What is claimed is:
1. A composition comprising a LT-ACT-B hybrid enterotoxin holotoxin, which holotoxin has immunologic adjuvant activity and is substantially less toxic than native E. coli heat-labile enterotoxin holotoxin as measured in the patent mouse assay.
2. The composition of claim 1 in which the holotoxin is recombinantly produced.
3. The composition of claim 1 in which the LT-A subunit of the LT-ACT-B hybrid enterotoxin holotoxin is a mutant LT-A.
4. The composition of claim 3 in which the mutant LT-A, if it were associated with LT-B would form a mutant holotoxin that is substantially less toxic than native heat-labile enterotoxin holotoxin as measured in the patent mouse assay.
5. A preparation comprising an antigen in combination with the composition according to claim 1.
6. The preparation according to claim 5, in which the antigen is selected from the group of antigens consisting of bacterial, fungal, protozoal, viral, helmenthal and other microbial pathogenic antigens.
7. The preparation according to claim 6, in which the antigen is selected from the group consisting of Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp., Helicobacter pylori, Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum, Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, variola virus, vaccinia virus, cowpox virus, varicella-zoster virus, Herpes Simplex virus 1, Herpes Simplex virus 2, influenza viruses, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, and Non-ANon-B Hepatitis virus antigens.
8. A composition useful in producing an immune response to a pathogen in a host comprising an admixture of an effective amount of an antigen and an adjuvant effective amount of the composition according to claim 1.
9. A kit useful in producing an immune response in a host to a pathogen comprising two components: (a) an effective amount of antigen and (b) an adjuvant effective amount of an LT-ACT-B hybrid enterotoxin holotoxin, which holotoxin has immunologic adjuvant activity and is substantially less toxic than native E. coli heat-labile enterotoxin holotoxin as measured in the patent mouse assay.
10. A method of creating or sustaining an immune response to an antigen in a host comprising administering an admixture of an effective amount of an antigen and an adjuvant effective amount of an LT-ACT-B hybrid enterotoxin holotoxin, which holotoxin has immunologic adjuvant activity and is substantially less toxic than native E. coli heat-labile enterotoxin holotoxin as measured in the patent mouse assay, in an orally acceptable pharmaceutical carrier.
11. The method of claim 10 where a serum response is produced.
12. The method of claim 10 where a cell-mediated immune response is produced.
13. The method of claim 10 where a mucosal response is produced.
14. The method of claim 10 further comprising administering a subsequent boost of the antigen.
15. The method of claim 10 wherein the antigen is derived from the group consisting of bacterial, viral, protozoal, fungal, helminthal, and other microbial pathogens.
16. The method of claim 10 wherein the administration is mucosal administration.
17. A method of inducing an immune response to an antigen in a host comprising administering an effective amount of an antigen in conjunction with an adjuvant effective amount of an LT-ACT-B hybrid enterotoxin holotoxin, which holotoxin has immunologic adjuvant activity and is substantially less toxic than native E. coli heat-labile enterotoxin holotoxin as measured in the patent mouse assay.
18. The method of claim 16 in which the antigen and the holotoxin are administered simultaneously.
19. The method of claim 16 in which the antigen and holotoxin are administered separately within a short time of each other.
20. A method of inducing an immune response against an enterotoxic bacterial organism comprising administering an adjuvant effective amount of an LT-ACT-B hybrid enterotoxin holotoxin, which holotoxin has immunologic adjuvant activity and is substantially less toxic than native E. coli heat-labile enterotoxin holotoxin as measured in the patent mouse assay, as a component of a vaccine directed against the enterotoxic bacterial organism.
21. The method of claim 19 wherein the enterotoxic bacterial organism is selected from the group consisting of enterotoxic bacterial organisms which express a cholera-like toxin.
22. The method of claim 19 wherein the enterotoxic bacterial organism is selected from the group consisting of Escherichia spp. and Vibrio spp.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A method for preparing biomass-based polymer emulsion, the method comprising:
1)
a) based on mole mass ratio, vacuum dehydrating one part of cardanol for between 1 and 3 hours to be anhydrous, dissolving the cardanol in between 10 and 100 parts of a solvent a, followed by addition of between 0.8 and 1.2 part of a sulphonating agent and between 0.8 and 1.2 part of a strong base;
b) allowing a resulting mixture to react at a temperature of between 70 and 150\xb0 C. for between 2 and 6 hours, cooling the mixture to room temperature, adjusting a pH value thereof using chlorhydric acid to between 1 and 5, allowing the mixture to react at a temperature of between 70 and 150\xb0 C. for between 1 and 3 hours, cooling to room temperature, allowing to stand overnight, filtering, washing using between 10 and 100 parts of the solvent a at a temperature of between 0 and 5\xb0 C. to yield a white solid;
c) drying and dissolving the white solid in between 10 and 100 parts of an anhydrous solvent b, followed by addition of between 0.8 and 1.5 part of a modifier and between 0.8 and 1.5 part of triethylamine, allowing a resulting solution to react at a temperature of between 0 and 5\xb0 C. for between 0.5 and 24 hours, to yield a cardanol-based polymerizable emulsifier;
2)
d) based on mole mass ratio, providing one part of dried castor oil with a temperature of between 0 and 20\xb0 C., adding dropwise to the castor oil 10-30 wt. % of acetone dissolved maleic anhydride in 5-30 min, heating a resulting mixture to a temperature of between 25 and 50\xb0 C. and allowing to react for between 0.5 and 24 hours;
e) continuing heating the mixture to a temperature of between 60 and 80\xb0 C. and allowing to react for between 4 and 5 hours, removing a solvent at a temperature of between 30 and 40\xb0 C. under vacuum, cooling the mixture to room temperature, to yield a red-orange maleic anhydride modified castor oil;
f) heating the maleic anhydride modified castor oil to 60\xb0 C., followed by addition dropwise of between 1.0 and 2.5 parts of an isocyanate, between 0.1 and 1 part of a catalyst, and between 0.1 and 1 part of a chain extendor, allowing to react for between 1 and 6 hours, to yield a viscous polymerizable castor oil-based polyurethane prepolymer;
3)
g) based on mole mass ratio, mixing one part of the castor oil-based polyurethane prepolymer obtained in 2), between 0.1 and 0.5 part of the cardanol-based polymerizable emulsifier obtained in 1), between 0.5 and 2 parts of an acrylate monomer, and between 5 and 50 parts of water;
h) allowing a resulting solution to swell at a temperature of between 50 and 60\xb0 C. for between 0.5 and 2 hours, heating the solution to a temperature of between 70 and 90\xb0 C., adding dropwise to the solution an aqueous solution comprising between 0.001 and 0.1 part of an initiator, and allowing the solution to react for between 2 and 4 hours, to yield biomass-based polymer emulsion;
wherein
the strong base is potassium hydroxide andor sodium hydroxide;
the solvent a is water, alcohol, methanol, tetrahydrofuran, acetone, dioxane, acetonitrile, dimethylformamide, dimethyl sulfoxide, or a mixture thereof;
the solvent b is dichloromethane, tetrahydrofuran, acetonitrile, or a mixture thereof;
the modifier is acryloyl chloride, methacryloyl chloride, allyl chloride, or a mixture thereof;
the sulphonating agent is Na2S2O5;
the isocyanate is 1,6-diisocyaate, 1,6-diisocyanate, or isophorone diisocyanate;
the catalyst is N,N,N\u2032,N\u2032-Tetramethyl-1,4-diaminobutane, triethylenediamine, dibutyltin dilaurate, or stannous octoate;
the chain extendor is ethylenediamine, diethylenetriamine, hexamethylene diamine, isophoronediamine, p-phenylenediamine, or a mixture thereof;
the acrylate monomer is methylmethacrylate or butyl acrylate; and
the initiator is potassium persulfate, ammonium persulfate, or a mixture thereof.