Classifications

• • 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
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • 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
  • A61K2039/106—Vibrio; Campylobacter; Not used, see subgroups
• • 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
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
• • 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
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2

Definitions

• Anû-Helicobacter vaccine composition comprising a Thl type adjuvant
• the present invention relates to the particular use of a vaccine preparation intended to induce in a mammal, a protective immune response against a pathogenic organism infecting mucous membranes, in particular against the Helicobacter bacteria.
• Helicobacter is a bacterial genus characterized by gram negative spiral bacteria. Several species colonize the gastrointestinal tract of mammals. We cite in particular H. pylori, H. heilmanii, H. felis and H. mustelae. Although H. pylori is the species most commonly associated with human infections, in some rare cases, H. heilmanii and H. felis have been isolated from humans. A Helicobacter type bacterium, Gastrospirillum hominis has also been described in humans.
• Helicobacter infects more than 50% of the adult population in developed countries and almost 100% of that of developing countries; making it one of the predominant infectious agents worldwide.
• H. pylori is found exclusively to date on the surface of the stomach lining in humans and more particularly around crater lesions of gastric and duodenal ulcers. This bacterium is currently recognized as the etiological agent of antral gastritis and appears as one of the cofactors required for the development of ulcers. Furthermore, it seems that the development of gastric carcinomas may be associated with the presence of H. pylori.
• a variant of these methods consists in carrying out a primary immunization by the systemic route before administering the antigen by the nasal route.
• the antigen most often a bacterial lysate or a purified protein, is combined with an appropriate adjuvant such as cholera toxin (CT) or heat-labile toxin (LT) of E. coli.
• CT cholera toxin
• LT heat-labile toxin
• the humoral response which is observed is predominantly of the IgA type. This indicates that there was a local immune response.
• IgA indicates the implementation of a response from T-helper type 2 lymphocytes (Th2 response).
• T-helper lymphocytes by a particular antigen makes it possible to obtain different sub-populations of T-helper cells, characterized by different synthesis profiles of cyto ines.
• Thl cells in particular selectively produce interleukin-2 (IL-2) and interferon- ⁇ (IFN- ⁇ ), while Th2 cells preferably secrete IL-4, -5, and -10 .
• IL-2 interleukin-2
• IFN- ⁇ interferon- ⁇
• Thl cells preferably secrete IL-4, -5, and -10 .
• cytokines these two types of T-helper cells have distinct roles: Thl cells promote cell-mediated immunity ia an inflammatory response, while Th2 cells stimulate the humoral response IgA, IgE and certain subclasses of IgG.
• cytokines produced by mouse Thl cells can stimulate the antibody response and in particular that IFN- ⁇ induces an IgG2a response.
• a pharmaceutical composition which comprises an immunogenic agent derived from Helicobacter and at least one compound (capable of promoting the induction of an immune response of the T-helper 1 (Thl) type against Helicobacter) selected among:
• lipids being weak inhibitors of protein kinase C and having a structure which includes a lipophilic group derived from cholesterol, a linking group selected from carboxyamides and carbamoyls, a spacer arm consisting of a linear alkyl chain, branched or not, from 1 to 20 carbon atoms, and a cationic amino group selected from primary, secondary, tertiary and quaternary amines, provided that these lipids are not presented in the form of liposomes when said composition does not contain saponin or glycolipopeptide of formula (I); and
• R ′ represents a saturated or unsaturated alkyl residue one or more times and comprising from 1 to 50 carbon atoms, preferably 1 to 20 carbon atoms,
• X represents -CH 2 -, -O- or -NH-
• R 2 represents a hydrogen atom or a saturated or unsaturated alkyl residue one or more times and comprising from 1 to 50 carbon atoms, preferably 1 to 20 carbon atoms,
• R 3 , R 4 and R 5 each represent, independently of one another, a hydrogen atom or an acyl-CO-R 6 residue, in which R 6 represents an alkyl residue having from 1 to 10 atoms carbon,
• R 7 represents a hydrogen atom, a C r C 7 alkyl group, hydroxymethyl, 1-hydroxyethyl, mercaptomephyl, 2- (methylthio) -ethyl, 3-aminopropyl, 3-ureido-propyl, 3-guanidylpropyl, 4- aminobutyl, carboxymethyl, carbamoylmethyl, 2-carboxethyl, 2-carbamoylethyl, benzyl, 4-hydroxybenzyl, 3-indolylmethyl or 4-imidazolylmethyl,
• R 8 represents a hydrogen atom or a methyl group
• R 9 represents a hydrogen atom, an acetyl, benzoyl, trichloracetyl, trifluoroacetyl, methoxycarbonyl, t-butyloxycarbonyl or benzyloxycarbonyl group, and
• R 7 and R 8 can, when taken together, represent a group
• a method for preventing or treating an infection promoted by a microorganism capable of infecting the gastro-duodenal mucosa of a mammal eg, a Helicobacter infection according to which the mammal is administered systemically, in one or more times , a composition containing at least one immunogenic agent derived from said microorganism eg from Helicobacter and at least one compound capable of promoting the induction of an immune response of the T-helper 1 (Thl) type against eg, Helicobacter .
• Thl T-helper 1
• a method for preventing or treating an infection promoted by a microorganism capable of infecting the gastro-duodenal mucosa of a mammal, eg a Helicobacter infection according to which a composition is administered to the mammal, in one or more times containing at least one immunogenic agent derived from said microorganism eg from Helicobacter and at least one compound selected from the compounds (i) to (iii) mentioned above, and by which an Thl type immune response is induced against eg d 'Helicobacter.
• Th1 response useful for the purposes of the present invention can be demonstrated by estimating the relative importance of the Th1 response with respect to the Th2 response, by comparing for example the levels of IgG2a and IgGl induced in mice against Helicobacter, which respectively demonstrate the implementation of the Th1 and Th2 responses.
• the Thl response that one seeks is generally accompanied by a Th2 response.
• the Th2 response should not be significantly predominant compared to the Th1 response.
• the levels of IgG2a and IgG1 induced in mice can be assessed in a conventional manner using an ELISA test, provided that the tests used for each of the two sub-isotypes are of the same sensitivity and in particular that the anti-IgG2a and anti-IgG 1 antibodies have the same affinity.
• the amounts of IgG2a and IgG1 can in particular be measured using an ELISA test identical or similar to that described below.
• the wells of a polycarbonate ELISA plate are coated with 100 ⁇ l of a bacterial extract of Helicobacter e.g. H. pylori at approximately 10 ⁇ g / ml in carbonate buffer.
• the ELISA plate is incubated for 2 hours at 37 ° C and then overnight at 4 ° C.
• the plate is washed with PBS buffer (phosphate buffered saline) containing 0.05% of Tween 20 (PBS / Tween buffer).
• the wells are saturated with 250 ⁇ l of PBS containing 1% bovine serum albumin in order to prevent the non-specific binding of the antibodies.
• the plate After one hour of incubation at 37 ° C, the plate is washed with PBS / Tween buffer.
• the plate is incubated for 90 minutes at 37 ° C, washed and evaluated according to standard procedures. For example, a goat anti-IgG2a or anti-mouse IgG1 antibody coupled to an enzyme such as peroxidase is used. Incubation in the presence of this antibody is continued for 90 minutes at 37 ° C.
• the plate is washed and the reaction is developed with the appropriate substrate (for example O-phenyldiamine dihydrochloride when the enzyme used is peroxidase).
• the reaction is evaluated by colorimetry (by measuring the absorbance by spectrophotometry).
• the titer of the antiserum in IgG2a or in IgG1 corresponds to the reverse of the dilution giving an absorbance of 1.5 to 490 nm.
• Th1 response useful for the purposes of the present invention is marked by a ratio of ELISA titers IgG2a: IgG1 in mice which must be greater than 1/100, 1/50 or 1/20, advantageously greater than 1 / 10, preferably greater than 1/3, most preferably, greater than 1/2, 5 or 10.
• this ratio is around 1, we then speak of a mixed Thl / Th2 response or balanced.
• the ratio is greater than or equal to 5
• Th1 (or Th2) response in mice is predictive of a Th1 (or Th2) response in humans. Although it is easier to assess the type of response in mice, it can also be done in humans by measuring the levels of cytokines specific for the Th1 response on the one hand and on the other hand the response Th2, which are subsequently induced. Thl and Th2 responses can be evaluated directly in humans relative to each other based on the specific cytokine levels of the two response types (see above) eg, based on the IFN- ⁇ / IL-4.
• the ELISA titer which reflects the amount of IgG2a, must be equal to or greater than 10,000, preferably equal to or greater than 100,000, of particularly preferably equal to or greater than 1,000,000; this then means that the Thl response is significant.
• the mammal for which the pharmaceutical composition or method is intended is advantageously a primate, preferably a human.
• Saponins useful for the purposes of the present invention are described in particular in US Pat. No. 5,057,540 with reference not to their structures but to the fractions in which they are present after fractionation of an aqueous extract of the bark of Quillaja saponaria Molina, by high pressure liquid chromatography (HPLC) and low pressure chromatography on silica.
• HPLC high pressure liquid chromatography
• QA-7, QA-17, QA-18 and QA-21 also known as QS-21.
• QS-21 is known to be an adjuvant which promotes the induction of an immune response mainly of the Thl type. We then speak of Thl type adjuvant.
• Cationic lipids useful for the purposes of the present invention are described in particular in US Patent No. 5,283,185.
• mention is made of cholesteryl-3 ⁇ -carboxyl-amido-ethylenetrimethylammonium iodide 1- dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide, cholesteryl-3 ⁇ -carboxyamidoethyleneamine iodide, cholesteryl-3 ⁇ -oxysyccinamidoethylenetrimethylammonium iodide, l-dimethylamino-3- iodide trimethylammonio-DL-2-propyl-cholesteryl-3 ⁇ -oxysuccinate, iodide of 2 - [(2- trimethylammonio) -ethylmethylamino] ethyl-cholesteryl-3 ⁇ -oxysuccinate, 3 ⁇ - [N- (polyethyleneimine) -carbamoyl] cholesterol,
• DC-chol (or its saline form) is known to be an adjuvant which promotes the induction of a balanced balanced response of the Thl / Th2 type. We then speak of a Thl / Th2 or Thl + Th2 type adjuvant.
• cationic lipids can be used in dispersion or else put in the form of liposomes.
• Liposomes can be produced as described in US Pat. No. 5,283,185, by combining the cationic lipids with a neutral phospholipid e.g., phosphatidylcholine or phosphatidylethanolamine.
• Glycolipopeptides useful for the purposes of the present invention are described in particular in US Patent No. 4,855,283 and EP 206,037. These are in particular glycolipids of general formula (I) in which a sugar residue is a 2-amino-2-deoxy-D-glucose or 2-amino-2-deoxy-D-galactose residue.
• the 2-amino group of the amino sugar can be linked to glycine, sarcosine, hippuric acid, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, ornithine, citrulline, arginine, aspartic acid, asparagine, glutamic acid, glutamine, phenylalanine, tyrosine, proline, tryptophan or histidine in D or L form or with aminocarboxylic acids such as ocaminobutyric acid, cc-aminovaleranic acid, cc-aminocaproic acid or oc acid
• glycolipopeptides are cited: N- (2-glycinamido-2-deoxy- ⁇ -deoxy- ⁇ -D-glucopyranosyl) -N-dodecyldodecanoyl- amide, N- (2-glycinamido-2-deoxy- ⁇ - D-glucopyranosyl) -N-dodecyl-actadecanoylamide, N- (2-glycinamido-2-deoxy- ⁇ -D-glucopyranosyl) -N-tetradecyl-dodecanoylamide, N- (2-L-alaninamido-2deoxy- ⁇ -D- glucopyranosyl) -N-dodecyl-dodecanoylamide, N- (2-D-alanimamido-2deoxy- ⁇ -D-glucopyranosyl) -N-dodecyl-octadecanoyl
• a composition according to the invention may contain one or more previously mentioned compounds.
• two compounds are used; (a) one being selected from saponins purified from an extract of Quillaja saponaria and (b) the other being selected (i) either from cationic lipids or a salt thereof, said lipids being inhibitors weak in protein kinase C and having a structure which includes a lipophilic group derived from cholesterol, a linking group selected from carboxyamides and carbamoyls, a spacer arm consisting of a linear alkyl chain, branched or not, from 1 to 20 atoms carbon, and a cationic amino group selected from primary, secondary, tertiary and quaternary amines, (ii) or from the glycolipeptides of formula (I).
• the mixtures QS21 + DC-Chol and QS-21 + Bay RI 005 are cited.
• adjuvants capable of promoting a Thl type immune response that is to say adjuvants of the Thl or Thl / Th2 type
• Thl type immune response that is to say adjuvants of the Thl or Thl / Th2 type
• adjuvants of the Thl or Thl / Th2 type exist in the prior art from which the person skilled in the art is able to select the one that best suits their needs.
• liposomes are mentioned in particular; ISCOMS; microspheres; protein chochleates; vesicles formed from nonionic surfactants; dispersions of cationic amphiphiles in water; oil / water emulsions; muramidyldipeptide (MDP) and its derivatives such as glucosyl muramidyldipeptide (GMDP), threonyl-MDP, murametide and murapalmitine; as well as various other compounds such as the monophosphoryl-lipid A (MPLA) major lipopolysaccharide of the wall of a bacterium, for example of E. coli, Salmonella minnesota, Salmonella typhimiirium or Shigella ⁇ exneri; algan-glucan; gamma-inulin; calcitriol and loxoribine.
• MDP muramidyldipeptide
• GMDP glucosyl muramidyldipeptide
• GMDP glu
• Liposomes useful for the purposes of the present invention can be selected in particular from pH-sensitive liposomes such as those formed by mixing cholesterol hemisuccinate (CH ⁇ MS) and dioleyl phosphatidyl ethanolamine (DOPE); liposomes containing cationic lipids known for their fusiogenic properties, such as 3 beta - (N- (N ', N'-dimethyl aminoethane) - carbamoyl) cholesterol (DC-chol) and its equivalents described in US Patent No.
• pH-sensitive liposomes such as those formed by mixing cholesterol hemisuccinate (CH ⁇ MS) and dioleyl phosphatidyl ethanolamine (DOPE); liposomes containing cationic lipids known for their fusiogenic properties, such as 3 beta - (N- (N ', N'-dimethyl aminoethane) - carbamoyl) cholesterol (DC-chol) and its equivalents described in US Patent No.
• ISCOMs useful for the purposes of the present invention can be selected in particular from those composed of QuilA or QS-21 associated with cholesterol and optionally also with a phospholipid such as phosphatidylcholine. These are particularly interesting for the formulation of lipid antigens.
• Microspheres useful for the purposes of the present invention can in particular be formed from compounds such as polylactide-co-glycolide (PLAGA), alginate, chitosan, polyphosphazene and many other polymers.
• PLAGA polylactide-co-glycolide
• alginate alginate
• chitosan alginate
• polyphosphazene polyphosphazene
• Protein chochleates useful for the purposes of the present invention can in particular be selected from those formed from cholesterol and optionally from an additional phospholipid such as phosphatidylcholine. These are especially advantageous for the formulation of lipid antigens.
• Vesicles formed from nonionic surfactants useful for the purposes of the present invention can in particular be formed by a mixture of 1-monopalmitoyl glycerol of cholesterol and of dicetylphosphate. They are an alternative to conventional liposomes and can be used for the formulation of all the immuno - , gt> è v -nes agents mentioned.
• Oil / water emulsions useful for the purposes of the present invention can in particular be selected from MF59 (Biocin-Chiron), SAF1 (Syntex) and the montanides ISA51 and ISA720 (Seppic).
• the immunogenic agent derived from Helicobacter is advantageously selected from a preparation of inactivated Helicobacter bacteria, a Helicobacter cell lysate, a peptide and a polypeptide of Helicobacter in purified form.
• a preparation of inactivated bacteria can be obtained according to conventional methods well known to those skilled in the art.
• a dose of inactivated bacteria or lysate cell, suitable for prophylactic or therapeutic purposes can be determined by those skilled in the art and depends on a number of factors such as the individual for whom the vaccine is intended, eg, age, of the antigen itself likewise, the route and mode of administration, the presence / absence or the type of adjuvant, as can be determined by those skilled in the art. In general, it is indicated that an appropriate dose is from about 50 ⁇ g to 1 mg to about 1 mg, of lysate.
• a peptide or a polypeptide derived from Helicobacter can be purified from Helicobacter or obtained by the techniques of genetic engineering or even by chemical synthesis. The latter method is advantageous in the case of peptides.
• Any chain of amino acids whose size is less than about 50 amino acids is called a "peptide".
• polypeptide is used which is also interchangeable with the term "protein”.
• a peptide or polypeptide useful for the purposes of the present invention can be identical or similar to that existing under natural conditions. It is similar in that it is capable of inducing an immune response of the same nature but it may include certain structural variations such as for example a mutation, the addition of a residue of lipid nature or else be in the form of a peptide. or fusion polypeptide.
• a dose of the peptide or of the polypeptide, suitable for prophylactic or therapeutic purposes can be determined by a person skilled in the art and depends on a certain number of factors such as the individual for whom the vaccine is intended, eg, age, the antigen itself, the route and mode of administration, the presence / absence or type of adjuvant, as can be determined by those skilled in the art. Generally, it is indicated that an appropriate dose is from about 10 ⁇ g to about 1 mg, preferably about 100 ⁇ g.
• the immunogenic agent derived from Helicobacter can be any Helicobacter polypeptide eg, from H. pylori. It can in particular be a polypeptide present in the cytoplasm, a polypeptide of the internal or external membrane or a polypeptide secreted in the external medium.
• Helicobacter polypeptides have already been described in the literature, either by reference to their amino acid sequence deduced from the sequence of the corresponding cloned or identified gene, or by reference to a purification process which makes it possible to obtain them in the form isolated from the rest of their natural environment.
• polypeptides are also described in WO 96/40893, WO 96/33274, WO 96/25430 and WO 96/33220.
• a polypeptide useful for the purposes of the present invention may be identical or similar to one of those cited in the reference insofar as it is capable of promoting an immune response against Helicobacter. Provided this latter condition is met, the immunogenic agent can also be a peptide derived from a polypeptide cited in reference.
• a polypeptide selected from the UreA and UreB subunits of Helicobacter urease is used (see WO 90/4030).
• they are used both, combined in the form of apoenzyme of urease or also in multimeric form (see WO 96/33732).
• a pharmaceutical composition useful for the purposes of the present invention may contain one or more immunogenic agents.
• an advantageous composition can comprise UreA and UreB e.g., in the form of an apoenzyme, as well as one or more other polypeptides in particular selected from those mentioned above.
• a pharmaceutical composition useful for the purposes of the present invention may also contain compounds other than the immunogenic agent itself and the adjuvant of the Thl or Thl / Th2 type; the nature of these compounds depending to a certain extent on the nature of the immunogenic agent, inactivated bacteria, cell lysate, peptide or polypeptide.
• a composition can also comprise an adjuvant capable of promoting the induction of a Th2 type immune response, e.g. an aluminum compound such as aluminum hydroxide, aluminum phosphate or aluminum hydroxy phosphate. This can be advantageous in the case where the adjuvant useful for the purposes of the present invention is a Thl type adjuvant such as QS-21.
• the therapeutic or prophylactic effectiveness of a method or use according to the invention can be evaluated according to standard methods eg, by measuring the induction of an immune response or the induction of therapeutic or protective immunity in using eg, the mouse / H. felis model and the procedures described in
• H. felis can be replaced in the mouse model by another species of Helicobacter.
• the efficacy of an immunogenic agent derived from H. pylori is preferably evaluated in a mouse model using a strain of H. pylori adapted to the mouse. Efficacy can be determined by comparing the degree of infection in the gastric tissue (by measuring urease activity, bacterial load, or gastritis status) to that of a control group. There is a therapeutic or protective effect when the infection is reduced compared to the control group.
• a pharmaceutical composition useful for the purposes of the present invention can be manufactured in a conventional manner.
• it can be formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or saline.
• a pharmaceutically acceptable diluent or carrier e.g., water or saline.
• the diluent or carrier can be selected depending on the mode and route of administration and according to standard pharmaceutical practices. Appropriate carriers or diluents as well as what is essential for the development of a pharmaceutical composition are described in Remington's Pharmaceutical Sciences, a standard reference book in this field.
• compositions useful for these purposes can be used to treat or prevent i.a. Helicobacter infections and therefore gastroduodenal diseases associated with these infections, including acute, chronic or atrophic gastritis, peptic ulcers e.g., gastric or duodenal ulcers.
• a pharmaceutical composition according to the invention can be administered in a conventional manner, in particular by the mucous route, eg by the ocular, oral route, eg buccal or gastric, pulmonary, intestinal, rectal, vaginal or urinary route, or by the systemic route, in particular parenteral, eg, intravenous, intramuscular route. , intradermal, intraepidermal and subcutaneous.
• parenteral eg, intravenous, intramuscular route. , intradermal, intraepidermal and subcutaneous.
• a site of administration is preferably chosen located under the diaphragm of an individual.
• the dorsolumbar region constitutes, for example, a suitable site of administration, in particular for the intraepidermal, intramuscular, intradermal and subcutaneous routes; the latter being chosen preferably by the intravenous route.
• the operation which consists in administering a pharmaceutical composition useful for the purposes of the present invention can be repeated one or more times, preferably at least twice, leaving a certain time interval between each administration; interval which is of the order of the week or the month. Its precise determination is within the reach of those skilled in the art and may vary depending on various factors such as the nature of the immunogenic agent, the age of the individual, etc.
• the vaccination protocol is implemented using the same route of administration during the primary immunization and boosters. In this case, we speak for example of strict system administration.
• a method in which the administration of the immunogenic agent is carried out by a strict systemic route is defined as a method which does not involve a route of administration other than the systemic route.
• a method in which the immunogenic agent is administered by the systemic route and by the mucous route does not meet the definition given above.
• a method in which the administration of the immunogenic agent is carried out by a strict systemic route must be understood as a method in which the immunogenic agent is administered by a systemic route excluding any other route, in particular the mucosal route.
• a vaccination scheme which consists in administering the urease apoenzyme in combination with QS-21, DC-chol or one of their equivalents, three times by subcutaneous route, in the dorso-lumbar region with an interval of two to four weeks between each administration.
• a pharmaceutical composition according to the present invention can be a simple step forming part of a more elaborate vaccination protocol.
• a pharmaceutical composition according to the present invention may be preceded or followed by the administration of a pharmaceutical composition containing an immunogenic agent derived from Helicobacter chosen independently from those listed above or from others such as a vaccine vector or a DNA molecule; but not containing QS-21, DC-chol or one of their equivalents; these can then be replaced by a whole other adjuvant; the two compositions can be administered by identical or different routes.
• Immunogens other than those described above and which can be used in a multistage vaccination protocol comprising an administration step involving a medicament useful for the purposes of the present invention or a composition according to the present invention can be selected from a polynucleotide molecule, in particular a DNA molecule comprising a sequence coding for a Helicobacter peptide or polypeptide placed under the control of the elements necessary for its expression in a mammalian cell; or alternatively a vaccine vector comprising a sequence coding for a Helicobacter peptide or polypeptide placed under the control of the elements necessary for its expression in a mammalian cell (if it is a viral vector) or in a prokaryote (if it is a bacterial vector).
• the DNA molecule can advantageously be a plasmid which is unable both to replicate and to integrate substantially in the genome of a mammal.
• the coding sequence cited above is placed under the control of a promoter allowing expression in a mammalian cell.
• This promoter can be ubiquitous or specific for a tissue.
• ubiquitous promoters mention is made of the early promoter of Cytomegalovirus (described in US Patent No. 4,168,062) and the promoter of Rous sarcoma virus (described in Norton & Coffin, Molec. Cell. Biol. (1985) 5: 281 ).
• the desmin promoter (Li et al, Gene (1989) 78: 244443; Li & Paulin, J. Biol. Chem. (1993) 268: 10403) is a selective promoter which allows expression in muscle cells and also in cells. skin.
• a specific promoter of muscle cells is for example the promoter of the myosin or dystrophin gene.
• plasmids which can be used for the purposes of the present invention are described i.a., in WO 94/21797 and Hartikka et al, Human Gene Therapy (1996) 7: 1205.
• the nucleotide molecule e.g. the DNA molecule can be formulated or not.
• the choice of formulation is very varied.
• the DNA can simply be diluted in a physiologically acceptable solution with or without a carrier. When the latter is present, it can be isotonic or weakly hypertonic and have low ionic strength. For example, these conditions can be fulfilled with a sucrose solution e.g. 20%.
• the polynucleotide can be combined with agents which promote entry into the cell. It can be (ii) a chemical agent which modifies cell permeability, such as bupivacaine (see for example WO 94/16737) or (ii) an agent which associates with the polynucleotide and acts as a vehicle facilitating the transport of the polynucleotide .
• the latter may in particular be cationic polymers e.g. polylysine or a polyamine e.g. derivatives of spermine (see WO 93/18759). They can also be fusogenic peptides e.g. GALA or Gramicidine S (see WO 93/19768) or even peptides derived from viral fusion proteins.
• Anionic or neutral lipids have been known for a long time as being able to serve as transport agents, for example in the form of liposomes, to a large number of compounds including polynucleotides. A detailed description of these liposomes, their constituents and their manufacturing processes is for example provided by Liposomes: A Practical Approach, RPC New Ed, IRL press (1990).
• Cationic lipids are also known and commonly used as transport agents for polynucleotides.
• Lipofectin I also known under the name of DOTMA (N- [l- (2,3-dioleyloxy) propyl] -N, N, N- trimethylammonium chloride), DOTAP (1,2-bis (oleyloxy ) -3- (trimethylammonio) propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidoglycyl spermine) and cholesterol derivatives such as DC-chol (3 beta - (N- (N ', N'-dimethyl aminoethane) ) -carbamoyl) cholesterol).
• Cationic lipids are preferably used with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine) as for example that is described in WO 90/1 1092.
• DOPE dioleyl phosphatidylethanolamine
• Gold or tungsten microparticles can also be used as transport agents, as described in WO 91/359, WO 93/17706 and Tang et al, Nature (1992) 356: 152.
• the polynucleotide is precipitated on the microparticles in the presence of calcium chloride and spermidine then the whole is administered by high-speed jet in the dermis or in the epidemic, using a device without a needle such as those described in US Pat. Nos. 4,945,050 and No. 5,015,580 and WO 94/24243.
• the amount of DNA that can be used to vaccinate an individual depends on a number of factors such as for example the strength of the promoter used to express the antigen, the immunogenicity of the product expressed, the condition of the mammal to which is intended for administration (eg, weight, age, and general health), mode of administration and type of formulation.
• a dose suitable for prophylactic or therapeutic use in an adult of the human species is from approximately 1 ⁇ g to approximately 5 mg, preferably from approximately 10 ⁇ g to approximately 1 mg, and very particularly preferably from about 25 ⁇ g to about 500 ⁇ g.
• vaccine vectors there are vaccine vectors.
• the vectors of viral origin are in particular adenoviruses and poxviruses.
• An example of a vector derived from an adenovirus as well as a method for constructing a vector capable of expressing a DNA molecule coding for a peptide or polypeptide useful for the purposes of the present invention are described in US Patent No. 4,920,209.
• Poxviruses which can also be used are, for example, vaccinia and canarypox viruses. They are described respectively in US patents Nos.
• Poxviruses capable of expressing a peptide or polypeptide useful for the purposes of the present invention can be obtained by homologous recombination as described in Kieny et al, Nature (1984) 312: 163, so that the DNA fragment encoding for the peptide or the polypeptide is placed under conditions suitable for its expression in mammalian cells.
• a bacterial vector such as the Biliated Bacillus of Calmette-Guérin can also be considered.
• the dose of a viral vector intended for prophylactic or therapeutic purposes can be from approximately 1x10 4 to approximately 1x10 ] , advantageously from approximately 1x10 7 to approximately 1x10 10 , preferably from approximately lxl 0 7 to approximately lxlO 9 units forming plates per kilogram.
• bacterial vectors there are in particular Shigella, Salmonella, Vibrio cholerae, Lactobacillus and Streptococcus.
• Non-toxic mutant strains of Vibrio cholerae which may be useful as a live vaccine are described in Mekalanos et al, Nature (1983) 306: 551 and US Patent No.
• the DNA sequence coding for a peptide or polypeptide ⁇ Helicobacter can be inserted into the bacterial genome or else remain in the free state, carried by a plasmid.
• a DNA molecule or a vaccine vector can comprise a sequence coding for any polypeptide or peptide described above.
• a DNA molecule, preferably a viral vaccine vector can also comprise a sequence coding for a cytokine, for example a lymphokine such as interleukin-2 or -12, under the control of elements suitable for expression in a cell. mammal.
• An alternative to this option also consists in adding to a pharmaceutical composition useful for the purposes of the present invention comprising a DNA molecule or a vector, another molecule or viral vector coding for a cytokine.
• the invention therefore also relates to a pharmaceutical composition intended for treating or preventing a Helicobacter infection which comprises for consecutive administration: (i) a first product containing (a) an immunogenic agent derived from Helicobacter independently selected from a preparation of inactivated Helicobacter bacteria, a Helicobacter cell lysate, a Helicobacter peptide and polypeptide in purified form, and (b) a compound capable of promoting the induction of an immune response from type Thl and (ii) a second product containing an immunogenic agent derived from Helicobacter independently selected from a preparation of inactivated Helicobacter bacteria, a Helicobacter cell lysate, a peptide and a polypeptide of Helicobacter in purified form, a DNA molecule comprising a sequence coding for a Helicobacter peptide or polypeptide placed under the control role of the elements necessary for its expression and a vaccine vector comprising a sequence coding for a Helicobacter peptide or polypeptide placed under the control of the elements
• Figure 1 refers to Example 1 and presents the levels of urease activity after challenge, measured 4 hrs after the sacrifice of the mice having received 3 times, on D0, D28 and D56: (a) a urease preparation approximately 80% encapsulated in DC-chol liposomes, in the dorso-lumbar muscles; or (b) an urease preparation adjuvanted by cholera toxin, intragastrically. Experiments (c) and (d) correspond respectively to the positive and negative controls.
• Figure 2 refers to Example 1 and presents the levels of urease activity after challenge, measured 4 hrs after the sacrifice of the mice having received 3 times, on D0, D28 and D56: (a) a urease preparation adjuvanted by cholera toxin, intragastrically or (b) a urease preparation adjuvanted by PCPP, subcutaneously in the left posterior sub-lumbar region; or (c) a urease preparation adjuvanted with QS-21, subcutaneously in the lower back. Experiments (c) and (d) correspond respectively to the positive and negative controls.
• Figure 3 shows the amounts of serum immunoglobulins induced in monkeys subjected to immunization protocols described in Example 2, and expressed as ELISA titer.
• a control group comprising 4 monkeys and three test groups are formed, each of the test groups comprising 8 monkeys; each test group is divided into two subgroups of 4 monkeys, one receiving only the preparation
• Group 1 and read corresponds to the administration protocol [nasal + intragastric, 4 times];
• group 2 and 2u corresponds to the administration protocol [intramuscular, 4 times];
• group 1 and read corresponds to the administration protocol [nasal + intragastric, intramuscular, nasal + intragastric, intramuscular].
• the ELISA titer is measured three times: a first time, on D0 (white stripe), a second time on D42 (gray stripe), a third time on D78 (black stripe).
• Figure 4 shows the quantities of serum immunoglobulins induced in mice subjected to the immunization protocols described in Example 3, and expressed in ELISA titer.
• O indicates the ELISA titer in IgG2a and indicates the titer
• mice Two control groups (positive and negative controls), four test groups (A1 to A4) as well as a reference group (LT) are formed; each of the groups including 10 mice. Measurements of the quantities of serum immunoglobulins are carried out for only 5 of the 10 mice.
• the mice of groups A1 to A4 received doses of 10 ⁇ g of urease subcutaneously in the left posterior sub-lumbar part, in the presence of QS-21 (Al), Bay R1005 (A2), DC-chol ( A3) or PCPP (A4).
• the reference group mice received doses of 40 ⁇ g of urease orally in the presence of the heat-labile protein of E. coli.
• FIG. 5 shows the levels of urease activity measured in the stomach mucosa, at DO550 4 hours after the mice subjected to the immunization protocols described in Example 3, have been sacrificed.
• the groups are as described for Figure 4.
• FIG. 6 shows the levels of urease activity measured in the stomach mucosa, at DO550 24 hours after the mice subjected to the immunization protocols described in Example 3, have been sacrificed.
• the groups are as described for Figure 4.
• FIG. 7 shows the bacterial load measured in the stomach mucosa, after the mice subjected to the immunization protocols described in Example 3, have been sacrificed.
• the groups are as described for Figure 4.
• Figures 8A and 8B show the urease activity (Fig. 6A) evaluated after 4 hrs (OD at 550 nm) with the Jatrox test (Procter & Gamble) and the bacterial load (Fig. 6B) in mice infected with H.
• mice 6/8 week old Swiss female mice were supplied by Janvier (France). For the duration of the experiment, sterilized equipment was used; the cages were protected by "isocaps"; the mice were fed filtered water and irradiated food.
• mice received 3 doses of the same product; each dose 28 days apart (days 0, 28 and 56).
• the administration of the product was carried out by the nasal route (up to 50 ⁇ l on awake mice), by oral route (300 ⁇ 1 in 0.2 M NaHC ⁇ 3 by gastric gavage), or by subcutaneous route (300 ⁇ l under the skin of the neck or under the skin on the left side of the lumbar region).
• an intramuscular inoculation was performed (50 ⁇ l) in the dorsolumbar muscles of the anesthetized mice.
• 10 ⁇ g of urease were administered by nasal, subcutaneous or intramuscular route, and 40 ⁇ g by oral route.
• 400 ⁇ g of cells were administered subcutaneously or orally.
• the apoenzyme of H urease. pylori was expressed in E. coli and purified as described in Example 5 of WO96 / 31235. In the following text to designate this apoenzyme, the simple term urease is used.
• DC-chol liposomes containing urease are prepared as follows: First, in order to obtain a dry lipid film containing 100 mg of DC-chol (R-Gene Therapeutics) and 100 mg of DOPC (dioleylphosphatidylcholine) ( Avanti Polar Lipids), these products are mixed in powder form in approximately 5 ml of chloroform. The solution is allowed to evaporate in vacuo using a rotary evaporator. The film thus obtained on the walls of the container is dried under high vacuum for at least 4 hrs. In parallel, 20 mg of a urease lyophilisate and 100 mg of sucrose are diluted in 13.33 ml of 20 mM Hepes buffer, pH 7.2.
• the lyophilisate is taken up in an appropriate volume of water or buffer and the suspension is purified on a discontinuous gradient of sucrose (steps of 0, 30 and 60%) so as to obtain a preparation in which the amount of encapsulated urease is greater than approximately 70% of the total amount of urease.
• Cholera toxin is used as a mucosal adjuvant at a rate of 10 ⁇ g / dose of urease or of bacterial preparation.
• QS-21 (Cambridge Biosciences) is used as an adjuvant at a rate of 15 ⁇ g / dose of urease.
• PCPP Polyphosphazene
• mice Two weeks after the second booster, the mice were subjected to gastric gavage with 300 ⁇ l of a suspension of an H. strain. pylori adapted to the mouse, the ORV2002 strain (1 x 10 7 live bacteria in 200 ⁇ l of PBS; OD 550 of approximately 0.5). A group having received no antigen back and serving as a control is tested the same.
• mice were sacrificed by rupture of the cervical vertebrae.
• the stomachs were removed to assess urease activity and to do histological analyzes.
• Urease activity was evaluated after 4 and 24 hours (OD at 550 nm) with the Jatrox test, Procter & Gamble) and after 24 hours the number of mice still negative (OD less than 0.1) was noted.
• the ELISPOTs were performed according to Mega et al, J. Immunol. (1992) 148: 2030.
• the plates were coated with an extract of H. proteins. pylori at a concentration of 50 ⁇ g / ml.
• the cells thus digested were filtered after each step using 70 ⁇ m filters (Falcon), then washed 3 times in a solution of RPMI 1640 (Gibco) supplemented with 5% of fetal calf serum (FCS), and incubated in the same solution for at least 4 hours in plates covered with nitrocellulose (Millipore) (100 ⁇ l / well, 4 wells). For each half of the stomach, 1 to 3, 10 5 cells are obtained (large cells and macrophages were not counted).
• Biotinylated IgA and the streptavidin-biotinylated peroxidase complex were from Amersham.
• the spots were revealed under the action of the AEC substrate (Sigma) and as soon as the plates were dry, they were counted under a microscope (magnification 16 or 40X).
• the mean values corresponding to the number of spots (spots) of IgA in four wells were calculated and expressed as the number of spots / 10 6 cells.
• Figure 1 shows that a urease preparation encapsulated in DC-chol liposomes gives as good results as those obtained in the standard reference experiment.
• Figure 2 shows that an urease preparation adjuvanted by QS-21 gives as good results as those obtained in the standard reference experiment.
• this figure shows that the results obtained using PCPP as an adjuvant are much worse than those obtained with QS-21. This is explained by the fact that the PCPP preferentially induces with the urease a Th2 type response while the QS-21 with the urease induces a balanced Th1 / Th2 response; as shown in the table below.
• experiments (a) to (e) whose results in terms of urease activity 4 hrs after the mice have been sacrificed are shown in FIG. 2 and it is indicated that the number of mice which are still negative for urease activity 24 hrs after being sacrificed is respectively (a) 1/8, (b) 0/8, (c) 5/8, (d) 0/8 and (e) 10/10. This is in accordance with what was concluded in the paragraph previously; namely that experiment (c) leads to results similar to those obtained during the standard reference experiment.
• the table below presents the quantities of serum IgA, IgG1 and IgG2a induced during the experiments whose results in terms of urease activity are reported in Figures 1 and 2 as well as the number of mice whose urease activity is characterized by an OD less than 0.1 after 4 and 24 hrs after sacrifice.
• the amounts of IgA, IgG 1 and IgG2a are expressed as ELISA titer.
• Example 1A Insofar as there is a cross-reactivity between the GPLOs and H. pylori, it was chosen to use a preparation of inactivated H. pylori bacteria, as described in Example 1A, alone or in combination with urease recombinant prepared according to the method referenced in Example 1A.
• DC-chol E. toxin heat-labile. coli (LT) (Sigma) or the cholera toxin B (CTB) subunit (Pasteur Mérieux sera & vaccines) was used as a mucosal adjuvant while DC-chol was used as a parenteral adjuvant.
• DC-chol powder is simply rehydrated by an antigen preparation. The doses used are as follows:
• Biopsies, urease test and bacteriological / histological study A biopsy was performed on each of the monkeys before and after immunization (one month after the third booster). From the biopsies, a urease test and a histological study were carried out.
• Urease activity is evaluated using the Jatrox kit (Procter & Gamble). The importance of this activity is estimated as follows, decreasingly: level 3, pink coloration appearing during the first 10 minutes; level 2, pink coloration appearing between 10 and 30 minutes after the addition of the reagents; level 1, pink coloring appearing between 30 min and 4 hrs and level 0, weak or non-existent coloring after 4 hrs.
• the table below relates to the bacterial load which, before and after immunization, is assessed using two tests: (i) by evaluating urease activity and (ii) by carrying out a histological study.
• the results relating thereto are presented in columns 3 to 6.
• the last three columns indicate for each group (control, 1, 2 or 3) the number of monkeys for which the bacterial load remains unchanged after immunization (_>) according to the two tests; or appears less (it) or increased (TJ) in at least one of the two tests, the other test indicating a stationary bacterial load.
• the results of the two tests go in the same direction, the upward or downward arrow is double.
• this table reveals that in the group having been subjected to a strict mucosal immunization protocol, the results are substantially identical to those obtained with the negative control group.
• an immunization protocol by mixed mucosal and intramuscular route or by the strict intramuscular route, a marked reduction in the bacterial load is observed.
• an adjuvant such as DC-chol, capable of promoting a balanced Thl and Th2 response, in order to obtain a protective effect.
• the immunization conditions enabling the desired effect to be obtained include the use of the targeted parenteral route in the sub-diaphragmatic region or that of a Thl adjuvant.
• mice 6/8 week old Swiss female mice were supplied by Janvier (France). For the duration of the experiment, sterilized equipment was used; the cages were protected by "isocaps"; the mice were fed filtered water and irradiated food. Administration protocol
• mice received 3 doses of the same product; each dose 21 days apart (days 0, 21 and 42).
• the administration of the product was carried out orally (300 ⁇ l in 0.2 M NaHCO ⁇ by gastric gavage), or by subcutaneous route (300 ⁇ l under the skin on the left side of the lumbar region).
• Ten ⁇ g of urease was administered subcutaneously and 40 ⁇ g orally.
• the apoenzyme of H urease. pylori was expressed in E. coli and purified as described in Example 5 of WO96 / 31235. In the following text to designate this apoenzyme, the simple term urease is used.
• E. toxin heat-labile. coli (Sigma) is used as a mucosal adjuvant at a rate of 1 ⁇ g / dose of urease.
• QS-21 (Cambridge Biosciences) is used as an adjuvant at a rate of 15 ⁇ g / dose of urease.
• Bay RI 005 (Bayer) is used as an adjuvant at a rate of 400 ⁇ g / dose of urease.
• DC-chol (R-Gene Therapeutics) is used as an adjuvant at a rate of 65 ⁇ g / dose of urease.
• PCPP Polyphosphazene
• mice Four weeks after the second booster, the mice were subjected to gastric gavage with 300 ⁇ l (3 x 10 6 live bacteria) of a suspension of a strain of H. pylori adapted to mice and resistant to Streptomycin, the strain ORV2001. A group having received no dose of antigen and serving as a control is likewise tested.
• test suspension is prepared as follows: H. pylori is cultured on Muller- ⁇ inton agar (Difco) containing 5% sheep blood (bioMérieux) (medium
• M ⁇ A which contains the following antibiotics from Sigma: Trimethoprim 5 ⁇ g / ml,
• the culture dishes are incubated for 3 days at 37 ° C. under microaerophilic conditions (Anaerocult C, Merck).
• This culture is harvested to inoculate a bottle provided with vents of 75 cm 2 (Costar) containing 50 ml of Brucella broth supplemented with 5% fetal calf serum and with the abovementioned antibiotics.
• the bottle is incubated under micro aerophilic conditions, with gentle shaking for 24 hours.
• the suspension is then diluted in Brucella broth to give an OD of 0.1 to 550 nm (or 10 7 CFU / ml).
• mice were sacrificed by rupture of the cervical vertebrae.
• the stomachs were removed to evaluate the urease activity and the bacterial load by quantitative culture.
• a longitudinal quarter of the stomach (antrum + corpus) is used for each of the tests.
• Urease activity was evaluated after 4 and 24 hours (OD at 550 nm) with the Jatrox test, Procter & Gamble) and after 24 hours the number of mice still negative (OD less than 0.1) was noted.
• the mucosa of a quarter of the stomach of each mouse is placed in the Portagem medium of bioMérieux and then within two hours, transferred to the culture chamber.
• the sample is then homogenized using a Dounce homogenizer (Wheaton, Millville USA) containing 1 ml of Brucella medium (Brucella broth) and diluted in series to 10 ⁇ 3 .
• the ELISA analyzes were carried out according to the standard protocol (the biotinylated conjugates and streptavidin peroxidase came from Amersham and the OPD substrate from Sigma).
• the plates were coated with H pylori extracts (5 ⁇ g / ml) in carbonate buffer.
• a mouse control serum directed against the extract of H. pylori was introduced in each experiment.
• the titer corresponds to the inverse of the dilution giving an OD of 1.5 to 490 nm.
• Serum response As shown in Figure 4, after three immunizations, all mice immunized subcutaneously show a significant serum IgG response.
• IgG 1 IgG2a ratios
• the PCPP induces a predominant response of the Th2 type (high IgG1 level, low IgG2a level).
• Bay RI 005 and DC-chol induce a more balanced Thl / Th2 response.
• QS-21 induces a predominant Thl-type response.
• the main difference between the four groups of mice A1 to A4 lies in their IgG2a titers, the IgG l titers all being similar.
• Post-test protection Figures 5 to 7 show that the level of protection in groups A1 and A2 is similar to or even better than that observed in the reference group (LT). These are the groups that received the urease doses in the presence of QS-21 and Bay RI 005 respectively.
• Group A3 DC-chol
• group A4 PCPP
• SC subcutaneous
• OF1 mice were infected with 10 6 plaque-forming colonies (cfu) of the H. strain. pylori ORV2001. After one month, it was verified that the infection was well established by randomly sacrificing 10/100 mice and testing urease activity on a quarter of the entire stomach. The results being all positive, we then immunized mice (10 per group) 3 times at 3 weeks apart, either subcutaneously using 10 ⁇ g of recombinant urease adjuvant with 15 ⁇ g of QS21 (Aquila) or 400 ⁇ g of Bay R1005 adjuvant (Bayer), or orally using 40 ⁇ g of urease mixed with 1 ⁇ g of LT.
• immunization was performed either in the neck, to reach the lymph nodes in the upper region of the body, or in the lumbar region, to reach the abdominal lymph nodes.
• Ten mice were left uninfected and unimmunized (negative control), while mice in the positive control group received saline, QS21 or Bay adjuvant subcutaneously (lumbar region).
• mice Performing histopathology on these same mice did not reveal any greater gastritis compared to the controls.
• protected mice had a high serum level of the two isotypes IgGl and IgG2, which is representative of a balanced Th2 / Thl response.
• mice immunized subcutaneously in the lumbar region had the highest serum IgA levels, demonstrating a mucosal response.

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