GB2290710A - Protease from Helicobacter pylori for use in vaccines/therapeutic compositions - Google Patents

Description

HELICOBACTER PYLORI VACCINES/ THERAPEUTIC COMPOSITIONS The present

invention relates to Helicobacter p lori (H.pvlori) vaccines/therapeutic compositions and, in particular, to vaccines/therapeutic compositions against H.Pylori infection comprising H. pylori haemagglutinin/protease (HAP) protein or a fragment thereof.

H.pylori (formerly Campylobacter pyloridis or C.pylori) is a spiralshaped Gram negative human gastric pathogen. H.Pylori is well documented in its association with gastric and duodenal ulcer disease as well as gastric cancer. The microorganism has been described as the most common chronic infectious agent of man.

A number of different determinants possessed by H.pYlori have been proposed as possible pathogenic factors of the microorganism including the urease, haemagglutinin and heat-shock proteins as well as its flagelli and cytotoxins. However, the precise roles of these determinants in the pathogenicity of the microorganism and therefore their usefulness in the prevention and/or treatment of H.pylori infection has yet to be resolved.

We surprisingly have found that the H.pylori genome includes a gene which is almost identical to the zinc metalloprotease gene of Vibrio cholerae (known as the haemagglutinin/protease (hap) gene). The H.pylori hap gene which we have identified has been found to be over 99% similar to the V. cholerae han gene. In V.cholerae the HAP enzyme has been shown to be important in the attachment and detachment of the organism during cholera infection.

The enzyme encoded by the H.pYlori hap gene has been identified as a soluble secreted zinc metalloprotease enzyme. Zinc metalloprotease enzymes are well known as important markers of pathogenicity in bacteria causing disease in mammals. Furthermore, and importantly, this protein is linked to the microorganism's pathogenicity by identification that patients suffering from H.Pylori infection have high-titre antibodies to the H.pYlori HAP protein.

Zinc metalloprotease enzymes are known to have rapid substrate turnover and broad substrate profiles. Reviews on the state of the art are by Frausto da Silva J.J.R and Williams R.J.P in, The Biological Chemistry of the Elements, 1993, Clarendon Press, Oxford, Chapter 11, and Vallee, B.L. and Auld D.S., Biochemistry, 29, 5647-5659.

The zinc metalloprotease enzyme of Pseudomonas aeruginosa (also known as the elastase enzyme) has been shown to be important in the lung tissuedestructive processes caused by this organism in cystic fibrosis patients, (Bever R.A. and Iglewski B.H., J.Bacteriol., 1988, 170, 4309-4314). Similarly, the zinc metalloprotease enzyme of Vibrio cholerae (V. cholerae) (also known as the mucinase enzyme or haemagglutinin/protease (HAP) enzyme) has been shown to be important in the attachment and detachment of these organisms during the disease cholera. References on the state of the art include: Hase C.C. and Finkelstein R.A., J. Bacteriol., 1991, 173, 3311-3317; and Finkelstein R.A. et al., Infect. Immunol. 1992, 60 472-478.

The above features and the known importance of the V.cholerae HAP enzyme in its pathogenicity in man make the H.Pylori HAP protein a highly suitable subject for a peptide or protein vaccine/therapeutic composition against H.pYlori infection.

Accordingly, the present invention provides a vaccine/therapeutic corpos. tion corprising H.nylori HAP protein or a fragment thereof, wherein the Helicobacter pylori HAP protein includes the amino sequence as shown in Figure 1 of the accompanying drawing.

By the term "protein" is meant the whole or substantially the whole H. pylori HAP protein.

By the term "fragment" is meant any portion of the H.Pylori protein which elicits an antibody response in a patient.

It is to be understood that the terms "protein" and "fragment" as used in the present invention include any protein and any protein fragment, respectively, with an amino acid sequence corresponding to the naturally occurring amino acid sequence or a derivative thereof. The term "derivative" includes any protein, polypeptide or peptide with amino acid variations from the naturally occurring amino acid sequence, but which display the same functionality as the naturally occurring amino acid sequence and/or elicit a protective antibody response in humans. such derivatives often include conservative amino acid replacements which take place within a family of amino acids.

The H.pylori HAP protein or fragment thereof in the vaccine/therapeutic composition may be substantially purified naturally occurring amino acid sequences or synthetic amino acid sequences. One advantage of synthetic vaccines/therapeutic compositions is that they can be made free of any competing antigens and of any contaminating and potentially harmful products. Preferably the H.pylori HAP protein or fragment thereof is a recombinant protein polypeptide or peptide. Recombinant DNA and protein technology is now able to provide high quality and quantity protein and polpeptide products.

only certain regions of proteins and polypeptides constitute antigenic sites which elicit an antibody response in the patient. It is therefore advantageous that the vaccine/therapeutic composition comprises one or more H.Pylori HAP protein fragments which correspond to identified antigenic sites of the H.pvlori HAP protein. Known antigenic sites of the H.Pylori HAP protein include the zinc binding region and the protease active site. These functional regions were identified by X-ray crystallography of purified zinc metallic proteases similar in amino acid sequence to the H.pvlori HAP protein, for example the P.aeruginosa elastase reviewed in Vallee, B.L. & Auld, D.S., (1990), Zinc Coordination, Function and Structure of Zinc Enzymes and other Proteins, Biochemistry 29: 5647-5659.

The vaccine/therapeutic composition according to the present invention may comprise two or more components selected from the H.Pvlori HAP protein or fragments thereof. For example, the vaccine may comprise two H. pYlori HAP protein fragments such as the H.pvlori HAP protein zinc binding region and the protease active site.

The vaccine/therapeutic composition is preferably formulated to comprise the selected H.Pylori HAP protein or fragment thereof together with a pharmaceutically acceptable carrier. Such carrier includes any substance which does not itself induce the production of antibodies harmful to the individual receiving the vaccine/therapeutic composition. suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive virus particles. Additionally, these carriers may function as immunostimulating agents or adjuvants. Furthermore, the antigen may be conjugated to a bacterial toxoid adjuvent, such as a toxoid from diphtheria, tetanus, cholera or H.Pylori.

Preferred adjuvants to enhance effectiveness of the vaccine/therapeutic composition include: aluminium salts (alum), such as aluminium hydroxide, aluminium phosphate, or aluminium sulphate; oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides or bacterial cell wall components), e.g. MF59, SAF and Ribi TM; Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); cytokines, such as interleukins; macrophage colony stimulating factor (M-CSF); tumour necrosis factor (TNF); and other substances that act as immunostimulating agents to enhance the effectiveness of the composition. The preferred known adjuvant is alum.

The vaccine/therapeutic composition may also comprise one or more additional antigens derived from an alternative source. Such antigens may comprise other H.pylori proteins or fragments thereof, for example the H. Pylori cytotoxin associated immunodominant (CAI) protein, heat shock protein (HSP), or urease protein. Other suitable antigens include the V. cholerae toxin A and/or B subunits.

The present invention further provides a vaccine/therapeutic composition as hereinbefore described for the prevention or treatment of H.pylori infection. Also provided is the use of H.Pylori HAP protein or a fragment thereof in the manufacture of a vaccine/therapeutic composition for the treatment of H.Pylori infection.

The present invention will now be described in more detail with reference to the acconpanying 6 drawing Fig. 1 which shows the nucleotide and corresponding amino acid sequence of a cloned 1.5kb H.Pylori hap gene fragment showing about 1 kb of coding sequence and about 500 bp of 31 flanking sequence. Numbering is from the first adenosine base of the EcoRI site. The three base in-frame deletion (marked AH) from the V.cholerae hap gene is at position 222 and the region identical to the V.cholerae han gene extends from position 1 beyond the coding region (double underlined) except for a single addition at position 798 and a single deletion (of a T) at position 843. The stop codon is indicated by.

Within the identified H.Pylori hap nucleic acid sequence two regions have been identified as comprising coding regions for the active site component of the protein. The two regions are around nucleic acids numbered 16-18 (inclusive), encoding a tyrosine residue and nucleic acids numbered 220-222 (inclusive), encoding a histidine residue.

The region around nucleic acids numbered: 43-45 (inclusive), encoding a glutamic acid residue have been identified as a putative zinc binding encoding region.

The amino acid sequence shown in Fig. 1 or any fragment thereof can be constructed by any polypeptide synthesis method known in the art. Similarly, the nucleic acid sequence shown in Fig. 1 or any fragment thereof can be constructed by known standard procedures in the art. Incorporation of the nucleotide sequence into a known expression vector such as lambda gt11 or Bluescript under appropriate conditions will result in recombinant H.Pylori HAP protein or fragments thereof.

The vaccine/therapeutic composition according to the present invention typically contains diluents such as water, saline, glycerol or ethanol. Additionally, auxillary substances, such as wetting or emulsifying agents, pH buffering substances and the like may be present in the vaccine/therapeutic composition.

The vaccine/therapeutic composition may be prepared in injectable form, either as a liquid solution or suspension or in a solid form which may also be suitable for the preparation of a solution for injection. The vaccine/therapeutic composition may also be emulsified or encapsulated in liposomes for enhanced adjuvant effect.

Administration of the vaccine/therapeutic composition may be prophylactic or therapeutic and may comprise a method of treatment of the patient.

The vaccine/therapeutic composition of the invention is administered in sufficient quantity to elicit a protective response in the host. This quantity depends on, amongst other factors, the size and health of the patient, the capability of the patient's immune system to provide an immune response, the degree and speed of protection desired and the formulation of the vaccine/therapeutic composition.

The vaccine/therapeutic composition is conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. oral formulations are most preferred for the H.pylori vaccines or therapeutic compositions of the present invention because of the almost exclusivity of H.pylori infection in the stomach. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine/therapeutic composition may be administered in conjunction with other immunoregulatory agents.

The present invention will now be described in more detail with reference to the following examples:

Example 1

Growth of H.pylori strains and extraction of DNA and proteins.

H.Pylori strains NCTC (National Collection of Type Cultures, London) 11637, 11638, 11916 and HP 34 (clinical isolate from a biopsy taken during endoscopy of a patient at Queen's Medical Centre, The University Hospital, Nottingham) were grown on Columbia blood agar with 5% horse blood (Oxoid) as a lawn for DNA extraction, for 2 days under microaerophilic conditions (Campypak, BBL) at 37 0 C. The resulting growth was harvested from four plates and was first Gram stained to identify the characteristic morphology. The cells were washed in iml of lysis buffer (5OmM EDTA, 10OmM NaCl), and resuspended in 400pl of lysis buffer to which 30pl of lysis buffer containing 20% N-lauroylsarcosine (Sigma) was added. After five minutes incubation at room temperature, the suspension was repeatedly extracted with phenol saturated with TE (1OmM Tris-HCI, pH 8.0, lmM EDTA) buffer until no interface was evident. The nucleic acids were then ethanol precipitated overnight, collected by centrifugation, washed in 70% ethanol and the pellet air dried for 10 minutes. The DNA was then treated with proteinase K and purified using a Qiagen minicolumn according to the manufacturer's instructions. H.Pylori strain NCTC 11638 was grown in liquid culture by adding one harvested plate of culture to 100m1 of Brucella broth (Difco) containing 2% P-cyclodextrin (Sigma) and 0.2m1 of reconstituted H.Pylori selective supplement (oxoid) in a 500 ml conical flask. The flask was incubated with gentle shaking (100 r.p.m) in an anaerobic jar for 3 days under microaerophilic conditions (Campypak, BBL) at 37 0 C. The H.Pylori cells were collected by centrifugation and the proteins in the supernatant precipitated as previously described in Milton D.L., Norqvist, A., and Wolf Watz, H., (1992), J. Bacteriol., 174:7235-7244. Cellular proteins were extracted by resuspending two plates of growth in 1.5 ml of protein-extraction buffer (1OmM Tris pH 7.5, lmm MgC1 2' 0.15mM EDTA, lmM DTT, lmM PMSF, 2pgm1_ 1 pepstatin A (Sigma) 0.5mgm1_ 1 leupeptin (Sigma)), adding 200M1 of 10% SDS and boiling for 5 minutes. The suspension was then cooled on ice for 5 minutes and the supernatant collected by centrifugation at 12500 r.p.m. for five minutes. The protein concentration was determined according to Bradford, M., (1976), Anal. Biochem, 72, 248-252, (1976).

Example 2

Identification of a H.pylori protease enzyme 40pg of total cell and supernatant proteins from H.Pylori NCTC 11638 and a clinical isolate of P.aeruginosa were separated on vertical minigels (Hoeffer Scientific), comprising a 5% acrylamide stacking gel and a 13% resolving gel, according to the procedure of Laemmli, U.K, (1970), Nature, 227:680-685. Electrophoresis was performed at 20mA. Half of the gel was stained directly and the other half was incubated before staining to reveal protease activity. The gel portion that was stained directly was placed in a solution of 0.1% Coomassie brilliant blue R-250 in destain solution (40% methanol, 10% acetic acid, 50% water) for 1h, and then placed in several changes of destain solution. The unstained gel was incubated, and the protease activity of this gel was detected by the procedures as described in Milton et al., (1992), J. Bacteriol., 174:7235-7244, except that the gel was finally stained with Coomassie brilliant blue R-250 as above rather than amido black. Protease activity was identified in the gel by cleared bands (digestion of the gelatin and other proteins). Protease activity was clearly present in the H.pylori cell, supernatant and P. aeruginosa tracks. In the H.Pylori tracks numerous proteolytic bands were present. An overloaded gel (10Ogg of total H.pylori protein) according to the above procedure showed a clear protease band at about 35kDa.

The electrophoretic separation was transferred on to a nitrocellulose membrane by semi-dry blotting using transfer buffer (48mM Tris pH 8, 30mM glycine, 20% methanol, 1.3mM SDS) and an ATTO AE-6675 Horizblot transfer unit (Genetic Research International) according to the manufacturer's instructions. The nitrocellulose membrane was then dried and placed in blocking solution (1% bovine serum albumin in wash buffer (1OmM Tris pH 7. 5, 10OmM NaC1,0.1% Tween 20)) for 1 hour at room temperature with constant rocking. The membrane was probed with either rabbit anti-P. aeruginosa elastase, Bever and Iglewski., (1988), J. Bacteriol 170:4309- 4314, or pooled human sera absorbed with E.coli according to techniques well known in the art. The primary antibodies were added to the blocking buffer at 1:1000 and incubation was continued for 1 to 4 hours. The membrane was then briefly washed twice with wash buffer, once for 15 minutes and once for 5 minutes with rocking. The HRP-labelled antibody (either anti-rabbit or anti-human) was added to the membrane at a concentration of 1:1000 in wash buffer and incubated for 1 hour as above. The membrane was then washed once for 15 minutes, and four times for 5 minutes with wash buffer as above. The membrane was then developed using ECL substrate reagents (Amersham) and exposed to Fuji RX X-ray film and developed according to the manufacturer's instructions. Re-probing of blots was performed after stripping by incubating in 20mM glycine pH 2.5, 0.055% Tween 20 overnight at room temperature with continuous shaking.

After probing with HRP labelled anti-rabbit antibodies, a strong band was visible in the P.aeruginosa track and a band of similar molecular weight to the P.aeruginosa elastase was seen in the H.Pylori cellular protein extracts. After probing with the pooled sera from five patients with hightitre antibodies against H.Pylori, a large number of bands were observed including a strong response to the band (35KDa) previously identified by the anti-P.aeruginosa elastase antibody. After probing with pooled sera from five patients not infected with H.Pylori no bands were visible. Thus, a protein of similar size and immuniological reactivity to the P. aeruginosa elastase protein was shown to be present in cellular and supernatant protein extracts of H.pylori NCTC 11638.

Example 3

Cloning of the H.Dylori hap gene Sgg of HindIII or BamHI digested H.Pylori NCTC 11638 genomic DNA were separated and blotted onto Hybond N (Amersham) as described in Smith et al. (1992), Gene, 114:211-216. The genomic blots were probed overnight with 20Ong of a 3.2kb HindIII fragment of V.cholerae hap gene DNA and 5ng of HindIII - digested phase lambda DNA directly labelled with HRP (Amersham ECL kit), washed, developed and exposed to Fuji RX X-ray film according to the manufacturer's instructions. A 4kb HindIII and a 1.5kb BamHI fragment of H.Pylori DNA hybridized strongly to the V.cholerae hap gene probe.

Both the 4kb (HindIII) and the 1.5kb (BamHI) fragments were cloned from H. Dylori NCTC 11638 genomic DNA by the following method:

Two 10Ag portions of genomic DNA were digested with either BamHI or HindIII and size separated on a 0.5 x TBE minigel. The 4kb HindIII fragment and the 1.5kb BamHI fragment were extracted from the gel and ligated separately into either HindIII or BamHI - digested and calf alkaline phosphatased pUC18, pUC19 and pAT153 as described in Smith,A.W., (1990), Ph.D. Thesis, University of Nottingham, Nottingham, United Kingdom. The resultant plasmids were transformed into E.coli strain DH5a and plated out onto LBroth plates containing 100 ng/1 ampillicin and incubated overnight at 37 0 C. The desired recombinants were indentified by colony lifts onto Hybond N and screened by colony hybridization using the 3.2kb HindIII 'li.cholerae (ECL) probe above according to manufacturer's instructions. The 1.5kb BamHI fragment was sequenced in both directions using custom made, universal and reverse sequencing primers and Sequenase Version 2 (United States Biochemical Corporation) according to the manufacturer's instructions.

Sequencing of the 1.5kb fragment did not reveal a start codon but did reveal a region of about 1kb that was over 99% identical to the V. cholerae hap gene sequence with only three base in-frame deletion of a histidine residue in the coding region (AH in Fig. 1). The sequences diverge completely about 50bp downstream of the stop codon, with the other bases differing before the complete divergence. The cloned sequence is then quite different from the 31flanking region of the V.cholerae hap locus. Such coding sequence conservation is highly unusual and difficult to explain either by a common precursor gene or by intrageneric gene transfer. The %G+C content for H.Pylori is 34-37% while for V.cholerae it is 46-48%, the subsequent difference in codon usage between the two genera should have allowed the DNA sequences to diverge even if the amino acid sequences were still conserved.

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Claims (11)

CLAIMS:

1. A vaccine/therapeutic composition comprising Helicobacter pylori HAP protein or a fragment thereof, wherein the Helicobacter Pylori HAP protein includes the amino acid sequence as shown in Figure 1 of the accompanying drawing.

2 A vaccine/therapeutic composition as claimed in claim 1 wherein the Helicobacter Pylori HAP protein or fragment thereof is a recombinant protein, polypeptide or peptide.

3. A vaccine/therapeutic composition as claimed in claim 1 or claim 2 wherein the Helicobacter Pylori HAP protein fragment comprises the zinc binding region of the protein.

4. A vaccine/therapeutic composition as claimed in claim 1 or claim 2 wherein the Helicobacter Pylori HAP protein fragment comprises the protease active site of the protein.

5. A vaccineltherapeutic composition as claimed in any one of claims 1 to 4 comprising a combination of two or more components selected from Helicobacter pylori HAP protein or fragments thereof.

6. A vaccine/therapeutic composition as claimed in any one of claims 1 to 5 further comprising a pharmaceutically acceptable carrier.

7. A vaccine/therapeutic composition as claimed in any one of claims 1 to 6 further comprising one or more additional antigens derived from an alternative source.

8. A method for the preparation of a vaccine/therapeutic composition as claimed in claim 6 comprising combining a Helicobacter Pylori HAP protein or fragment thereof, in accordance with any one of claims 1 to 5 together with a pharmaceutically acceptable carrier.

9. A vaccine/therapeutic composition as claimed in any one of claims 1 to 7 for the prevention or treatment of H.pylori infection.

10. Use of Helicobacter Pylori HAP protein or a fragment thereof in the manufacture of a vaccine/therapeutic composition for the treatment or prevention of Helicobacter pylori infection.

11. A vaccine-therapeutic composition for the prevention or treatment of Helicobacter pylori infection substantially as hereinbefore described.