United States Patent:  6,103,243

Assignee:  Biotechnology Australia PTY, LTD (New South Wales, AU)

Filed:  June 2, 1995

The present invention relates to the specific stimulation of serum and secretory antibodies through mucosal presentation of antigens.

DISCLOSURE OF INVENTION

In a first form the invention provides a complex comprising: an immunogen; linked to a carrier molecule which is capable of specifically interacting with the mucosal epithelium of a vertebrate host; wherein both the immunological activity of the immunogen and the capacity of the carrier molecule to specifically interact with the mucosal epithelium of the vertebrate host is substantially maintained, and said 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, part, analogues, homologues, derivatives or combinations thereof and a hormone, thereapeutic agent, antigen or hapten. These immunogens include hormones such as LHRH (luteinising hormone releasing hormone) FSH, HGH and Inhibin; allergens such as grass pollens (for instance barley and couch), weed pollens (eg. clover, dock), tree pollens (eg. ash, cyprus), plant pollens (eg. brcom), epithelia (eg. cat hair, dog hair, pig hair) and house dust, wheat chaff and Kacok; immunogens for vaccines against agents such as influenza, measles, Rubella, smallpox, yellow fever, diphtheria, tetanus, cholera, plague, typhus, BCG, haemophilus influenze, Neisseria catarrhalis, Kelbsiella pneumonia, pneumococci and streptococci especially S. mutans; and pili including pili derived from E. Coli, N. gonorrhoeae, N. meningitis, N. catarrhalis, Yersinia, 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 haemagglutinins such as from influenza, measles, Rubella, smallpox or yellow fever viruses; toxins or binding subunits thereof such as LTB ricin, abrin, diphtheria toxin, modecin, tatanus toxcin and others of similar structures; and lectins whether from plant or other origin. Lectins include for example conconavalin A, Pokeweed mitogen or lectins from Lens culinaris, Helix pomatia, Glycine max, Arachis hypogea, or Ulex europeus or Abrin, Asparagus pea, Broad bean, Camel's foot tree, Castor bean, Fava bean, Green marine algae, Hairy vetch, Horse gram, Horse shoe crab, Jack bean, Japanese wisteria, Jequirity, Scotch laburnum, Lima bean, Limulin, Lotus, European mistletoe, Mung bean, Osage orange, Pagoda tree, Garden pea, Potato, Red kidney bean, Red marine algea, Siberian pea tree, edible snail, garden snail, Spindle tree, Sweet pea, Tomato, wheat germ or winged pea.

In a preferred embodiment of the invention there is provided a complex which comprises luteinising hormone releasing hormone and LTB.

In another form the present invention provides a process for the production of a complex as described above which process comprises;

(a) reacting the immunogen with the carrier molecule to form said complex;

(b) chemically modifying the immunogen to provide at least one functional group capable of forming a chemical linkage, and reacting the immunogen and carrier molecule to form said complex; or

(c) chemically modifying the carrier molecule to provide at least one functional group capable of forming a chemical linkage, and reacting the immunogen and carrier molecule to form said complex;

(d) chemically modifying the immunogen and the carrier molecule to provide functional groups capable of forming a chemical linkage, and reacting the immunogen and carrier molecule to form said complex;

(e) reacting the immunogen with at least one liking agent, and reacting the immunogen and the carrier molecule to form said complex;

(f) reacting the carrier molecule with at least one linking agent and reacting the immunogen and the carrier molecule to form said complex;

(g) reacting the immunogen and the carrier molecule with at least one linking agent, and reacting the immunogen and the carrier molecule to form said complex; or

(h) a combination of any of the preceding process steps.

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

Alternatively the invention provides a process for the production of a complex which process comprises

(a) chemically synthesising the immunogen and/or the carrier molecule, and forming the complex by chemical reaction; or

(b) synthesising a hybrid peptide comprising amino acid sequences of the immunogen and the carrier molecule. Preferably the peptide is prepared by solid phase, enzymatic or manual peptide synthesis.

In a preferred from of the invention the synthesised immunogen or carrier molecule whilst bound to the resin of the solid phase peptide synthesiser is coupled to the carrier molecule or immunogen respectively.

In another form of the present invention there is provided a transformant host transformed with a recombinant DNA molecule comprising a first DNA sequence which on expression codes for the amino acid sequences of all, part, an analogue, homologue, derivative or combination thereof, of the immunogen, a second DNA sequence which on expression codes for the amino acid sequence of all; part, an analogue, homolgue, derivative or combination thereof of the carrier molecule, and vector DNA.

In a preferred form of the invention the transformant host is a Gram negative or Gram positive baceterium, a yeast, fungus or a higher eukaryotic cell. In one preferred form of the invention, said host is E. coli. A culture of a transformant microorganism falling within the scope of the present invention had been deposited with the American type culture collection and has been designated the number:

In further form the invention provides a recombinant DNA molecule comprising a first DNA sequence which on expression codes for the amino acid sequence of the immunogen, a second DNA sequence which on 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 alternative vectors are envisaged within the scope of the present invention and these include viruses, bacteriophages and cosmids. In one preferred form of the invention there is provided plasmid pBTAK66 which when a host cell is transformed with said plasmid will yield a proteinceous product which includes a polypeptide falling with the scope of the present invention.

In a further form of the invention there is provided a polynucleotide sequence which comprises a first hybrid polynucleotide sequence which acts as a coding sequence for a fusion product comprising an amino acid sequence of an immunogen fused to an amino acid sequence of a carrier molecule, a polynucleotide sequence sufficiently related to said first hybrid polynucleotide sequence so as to hybridize to said first hybrid polynucleotide sequence, a polynucleotide sequence related by mutation, including single or multiple base substitutions, deletions, insertions and inversions, to said first hybrid polynucleotide sequence or hybridizing sequence or a polynucleotide sequence which on expression codes for a polypeptide which deplays similar biological or immunological activity to said fusion product.

Preferably the polynucleotide sequence is one wherein the first hybrid polynucleotide sequence acts as a coding sequence for the amino acid sequence of all, part, an analogue, homologue, derivative or combination thereof of LHRH fused to the amino acid sequence of a carrier molecule, more preferably LTB.

In a further form of the invention there is provided a medicament which comprises a complex according to the invention together with a pharmaceutically acceptable carrier or diluent. Examples of pharmaceutically acceptable carriers and diluents include typical carriers and diluents such as tablets, aquecs solutions, sodium bicarbonate solutions and similar diluents which neutralise stomach acid or have similar buffering capacity, glycols, oils, oil-in-water or water-in-oil emulsions, and include medicaments in the form of emulsions, gels, pastes and viscous colloidal dispersions. The medicament may be presented in capsule, tablet, slow release or elixir form or as a gel or paste or may be presented as a nasal spray and in this form may be in the presence of an aerosol. Furthermore, the medicament may be provided as a live stock feed or as food suitable for human consumption.

The present inventors have also found that co-administration of certain dietary molecules with a complex of the present invention can selectively modulate the magnitude and/or type of the immune response to the immunogen of the complex.

Accordingly the present invention further provides a medicament which comprises the complex of the present invention together with a dietary molecule which dietary molecule can selectively modulate the magnitude and/or type of the immune response to the immunogen of the complex.

The dietary molecule envisaged by the present invention include basic, neutral and acidic amino acids, such as argenine, 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 thiamine, riboflavin, pyridoxal, cyanocobalamin (V.B₁₂) ascorbic acid (V.C). Vit D₂, etc--Ergosterol, Vit.E, Vit.A, Vit K etc; sugars including monosaccharides e.g. galactose, mannose, mannitol, sorbitol, glucose, xylose, Allose, altrose, arabinose, digitoxose, arythrose, fructose, lyxose, muramic acid, mannose, pyruvic acid, ribose, tagatose, talose and the amidatec and N acetylated, derivatives thereof; oligosaccharides e.g. lactose, maltose, melibiose, sucrose, cellubiose, N,N diacetyl chitoblose, 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 of presenting a complex of the present invention which method comprises the mucosal administration of a complex of the present invention together with a dietary molecule capable of modulating the magnitude and/or type of immune response of the immunogen.

The invention also provides the oral administration of the medicaments of the invention, in order to elicit a response to the active molecule in the host. Such a response, in the case where the active molecule is an antigen or hapten may be a systemic and/or a mucosal immune response. In the case where the active molecule is LHRH or a derivative, analogue, homologue, part or combination thereof, of LHRH, the response will be inhibition of gonadal function in the host. Where the oral medicament incorporates a dietary molecule according to the invention, the invention provides a method for enhancing the host's response to the active molecule which comprises administering such an oral medicament to the host.

Claim 1 of 11 Claims

1. A protein conjugate comprising an immunogen covalently linked to a carrier molecule,

wherein said carrier molecule is capable of specifically interacting with the mucosal epithelium of a vertebrate host, and

wherein said protein conjugate retains both the ability of the immunogen to elicit an immune response in said vertebrate host and the capacity of the carrier molecule to specifically interact with the mucosal epithelium of said host, and wherein said protein conjugate elicits a serum and secretory immune response in said host following oral administration, without non-oral administration in Freund's adjuvant.

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