WO2002045741A2 - Helicobacter pylori prime and boost vaccination comprising caga and nap antigens - Google Patents

Abstract

A method for immunising a mammal against H.pylori infection, comprising the administration of a first immunogenic composition as a mucosal priming dose and a second immunogenic composition as a parenteral boosting dose, wherein the first and second immunogenic compositions each comprise CagA and NAP antigens. The combination of CagA and NAP has been found to be particularly effective in raising mucosal and systemic humoral immune responses when administered according to a mucosal prime/parenteral boost regime.

Description

HELICOBACTER PYLORI PRIME AND BOOST VACCINATION

All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

This invention is in the field of vaccination, more particularly in the field of vaccination (prophylactic or therapeutic) against Helicobacter pylori infection.

BACKGROUND ART

For many years, vaccination against Helicobacter pylori infection focused solely on mucosal administration, and successful parenteral vaccination was reported with surprise [e.g. reference 1]. A vaccination regime based on mucosal priming and parenteral boosting with urease antigen has also been disclosed [e.g. references 2 and 3].

It is an object of the invention to provide alternative and improved methods which use parenteral administration for vaccinating against Hpylori infection.

DISCLOSURE OF THE INVENTION

The invention provides a method for raising an immune response in a mammal, the method comprising the mucosal administration of a first immunogenic composition followed by the parenteral administration of a second immunogenic composition, wherein the first and second immunogenic compositions each comprise Helicobacter pylori CagA and NAP antigens. Combinations including CagA and NAP have been found to be particularly effective in raising mucosal and systemic humoral immune responses when administered according to a mucosal prime/parenteral boost regime.

The first immunogenic composition (priming dose)

The first immunogenic composition is given mucosally. Suitable routes of mucosal administration include oral, intranasal (IN), intragastric, pulmonary, intestinal, rectal, ocular, and vaginal routes. Oral or intranasal administration is preferred. The first immunogenic composition is preferably adapted for mucosal administration [e.g. see refs. 4, 5 & 6]. Where the composition is for oral administration, for instance, it may be in the form of tablets or capsules (optionally enteric-coated), liquid, transgenic plants etc. [see also ref. 7, and Chapter 17 of ref. 19].

Where the composition is for intranasal administration, it may be in the form of a nasal spray, nasal drops, gel or powder [e.g. ref. 8].

The first immunogenic composition may comprise a mucosal adjuvant. Suitable mucosal adjuvants include: (A) E.coli heat-labile enterotoxin ("LT"), or detoxified mutants thereof, such as the K63 or R72 mutants [e.g. Chapter 5 of ref. 9]; (B) cholera toxin ("CT"), or detoxified mutants thereof [e.g. Chapter 5 of ref. 9]; or (C) microparticles {i.e. a particle of ~100nm to ~150μm in diameter, more preferably ~200nm to ~30μm in diameter, and most preferably ~500nm to ~10μm in diameter) formed from materials that are biodegradable and non-toxic {e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone etc.); (D) a polyoxyethylene ether or a polyoxyethylene ester [10]; (E) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol [11] or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol [12]; (F) chitosan [e.g. 13] and (G) an immunostimulatory oligonucleotide {e.g. a CpG oligonucleotide) and a saponin [14]. Other mucosal adjuvants are also available [e.g. see chapter 7 of ref. 19].

Mutants of LT are preferred mucosal adjuvants, in particular the "K63" and "R72" mutants [e.g. see reference 15], as these result in an enhanced immune response.

Microparticles are also preferred mucosal adjuvants. These are preferably derived from a poly(α-hydroxy acid), in particular, from a poly(lactide) ("PLA"), a copolymer of D,L-lactide and glycolide or glycolic acid, such as a poly(D,L-lactide-co-glycolide) ("PLG" or "PLGA"), or a copolymer of D,L-lactide and caprolactone. The microparticles may be derived from any of various polymeric starting materials which have a variety of molecular weights and, in the case of the copolymers such as PLG, a variety of lactide: glycolide ratios, the selection of which will be largely a matter of choice, depending in part on the coadministered antigen. Antigen may be entrapped within the microparticles, or may be adsorbed to them.

Entrapment within PLG microparticles is preferred. PLG microparticles are discussed in further detail in ref. 16, in chapter 13 of ref. 17, and in chapters 16 & 18 of ref. 19.

The CagA-containing and NAP-containing microparticles may be a mixture of two distinct populations of microparticles, the first containing NAP and the second containing CagA. Alternatively, the microparticles may be present as a single population, with CagA and NAP (and any further antigens) distributed evenly.

LT mutants may advantageously be used in combination with microparticle-entrapped antigen, resulting in significantly enhanced immune responses.

The second immunogenic composition (boosting dose) The second immunogenic composition is given parenterally. Suitable parenteral routes of administration include intramuscular (IM), subcutaneous, intravenous, intraperitoneal, intradermal, or transdermal [e.g. ref. 18] routes, as well as or delivery to the interstitial space of a tissue. The intramuscular route is preferred. The second immunogenic composition is preferably adapted for parenteral administration (e.g. in the form of an injectable, which will typically be sterile and pyrogen-free).

The second immunogenic composition may comprise a parenteral adjuvant. Suitable parenteral adjuvants include: (A) aluminium compounds (e.g. aluminium hydroxide, aluminium phosphate, aluminium hydroxyphosphate, oxyhydroxide, orthophosphate, sulphate etc. [e.g. see chapters 8 & 9 of ref. 19]), or mixtures of different aluminium cmopounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous etc.), and with adsorption being preferred; (B) MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer) [see Chapter 10 of ref. 19; see also ref. 20]; (C) liposomes [see Chapters 13 and 14 of ref. 19]; (D) ISCOMs [see Chapter 23 of ref. 19]; (E) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion [see Chapter 12 of ref. 19]; (F) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox™); (G) saponin adjuvants, such as QuilA or QS21 [see Chapter 22 of ref. 19], also known as Stimulon™; (H) ISCOMs, which may be devoid of additional detergent [21]; (I) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (J) cytokines, such as interleukins (e.g. JL-1, JL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colony stimulating factor, tumor necrosis factor, etc. [see Chapters 27 & 28 of ref. 19]; (K) microparticles [see above]; (L) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) [e.g. chapter 21 of ref. 19]; (M) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions [22]; (N) oligonucleotides comprising CpG motifs [23] i.e. containing at least one CG dinucleotide, with 5-methylcytosine optionally being used in place of cytosine; (O) a polyoxyethylene ether or a polyoxyethylene ester [10]; (P) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol [11] or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional nonionic surfactant such as an octoxynol [12]; (Q) an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) and a saponin [14]; (R) an immunostimulant and a particle of metal salt [24]; (S) a saponin and an oil-in-water emulsion [25]; (T) a saponin (e.g. QS21) + 3dMPL + IL-12 (optionally + a sterol) [26]; and (U) other substances that act as immunostimulating agents to enhance the effectiveness of the composition [e.g. see Chapter 7 of ref. 19].

Aluminium compounds and MF59 are preferred adjuvants for parenteral use. Administration regimes

The interval between priming and boosting will vary according to factors such as the age of the mammalian patient and the nature of the composition (antigen, adjuvant etc.), and these factors can readily be assessed by a physician. Administration of the first priming and boosting doses is generally separated by at least 2 weeks, typically at least 4 weeks.

The method of the invention may comprise more than one mucosal priming dose e.g. two or more (e.g. 3, 4, 5 or more) priming doses, followed by parenteral booster dose(s). Identical immunogenic compositions will generally be used for each priming dose.

The method of the invention may comprise more than one parenteral boosting dose e.g. mucosal priming, followed by two or more (e.g. 3, 4, 5 or more) parenteral doses. Identical immunogenic compositions will generally be used for each booster dose.

Priming and boosting doses may therefore be distinguished by the route of administration, rather than by their timing.

The boosting dose(s) may be followed by further doses ('late' boosting), which may be delivered parenterally or mucosally.

The mammal

The mammal to whom the compositions are administered is typically primate, such as a human. The human may be a child or an adult. Suitable lower mammals (e.g. for research or testing purposes) include mice [e.g. ref. 27] and dogs [e.g. ref. 28, which uses beagles].

The antigens

CagA is a cytotoxin-associated antigen, and is disclosed in, for instance, references 29, 30 and 31. NAP antigen is associated with neutrophil activation, and is disclosed in, for instance, references 32 and 33. Identifying CagA and NAP antigens in any given strain of H.pylori is straightforward, particularly in light of the available genomic sequences for H.pylori [e.g. refs. 34 & 35]. The antigens for use according to the invention include alleles and polymorphic forms, as well as variants, mutants and fragments which retain antigenicity.

The antigens in the immunogenic compositions will typically be in the form of proteins. The proteins can, of course, be prepared by various means (e.g. native expression, recombinant expression, purification from H.pylori culture, chemical synthesis etc.) and in various forms (e.g. native, fusions etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other bacterial or host cell proteins). As an alternative to protein-based vaccination, the antigens in the immunogenic compositions may be in the form of nucleic acid [e.g. refs. 36 & 37]. Exemplary naked DNA introduction methods are described in references 38 and 39. Uptake efficiency may be improved using biodegradable latex beads, which are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.

Nucleic acid can, of course, be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the H.pylori bacterium itself etc.) and can take various forms (e.g. single stranded, double stranded, vectors etc.). In addition, the term "nucleic acid" includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc. Gene guns can be used to administer nucleic acid antigens.

In addition to the CagA and NAP antigens, the immunogenic compositions may comprise further H.pylori antigens (e.g. the vacuolating cytotoxin NacA [e.g. ref. 29], urease, HopX [e.g. 40], HopY [e.g. 40] etc.), as well as further antigens from viruses (e.g. hepatitis B virus surface antigen) and/or other bacteria (e.g. the H.influenzae Hib antigen, diphtheria toxoid, tetanus toxoid, B. pertussis antigens etc.). The presence of NacA is preferred.

Other antigens which may advantageously be included in compositions of the invention are: - a protein antigen from N.meningitidis serogroup B, such as those in refs. 41 to 47, with protein '287' (see below) and derivatives (e.g. 'ΔG287') being particularly preferred.

- an outer-membrane vesicle (OMN) preparation from N.meningitidis serogroup B, such as those disclosed in refs. 48, 49, 50, 51 etc.

- a saccharide antigen from N.meningitidis serogroup A, C, W135 and/or Y, such as the oligosaccharide disclosed in ref. 52 from serogroup C [see also ref. 53].

- a saccharide antigen from Streptococcus pneumoniae [e.g. 54, 55, 56].

- an antigen from hepatitis A virus, such as inactivated virus [e.g. 57, 58].

- an antigen from hepatitis B virus, such as the surface and/or core antigens [e.g. 58, 59].

- an antigen from hepatitis C virus [e.g. 60]. - an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B.pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3 [e.g. refs. 61 & 62].

- a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter 3 of ref. 63] e.g. the CRM₁₉₇ mutant [e.g. 64]. - a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of ref. 63]. - a saccharide antigen from Haemophilus influenzae B [e.g. 53].

- an antigen from N.gonorrhoeae [e.g. 41, 42, 43].

- an antigen from Chlamydia pneumoniae [e.g. 65, 66, 67, 68, 69, 70, 71].

- an antigen from Chlamydia trachomatis [e.g. 72]. - an antigen from Porphyromonas gingivalis [e.g. 73].

- polio antigen(s) [e.g. 14, 75] such as IPN or OPN.

- rabies antigen(s) [e.g. 76] such as lyophilised inactivated virus [e.g.77, RabAvert™].

- measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11 of ref. 63].

- influenza antigen(s) [e.g. chapter 19 of ref. 63], such as the haemagglutinin and/or neuraminidase surface proteins.

- an antigen from Moraxella catarrhalis [e.g. 78].

- an antigen from Streptococcus agalactiae (group B streptococcus) [e.g. 79, 80].

- an antigen from Streptococcus pyogenes (group A streptococcus) [e.g. 80, 81, 82].

- an antigen from Staphylococcus aureus [e.g. 83]. The composition may comprise one or more of these further antigens.

Where a saccharide or carbohydrate antigen is included, it is preferably conjugated to a carrier protein in order to enhance immunogenicity [84]. Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. The CRM₁ diphtheria toxoid is particularly preferred. Other suitable carrier proteins include the N.meningitidis outer membrane protein [85], synthetic peptides [86], heat shock proteins [87], pertussis proteins [88], protein D from Hinfluenzae [89], toxin A or B from C.difficile [90], etc. Any suitable conjugation reaction can be used, with any suitable linker where necessary.

Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means). Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens.

Compositions for use according to the invention The invention also provides a vaccine comprising H.pylori CagA and NAP antigens, for use in raising an immune response against H.pylori, wherein the vaccine is administered as a mucosal priming dose and a parenteral boosting dose. The invention also provides the use of H.pylori CagA and NAP antigens in the manufacture of a vaccine for use in raising an immune response against H.pylori, wherein the composition is administered as a mucosal priming dose and a parenteral boosting dose.

The invention also provides a kit comprising a first immunogenic composition and a second immunogenic composition, each immunogenic composition comprising H.pylori CagA and NAP antigens, wherein the first immunogenic composition is a mucosal priming dose and the second immunogenic composition is a parenteral boosting dose.

The compositions will include "immunologically effective amounts" of CagA and NAP antigens i.e. amounts sufficient to raise a specific immune response or, more preferably, to treat, reduce, or prevent H.pylori infection. An immune response can be detected by looking for anti-NAP or anti-CagA antibodies (e.g. IgG or IgA) in patient samples (e.g. in blood or serum, in mesenteric lymph nodes, in spleen, in gastric mucosa, and/or in faeces). Reduction and eradication of H.pylori infection can be detected by monitoring bacterial colonisation of the gastric mucosa. The precise effective amount for a given patient will depend upon the patient's age, size, health, the nature and extent of the condition, the precise composition selected for administration, the patient's taxonomic group, the capacity of the patient's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating physician's assessment of the medical situation, and other relevant factors. Thus, it is not useful to specify an exact effective amount in advance, but the amount will fall in a relatively broad range that can be determined through routine trials, and is within the judgement of the clinician. For purposes of the present invention, an effective dose will typically be from about O.Olmg/kg to 50mg kg in the individual to which it is administered.

The compositions of the invention will typically be formulated with pharmaceutically acceptable carriers or diluents. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of the antigens which does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of acceptable excipients is available in the well-known Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Techniques and definitions The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature eg. Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D.N Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed, 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Transcription and Translation (B.D. Hames & S.J. Higgins eds. 1984); Animal Cell Culture (R.I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J.H. Miller & M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer & Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (Weir & Blackwell eds 1986).

The term "comprising" means "including" as well as "consisting", so a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

A composition containing X is "substantially free" from Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least -90% by weight of the total of X+Y in the composition, more preferably at least ~95% or even 99% by weight.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 to 3 show results from example 1. Figure 1 shows the immune responses in spleen, mesenteric lymph nodes and gastric mucosa, with figure 1A showing results for CagA, and figure IB showing results for NAP. Figure 2 shows serum titres. Figure 3 shows IgA titres in fecal extracts. The X axis indicates the group number.

Figures 4 to 6 show results from example 2. Figure 4 shows the immune responses in spleen, mesenteric lymph nodes and gastric mucosa, with figure 4A showing results for CagA, and figure 4B showing results for NAP (erroneously, results were not measured for the boosted animals). Figure 5 shows serum titres. Figure 6 shows IgA titres in fecal extracts. Figures 7 to 10 show results from example 3. Figure 7 shows serum IgG responses against NAP and CagA. Figure 8 shows splenic immune responses against NAP (Figure 8A) and CagA (Figure 8B). Figure 9 shows anti-CagA IgA titres in fecal extracts. Figure 10 shows anti-CagA/NAP B -4 secreting cells following oral prime/IM boost. Figure 11 gives results from example 4 and shows serum anti-CagA IgG titres.

Figures 12 to 16 show results from example 5. Figure 12 shows serum IgG responses against NAP (Figure 12A) and CagA (Figure 12B). Figure 13 shows splenic immune responses against NAP (Figure 13A) and CagA (Figure 13B). Figure 14 shows anti-CagA IgA titres in SLN and MLN. Figure 15 shows anti-NAP IgA titres in fecal extracts. Figure 16 shows anti-CagA/NAP TL-4 secreting cells following IN prime/IM boost.

Figure 17 shows total anti-NAP and anti-cagA ASCs in spleen and MLN in 'late' boosting experiments, and Figure 18 shows anti-NAP and anti-CagA IgG and IgA ASC for the same experiments. Black bars show anti-CagA values; white bars show anti-NAP values.

Note: "ASC" refers to "antibody secreting cells", and "MNC" refers to "mononuclear cells".

MODES FOR CARRYING OUT THE INVENTION

Example 1 - Comparison of immunisation routes

The effect of parenteral (intramuscular) boosting after mucosal (oral) priming was investigated. For comparison, the effect of mucosal boosting after parenteral priming was also investigated. Four groups of 10 female BALB/c mice each were used:

For intramuscular injection, each 50μl dose used lOμg each of NAP and CagA, adjuvanted with alum, and administered in the right thigh. For oral administration, each 500μl dose used lOOμg each of NAP and CagA, adjuvanted with lOμg LT-R72, in 3% bicarbonate/PBS, and administered through a ball-end steel feeding needle. Immunisations were performed at 0, 10, 20, 30, 40 and 50 days. Sera and fecal pellets were collected 6 days after the final immunisation, and the mice were sacrificed the next day to prepare single cell suspensions of the gastric mucosa, mesenteric lymph nodes (MLN) and spleen. The results of an ELISPOT assay for 5-10 mice per group are shown in Figure 1. Standard deviations from the average values shown were below 30%. It is immediately evident from Figure 1 A that the only effective regime for eliciting a local anti-CagA and anti-NAP humoral responses in the mucosal effector site of the stomach as well as in the local inductive site of the MLN was that given to group 4 i.e. mucosal priming followed by parenteral boosting. Moreover, a strong systemic response was detected in the spleen. Parenteral immunisation alone (group 1) failed to induce local antibody responses, although a systemic anti-CagA response was observed in the spleen (Figure 1A, group 1). Groups 2 and 3 failed to show any detectable ELISPOT responses at any site to either antigen. Figure 2 shows serum titres against both antigens. Again, it is immediately evident that the best responses were observed in group 4 i.e. mucosal priming followed by parenteral boosting. Anti-NAP titres were significantly higher that anti-CagA titres in all groups.

As an additional measure of mucosal immunity, NAP-specific IgA responses were measured in fecal extracts (Figure 3). The titres were highest in group 4 (mucosal priming followed by parenteral boosting), followed by group 2 (mucosal only). As expected, intramuscular immunisation alone (group 1) did not induce specific IgA responses. This is further evidence of the potency enhancement resulting from adding parenteral boosting to mucosal priming.

Example 2 - Use of PLG microparticles

The effect of entrapment within poly(lactide-co-glycolide) microparticles was investigated. Microparticles with 1% w/w loading levels of CagA and NAP were prepared by emulsifying 2ml antigen solution (2.5mg/ml) with 8.5ml 6% w/v polymer solution (RG 504) in methylene chloride. The primary emulsion was homogenised for 3 minutes as 12,000rpm and then 40ml of a 10% w/v solution of poly vinyl alcohol in distilled water was added. The resulting multiple emulsion was further homogenised at 12,000rpm for 3 minutes. The W/O/W emulsion was then stirred at lOOOrpm on a magnetic stirrer overnight at room temperature. This resulted in complete evaporation of the solvent and formation of PLG-CagA and PLG-NAP microparticles. The resulting microparticles were washed twice with distilled water and freeze-dried for further use.

The size-distribution of the microparticles was determined using a Malvern particle size analyser. The loading levels of the two formulations were estimated by hydrolysing lOmg microparticles with 1.0ml 0.2N NaOH/1% SDS solution overnight, followed by a BCA assay:

Eight groups of 10 female BALB/c mice each were used for immunological studies:

For intramuscular administration, each 50μl dose used lOμg each of NAP and CagA, adjuvanted with alum (adsorbed) or PLG (entrapped), and administered in the right thigh. For oral administration, each 500μl dose used 50μg each of NAP and CagA (half that of example 1), adjuvanted with lOμg LT-R72 and (optionally) PLG, in 3% bicarbonate/PBS, and administered through a ball-end steel feeding needle. Immunisations were performed at 0, 10, 20, 30, 40 and 50 days. Sera and fecal pellets were collected 6 days after the final immunisation, and the mice were sacrificed the next day to prepare single cell suspensions of the gastric mucosa, mesenteric lymph nodes (MLN) and spleen. The results of an ELISPOT assay for 5-10 mice per group are shown in Figure 4. Standard deviations from the average values shown were below 25%. As in example 1, strong local and systemic humoral anti-CagA responses in spleen and MLN were seen in the mucosal prime / parenteral boost groups (6 & 8). The enhancement provided by parenteral boosting is evident when group 8 is compared with group 4, and when group 6 is compared with group 3. The benefit of using mucosal administration before parenteral administration is evident when group 8 is compared with group 7, and when group 6 is compared with group 5.

Figure 5 shows serum IgG responses measured by ELISA. A comparison of groups 3 and 6 shows that parenteral boosting significantly enhances antibody titres against CagA and NAP. The same is seen when comparing groups 4 and 8. The benefit of using PLG as a mucosal adjuvant are evident from a comparison of groups 6 and 8. No groups showed a mucosal response in the stomach, probably because the antigen dose was half that used in example 1.

NAP-specific and CagA-specific IgA responses were measured in fecal extracts. Anti-CagA responses were negligible in all groups; anti-NAP responses are shown in Figure 6. The titres were highest in groups 6 and 8 (mucosal priming / parenteral boosting). A comparison of groups 3 and 6 shows that parenteral boosting significantly enhances IgA titres, and the same is seen when comparing groups 4 and 8 - oral immunisation alone is ineffective. The benefit of using PLG as an adjuvant are evident from a comparison of groups 6 and 8.

Figures 5 and 6 thus give further evidence of the enhancement of local and systemic immune responses resulting from the addition of parenteral boosting to mucosal priming, and also show the advantages of entrapment within PLG microparticles for mucosal administration.

Example 3 - Use of PLG microparticles and different LT adjuvants

In further experiments involving oral priming and intramuscular boosting, eight groups of mice were used to assess the effect of PLG-entrapment of CagA/NAP antigens, the use of LTK63 vs. LTR72 adjuvant, and a two-fold increase in dosage.

Oral doses were given three times; intramuscular doses were given twice with lOμg antigens.

Serum anti-NAP titres were induced at levels similar to the previous experiments (Figure 7). The highest titres were induced in the groups receiving PLG-entrapped antigens and in the groups receiving the higher of the two doses of antigen. However, the magnitude of the responses was generally the same.

Measuring systemic anti-NAP and anti-CagA responses by the ELISPOT assay was more informative in differentiating between the potency of the various immunization groups. LTK63 as an adjuvant (groups 7 & 8) failed to induce any splenic anti-NAP (Figure 8A) or anti-CagA (Figure 8B) ASC response. In contrast, LTR72 (groups 1 to 6) was effective as an oral adjuvant and there was no significant difference between doses of 10 or 50 μg.

Entrapment of antigens in PLG for both oral priming and IM boosting immunizations significantly increased antigen-specific splenic ASCs. These data indicate that PLG- entrapment of NAP and CagA antigens together with LTR72 as an adjuvant for oral priming immunizations and PLG-entrapment of NAP and CagA antigens for BVI boosting immunizations represent the preferred regimen, adjuvant and delivery systems.

In terms of anti-NAP responses in fecal extracts (Figure 9), oral immunisations with LTK63 adjuvant (groups 7 & 8) did not induce anti-NAP IgA titres. In contrast, oral immunisations with LTR72 adjuvant (groups 1 to 6) and with PLG-entrapped antigens at the highest dose (lOOμg) induced strong fecal IgA responses (Figure 9). These data further substantiate the earlier data — LTR72 is a preferred adjuvant and PLG entrapment is preferred as a delivery system instead of alum precipitation. Correlates of protection against H.pylori challenge are not well established. However, it has been shown in a gene knockout model that CD4+ cells are essential for protection. CD4+ T cell responses were therefore measured in mice following oral prime/TM boost. Splenocytes isolated from mice following the prime/boost regimen were analysed and significant numbers of CagA/NAP-specific IL-4 secreting cells were induced (Figure 10). In contrast, few or no CagA/NAP-specific IFN-γ secreting cells were detected following the prime/boost regimen. No cytokine secreting cells (IL-4 or IFN-γ) were detectable in MLN or SLN. Oral prime/IM boost immunisations with Cag A/NAP thus selectively induce TH2 (not TH1) type cytokines.

Example 4 - alternative routes of priming and adjuvants

The benefits of following mucosal immunisation by parenteral immunisation were established in the previous examples. The use of the intranasal route for priming was investigated and compared with the oral route.

The benefits of using PLG as a parenteral adjuvant instead of alum are evident from Figures 4 and 5. In Figures 4 A & 4B, 10-fold increases in local (MLN) and systemic (spleen) responses is seen when comparing groups 1 and 2. A 3-fold increase is seen in Figure 5. Therefore the use of PLG microparticles in the parenteral booster dose was also investigated.

LT-R72 is clearly an effective adjuvant, but alternative forms of LT were also investigated, in particular LT-K63 and wild-type LT. Different LT doses were also tested. Different antigen doses were also investigated to determine whether, as suggested by examples 1 and 2, antibody responses in the gastric mucosa favour high doses.

For the above investigations, eight groups (each of 10 female BALB/c mice) were used:

Mucosal doses were given three times and intramuscular doses were given twice. All IM doses were given at lOμg either in alum or in PLG as indicated. Immunisations were given at 10 day intervals. Sera and fecal pellets were collected at 6 days post final immunisation and the mice were sacrificed at 7 days post final immunisation to prepare single cell suspensions from the stomach mucosa and MLN & SP for the ELISPOT assay. Intranasal priming immunisations with soluble NAP+CagA plus LTK63 adjuvant induced 10 fold higher serum antibody titres than the oral priming immunisations with NAP+CagA plus LTK63 or LTR72 (Figure 11). In agreement with the earlier results, PLG-entrapment enhanced serum antibody responses several-fold. Intranasal priming immunisations are thus superior to oral priming immunisations for the induction of specific serum antibody responses.

Example 5 -further investigations on intranasal priming

Examples 1 to 4 show that oral priming with NAP+CagA with LTR72 adjuvant, followed by IM boosting with NAP+CagA, significantly enhanced systemic and mucosal humoral responses to the two antigens. Humoral responses were several folds higher than IM or oral immunisations alone. Example 4 also shows that, compared to the oral route of mucosal immunisation, the IN route can induce higher serum antibody responses and to permit doses several orders of magnitude lower. Example 5 deals with intranasal delivery. The intranasal priming route was further investigated, together with comparisons of (a) LTK63 vs. LTR72 as adjuvant and (b) PLG- entrapped vs. soluble antigens. It was also investigated whether IN or IM immunisations alone, totalling the same number of prime/boost immunisations, could induce comparable immune responses to prime/boost immunisations.

Ten groups of mice were immunised with NAP+CagA as follows:

All priming doses were given 3 times IN, except for groups 7 & 8 which received 5 IM doses. Where administered, boosting doses were given twice intramuscularly. Intranasal dosages were lOμg; intramuscular doses were 25μg. Where given, LT dose was lOμg. Figure 12 shows that three IN priming immunisations followed by two IM boosts (groups 1 to 6) significantly enhanced the serum anti-NAP (Figure 12A) and anti-CagA (Figure 12B) responses compared to five IN or five IM immunisations alone. The IN prime/IM boost strategy therefore significantly enhanced systemic humoral responses compared to immunisations by IM or IN alone (groups 7 to 10). There were no significant differences between the various groups receiving LTK63 or LTR72 as an adjuvant, indicating that the non-toxic LTK63 adjuvant is as effective as LTR72 (with residual toxicity). There were no significant differences between the groups receiving PLG-entrapped or soluble antigens for the IN immunisations. For the BVI boost or IM only immunisations there was no difference between PLG-entrapment vs. alum precipitation of the protein antigens. IN prime/IM boost immunisations induced an average of two fold higher anti-NAP titres compared to anti-CagA titres, suggesting that the NAP antigen is a stronger immunogen for the induction of serum humoral responses.

Data obtained by ELISPOT assay (Figure 13) showed a clearer difference between the various immunisation groups. Anti-NAP ASC numbers (Figure 13 A) in spleen were highest in the IN only group which were immunised with PLG-entrapped CagA+NAP and LTK63 (group 10); the lowest number of anti-NAP ASC was measured after IM-only immunisations (groups 7 and 8). The groups that received PLG-entrapped CagA+NAP plus LTR72 IN followed by PLG-entrapped CagA+NAP (group 4) or alum-precipitated CagA+NAP (group 5) also showed high numbers of anti-NAP ASC. Only three groups had measurable local responses in SLN, namely the groups that were immunised with PLG-entrapped CagA+NAP plus LTK63 IN followed by PLG-entrapped CagA+NAP or alum-precipitated CagA/NAP IM (groups 1 and 2), and the group that was immunised with PLG-entrapped CagA+NAP plus LTR72 IN followed by alum-precipitated CagA+NAP (group 5). Of these, the only group that also showed a response in MLN was group 5. Thus, based on the ELISPOT data, the best immunisation strategy for the induction of systemic as well as local responses is IN priming with PLG-entrapped CagA+NAP plus LTR72, followed by IM boosting with alum- precipitated CagA NAP.

Figure 14 shows local responses. Significant numbers of CagA-specific ASC were found in lymph nodes draining the stomach (SLN) mucosa following IN prime/IM boost. No specific responses were detectable in SLN of mice immunized IN-only or IM-only. MLN responses were low or non-specific.

IM immunisations alone did not induce any anti-NAP IgA titres in fecal extracts (Figure 15). In contrast, IN immunisations alone induced strong anti-NAP IgA titres. There was a significant enhancement of anti-NAP IgA titres in mice immunised with PLG-entrapped vs. soluble antigens. IN priming with PLG-entrapped CagA+NAP plus LTR72 followed by boosting with alum-precipitated CagA+NAP gave the highest anti-NAP IgA titres in fecal extracts, further supporting the ELISPOT data on the induction of local and systemic responses with this immunisation group. Anti-CagA responses in the spleen were significantly enhanced following the prime/boost regimen compared to the IN- or IM-only immunisations. Therefore the IN prime/IM boost regimen induced strongly-enhanced systemic and local humoral responses.

CD4+ T cell responses were measured in mice following IN prime/IM boost. Splenocytes isolated from mice following the prime/boost regimen contained significant numbers of CagA/NAP-specific IL-4 secreting cells (Figure 16). In contrast, few or no CagA/NAP- specific IFN-γ secreting cells were detected following the prime/boost regimen. No cytokine secreting cells (IL-4 or IFN-γ) were detectable in MLN or SLN. There did not appear to be a significant difference between the IN-only, IM-only or IN prime/IM boost groups. Thus it appears that IN prime/IM boosting immunisations with CagA/NAP selectively induce TH2- type cytokines rather than and not THl-type cytokines.

Example 6 -parenteral or mucosal administration after boosting dose(s) - 'late' boosting

Mice immunised with 3x intranasal priming and 2x intramuscular boosting were rested for three months and then given a 'late' boost either intranasally or intramuscularly.

Total anti-NAP and anti-cagA antibody secreting cells in spleen and mesenteric lymph nodes are shown in Figure 17. Spleen anti-NAP and anti-CagA IgG and IgA ASC responses are shown in Figure 18. The results show that late boosting by both routes is effective.

Conclusions

Oral or intra-muscular immunisation alone does not induce local antibody responses in stomach or MLN against CagA or against NAP. Oral priming followed by intramuscular boosting is , however, a potent strategy for induction of mucosal, local and systemic humoral responses — it induces local humoral responses in the mucosal effector site of the stomach and in the local inductive site, the mesenteric lymph nodes, and more potent systemic responses were detected in spleen and serum with this combined regimen.

Intranasal priming is superior to oral priming for the induction of NAP and CagA specific antibody responses. Intranasal priming enhances systemic antibody responses several-fold compared to oral priming, and there is evidence of enhanced local responses in the stomach. IN priming followed by IM boosting induces significantly higher local and systemic antibody and T cell responses compared to IN or IM alone. IN or oral priming followed by IM boosting selectively induces antigen-specific IL-4 but not IFN-γ secreting cells in spleen, suggesting that TH2-type, but not TH1 type, cytokine responses are selectively induced.

Encapsulation of antigens in PLG enhances local and systemic responses as measured by the ELISPOT assay, and serum responses are also enhanced (compared to adsorption on alum) following oral or IM immunisations.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. REFERENCES (the contents of which are hereby incorporated in full)

[I] International patent application WO99/57278. [2] Lee et al. (1999) Vaccine 17:3072-82.

[3] International patent application WO98/48835.

[4] Walker (1994) Vaccine 12:387-400.

[5] Clements (1997) Nature Biotech. 15:622-623.

[6] McGhee et al. (1992) Vaccine 10:75-88.

[7] Michetti (1998) I.Gastroenterol [Suppl X]:66-68.

[8] Almeida & Alpar (1996) J. Drug Targeting 3:455-467.

[9] Del Giudice et al. (1998) Molecular Aspects of Medicine, vol. 19, number 1.

[10] International patent application WO99/52549.

[II] International patent application WO01/21207. [12] International patent application WOO 1/21152. [13 International patent application WO99/27960. [14] International patent application WO00/62800. [15] International patent application WO98/18928. [16] Morris et al. (1994) Vaccine 12:5-11.

[17] Mucosal Vaccines, eds. Kiyono et al, Academic Press 1996 (ISBN 012410587).

[18] International patent application WO98/20734.

[19] Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995 (ISBN 0-306-44867-X.

[20] International patent application WO90/14837.

[21] International patent application WO00/07621.

[22] European patent applications 0835318, 0735898 and 0761231.

[23] Krieg (2000) Vaccine 19:618-622; Krieg (2001) Curr opin Mol Ther 2001 3:15-24; WO96/02555, WO98/16247, WO98/18810, WO98/40100, WO98/55495, WO98/37919 and WO98/52581 etc.

[24] International patent application WO00/23105.

[25] International patent application WO99/11241.

[26] International patent application WO98/57659.

[27] Marchetti et al. (1995) Science 267:1655-1658.

[28] International patent application WO99/35907.

[29] International patent application WO93/18150. [30; Covacci et al. (1993) Proc. Natl. Acad. Sci. USA 90:5791-5795.

[31 Tummuru et al. (1994) Infect. Immun. 61:1799-1809.

[32; International patent applications WO96/01272 & WO96/01273, especially SEQ ID NO:6.

[33 Evans et al. (1995) Gene 153:123-127. [34 Tomb et al. (1997) Nature 388:539-547. [35 Aim et al. (1999) Nature 397:176-180. [36 Robinson & Torres (1997) Seminars in Immunology 9:271-283. [37 Donnelly et al. (1997) Annu. Rev. Immunol. 15:617-648. [38 International patent application WO90/11092. [39 US patent 5,580,859. [40 International patent application WO98/04702. [41 International patent application WO99/24578.

[42; International patent application WO99/36544.

[43 International patent application WO99/57280. [44; International patent application WO00/22430. [45. Tettelin et al. (2000) Science 287:1809-1815. [46 International patent application WO96/29412. [47 Pizza et al. (2000) Science 287:1816-1820. [48 International patent application PCT/IBOl/00166. [49; Bjune et al. (1991) Lancet 338(8775): 1093-1096. [50; Fukasawa et al. (1999) Vaccine 17:2951-2958. [51 Rosenqvist et al. (1998) Dev. Biol. Stand. 92:323-333. [52 Costantino et al. (1992) Vaccine 10:691-698. [53 Costantino et al. (1999) Vaccine 17:1251-1263.

[54; Watson (2000) Pediatr Infect Dis 19:331-332.

[55 Rubin (2000) Pediatr Clin North Am 47:269-285, v.

[56; Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207.

[57 Bell (2000) Pediatr Infect Dis 19: 1187-1188. [58 Iwarson (1995) APMIS 103:321-326. [59; Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.

[6o; Hsu et al. (1999) Clin Liver Dis 3:901-915.

[61 Gustafsson et al. (1996) N Engl. I. Med. 334:349-355. [62] Rappuoli et /. (1991) TIBTECH 9:232-238.

[63] Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0.

[64] Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70.

[65] International patent application PCT/IB01/01445.

[66] Kalman et al. (1999) Nature Genetics 21:385-389.

[67] Read et al. (2000) Nucleic Acids Res 28: 1397-406.

[68] Shirai et al. (2000) J. Infect. Dis. 181(Suppl 3):S524-S527.

[69] International patent application WO99/27105.

[70] International patent application WO00/27994.

[71] International patent application WO00/37494.

[72] International patent application WO99/28475.

[73] Ross et al. (2001) Vaccine 19:4135-4142.

[74] Sutter et al. (2000) Pediatr Clin North Am 47:287-308.

[75] Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126.

[76] Dreesen (1997) Vaccine 15 Suppl:S2-6.

[77] MMWRMorb Mortal Wkly Rep 1998 Jan 16;47(1):12, 19.

[78] McMichael (2000) Vaccine 19 Suppl LS101-107.

[79] Schuchat (1999) Lancet 353(9146):51-6.

[80] International patent application PCT/GB01/04789.

[81] Dale (1999) Infect Dis Clin North Am 13:227-43, viii.

[82] Ferretti et al. (2001) PNAS USA 98: 4658-4663.

[83] Kuroda et al. (2001) Lancet 357(9264): 1225-1240; see also pages 1218-1219.

[84] Ramsay et al. (2001) Lancet 357(9251): 195-196. See also: Lindberg (1999) Vaccine 17 Suppl 2:S28-36; Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114 etc.

[85] European patent application 0372501.

[86] European patent applications 0378881 & 0427347.

[87] International patent application WO93/17712.

[88] International patent application WO98/58668; see also EP-0471177.

[89] International patent application WOOO/56360.

[90] International patent application WO00/61761.

Claims

1. A method for raising an immune response in a mammal, the method comprising the mucosal administration of a first immunogenic composition followed by the parenteral administration of a second immunogenic composition, wherein the first and second immunogenic compositions each comprise Helicobacter pylori CagA and NAP antigens.

2. The method of claim 1, wherein the first immunogenic composition is administered orally or intranasally.

3. The method of claim 1 or claim 2, wherein the second immunogenic composition is administered intramuscularly.

4. The method of claim 3, wherein the second immunogenic composition is administered by injection.

5. The method of any preceding claim, wherein the first immunogenic composition comprises one or more mucosal adjuvants.

6. The method of claim 5, wherein the mucosal adjuvant is a non-toxic form of E.coli heat- labile enterotoxin.

7. The method of claim 6, wherein the non-toxic form is the K63 or R72 mutant.

8. The method of any preceding claim, wherein the first immunogenic composition comprises poly(lactide-co-glycolide) microparticles.

9. The method of any preceding claim, wherein the second immunogenic composition comprises a parenteral adjuvant.

10. The method of claim 9, wherein the parenteral adjuvant is an alum.

11. The method of claim 9, wherein the parenteral adjuvant comprises poly(lactide-co-glycolide) microparticles .

12. The method of any preceding claim, wherein the interval between administering the first and second immunogenic compositions is at least 2 weeks.

13. The method of any preceding claim, wherein the mammal is a human.

14. The method of any preceding claim, wherein the antigens in the immunogenic compositions are in the form of proteins.

15. The method of any preceding claim, wherein (i) the first immunogenic composition is for intranasal administration and (ii) the second immunogenic composition is for intramuscular administration.

16. The method of any preceding claim, wherein (i) the first immunogenic composition is for oral administration and comprises LTR72 and (ii) the second immunogenic composition is for intramuscular administration and comprises antigen encapsulated in poly(lactide-co-glycolide) microparticles .

17. The method of any preceding claim, wherein (i) the first immunogenic composition is for oral administration and comprises LTR72 and poly(lactide-co-glycolide) microparticles and (ii) the second immunogenic composition is for intramuscular administration and comprises an alum.

18. The method of any one of claims 1 to 17, wherein the antigens in the immunogenic compositions are in the form of nucleic acid.

19. The method of any preceding claim, wherein the first and/or second immunogenic compositions comprise further H.pylori antigens and/or further antigens from viruses and/or bacteria other than H.pylori.

20. The method of claim 19, wherein the first and/or second immunogenic compositions further comprise(s) H.pylori NacA antigen.

21. A kit comprising a first immunogenic composition and a second immunogenic composition, each immunogenic composition comprising H.pylori CagA and NAP antigens, wherein the first immunogenic composition is a mucosal priming dose and the second immunogenic composition is a parenteral boosting dose.

22. A vaccine comprising H.pylori CagA and NAP antigens, for use in raising an immune response against H.pylori, wherein the vaccine is administered as a mucosal priming dose and a parenteral boosting dose.

23. The use of H.pylori CagA and NAP antigens in the manufacture of a vaccine for use in raising an immune response against H.pylori, wherein the vaccine is administered as a mucosal priming dose and a parenteral boosting dose.