of the efficacy of potential vaccine candidates. However, the suckling mouse model, described below is adequate for evaluation of the efficacy of antibodies induced by vaccine candidates. The model is based on the assumption that antibodies prevent the bacteria to colonize the intestine.
Several groups have reported identification of carbohydrate mimicking peptides for different bacteria and viruses. There are several advantages of using short peptides as vaccine candidates, they are relatively cheap to produce, easy to store and handle, and most importantly, safe. (Meloen, R.H., Puijk, W.C. & Slootstra, J. W., J. Mol. Recognit. 13, 352- 9. (2000); Charalambous, B.M. & Feaυers, I.M., J. Med. Microbiol 50, 937-9. (2001)).
Abstract of the invention
Object of the present invention are peptide mimics against the capsular polysaccharide of V cholerae 0139.
Also disclosed are monoclonal antibodies (mAbs) against the capsular polysaccharide of V. cholerae 0139. Said antibodies are used to screen different phage-displayed random peptide libraries.
A further object of the present invention is the use of the above peptide as medicaments, in particular for the preparation of medicaments for the prevention of cholera infections.
Still another object of the present invention are compositions comprising the above peptides, in particular pharmaceutical compositions.
These and other objects of the present invention shall be disclosed in detail, also by means of examples. Detailed description of the invention and of the preferred embodiments.
All the definitions used herein are part of the common knowledge of the person skilled in this art and reference is made to the general literature. Specific reference can be made to the above mentioned WO 99/11660 also for the general technical problem of dealing with polysaccharide antigens.
We have produced monoclonal antibodies (mAbs) against the capsular polysaccharide of V. cholerae 0139 and used them to screen different phage-displayed random peptide libraries. Peptide libraries displayed on the surface of filamentous phages have been used to produce mimics to several different bacterial surface polysaccharides (Meloen, R.H., Puijk, W.C. & Slootstra, J.W. J. Mol. Recognit. 13, 352-9. (2000)) that when used for immunization in animals, induce antibodies with the same biological activity as the antibody used as template. We have selected eight different phage clones, characterized them using enzyme immunoassay (EIA) with the mAbs, and then tested the specificity by competition with the V. cholerae 0139 capsular polysaccharide. The peptide sequences were then determined and peptides were synthe- sised and conjugated before immunization of mice.
The preferred peptides are
Ncl-1 : AEGEFSPGVWKAAFQGDKLPDPAK - SEQ ID No. 1; Vcl-4 : AEGEFYCSGPPDRVCWGPDPAK - SEQ ID No. 2; Vcl-10 : AEGEFPSRMQFHGDQDYGDPAK - SEQ ID No. 3; Vcl-12 : AEGEFMGDICSWCYSSAPDPAK - SEQ ID No. 4; Vcl-22 : AEGEFCSPPDSPGNCGDPAK - SEQ ID No. 5; Vc2-1 : AEGEFSYDMLWMMTPQTGDPAK - SEQ ID No. 6; Vcl2-1 : AEGEFCGFVWPFKCHRIGDPAK - SEQ ID No. 7; Vcl2-2 : AEGEFKVPFRMLNRQVNGDPAK - SEQ ID No. 8.
Due to the presence of cys residues, the peptides can also be in cyclic form.
Advantageously, the peptides of the present invention are relatively short and can be synthesized and conjugated to different carrier proteins and be used for oral or nasal immunization in order to give rise to a protective mucosal immunity. The application of such a vaccine for the prevention of cholera is novel for the field of enteric vaccines.
The present invention comprises also antigenic portions and equivalent of the peptides. In this context, antigenic portion and equivalent are well understood by the skilled person. However, just for sake of clarity, antigenic portion means portions which generate an antibody response and the definition of equivalent is given in the above mentioned WO 99/11660. A MAP construct can be an andvantageous embodiment for the purposes of therapeutic application. In any case, the whole technology of vaccines available can be used in the present invention.
The peptides of the present invention can be also conjugated with another substance useful in immunization, preferably a protein, such as for example Bovine Serum Albumin or Keyhole Limpet Hemo- cyanin, tetanus toxoid and diphteria toxoids.
The peptides of the present invention can be prepared by a process comprising the following steps: a) producing a monoclonal antibody against the capsular polysaccharide of V. cholerae 0139; b) using said monoclonal antibody to screen different phage-displayed random peptide libraries; c) isolating the peptides displayed on the surface of the filamentous phages selected in step b).
Conveniently, the peptides of the present invention, once characterized by their aminoacid sequence, can be scaled up to industrial prepa- ration by classical chemical synthesis, such as disclosed for example Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis by Gregg B. Fields, Sidney P. Colowick, Melυin I. Simon (Editors) 1997, Academic Press, Inc., New York.
Given their amino acid sequence, the peptides of the present invention can be also obtained by polynucleotides coding for them.
Said polynucleotides can be derived from the amino acid sequence and, once obtained, they can be used for producing the peptide by means of conventional genetic engineering techniques currently used in the field.
Reference can be made to the above mentioned WO 99/11660 and the literature referenced therein.
The peptides of the present invention are used to immunize subjects against Vibrio cholerae 0139.
The present invention also provides a diagnostic method for detecting antibodies against Vibrio cholerae 0139 in a subject, for example a subject suspected to be infected or having been infected, the method comprising: a) contacting a biological sample of the subject with at least one peptide of the present invention; b) detecting peptide-antibody complex formation.
In the above method, the peptide can optionally be isolated.
The carrying out of this method is performed with conventional techniques and does not need further explanation, being normal practice in clinical laboratories.
Conveniently, in one possible embodiments of the present invention, the peptides used in the above method will be contained in a suitable diagnostic kit, which is comprised in the scope of this invention.
The following example further illustrates the present invention.
Example
Materials and methods
Bacterial strains
CPS was purified from V. cholerae 0139 AI-1838, a patient isolate from the strain collection at the Laboratory Science Division, International Centre of Diarrhoeal Diseases Research, Bangladesh, Dhaka, Bangladesh, Centre for Health and Population Research, ICDDR,B. As negative control, in EIA experiments, LPS from E. coli 0172 CCUG 36540 (Culture Collection, University of Gothenborg, Sweden) was used.
For the suckling mouse experiments we used V. cholerae 0139 strain 1852, also a patient isolate from the strain collection at ICDDR,B.
Production of monoclonal antibodies
The monoclonal antibodies were prepared basically as described in de St. Groth, S.F. and Scheidegger, D., (1980) J. Immunol Methods 35:1- 21. Briefly, 8-12 weeks old BALB/c female mice were immunized intraperitoneally with 2 x 107 heat killed V. cholerae AI-1838 bacteria. After twice booster, fusion of spleen cells from the immunized mice and mouse Sp2/0 cells was carried out three days after last booster dose, using polyethyleneglycol 4000 (PEG 4000, VWR International, Stockholm, Sweden) as the fusing agent. The first screening after 2 weeks was performed by enzyme immunoassay (EIA) against the homologous CPS (V. cholerae AI-1838). The hybridomas secreting antibodies that reacted with the CPS were cloned twice by the limiting dilution method. Antibody classes and subclasses were determined by double radial immunodifusion against rabbit anti-mouse immunoglobulins γl, γ2a, γ2b, γ3 and μ heavy-chain antisera and rabbit anti-mouse and λ light- chain antiserum (Litton Bionetics, Kensington, MD). The culture supernatants were concentrated 10 times by ammonium sulfate precipitation, followed by intensive dialysis against PBS.
Isolation and purification of lipopolysaccharide and capsular polysaccharide from V cholerae 0139
V. cholerae strain AI-1838 was grown in a 30 1 fermentor (Belach AB, Stockholm, Sweden) in a rich tryptone-yeast extract (TY-medium) as described in Gustafsson, B., Rosen, A., Holme, T., Infect. Immun. 1982; 38: 449-54 to late logarithmic phase. The culture was checked for purity by Gram-stain and inoculation on blood- and L-agar at the end of the growth cycle and the bacteria killed with formaldehyde (1% wt/vol). After incubation at 4°C overnight, the bacteria were collected by centrifugation (8,000 x g, 4°C, 20 min) and washed with phosphate buffered saline (PBS, 0.01M, pH 7.3). Pelleted bacteria were then suspended in water and extracted with hot phenol-water (Westphal, O., Lύderitz, O., Bister, F., Z. Naturforsch., 1952; 7: 148-55).
For isolation of the LPS, the lyophilised aqueous phase after the extraction was subjected to a phenol-chloroform-petroleum ether (PCP) extraction as described earlier (Galanos, C, et al, Eur. J. Biochem., 1969; 9: 245-9; Kasper, D.L., Weintraub, A., Lindberg, A.A., Lόnngren, J., J. BacterioL, 1983; 153: 991-7).
The PCP-soluble fraction (containing LPS) was further processed as follows. The chloroform and petroleum ether were removed by evaporation and the LPS was precipitated from the phenol phase by 6 volumes of diethyl ether-acetone (1:5 v/v) (Qureshi, N., et al, J. Biol. Chem. 1982; 257: 11808-15). The CPS was purified from the crude phenol-water extract containing both LPS and CPS. The extract was dissolved in 0.1 M sodium-acetate buffer (pH 4.2), heated for 4 h at 100°C, the precipitate removed by centrifugation, and the CPS isolated by gel-permeation chromatography on Sephadex G-50. The E. coli 0172 was grown as above and the LPS extracted with hot phenol- water extraction (Westphal, O., et al, see above). The LPS was purified from the aqueous phase by treatment with RNase, DNase and Proteinase K.
Selection of peptides
Peptide mimics were selected by panning six different phage libraries: pVIII9aa, pVIII9aa. Cys, pVIII12aa, pVHI12aa.Cys, pVIIIlδaa and pVIII-28aa. All libraries have been constructed in the pVIII two- gene/phagemid vector pC89 (Felici, F., et al., J. Mol. Biol,. (1991) 222: 301-310). The libraries are carrying random inserts encoding peptides of various sizes fused into the N-terminal region of the major coat protein (protein VIII) of filamentous phage. pVIII9aa, pVIII12aa pNIIIlδaa and pVIII-28aa are four different libraries composed of random 9-mers, 12-mers, 15-mers and 28-mers, respectively, whereas pVIII9aa.Cys and pVHI12aa.Cys are libraries in which the random insert contains two cysteine residues (Luzzago, A., et al, (1993) Gene 128, 51-57). In this latter libraries, cysteines promote the formation of a disulfide bridge that constrains to some extent the conformation of the displayed peptide. All libraries have been generated as described (A. Luzzago and F. Felici "Construction of disulfide-constrained random peptide libraries displayed on phage coat protein VIII" in: Methods in Molecular Biology, Vol. 87: "Combinatorial peptide library protocols", Ed. S. Cabilly; Humana Press, Totowa, NJ, 155-164 - 1997).
Specific phage clones were isolated from the libraries by two rounds of affinity selection as described previously (Felici et al, 1991). In the first round the monoclonal antibody was incubated at 1 μM concentration overnight at 4°C with 1010 transducing units of library in a total volume of 10 μl. The mixture was incubated with 0.25 μg of a biotin-conjugated goat anti-mouse IgG secondary antibody (Fc specific, Sigma, St. Louis, MO), which had been pre-adsorbed overnight at 4°C with 2 x 1011 phage particles of UV-killed M13K07 to prevent nonspecific binding. The phage-mAb-secondary antibody complexes were bound to streptavidin-coated dishes and bound complexes eluted after a washing step. The second round of biopanning was carried out in the same way, but using 10 nM concentration of mAb and proportionally lower amounts of the secondary antibody. Positive phage clones were identified through plaque immunoscreening (Luzzago, et al (1993)).
The interaction of the selected phage clones with the antibody was tested in ELISA. Ninety-six wells ELISA plates were coated with 100 μl per well of rat anti-pill (coat protein III) monoclonal antibody (Dente, L., et al, Gene 148, 7-13) (concentration 1 μg/ml) in 50 mM NaHCOs, 0.02% (w/v) NaN3, pH 9.6, and washed 8 times with TBST (50 mM Tris/Cl, 150 mM NaCl, pH 7.5, 0.05% (v/v) Tween20). 100 μl per well of cleared phage supernatant was added. After washing as above and incubation with antibody (concentration 1 μg/ml) in blocking buffer (5% (w/v) non-fat dry milk, 0.05% Tween20 in PBS), and washing, the binding of the monoclonal antibody was detected by alkaline-phosphatase conjugated goat anti-mouse IgG antibody (Sigma, dilution 1:5000) using p-nitrophenyl phosphate substrate tablets (Sigma). Specific inhibition of recognition of phage clones by the antibody was performed under the same conditions, except that the mAb was preincubated with a preparation of 0139 capsular polysaccharide in blocking buffer.
The phage DNA of the positive clones was sequenced by the dideoxy mediated chain termination method (Sanger, F., et al, (1977) Proc. Natl. Acad. Scl, 74 5463-5467).
Peptides were synthesized and conjugated to Bovine Serum Albumin (BSA) and Keyhole Limpet Hemocyanin (KLH) by Thermo Hybaid, Thermo Biosciences GmbH, Ulm, Germany.
Immunizations 6 week old, female, Balb/c mice (n=8 per peptide) were immunized with the Vcl-1; Vcl-4; Vcl-10; Vcl-12, Vcl-22, Vc2-1, Vcl2-1 and Vcl2-2 KLH-peptide conjugates. 30 μg/mouse (corresponds to 5-7 μg peptide) in AlumGel, were immunized intraperitoneally at day 1, 42 and 63. Blood was taken from the tail artery at day 0, 56 and at day 77 by heart puncture. Negative control mice received KLH alone or a peptide selected with Mabs for Shigella flexneri, according to the same immunization schedule. The blood was left in room temperature to coagulate and the centrifuged at 5,000g for 10 minutes. The seras were pipetted off and kept in -20 °C until analysis.
Enzyme immunoassay
The sera from the immunized mice were tested individually. EIA plates (Costar® 3590, Corning Inc. Corning, NY) were coated at 4 °C overnight with 100 μL/well of BSA conjugated peptide, V. cholerae 0139 capsular polysaccharide, and E. coli 0172 lipopolysaccharide (5 μg/mL in phosphate buffered saline, PBS (10 mM phosphate buffer [pH 7.2], 140 mM NaCl), all in duplicates. After blocking with 0,25% skimmed milk, the plates were washed three times in washing buffer (0,15 M NaCl, 0,05% Tween 20). 100 μL of sera in ten-fold dilutions in PBST (10 mM phosphate buffer [pH 7.2], 140 mM NaCl.0,05% Tween 20) was added and incubated in 20 °C for two hours. After washing as above, 100 μL of alkaline-phosphatase-conjugated rabbit anti mouse immunoglobulins (Dako A/S, Denmark) diluted in PBST were added and incubated in 20 °C over night. For developing, plates were washed as above, and 100 μL of a 1 M diethanolamine, 0.5 M MgCl2, pH 9.8, 1 mg-mL_1 sodium p-nitrophenol phosphate was added. Plates were incubated at 20 °C and read in a Multiskan Plus EIA reader (Labsystems) at 405 nm after 10, 25, 50 and 100 min. Absorbance values between 0,2 and 1 was chosen and extrapolated to absorbance after 100 min of incubation.
Competition EIA Sera from day 77 was pooled for each group and titrated in EIA against the corresponding BSA conjugated peptide. Sera concentration that gave an absorption value between 0,6 and 1 at 100 minutes were chosen. Coating of the plates was done as described above. The sera was preincubated in 20 °C for two hours with different concentrations of BSA conjugated peptide, free peptide, V. cholerae 0139 capsular polysaccharide, and E. coli 0172 lipopolysaccharide. 100 μL was added and the plates were further treated as above. The absorption was read after 100 minutes. The inhibition was calculated as percentage of the absorbance of sera without inhibitor added.
Protection studies
2-5 day old Swiss albino mice were separated from the mother two hours before the experiments. The sera were deactivated in 56 °C for 30 minutes. 25 μl of sera and 104 bacteria per mouse were preincubated in room temperature for 30 minutes prior to tube feeding. The mice were then observed for 48 hours.
Results
Monoclonal antibodies
Three different murine monoclonal antibodies (Vc 1, Vc 2 and ICL 12) have been used in this study. All mAbs, have been tested for specificity using EIA and shown to react only with the homologous antigens, i.e. the CPS and LPS isolated from V. cholerae 0139. Both Vcl and Vc2 are of the IgGβ isotype, whereas the ICL 12 is an IgG2b. All three mAbs have been tested in the suckling mouse model described above. The protective efficacy was 100%, 75%, and 83%, respectively.
Peptides
The following peptides were selected: Vcl-1 : AEGEFSPGVWKAAFQGDKLPDPAK - SEQ ID No. 1;
Vcl-4 : AEGEFYCSGPPDRVCWGPDPAK - SEQ ID No. 2;
Vcl-10 : AEGEFPSRMQFHGDQDYGDPAK - SEQ ID No. 3;
Vcl-12 : AEGEFMGDICSWCYSSAPDPAK - SEQ ID No. 4;
Vcl-22 : AEGEFCSPPDSPGVCGDPAK - SEQ ID No. 5;
Vc2-1 : AEGEFSYDMLWMMTPQTGDPAK - SEQ ID No. 6;
Vcl2-1 : AEGEFCGFVWPFKCHRIGDPAK - SEQ ID No. 7;Vcl2-2 :
AEGEFKVPFRMLNRQVNGDPAK - SEQ ID No. 8.
The selected peptides have no consensus sequence, they are of different length, some are linear and some have cystein inserts that will form loops. Selected peptides have been sequenced, synthesized and conjugated to Bovine Serum Albumin (BSA) and Keyhole Limpet Hemocyanin (KLH).
Immune response in mice
To investigate the ability of the eight selected peptides to elicit an immune response against V. cholerae 0139 capsular polysaccharide, we immunized mice with peptides conjugated to KLH. After immunization, we tested the sera for reaction against peptides conjugated to BSA, V. cholerae 0139 capsular polysaccharide and E. coli 0172 LPS, which is structurally unrelated to V. cholerae 0139 CPS and E. coli 0172 LPS, as negative control. The immune response against the corresponding peptide BSA conjugate was very good for all eight peptides. The response against V. cholerae 0139 was lower, but still higher than the negative control, in any case sufficient to demonstrate protection, as is shown below. This pattern was the same for all peptides. Although there are no obvious common structural features, the peptides show striking functional similarities, mimic the same antigen and have been identified by using the same procedure, therefore they can be here considered as variants of the same product, and useful for an anti-V. cholera 0139 vaccine.
Competition of immune response To investigate the specificity of the immune response, we performed a competition EIA. The plates were coated with BSA conjugated peptides and the seras were preincubated with BSA conjugated peptide, un- conjugated peptide, V. cholerae 0139 CPS and E. coli 0172 LPS. The results show that the corresponding BSA conjugated peptide inhibited the reaction almost completely. The unconjugated peptide could inhibit the reaction partially. Antibodies against some of the peptides could be inhibited by V cholerae 0139 capsular polysaccharide. Vcl-1, Vcl-10, Vc2-1, Vcl2-1 and Vcl2-2 reached 50% inhibition after addition of 100- 200μg of V. cholerae 0139 CPS. Antibodies against the Vcl-4, Vcl-12 and Vcl-22 peptides were also inhibited but the inhibition did not reach 50% with the highest concentration of the CPS. The structurally unrelated Escherichia coli 0172 LPS used as negative control was unable to inhibit the specific antibodies
Suckling Mouse assay
The mouse sera against the peptides were further tested for protection in the suckling mouse assay. The results showed that the peptides could induce an antibody response in mice with a protective effect. Vcl-1, Vcl-12 and Vc2-1 showed a protection of 30% or less, Vcl-10, Vcl2-1 and Vcl2-2 a protection of 40-60%, and Vcl-22 and Vcl-4 a protection of 60-70%. Sera against two of the peptides, Vcl-22 and Vcl- 4, were further investigated, and showed a dose dependent decrease in protection when tested in different dilutions in the suckling mouse model. The protective effect could also be neutralised when the sera against the KLH conjugate were adsorbed with the corresponding BSA conjugate. In addition, sera from mice immunized with KLH alone or with an S. flexneri peptide conjugate did not show any protective efficacy against V. cholerae 0139 in the suckling mouse model.
The results are shown in the following Table. Table
Survival of mice in the suckling mouse model after 48 h exposure for V. cholerae 0139 strain
1852 incubated with different antibodies
Antibody Surviva Total (%)
Anti-KLH 1/14 (7%)
ICL- 12 32/41 (83%)
Anti-Vcl-1 3/11 (27%)
Anti-Vcl-4 15/23 (65%)
Anti-Vcl-10 10/18 (56%)
Anti-Vcl-12 3/12 (25%)
Anti-Vcl-22 12/17 (71%)
Anti-Vc2-1 4/13 (31%)
Anti-Vcl2-1 8/14 (57%)
Anti-Vcl2-2 6/13 (46%)
Anti-S. flexneri peptide 1/4 (25%)
The results indicate that peptide mimics can induce protective humoral immune response against V. cholerae 0139. The antibodies against the peptides react with V. cholerae 0139 CPS and show protective effect against V. cholerae 0139 in the suckling mouse model. The antibody response after immunization is relatively weak against the V. cholerae 0139, explanations for that might be that intrape- ritonally immunization is not ideal or that another adjuvant can be used.
The results clearly show that the peptides induce a protective immune response in mice. It can therefore, be surmised that these peptides can be used for the preparation of vaccines against the V. cholerae 0139.
As regards industrial applicability, one possible realisation of the present invention is in the form of diagnostic kits containing the peptides.
The diagnostic kits which are the object of the present invention are known to the expert in the field and do not require any particular description. By way of an example, the reader is referred to the patent literature cited above, to which may be added US 6,265,176 and WO 01/63283 as further references.
As regards industrial applicability, one possible realisation of the present invention is in the form of vaccines containing the peptides, whose preparation comes within the framework of general knowledge; for further reference purposes the reader is referred to the patent literature cited in the present description.
The pharmaceutical compositions, in particular in the form of a vaccine, contain at least one peptide or its derivative as an active ingredient, in an amount such as to produce a significant prophylactic and/or therapeutic effect. The compositions covered by the present invention are entirely conventional and are obtained with methods which are common practice in the pharmaceutical industry, such as, for example, those illustrated in Remington's Pharmaceutical Science Handbook, Mack Pub. N. Y. — latest edition. According to the administration route chosen, the compositions will be in solid or liquid form, suitable for oral, nasal, parenteral or intravenous administration. The compositions according to the present invention contain, along with the active ingredient, at least one pharmaceutically acceptable vehicle or excipient. These may be particularly useful formulation coadjuvants, e.g. solubilising agents, dispersing agents, suspension agents, and emulsifying agents. Particular interest is put on adjuvants used in the formulation of vaccines.

Claims

1. Peptide mimics against the capsular polysaccharide of V. cholerae 0139, their antigenic portions, their equivalents selected from the group consisting of:
NH2-AEGEFSPGVWKAAFQGDKLPDPAK-COOH; NH2-AEGEFYCSGPPDRVCWGPDPAK-COOH; NH2-AEGEFPSRMQFHGDQDYGDPAK-COOH; NH2-AEGEFMGDICSWCYSSAPDPAK-COOH; NH2-AEGEFCSPPDSPGVCGDPAK-COOH; NH2-AEGEFSYDMLWMMTPQTGDPAK-COOH, NH2-AEGEFCGFVWPFKCHRIGDPAK-COOH;NH2- AEGEFKVPFRMLNRQVNGDPAK-COOH
2. A peptide according to claim 1, which is contained in a MAP construct.
3. A peptide according to claim 1 or 2, conjugated with another substance useful in immunization.
4. A peptide according to claim 3, wherein said another substance is a protein.
5. A peptide according to claim 4, wherein said protein is Bovine Serum Albumin or Keyhole Limpet Hemocyanin, tetanus toxoid and diphteria toxoids.
6. A process for the preparation of the peptides of claims 1-2, comprising the following steps: a) producing a monoclonal antibody against the capsular polysaccharide of V. cholerae 0139; b) using said monoclonal antibody to screen different phage-displayed random peptide libraries; c) isolating the peptides displayed on the surface of the filamentous phages selected in step b).
7. A polynucleotide coding for a peptide of claim 1.
8. The use of the peptides of any one of claims 1-5 and/or of the polynucleotide of claim 7 as medicaments.
9. The use of the peptides of any one of claims 1-5 and/or of the polynucleotide of claim 7 for the preparation of a medicament for the prophylaxis of cholera .
10. A pharmaceutical composition comprising at least one peptide of any one of claims 1-5 and/or at least a polynucleotide of claim 7, in admixture with at least one pharmaceutical carrier or vehicle.
11. The composition of claim 10, in the form of a vaccine.
12. The composition of claim 13, for oral, nasal, parenteral or intravenous administration.
13. A diagnostic method for detecting the presence of Vibrio cholerae 0139 in a subject comprising: a) contacting a biological sample of the subject with at least one peptide of any one of claims 1-5; b) detecting peptide-antibody complex formation.
14. A diagnostic kit comprising at least one peptide of any one of claims 1-5.
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EP2108656A1 (en) 2008-03-19 2009-10-14 Beninati, Concetta Antigenic protein fragments of streptococcus pneumoniae
US9044511B2 (en) 2010-10-26 2015-06-02 Marealis As Peptide

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