NO870148L - ORAL VACCINES. - Google Patents

Description

TECHNICAL AREA

The present invention relates to the specific stimulation of serum and secretion antibodies through the delivery of antigens to the mucous membrane.

BACKGROUND TECHNOLOGY

Several infections in mammals have sufficiently harmful effects on the mammal to warrant vaccination against the particular antigen responsible for the infection. Vaccination programs have therefore been implemented where the mammal is antigenically challenged with an antigen so that a response is triggered which gives the mammal immunity.

Antigen administration to the mammal can take place in several ways, including injection intramuscularly (i.m.), subcutaneously (s.c.) or through oral administration (per os). I.m.-

or s.c. injection of antigen suffers from the disadvantages that relatively specialized knowledge is required, it is difficult to perform on a large scale, it is expensive and a number of side effects can occur either against the immunizing antigen or against the emulsifying reagent in which it is present. Oral administration of vaccines, on the other hand, is relatively problem-free, except that oral administration of a number of antigens requires relatively large amounts of antigen, as the amount of substance that is actually absorbed and is capable of stimulating an effective immune response is usually low. Thus, the amount of antigen required for oral immunization usually far exceeds that required for systemic induction of immunity. There is also a main disadvantage of the oral administration of the large amounts of antigen that are required to produce an anti-substance response, and that is that this administration of these large amounts of antigen often leads to the induction of systemic tolerance (Tomasi, 1980; Mowat , 1985; Mowat and Parrot, 1983; Ngan&Kind, 1978, Hanson et al, 1979, Richman et al, 1978, Rothberg et al, 1973).

The evidence available today suggests that

the mechanism by which antigen is taken up by the small intestine after oral administration, usually primarily via non-specific uptake of the contents of intestinal tract cavities by 11M" cells overlying Peyer's Patches and other lymph nodes

in GALT (gut-associated-lymphatic tissue) (Bland and Britton, 1984). The subsequent sensitization of local lymphocyte populations leads to the generation of local IgA immune responses plus the sensitization of IgG suppressor cells with accompanying suppression of serum IgG responses (Tomasi, 1980; Mowat, 1985; Mowat and Parrot, 1983, Ngan&Kind, 1978 ,

Hanson et al, 1979, Richman et al, 1978; Rothberg et al, 1973).

It is therefore obvious that the site of antigen uptake, whether through Peyer's Patches or the gut epithelium, and very likely the amount of antigen administered, dictates the type of immune response generated by orally administered antigen. The question then is whether there are any other antigens apart from cholera toxin that exhibit the ability to specifically trigger the mucosal immune system after oral challenge and/or to stimulate the humoral immune response in a dose-dependent manner without inducing systemic tolerance and without the need for unreasonably large doses of antigen.

With this overview in mind, we decided to investigate the possible potential of certain binding molecules, which have been implicated in the initial binding of a number of enteric pathogens, in stimulating the immune response after being administered orally. These surface antigens which confer binding properties to a variety of strains of enterotoxigenic E. Coli (ETEC) have been identified as non-flagellar, filamentous proteinaceous appendages or pili (Gaastra & de Graaf, 1982). Examples include the antigens CAF I and CFA II of human ETEC strains and the K88, K99, F41 and 987P pili of animal ETEC strains (Gibbons et al, 1975; Evans&Evans, 1978; Levine et al, 1980; Morgan et al al, 1978; de Graaf&Roorda, 1982) to animal ETEC strains. In addition, we have investigated the ability of a number of other proteins, which have no obvious role in colonization, to trigger the immune system after oral administration. These antigens include several lectins,

a serotypic antigen of S. typhimurium (type "i" flagella) inactivated influenza virus and S. typhimurium endotoxin (LPS). Oral ejaculation was compared with the response elicited against full-

constant intramuscular challenge (i.m.).

The aim of these investigations was thus to provide a method by which the uptake of an immunogen or antigen by means of the mucous membrane in the gastrointestinal tract is improved to such an extent that it is possible to produce serum and secretion antibodies by oral administration of small doses of the immunogen without induction of oral tolerance.

Accordingly, the invention describes a group of molecules (mucosal immunogens) which, after administration, lead to the production of serum antibodies against the proteins at levels comparable to those obtained by intramuscular injection of the molecules. When larger amounts of these antigens are added, there is an accompanying stimulation of the production of mucosal antibodies against the immunizing molecules.

In a further aspect of the invention, a method is described whereby the produced antibody response against the orally supplied molecules can be increased or changed by means of the simultaneous supply of several dietary molecules.

Another aspect of the invention describes a method by which a hapten or protein can be linked to a mucosal immunogen and where the complex of these, when added, results in the production of antibodies against the hapten or the linked protein.

DESCRIPTION OF THE INVENTION

In a first form, the invention provides a complex comprising: an immunogen, bound to a carrier molecule capable of specifically reacting with the mucosal epithelium of a vertebrate host, where both the immunological activity of the immunogen and the capacity of the carrier molecule to specifically react with the mucosal epithelium of the vertebrate host substantially maintained, and wherein the complex is capable of eliciting a systemic, cellular and/or mucosal immune response in the vertebrate host.

Preferred immunogens according to the invention include: all, some, analogues, homologues, derivatives or combinations thereof and a hormone, a therapeutic agent, antigen or hapten. These immunogens include such hormones as LHRH (hormone that releases luteinizing hormone), FSH, HGH and Inhibin, such allergens as grass pollen (e.g. barley and vetch), weed pollen (e.g. clover, hay moth), tree pollen (e.g. e.g. ash, cypress), plant pollen (e.g. broom), epithelia (e.g. cat hair, dog hair, pig bristles) and house dust, wheat chaff and Kapok, immunogens for vaccines against such agents as influenza, measles, Rubella, smallpox, yellow fever, diphtheria, tetanus, cholera, plague, typhus, BCG, haemophilus influenzae, Neisseria catarrhalis, Klebsiella pneumonia, pneumococci and streptococci, especially S. mutans; and pili, including pili derived from E. coli, N. gonorrheae, N. meningitis, N. catarrhalis, Yersinia spp, Pseudomonas aeruginosa, Pseudomonas spp, Moraxella bovis, Bacteroides nodosus, Staphylococci spp, Streptococci spp and Bordetella spp.

Preferred carrier molecules include bacterial adhesins, such as 987P, K99, CFAI, CFAII, K88 or F41, viral hemagglutinins such as those from influenza, measles, rubella, smallpox or yellow fever viruses, toxins or binding subunits thereof, such as LTB- ricin, abrin, diphtheria toxin, modecin, tatanus toxin and others with similar structures, and lectins, whether of plant or other origin. Lectins include e.g. conconavalin A, kermesberry mitogen or lectins from Lens culinaris, Helix pomatia, Glycine max, Arachis hypogea or Ulex europeus or Abrin, sloe asparagus, bean vetch, camel's foot tree, castor seed, fava bean, green algae, hairy vetch, horse gram, horseshoe crab, hunting tree bean, Japanese wisteria, paternoster , Scottish golden rain, lima bean, limulin, lotus, European mistletoe, mung bean, yellow tree, pagoda tree, garden pea, potato, red cut bean, red algae, Siberian bush, edible snail flower, garden snail flower, common bone wood, sweetened, tomato, wheat germ or asparagus.

In a preferred embodiment of the invention, a complex comprising hormone which releases luteinizing hormone and LTB is provided.

In another form, the present invention provides a method for producing a complex as described above, which comprises: (a) Reaction of the immunogen with the carrier molecule, whereby the complex is obtained, (b) chemical modification of the immunogen to provide at least one functional group capable of forming a chemical bond, and reacting the immunogen and the carrier molecule to the complex, or (c) chemically modifying the carrier molecule to provide at least one functional group capable of forming a chemical bond, and reacting the immunogen and the carrier molecule of the complex, (d) chemically modifying the immunogen and carrier molecule to provide functional groups capable of forming a chemical bond, and reacting the immunogen and carrier molecule to the complex, (e) reacting the immunogen and at least one binding agent, and reacting the immunogen and the carrier molecule to the complex, (f) reacting the carrier molecule with at least one binding agent and reacting the immunogen and the carrier molecule to the complex, (g) reacting the immunogen and the carrier molecule with at least one binding agent, and reacting the immunogen and the carrier molecule to the complex, or (h) a combination of any of the above process steps.

In another form, the invention provides a method which comprises providing a recombinant DNA molecule comprising a first DNA sequence which, after expression, codes for the amino acid sequence of the immunogen, a second DNA sequence which, after expression, codes for the amino acid sequence of the carrier molecule, and vector DNA , transforming a host with the recombinant DNA molecule so that the host is capable of expressing a hybrid proteinaceous product comprising the complex, culturing the host to achieve the expression, and collecting the hybrid proteinaceous product.

Alternatively, the invention provides a method for producing a complex comprising (a) chemically synthesizing the immunogen and/or the carrier molecule, and forming the complex by means of chemical reaction, or (b) synthesizing a hybrid peptide comprising the amino acid sequences of the immunogen and the carrier molecule. Preferably, the peptide is prepared using peptide synthesis in solid phase, enzymatic or manual peptide synthesis.

In a preferred embodiment of the invention, the synthesized immunogen or carrier molecule, while bound to the resin in the peptide synthesis mixture in solid phase, is connected to the carrier molecule and the immunogen, respectively.

In another form of the present invention, a transformant host is provided which has been transformed with a recombinant DNA molecule comprising a first DNA sequence which, after expression, codes for the amino acid sequences of the whole, a part, an analogue of, a homologue of, a derivative of or a combination of the immunogen, a second DNA sequence which, after expression, codes for the amino acid sequence of all, part of, an analogue of, a homologue of, a derivative of or a combination of the carrier molecule, as well as vector DNA.

In a preferred embodiment of the invention

is the transformant host a gram negative or gram positive bacterium, a yeast, fungus or a higher eukaryotic cell. In a preferred embodiment of the invention, the host is E. coli. A culture of a transformant microorganism falling within the scope of the present invention has been deposited with the American Type Culture Collection and has been designated with the number ATCC 67114.

In a further form, the invention provides a recombinant DNA molecule comprising a first DNA sequence which after expression codes for the amino acid sequence of the immunogen, a second DNA sequence which after expression codes for the amino acid sequence of the carrier molecule, and vector DNA. In a preferred embodiment of this form of the invention, the vector DNA is plasmid DNA, but within the scope of the present invention alternative vectors are considered and these include viruses, bacteriophages and cosmids. In a preferred form of the invention, plasmid pBTAK66 is provided as,

when a host cell is transformed with the plasmid, will yield a protein-like product comprising a polypeptide that falls within the scope of the present invention.

In a further form of the invention, there is provided a polynucleotide sequence comprising a first hybrid polynucleotide sequence which acts as a coding sequence for a fusion product comprising an amino acid sequence of an immunogen bound to an amino acid sequence of a carrier molecule, a polynucleotide sequence which is sufficiently related to the first hybrid polynucleotide sequence to to hybridize to the first hybrid polynucleotide sequence, a polynucleotide sequence derived by mutation, including substitutions, deletions, insertions and conversions of one or more bases, from the first hybrid polynucleotide sequence or hybridizing sequence, or a polynucleotide sequence which, after expression, codes for a polypeptide that exhibits similar biological or immunological activity as the fusion product. Preferably, the polynucleotide sequence is one in which the first hybrid polynucleotide sequence acts as a coding sequence for the amino acid sequence of all, part, an analog,

a homologue, derivative or combination of LHRH linked to the amino acid sequence of a carrier molecule, preferably LTB.

In a further form of the invention, a drug is provided which comprises a complex according to the invention together with a pharmaceutically acceptable carrier or diluent. Examples of pharmaceutically acceptable carriers and diluents include such typical carriers and diluents as tablets, aqueous solutions, sodium bicarbonate solutions and similar diluents that neutralize gastric acid or have similar buffer capacity, glycols, oils, oil-in-water or water-in- oil emulsions, and includes drugs in the form of emulsions, gels, masses and viscous colloid dispersions. The medication can be presented in capsule, tablet, slow release or elixir form, or as a gel or mass, or can be presented as a nasal spray and in this form can be present in the presence of an aerosol. Furthermore, the drug can be provided as a livestock feed or as food suitable for human consumption.

The present inventors have also found that the simultaneous administration of certain dietary molecules together with a complex according to the present invention can selectively modulate the size and/or type of the immune response against the immunogen in the complex.

Consequently, the present invention further provides a drug comprising the complex according to the present invention together with a dietary molecule which can selectively modulate the size and/or type of the immune response against the immunogen in the complex.

The dietary molecule that comes into consideration in the present invention includes basic, neutral and acidic amino acids, such as arginine, histidine, lysine, alanine, cysteine, cystine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine , valine, aspartic acid, glutamic acid, water-soluble and insoluble vitamins, such as thiamin, riboflavin, pyridoxal, cyanocobalamin (V.B.12) ascorbic acid (V.C.) Vit D2, etc. - Ergosterol, Vit. E, Wit. A, White. K, etc; sugars include monosaccharides, e.g. galactose, mannose, mannitol, sorbitol, glucose, xylose, allose, altrose, arabinose, digitoxose, erythose, fructose, lyxose, muramic acid, mannose, pyruvic acid, ribose, tagatose, talose and the amidated and N-acetylated derivatives thereof; oligo-saccharides, e.g. lactose, maltose, melibiose, sucrose, cellibiose, N,N-diacetyl chitobiose, gentobiose, isomaltose, lactobionic acid, trehalose, turanose; and dietary minerals and co-factors, such as manganese, magnesium, zinc, calcium and iron.

The invention also provides a method

for the administration of a complex according to the present invention which comprises mucosal administration of a complex according to the present invention together with a dietary molecule capable of modulating the size and/or type of the immunogen's immune response.

The invention also provides for the oral administration of the drug according to the invention to produce a response to the active molecule in the host. Such a response can, in the case where the active molecule is an antigen or hapten, be a systemic and/or mucosal immune response. In the case where the active molecule is LHRH or a derivative, an analogue, a homologue, a part or a combination thereof, the response will be inhibition of gonadal function in the host. When the oral drug contains a dietary molecule according to the invention, the invention provides a method for increasing the host's response to the active molecule which comprises administering such an oral drug to the host.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the N-terminal amino acid sequence of the pilin subunit 987P compared to the N-terminal amino acid sequence of other pilin proteins.

EMBODIMENTS OF THE INVENTION

Materials

Lectins were purchased from Sigma Chemical Company. Inactivated flu vaccine was acquired from Commonwealth Serum Labs.

(Australia). Sugars and vitamins were obtained from the following sources: Lactose (AR grade) - Ajax Chemicals, Sydney, Australia; Fructose D(-), Mannose D(+), Sorbitol and Xylose D( + ) (all

AR degree) - B.D.H. Chemicals Ltd. Poole, England; Melibiose D(+) - Sigma Chemicals Co., St. Louis, Miss., Retinal (Vit.

A. - aldehyde) - Fluka AG, Chemicals, Fabrik Buchs, Switzer-land - HC1 (Vit. B±), Riboflavin (Vi. B2), Pyridoxal (Vit. Bg), Cyancobalamin (Vit. B^2>, L -ascorbic acid (Vit. C), ergosterol (Pro Vit. D) and dl-α-tocopherol (Vit. E) - Sigma Chemical Co., St. Louis, Miss.

Bacterial strains and media

The strains of E. coli K99, 987P and LTD used in these experiments are listed in Table 1 and were generous gifts from Dr Susan Clark (Molecular Biology Laboratory Biotechnology Australia). Cultures were grown at 37°C (unless otherwise stated) with shaking in Luria culture medium (LB)

with or without 1 mM isopropylthio-n-D-galactopyranoside

(IPGT) as indicated (Table 1). Salmonella thyphimurium was grown at 37°C with shaking in LB plus 0.2% glycerol.

PURIFICATION OF ANTIGENS

Production of pili

E. coli expressing the cloned pili, as determined by using radiolabeled antiserum, were harvested during logarithmic growth phase. Cultures were heated at 60°C

for 30 minutes, after which the organisms were pelleted by centrifugation (3000 x g, 30 minutes, 4°C). The supernatant was examined for pili content by 12.5% SDA-PAGE using a modification of the method of Laemmli (Laemmli, 1970; Salit et al., 1980).

Purification of K99: The culture supernatant was adjusted to pH 9.7 with 10 N NaOH and stirred at room temperature (R.T.)

for 10 minutes. The resulting precipitate containing pili was recovered by centrifugation (3000 x g, 30 min, 4°C)

and resuspended in 100 ml of distilled E₂O (dr₂O) pH 7.2. This procedure was repeated twice.

Purification of 9 8 7P: The procedures used were as detailed above, except that pili precipitation was achieved by adjusting the pH to 3.9 with glacial acetic acid.

Hydroxyapatite chromatography

Hydroxyapatite (HA) (DNA-gra Bio-Gel HTP, Bio-Rad) was gently swollen in an excess of dE₂O, and after a short time (less than 2 minutes) small grains were gently decanted. Fresh dH 2 O was added and used to gently resuspend the gel, after which small grains were again decanted. This procedure was repeated several times. A column

(30 x 5 cm) was filled with a slurry of approximately 30% HA and allowed to settle by gravity. Tight packing was then achieved by passing dH 2 O through the column at a flow rate of 16 ml/hr until the surface of the gel layer was stationary. Samples (100 ml) of either K99 or 987P were added at flow rates not exceeding 30 ml per minute. hour. The column was then washed with until no protein was detected in the flow-through by detection at 280 nm. Pili were eluted at a flow rate of 30 ml/hr using a linear gradient of 15 - 250 mm sodium phosphate, pH 7.5. Fractions were collected and examined using SDS-PAGE. Pili peak was mined and merged.

Ion exchange chromatography

Pooled fractions of K99 and 987P pili (from the HA chromatography) were reprecipitated with NaOH (pH 9.7) or glacial acetic acid (pH 3.9), respectively. After centrifugation (3000 x g, 10 minutes), the pellets containing pili were resuspended in 50 mm citrate buffer, pH 5.5 (K99) and 50 mm Tris-HCl, pH 8.5 (987P) before loading into the ion exchange columns equilibrated with the same buffers . K99 and 987P were loaded onto CM and DEAE columns, respectively, at a flow rate of 100 ml/hr, washed with 2 volumes of loading buffer and the pili were eluted using a linear gradient from 10 mm to 0.5 M NaCl in the equilibration buffers. Fractions were examined by SDS-PAGE for protein content and LPS contamination according to the method of Tsai and Frasch (1982). LTB cleaning

three liters of LTD supernatant was diluted to 6 liters with dH 2 O. The pH was adjusted to 6.5 with glacial acetic acid and a 5 x 30 cm column was loaded with fast flow CM-Sepharose<®>equilibrated with 10 mM phosphate buffer, pH 6.5, at a flow rate of 1.2 liters/ hour. The column was then washed with 400 ml of 10 mM phosphate buffer, pH 6.5, and bound protein was eluted by a linear gradient of 10 - 500 mM NaCl in 10 mM phosphate, pH 5.5. Fractions were collected and analyzed by SDS-PAGE, the LTD peak was pooled.

Isolation of flagella

Bacterial cultures in the last part of the logarithmic growth phase were pelleted by centrifugation (3000 x g for 15 minutes at 4°C). The cells were resuspended in saline and heated at 60°C for 30 minutes, followed by centrifugation (3000 x g, 10 minutes, 4°C). The supernatant was precipitated by adding a solution of 100% TCA (w/v), whereby a final concentration of 10% (w/v) was obtained, and centrifuged for 10 minutes at 1500 x g, 4°C. The pellet was resuspended in a small volume of 1 M Tris, pH 8.8, and sonicated until dissolved. Ethanol was added to a final concentration of 80% (vol/vol) and the flagella were spun down at 2000 x g, 10 min at 4°C. The pellet was resuspended in acetone, sonicated until suspension and re-precipitated by centrifugation (5000 x g). Finally, the pellet was dissolved by boiling in 10% SDS and 50 mM EDTA in 10 mM Tris. HCl pH 8.0, before chromatography with Sephacryl<®>S-200.

Purification of flagella

After boiling for 15 minutes, the flagella were clarified by centrifugation for 15 minutes in a Beckman tabletop microcentrifuge to remove unsolubilized material. The supernatant was applied to a 2.5 x 80 cm column of Sephacryl<®>S-200 (Pharmacia, Fine Chemicals) equilibrated with 20 mM Tris, pH 8.8, 0.1% SDS, and 10 mM EDTA, and eluted using the same buffer. Fractions were collected and analyzed by SDS-PAGE. Finally, the flagellar tip was pooled and precipitated with 10% (final concentration) TCA, followed by centrifugation, ethanol and acetone washes as previously described. The final pellet was resuspended in dE₂O.

Purification of lipopolysaccharide (LPS)

Cultures of S. typhimurium grown overnight were extracted (30 min, room temperature) with 0.5 M CaC^ in 20% ethanol (v/v) containing 100 mM citrate, pH 3.0, and Zwittergent 3, 12 (w/v) (Calbiochem.). Bacteria were pelleted by centrifugation (3000 x g, 10 min at 4°C) and the pellet resuspended in 50 mM EDTA, pH, 8.0. The suspension was vigorously stirred for 30 minutes at room temperature. After removal of the bacteria by centrifugation, ethanol was added to the supernatant until a final concentration of 75%. Protein material was pelleted and the supernatant adjusted to 90% ethanol. The precipitate formed was pelleted and washed with acetone, reprecipitated and finally resuspended in dE 2 O. The preparation was analyzed for sugar content using the Anthrone reagent (Herbert et al, 1985) and checked for the presence of contaminating proteins using SDS-PAGE. Commercial E. coli LPS (Sigma Chemical Co:, ) was used as a standard

in both analyses. Gels were stained for LPS using a silver stain according to the method of Tsai and Frasch

(1982).

Production of polysaccharide (PS)

Lipid A was cleaved from the S. Typhymurium LPS preparation by incubating the LPS with 1 M glacial acetic acid and heating at 100°C

for 2 - 5 hours. Lipid A was then removed by centrifugation at 3000 x g for 10 min at 4°C.

Description of purified antigens

SDS-PAGE analysis of purified K99 and 987P pilipreparates revealed the presence of a single band at 17500

and 20,000 molecular weight (respectively) under reducing conditions (Fig. 1). This agrees with the published data of Isaacson and others (Isaacson & Richter, 1981; Morris et al, 1980; de Graaf et al, 1981; Fusco et al, 1978). The ease with which these proteins precipitate at pH 9.7 and 3.9 (for K99 and 987P, respectively) suggests that the pI of these two proteins

is approximately at these areas (see Isaacson & Richter, 1981;

de Graaf et al, 1981).Silver staining of these preparations showed that they contained little (less than 1 µg/100 µg protein) or no contamination with LPS.

Determination of the amino-terminal sequence in 987P

Amino-terminal microsequencing was performed for us

by Biotechnology Research Enterprises S.A. Pty. Ltd., Adelaide, South Australia. A 100 nmol sample of 987P purified as described above was analyzed. The amino-terminal sequence of 987P was compared with the published sequence of K99 and revealed homology between these two molecules (Fig. 1.)

(Gaastra and de Graaf, 1982).

Purified LTB and S. typhimurium flagella were also found to be free of contaminating LPS, and to move as monomers at apparent molecular weights of 12,500 and 52,000, respectively, when examined by SDS-PAGE under reducing conditions.

Silver-stained SDS-PAGE gels of purified LPS revealed no detectable protein contamination. Complex sugar content, analyzed by Anthron reaction, was found to be 2 mg/ml. Polysaccharide free of lipid A was also found to contain 2 mg/ml polysaccharide and its failure to migrate on SDS-PAGE (as revealed in the silver gels) indicated that it was free of contaminating lipid.

Dinitrophenylation of antigens

K99, LTB and lectins were dinitrophenylated according to the procedure of Little and Eisen (1967). Briefly, carriers (in 0.1 M carbonate/bicarbonate buffer, pH 9.5) were reacted with a 0.1 M solution of DNFB 8 (in acetone) overnight at room temperature. The proteins were then dialyzed thoroughly against the coupling buffer. Previous investigations by us have shown that 987P has no free amino groups exposed for coupling, so a diamino intermediate was first bound to the free carboxyl residues in the protein as follows: 10 mg of purified 987P was precipitated at pH 3.9 by the addition of glacial acetic acid. The pili were removed by centrifugation at 3000 x g, 10 minutes at 4°C. The pellet was resuspended in dH 2 O and the pH raised to 6.5 with 1 N NaOH. The pellet solution was then reacted with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-HCL (EDAC, Bio Rad Laboratories, Richmond, Calif.), to a final concentration of 0.5 mM in the presence of 20 mM 1, 2-diaminoethane (BDH Chemicals Ltd., Poole, England), overnight at room temperature (20 - 23°C). The amino-substituted 987P was dialyzed for 24 hours against two changes of 0.1 M carbonate/bicarbonate buffer, pH 9.5, before being used in subsequent conjugation steps. Lectin-binding sites were protected during reaction with DNFB by addition of the lectin-specific sugars. Thus, a 50 mM solution of D-glucose, D-mannose, N-acetyl-D-galactosamine, D-galactose, N-acetyl-D-galactosamine, D-gal(1-3)-D-galN-Ac was added and L-fucose to the following lectins: respectively Conconavalin A, kermesbær mitogen, Lens culinaris, Helix pomatia, Phaseolus vulgaris, Glycin max, Arachis hypogea and Ulex eruopeus.

Administration of antigen

Female C57BL/6J mice (18 - 22 g) were obtained from the Animal Resources Center (Perth, Western Australia). Everyone

the mice were starved for 3-4 hours before oral or intramuscular (i.m.) administration of antigens. The mice were infused with antigen at appropriate concentrations in 0.5 ml of 0.1 M carbonate/bicarbonate buffer, pH 9.5, using a specially prepared delivery needle. Parallel doses of antigen were injected i.m. in 0.1 ml of sterile physiological saline in the left hind leg. Groups of 5 mice received antigen either orally or intramuscularly, were given two identical doses of antigen, on day 0 and day 14. A blood sample was taken (approximately 0.5 ml)

from the retroorbital plexus on day 14 and day 21. The mice were then euthanized by cervical dislocation and intestinal lavages performed in the small intestine in the following manner. The small intestine was carefully removed and a small amount of wash buffer (1.0 ml, 30 mM Tris.HCl, pH 8.8, 0.9% NaCl, 50 mM EDTA plus 1.0% Tween<®>20) introduced into the cavity of the intestine via a delivery needle with a blunt end. After gently massaging the intestine, the contents were squeezed out between the index finger and thumb. The intestinal washings thus obtained were immediately centrifuged to remove degradation debris and stored at -20°C until assayed.

Blood samples were allowed to clot at 4°C before removal of the serum,

and stored at -20°C.

Enzyme-linked immunosorbent assay (ELISA)

ELISA for determination of antibody titers was performed as previously described by Russell-Jones et al, (1984).

EXAMPLE 1

Identification of molecules active as mucosal immunogens

The potential ability of a number of molecules known to bind to the intestinal mucosa and stimulate the production of an immune response following oral administration of the molecules was investigated. The response produced by these molecules was compared with the response obtained after similar administration of other molecules without any mucosa-binding groups.

As will be seen from Table 1.1, three large classes of proteins were detected in these experiments: those that produced a serum and intestinal response, K99, 987P, LTB, flu vaccine and the various lectins (class I) (these will hereinafter referred to as mucosal immunogens), those that elicited only a serum response (LPS) (class II) and those that failed to elicit either a serum or intestinal response at the doses tested (flagellates, BSA and P.S.) (class III) . Among the class I antigens, 987P was a significantly better stimulator of IgA antibody (ab) (48.5 h 1.8) compared to LTB (12.2 - 4.4), or K99 (3.2 - 4.9 ). In addition, 987P also stimulated gastro-intestinal IgG (10.8 - 1.76) to a greater extent than both K99 (3.0 - 5.3) and LTB (1.0), and only 987P was able to stimulate serum IgA (10.8 8.8). All four class I antigens stimulated serum IgG to an equal extent (Table 1.1.) The class II antigen, LPS, stimulated a small serum IgG response (12.8 - 1.0) without any accompanying IgA or gastro -intestinal reactivity. Finally, BSA, flagella and the polysaccharide residue from LPS, the class III antigens, failed to induce both serum and intestinal IgG or IgA. Representative samples from all three classes: K99, 987P, LTP, LPS and flagella, were administered intramuscularly and examined for both serum and intestinal response (Table 1.2). Class I and II antigens elicited similar serum IgG responses but failed to elicit serum IgA or intestinal IgG/IgA Ab responses. Only the anti-LPS serum IgG response was found to be significantly enhanced by intramuscular injection. Both 987P, K99 and LTB were further examined in dose response studies after both oral and intramuscular administration. As seen in Tables 1.3 and 1.4, 987P consistently produced higher titers than both K99 and LTB, regardless of route of administration. Interestingly, the class I antigen, LTB, showed a bell-shaped dose response with a plateau maximum between 10 and 50 pg. None of the other antigens from class I produced this effect, nor did LTB when administered intramuscularly. Oral administration of all class I antigens (Ag) produced higher levels of intestinal IgA Ab (S-IgA) over the wide range of doses tested. Comparison between the two routes of administration suggests that although intramuscular injection consistently produced higher titers, oral administration of mucosal immunogens resulted in antibody production levels comparable to those obtained by the intramuscular route between 10 and 100 µg of antigen for class I antigens and II.

Example 2

Effect of dietary molecules on the immune response to mucosal immunogens after oral administration.

Initial investigations in our laboratory using outbred male Swiss mice suggested that it was possible to alter the immune response to orally administered antigens by coadministration of certain dietary molecules. Consequently, the mucosal immunogens K99, 987P and LTB were administered to the mice in the presence of several dietary sugars and vitamins. It was concluded that as the different antigens were known to bind to different molecules on the surface of the intestinal epithelium, and as it is known that there is a change in the distribution of glycoproteins and glycolipids outside the intestine, as well as a change in the distribution of absorbent cells, it may be possible to stimulate the uptake of molecules bound to these cells by adding the antigens in the presence of the particular food molecule which is normally taken up by these cells. An addition to this argument would be that the profile of stimulating molecules changed from antigen to antigen.

The results presented in Tables 2.1 and 2.2 and 2.3 show that although most of the vitamins and sugars have had some effect in modulating the immune response to K99, 987P and LTB, some dietary molecules appear to be selective as to which mucosal immunogen they were shown to affect, but also with regard to whether they induced primarily a secretory response or a serum response. Thus, the serum antibody response to K99 was significantly increased (p < 0.05) by co-administration of vitamin B^°9melibiose, unchanged when given together with vitamin B^, vitamin B, vitamin E, fructose or mannose, and decreased in varying degrees of vitamin A, vitamin B ± ,, vitamin Brfa, vitamin C, lactose, sorbitol and xylose (table 2.1.).

In contrast, the serum Ab response to oral 987P (Table

2.2) elevated when 987P was co-administered with vitamin Bg, vitamin B, o, vitamin C, vitamin E, fructose or mannose, it remained unchanged with vitamin A, vitamin B^, vitamin B^ > lactose, melibiose, sorbitol and xylose, and reduced in the presence of vitamin D. LTB, on the other hand, showed a unique profile for the effect of co-administration of dietary molecules on serum Ab levels. The results in table 2.3 clearly show an increased serum titer against LTB in the presence of vitamin A, vitamin B^, vitamin D, fructose, mannose and xylose. Little or no change with vitamin B^, vitamin B^» vitamin C or melibiose, and almost complete inhibition with vitamin B^, vitamin E, lactose. melibiose and sorbitol. The inhibition of an immune response observed with vitamin Bfa, lactose, melibiose, and sorbitol was expected because of the similarities in structure between these compounds and galactose, which is claimed to be the specific sugar determinant on the GM1 ganglioside that LTB is known to bind to. These results broadly suggest that K99, 987P and LTB bind to and are taken up by the microvilli epithelium.

Dose response experiments (tables 2.4, 2.5 and 2.6) show

that it is possible to stimulate the secretory branch of the immune system without simultaneous stimulation of serum antibodies, or conversely to increase the serum response without affecting the level of secretory Ab, simply by adding the dietary molecule to those orally

added mucosal immunogens. Thus, simultaneous administration of large doses of vitamin B2 or melibiose and K99 leads to a two-fold and eight-fold increase in serum Ab, respectively, with a small accompanying increase in secretory Ab. Conversely, simultaneous administration of vitamin D in increasing doses leads to a fall in serum Ab and an increase in secretory Ab. Certain dietary molecules, on the other hand, also result in stimulation of both secretion and serum Ab titers, as shown by an eight-fold increase in serum Ab and a 1000-fold increase in S-IgA after simultaneous administration of vitamin C and 987P.

Experiments in which the mucosal immunogens were injected intramuscularly together with vitamins or sugars showed little, if any, effect on the immune response, thereby demonstrating that the change in response due to co-administration of these molecules and the mucosal immunogens must occur at or near the site of absorption of the molecules,

and not directly in the immune system (table 2.7).

Example 3

The above two examples showed that small doses of orally administered mucosal immunogens are capable of inducing significant serum IgG titers with or without a concomitant increase in secretory IgA antibody levels. Furthermore, it was shown that the immune response can be prolonged by the simultaneous administration of dietary molecules. The finding that the lectins used in the first study were capable of acting as carriers to elicit an anti-DNP response suggested the possibility that at least some of these mucosal immunogens act as "carriers" for other antigens, and therefore improves the relatively poor absorption of most antigens through the intestinal epithelium.

The following investigations were designed to investigate whether this carrier possibly exists and to determine some of the various parameters for its successful use as a means of designing new and more effective oral vaccines and/or orally administered drugs.

Materials and methods

Conjugation of antigens to the mucosal immunogens Dinitrophenylation of carriers

DNFB was reacted with lectins and carrier as described above (see general methods)

Conjugation of lipopolysaccharide (LPS) and polysaccharide (PS)

S. Typhimurium LPS and PS were purified as described previously (accompanying examples). LPS and PS were linked to MI using the periodate method (Avrameus and Ternynck, 1971).

Bonding with glutaraldehyde

Lutinizing hormone-releasing hormone (LHRH), bovine serum albumin (BSA) (from Sigma Chemical Co., St. Louis, Miss.), and S. typhimurium flagella were individually bound to MI using the two-step glutaraldehyde method of Ayrameus et al (1978). Briefly, the required protein was reacted with 0.2% glutaraldehyde for 2 hours at room temperature. The proteins were dialyzed overnight against carbonate/bicarbonate buffer, pH 9.5, followed by the addition of MI at molar ratios of 5, 10, 20 and 40:1, antigen:MI, as appropriate, and reacted for 24 hours at room temperature . Finally, ethaneclamin (Sigma) was added to a final concentration of 0.1 M (1 hour, room temperature), followed by dialysis overnight at 4°C against 0.1 M carbonate/bicarbonate buffer, pH 9.5.

Peroxidase-conjugated lectins

Commercial preparations of peroxidase conjugated to the lectins from Glycine max, Arachis hypogea, Tetragonolobus purpureas and conconavalin A were acquired from Sigma.

Chemical synthesis of LHRH conjugates

LHRH was conjugated to LTB using the glutaraldehyde procedure outlined above. Glutaraldehyde-activated LHRH was added to LTB at a ratio of 20:1, LHRH:LTB, and allowed to bind overnight at room temperature. The resulting conjugate was dialyzed thoroughly against 0.1 M carbonate/bicarbonate buffer, pH 9.5, before administration. Controls consisted of LHRH or LTB that had been treated with glutaraldehyde alone.

Genetic fusion of LHRH to [ 3-galactosidase and LTB

CONSTRUCTION OF A PLASMID VECTOR EXPRESSING A FUSION LTB/LHRH HYBRID POLYPEPTIDE

1• DNA fragment containing LTB coding sequences

A Hind III fragment was obtained from a pBR322-based plasmid containing the cloned LTB gene (Leong et al, 1985), from plasmid NP307 in E. coli strain RC411 (Dallus et al, 1970) which had been modified, by using a Spe I site near the stop codon for LTB, and then digestion with mung bean nuclease using standard conditions. (Unless otherwise noted, the conditions used are for standard recombinant DNA techniques and nucleic acid modifying enzymes, as described in Molecular Cloning, A Laboratory Manual, Maniatis, Fritsch and Sambrook, Cold Spring Harbor Laboratory, 1980). This eliminated the stop codon normally found after amino acid 123 in LTB, removed 9 base pairs of DNA and randomly generated a Hind II site as shown below:

This fragment was ligated into the vector pUC13 (Messing, 1983) after resolution with Hind III and phosphatase treatment of the plasmid using standard conditions. This served to position the remaining polylinker region from pUCl3, including a Pstl, SalI, XbaI, BamHI, SmaI, Sstl and EcoRi site downstream of the LTB sequence containing the DNA insert.

2. Preparation of synthetic LHRH-encoding oligonucleotides

Two oligonucleotides 30 bases in length, with sequences described in A and B below, were designed to form overlapping hybrid duplexes, as shown in C, resulting in a duplex that would encode straight-chain end-to-end repeats of the 10 amino acids that code for the peptide hormone LHRH (Schally and Coy, 1983, Role of Peptides and Proteins in Control of Reproduction, McCann and Dhindsa eds. Elsevier Science Publishing Co. Inc. pp. 89-110). In this sequence, glutaric acid replaces the normal N-terminal pyroglutamic acid.

The oligonucleotides were heat-treated together for 1 h at 40°C in 50 mM NaCl, 10 mM Tris pH 7.5, end-capped with Klenow, and then the mixture was ligated into Smal-cut M13 mpl8 using standard procedures. Ml3 phage containing inserts were isolated, and the DNA sequences of the inserts were determined by the dideoxy technique. A recombinant, designated P29, was selected for the fusion construct. Its DNA sequence together with the amino acids it encodes in the region of the insertion at the Smal site is reproduced below.

N-terminus in 3-galactosidase, α-fragment

DNA sequencing confirmed the introduction of DNA from the synthetic oligonucleotides to place a fusion of approximately 3 and a half repeats of the LHRH coding sequence (34 amino acids) in frame. The complete LHRH coding blocks are indicated by the arrows.

The copies of DNA from P29 were digested with EcoRI and HincII (sites indicated in the DNA sequence above), and end filled using standard conditions. The small fragment with 140 base pairs was isolated from a polyacrylamide gel.

3. Construction of the LTB/LHRH fusion vector

Plasmid pUC13 containing the LPB coding sequence inserted into the Hind III site (section 1 above) was resolved with SalI, end filled and phosphatase treated using standard conditions. Vector DBA was then ligated to the end-filled EcoRI/HincII fragment from P29 (section 2 above). The fusion should have the amino acid sequence below.

There is a stop codon 24 bases further from the LHRH sequence that determines the production of a 176 amino acid polypeptide, with 122 amino acids from the LTB, 34 amino acids encoding the LHRH repeat, and a further 20 amino acids derived from the associated regions.

The correct construct was sorted out by resolving DNA from minipreparations with EcoRI and selecting plasmids with the appropriate large EcoRI fragment. The presumed positive plasmids were further sorted with regard to expression of this polypeptide, driven from the lac promoter in pUC13. Bacterial extracts were analyzed on polyacrylamide gels followed by transfer to nitrocellulose paper and Western blotting with rabbit antisera directed against an LTB and LHRH conjugate. A peptide of the expected size was detected using both antisera.

4. Construction of expression plasmid K66 and expression strain BTA1185

A 573 bp EcoRI fragment from the pUC13 LTB-LHRH fusion plasmid described in 3, containing the LTB-LHRH fusion coding region in its entirety, was isolated from an agarose gel and ligated into the EcoRI-cut, phosphatase-treated expression vector pKK233-3 (from Pharmacia). The resulting expression plasmid PBTA K66 placed the expression of the fusion protein under the control of the tac promoter, where expression is induced with IPTG. The plasmid was transformed into E.coli host strain JM101 (SupE, thi, (lac-pro AB) [F<1>traD36, pro AB lacl^ Z M 15), thereby obtaining the host vector expression system BTA1185.

5. Production and purification of LTB/LHRH fusion protein for animal experiments

The LTB(LHRH). -producing strain was grown as described above. After induction with IPTG for 2 hours, bacteria were pelleted by centrifugation (3000 x g, 10 minutes at 4°C). Bacteria were then resuspended in 3-^ 2®°9lysed in a French press. After removal of the bacterial remains by centrifugation (18000 x g, 10 minutes at 4°C), the supernatant was loaded onto a column of agthio-galactose (Sigma). The fusion protein was then eluted with 0.5 M galactose and dialyzed against 0.1 M carbonate/bicarbonate buffer, pH 9.5.

Antigen administration and measurement of the immune response

All oral administration procedures, antibody collection and ELISA determinations were as previously described.

RESULTS

Demonstration of the carrier potential of the mucosal immunogens

All of the mucosal immunogens tested demonstrated the ability to efficiently transport the covalently bound hapten-DNP across the intestinal mucosa and to elicit a serum anti-DNP Ab response following administration of the dinitrophenylated MI. However, DNP-modified BSA was completely ineffective in eliciting an anti-DMP or anti-BSA response when added at the concentrations tested (Table 3.1). Initial experiments in which K99 and 987P were complexed with much larger molecules than DNP failed to elicit immune responses against either the mucosal immunogen or molecules linked to it (Tables 3.2 and 3.3), possibly due to steric effects of the binding of the pili of the mucosal epithelium. It was therefore decided to vary the ratio between antigen and MI. When different ratios of BSA:pili were tested, it was found that when ratios greater than 1:20, BSA:pili, were added, it was not possible to elicit either anti-BSA or anti-pili responses, even with a amount of 500 µg, which showed that it was not possible for the complexes to be effectively bound to the mucosal epithelium, and therefore to produce an immune response. However, when the ratios 1:20 or 1:40 were used, good responses were observed both to BSA and pili (tables 3.2 and 3.3). The magnitude of the immune response was easily varied by changing the amounts of added complex (table 3.4).

Oral administration of LHRH coupled to LTB resulted in a significant reduction in total uterine and ovarian weights in female mice receiving either 20 or 50 µg LHRH-LTB

(P 0.05), (Table 3.5, 3.6). No such weight loss was observed either with LHRH or LTB administered alone or together, or against intramuscular injection of LHRH-(3-galactosidase, LHRH-LTB or free LHRH). The effect of the weight loss was also observed developmentally as there was a complete absence of mature follicles in the ovaries, the animals were thus effectively “castrated.” There was a small reduction in reproductive tract weights when mice were fed the genetically engineered LTB-(LHRH)^ ^ fusion protein (Table 3.6), but in this experiment the reduction was not significant at the amounts that were tried.

Example 4

Induction of cell-mediated immunity after oral administration of antigen

Delivery of mucosal immunogens was shown to be effective in eliciting humoral responses, as measured by the production of serum and intestinal antibodies. However, it was not known whether a joint-induced stimulation of a cell-mediated immune response (CMI) against the mucosal immunogens occurred.

The following studies were designed to compare CMI generated by oral administration of a mucosal immunogen with that generated by classical subcutaneous (s.c.) injection of antigen in complete Freund's adjuvant (CFA).

Methods

Male C57B1/6J mice were immunized by infusing 20 µg antigen in 0.1 M carbonate/bicarbonate buffer, pH 9.65, or by injecting 20 µg antigen in CFA s.c. Controls received only buffer or excipient. 7 days after immunization, the mice were injected in the left hind foot pad with 10 µg antigen in 20 µl saline, and injected with 20 µl saline alone in the right hind foot pad. After an additional 24 hours, the difference in thickness between the left and right hindfoot pads was measured using a micrometer.

Results

The results shown in table 4.1 show that a good cellular immune response was produced after oral administration of either 987P or LTB mucosal immunogens. The response was actually surprisingly high as it was only slightly less than that produced by subcutaneous injection of these antigens into CFA.

INDUSTRIAL APPLICABILITY

Industrial application of the invention includes the production of oral medicaments for administration to vertebrate hosts.

Potential vaccine subjects for oral vaccine

Allergens: Various grass pollen: barley, ground Weed pollen: clover, haymole

Tree pollen: ash, cypress

Plant pollen: broom

Epithelial: cat hair, dog hair, pig bristles Miscellaneous: house dust, wheat bait, Kapok.

Hormones: LHRH, FSH, HGH, Inhibin

Vaccines: Hemagglutinins from

Influenza, measles, Rubella, smallpox, yellow fever, diphtheria, tetanus, cholera, plague, typhoid, BCG, Haemophilis influenzae, Neisseria catarrhalis, Klebsiella pneumonia,

Pneumococci, streptococci, especially S. mutans. Pili from: E. coli, N. gonorrheae, N. meningitis,

N. catarrhalis, Yersinia spp., Pseudomonas aeruginosa, Pseudomonas spp., Moraxella bovis, Bacterioides nodosus, Staph. spp., Strep. spp., Bordetella spp.?

LITERATURE REFERENCES

Avremeas, S., Ternynck, T.&Geudson, J.-L- 1978 Coupling of Enzymes to antibodies and antigens. Scand. J. Immunol., 8th suppl. 7, 7-23.

Avrameas, S. & Ternynck, T. 1971 Peroxidase labeled antibody and Fab conjugate with enhanced intracellular penetration. Immunochem., 8 1175-1179.

Befus, A.D., and J. Bienenstock, 1982. Factors involved in symbiosis and host resistance at the mucosa-parasite inter- 8IENENST0CK, J., and'A.0. Se<r>us. 1980. Mucosal Immunology. Immunology. 41 : 249.

8LA<E. M.S., and E.C Gotscnlich. 1984. Purification and partial characterization of the opac:ty-associated proteins of Neisseria gonorrhoeae. 159 : 452.

BLAND, P.W. & Britton, O.C. (1984) Morphological study of antigen-sampling structures in the rat large intestine. Infact. Immun., 43, 693-699.

CLARK, S., A. Cahill, C. Stirzaker, P. Greenwood and R. Gregson. 1985. Prevention by vaccination-animal bacteria, p. 481-487. In S. Tzipori (ed.) Infectious diarrhea in the young. Elsevier Science Publishers 8.V.

CONTEY, J.R., and L.R. Inman. 1981. Oiarrhea due to Escherichia coli strain RDEC-1 in the rabbit. The Peyer's patch as the initial site of attachment and colonization. J. Infect. Oops. 43 : 440.

DALLAS et. eel. (1979) Cistrons encoding Escherichia coli heat labile toxin J. Bact. 139 850-858.

AVREMEAS, S., Ternynck, T. 4 Geudson, J.-L. 1978 Coupling of enzymes to antibodies and antigens. Scand. J. Immunol., 8th suppl. 7, 7-23.:AVRAMEAS, S. i Ternynck.. T. 1971 Peroxidase labeled antibody and Fab conjugate with enhanced intracellular penetration. Immunochem., 8, 1175-1179.

BEFUS, A.D., and J. Bienenstock.. 1982. Factors involved in symbiosis and host resistance at the nucosa-parasite interface. Prog. Allergy. 31 : 76.

81ENENSTOCK, J., and A.D. 3 ephesus. 1980. Mucosal Immunology. Immunology. 41 : 249.

BLAKE. M.S., and E.C. Gotschnlich. 1984. Purification and partial characterization of the opacity-associated proteins of Neisseria gonorrhoeae. 159 : 452.

BLAND, P.W. & Britton, D.C. (1984) Morphological study of antigen-sampling structures in the rat large intestine. Infect. Immun., 43, 693-699.

CLARK, S., A. Cahill, C. Stirzaker, P. Greenwood and R. Gregson. 1985. Prevention by vaccination-animal bacteria, p. 481-487. In S. Tzipori (ed.) Infectious dlarrhoea in the young. Elsevier Science Publishers B.V.

CONTEY, J.R., and L.R. Inman. 1981. Diarrhea due to Escherichia coli strain RDEC-1 in the rabbit. The Peyer's patch as the initial site of attachment and colonization. J. Infect. D1s. 43 : 440.

DALLAS et. eel. (1979) Clstrons encoding Escherichia coli heat labile toxln J. Bact. 139 850-858.

ELSON, CO. & EALDING, W. (1984a) Generalized systemic and mucosal Immunity in mice after mucosal stimulation with cholera toxin. J. Immunol.. 132, 2736-2741.

ELSC, CO. & EALDING, ' A. (1984b) Cholera toxin did not induce oral tolerance in mice and abrogated oral tolerance to an unrelated antigen. J. Immunol. 133, 2892-2897.

EVANS, D.G. & Evans, D.J. (1978) New surfaca-associatad heat-1 aoi le colonization factor antigen (CFA/11) producad by enterotoxigeni; Escherichia coli of sercgroups 06 and 08. Inf. Immun., 21, 633-547.

De GRAAF, F.K., P. Klemm, and W. Gaastra. 1981. Purification, characterization and partial covalent structure of the adhesive antigen <99 of Escherichia coli. Infect. Immune. 33 : 377.

De GRAAF,<F.>K. & Roorda, I. (1882) Production, pur i f i eat i on, ane charac:er'zation of the f;, m b r i al adhesive antigen F41 isolatad~'om calf enterooathogenic EsCari chia coli strai.n 341M. Infect. Immun., 36 , 751-753.

FUJITA, (<. i Finkelstein, R.A. 1972 Antitoxic immunity in axper:-nentai Cholera. j. Infect. Dis., 125, 647-655.

FUSCO, ?., A. To, S. To, and C. Brinton, Jr. 1978. The purification and characterization of four types of E. Coli pili and the specificity of E. coli pili for immunity colonization and adhesion, p. 60-70. In C. Miller (ed.), XIIIth U.S. Japan Conference on Cholara, Atlanta, ' Ga., 1977. National institution of Health, 3ethesda, Md.

GAASTRA, 'A., and F.K. de Graaf. 1982. Host specific fimbrial adhesion of noninvasive enterotoxigenic Escherichia Coli strains< Microbiol. Fox. 46 : 129.

GAASTRA, R.A. (1975) An attempt to identify the intestinal receptor for the K88.ashes1n by means of a haemagglutination inhibition test using glycoproteins and fractions and sow colostrum. J.Gen. Microbiol., 86, 228-240.

GIBBONS, R.A. (1975) An attempt to identify the intestinal receptor for the K88 ashesin by means of a haemagglutination inhibition test using glycoproteins and fractions from sow colostrum. J.Gen. Microbiol., 86, 228-240.

HANSON, D.G.. VA7., N.M. , MAYA,. L.C.S. 4 LYNCH, J.M..U 979) Inhibition of specific Immune responses by feeding protein antigens. J. Immunol., 123, 2337-2343.

HERBERT, D., P.J. Phipps, and R.E. Strange. 1985. Carbohydrate analyses, determination of total carbohydrate, p. 265-279. In J.R. Norris, and D.W. Ribben (ed.), Methods in Microbiology. v. 5B. ISAACSON, R.E., J. Colmenero, and P. Richter. 1981. Escherichia coli K99 pili are composed of one subunit spee i es. FEMS Microbiol. Easy. 12 : 229.

ISAACSON, R.E., and P. Richter. 1981. Escherichia Coli 987P Pi lice. Pur i f ication-and partial characterization. J. Bacteriol. 146 : 784.

KLIPSTEIN, F.A., ENGERT, R.F., & SHORT, H.3. (1980) Protective effect of immunization with heat-labile antertoxin in gnotobiotic rats monocentamintaed with enterotoxigenic Escnerichia coli. Inf. Immunol., 28, 163-170.

LAEMMLI, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227 : 530.

LEONG et. eel. (1985) Nucleotide sequence comparison between heat labile toxin B-subunit cistrons from Escherichia coli of human and porcine origin Infection and Immunity 48 73-77.

LEVINE, M.M., Rennels, M.3., Oaya, V. 4 Hughes, T.P. (1980) Haemagglutination and colonizatlon factors in enterotoxigenic and enteropathogenic Escherichia coli that cause diarrhea. J. Inf. Dis., 141, 733-737.

LITTLE, J.R. & Eisen, H.N. (1967) Preparation of immunogenlc 2,4-dlnitrophenyl and 2,4,6-tri itrophenyl proteins. In "Methods in Immunology and Immunochemistry" (Ed. Williams, CA. & Chase, M.W.). I, p. 128. Academic Press, New York.

MESSIAH, 3rd, and'CP. Laugh Blonde (1960). Ce 11 proliferation and migration as revealed by radioautography after injection of thymidine

-H3 into male rats and mice. An. J. Anat. 106 : 247.

MESSING (1983) New M13 Vectors for donating Methods in Enzymologv 101 20-78

MORGAN, R.-., Isaacson, R.E., Moon, H.W., Br in ton, CC. & Two, C-C

(1978) Immunization of suckling pigs against enterotoxigenic Escherichia coli-induced diarrheal disease by vaccinating dams -»ith purified 937 or ;<99 pili. Inf. Immun., 22, 771-777.

MORRIS, J.A., C.J. Thorns, and W.J. Sojka. 1980. Evidence for :wo adhesive a.ntigens cn the <99 reference strain Escherichia coli 5-1.

J.Gen. Microbiol. 113 : 107.

MOWAT, A. Mc I., (1985) The role of antigen recognition and suppressor cells in mice with oral tolerance to ovalbumin. Immun., 56, 252-250.

MOWAT, A. Mc!. & PARROT, D.M.V.U 983) Immuno 1 cg i ca 1 responses to -"ed protein an:<;>gens in mice. Immun., 50, 547-554.

NGAN, J. 4 <IND, L.S.,<1978) Suppressor T cells for IgE and IgG in peyer's patches of mice tolerant by the oral administration of ovalbumin. J. Immunol., 120, 861-865.

PIERCE, N.F. & KOSTER, F.T./(1980) Priming and suppression of the intestinal immune response to cholera toxoid/toxin by parenteral toxoid in rats. J. Immunol., 124, 307-311.

PIERCE, N.F., SACK, R.B., & SIRCAR, B.K., (1977) Immunity to experimental cholera. J. Inf. Dis., 135, 888-896.

RICHMAN, L.K., CHILLER, J.M., BROWN, W.R., HANSON, D.G. & NELSON, N.M.

(1978) Enterlcally induced immunology cal tolerance. J. Immunol., 121, 2429-2434.

RICHMAN, L.K., A.S. Graeff, and W. Strober. 1981 Antigen presentation by macrophage enriched cells from the mouse Peyer's patch. Cell Immunol. 62 : 1100.

ROTHBERG R.M., KRAFT, S.C, £ MICHALEK, S.M., (1973) Systemic immunity after local antigenic stimulation of the lymphoid

tissue of the gastrointestinal tract. J. Immunol., 111, 1906-1913.

RUSSELL-JONES, G.J., E.C. Gotschnlich, and M.S. Blake. 198*. A surface receptor specific for human IgA on group B streptococci possessing the Ibc protein antigen. J. Exp. Med. 160 : 1467.

RUSSELL-JONES, G.J., and E.C. Gotschlich. 1984. Identification of protein antigens of group B streptococci, with special reference to the Ibc antigen. J. Exp. Med. 160 1476.

SALIT, I.E., M. Blake, and E.C. Gotschlich. 1980. Intrastrain heterogeneity of gonococcal pili is related to capacity colony variance. J. Exp. Med. 151 : 716.

SCHALLY AND COY (1983). Stimulatory and inhibitory analogues of LH-releasing hormone: 3asic and clinical studies in Role of Peorides and Proteins in Control of Reproduction McCann and Dhindsa eds. Elsevie Science Publishing Co. Inc. ppS9-110. Maniatis, Fritsch and Sambrook (1982) Molecular Cloning, Laboratory Manual. Cold Spring Harbor Laboratory.

SEVENNERHOLM, A.M. & Holmgren, J. 1978 Inditi fication of E. coli heat-labile enterotoxin by means of a gang 1 in os in the immunosorbent assay CGM-1 ELISA) procedure. Curr. Microbiol. 1, 19-23.

TOMASI, T.B., (1980) Oral tolerance. Transplantation, 29, 353.

TONER, P.G., K.E. Carr, and G.M. Wyburn (1971). Intestines. In The Digestive System - An Ultrastructural Atlas and Review, Butterworths, London.

TSAI, C.M., and Frasen, CE. 1982. A sensitive Silver stain for detecting 1 ipopofysaccharide in polyacrylamide gels. An 1 al. Biochem., 119, 115-119.

175

Claims (63)

1. Complex, characterized in that it comprises an immunogen bound to a carrier molecule capable of specifically reacting with the mucosal epithelium of a vertebrate host, where both the immunological activity of the immunogen and the ability of the carrier molecule to specifically react with the mucosal epithelium of the vertebrate host are essentially maintained, and the complex is capable of eliciting a systemic, cellular and/or mucosal immune response in the vertebrate host. 2. Complex according to claim 1, characterized in that the immunogen is selected from all, part(s), an analog, homolog, a derivative or a combination thereof of a hormone, therapeutic agent, antigen or hapten. 3. Complex according to claim 1, characterized in that the complex is capable of producing a systemic or cellular immune response in the vertebrate host. 4. Complex according to claim 1, characterized in that the complex is capable of producing a mucosal immune response in the vertebrate host. 5. Complex according to one of claims 1-4, characterized in that the immunogen is all, part, an analog, homolog, a derivative or combination thereof of an antigen or hapten. 6. Complex according to one of claims 1-5, characterized in that the immunogen is all, part, an analog, homolog, a derivative or a combination thereof of a hormone. 7. Complex according to one of claims 1-6, characterized in that the immunogen is all, part, an analog, homolog, a derivative or a combination thereof of a hormone that releases luteinizing hormone. 8. Complex according to one of claims 1-6, characterized in that the immunogen is FSH, HGH, inhibin. 9. Complex according to one of claims 1-5, characterized in that the immunogen is an allergen gene. 10. Complex according to claim 9, characterized in that the allergen is grass pollen, weed pollen, tree pollen, plant pollen, cat hair, dog hair, pig bristles or other epithelium or house dust, wheat bait or Kapok. 11. Complex according to one of claims 1-4, characterized in that the immunogen is a surface protein derived from influenza, measles, rubella, smallpox, yellow fever, diphtheria, tetanus, cholera, plague, typhus - or BCG-causing agents, Haemophilus influenzae, Neisseria catarrhalis, Klebsiella pneumoniae, pneumococci and streptococci; or pilus derived from E. coli, N. gonorrhoeae, N. meningitidis, N. catarrhalis, Yersinia spp, Pseudomonas aeruginosa, Pseudomonas spp, Moraxella bovis, Bacteroides nodosus, Staphylococci spp, Streptococci spp and Bordetella spp. 12. Complex according to one of claims 1-4, characterized in that the immunogen is a surface polysaccharide derived from diphtheria, tetanus, cholera, plague, typhoid or BCG-causing agents, Haemophilus influenzae, Neisseria catarrhalis, Klebsiella pneumoniae , pneumococci and streptococci or a pilus derived from E. coli, N. gonorrhoeae, N. meningitidis, N. catarrhalis, Yersinia spp, Pseudomonas aeruginosa, Pseudomonas spp, Moraxella bovis, Bacteroides nodosus, S taphylococci spp, Strepto cocci spp and Bordetella spp . 13. Complex according to one of claims 1-4, characterized in that the immunogen is a secretion product derived from diphtheria, tetanus, cholera, plague, typhoid or BCG-causing agents, Haemophilus influenzae, Neisseria catarrhalis, Klebsiella pneumoniae , pneumococci and streptococci or a pilus derived from E. coli, N. gonorrhoeae, N. meningitidis, N. catarrhalis, Yersinia spp, Pseudomonas aeruginosa, Pseudomonas spp, Moraxella bovis, Bacteroides nodosus, Staphylococci spp, Streptococci spp and Bordetella spp. 14. Complex according to one of claims 11 - 13, characterized in that the surface protein, -the polysaccharide or secretion product is derived from Streptococcus mutans. 15. Complex according to one of claims 1-14, characterized in that the carrier molecule is all, part, an analog, homolog, a derivative or a combination thereof of a bacterial adhesin, virus hemagglutinin, a toxin or the binding subunit thereof, or a lectin either of plant origin or other origin. 16. Complex according to claim 15, characterized in that the carrier molecule is all, part, an analog, homolog, a derivative or a combination thereof of the heat-labile toxin from enterotoxigenic E. coli. 17. Complex according to claim 15, characterized in that the carrier molecule is all, part, an analog, homolog, a derivative or a combination thereof of a bacterial pilus. 18. Complex according to claim 17, characterized in that the bacterial pilus is K99 or 987P pilus from E. coli. 19. Complex according to claim 17, characterized in that the bacterial pilus is CFAI, CFAII, K88 or F41. 20. Complex according to claim 15, characterized in that the carrier molecule is all, part, an analog, homolog, a derivative or a combination thereof of a lectin. 21. Complex according to claim 20, characterized in that the lectin is Conconavalin A, kermesberry mitogen or the lectin from Lens culinaris, Helix pomatia, Glycine max, Arachis hypogea or Ulex europeus. 22. Complex according to claim 20, characterized in that the lectin is a lectin from abrin, veil asparagus, vetch, camel's foot tree, castor bean, fava bean, green seaweed, plumeria, horse gram, autumn shoe crab, hunting bean, Japanese wisteria, paternoster, Scottish golden rain, lima bean, limulin, lotus, European mistletoe, mung bean , yellow tree, pogada tree, garden pea, potato, red bean, red seaweed, Siberian pea bush, edible snail flower, garden snail flower, common bone wood, sweetened, tomato, wheat germ or asparagus. 23. Complex according to claim 15, characterized in that the carrier molecule is all, part, an analog, homolog, a derivative or combination thereof of a viral hemagglutinin. 24. Complex according to claim 23, characterized in that the viral hemagglutinin is a hemagglutinin derived from influenza, measles, rubella, smallpox or yellow fever viruses. 25. Complex according to one of claims 1-4, characterized in that the immunogen is all, part, an analog, homolog, derivative, or combination thereof of LHRH, and the carrier molecule is all, part, an analog, homolog, a derivative or a combination thereof of LTB. 26. Method for producing a complex according to one of claims 1-25, characterized by: (a) the immunogen reacts with the carrier molecule so that the complex is formed, (b) the immunogen is chemically modified so that at least one functional group capable of forming a chemical bond is obtained, and the immunogen and the carrier molecule are reacted so that the complex is formed, or (c) the carrier molecule is chemically modified so that at least one functional group capable of forming a chemical bond is obtained, and the immunogen and the carrier molecule are reacted so that the complex is formed, (d) the immunogen and the carrier molecule are chemically modified so that functional groups capable of forming a chemical bond are obtained, and the immunogen and the carrier molecule are reacted so that the complex is formed, (e) the immunogen is reacted with at least one binding agent, and the immunogen and the carrier molecule are reacted so that the complex is formed, (f) the carrier molecule is reacted with at least one binding agent, and the immunogen and the carrier molecule are reacted so that the complex is formed, (g) the immunogen and the carrier molecule are reacted with at least one binding agent, and the immunogen and the carrier molecule are reacted so that the complex is formed, or (h) a combination of any of the above method steps is performed. 27. Method for producing a complex according to one of claims 1 - 25, characterized in that a recombinant DNA molecule is provided which comprises a first DNA sequence which after expression codes for the amino acid sequence of the immuno gene, a second DNA sequence which after expression codes for the amino acid sequence of the carrier molecule, and vector DNA, a host is transformed with the recombinant DNA molecule so that the host is able to express a hybrid protein product comprising the complex, the host is cultured so that expression is achieved and the hybrid protein product is recovered. 28. Method for producing a complex according to one of claims 1 - 25, characterized by : (a) the immunogen and/or carrier molecule is chemically synthesized and in the complex is formed by means of reactions according to the method according to claim 26, or (b) a hybrid peptide comprising the amino acid sequences of the immunogen and the carrier molecule is synthesized. 29. Method according to claim 28, characterized in that the peptide is produced using peptide synthesis in solid phase, enzymatic or manual peptide synthesis. 30. Method according to claim 29, characterized in that the peptide is produced by peptide synthesis in solid phase. 31. Method according to claim 30, characterized in that the synthesized immunogen or carrier molecule, while bound to the resin in the solid-phase peptide synthesizer, is connected to the carrier molecule or the immunogen, respectively. 32. Method according to one of claims 28 - 31, characterized in that the synthetic peptide is all, part, an analog, homolog, a derivative or a combination thereof of LHR. 33. Method according to claim 26, characterized in that the carrier molecule is LTB and the binding agent is glutaraldehyde. 34. Complex according to one of claims 1-25, characterized in that it is produced using a method according to one of claims 26-31. 35. Expression product from a transformant host, characterized in that it comprises a complex according to one of claims 1-25. 36. Transformant host, characterized in that it is transformed with a recombinant DNA molecule comprising a first DNA sequence which, upon expression, codes for the amino acid sequences of all, part, an analog, homolog, a derivative or a combination thereof, of the immunogen, a second DNA sequence which, upon expression, encodes the amino acid sequence of all, part, an analog, homolog, derivative, or combination thereof, of the carrier molecule, and vector DNA. 37. Transformant host according to claim 30, characterized in that the host is a gram negative bacterium, a gram positive bacterium, a yeast, a fungus or a higher eukaryotic cell. 38. Transformant host according to claim 36 or 37, characterized in that the host is E. coli. 39. Culture, characterized in that it consists of the transforming microorganism designated ATCC 67114. 40. Recombinant DNA molecule, characterized in that it comprises a first DNA sequence which upon expression codes for the amino acid sequence of the immunogen, a second DNA sequence which upon expression codes for the amino acid sequence of the carrier molecule, and vector DNA. 41. Recombinant DNA molecule according to claim 40, characterized in that the vector DNA is plasmid DNA. 42. Plasmid, characterized in that it is pBTAK6 6. 43. Recombinant DNA molecule according to claim 40, characterized in that the vector DNA is virus, bacteriophage or cosmid DNA. 44. Polynucleotide sequence, characterized in that it comprises a first hybrid polynucleotide sequence that acts as a coding sequence for a fusion product comprising an amino acid sequence from an immunogen fused with an amino acid sequence from a carrier molecule, a polynucleotide sequence that is sufficiently related to the first hybrid nucleotide sequence to be able to hybridize to the first hybrid polynucleotide sequence , a polynucleotide sequence related by mutation, comprising single or multiple base substitutions, deletions, insertions and substitutions, to the first hybrid polynucleotide sequence or hybridizing sequence, or a polynucleotide sequence which, upon expression, codes for a polypeptide that provides similar biological or immunological activity as the fusion product. 45. Polynucleotide sequence according to claim 44, characterized in that the first hybrid polynucleotide sequence acts as a coding sequence for the amino acid sequence in its entirety, part, an analog, homolog, a derivative or a combination thereof, of an antigen or hapten fused with the amino acid sequence of a carrier molecule. 46. Polynucleotide sequence according to claim 44, characterized in that the first hybrid polynucleotide sequence acts as a coding sequence for the amino acid sequence of all, part, an analog, homolog, a derivative or a combination thereof, of LHRH fused with the amino acid sequence of a carrier molecule. 47. Medication, characterized in that it comprises a complex according to one of claims 1-25 together with a pharmaceutically acceptable carrier or diluent. 48. Medicine according to claim 47, characterized in that it is adapted for oral administration. 49. Medicine according to claim 47, characterized in that it is adapted for nasal administration. 50. Medicine according to one of claims 47 - 49, characterized in that the medicine is available in capsule, tablet, slow-release, elixir, gel, paste or nasal spray form. 51. Medicine according to one of claims 47 - 50, characterized in that it additionally comprises a dietary molecule which can selectively modulate the size and/or type of immune response against the immunogen in the medicine. 52. Medicine according to claim 51, characterized in that the dietary molecule is selected from amino acids, vitamins, monosaccharides and oligosaccharides. 53. Medicine according to claim 51 or claim 52, characterized in that the dietary molecule is selected from vitamin A, vitamin B^, vitamin B^, vitamin B^, vitamin B12, vitamin C, vitamin D, vitamin E, fructose, lactose, mannose, melibiose, sorbitol and xylose. 54. Method for supplying a complex according to one of claims 1-25 to a vertebrate host, characterized in that it comprises mucosal administration of a drug according to one of claims 4 7 - 51. 55. Method for supplying a complex according to one of claims 1-25 to a vertebrate host, characterized in that it comprises oral administration of a drug according to one of claims 47, 48, 50" and 51. 56. Procedure for supplying a komlex according to et of claims 1-25 to a vertebrate host, characterized in that it comprises nasal administration of a drug according to one of claims 47, 49, 50 or 51. 57. Method for inhibiting gonadal function in a mammal, characterized in that it comprises mucosal administration of a drug according to one of claims 47 - 51. 58. Method for selectively modulating the size and/or type of immune response against the immunogen in a complex according to one of claims 1-25, characterized in that it comprises mucosal administration of a drug according to claim 51 to a vertebrate host. 59. Method for selective modulation of cellular immune responses against a complex according to one of claims 1 - 25, characterized in that it comprises mucosal administration of a drug according to claim 45 in such a way that the cellular immune response against the complex in the vertebrate host specifically increases or decreases . 60. Method according to claim 58 or claim 59, characterized in that it comprises the administration of a drug according to one of claims 47 - 50 and a dietary molecule. 61. Complex, characterized in that it is mainly as described above with reference to the examples. 62. Method for inducing or modulating an immune response in a host, characterized in that it is mainly as described above with reference to the examples. 63. Carrier molecule, characterized in that it is mainly as described above with reference to the figure.