WO2001092336A1

WO2001092336A1 - Lactoferrin polypeptides from h. pylori and vaccine compositions thereof

Info

Publication number: WO2001092336A1
Application number: PCT/SE2001/001176
Authority: WO
Inventors: Carol Nilsson; Karl-Anders Karlsson; Jörgen Bergström; Thomas Larsson
Original assignee: A+ Science Invest Ab
Priority date: 2000-05-29
Filing date: 2001-05-25
Publication date: 2001-12-06
Also published as: AU2001262839A1; SE0001988D0

Abstract

Lactoferrin binding polypeptides derived from Helicobacter pylori are disclosed. Said polypeptides comprise or consist of the polypeptide with SEQ. ID. No. 1, or a fragment thereof. Also nucleotide sequences encoding such polypeptides are disclosed, as well as use of such polypeptides in the treatment or prophylaxis of diseases caused by Helicobacter, and in the production of vaccines or pharmaceutical compositions for the prevention or treatment of diseases.

Description

LACTOFERRINPOLYPEPTIDES FROMH. PYLORIANDVACCINE COMPOSITIONS THEREOF

Field of the invention The present invention relates to novel polypeptides and use thereof. More specifically, the invention relates to novel lactoferrin binding polypeptides derived from 5 Helicobacter pylori, and the use thereof in treatment of diseases and conditions caused by Helicobacter pylori and in the production of vaccines against Helicobacter pylori .

10 Background of the invention

Helicobacter pylori is a gram-negative spiral bacterium which colonizes the gastric epithelium of humans. It is a known gastric pathogen which has been implicated in diseases such as peptic ulcers, gastritis and gastric

15 cancer. Many people all over the world, both in western countries and the developing world, are affected by diseases and conditions caused by Helicobacter pylori. Even though treatment strategies for such diseases have been developed, there is a need for new, more effective, more

20 general or less expensive pharmaceuticals and vaccines for treatment, both in curative and prophylactic purposes, of such Helicobacter pylori dependent diseases.

It is known that lactoferrin, which is found in the stomach and intestines of mammals including humans, is a 25 very important source of iron for Helicobacter pylori. It is also known that Helicobacter pylori needs iron in order to maintain its virulence.

A 98 kD and a 70 kD Helicobacter protein are described in WO 97/13784, as well as the use of these pro-

30 teins for the production of a pharmaceutical preparation for treatment of Helicobacter infections. It is stated that these proteins are lactoferrin-binding proteins. No description of the sequences of these proteins is given. The complete genome of Helicobacter pylori has been described earlier, see e.g. Tomb, J-F et al . Nature 388 (1997) , 539-547 describing the genome of Helicobacter pylori strain 26695. Also the sequences of several genes of this genome have been published. For example, WO 98/43478 describes isolated Helicobacter polynucleotides, which are said to be useful in diagnosis, prevention and treatment of Helicobacter infections and gastrointestinal diseases and genes from Helicobacter pylori for treatment of infection caused by Helicobacter pylori, WO 99/21959 describes vaccine formulations comprising one or more isolated polypeptides from Helicobacter pylori for treatment of Helicobacter pylori infections, and WO 97/37044 describes Helicobacter pylori polypeptides and preparations and vaccines thereof .

All these three PCT-publications contain very many sequences. It seems as if all possible polypeptides from Helicobacter pylori have been listed, and it has then been stated that they may be used in vaccines. However, man skilled in the art would easily realize that it is highly unlikely that all these polypeptides will have a pharmaceutical effect needed for vaccine purposes. There is nothing in any of these PCT-publications that indi- cates how one would be able to select appropriate polypeptides, and not even the functions of the different polypeptides are given. It would thus be very difficult to produce a suitable, effective vaccine for treatment of Helicobacter pylori-infections based on the teachings in these PCT-publications.

Summary of the invention In the research work leading to the present invention it was found that a specific polypeptide, different from the ones described in WO 97/13784, has a lactoferrin binding property and that this polypeptide can be used i.a. in the production of vaccines for treatment of Helicobacter infections.

This lactoferrin binding outer membrane protein or polypeptide was identified in different strains of Heli- cobacter pylori, and it was found that they all shared a lactoferrin binding polypeptide consisting of 528 amino acid residues, with only minor variations between the different strains, and the sequence of this protein is given in the appended sequence listing. The invention thus relates to lactoferrin binding polypeptides comprising or consisting of the polypeptide with SEQ. ID. No. 1, 2 or 3 according to the appended sequence listing. The invention also relates to fragments of these polypeptides retaining the lactoferrin binding ability.

The invention also relates to nucleotide sequences encoding the above mentioned polypeptides.

Furthermore, the invention relates to vaccines or pharmaceutical compositions for the prevention or treat- ment of a disease caused by Helicobacter produced by the use of at least one of the above mentioned polypeptides.

The invention also relates to a method for the prevention or treatment of a disease caused by Helicobacter wherein a pharmaceutically effective amount of a vaccine or pharmaceutical composition as mentioned above is administered to a patient.

The invention also relates to the diagnosis of diseases caused by Helicobacter, and especially of Helicobacter pylori by use of at least one of the above men- tioned polypeptides with SEQ. ID. Nos. 1, 2 and 3, or at least one antibody against at least one of these polypeptides .

Finally, the invention relates to methods for the production of vaccine for the prevention or treatment of a disease caused by Helicobacter based on the use of at least one of the above mentioned polypeptides. The characterizing features of the invention will be evident from the following description and the appended claims .

Detailed description of the invention

As stated above the invention relates to a polypeptide consisting of 528 amino acid residues. The nucleo- tide sequence encoding this protein has been identified in i.a. Helicobacter pylori strains 26695 and J99, and it is essentially the same in these strains - only 26 amino acid residues differ. The amino acid residues in positions 11, 16, 42, 48, 84, 92, 95, 97, 121, 134, 139, 141, 147, 148, 170, 175, 197, 230, 252, 301, 306, 349, 351, 385, 394 and 491 seem thus to be of less importance, and they may thus be any amino acid, provided that they do not significantly reduce the lactoferrin-binding ability of the polypeptide, as shown in SEQ. ID. No. 1.

The exact sequence for the lactoferrin binding protein according to the invention found in Helicobacter py- lori strain 26695 is given in SEQ. ID. No. 2, and the exact sequence for the lactoferrin binding protein according to the invention found in Helicobacter pylori strain J99 is given in SEQ. ID. No. 3.

The expression "homologue" used herein relates to polypeptides that are structurally similar to the polypeptides according to the invention,^' with SEQ. ID. No. 1, 2 or 3 given in the appended sequence listing, with essentially the same lactoferrin binding ability as the polypeptides with SEQ. ID. No. 1, 2 or 3. These three polypeptides are approximately 60 kD polypeptides.

Nucleotides encoding these polypeptides may be used for production of the desired polypeptide.

The polypeptides according to the invention, optionally produced by the use of the nucleotide sequences ac- cording to the invention, may be used for the production of vaccines or pharmaceutical preparations for treatment of diseases caused by Helicobacter, and in particular disease caused by Helicobacter pylori, such as chronic gastritis, gastric duodenal ulcer, gastric cancer, and MALT lymphoma.

For the production of a vaccine one of the polypep- tides according to the invention, or a fragment thereof, is coupled to an adjuvant. When such a vaccine is administered to a patient, the immune response of the patient will lead to the production of antibodies against the lactoferrin binding protein according to the invention. Preferably a pharmaceutical preparation according to the invention comprises antibodies against the lactoferrin binding protein according to the invention.

The administration of a vaccine or a pharmaceutical preparation according to the invention leads to an inhib- iting effect on the lactoferrin binding by the Helicobacter bacteria present in the patient, and will thus reduce or completely eliminate the virulence of the bacteria.

The vaccine or pharmaceutical composition according to the invention, used according to the invention or pro- duced according to the invention may also comprise other substances, such as an inert vehicle, or pharmaceutical acceptable adjuvants, carriers, diluents, preservatives etc., which are well known to persons skilled in the art.

The term "patient", as it is used herein, relates to any human or non-human mammal in need of curative or prophylactic treatment according to the invention.

The invention also relates to a method for the production of a vaccine composition for vaccination against Helicobacter pylori. This production may comprise the in- troduction of at least one protein or polypeptide according to the invention, i.e. a polypeptide with a sequence according to SEQ. ID. No. 1, 2 or 3 in the appended sequence listing, or a lactoferrin binding ho ologue or fragment thereof into a pharmaceutically acceptable car- rier. Said pharmaceutically acceptable carrier may e.g. be a bacterial vector. Protective antibodies may also be produced by immunization of a mammal with a protein or polypeptide according to the invention, i.e. a polypeptide with a sequence according to SEQ. ID. No. 1, 2 or 3 in the ap- pended sequence listing, or a lactoferrin binding homo- logue or fragment thereof.

The vaccines according to the invention may be used for both prophylactic and curative vaccination, i.e. for protection against or treatment of a disease caused by Helicobacter pylori.

The invention will now be further explained in the following examples. These examples are only intended to illustrate the invention and should in no way be considered to limit the scope of the invention.

Brief description of the drawings In the examples, reference is made to the accompanying drawings on which: Fig. 1 shows electrophoresis gels with a clear band formed of the lactoferrin binding protein according to the invention.

The abbreviations in the figure have the following meaning :

BSA = bovine serum albumin Cat = H. pylori catalase

LBP = H. pylori lactoferrin binding protein SA = streptavidin

BCC = H. pylori biotin carboxyl carrier protein SA₂ = SA dimer SA/BCC = SA and BCC complex.

The lactoferrin used was obtained from Sigma, Catalogue # L0520, batch # 61H3905.

Examples The following materials and methods were used for the identification of the lactoferrin-binding protein of Helicobacter pylori according to the invention. Bacterial strains and growth condi tions

Three strains of H. pylori were used, 26695, CCGU 17874 and 17875 from the Culture Collection, Goteborg University. The cells were stored at -80°C in soy broth containing 15% glycerol by volume, and were grown on agar (14 g/L) containing 10% heat- inactivated fetal calf serum, brucella broth (28 g/L; Difco Laboratories, Detroit, MI) and 1 ml/L IsoVitale X enrichment vitamins (Becton Dickson Europe, Merlyan, France) in a humid (98%) micro- aerophilic chamber (0₂: 5-7%, C0₂ : 8-10%, N₂ : 83-87%, and H₂: less than 2%) at 37°C. After 48 hours, the cells were scraped off and washed three times in phosphate-buffered saline (PBS) .

Labeling of receptor conjugate

The trifunctional reagent Sulfo-SBED (sulfosucin- imidyl [2-6- (biotinamido) -2- (p-azidobanzamido) -hexano- amido] ethyl-1, 3 ' -dithiopropionate, Pierce, USA) was con- jugated to human lactoferrin (Sigma #L0520) , following the instruction of the manufacturer. Briefly, 5 μl Sulfo- SBED (10 μg/μl) in dimethylsulfoxide was added to 100 μl lactoferrin (1 μg/μl) in 0.1 M potassium phosphate buffer, pH 7.2. The mixture was incubated at room tem- perature in the dark for 1 hour. The product was desalted on a HiTrap Desalt column (Amersham Pharmacia Biotech, Sweden) in a 1 ml fraction and stored in the dark to be further used the same day.

Tagging of H. pylori lactof err in-binding protein

The protein was tagged using a procedure previously described for cloning [D. liver et al, Science 279, p. 373-377 , 1998] . In short, freshly harvested bacteria from approximately one-third of a Petri dish were incubated for 30 minutes at ambient temperatures with the labeled lactoferrin conjugate and irradiated under an ultraviolet lamp. The disulfide bond on the linker was reduced by the addition of dithiothreitol to give a final concentration of 50 mM. The tagged bacteria were washed three times in PBS, pH 7.4, and frozen.

Enrichment of biotinylated bacterial proteins wi th strep- tavidin- coated magnetic beads

Tagged bacterial pellets were dissolved in 2% SDS containing 25 mM Tris, pH 8.0, followed by dilution to 0.5% SDS with 25 mM Tris. 200 μl Bio Mag streptavidin- coated beads (PerSeptive Biosystems, Framingham, MA, USA) were washed three times in PBS. Bacterial extracts were incubated with beads for 16 hours at 4°C and non-bound material was washed away from the beads using 0.5% SDS. The beads were finally heated to 95 °C in SDS-PAGE sample buffer for 15 minutes. The result is shown in Figure 1.

Electrophoresis and detection of biotinylated proteins

SDS PAGE and Coomassie staining were carried out with Pharmacia PhastSystem™ (Pharmacia Biotech, Uppsala, Sweden) or Bio-Rad Mini-PROTEAN II (Bio-Rad, Hercules,

CA, USA) according to the protocols of the manufacturers. Briefly, samples (4 μl or 25 μl, respectively) extracted from beads were applied on a homogeneous gel of 12.5%. After electrophoresis the gel was either stained with GelCode^® Blue Stain Reagent (Pierce, USA) or electroblot- ted to a PVDF (0.2 μm) membrane according to the manuals. The transfer buffer consisted of 20% methanol, 192 mM glycine, and 25 mM Tris at pH 8.3.

The PVDF membrane was preincubated in blocking solu- tion. 3% BSA, 50 mM HEPES-ΝaOH, 100 mM ΝaCl, pH 7.3 for 1.5 hours and washed with 0.05% Tween-20, 50 mM HEPES- ΝaOH, 100 mM ΝaCl, pH 7.3 (washing buffer) . The membrane was then incubated with horseradish peroxidase-conjugated streptavidin (HRP-ΝeutrAvidin™, Pierce, USA) in washing buffer with 1% bovine serum albumin (BSA) added. After 1.5-2 hours the membrane was washed 5 times in washing buffer, and biotin-containing protein bands were devel- oped with 0.1% H₂0₂ and 0.02% DAB (3 , 3 ' -diaminobenzidine hydrochloride, Pierce, USA) in washing buffer. The membranes were then washed with water and dried at room temperature .

Enzymatic digestion

Each protein band from the Coomassie-stained gel was cut out, placed in separate siliconized tubes and gently macerated with a hand-hold mixer. The dye was removed by adding 85 μl 25 mM NH₄HC0₃ in 50% CH₃CN and the tube was vortexed for 30 minutes before the supernatant was removed. This procedure was repeated twice. Destained gel pieces were dried for 40 min in a vacuum concentrator (SpeedVac, Savant Instruments, USA) . 15 μl trypsin (por- cine, sequencing grade, modified, Promega, USA) in 25 mM NH4HCO₃, pH 8, was added. The tubes were incubated for 16 hours at 37°C. Peptides were extracted from the gel by adding 15 μl 5% CF₃COOH either in 75% CH₃CN, or in water, to the tube. The tubes were then vortexed for 30 minutes and centrifuged for 2 minutes at 5000 x g.

Mass spectrometry

Mass spectra were obtained using a TofSpecE (Micro- mass, Manchester, England) time-lag focusing MALDI-TOF mass spectrometer equipped with a reflectron. Samples were prepared using the dried-drop method, by mixing 0.5 μl tryptic digest with 0.5 μl matrix directly on the MALDI target. The matrix used was α-cyano-4-hydroxy- cinnamic acid (Aldrich Chemie, Steinheim, Germany) , 10 mg/ml in CH₃CN:water 1:1. Sample purification and concentration were achieved by using ZipTips (Millipore, Bedford, MA, USA) containing C₁₈ material according to the manufacturer's instruction. One μl of the eluate was added to 0.5 μl of matrix solution and prepared using the dried-drop method as above. Peptide masses obtained were used to identify known proteins through database searches using MS-Fit (http://prospector.ucsf.edu), which lead to the identification of the polypeptides according to the invention (SEQ. ID No . 2 with accession # 2313598 and SEQ. ID No. 3 with accession # 4154973) .

Claims

1. A lactoferrin binding polypeptide comprising the polypeptide with SEQ. ID. No. 1. 2. A lactoferrin binding polypeptide comprising the polypeptide with SEQ. ID. No.

2.

3. A lactoferrin binding polypeptide comprising the polypeptide with SEQ. ID. No. 3.

4. A lactoferrin binding polypeptide consisting of the polypeptide with SEQ. ID. No. 1 or a homologue thereof .

5. A lactoferrin binding polypeptide consisting of the polypeptide with SEQ. ID. No. 2 or a homologue thereof .

6. A lactoferrin binding polypeptide consisting of the polypeptide with SEQ. ID. No. 3 or a homologue thereof .

7. A lactoferrin binding polypeptide consisting of a fragment of the polypeptide with SEQ. ID. No. 1 or a homologue thereof.

8. A lactoferrin binding polypeptide consisting of a fragment of the polypeptide with SEQ. ID. No. 2 or a homologue thereof.

9. A lactoferrin binding polypeptide consisting of a fragment of the polypeptide with SEQ. ID. No. 3 or a homologue thereof.

10. A fragment of a polypeptide with SEQ. ID. No. 1, 2 or 3 capable of inducing an immune response against Helicobacter pylori .

11. A nucleotide sequence encoding a polypeptide according to any one of the claims 1-10.

12. A vaccine or a pharmaceutical preparation for the prevention or treatment of a disease caused by Helicobacter produced by the use of at least one polypeptide according to any one of the claims 1-10.

13. A vaccine or a pharmaceutical preparation for the prevention or treatment of a disease caused by Heli- cobacter comprising at least one polypeptide according to any one of the claims 1-10 inserted in a pharmaceutically acceptable carrier.

14. A vaccine or a pharmaceutical preparation for the prevention or treatment of a disease caused by Helicobacter inducing or comprising an antibody that binds to a compound according to any one of the claims 1-10.

15. A vaccine or pharmaceutical preparation, according to any one of the claims 12-14, wherein said disease is caused by Helicobacter pylori.

16. A pharmaceutical preparation for treatment of a disease caused by Helicobacter pylori comprising a substance that prevents a polypeptide with SEQ. ID. No. 1, 2 or 3 from binding to lactoferrin.

17. A vaccine or pharmaceutical preparation according to any one of the claims 12-16, further comprising at least one substance selected from the group consisting of an inert vehicle, a pharmaceutical acceptable adjuvant, a pharmaceutical acceptable carrier, a pharmaceutical ac- ceptable diluent, and a pharmaceutical acceptable preservative.

18. Use of a substance that prevents a polypeptide with SEQ. ID. No. 1, 2 or 3 from binding to lactoferrin for the production of a pharmaceutical preparation for treatment of a disease caused by Helicobacter pylori.

19. Use of a polypeptide according to any one of the claims 1-10 or a nucleotide sequence according to claim 11, for the production of a vaccine or a pharmaceutical preparation for the prevention or treatment of a disease caused by Helicobacter.

20. Use according to claim 19, wherein said disease is a Helicobacter pylori infection.

21. A method for the prevention or treatment of a disease caused by Helicobacter wherein a pharmaceutically effective amount of a vaccine or pharmaceutical preparation according to any one of the claims 12-17 or produced according to any one of the claims 18-20 is administered to a patient .

22. A method for production of antibodies for the prevention, treatment or diagnosis of a disease caused by Helicobacter wherein a mammal is immunized with a polypeptide according to any one of the claims 1-10, and antibodies raised in the mammal against said polypeptide then are isolated from said mammal .