WO1997003360A1

WO1997003360A1 - HELICOBACTER VACCINE ANTIGEN RECOGNIZED BY A PROTECTIVE MONOCLONAL ANTIBODY (IgG 50)

Info

Publication number: WO1997003360A1
Application number: PCT/US1996/011245
Authority: WO
Inventors: John G. Nedrud; Steven J. Czinn
Original assignee: Oravax, Inc.
Priority date: 1995-07-07
Filing date: 1996-07-02
Publication date: 1997-01-30
Also published as: AU6345396A; ZA965734B

Abstract

The invention features Helicobacter polypeptides which are recognized by monoclonal antibody IgG 50, monoclonal antibodies (e.g., IgG 50) which recognize these polypeptides and methods of preventing and/or treating Helicobacter infection using these polypeptides and monoclonal antibodies. The figure is a graph showing that monoclonal antibody IgG 50 is effective at passive protection of mice against Helicobacter felis infection.

Description

HELICOBACTER VACCINE ANTIGEN RECOGNIZED BY A PROTECTIVE MONOCLONAL ANTIBODY (IgG 50)

Background of the Invention

This invention relates to methods and compositions for preventing and/or treating Helicobacter infection.

Helicobacter is a genus of spiral, gram-negative bacteria which colonize the gastrointestinal tracts of mammals. Several species colonize the stomach, most notably, H. pylori , H. heilmanii , H. felis, and H.

mustelae . Although H. pylori is the species most

commonly associated with human infection, H. heilmanii and H. felis have also been found to infect humans, but at lower frequencies than H. pylori .

Helicobacter infects over 50% of adult populations in developed countries, and nearly 100% in developing countries and some Pacific rim countries, making it one of the most prevalent infections of humans worldwide.

Infection with H. pylori results in chronic stomach inflammation in all infected subjects, although the clinical gastroduodenal diseases associated with

Helicobacter generally appear from several years to several decades after the initial infection. H. pylori is the causative agent of most peptic ulcers and chronic superficial (type B) gastritis in humans. H. pylori infection is also associated with atrophy of the gastric mucosa, gastric adenocarcinoma, and non-Hodgkin's

lymphoma of the stomach (see, e.g., Blaser, J. Infect. Dis. 161:626-633, 1990; Scolnick et al . , Infect. Agents Dis. 1:294-309, 1993; Goodwin et al . ,

"Helicobacter pylori , " Biology and Clinical Practice, CRC Press, Boca Raton, FL, 465 pp, 1993; Northfield et al . , "Helicobacter pylori , " Infection , Kluwer Acad. Pub., Dordrecht, 178 pp, 1994). If untreated, H. pylori infection and the

associated gastritis persist lifelong, despite systemic and local immune responses to the bacterium in the infected host (Crabtree et al . , "Host responses," in Helicobacter pylori Infection , Northfield et al . (Eds.), Kluwer Acad. Pub., Dordrecht, pp. 40-52, 1991; Kist

"Immunology of Helicobacter pylori , " in Helicobacter pylori in peptic ulceration and gastritis , Marshall et al . (Eds.), Blackwell Sci. Pub., Oxford, pp. 92-110, 1991; Fox et al . , Infect. Immun. 61:2309-2315, 1993).

Conventional treatment of peptic ulcer disease associated with H. pylori infection involves the use of one or more antibiotics combined with a proton pump inhibitor or an H₂-receptor antagonist. Such treatment regimens are unsuccessful in 10% to 70% of patients. Moreover, successful eradication of H. pylori infection with antibiotics does not prevent subsequent reinfection. The most effective conventional treatment is a triple therapy with bismuth, metronidazole, and either amoxicillin or tetracycline. The triple therapy treatment is

complicated by a complex and prolonged dosing regimen, a high incidence of side-effects, poor compliance, and emergence of resistant bacterial strains (Hentschel et al . , N. Engl. J. Med. 328:308-312, 1993). Summary of the Invention

We have identified a Helicobacter polypeptide (hereinafter designated "IgG 50 ligand"), which is recognized by monoclonal antibody IgG 50, and may be used, e .g. , in methods and compositions for preventing and/or treating Helicobacter infection. In addition, we have shown that monoclonal antibody IgG 50 is effective in imparting passive immunity against Helicobacter infection. Accordingly, the invention features a method of preventing or treating Helicobacter (e .g. , H. pylori , H. felis, or H. Heilmanii) infection in a mammal

involving administering (e.g., to a mucosal (e.g., oral or intranasal) surface) to the mammal a Helicobacter (e .g. , H. pylori or H. felis) antigen which is recognized by monoclonal antibody IgG 50 (e.g., a Helicobacter antigen which is a polypeptide having a molecular weight of 16-19 kD, as measured by SDS-PAGE, e .g. , IgG 50 ligand, or a fragment or derivative thereof). Mammals that may be treated using the methods of the invention include, but are not limited to, mammals such as humans, cows, horses, pigs, dogs, cats, sheep, and goats.

The invention also features a substantially pure Helicobacter (e .g. , H. pylori or H. felis) polypeptide which is recognized by monoclonal antibody IgG 50, e . g. , a Helicobacter polypeptide having a molecular weight of 16-19 kD, as measured by SDS-PAGE, e .g. , IgG 50 ligand, or a fragment or derivative thereof. Vaccine

compositions containing a polypeptide recognized by IgG 50 (or immunogenic fragments or derivatives thereof), as is described above, in a pharmaceutically acceptable carrier or diluent, are also included in the invention. The vaccine composition may also include an adjuvant (e .g. , a cholera toxin, the heat-labile enterotoxin of Escherichia coli , or a fragment or derivative thereof having adjuvant activity).

The invention also features a monoclonal antibody (e.g., IgG 50) that recognizes a Helicobacter antigen (e .g. , a Helicobacter antigen which has a molecular weight of approximately 16-19 kD, as measured by SDS-PAGE, e.g., IgG 50 ligand) which is recognized by

monoclonal antibody IgG 50. Such a monoclonal antibody may be used in a method for preventing or treating

Helicobacter infection in a mammal involving administering (e.g., to a mucosal surface, e .g. , orally) the monoclonal antibody to the mammal. Such an antibody may also be used in a method of detecting a Helicobacter antigen in a sample. In this method, the sample is contacted with the monoclonal antibody and detection of the antibody bound to the sample is used as an indication of the presence of the antigen in the sample. This method may employ standard immunological assays, e.g., Western blot analysis or ELISA.

The invention also features a substantially pure nucleic acid (DNA or RNA) comprising a nucleotide sequence encoding a Helicobacter antigen recognized by monoclonal antibody IgG 50 (e.g., a Helicobacter antigen which has a molecular weight of approximately 16-19 kD, as measured by SDS-PAGE, e .g. , IgG 50 ligand).

By "protein" or "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

By "substantially pure" is meant a preparation which is at least 60% by weight (dry weight) the compound of interest, e.g., a IgG 50 ligand polypeptide or IgG 50 ligand-specific antibody. Preferably the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

By "purified DNA" is meant DNA that is not immediately contiguous with both of the sequences (e . g. , coding sequences) with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the

naturally-occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Detailed Description

The drawings are first described.

Drawings

Pig. 1 is a graph showing the percentages of mice protected against H. felis challenge by passive oral immunization with the indicated monoclonal antibodies, including IgG 50 (50), which were raised against H. felis lysates. Protection was measured by urease broth assays carried out on gastric biopsies obtained one week after inoculation.

Pig. 2 is a photograph of a silver-stained SDS-polyacrylamide gel (12%) upon which the following samples were fractionated: lane A = molecular weight markers; lane B = H. felis lysate; lane C = an immunoprecipitate of an H. felis lysate using monoclonal antibody IgG 50; and lane D = a control immunoprecipitation reaction lacking antigen.

Methods and Compositions for Preventing and/or Treating Helicobacter Infection The Helicobacter antigen recognized by monoclonal antibody IgG 50 (IgG 50 ligand) can be used in

vaccination methods for preventing and/or treating

Helicobacter (e .g. , H. pylori , H. felis , or H. heilmanii) infection. In these methods, IgG 50 ligand, or an immunogenic fragment or derivative thereof, is

administered to a mucosal (e.g., intranasal, oral, ocular, gastric, rectal, vaginal, intestinal, or urinary tract) surface of a mammal, or is administered

parenterally (e .g. , by intravenous, subcutaneous, intraperitoneal, or intramuscular routes). Any of a number of adjuvants that are known to one skilled in the art may be co-administered with the IgG 50 ligand vaccine antigen. For example, a cholera toxin (CT), the heat- labile enterotoxin of Escherichia coli (LT), or a

fragment or derivative thereof having adjuvant activity, may be used in mucosal administration. An adjuvant such as RIBI (immunoChem, Hamilton, MT) or aluminum hydroxide may be used in parenteral administration. Recombinant attenuated vectors derived from microorganisms, e.g., bacteria or viruses, such as Salmonella, Shigella, vaccinia virus, rotavirus, adenovirus, BCG virus, and poliovirus, may also be used for administration of the vaccines of the invention (see, e . g. , Lagranderie et al . , Vaccine 11:1283, 1993; Morris et al . , Gastroenterology 103:699-701, 1992; Hacket, Vaccine 8:5-11, 1990; Cardenas et al . , Clinical Microbiology Reviews 5:328-342, 1992; Dedieu et al . , J. Virol. 66:3161-3167, 1992; Jenkins et al . , J. Virol. 64:1201-1206, 1990; Karnell et al . ,

Vaccine 11:830-836, 1993; Schδdel et al . , Sem. Immunol. 2:341-349, 1990; Tacket et al . , Vaccine 10:673-676,

1992).

IgG 50 ligand polypeptides which may be used in the vaccination methods of the invention may be prepared using any of several standard methods. For example, standard recombinant DNA methods may be employed (see, e.g., Ausubel et al., Eds., Current Protocols in

Molecular Biology, John Wiley & Sons, Inc., 1994). In these methods, a suitable host cell is transformed with an appropriate expression vector containing all or part of an IgG 50 ligand-encoding nucleic acid (e.g., DNA or RNA) fragment. Nucleic acids encoding IgG 50 ligand are isolated using standard methods (see, e.g., Ausubel et al . , supra). For example, monoclonal antibody IgG 50, which specifically recognizes IgG 50 ligand, may be used to immunoprecipitate IgG 50 ligand from Helicobacter lysates (see Fig. 2 and below). The immunoprecipitated polypeptide may be further purified by, e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), after which it is transferred onto a membrane (e.g., a PVDF or nitrocellulose membrane), from which it is eluted for microsequence analysis (see, e.g., Ausubel et al . , supra). Based on the amino acid sequence of the purified polypeptide, degenerate primers may be designed for use in polymerase chain reaction (PCR) methods for generating probes which may be used for screening

Helicobacter (e .g. , H. pylori or H. felis) DNA libraries (e.g., cDNA libraries), in order to isolate clones which encode IgG 50 ligand (see, e.g., Ausubel et al . , supra).

In addition to immunoprecipitation and SDS-PAGE, other standard protein purification methods (e.g., conventional column chromatography, high-performance liquid chromatography, and two-dimensional gel

electrophoresis), and combinations of any of these methods, may be used to obtain purified IgG 50 ligand for microsequence analysis. A nucleic acid clone encoding IgG 50 ligand may also be obtained by screening an expression library, e.g., a λGT11 expression library, prepared from Helicobacter (e .g. , H. pylori or H. felis) nucleic acid (see, e.g., Blanchard et al . , Infection and Immunity 63 (4) :1394-1399, 1995; Ausubel et al . , supra).

Any of a variety of expression systems may be used to produce recombinant IgG 50 ligand polypeptides, once nucleic acid molecules which encode them are obtained. For example, IgG 50 ligand polypeptides may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., yeast cells (e.g., Saccharomyces cerevisiae), mammalian cells (e.g., COS1, NIH3T3, or JEG3 cells), or arthropod cells (e.g., Spodoptera frugiperda (SF9) cells)). Such cells are available from a number of different sources known to those skilled in the art, e.g., the American Type Culture Collection (ATCC),

Rockville, MD (also see, e .g. , Ausubel et al . , supra). The transfection/transformation method used, and the choice of expression vector, will depend on the host system selected, as is described by, e.g., Ausubel et al . , supra . Expression vectors (e.g., plasmid or viral vectors) can be chosen from, e.g., those described in Cloning Vectors: A Laboratory Manual (Pouwels et al . , 1985, Supp. 1987; also see, e.g., Ausubel et al., supra).

IgG 50 ligand polypeptides, particularly short fragments, may also be produced by chemical synthesis, e.g., by the method described in Solid Phase Peptide

Synthesis , 1984, 2nd ed., Stewart and Young, eds., Pierce Chemical Co., Rockford, IL, and by standard in vitro translation methods. In addition, IgG 50 ligand may be purified from Helicobacter cultures, using standard methods.

In addition to native, full length, Helicobacter IgG 50 ligand, polypeptide fragments of IgG 50 ligand, or IgG 50 ligand polypeptides (or polypeptide fragments of IgG 50 ligand) containing mutations, may be used in the invention, provided that antigenicity is retained.

Fragments of IgG 50 ligand polypeptides are made by standard methods, including, e.g., recombinant, chemical synthetic, or proteolytic methods (see, e.g., Ausubel et al . , supra). Generally, IgG 50 ligand fragments should be at least 10 amino acids in length, for example 50-200 amino acids in length, in order to maintain antigenicity. Genes encoding fragments of IgG 50 ligand, and/or IgG 50 ligand containing mutations, are made using standard methods (see, e.g., Ausubel et al . , supra). Fragments and derivatives of IgG 50 ligand which are included in the invention may be screened for antigenicity using standard methods in the art, e.g., by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity (see below).

Fusion proteins containing IgG 50 ligand (or a fragment or derivative thereof) fused to, e.g., an adjuvant (e.g., CT, LT, or a fragment or derivative thereof having adjuvant activity), are also included in the invention, and can be prepared using standard methods (see, e.g., Ausubel et al . , supra). In addition, the vaccines of the invention may be covalently coupled or cross-linked to adjuvants (see, e .g. , Cryz et al . ,

Vaccine 13:67-71, 1994; Liang et al . , J. Immunology

141:1495-1501, 1988; and Czerkinsky et al . , Infection and Immunity 57:1072-1077, 1989).

The amount of vaccine administered depends on, e.g., the particular vaccine antigen, whether an adjuvant is co-administered with the antigen, the type of adjuvant co-administered, the mode and frequency of

administration, and the desired effect (e.g., protection and/or treatment), as can be determined by one skilled in the art. In general, the vaccine antigens of the

invention are administered in amounts ranging between, e.g., 1 μg and 100 mg. If adjuvants are administered with the vaccines, amounts ranging between, e.g., 1 ng and 1 mg may be used. Administration is repeated as necessary, as can be determined by one skilled in the art. For example, a priming dose can be followed by 3 booster doses at weekly intervals. Antibodies against IgG 50 ligand (e.g., monoclonal antibodies such as IgG 50) may be used in passive

immunization methods for protecting and/or treating mammals (e.g., humans) from Helicobacter (e .g. , H.

pylori , H. felis, or H. heilmanii) infection. Monoclonal antibodies against IgG 50 ligand are produced using standard immunological methods (see, e . g. , Coligan et al., Eds., Current Protocols in Immunology, John Wiley & Sons, Inc., New York, New York, 1994). Antigens for use in these methods may be obtained, e.g., by

immunoprecipitation of Helicobacter lysates with IgG 50, followed by fractionation of the immunoprecipitate by SDS-PAGE. Antibodies of any isotype, e.g., IgA and IgG, may be used in the invention. In addition to monoclonal antibodies, purified polyclonal antibodies, single chain antibodies, chimeric antibodies (e.g., human/murine chimeric antibodies), humanized antibodies (e.g.,

humanized murine monoclonal antibodies), and Fab

fragments which recognize IgG 50 ligand may be used in the invention.

In the passive immunization methods of the invention, antibodies (e.g., monoclonal antibodies) which recognize IgG 50 ligand, e.g., IgG 50, are administered to a mucosal (e.g., oral or intragastric) surface of a mammal. The amount of antibody used in this method can be determined by one skilled in the art.

The IgG 50 ligand polypeptides, nucleic acids, and antibodies of the invention may also be used for

detecting the presence of anti-Helicobacter antibodies, Helicobacter nucleic acids, or Helicobacter polypeptides, respectively, in biological samples, using standard methods (e.g., Western blot analysis, ELISA, and nucleic acid hybridization methods; see, e.g., Ausubel et al . , supra; Coligan et al . , supra). The following examples are meant to illustrate, but not to limit, the methods of the invention.

Modifications of the conditions and parameters set forth below that are apparent to one skilled in the art are included in the invention.

EXAMPLES

Example I - Production and Isolation of Monoclonal

Antibodies Against H. Felis

The H. felis mouse model is an accepted model for H. pylori infection of humans (see, e .g. , Lee et al . , European Journal of Gastroenterology and Hepatology

7:303-309, 1995). This model has been used to identify other candidate Helicobacter vaccine antigens, e .g. , urease (Czinn et al . , Vaccine 11:637-642, 1993; Blanchard et al . , supra). In order to identify and characterize Helicobacter antigens that may be useful vaccines, monoclonal antibodies against sonicated H. felis lysates were generated, as follows.

Monoclonal antibodies (including IgG 50) against sonicated H. felis lysates were produced by a

modification of the procedure of Mazanec et al . (J.

Virol. 61:2624-2626, 1987; Czinn et al . , supra). BALB/c mice obtained from the Jackson Laboratory (Bar Harbor, Maine) were immunized intragastrically four times over a 6-week period. The first three times, the mice were immunized with 2 mg of sonicated H. felis plus 10 μg cholera toxin (Sigma Chemical Co., St. Louis, MO). For the last immunization, the cholera toxin was omitted, and, in addition to the intragastric immunization, the mice received an intravenous boost of 2 mg of H. felis sonicate. Three days following the final immunization, the mice were sacrificed, and their spleen cells were hybridized to SP2/01-Ag myeloma cells (ATCC accession number CRL 8006), using standard methods (see, e.g., Harlow et al ., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Coligan et al . , supra). Clones obtained by limited dilution were screened for secretion of anti-H. felis monoclonal antibodies by an enzyme-linked immunosorbent assay (ELISA) using an H. felis outer-membrane

preparations (OMPs) as an antigen (Blanchard et al . , supra). Several monoclonal antibodies which recognize H. felis antigens, including IgG 50, were isolated using this method.

Example II - Passive Protection Against H. felis

Challenge Imparted by Monoclonal Antibodies Against

H. felis

Monoclonal antibodies which recognize Helicobacter antigens, and were isolated using the methods described above, were analyzed in passive protection studies, as described by Czinn et al . (supra) and Blanchard et al . (supra). Briefly, H. felis harvested from a fresh plate was brought to a concentration of 5 × 10⁶ colony-forming units (CFU)/ml in phosphate-buffered saline (PBS). Two ml aliquots of the bacteria were combined with 2 ml of ascites fluid containing the anti-H. felis monoclonal antibodies, and incubated at 37°C for 30 minutes. Four hundred μl (~10⁶ organisms) of the bacteria/antibody mixture was administered to mice by gastric intubation. The mice received an additional 200 μl of ascites fluid at 4, 8, and 24 hours. Mice were necropsied on day 9, and their gastric tissues were examined for H. felis colonization by urea broth assays (see, e . g. , Blanchard et al . , supra). Using these methods, monoclonal antibody IgG 50 was found to be effective at passively protecting mice from H. felis challenge (see Fig. 1 and Table 1, below).

Monoclonal antibody IgG 50 is produced by

hybridoma cell line #50-G₆-B₇, which was deposited with the ATCC (Rockville, MD) on June 30, 1995, and assigned ATCC accession number HB-11952.

Example III - Characterization of the Helicobacter

Antigen Recognized by IgG 50

In order to characterize further the Helicobacter antigen (IgG 50 ligand) recognized by IgG 50,

radiolabeled H. felis OMPs were immunoprecipitated with IgG 50, and the immunoprecipitates were analyzed by SDS-PAGE (12%), followed by autoradiography (Blanchard et al . , supra). These experiments revealed that IgG 50 recognizes a Helicobacter polypeptide of approximately 16-19 kD molecular weight (Blanchard et al . , supra ; Fig. 2, lane C). Further characterization of IgG 50 ligand by metal chelate chromatography has revealed that it binds nickel.

IgG 50 immunoprecipitates two bands (Fig. 2, lane C) from H. felis lysates which are close to each other in molecular weight. Amino acid analysis of the polypeptides in these bands shows that their amino acid contents are very similar (Table 2 (upper band) and Table 3 (lower band)).

Other embodiments are in the following claims. What is claimed is:

Claims

1. A substantially pure Helicobacter polypeptide which is recognized by monoclonal antibody IgG 50.

2. The polypeptide of claim 1, wherein said polypeptide has a molecular weight of approximately 16-19 kD, as measured by SDS-PAGE.

3. A vaccine comprising the polypeptide of claim 1, or an immunogenic fragment or derivative thereof, in a pharmaceutically acceptable carrier or diluent.

4. The vaccine of claim 3, further comprising an adjuvant.

5. The vaccine of claim 4, wherein said adjuvant is a cholera toxin, or a fragment or derivative thereof having adjuvant activity.

6. The vaccine of claim 4, wherein said adjuvant is the heat-labile enterotoxin of Escherichia coli , or a fragment or derivative thereof having adjuvant activity.

7. A composition for treating Helicobacter infection in a mammal, said composition comprising a monoclonal antibody that recognizes a Helicobacter antigen recognized by IgG 50, formulated for

administration.

8. The composition of claim 7, wherein said mucosal surface is oral.

9. The composition of claim 7, wherein said monoclonal antibody is IgG 50.

10. A monoclonal antibody that recognizes a Helicobacter antigen recognized by monoclonal antibody IgG 50.

11. The monoclonal antibody of claim 10, wherein said Helicobacter antigen has a molecular weight of approximately 16-19 kD, as measured by SDS-PAGE.

12. The monoclonal antibody of claim 10, wherein said monoclonal antibody is IgG 50.

13. A substantially pure nucleic acid comprising a nucleotide seguence encoding a Helicobacter antigen recognized by monoclonal antibody IgG 50.

14. A method of detecting a Helicobacter antigen in a sample, said method comprising the steps of:

(i). contacting said sample with the monoclonal antibody of claim 10; and

(ii). detecting said antibody bound to said sample as an indication of the presence of said antigen in said sample.