WO1998022135A1 - Agregat lipidique pharmaceutique presentant un antigene d'helicobacter pylori et un lipide a charge negative - Google Patents

Agregat lipidique pharmaceutique presentant un antigene d'helicobacter pylori et un lipide a charge negative Download PDF

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Publication number
WO1998022135A1
WO1998022135A1 PCT/SE1997/001927 SE9701927W WO9822135A1 WO 1998022135 A1 WO1998022135 A1 WO 1998022135A1 SE 9701927 W SE9701927 W SE 9701927W WO 9822135 A1 WO9822135 A1 WO 9822135A1
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pharmaceutical formulation
pylori
polypeptide
lipid
formulation according
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PCT/SE1997/001927
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English (en)
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Thomas Berglindh
Jan-Erik Löfroth
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Astra Aktiebolag
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Priority to AU51418/98A priority Critical patent/AU5141898A/en
Publication of WO1998022135A1 publication Critical patent/WO1998022135A1/fr

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    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
    • 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
    • A61K47/54—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 an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

Definitions

  • the present invention provides a new pharmaceutical formulation comprising polypeptides, which constitute Helicobacter pylori antigens, or nucleic acid molecules coding for the said polypeptides. More specifically the present invention relates to a pharmaceutical formulation in the form of a lipid aggregate comprising these polypeptides or antigens. Furthermore, the invention provides a process for the preparation of such a pharmaceutical formulation, and the use of such a formulation in the treatment of Helicobacter pylori infections, as well as a method of treating a patient to elicit a protective immune response against Helicobacter pylori infections, said formulation being suitable for both therapeutic and prophylactic use.
  • the pharmaceutical formulation shall provide an improved immune response to the polypeptides or antigens delivered by the formulation.
  • Helicobacter pylori is an important human pathogen, involved in several gastroduodenal diseases. Colonization of gastric epithelium by the bacterium leads to active inflammation and progressive chronic gastritis, with a greatly enhanced risk of progression to peptic ulcer disease.
  • H. pylori In order to colonize the gastric mucosa, H. pylori uses a number of virulence factors. Such virulence factors comprise several adhesins, with which the bacterium associates with the mucus and /or binds to epithelial cells; ureases which helps to neutralize the acid environment; and proteolytic enzymes which makes the mucus more fluid.
  • adhesins with which the bacterium associates with the mucus and /or binds to epithelial cells
  • ureases which helps to neutralize the acid environment
  • proteolytic enzymes which makes the mucus more fluid.
  • the pathogen persists in the gastric mucosa, normally for the life of the host. The reason for this is probably that the spontaneously induced immune-response is inadequate or directed towards the wrong epitopes of the antigens.
  • hpaA gene which codes for a 20 kDa receptor-binding subunit of the N-acetymeuraminyllactose-binding fibrillar hemagglutinin (NLBH) of H. pylori, has been disclosed by Evans et al. (1993) J. Bacteriol. 175, 674-683.
  • MAbs Monoclonal antibodies against membrane preparations of H. pylori have been disclosed by B ⁇ lin et al. (1995) J. Clin. Microbiol. 33, 381-384.
  • MAbs Monoclonal antibodies against membrane preparations of H. pylori
  • MAbs designated HP30-1:1:6, reacted with a 30 kDa protein which was shown to be exposed on the surface of intact bacteria and to have properties like that of an adhesin.
  • the H. pylori cell transforms from a bacillary to a coccoid form.
  • the H. pylori cell is much less sensitive to antibiotics and other anti-bacterial agents. Circumstantial evidence indicate the H. pylori might be transmitted between individuals in this form, possibly via water or direct contact.
  • An efficient vaccine composition should therefore elicit an immune response towards both the coccoid and the bacillary form of H. pylori. Since systemic immunity probably only plays a limited role in protection against mucosal infections, it is also important that the vaccine composition will enhance protective immune mechanisms locally in the stomach.
  • vaccines have been formulated in known delivery systems such as liposomes, ISCOMs, cochleates, etc, or have been attached to or included into polymer microspheres of degradable or non-degradable nature.
  • Antigens have been associated with live attenuated bacteria, viruses or phages or with killed vectors of the same kind.
  • Other acceptable carriers or diluents known to those skilled in the art, are e g phosphate buffered saline, enterically coated powder formulations, surface active substances and polymers, etc.
  • the purpose of this invention is to provide a new pharmaceutical formulation of an antigenic Helicobacter pylori polypeptide or a pharmaceutical formulation of a nucleic acid molecule coding for such a polypeptide. More specifically, the present invention relates to a new pharmaceutical formulation based on a lipid aggregate of a negatively charged lipid or mixture of lipids, and at least one polypeptide which is a Helicobacter pylori antigen, or nucleic acid molecules coding for such polypeptides, and optionally a pharmaceutically acceptable carrier.
  • One suitable antigen for the lipid aggregate is a 29 kDa polypeptide, obtained by the recombinant cloning of a H. pylori gene which encodes a surface exposed protein.
  • the 29 kDa polypeptide is described below as well as in the Applicant's co-pending patent application W096/38475, published 5 December 1996.
  • the nucleic acid sequence of the 29 kDa polypeptide is similar to the sequence of the hpaA gene as published by Evans et al. (1993) in Journal of Bacteriology, vol 175, 674-683.
  • the pharmaceutical formulation according to the present invention is also suitable for the hpaA gene.
  • the new pharmaceutical formulation is also suitable for other H. pylori antigens, especially membrane proteins.
  • suitable antigens for the formulation are the 29 kDa polypeptide described below, or the hpaA gene described by Evans et al.
  • the different names 29 kDa and hpaA gene or HpaA protein are used interchangable in the present specification as suitable antigens for the present formulation, even if the sequence of the hpaA gene from different H. pylori strains shows some differences (P.W. Toole et al, Bacteriology Vol. 177, No. 21, Nov. 1995).
  • the 29 kDa polypeptide is described in the Applicant's co-pending W096/38475, published 5 December 1996, as an antigenic protein which is expressed in all strains of H. pylori, also in coccoid forms of the bacterium, and which is able to induce a mucosal as well as a systemic immune-response in a host measured as antibody production.
  • the 29 kDa polypeptide is expressed by all H. pylori strains tested, and antibodies created towards this protein do not cross-react with common endogenous human bacteria of other species or with selected human tissues including the gastric mucosa.
  • the 29 kDa polypeptide is useful both for the detection of H. pylori infections as well as for the manufacture of vaccine compositions.
  • the 29 kDa polypeptide elicits a protective or therapeutic immune response against such H. pylori infections.
  • the 29 kDa polypeptide is described in W096/38475 hereby incorporated in whole by reference. It has an amino sequence substantially similar to a Helicobacter pylori surface-exposed antigen and has an approximate molecular weight of 29 kDa.
  • the said surface-exposed antigen has i.a. the following important properties:
  • the 29 kDa polypeptide is lipidated and thus post-translationally modified. This feature of the polypeptide may be of importance for its immunogenicity and for its proper exposure on the surface of H. pylori. It is known in the art that lipid modification can be essential for the immunological properties of bacterial lipoproteins (see Weis, J.J. et al. (1994) Infection and Immunity, vol. 62, 4632-4636).
  • virulence factor a molecule specifically involved in adherence of H. pylori to the epithelial surface of the gastric mucosa and / or in the establishment and maintenance of H. pylori infection.
  • the said polypeptide has an amino acid sequence according to positions 1-260, or 28-260, in SEQ ID NO: 2 or 4 of the Sequence Listing. Further, it is believed that positions 1-260 in SEQ ID NO: 2 and 4 represent the uncleaved protein, while positions 1-27 represent a signal sequence and positions 28-260 represent the mature polypeptide. The only difference between SEQ ID NO: 2 and SEQ ID NO: 4 is that SEQ ID NO: 2 has a Ser residue in position 222, while SEQ ID NO: 4 has an Arg residue in the same position.
  • polypeptides which can be formulated according to the invention is not to be limited strictly to the 29 kDa polypeptide with an amino acid sequence identical with the above mentioned positions in SEQ ID NO: 2 or 4 in the Sequence Listing. Rather the invention encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the properties of the 29 kDa polypeptide. Such properties include the ability to elicit a mucosal as well as systemic immune-response against H. pylori in a mammal host; the ability to work as an adhesin; and the presence of the polypeptide in both bacillary and coccoid forms of H. pylori.
  • one purpose of the invention is to present a formulation based on a lipid aggegate of polypeptides, the amino acid sequence of which is at least 90% homologous, preferably at least 95% homologous, with the amino acid sequence shown as positions 1-260, or positions 28-260, in SEQ ID NO: 2 or 4, in the Sequence Listing, which polypeptides nevertheless have substantially the biological activities of the 29 kDa polypeptide.
  • the purpose of the invention is to provide a formulation based on a lipid aggregate of peptides, with a length of at least 5 amino acids, which comprise an immunogenic epitope of the 29 kDa polypeptide and retains the ability to elicit an immune response against H. pylori bacteria in a mammal host.
  • Such epitope(s) can be presented alone or in the form of fusion proteins, where the epitope is fused to an inert or immunologically active carrier polypeptide. The identification of these epitopes will be based on the presence of host-generated antibodies towards different segments of the 29 kDa polypeptide.
  • One way of obtaining structural information on the epitopes of the 29 kDa polypeptide is the production and characterisation of monoclonal antibodies binding to the polypeptide, followed by mapping of epitopes by e.g. Pepscan analysis.
  • Monoclonal antibodies can be produced by standard methods, such as those described by De St. Groth (1980) in J. Immunol. Methods, vol. 35, 1-21.
  • the invention provides a formulation based on a lipid aggregate of an isolated and purified nucleic acid molecule which has a nucleotide sequence coding for a polypeptide as defined above.
  • the said nucleic acid molecule is a DNA molecule which has a nucleotide sequence identical with SEQ ID NO: 1 or 3 of the Sequence Listing.
  • the DNA molecule is not to be limited strictly to the sequence shown as SEQ ID NO: 1 or 3. Rather the invention encompasses formulations of DNA molecules carrying modifications like substitutions, small deletions, insertions or inversions, which nevertheless encode polypeptides having substantially the biochemical activity of the 29 kDa polypeptide.
  • SEQ ID NO: 1 has an A residue in position 1458, while SEQ ID NO: 3 has a C residue in the same position.
  • the invention provides a formulation of DNA molecules which nucleotide sequences are degenerate, because of the genetic code, to the nucleotide sequence shown as SEQ ID NO: 1 or 3. Since there are 64 possible codons, but only 20 natural amino acids, most amino acids are coded for by more than one codon. This natural "degeneracy", or “redundancy”, of the genetic code is well known in the art. It will thus be appreciated that the DNA sequence shown in the Sequence Listing is only an example within a large but definite group of DNA sequences which will encode the polypeptide as described above.
  • the inventions includes also formulations based on a lipid aggregate of an isolated nucleic acid molecule selected from: (a) nucleic acid molecules comprising a nucleotide sequence which is identical with, or substantially similar to, positions 796-1572 or 874-1572 in SEQ ID NO: 1 or 3 in the Sequence Listing;
  • nucleic acid molecules comprising a nucleotide sequence capable of hybridizing to a nucleotide sequence complementary the polypeptide coding region of a DNA molecule as defined in (a) and which codes for a polypeptide as described above, or a functionally equivalent modified form thereof; and (c) nucleic acid molecules comprising a nucleic acid sequence which is degenerate as a result of the genetic code to a nucleotide sequence as defined in (a) or (b) and which codes for a polypeptide as described above, or a functionally equivalent modified form thereof.
  • Figure la shows the result of eradication of H. pylori infection in mice after oral administration of different agents, such as a Helicobacter antigen alone or together with an adjuvant; the antigen is administered either directly (control) or incorporated in lipid aggregates according to the present invention.
  • agents such as a Helicobacter antigen alone or together with an adjuvant; the antigen is administered either directly (control) or incorporated in lipid aggregates according to the present invention.
  • H. pylori (as CFU, geometric mean values) in 25mm 2 scrapings of antrum and corpus, following oral administration of different agents.
  • CFU Colony forming units
  • CT Cholera toxin
  • LA Lipid aggregates
  • LA(29 kDa) 29 kDa polypeptide formulated in Lipid aggregates.
  • Figure lb shows the same as Figure la) but displayed as the total number of H. pylori in the stomach. Abbrevations see: Figure la) above.
  • Figure 2 shows mucosal antibody response towards a Helicobacter antigen incorporated in a lipid aggregate according to the present invention.
  • the figure 2) shows specifically the total amount of mucosal immunoglobulin (Ig) with a specificity for the antigen preparation comprising the 29 kDa polypeptide, detected by ELISA technique.
  • Ig mucosal immunoglobulin
  • Figure 3) shows mucosal antibody response towards a Helicobacter antigen incorporated in a lipid aggregate according to the present invention.
  • the figure 3) shows specifically the amount of mucosal IgA antibodies with a specificity for the antigen preparation comprising the 29 kDa polypeptide. Abbrevations see: Figure la) above.
  • Figure 4) shows serum antibody response towards a Helicobacter antigen incorporated in a lipid aggregate according to the present invention.
  • the figure 4) shows specifically the amount of Helicobacter specific IgG antibodies in sera with a specificity for an antigen preparation comprising a membrane protein. Abbrevations see: Figurela) above.
  • FIG 5) shows serum antibody response towards a Helicobacter antigen incorporated in a lipid aggregate according to the present invention.
  • the figure 4) shows specifically the amount of 29 kDa specific IgG antibodies in sera with a specificity for an antigen preparation comprising 29 kDa polypeptide. Abbrevations see: Figure la) above.
  • Means of obtaining the improved formulations are based on the use of negatively charged lipids, either alone or in combination with other lipid material such as, but not limited to phospholipids, glycerides, etc, and in combination with a cation component, such as positively charged ions.
  • the use of the formulations result in an improved immune response to the antigens described above when given by different administration routes, such as, but not limited to, the oral, the rectal, the tonsillar, the buccal, the nasal, the vaginal etc, administration route.
  • the preferred administrations are the peroral, rectal and nasal routes.
  • the negatively charged lipids can be, but are not limited to: phosphatidylserine, phosphatidylinositol, phosphatidic acid, and phosphatidylglycerol.
  • the said negatively charged lipids might be combined with one or more additives in the formulation, such as, but not limited to: - other lipids, e g cholesterol, phosphatidylcholine, phosphatidyletanolamine, etc;
  • lipid A and its derivatives e g lipid A and its derivatives, saponins like QS-21, cholera toxin (CT), etc.
  • lipid film composed of the negatively charged lipids with or without other lipids, e g cholesterol; the addition of the antigen in a surfactant solution which then also produces mixed micelles with the lipids with incorporated antigen, preferably a nonionic surfactant with high critical micelle concentration is used; the exchange of the surfactant with the positively charged ion.
  • the last step can be carried out in different ways.
  • One way is to first remove the surfactant, e g by dialysis, and then add for example Ca in a second dialysis step, or directly from a concentrated Ca solution.
  • Another way is to exchange the surfactant with the positively charged ion in one dialysis step.
  • a modification of these processes can be utilized if the antigen is insensitive to organic solvents.
  • the antigen is dissolved together with the lipids in an organic solvent.
  • the formation of liposomes with incorporated antigen is then formed after evaporation of the solvent and after the dispersion in a buffer solution.
  • the dosage form used may be a solid, semisolid, liquid dispersion, or solutions prepared by use of well known pharmaceutical techniques, such as blending, granulation, milling, spray drying, compaction, coating, etc.
  • the formulations may be monolithic, such as tablets, or capsules, or in the form of multiple formulations administered in a tablet, capsule or sachets.
  • the antigen is a polypeptide for use in therapy of Helicobacter pylori infection in a mammal, including man, and for use as a therapeutic or prophylactic vaccine as described above.
  • a vaccine formulation for inducing a protective immune response in a mammal, including humans, against the bacillary and /or coccoid form of Helicobacter pylori.
  • a vaccine formulation comprises a lipid aggregate and an immunogenically effective amount of a polypeptide as defined above.
  • the polypeptide includes at least a part of the 29 kDa polypeptide comprising an immunogenic epitope, or a modified form of said polypeptide which retains the capability to induce protective immunity against Helicobacter pylori infection.
  • modified form includes, but is not restricted to, forms of the polypeptide which are post-translationally modified, e.g. lipidated.
  • the vaccine formulation according to the invention can be used for both therapeutic and prophylactic purposes.
  • the vaccine formulation according to the invention is preferably administered to any mammalian mucosa exemplified by the buccal, the nasal, the tonsillar, the gastric, the intestinal (small and large intestine), the rectal and the vaginal mucosa.
  • the mucosal vaccines can be given together with for the purpose appropriate adjuvants.
  • the vaccine can also be given parenterally, by subcutaneous, intracutaneous or intramuscular route, optionally together with the appropriate adjuvant.
  • adjuvant is meant a component which in general enhances the effect of the vaccine.
  • Such adjuvants are for instance cholera toxin and E.coli heat- labile toxin and their non-toxic derivatives saponins, cytokines and chemokines.
  • Non-toxic derivatives of cholera toxin and E.coli heat labile toxin includes e.g. mutations with reduced ADP-ribosylating activity or CTA1-DD which binds only to lg cells.
  • An alternative approach for creating an immune response against the 29 kDa polypeptide is to use the approach known as "nucleic acid vaccination” or "naked DNA” vaccination. It is known in the art that injection into muscle of plasmid DNA encoding an antigen of interest can result in sustained expression of the antigen and generation of an immune response (see e.g. Rabinovich et al. supra). Several routes of administration are possible, such as parental, mucosal or via a "gene-gun” that delivers tiny amounts of DNA-coated gold beads (Fynan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11478-11482).
  • nucleic acid molecule can be expressed in plasmid comprising a suitable eukaryotic promoter. This "naked DNA” can then be given, e g orally with the formulation. Epitopes of the expressed protein will be expressed by MHC molecules on the surface of the cells and trigger an immune response. Consequently, nucleic acid molecules and vectors as disclosed in the previous paragraphs for use in therapy, in particular for use as a vaccine, are further aspects of the invention. The use of such nucleic acid molecules and vectors in the manufacture of compositions for treatment, prophylaxis or diagnosis of Helicobacter pylori infection are also further aspects of the invention.
  • the invention provides a method of eliciting in a mammal, including man, a protective immune response against Helicobacter pylori infection, said method comprising the step of administering to the said mammal an immunologically effective amount of a vaccine formulation according to the present invention.
  • immunologically effective amount is intended to mean an amount which elicit a significant protective Helicobacter pylori response, which will eradicate a H. pylori infection in an infected mammal or prevent the infection in a susceptible mammal.
  • an immunologically effective amount would comprise approximately 1 ⁇ g to 1 000 mg, preferably approximately 100 ⁇ g to 100 mg, of a H.
  • pylori antigen for oral administration or approximately less than 100 ⁇ g for parenteral administration.
  • An immunologically effective amount of the antigen is typically administered in a formulation with the antigen in a range from 0.01 % w/w to 99 % w/w.
  • Example 1 The preparation of the lipid aggregate comprising a H. pylori antigen is described in Example 1 and its use is illustrated in Example 2.
  • Example 3 to 7 describe the preparation and tests of a suitable antigen for the pharmaceutical formulation prepared in Example 1, i.e. an antigen for the formulation according to the present invention.
  • EXAMPLE 1 Formulation of the 29 kDa polypeptide rHpaA 1.1 Materials & Methods
  • BPS brain phosphatidylserine, Avanti
  • CH cholesterol
  • TES N- tris(Hydroxymethyl)methyl-2-aminoethanesulfonic acid
  • L-histidine NaCl
  • NOG N-octyl- ⁇ -D-glucopyranoside
  • CaCl 2 2H 2 0, NaOH, chloroform, and methanol were used as purchased.
  • the water was ELGA quality (18.2 M ⁇ ).
  • the 29 kDa polypeptide and polyclonal rabbit-anti-29 kDa were obtained inhouse according to the examples described below.
  • the final composition of the formulation was intended to contain 1 mg/mL of the antigen, with a lipid /antigen weight ratio of 4/1, and with a total lipid concentration in the range of 2.5 - 3 mg/mL.
  • a lipid film of BPS and CH ratio 9/1 w/w was obtained in a rotavapor system by evaporation of the chloroform/methanol (9/1 v/v) solvent that had been used for mixing the lipids.
  • the lipid film was dissolved in a buffer solution (2 mM TES, 2 mM L-histidine, 100 mM NaCl, pH adjusted to 8.0 with NaOH(aq)) containing 2% w/w NOG (the NOG/lipid ratio intended to be 7.5/1 w/w) with or without the antigen.
  • NOG was exchanged for Ca 2+ during dialysis (Spectrapoor no 1, MW cutoff 6000- 8000), twice versus a 3mM Ca 2+ buffer solution (overnight and four hours), and twice versus a 6 mM Ca 2+ solution (overnight and four hours).
  • the concentration of the centrifugated formulation was adjusted with 6mM Ca + buffer (vortex and 20 seconds in an ultrasonic bath) to give a composition in the formulation to be administered corresponding to 0.33 mg/mL of 29 kDa polypeptide. Administration of 300 ⁇ L would then give 100 ⁇ g administered 29 kDa polypeptide to each animal. Formulations manufactured without antigen were adjusted in the same way.
  • EXAMPLE 2 Analysis of the H. pylori 29 kDa protein for use as a vaccine, when used directly and when incorporated in a lipid aggregate formulation.
  • mice Female SPF BALB/c mice were purchased from Bomholt Breeding centre (Denmark). They were kept in ordinary makrolon cages with free supply of water and food. The animals were 4-6 weeks old at arrival.
  • H. pylori strain 244, originally isolated from an ulcer patient. This strain has earlier proven to be a good colonizer of the mouse stomach.
  • the bacteria were grown overnight in Brucella broth supplemented with 10% fetal calf serum, at +37°C in a microaerophilic atmosphere (10% C0 2 , 5% 0 2 ).
  • the animals were given an oral dose of omeprazole (400 ⁇ mol /kg) and after 3-5 h an oral inoculation of H. pylori (approximately 10 7 -10 8 cfu/animal). Infection was checked in control animals 2-3 weeks after the inoculation.
  • mice The 7 groups of mice, with 6 animals /group were immunized 4 times over a 34 day period (day 1, 15, 25 and 35).
  • Recombinant purified Helicobacter 29 kDa polypeptide was given at a dose of 100 ⁇ g/mouse either directly suspended in TES buffer or as incorporated into lipid aggregates (see above) .
  • the groups are listed in Table I.
  • CT cholera toxin
  • mice were sacrificed by C0 2 and cervical dislocation. The abdomen was opened and the stomach removed. After cutting the stomach along the greater curvature, it was rinsed in saline. In in stomach half, an area of 25 mm 2 of the mucosa from the antrum and corpus was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth and plated onto Blood Skirrow plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted. The identity of H. pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining.
  • Mucosal antibodies were collected by the following technique. One half of the rinsed stomach was placed mucosal side up on a piece of paper. Likewise the duodenum was cut open and placed mucosal side up. One standardized round filter paper (30.4 mm 2 ) was placed on the antrum and one on the corpus musosa. After 10 minutes the papers were transferred to one tube with 200 ⁇ l special buffer containing protease inhibitors. A paperstrip 4.8x19 mm (91.2 mm 2 ) was placed in the same way on the duodenum mucosa and subsequently treated in the same way. The buffer solution was, after a minimum of one hour extraction of the filter papers, used directly for ELISA measurements of antibody concentration or keep frozen at -20°C.
  • Serum antibodies were collected from blood drawn by heart-puncture under - anesthesia. Prior to centrifugation, the blood was diluted with equal amount of PBS. The serum was kept at -20°C until analysis.
  • Mucosal antibodies were measured using an ELISA were 29 kDa polypeptide was plated followed by addition of mucosal extract. The response was developed by using Alkaline Phospahatase labelled anti-IgA and anti-Ig. Standad curves were created from plating known amounts of mouse IgA and Ig instead of the unknown sample. Serum antibodies were measured in a similar way but in this case both membrane proteins of the infecting strain i.e. 244 as well as 29 kDa polypeptide was plated.
  • mice in this study were infected with H. pylori strain 244 one month prior to immunizations. Mice in groups of six were then immunized with either cholera toxin (CT) or CT together with the Helicobacter 29 kDa polypeptide either directly or formulated in lipid aggregates (LA). Lipid aggregates were also tested without any protein incorporation with and without CT (see Table I) Control animals received vehicle only (TES buffer). One week after the final immunization, the animals were sacrificed and colony forming units (CFU) was determined (see Fig. la, and Fig. lb). All control animals, as well as those receiving CT, LA and CT+LA were heavily infected in both antrum and corpus. Animals actively immunized with 29 kDa polypeptide plus CT, had significantly decreased bacterial content (CFU values) compared with the controls. In 2/5 mice no bacteria could be detected in the corpus.
  • CFU colony forming units
  • the degree of H. pylori colonization in the gastric mucosa decreased compared to administration of lipid aggregate alone.
  • the addition of CT to 29 kDa polypeptide containing lipid aggregate further decreased the bacterial content, especially in the antrum.
  • Serum antibody response was measured towards a membrane prepartion of H. pylori strain 244 (the strain used for infection of the mice) and towards 29 kDa polypeptide.
  • 29 kDa polypeptide formulated in the lipid aggregates according to the present invention could, even in the absence of CT, induce an immune response of such nature that the degree of H. pylori colonization of the gastric mucosa of mice decreased.
  • the H. pylori surface located protein 29 kDa polypeptide has been shown to be a strong and consistent antigen when in a purified recombinant form is presented to a mucosal surface in a physiological buffer solution. • The 29 kDa polypeptide will stimulate a competent local immune response capable of eradicating H. pylori colonizing the gastric mucosa.
  • 29 kDa polypeptide will induce an adequate local immune response even in the absence of CT, when it is formulated in lipid aggregates. It is concluded that 29 kDa polypeptide can evoke a relevant immune response locally in the gastric mucosa both when administered together with cholera toxin as well as when it is formulated in a lipid aggregate.
  • This lipid aggregate formulation obviously has the capability to present H. pylori antigens to the local immune system in such a way that an adequate eradicating immune response is obtained.
  • Lipid aggregates (LA) 0,3 ml /dose p.o 4
  • a suitable antigen for the formulation according to the present invention is the 29 kDa polypeptide described in the Applicant's co-pending W096/38475.
  • the 29 kDa polypeptide used in examples 1 and 2 was prepared according to the published W096/38475.
  • the plasmid pAEl has been deposited under the Budapest Treaty at the National Collections of Industrial and Marine Bacteria (NCIMB), Aberdeen, Scotland, UK, and under accession number NCIMB 40732. The date of deposit is 16 May 1995.
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  • TELEX 19237 astra s
  • TTT TCT TTT GCA CAA AAA AAA GAA GGG TAT TTG GCG GTT GCT ATG AAT 1164 Phe Ser Phe Ala Gin Lys Lys Glu Gly Tyr Leu Ala Val Ala Met Asn 110 115 120
  • Lys Val lie Ser Val Asp Ser Ser Asp Lys Asp Asp Phe Ser Phe Ala 100 105 110
  • Asn Lys lie Phe Ala Asn lie Met Gin Glu lie Asp Lys Lys Leu Thr 225 230 235 240
  • TTT TCT TTT GCA CAA AAA AAA GAA GGG TAT TTG GCG GTT GCT ATG AAT 1164 Phe Ser Phe Ala Gin Lys Lys Glu Gly Tyr Leu Ala Val Ala Met Asn 110 115 120
  • Lys Val lie Ser Val Asp Ser Ser Asp Lys Asp Asp Phe Ser Phe Ala 100 105 110
  • Asn Lys lie Phe Ala Asn lie Met Gin Glu lie Asp Lys Lys Leu Thr 225 230 235 240
  • Fig.la Number of Heliocobacter pylori (as CFU, geometric mean values) in 25mm 2 scrapings of antrum and corpus, following oral administration of different agents.
  • Fig.lb As la) but displayes as total number of Heliocobacter pylori in the stomach. Abbrevations see: Fig.la)
  • Fig.2 Total amount of mucosal immunoglobulin (Ig) with a specificity for 29kDa polypeptide, detected by ELISA technique. Abbrevations see: Fig.la
  • Fig.3 Amount of 29kDa specific mucosal IgA antibodies. Abbrevations see: Fig.la
  • Fig.4 Heliocobacter specific IgG antibodies in sera. Abbrevations see: Fig.la
  • Fig.5 29kDa specific IgG antibodies in sera. Abbrevations see: Fig.la

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  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une nouvelle formulation pharamceutique comprenant un agrégat lipidique d'un lipide à charge négative ou d'un mélange lipidique, et au moins un polypeptide antigénique, natif ou recombiné, qui constitue un antigène d'Helicobacter pylori, et éventuellement un support pharmaceutiquement acceptable. Cette invention concerne plus particulièrement un agrégat lipidique comprenant un antigène exposé en surface, qui présente un poids moléculaire d'environ 29 kDa. Cette invention concerne également une formulation d'une molécule d'acide nucléique codant pour ledit polypeptide. Ces nouvelles formulations, qui sont utiles en tant que formulations de vaccins, permettent de mettre en lumière une réponse immunitaire contre les infections dues à l'Helicobacter pylori; ces formulations de vaccins peuvent convenir à la fois à une utilisation thérapeutique et à une utilisation prophylactique.
PCT/SE1997/001927 1996-11-22 1997-11-18 Agregat lipidique pharmaceutique presentant un antigene d'helicobacter pylori et un lipide a charge negative WO1998022135A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU51418/98A AU5141898A (en) 1996-11-22 1997-11-18 Pharmaceutical lipid aggregate with helicobacter pylori antigen and negatively charged lipid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9604296-5 1996-11-22
SE9604296A SE9604296D0 (sv) 1996-11-22 1996-11-22 New pharmaceutical formulation of polypeptides

Publications (1)

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WO1998022135A1 true WO1998022135A1 (fr) 1998-05-28

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Country Link
AU (1) AU5141898A (fr)
SE (1) SE9604296D0 (fr)
WO (1) WO1998022135A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001076625A1 (fr) * 2000-04-12 2001-10-18 Astrazeneca Ab Systeme d'apport de polypeptide iscom
WO2001076623A1 (fr) * 2000-04-12 2001-10-18 Astrazeneca Ab Systeme de diffusion de polypeptide iscom pour antigenes de helicobacter pylori
EP1150710A1 (fr) * 1999-02-17 2001-11-07 Csl Limited Complexes immunogenes et methodes y relatives
WO2001083533A1 (fr) * 2000-04-29 2001-11-08 Astrazeneca Ab Antigene de helicobacter pylori : fusion de hpaa et de hpa44
WO2010041143A3 (fr) * 2008-10-08 2010-07-08 Immune Solutions Limited Vaccins oraux pour produire une immunite mucosale
US8258106B2 (en) 1994-07-15 2012-09-04 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
WO2017102779A1 (fr) * 2015-12-14 2017-06-22 Technische Universität München Vaccins contre helicobacter pylori
US10232022B2 (en) 2008-10-21 2019-03-19 Baxalta Incorporated Lyophilized recombinant VWF formulations
US11191837B2 (en) 2007-12-28 2021-12-07 Takeda Pharmaceutical Company Limited Recombinant VWF formulations
US11197916B2 (en) 2007-12-28 2021-12-14 Takeda Pharmaceutical Company Limited Lyophilized recombinant VWF formulations

Citations (1)

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WO1995009648A1 (fr) * 1993-10-04 1995-04-13 Gould Fogerite Susan Vaccins a base de cochleate proteique ou peptidique et procedes d'immunisation faisant appel a ces vaccins

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WO1995009648A1 (fr) * 1993-10-04 1995-04-13 Gould Fogerite Susan Vaccins a base de cochleate proteique ou peptidique et procedes d'immunisation faisant appel a ces vaccins

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Title
EMBL, DATABAS GENBANK/DDBJ, Accession No. X92502, JONES A.C. et al., "Gene Cloning of Flagellar Sheath Protein of Helicobacter Pylori"; & J. BACTERIOL., 175(3), 674-683 (1993). *
GUT, Volume 37, No. 1, 1995, A. JONES et al., "Gene Cloning of a Flagellar Sheath Protein of Helicobacter Pylori", page A63. *
MICROBIOLOGY, Volume 141, 1995, CATHERINE J. LUKE et al., "Identification of a 29 kDa Flagellar Sheath Protein in Helicobacter Pylori Using a Murine Monoclonal Antibody", pages 597-604. *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8258106B2 (en) 1994-07-15 2012-09-04 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
EP1150710A1 (fr) * 1999-02-17 2001-11-07 Csl Limited Complexes immunogenes et methodes y relatives
JP2002537271A (ja) * 1999-02-17 2002-11-05 シーエスエル、リミテッド 免疫原複合体およびそれに関する方法
EP1150710A4 (fr) * 1999-02-17 2003-09-03 Csl Ltd Complexes immunogenes et methodes y relatives
EP2204186A1 (fr) * 1999-02-17 2010-07-07 Csl Limited Complexes immunogènes et méthodes y relatives
US8173141B2 (en) 1999-02-17 2012-05-08 Csl Limited Immunogenic complexes and methods relating thereto
US7776343B1 (en) 1999-02-17 2010-08-17 Csl Limited Immunogenic complexes and methods relating thereto
WO2001076625A1 (fr) * 2000-04-12 2001-10-18 Astrazeneca Ab Systeme d'apport de polypeptide iscom
WO2001076623A1 (fr) * 2000-04-12 2001-10-18 Astrazeneca Ab Systeme de diffusion de polypeptide iscom pour antigenes de helicobacter pylori
WO2001083533A1 (fr) * 2000-04-29 2001-11-08 Astrazeneca Ab Antigene de helicobacter pylori : fusion de hpaa et de hpa44
US11191813B2 (en) 2007-12-28 2021-12-07 Takeda Pharmaceutical Company Limited Lyophilized recombinant VWF formulations
US11197916B2 (en) 2007-12-28 2021-12-14 Takeda Pharmaceutical Company Limited Lyophilized recombinant VWF formulations
US11191837B2 (en) 2007-12-28 2021-12-07 Takeda Pharmaceutical Company Limited Recombinant VWF formulations
CN102176907A (zh) * 2008-10-08 2011-09-07 免疫解决方案有限公司 用于产生粘膜免疫的口服疫苗
EP2344152A4 (fr) * 2008-10-08 2013-04-03 Immune Solutions Ltd Vaccins oraux pour produire une immunite mucosale
EP2344152A2 (fr) * 2008-10-08 2011-07-20 Immune Solutions Limited Vaccins oraux pour produire une immunite mucosale
WO2010041143A3 (fr) * 2008-10-08 2010-07-08 Immune Solutions Limited Vaccins oraux pour produire une immunite mucosale
US10232022B2 (en) 2008-10-21 2019-03-19 Baxalta Incorporated Lyophilized recombinant VWF formulations
WO2017102779A1 (fr) * 2015-12-14 2017-06-22 Technische Universität München Vaccins contre helicobacter pylori
US10828358B2 (en) 2015-12-14 2020-11-10 Technische Universität München Helicobacter pylori vaccines
AU2016374289B2 (en) * 2015-12-14 2023-08-03 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Helicobacter pylori vaccines

Also Published As

Publication number Publication date
SE9604296D0 (sv) 1996-11-22
AU5141898A (en) 1998-06-10

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