KR20010020418A - Anti-Helicobacter vaccine composition comprising a Th1-type adjuvant - Google Patents

Abstract

The present invention is derived from Helicobacter and relates to adjuvants such as QS-21, DC-chol or Bay R1005, which induces an immune response of T helper type 1 (Th1) in mammals. The present invention relates to the use of an immune substance for preparing a pharmaceutical composition and for preventing or treating a Helicobacter infection.

Description

Anti-Helicobacter vaccine composition comprising a Th1-type adjuvant}

Helicobacter is a genus of bacteria characterized by gram negative spiral bacteria. Several species cluster in the gastrointestinal tract of mammals. Specifically, there are H. pylori, H. heilmanii, H. felis and H. mustelae. H. pylori is the most common species involved in human infection, and in rare cases it is possible to separate H. heilmanii and H. felis in humans. Also bacteria of Helicobacter and Gastrospirillum hominis are found in humans.

More than 50% of the adult population in developed countries is infected with Helicobacter and nearly 100% of the adult population in developing countries has become one of the major infectious agents in the world.

H. pylori is so far found only on the surface of human gastric mucosa, and more particularly around crater lesions of gastric and duodenal ulcers. The bacterium is currently known as a causative agent of vestibularitis and is one of the co-factors necessary for the development of ulcers. Moreover, the development of gastric cancer is believed to be related to H. pylori.

Therefore, there is a great need to develop vaccines that prevent or treat Helicobacter infection.

To date, several Helicobacter proteins have already been proposed as vaccine antigens and the generally recommended vaccine method consists of delivering the antigen to the level of the gastric mucosa, i.e. to the location where the immune response should occur. Thus, for this purpose, oral administration was chosen.

For example, it has been proposed to deliver antigens to locations other than the gastric mucosa, such as the nose or rectal mucosa (International Patent Publication No. 96/31235). Lymphocytes stimulated by antigens in the mucus region, the so-called inducer, selectively migrate and circulate to other mucus regions, the so-called effectors, to induce an immune response.

A variation of this method consists in performing the first immunization via a systemic route prior to administering the antigen through the nose.

In administration via the mucus route, antigens that are predominantly bacterial lysate or purified protein are mixed with a suitable adjuvant such as E. coli cholera toxin (CT) or thermo-variable toxin (LT).

When administration via the mucus route is used, the humoral response observed is predominant in the IgA type. This indicates that a local immune response occurs.

Some very early thought that there was a close connection between the strong response of the IgA type and the protective effect [Czinn et al., Vaccine (1993) 11: 637]. Others had more limited opinion [Bogstedt et al., Clin. Exp. Immunol. (1996) 105: 202. Although there is no certainty on this task so far, the induction of antibodies of the type IgA in particular is what most people hope for.

In general, the appearance of IgA indicates a response (Th2 response) to type 2 T helper lymphocytes.

Stimulation of T helper lymphocytes by specific antigens makes it possible to obtain various subpopulations of T helper cells that synthesize various cytokines.

In particular Th1 cells selectively produce interleukin-2 (IL-2) and interleukin-γ (IFN-γ), while Th2 cells preferably secrete IL-4, -5 and -10. Due to the differentiated production of cytokines, these two types of T helper cells have a distinct role: Th1 cells promote cell-mediated immunity, ie infectious response, whereas Th2 cells promote IgA, Stimulates humoral responses of IgE and certain IgG subtypes. It is also known that cytokines produced by mouse Th1 cells can stimulate antibody responses and in particular INF-γ can induce IgG2a responses.

Therefore, from various studies of the prior art, there is a view that the induction of Th2 response characterized by the appearance of IgA is not sufficient but is essential for obtaining a preventive effect.

Surprisingly, even though the Th2 response was not dangerous, we found that it was also essential to induce a high Th1 response. Experimental results demonstrate that prophylactic effects are more easily related to Th1 responses than Th2 responses.

Contrary to what was initially discovered [D'Elios et al., J. Immunol., (1997) 158: 962], the present invention has found the importance of inducing an infectious Th1 response that has a prophylactic effect upon immunization.

For example, it is possible to induce a Th1 response to Helicobacter by applying many factors, such as an adjuvant type. By using a specific adjuvant, it has been demonstrated that a level of protection similar to or greater than that observed when an adjuvant such as mucus route and bacterial toxins is used.

The present invention relates to the specific use of vaccine preparations that induce a prophylactic immune response against pathogens, in particular Helicobacter bacteria, that infect mammalian mucosa.

1 is for Example 1 and shows the measurement of urease activity 4 hours after killing mice received three times in D0, D28 and D56: (a) about 80% via dorsolumbar muscle was DC- preparation of urease encapsulated with chol liposomes; Or (b) preparation of urease using cholera toxic adjuvant via the intestinal route. Experiments (c) and (d) correspond to positive and negative controls, respectively.

FIG. 2 is for Example 1 and shows the measurement of urease activity 4 hours after killing mice received 3 times in D0, D28 and D56: (a) cholera toxic adjuvant via intestinal route Preparation of urease or (b) preparation of urease using PCPP adjuvant via the subcutaneous route in the lower left lumbar region; Or (c) preparation of urease using QS-21 adjuvant via the subcutaneous route below and below. Experiments (c) and (d) correspond to positive and negative controls, respectively.

Figure 3 shows the amount of serum immunoglobulins induced in monkeys subjected to the immunological procedure described in Example 2 and expressed by ELISA titration. A control group comprising three monkeys and three test groups were prepared, each of which included eight monkeys; Each test group is divided into two subgroups of four monkeys, one with only inactivated H. pylori (1, 2, and 3) and the other with inactivated Helicobacter pillow (H.pylori) and recombinant urease (1u, 2u and 3u). Groups 1 and 1u correspond to the course of administration [nose + gut, 4]; Groups 2 and 2u correspond to the course of administration [intramuscular, 4]; Groups 1 and 1u correspond to the administration procedure [nose + intestinal, intramuscular, nasal + intestinal, intramuscular]. ELISA titrations were measured three times: the first at D0 (white band), the second at D42 (shaded band) and the third at D78 (black band).

Figure 4 shows the amount of serum immunoglobulins derived from mice subjected to the immunomodulatory procedure described in Example 3 and expressed by ELISA titration. ○ indicates ELISA IgG2a titration and ◆ indicates ELISA IgG1 titration. In addition to the reference group, two controls (positive and negative control), four test groups (A1 to A4) were formed, each group containing 10 mice. Determination of serum immunoglobulin amounts was performed with only 5 of 10 animals. Mice in groups A1 to A4 are QS-21 (A1), Bay R1005 (A2) via the subcutaneous route to the left rear lumbar sublumber portion. 10 μg of urease added with DC-chol (A3) or PCPP (A4) was administered. Mice in the control group received 40 μg of urease to which E. coli thermo-variable protein was added via the oral route.

FIG. 5 shows urease activity levels measured at the level of gastric mucosa at OD ₅₅₀ 4 hours after killing mice subjected to the immunomodulatory procedure described in Example 3. FIG. The group is as described in FIG.

FIG. 6 shows urease activity levels measured at the level of gastric mucosa at OD ₅₅₀ 24 hours after killing mice subjected to the immunomodulatory procedure described in Example 3. FIG. The group is as described in FIG.

FIG. 7 shows bacterial loads measured at the level of gastric mucosa after killing mice subjected to the immunomodulatory procedure described in Example 3. FIG. The group is as described in FIG.

Figures 8A and 8B show the Jaxrox test (Procter & Gamble) and bacterial load in mice infected with H. pylori and undergoing various treatments from A to H (Fig. 6B). Urea activity measured after time is shown (FIG. 6A) [A: LT + urease via oral route; B: QS21 + urease via the parenteral route of the neck; C: QS 21 + urease via the parenteral route of the lumbar region; QS 21 alone through the subcutaneous route of the lumbar region; E: Bay R1005 + urease via the parenteral route of the neck; F: Bay R1005 + urease via the parenteral route of the lumbar region; G: Bay R1005 alone (control) through the subcutaneous route of the lumbar region; H: Saline (positive control) through the subcutaneous route of the lumbar region] I represents the negative control.

As a result, the object of the present invention is to:

(a) In the manufacture of a medicament that tends to be administered via a tissue route to prevent or treat a Helicobacter infection, the immune response derived from Helicobacter and the T helper 1 (Th 1) type immune response against Helicobacter Concurrent use of compounds that may enhance induction.

(b) a pharmaceutical composition comprising an immune material derived from Helicobacter and at least one compound from the following (which may enhance the induction of a T helper 1 (Th1) type immune response to Helicobacter):

(i) saponins isolated from extracts of Quillaja saponaria;

(ii) a cationic lipid or a salt of the latter; Wherein the composition does not contain saponin or the glycolipid peptide represented by the following formula (I), and in the condition that the lipid is not provided in the form of liposomes, the lipid is a weak inhibitor of protein kinase C, a lipid derived from cholesterol Spacer arm comprising a linking group selected from affinity groups, carboxamides and carbamoyls, linear alkyl chains without C _{1 to} C ₂₀ side chains or without side chains, and primary, secondary, tertiary and quaternary A cationic amine group selected from amines; And

(iii) glycolipid peptides represented by the following structural formula (I):

At this time:

R ¹ represents a saturated or one or more unsaturated C ₁ -C ₅₀ , preferably C ₁ -C ₂₀ alkyl group,

X represents -CH _2- , -O- or -NH-,

R ² represents a hydrogen atom, a saturated or one or more unsaturated C ₁ -C ₅₀ alkyl group,

R ³ , R ⁴ and R ⁵ each independently represent a hydrogen atom, acyl-CO-R ⁶ (wherein R ⁶ is an alkyl group of C ₁ to C ₁₀ ),

R ⁷ is a hydrogen atom, a C _{1 to} C ₇ alkyl group, hydroxymethyl group, 1-hydroxyethyl group, mercaptomethyl group, 2- (methylthio) ethyl group, 3-aminopropyl group, 3-ureidopropyl group, 3 -Guanidylpropyl group, 4-aminobutyl group, carboxymethyl group, carbamoylmethyl group, 2-carboxyethyl group, 2-carbamoylethyl group, benzyl group, 4-hydroxybenzyl group, 3-indolylmethyl group or 4-imid Represents a dazolylmethyl group,

R ⁸ represents a hydrogen atom or a methyl group, and

R ⁹ is a hydrogen atom, an acetyl group, benzoyl group, trichloroacetyl group, trifluoroacetyl group, methoxycarbonyl group, t-butyloxycarbonyl group or benzyloxycarbonyl group, and

R ⁷ and R ⁸ may be bonded to each other to represent a —CH ₂ —CH ₂ —CH ₂ — group.

(c) in the preparation of a pharmaceutical composition capable of inducing a T helper 1 (Th1) type immune response against Helicobacter, the immunological substance derived from Helicobacter and the compounds mentioned above (i) to (iii) Use of at least one compound selected from; And

(d) at least one immune substance derived from said microorganism such as Helicobacter and at least one compound capable of enhancing the induction of a T helper 1 (Th1) type immune response against eg Helicobacter; A method comprising administering a composition to a mammal via a tissue route, thereby preventing or treating enhanced infection by a microorganism capable of infecting the gastroduodenal mucosa of the mammal, eg, Helicobacter infection.

(e) administering to a mammal a composition comprising a microorganism, for example at least one immune substance derived from Helicobacter, and at least one compound selected from the aforementioned compounds (i) to (iii), eg A method for preventing or treating an infection enhanced by a microorganism that can infect a gastroduodenal mucosa of a mammal, for example by inducing a Th1-type immune response against Helicobacter.

Induction of useful Th1 responses aims at the present invention by measuring the relative levels of Th1 responses to Th2 responses by comparing IgG2a and IgG1 levels induced in mice against Helicobacter, indicating each of the Th1 and Th2 responses, respectively. I can prove it. Indeed, the Th1 response found is usually accompanied by a Th2 response. However, the Th2 response is not significantly superior to the Th1 response. IgG2a and IgG1 levels induced in mice were determined using the ELISA test if the test used for each of the two subisotypes had equal sensitivity, especially if the anti-IgG2a and anti-IgG1 antibodies had equal affinity. It can be confirmed universally.

The amount of IgG2a and IgG1 is measured in particular using an ELISA test identical or similar to the following description. The wells of the polycarbonate ELISA plate are coated with 100 μl of bacterial extract of Helicobacter, for example H. pylori, which is about 10 μg / ml in carbonate buffer. ELISA plates were incubated at 37 ° C. for 2 hours and then left overnight at 4 ° C. Plates were washed with PBS (phosphate buffered saline) buffer (PBS / Twin buffer) containing 0.05% Tween 20. Wells were saturated with 250 μl of PBS containing 1% bovine serum albumin to prevent nonspecific binding of the antibody. After incubation at 37 ° C. for 1 hour, the plates were washed with PBS / Twin buffer. A few days after receiving a composition that tended to induce a Th1-type immune response against Helicobacter, antiserum obtained from mice was serially diluted with PBS / Twin buffer. 100 μl of dilution was added to the wells. Plates were measured according to incubation, washing and standard procedures for 90 minutes at 37 ° C. For example, goat antibodies against mouse IgG2a or IgG1 bound to enzymes such as peroxidase are used. Incubation continued in the presence of these antibodies for 90 minutes at 37 ° C. The plate was washed and then reacted with a suitable substrate (eg O-phenyldiamine dihydrochloride when the enzyme used was peroxidase). The response was measured by colorimetry (by measuring absorbance by spectrometry). IgG2a or IgG1 titre for antiserum corresponds to the inverse of the dilution with absorbance 1.5 at 490 nm.

Induction of a useful Th1 response for the purposes of the present invention is characterized in that the ELISA IgG2a: IgG1 titration ratio in mice is at least 1/100, 1/50 or 1/20, advantageously at least 1/10, preferably at least 1/3, most Preferably at least 1/2, 5 or 10. When the ratio is about 1, the Th1 / Th2 reaction is mixed or balanced. When the ratio is 5 or more, the Th1 reaction prevails.

Generation of a Th1 (or Th2) response in mice is equivalent to a Th1 (or Th2) response in humans. Although it is easier to assess the form of the response in mice, it can be assessed by measuring the level of cytokines specific for the Th2 response to the Th1 response, which in turn is induced in humans. Th1 and Th2 responses can be directly assessed in humans relative to each other based on the level of cytokines specific to the two forms of response (described above), for example based on the IFN-γ / IL-4 ratio. Can be.

Alternately, if the above-mentioned assay method is used, it is possible to predict that the ELISA titration reflecting the amount of IgG2a should be at least 10,000, preferably at least 100,000, more preferably at least 1,000,000. ; This means that the Th1 response is important.

Mammals for the intended pharmaceutical compositions or methods are preferred primates, preferably humans.

Saponins useful for the purposes of the present invention are Quilaja saponaria Molina by high-performance liquid chromatography (HPLC) and low-pressure chromatography with silica, not for structures in US Pat. No. 5,057,540. The fractions present after fractionating the liquid extract of the bark are described. In particular, QA-21, also referred to as fractions QA-7, QA-17, QA-18 and QS-21, was mentioned. Use of the latter is particularly preferred. QS-21 is known primarily as an adjuvant to enhance the induction of Th1-type immune responses. This adjuvant is called Th1 type.

Useful cationic lipids for the purposes of the present invention are described in detail in US Pat. No. 5,283,185. For example, cholesteryl-3β-carboxyamidoethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylamino-DL-2-propylcholesterylcarboxylate iodide, cholesteryl -3β-carboxamidoethyleneamine iodide, cholesteryl-3β-oxysucciamidoethylenetrimethylammonium iodide, 1-dimethylamino-3trimethylammonio-DL-2-propyl-cholesteryl- 3β-oxysuccinate iodide, 2-[(2-trimethyl-ammonio) ethylmethylamino] ethylcholesteryl-3β-oxysuccinate iodide, 3β- [N- (polyethyleneimine) carbamoyl] -Cholesterol and 3β- [N- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-chol) or salts thereof. DC-chol (or salt form thereof) is known as an adjuvant that promotes the induction of a mixed balanced response of the Th1 / Th2 type. The adjuvant consists of Th1 / Th2 or Th1 + Th2 types.

Such cationic lipids can be used in dispersion or can optionally be prepared in the form of liposomes. Liposomes can be prepared by binding to cationic lipids using neutral phospholipids such as phosphatidylcholine or phosphatidylethanolamine, as disclosed in US Pat. No. 5,283,185.

Glycolipid peptides useful for the purposes of the present invention are known in detail in US Pat. No. 4,855,283 and EP 206,037. These are in particular glycolipids represented by the general formula (I), wherein the residue of sugar is 2-amino-2-deoxy-D-glucose or 2-amino-2-deoxy-D-galactose residue to be. 2-sugar groups of amino sugars are in the form of D or L together with aminocarboxylic acids such as α-aminobutyric acid or α-amino valerianic acid, α-aminocaproic acid or α-aminoheptanoic acid , Sarcosine, hippuric acid, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, ornithine, citrulline, arginine. To aspartic acid, asparagine, glutamic acid, glutamine, phenylalanine, tyrosine, proline, tryptophan or histidine.

More specifically, it refers to the following glycolipid peptides:

N- (2-glycineamido-2-deoxy-β-deoxy-β-D-glucopyranosyl) -N-dodecyldodecanoylamide,

N- (2-glycineamido-2-deoxy-β-D-glucopyranosyl) -N-dodecylactadecanoylamide,

N- (2-glycineamido-2-deoxy-β-D-glucopyranosyl) -N-tetradecyldodecanoylamide,

N- (2-L-alanineamido-2-deoxy-β-D-glucopyranosyl) -N-dodecyldodecanoylamide,

N- (2-D-alanineamido-2-deoxy-β-D-glucopyranosyl) -N-dodecyloctadecanoylamide,

N- (2-D-phenylalanineamido-2-deoxy-β-D-glucopyranosyl) -N-dodecyloctadecanoylamide,

N- (2-L-valineamido-2-deoxy-β-D-glucopyranosyl) -N-octadecyldodecanoylamide,

N- (2-L-valineamido-2-deoxy-β-D-glucopyranosyl) -N-octadecyltetradecanoylamide,

N- (2-L-leucineamido-2-deoxy-β-D-glycopyranosyl) -N-dodecyldodecanoylamide,

N- (2-L-leucineamido-2-deoxy-β-D-glucopyranosyl) -N-octadecyldodecylamide (Bay R1005), and

N- (2-sarcosinamido-2-deoxy-β-D-glucopyranosyl) -N-octadecyldodecanoylamide.

The composition according to the invention comprises one or more of the compounds mentioned above. According to an advantageous embodiment, the composition uses two compounds; (a) one compound is selected from saponins purified from extracts of Quillaja saponaria, and (b) another compound is selected from (i) cationic lipids or salts thereof, The lipid is a spacer arm consisting of a weak inhibitor of protein kinase C and a linkage group selected from cholesterol-derived lipid affinity groups, carboxamides and carbamoyls, linear or non-branched alkyl chains of 1 to 20 carbon atoms. And, it means having a structure comprising a cationic amine group selected from primary, secondary, tertiary and quaternary amine, and is a compound selected from glycolipid peptide represented by (ii) or the above formula (I). For example, mention may be made of a mixture of QS21 + DC-Chol and QS-21 + Bay R1005.

Other adjuvant capable of promoting Th1-type immune response (referred to as Th1 or Th1 / Th2 form of adjuvant) from those of ordinary skill in the art who can choose the one that most closely matches the needs of those skilled in the art. There is a technical level of. As a guide, a wall of bacteria such as E. coli, Salmonella Minnesota, Salmonella typhimurium or Shigella flexneri; Algan-glucans; Gamma-inulin; Several other compounds, such as monophosphoryl-lipid A (MPLA), the major lipopolysaccharides from calcitriol and loxoribine, are of course particularly liposomes; ISCOMS; Microspheres; Protein chochleates; Vesicles consisting of nonionic surfactants; Cationic amphoteric dispersion in water; Oil / water emulsions; Mention is made of muramidyldipeptides (MDP) and derivatives thereof, such as glucosyl muramidyldipeptide (GMDP), threonyl-MDP, muramide and murapalmitin.

Useful liposomes for the purposes of the present invention include, in particular, cholesterol hemisuccinate (CHEMS) and dioleyl phosphatidyl ethanolamine (DOPE); 3-β- (N- (N ', N'-dimethylaminoethane) carbamoyl) cholesterol (DC-chol) and dimethyl as known from US Pat. No. 5,283,185 and WO 96/14831. Dioctadecylammonium bromide (DDAB), bay compounds known from EP 91645 and EP 0206 037, namely Bay R1005 (N- (2-deoxy-2-L-leucine) Liposomes comprising cationic lipids recognized for their fusiogenic properties such as amino-beta-D-glucopyranosyl) -N-octadecyldodecanoylamide acetate; and liposomes comprising MTP-PE, It is selected from pH-sensitive liposomes formed by the mixing of lipid affinity derivatives of MDP (muramidyldipeptide) Such liposomes are useful for addition as an adjuvant of all the above mentioned immune substances.

Useful ISCOMs for the purposes of the present invention can be selected in particular from compounds of QuilA or QS-21 that bind cholesterol and optionally bind phospholipids such as phosphatidylcholine. They are particularly useful for the preparation of lipid-containing antigens.

Useful microspheres for the purposes of the present invention can be formed from compounds such as polyactide-co-glycolide (PLAGA), alginates, chitosan, polyphosphazenes and many other polymers in particular.

Useful protein chochleates for the purposes of the present invention may be chosen, in particular, from those formed from additional phospholipids such as cholesterol and optionally phosphatidylcholine. These are especially used for the preparation of lipid-containing antigens.

Useful vesicles consisting of useful nonionic surfactants for the purposes of the present invention can be formed, in particular, by mixing 1-monopalmitoyl glycerol, cholesterol and disetylphosphate. These are selected from conventional liposomes and can be used for the preparation of all the above mentioned immune substances.

Useful oil / water emulsions for the purposes of the present invention can be chosen in particular from MF59 (Biocin-Kyron), SAF1 (Syntex) and Montanide ISA51 and ISA720 (Seppic).

Helicobacter-derived immune material is advantageously selected from preparations of purified Helicobacter bacteria, Helicobacter cell lysates, peptides and polypeptides.

For the purposes of the present invention, the preparation of inactivated bacteria is obtained according to universal methods well known in the art. As for bacterial lysates, the dose of inactivated bacterial or cellular lysates suitable for prophylactic or therapeutic purposes can be determined by one skilled in the art and can be determined by a variety of factors intended by the vaccine, such as age, antigen itself, administration. It depends on the route and method of, the presence / absence or form of the adjuvant. Generally, a suitable dose is 50 μg to 1 mg for about 1 mg of lysate.

Peptides or polypeptides derived from Helicobacter can be obtained by purified separation from Helicobacter or by chemical synthesis by genetic engineering techniques or alternatives. The latter process is beneficial in the case of peptides. A "peptide" is an amino acid chain that is about 50 amino acids in size or less. When larger, the term "polypeptide" may also be used, which may also be used as the term "protein". Peptides or polypeptides useful for the purposes of the present invention are the same or similar to those present in nature. It is similar in that it can induce the same type of immune response but includes structural diversity such as, for example, mutations, addition of residual groups of lipid properties or alternatively fusion of polypeptides or peptide forms.

Appropriate doses of peptides or polypeptides for prophylactic or therapeutic purposes can be determined by one skilled in the art and include various factors intended by the vaccine, such as age, antigen itself, route and method of administration, adjuvant. Depends on the presence / absence or form of In general, a suitable dose is about 10 μg to 1 mg and preferably about 100 μg.

An immune substance derived from Helicobacter is a polypeptide of Helicobacter, for example H. pylori. It is particularly present as a polypeptide present in the cytoplasm, a polypeptide of the inner and outer membranes or a polypeptide secreted into an external medium. Numerous polypeptides of Helicobacter have been described in reference to amino acid sequences that have already been described or cloned or identified from sequences of corresponding genes, or that purification procedures can be obtained in a form selected from the natural environment. As guidance, the following documents are specifically mentioned: International Patent Publication No. 94/26901 and International Patent Publication No. 96/34624 (HspA), International Patent Publication No. 94/09023 (CagA), International Patent Publication No. 96 / 38475 (HpaA), International Patent Publication No. 93/181150 (cytotoxin), International Patent Publication No. 95/27506 and Hazell et al., J. Gen. Microbiol. (1991) 137: 57 (catalase), French Patent No. 2 724 936 (membrane receptor for human lactoferrin), International Patent Publication No. 96/41880 (AlpA), European Patent No. 752 473 (FibA) and O ' Toole et al., J. Bact. (1991) 173: 505 (TsaA). Other polypeptides are also described in WO 96/40893, WO 96/33274, WO 96/25430, and WO 96/33220. Polypeptides useful for the purposes of the present invention are the same or similar to any of those cited in the reference for being able to enhance an immune response against Helicobacter. To satisfy this last condition, the immune material is also a peptide derived from the polypeptides cited by reference.

Advantageously, polypeptides selected from the UreA and UreB subunits of Helicobacter urease are used (WO 90/4030). Preferably, both mixed in urease apoenzyme form or alternatively in multimer form are used.

Useful pharmaceutical compositions for the purposes of the present invention comprise one immune substance or several immunomaterials. For example, advantageous compositions include UreA and UreB, eg, one or more other polypeptides selected from those mentioned above, as well as apoenzyme forms.

In addition, useful pharmaceutical compositions for the purposes of the present invention include compounds other than the immune substance itself and Th1 or Th1 / Th2 type adjuvant, the nature of these compounds being immune substances, inactivated bacteria, cell lysates, It depends to some extent on the nature of the peptide or polypeptide. For example, the composition also includes an adjuvant that can enhance the induction of a Th2-type immune response, such as aluminum compounds such as aluminum hydroxide, aluminum phosphate or aluminum hydroxyphosphate. This is an adjuvant useful for the purposes of the present invention is advantageous for Th1-type immunoadjuvant such as QS-21.

The therapeutic or prophylactic efficacy of a method or use according to the present invention may be achieved by standard methods such as Lee et al., Eur. J. Gastroenterology & Hepatology (1995) 7: 303 or Lee et al., J. Infect. Dis. (1995) 172: 161: The mouse / Helicobacter Felice model and process described in 161 are used to measure induction of an immune response or induction of therapeutic or prophylactic immunity. Those skilled in the art have realized that Helicobacter Fellis can be replaced by another Helicobacter species in a mouse model. For example, the efficacy of an immune substance derived from Helicobacter pylori is preferably assessed in a mouse model using Helicobacter pylori suitable for mice. Efficacy is determined by comparing the level of infection in gastric tissue (by measuring conditions of urease activity, bacterial load or gastritis) with a control. There is a therapeutic or prophylactic effect when the infection is reduced compared to the control.

Useful pharmaceutical compositions for the purposes of the present invention are prepared by universal methods. In particular, it is formulated with a pharmaceutically acceptable carrier or diluent, for example water or saline. Generally, diluents or carriers are selected according to the method and route of administration and according to standard pharmaceutical experiments. Suitable carriers or diluents, as well as those necessary for the preparation of pharmaceutical compositions, are described in the standard reference book Remington's Pharmaceutical Sciences in this field.

Compositions useful for this purpose, as well as methods according to the invention, include gastroduodenal diseases, including acute, chronic or atrophic gastritis, peptic ulcers, such as gastric or duodenal ulcers associated with Helicobacter infection and consequent such infection It is used to treat or prevent it.

The pharmaceutical compositions according to the invention are administered universally, in particular via the mucus route, for example into the eye, oral cavity, for example through the mouth, lungs, intestines, rectum, vaginal or urinary route or tissue and Parenterally, for example, via the intravenous, intramuscular, intradermal, epithelial and subcutaneous routes. Preferably, parenteral routes are used. When the parenteral route is used, below the diaphragm is selected as the preferred location for administration. The dorsolumbar portion constitutes a suitable site of administration, in particular the intraepithelial, intramuscular, intradermal and subcutaneous routes, and this latter route is preferably selected as the intravenous route.

In order to obtain a prophylactic or therapeutic effect, the practice consisting of administering a useful pharmaceutical composition for the purposes of the present invention is repeated once or several times, preferably at least twice, giving a time interval between each administration This interval is in the order of weeks or months. The exact determination of this is the ability of a person skilled in the art and varies depending on several factors such as the nature of the immune substance and the age of the subject.

According to a specific method, the vaccination process was performed using the same route of administration during the first and second boosters. In this particular case, the administration can be in strict tissue form, for example.

"Method of administering an immune substance is performed via a strict tissue route" is defined as a method that does not use a route of administration other than the tissue route. For example, the way in which the immune agent is administered by the tissue route or the mucus route does not fit the above definition. That is, "the method of administering an immune substance is to be carried out via a strict tissue route" should be understood to mean the way in which the immune substance is administered via other routes, in particular the tissue route which excludes the mucous route.

In a non-limiting example, urease apoenzyme mixed with QS-21, DC-chol or one of their equivalents is administered three times at intervals of 2-4 weeks via the subcutaneous route of the dorsolumber region. There is a vaccine plan consisting of:

It is also possible to foresee that administration of the pharmaceutical composition according to the invention is one step which forms part of a more sophisticated vaccine process. For example, a pharmaceutical composition according to the present invention may comprise an immune material derived from Helicobacter selected first or independently of those mentioned above, or other immune material such as a vaccine vector or DNA molecule, and QS-21, DC-chol or It is possible to later administer pharmaceutical compositions which do not include equivalents to them, in which case it is possible to replace the latter with completely different adjuvant and the two compositions can be administered via the same or different route.

By way of example and without limitation, the following procedure is mentioned:

First injection via the tissue pathway of urease apoenzyme in the presence of QS-21, followed by second immunization of urease apoenzyme with addition of QS-21 or LT via the mucus pathway; And

First immunization via the tissue pathway of the poxvirus encoding UreA and UreB, followed by a second immunization of urease apoenzyme with QS-21 added via the tissue or mucous pathway.

It can be used in a multistage vaccine procedure comprising a step of administering using a medicament or composition according to the invention useful for the purposes of the present invention, wherein an immune substance other than the one described above can be expressed in the expression of a polynucleotide molecule, in particular a mammalian cell. A DNA molecule comprising a sequence encoding a peptide or polypeptide of Helicobacter under the control of elements required for; Or alternatively is selected from a vaccine vector comprising a sequence encoding a peptide or polypeptide of Helicobacter under the control of elements necessary for the expression of a mammalian cell (if a viral vector) or a prokaryote (if a bacterial vector). .

DNA molecules are advantageous for plasmids that cannot replicate and substantially integrate into the mammalian genome. The coding sequence mentioned above is under the control of a promoter expressed in mammalian cells. Such promoters are universal or specific for tissues. Common promoters include the cytomegalovirus early promoter (US Pat. No. 4,168,062) and the Rous sarcoma virus promoter [Norton & Coffin, Molec. Cell. Biol. (1985) 5: 281. An optional promoter, the desmin promoter [Li et al., Gene (1989) 78: 244443; Li & Paulin, J. Biol. Chem. (1993) 268: 10403 to be expressed in muscle cells and skin cells. Promoters specific for muscle cells are, for example, promoters of myosin or dystrophin genes. Plasmid vectors that can be used for the purposes of the present invention are described in WO 94/21797 and Hartikka et al., Human Gene Therapy (1996) 7: 1205.

In pharmaceutical compositions useful for the purposes of the present invention, nucleotide molecules, such as DNA molecules, are formulated. The choice of formulation varies greatly. DNA is simply diluted in a pharmaceutically acceptable solution with or without a carrier. In the absence of a carrier, it is an isotonic solution or slightly hyperglycemic and has a low ionic force. For example, these conditions are filled with 20% sucrose solution.

Alternatively, the polynucleotides are mixed with a substance that promotes entry into the cell. It is (i) a chemical that regulates cell permeability, such as bupivacaine, or (ii) a substance that is mixed with polynucleotides and acts as a vehicle to promote the transport of polynucleotides. The latter are derivatives of cationic polymers, for example polylysine or polyamines, for example spermine (WO 93/18759). It is also a fusogenic peptide, for example a peptide derived from GALA or Gramicidin S (International Patent Publication 93/19768) or alternatively from a viral fusion protein.

It is also an anionic or cationic lipid. It is known that anionic or neutral lipids can act in the form of transporting substances, for example liposomes, for many compounds including polynucleotides for a long time. A detailed description of the liposomes, their components and the manufacturing process is provided by Liposome: A Practical Approach, RPC New Ed., Published by IRL (1990).

Cationic lipids are also known and are commonly used as transport materials for polynucleotides. DOTMA (N- [1- (2,3-diorayoxy) propyl] -N, N, N-trimethylammonium chloride), DOTAP (1,2-bis (oleoxyoxy) -3- (trimethyl ammonio) propane ), DDAB (dimethyldiooctadecylammonium bromine), DOGS (diooctadecylamidoglyl spermine) and DC-chol (3-beta- (N- (N ', N'-dimethylaminoethane) Lipofectin ™, known as a cholesterol derivative), is mentioned. A description of the lipids is provided by European Patent No. 187,702, International Patent Publication No. 95/26356, and US Patent No. 5,527,928. Cationic lipids are preferably used as neutral lipids such as, for example, DOPE (diorailylphosphatidylethanolamine) described in WO 90/11092.

Gold or tungsten microparticles are also used as the transport materials described in Nature (1992) 356: 152, including WO 91/359, WO 93/17706, and Tang. In this particular case, the polynucleotides are precipitated in the form of microparticles by the addition of calcium chloride and spermidine, and then entirely by high-speed jets to U.S. Pat. Administration is to the epidermis or epidermis using a needleless instrument such as that described in the heading.

The amount of DNA used to vaccinate the test article may be, for example, the strength of the promoter used to express the antigen, the immunity of the product being expressed, the conditions of the mammal being administered (e.g., weight, age and general health), administration It depends on many factors such as the method and formulation. In general, a suitable dose for prophylactic or therapeutic use in adults is 1 μg to 5 mg, preferably 10 μg to 1 mg, most preferably 25 μg to 500 μg.

Vaccine vectors are the aforementioned immune substances. Adenoviruses and poxviruses in particular are vectors of viral origin. Examples of vectors derived from adenoviruses, as well as methods of preparing vectors capable of expressing peptides or DNA molecules encoding polypeptides useful for the purposes of the present invention, are described in US Pat. No. 4,920,209. The poxviruses used as such are, for example, vaccinia and canarypox viruses. These are described in US Pat. Nos. 4,722,848 and 5,364,773, respectively (Tartaglia et al., Virology (1992) 188: 217 and Taylor et al., Vaccine (1995) 13: 539). Poxviruses capable of expressing peptides or polypeptides useful for the purposes of the present invention include DNA fragments encoding peptides or polypeptides suitable for expression in mammalian cells, as described in Kieny et al., Nature (1984) 312: 163. Obtained by homologous recombination placed under conditions. Bacterial vectors, such as the bile Calmette-Guerin bacillus, are also conceivable.

In general, the dose of viral vector intended for prophylactic or therapeutic purposes is about 1 × 10 ⁴ to 1 × 10 ¹¹ plaque forming units per kg, advantageously 1 × 10 ⁷ to 1 × 10 ¹⁰ , preferably Preferably 1 × 10 ⁷ to 1 × 10 ⁹ .

Among the bacterial vectors, Shigella, Salmonella, Vibrio cholerae, Lactobacillus and Streptococcus are mentioned. Non-toxic mutant strains of Vibrio cholerae as live vaccines are strains in which Mekalanos et al., Nature (1983) 306: 551 and US Pat. ); International Patent Publication No. 92/11354 (strains in which the irgA locus has been inactivated by mutation; these mutations are mixed in the same strain with the ctxA mutation); And International Patent Publication No. 94/1533 (mutations without functional ctxA and attRS1 sequences by deletion). This strain has been genetically engineered to express the heterologous antigens described in WO 94/19482.

Toxicized strains of Salmonella typhimurium, genetically engineered or otherwise used to express heterologous antigens as well as use as vaccines, are described in Nakayama et al., Biotechnology (1988) 6: 693 and internationally. Patent Publication No. 92/11361.

Other bacteria useful as vaccine vectors include High et al., EMBO (1992) 11: 1991 and Sizemore et al., Science (1995) 270: 299 (Shigella flexneri); Medaglini et al., Proc. Natl. Acad. Sci. USA (1995) 92: 6868 (Streptococcus gordonii); And Flynn J. L., Cell. Mol. Biol. (1994) 40 (suppl. I); International Patent Publication No. 88/6626, International Patent Publication No. 90/0594, International Patent Publication No. 91/13157, International Patent Publication No. 92/1796 and International Patent Publication No. 2/21376 (Calmete-Guerin Calmette-Guerin bacillus.

In bacterial vectors, DNA sequences encoding peptides or polypeptides of Helicobacter are carried by the plasmid to be inserted into the bacterial genome or alternatively remain free.

The DNA molecule or vaccine vector comprises the polypeptides encoding the polypeptides and peptides described above.

In addition, DNA molecules, preferably viral vaccine vectors, contain sequences encoding cytokines, such as lymphokines, such as interleukin-2 or -12, under the control of elements suitable for expression in mammalian cells. Alternatives to this selection also consist in adding pharmaceutical compositions useful for the purposes of the present invention comprising DNA molecules or vectors, another molecule encoding a cytokine or viral vector.

In general, an object of the present invention is also a pharmaceutical composition for treating or preventing a Helicobacter infection for continuous administration, comprising: (i) (a) inactivated Helicobacter bacteria, Helicobacter cells of Helicobacter in purified form; The first product comprising a helicobacter-derived immune material independently selected from the preparation of lysate, peptide and polypeptide, and (b) a compound capable of enhancing the induction of a Th1-type immune response, and (ii) in purified form Helicobacter bacteria, Helicobacter cell lysates, immune materials derived from Helicobacter independently selected from preparations of peptides and polypeptides, comprising a sequence encoding a peptide or polypeptide of Helicobacter under the control of an element required for expression DNA molecules and elements necessary for expression A second product comprising a vaccine vector comprising a sequence encoding a peptide or polypeptide of Helicobacter under the control of, preferably the first product comprises a peptide or polypeptide and the second product is a DNA molecule or vaccine vector When comprising, the coding sequence of said DNA molecule or vaccine vector provides for encoding the peptide or polypeptide contained in the first product.

In the above description, there is an essential reference to prevention and treatment, which is a means of combating Helicobacter infection and Helicobacter infection. However, the principles and methods described above can be applied to infections induced by microorganisms causing mutatis mutandis to cause gastric, duodenal or intestinal infections.

In addition, all documents cited and published herein are incorporated by reference.

Example 1 Study of Immune Action in Mice

1A- Materials and Methods

mouse

Swiss mice, 6 / 8-week-old females, were purchased from Janvier, France. Sterilized material was used during the entire experiment; The cages were protected by “isocaps”; Mice were fed filtered water and irradiated food.

Dosing process

During each experiment, mice received three doses of the same product, each administered at 28 day intervals (0, 28 and 56 days). Product administration can be achieved by nasal route (up to 50 μl in awake mice), oral route (300 μl in 0.2 M NaHCO ₃ through the gastric glands), or subcutaneous route (300 μl in the skin of the neck or on the left side of the lumbar region) Was performed through. In some cases intramuscular inoculation was performed in the dorsolumbar muscle of anesthetized mice (50 μl). 10 μg of urease was administered via the nasal, subcutaneous or intramuscular route and 40 μg was administered via the oral route. For inactivated bacterial preparations, 400 μg of cells were administered via the subcutaneous or oral route.

Antigens and Adjuvant

H. pylori urease apoenzyme was expressed in E. coli and purified as disclosed in the examples of WO 96/31235. In the rest of the text, the simple term urease is represented by these apoenzymes.

DC-chol liposomes containing urease were prepared as follows; First, in order to obtain a dry lipid film comprising 100 mg of DC-chol (R-gene therapy) and 100 mg of DOPC (daoleylphosphatidylcholine) (Avanti polar lipid), this product was powdered in about 5 ml of chloroform. Mixed. The solution was evaporated under vacuum using a rotary evaporator. The film obtained on the vessel wall was dried under high vacuum for at least 4 h. At the same time, 20 mg of urease lyophilizer and 100 mg of sucrose were diluted in 13.33 ml of 20 mM Hepes, pH 7.2 buffer. 10 ml of the solution prepared above (containing 1.5 mg of urease and 0.75% sucrose) was filtered through a 0.220 μm Millex filter and the lipid film was used for rehydration. The suspension was stirred for 4 h and then extruded (passed through a 0.2 μm polycarbonate separator to 10) or superfluidized (passed to 10 at a pressure of 500 kPa in a superfluidic Co Y10 superfluidizer). In the liposome suspension obtained above, the encapsulated urease was 10-60%. The sucrose concentration of the suspension was adjusted to 5% (addition of 425 mg of sucrose per 10 ml) followed by lyophilization. Prior to use, the lyophilizer is dissolved in a suitable volume of water or buffer, and the suspension is prepared at discrete sucrose concentrations (0, 30 and On 60% steps).

Cholera toxin was used as a mucosal adjuvant in a content of 10 μg per dose of urease or bacterial preparation.

QS-21 (Cambridge Bioscience) was used as an adjuvant of 15 μg per dose of urease.

Polyphosphazene (PCPP) (Virus Research Institute) was used as an adjuvant at a content of 100 μg per dose of urease.

Experiment

Two weeks after the second immunization, strain ORV2002 (1 × 10 ⁷ live bacteria in 200 μl PBS; OD ₅₅₀ of about 0.5) was added to the mouse along with 300 μl of a suspension of H. pylori strain suitable for the mouse. Administration was via gavage. One control group without antigen was tested as above.

Analysis of the Challenge

Four weeks after the experiment, the mouse's cervical vertebrae were shocked and killed. The stomach was removed for measuring urease activity and for histological analysis. Urease activity was measured by the Jatrox test after 4 and 24 hours (OD550 nm) [Procter & Gamble]. Several mice were still negative (OD values below 0.1) after 24 hours.

Measurement of partial antibody response by ELISPOT (salivary glands and stomach).

ELISPOTs are described in Mega et al., J. Immunol. (1992) 148: 2030. Plates were coated with extracts of H. pylori protein at a concentration of 50 μg / ml.

To test the antibody response at the gastric level, the following modified method was used: The stomach was cut into 1 mm ² size using an automatic instrument that cuts human tissue [McIllwain Laboratorious, Gilford, UK] and modified. 10% horse serum (Gibco), glutamine and antibiotics were added to the digestion of Dispase (2 mg / ml, Boeringer Mannheim) in 2 ml of Joklil solution. 4 and a half hours of digestion was performed with gentle mixing at 37 ° C. The digested cells were then filtered using 70 μm filter paper (Falcon) at each stage, then washed three times with RPMI 1640 (Gibco) solution with 5% fetal bovine serum (FCS), followed by nitrocellulose (Millipore) ( 100 μl / well, 4 wells) were incubated in the same solution for at least 4 hours. Stomach (stomach) the bandang 1 ~ 3 × 10 ⁵ cells are obtained (large cells and macrophages were counted in size).

Biotinylated IgA and streptavidin-biotin-coupled peroxidase complexes were purchased from Amersharm. Spots were found under the activity of AEC substrate (Sigma) and counted under the microscope as soon as the plates were dried (magnification x16 or x40). The average value corresponding to the number of IgGA spots in four wells was calculated and expressed as the number of spots per 10 ⁶ cells.

Response analysis by ELISA

ELISA analysis was performed according to standard procedures (biotin-conjugated conjugates and streptavidin-peroxidase were purchased from Amersham and OPD (O-phenyl-diamine dihydrochloride substrate was obtained from Sigma). The plates were coated with H. pylori extract in carbonate buffer A control serum from mice for H. pylori extract was introduced into each experiment. corresponds to the inverse of the dilution corresponding to 1.5 nm.

1B- result

The results are shown in Figures 1 and 2 described above and described below:

Before making any criticism for the purposes of Figures 1 and 2, it should be noted that the figure shows the results obtained with antigens in which choleratoxic adjuvant was used and administered via the intestinal route. This experiment is called a standard reference experiment because the prior art CT / IG mix gives the best results so far.

1 shows that urease preparations encapsulated in DC-chol liposomes show good results as obtained in standard reference experiments.

Furthermore, in the view of urease activity after 4 hours of death of the mice, the results of experiments (a) to (d) are shown in FIG. 1, which indicates that the number of mice still negative for urease activity until 24 hours after death was ( a) 5/10, (b) 4/10, (c) 0/10 and (d) 10/10. This is the same as the one concluded previously; That is, experiment (a) leads to results similar to those obtained with standard reference experiments.

Figure 2 shows that urease preparations using QS-21 adjuvant were as good as those obtained in standard reference experiments. In addition, the figure shows that the results obtained using PCPP as an adjuvant are much less satisfactory than those obtained using QS-21. This can be explained by the fact that PCPP preferably induces Th2-type response, whereas PCPP with urease induces Th2-type response as demonstrated in the table below, while QS-21 with urease induces Th1 / Th2 balanced response.

Moreover, the results of experiments (a) to (e) in the view of urease activity 4 hours after the death of the mouse are recorded in FIG. 2, which indicates that the number of mice still negative for urease activity until 24 hours after death. Respectively, (a) 1/8, (b) 0/8, (c) 5/8, (d) 0/8 and (d) 10/10, respectively. This is the same as the one concluded previously; In other words, experiment (c) leads to results similar to those obtained with standard reference experiments.

The amount of serum IgA, IgG1 and IgG2a derived during the experimental results for the urease activity shown in FIGS. 1 and 2 as well as the number of mice characterized by an OD value of 0.1 or less 4 and 24 hours after death are shown in the following table. Indicated. The amounts of IgA, IgG1 and IgG2a are shown by ELISA titrations.

The results shown in the table above show high serum antibody levels when the subcutaneous route is used (as well as the adjuvant suitable for such a route) rather than after the intestinal route (and appropriate adjuvant for this route). Moreover, these results show that, when DC-chol or QS-21 are used, higher IgG2a levels are obtained compared to IGG1 levels. This indicates that such adjuvant has the ability to induce Th1 response as well as Th2 response. On the other hand, when PCPP is used, the IgG2a level obtained is substantially lower than the IgG1 level. It can be concluded that the latter adjuvant cannot necessarily be a useful adjuvant for the purposes of the present invention by inducing a Th2 response.

Example 2: Immune Action Studies in Monkeys

2A- Materials and Methods

monkey

Twenty-eight biennial monkeys (Macaca fascicularis) purchased from Mauritius were used in this study. Before treating the monkeys with the various immunomodulatory procedures described below, most of them are biopsied chronically similar to Gastrospirillum homonis (GHLO) or Helicobacter H. heilmanii. Shows infection with organisms.

Dosing process

Since almost all monkeys were infected with GHLOs, they decided to test the efficacy of various processes in the treatment. Three procedures were used and summarized in the table below:

Administration via the intramuscular route is characterized as being performed in the dorsolumbar muscle.

Antigens and Adjuvant

Since there is a cross reaction between GPLOs and H. pylori, inactivated Helicobacter pylori (H. pylori) prepared as described in Example 1A alone or in accordance with the method referenced in Example 1A It was selected for use in combination with presynthetic urease.

E. coli thermo-variable toxin (LT) (Sigma) or cholera toxin B subunit (CTB) (Pasteur Meriux serums & vaccine) is used as a mucin adjuvant, while DC-chol is used as a parenteral adjuvant. DC-chol powders are simply rehydrated using antigen preparations.

The dose used is as follows:

Biopsies, urease tests and bacteriological / histologic studies

Biopsies were performed on each monkey before and after immunization (one month after the third immunization). Using biopsies, urease tests and histological studies were performed.

Urease activity was measured using a Jatrox kit (Procter & Gamble). The level of this activity was assessed in a decreasing manner as follows: level 3, pink appearing during the first 10 minutes; Level 2, pink appearing between 10 and 30 minutes after addition of reagents; Pink appears between level 1, 30 minutes and 4 hours, and weak or colorless after level 0 and 4 hours.

Histological studies were performed using biopsies immobilized on formalin and bacterial loads were quantified as follows: absence of bacteria (0); Some bacteria of Helicobacter type (0.5); As such many bacteria (1); Numerous bacteria (2); Quite a lot of bacteria (3). One level difference (eg 1 to 2) corresponds to 5 times the number of bacteria.

Response analysis by ELISA test

ELISA testing was performed as described in Example 1A.

1B- result

The following table relates to bacterial loads identified using two tests before and after immunization: (i) by measuring urease activity and (ii) by performing histological studies. The results on this are shown in columns 3-6. The last three columns point to each group (control, 1, 2 or 3). The number of monkeys in which the bacterial load remained unchanged (→) after immunization with two tests; Or one of the two tests is lower (↘) or increased (↗) and the other test indicates a bacterial load that does not change.

When the results of the two tests showed similar diversity, the up or down arrows were doubled.

Thus, the table shows that for groups undergoing immune measures via a strict mucus pathway, the results are substantially the same as those obtained in the negative control. On the other hand, in groups subjected to immunization via mixed mucus and intramuscular or strict intramuscular routes, a significant reduction in bacterial load is observed. This confirms the importance of immune action conditions, especially the adjuvant used; As a result, the use of an adjuvant such as DC-chol is recommended to promote a balanced Th1 and Th2 response in order to achieve a prophylactic effect.

These results are in line with other results for serum antibody levels shown in FIG. 3. 3 shows that immunization schemes through strict mucus pathways (1 and 1u) lead to results very similar to those of the negative control group. On the other hand, immunization schemes through mucosal and intestinal mixing pathways (2 and 2u) and stringent intramuscular pathways allow for antibody levels to be induced more substantially than controls.

Thus, high serum responses are associated with a preventive effect while low responses are associated with a lack of a preventive effect. Immunological conditions that are possible to achieve the desired effect (high serum response and prophylactic effect) require the use of a parenteral route or Th1 adjuvant that targets the diaphragm.

Example 3: Other Immunological Studies in Mice

3A- Materials and Methods

mouse

6 / 8-week female Swiss mice were provided by Janvier (France). In the entire experiment, sterile materials were used; The cage was protected by "isocaps"; Mice were fed filtered water and irradiated food.

Dosing process

In each experiment, mice received three doses of the same product; Each was administered 21 days apart (0, 21 and 42 days). Administration of the product was performed via the oral route (300 μl in 0.2 M NaHCO ₃ via the gavage) or the subcutaneous route (300 μl under the left skin of the lumbar region). 10 μg of urease was administered subcutaneously and 40 μg was administered via the oral route.

Antigens and Adjuvant

H. pylori urease apoenzyme was expressed in Escherichia coli and purified as described in Example 5 of WO 96/31235. In the remainder of the invention, the simple term urease refers to the apoenzyme.

E. coli thermo-variable toxin (Sigma) is used as a mucin adjuvant in an amount of 1 μg per serving of urease.

QS-21 (Cambridge Bioscience) is used as a mucin adjuvant in an amount of 15 μg per serving of urease.

Bay R1005 (Bayer) is used as a mucin adjuvant in an amount of 400 μg per urease.

DC-chol (R-Gene Therapeutics) is used as a mucus adjuvant in a content of 65 μg per urease.

Polyphosphazene (PCPP) (Virus Research Institute) is used as a mucosal adjuvant in an amount of 100 μg per urease.

Challenge

Four weeks after the second immunization, 300 μl (3 μL) of a suspension of H. pylori strain adapted to the mouse via a gavage and resistant to Streptomycin, strain ORV2001. 10 × ⁶ live bacteria). The control group was tested in the same manner without receiving the antigen.

The suspension was prepared as follows: Muller-Hinton medium (Biomeri) with 5% sheep blood containing H. pylori purchased from Sigma followed by antibiotics Cultured in bioMerieux) (MHA medium): 5 μg / ml Trimethoprim, 10 μg / ml Vancomycin, 1.3 μg / ml Polymixin B, Amhotericin 5 μg / ml and Steptomycin 50 μg / ml. Culture dishes were incubated for 3 days at 37 ° C. under microaerophilic conditions (amaerocult C, Merck). This culture was recovered for inoculation into a perforated 75 cm ² flask containing 50 ml of Brucella broth supplemented with 5% fetal bovine serum and the aforementioned antibiotics. The flasks were incubated under aerobic conditions with gentle mixing for 24 hours. The suspension was then diluted with Brucella broth to 0.1 at 550 nm OD.

Challenge Analysis

Four weeks after the experiment, mice were killed by impacting the cervical vertebrae. The stomach was removed to determine bacterial load through urease activity and quantitative culture. The upper quarter (antrum + corpus) above was used for each test. Urease activity was measured after 4 hours and 24 hours by Jatrox test (OD550 nm) [Procter & Gamble]. Several mice were still negative (OD values below 0.1) after 24 hours.

Infection measurement through quantitative culture of H. pylori

When the mice died, the upper 1/4 mucosa of each mouse was placed in Portagem medium purchased from bioMerieux and transferred to the incubation chamber within 2 hours. Samples were homogenized using a Dounce homogenizer (Weaton, Millville USA) containing 1 ml of Brucella medium (Brucella gravy) and diluted serially to 10 ⁻³ . 100 μl of each dilution (10 ⁰ , 10 ⁻¹ , 10 ⁻² and 10 ⁻³ ) was sprayed into Petri Dish containing NHA medium with the aforementioned antibiotics and incubated at 37 ° C. under aerobic conditions for 4 or 5 days. It became. Then, the number of living bacteria was counted. H. pylori was confirmed morphology using Gram staining and tested positive for urease, catalase and oxidase tests.

Response analysis by ELISA

ELISA analysis was performed according to standard procedures (biotin-conjugated conjugates and streptavidin-peroxidase were purchased from Amersham and OPD substrate from Sigma). The plate was coated with H. pylori extract in carbonate buffer. Control serum of mice against H. pylori extract was introduced in each experiment. The titration corresponds to the inverse of the dilution corresponding to 1.5 of OD490 nm.

3B- result

Before making any comment on the task of FIGS. 4-7, it should be noted that the figure shows the results obtained with antigens in which LT adjuvant was used and administered via the intestinal route. This experiment is called a standard reference experiment because the prior art LT / IG mixing gives the best results so far.

Serum reaction

As shown in FIG. 4, after three immunizations, mice immunized via the subcutaneous route show high serum IgG responses. Based on the IgG1: IgG2a ratio, it can be seen that PCPP induces a predominant response of the Th2 type (high IgG1 level, low IgG2a level). Bays R1005 and DC-chol induce a more balanced response of Th1 / Th2. Finally, QS-21 induces a predominant response of type Th1. In fact, the major difference between the four groups of mice A1 to A4 is in IgG2a titration, and the IgG1 titrations are all similar.

Prevention after experiment

5-7 show that the preventive levels of groups A1 and A2 are similar or better than those observed in the reference group (LT). Each of these groups received urease and bay R1005 with QS-21 added. Group A3 (DC-chol) showed a slightly lower level of prevention. On the other hand, in group A4 (PCPP), it is not possible to demonstrate a high prophylactic effect. It is found that the results shown in FIGS. 5 to 7 agree with each other.

Comparing the results shown in FIG. 4 with the results shown in FIGS. 5-7, the use of an adjuvant capable of inducing a Th1 or Th1 / Th2 response (QS-21. Bay R1005 or DC-chol) is a Th2-type immunity. It can be concluded that it enhances the protective effect as opposed to the use of adjuvant (PCPP).

Example 4 Treatment of H. pylori Infection in Mice

The present invention compared the efficacy of immunosuppressive actions via the subcutaneous route (SC) and the mucus route to treat H. pylori infection in mouse models.

OF1 mice were infected with 10 ⁶ plaque forming colonies (cfu) of the H. pylori ORV2001 strain. One month later, infection was checked by randomly killing 10/100 mice and testing urease activity against a quarter of the entire stomach. All results were positive, and mice (10 per group) were given either subcutaneous route or LT 1 using 15 μg of QS21 or 10 μg of recombinant urease with Bay R1005 adjuvant (Bayer) as an adjuvant. 40 μg of urease mixed with μg was immunized three times at three week intervals via the oral route. For each of the two adjuvant administered via the parenteral route, immune measures are performed to the neck to reach the upper lymph node or to the lumbar region to reach the abdominal lymph point. Ten mice (negative controls) remained uninfected and left unimmunized, whereas mice in the positive controls received saline, QS21 or bay immunoadjuvant via the subcutaneous route (lumbar region).

One month after the third immunization, the degree of colonization was measured by killing all mice, measuring urease activity (10/10 mice analyzed in each group) and performing quantitative culture (5/10 mice analyzed). To remove the stomach. 6A (tested for urease) and FIG. 6B (culture) show that in mice immunized with urease to which QS21 was added as an adjuvant via the subcutaneous route of the lumbar region, infection substantially disappeared (4/5 mice were quantitative). Negative in culture). In the presence of QS21 mice infected with urease through the subcutaneous route of the neck and mice receiving urease and LT via the oral route showed a 10-100 reduction in infection compared to uninfected mice. Bay immunoadjuvant induced the same reduction, which was more evident in mice immunized with lumbar region.

As observed in a previous prophylactic study of the present invention (Example 1), the prevented mice showed high serum levels for two isotypes IgG1 and IgG2 and represent a balanced Th2 / Th1 response. Moreover, mice immunized through the subcutaneous route of the lumbar region had the highest serum IgA levels, demonstrating the mucous response.

These results suggest that targeted tissue immunity can treat H. pylori infection in mice, and the use of adjuvant to induce Th1 / Th2-type mucosal response is desirable for this purpose. To indicate that

It is an object of the present invention to provide a compound capable of preventing or treating a Helicobacter infection and the use thereof.

Claims (28)

A pharmaceutical composition comprising an immune material derived from Helicobacter and at least one compound selected from: (i) saponins purified from extracts of Quillaja saponaria; (ii) a cationic lipid or a salt of the latter; Wherein the composition does not contain saponin or the glycolipid peptide represented by the following formula (I), and in the condition that the lipid is not provided in the form of liposomes, the lipid is a weak inhibitor of protein kinase C, a lipid derived from cholesterol Spacer arm comprising a linking group selected from affinity groups, carboxamides and carbamoyls, linear alkyl chains without C _{1 to} C ₂₀ side chains or without side chains, and primary, secondary, tertiary and quaternary A cationic amine group selected from amines; And (iii) glycolipid peptides represented by the following structural formula (I): At this time: R ¹ represents a saturated or one or more unsaturated C ₁ -C ₅₀ , preferably C ₁ -C ₂₀ alkyl group, X represents -CH _2- , -O- or -NH-, R ² represents a hydrogen atom, a saturated or one or more unsaturated C ₁ -C ₅₀ alkyl group, R ³ , R ⁴ and R ⁵ each independently represent a hydrogen atom, acyl-CO-R ⁶ (wherein R ⁶ is an alkyl group of C ₁ to C ₁₀ ), R ⁷ is a hydrogen atom, a C _{1 to} C ₇ alkyl group, hydroxymethyl group, 1-hydroxyethyl group, mercaptomethyl group, 2- (methylthio) ethyl group, 3-aminopropyl group, 3-ureidopropyl group, 3 -Guanidylpropyl group, 4-aminobutyl group, carboxymethyl group, carbamoylmethyl group, 2-carboxyethyl group, 2-carbamoylethyl group, benzyl group, 4-hydroxybenzyl group, 3-indolylmethyl group or 4-imid Represents a dazolylmethyl group, R ⁸ represents a hydrogen atom or a methyl group, and R ⁹ is a hydrogen atom, an acetyl group, benzoyl group, trichloroacetyl group, trifluoroacetyl group, methoxycarbonyl group, t-butyloxycarbonyl group or benzyloxycarbonyl group, and R ⁷ and R ⁸ may be bonded to each other to represent a —CH ₂ —CH ₂ —CH ₂ — group. The method of claim 1, wherein the composition comprises at least two kinds of compounds, wherein the first compound is selected from saponins purified from extracts of Quillaja saponaria, and the second compound is a cation. Selected from sex lipids or the following salts; The lipid is a spacer containing a linear alkyl chain does not contain a weak inhibitor and the side chain or the side chain of the coupler, C ₁ ~C ₂₀ selected from a lipid affinity group, carboxy amide and carbamoyl cholesterol derived from the Chi protein C Nez cancer (spacer arm) and a composition comprising a cationic amine group selected from primary, secondary, tertiary and quaternary amines. The composition of claim 1 or 2, wherein the compound is a QS-21 fraction purified from the extract of Quillaja saponaria as saponin. A composition according to claim 1 or 2, wherein the compound is a cationic lipid prepared in dispersed form. The compound according to claim 1, 2 or 4, wherein the compound is 3-β- [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-chol) or a salt of the latter. the latter) is a cationic lipid. The compound of claim 1, wherein the compound is N- (2-L-leucine-amido-2-deoxy-β-D-glycopyranosyl) N-octadecyl-dodecanoylamide (Bay) as a glycolipid peptide. R1005). The method according to any one of claims 1 to 6, wherein the Helicobacter-derived immune material is selected from inactivated Helicobacter bacteria, Helicobacter cell lysates, peptides and polypeptides prepared from purified Helicobacter. Composition. 8. The composition of claim 7, wherein the Helicobacter-derived immune material is Ureb or UreA subunit of Helicobacter urease. 9. The composition of claim 1, wherein the immune material is from Helicobacter pylori. 10. At least one or more of the following compounds and a helicobacter-derived immune material are selected for use in the manufacture of a pharmaceutical composition capable of inducing a T helper 1 (Th1) type immune response against Helicobacter . (i) saponins purified from extracts of Quillaja saponaria; (ii) a cationic lipid or a salt of the latter; Wherein the composition does not contain saponin or the glycolipid peptide represented by the following formula (I), and in the condition that the lipid is not provided in the form of liposomes, the lipid is a weak inhibitor of protein kinase C, a lipid derived from cholesterol Spacer arm comprising a linking group selected from affinity groups, carboxamides and carbamoyls, linear alkyl chains without C _{1 to} C ₂₀ side chains or without side chains, and primary, secondary, tertiary and quaternary A cationic amine group selected from amines; And (iii) glycolipid peptides represented by the following structural formula (I): At this time: R ¹ represents a saturated or one or more unsaturated C ₁ -C ₅₀ , preferably C ₁ -C ₂₀ alkyl group, X represents -CH _2- , -O- or -NH-, R ² represents a hydrogen atom, a saturated or one or more unsaturated C ₁ -C ₅₀ alkyl group, R ³ , R ⁴ and R ⁵ each independently represent a hydrogen atom, acyl-CO-R ⁶ (wherein R ⁶ is an alkyl group of C ₁ to C ₁₀ ), R ⁷ is a hydrogen atom, a C _{1 to} C ₇ alkyl group, hydroxymethyl group, 1-hydroxyethyl group, mercaptomethyl group, 2- (methylthio) ethyl group, 3-aminopropyl group, 3-ureidopropyl group, 3 -Guanidylpropyl group, 4-aminobutyl group, carboxymethyl group, carbamoylmethyl group, 2-carboxyethyl group, 2-carbamoylethyl group, benzyl group, 4-hydroxybenzyl group, 3-indolylmethyl group or 4-imid Represents a dazolylmethyl group, R ⁸ represents a hydrogen atom or a methyl group, and R ⁹ is a hydrogen atom, an acetyl group, benzoyl group, trichloroacetyl group, trifluoroacetyl group, methoxycarbonyl group, t-butyloxycarbonyl group or benzyloxycarbonyl group, and R ⁷ and R ⁸ may be bonded to each other to represent a —CH ₂ —CH ₂ —CH ₂ — group. The method according to claim 10, wherein the Helicobacter-derived immune substance and at least two or more compounds, the first compound is selected from saponins purified from extracts of Quillaja saponaria, the second compound is At least two compounds selected from cationic lipids or the following salts; The lipid is a weak inhibitor of protein kinase C and is a spacer arm comprising a linear alkyl chain that does not contain a linking group selected from alipophilic groups derived from cholesterol, carboxamides and carbamoyl, C _{1 to} C ₂₀ side chains or side chains. and a structure comprising a cationic amine group selected from primary, secondary, tertiary and quaternary amines. Use according to claim 10 or 11, characterized in that the compound is saponin, a QS-21 moiety purified from extracts of Quillaja saponaria. Use according to claim 10 or 11, characterized in that the compound is a cationic lipid prepared in dispersed form. 14. The compound of claim 10, 11 or 13, wherein the compound is 3-beta- [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-chol) or the latter salt is a cationic lipid. Characteristic uses. The compound of claim 10, wherein the compound is N- (2-L-leucine-amido-2-deoxy-β-D-glycopyranosyl) N-octadecyl-dodecanoylamide (Bay R1005) Phosphorus glycolipid peptide, characterized in that the use. The method according to any one of claims 10 to 15, wherein the Th1 type immune response is measured in a mouse, and the titration ratio of (i) ELISA IgG2a: IgG1 is at least 1: 100 or (ii) titration of ELISA IgG2a: IgG Use characterized in that the ratio is 1: 100 or more. The method according to claim 16, wherein the Th1 type immune response is measured in a mouse, characterized in that (i) the titration ratio of ELISA IgG2a: IgG1 is at least 1:10 or (ii) the titration ratio of ELISA IgG2a: IgG is at least 1:10. Use. 18. The method according to claim 17, wherein the Th1 type immune response is measured in mice, characterized in that (i) the appropriate ratio of ELISA IgG2a: IgG1 is at least 1: 2 or (ii) the appropriate ratio of ELISA IgG2a: IgG is at least 1: 2. Use. 19. The use according to any one of claims 10 to 18, wherein the Helicobacter-derived immune material is selected from inactivated Helicobacter bacteria, Helicobacter cell lysates, peptides and polypeptides prepared from purified Helicobacter. . 20. The use according to claim 19, wherein the Helicobacter-derived immune material is an Ureb or UreA subunit of Helicobacter urease. 21. The use according to any one of claims 10 to 20, wherein said immune substance is derived from Helicobacter pylori. 22. The use according to any one of claims 10 to 21, wherein said pharmaceutical composition is administered via a tissue route. Use according to claim 22, characterized in that the pharmaceutical composition is administered via a strict tissue route. 24. Use according to claim 22 or 23, characterized in that the pharmaceutical composition is administered via the diaphragm tissue route of a mammal, in particular a primate. Use according to any of claims 22 to 24, characterized in that the pharmaceutical composition is administered via the tissue route of the dorsolumbar portion of a mammal, in particular a primate. Use according to any one of claims 22 to 25, characterized in that the pharmaceutical composition is administered via the subcutaneous route, intramuscular route and intradermal route. 27. The use according to any one of claims 10 to 26, wherein said pharmaceutical composition is administered twice or three times through the tissue route during the same treatment to prevent or treat a Helicobacter infection. In the manufacture of a pharmaceutical composition administered via a tissue route for preventing or treating a Helicobacter infection, it is possible to enhance the induction of a T helper 1 (Th1) type immune response against Helicobacter-derived immune material and Helicobacter. Common uses of the compounds.