Pre-S gene coded peptide hepatitis B immunogens, vaccines, diagnostics, and synthetic lipid vesicle carriers

Abstract: A hepatitis B vaccine containing a peptide with an amino acid chain of at least six consecutive amino acids within the pre-S gene coded region of the envelope of hepatitis B virus. The vaccine being free of an amino acid sequence corresponding to the naturally occurring envelope proteins of hepatitis B virus and a physiologically acceptable diluent. The peptide being free or linked to a carrier. The carrier being a conventional carrier or a novel carrier including a lipid vesicle stabilized by cross-linking and having covalently bonded active sites on the outer surface thereon. Such novel carrier being useful not only to link the novel peptide containing an amino acid chain with amino acids within the pre-S gene coded region of the surface antigen of hepatitis B virus, but can also be used to bind synthetic peptide analogues of other viral proteins, as well as bacterial, allergen and parasitic proteins of man and animals. The peptides of the invention can be utilized in diagnostics for the detection of antigens and antibodies.

Description: The invention is directed to a novel carrier for peptides. For commonly used protein carriers there is a strong immune response to the carrier, as well as the synthetic peptide. Thus,it would be advantageous to evoke an anti-HBs response with peptides by use of non-protein carriers, which themselves do not evoke an antibody response. The possible use of several distinct vaccines in prophylaxis would be facilitated by the availability of fully synthetic immunogens. Biochem. Biophys. Research Communications 85/3 (1978, 983-990) describes in the last paragraph on page 985 covalent coupling of α-chymotrypsin to the lipids of liposomes via dimethyl suberimidate and via glutaraldehyde. The liposomes are prepared on the basis of egg yolk phosphatidyl choline, cholesterol, dipalmitoyl-L-α-phosphatidyl ethanolamine in a molar ratio of 7:2:1. This document shows the common preparation of a liposome. J. Imunolog. Methods 44 (1981) 79-85 discloses the modification of liposome surfaces with normal rabbit IgG by adsorption or by coupling with a water soluble cross-linking reagent in order to reduced the non-specific binding of the liposomes to erythrocytes. The liposomes are prepared by common methods and subsequently coated with IgG whereby coating of IgG is carried out with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Biochemica et Biophysica Acta 716 (1982) 140-150 discloses the use of liposomes as carriers of drug, enzymes, hormones and other biologically and medically important substances, in particular covalently coupling of antibody to liposomes. This document teaches, for example, coupling of IgG with palmitic acid and subsequently incorporating reaction product into the liposome. It is the object of the present invention to provide a carrier for peptides, in particular, for peptide immunizations which carrier does not itself provoke an immun response and does permit a strong antigenicity of the peptide. This problem is solved by a non protein lipid vesicle carrier for a peptide, said lipid vesicle carrier having active sites on the outer surface thereof consisting of -COOH, -CHO, -NH 2 and -SH active sites, said lipid versicle carrier comprising a lipid compound selected from the group consisting of a) aminoalkanes, diaminoalkanes, aminoalkenes and diaminoalkenes, each having 10 to 18 carbon atoms in the alkan or alkene part, b) fatty acids having 12 to 18 carbon atoms, c) fatty acid aldehydes and/or d) fatty acid mercaptans, said active sites being provided by said lipid compound and thereby enhancing immunogenicity of such peptides by covalent linking to said lipid vesicle carriers, which themselves do not provoke an antibody response and said lipid vesicle carrier being stabilized by cross-linking. The carrier of the present invention includes a lipid vesicle stabilized by cross-linking and having covalently bonded active sites on the outer surface thereof. The synthetic peptide is bonded via such active sites on the carrier to the outer surface of the lipid vesicle. Such active sites include -COOH, -CHO, -NH 2 and -SH. Such stabilization by cross-linking is accomplished by a stabilizing agent such as an aldehyde having at least two functional groups, such as a bifunctional aldehyde, for example, glutaraldehyde. The carrier of the present invention is chemically cross-linked with pendant functional groups. The novel carrier of the present invention includes a lipid vesicle having active sites on the outer surface thereof. Such active sites include -COOH, -CHO, -NH 2 and -SH. The lipid carrier can be stabilized by cross-linking by a stabilizing agent such as an aldehyde having at least two functional groups, such as a bifunctional aldehyde, e.g., glutaraldehyde. The bonding of the peptide to the lipid vesicle carrier occurs at the active sites on the lipid vesicle on the exterior surface of the carrier. Without wishing to be bound by any theory of operability, it is believed that such bonding is at least covalent bonding. It is possible to bind a peptide to two active sites on the outer surface of the lipid vesicle. For example, a -NH 2 group at one end of a peptide can bind with a -COOH active site on the outer surface of the lipid vesicle. The other end of the peptide can then bind with another active site on the lipid vesicle, for example, a -COOH group on the other end of the peptide can bind with a -NH 2 active site on the lipid vesicle. The preferred carrier to support the synthetic peptides of the present invention is a lipid vesicle. Lipid vesicles can be formed by sonicating a lipid in an aqueous medium, by resuspension of dried lipid layers in a buffer or by dialysis of lipids dissolved in an organic solvent against a buffer of choice. The latter procedure is preferred. Lipid vesicles consist of spheres of lipid bilayers that enclose part of the aqueous medium. Lipid vesicle (non-protein) carriers according to the present invention can be produced in a variety of ways. The preferred method to produce such carriers would be to treat a lipid vesicle containing aminoalkanes and diaminoalkanes having 10 to 18 carbon atoms, for example stearylamine, cetylamine and myrististylamine with a polyaldehyde, such as a dialdehyde, for example, butanedial (succinaldehyde), pentanedial (glutaraldehyde), hexanedial (adipoldehyde), heptanedial (pimelicaldehyde) and octanedial (suberaldehyde). Alternatively, a liposome containing aminoalkenes and diaminoalkenes having 10 to 18 carbon atoms, for example, oleylamine, can be treated with the aforementioned polyaldehydes. The lipid vesicle carrier thus formed has active aldehyde groups on the surface thereof allowing the direct linking of peptides via their N-terminal or lysine groups. Peptides linked to lipid vesicle carriers according to the present invention can also be prepared by treating an amino containing lipid vesicle as described above with a peptide activated by carbodiimide, for example, N-ethyl-N' (dimethylaminopropyl) carbodiimide. Alternatively a carbodiimide activated peptide is linked to polyaldehyde, e.g., dialdehyde, treated lipid vesicles which have been further derivatized by reaction with a water-soluble diaminoalkane, e.g., ethylene diamine and propylene diamine. Still further, lipid vesicles containing fatty acids (saturated and unsaturated) having 12 to 18 carbon atoms, e.g., stearic acid, oleic acid, palmitic acid and myristic acid, are activated with carbodiimide. Thereafter, the activated lipid vesicle is reacted with a peptide. Another approach to form a carrier according to the present invention involves using a fatty acid aldehyde as a component of the lipid vesicle and treating such lipid vesicle as described for glutaraldehyde treated lipid vesicles. Such lipid vesicle reacts directly with amino groups of peptides. In a preferred embodiment of a carrier according to the present invention, the aforementioned lipid vesicle carrier formed by treating a amino or diaminoalkane (or amino or diaminoalkene) having 10 to 18 carbon atoms with a polyaldehyde is further reacted with cysteine (L-or D- or ID- cysteine). These lipid vesicles are then reacted with a peptide having -SH groups, i.e., cysteine containing peptides. The link between the lipid vesicle and the peptide is mediated by a disulfide bond. Alternatively, a fatty acid mercaptan is used as a component of the lipid vesicle, for example, octadecanethiol. A cysteine containing peptide is directly linked to such lipid vesicle. Another approach to form carriers according to the present invention involves the preparation of the above described fatty acid mercaptan containing lipid vesicles which are further reacted with a dimaleimide, for example, para or ortho N-N'-phenylenedimaleimide. Such lipid vesicle is then reacted with a cysteine containing peptide. Alternatively, the link between the appropriate lipid vesicle and the appropriate peptide can be accomplished by commercially available cross-linking reagents such as dimethyl adipimidate; dimethyl 3,3'-dithiobis-propionimidate; 2-iminothiolane; di-succinimidyl suberate; bis[2-(succinimidooxy carbonyloxy)-ethyl] sulfone; disuccinimidyl tartarate; dithiobis (succinimidyl propionate); ethylene glycol bis(succinimidyl succinate); N-5-azido-2-nitrobenzoyloxysuccinimide; p-azidophenacyl bromide; p-azido-phenylglyoxal; 4-fluoro-3-nitrophenyl azide; N-hydroxysuccinimidyl-4-azide-benzoate; N-hydroxysuccinimidyl-4-azidosalicylic acid; m-maleimidobenzoyl N-hydroxy succinimide ester; methyl-4-azidobenzoimidate; p-nitrophenyl 2-diazo-3,3,3-trifluoroproprionate; N-succinimidyl-6 (4'-azido-2'-nitrophenylamino) hexanoate; succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate; succinimidyl 4-(p-maleimidomethyl) butyrate; N-(4-azidophenylthio)phthalimide; ethyl 4-aziodophenyl 1, 4-dithiobutyrimidate; N-succinimidyl (4-azidophenyldithio) propionate; 1-5-difluoro-2, 4-dinitrobenzene; 4,4'-difluoro-3,3'-dinitrodiphenyl-sulfone; 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene; p-phenylenediisothiocyanate; 4,4'-dithiobisphenylazide; erythritolbiscarbonate; N-succinimidyl 3-(2-pyridyldithiol) propionate; dimethyl pimelimidate and dimethyl suberimidate. The lipid vesicles according to the present invention act not only as carriers, but also as adjuvants. The lipid vesicle synthetic carriers of the present invention can be utilized to bind synthetic peptide analogues (eliciting protective antibodies) of various viral, bacterial, allergen and parasitic proteins of man and animals, besides synthetic peptide analogues of hepatitis B surface antigen, and especially the novel synthetic peptide analogue of hepatitis B surface antigen containing amino acid sequences corresponding to amino acid sequences in pre-S gene coded region of the HBV. Accordingly, the lipid vesicle synthetic carriers of the present invention can be used to bind with synthetic peptide analogues of the following viruses: influenza hemagglutinin (A/memphis/102/72 strain, A/Eng 1878/69 strain, A/NT/60/68/29c strain, and A/Qu/7/70 strain), fowl plague virus hemagglutinin, vaccinia, polio, rubella, cytomegalovirus, small pox, herpes simplex types I and II, yellow fever, Infectious ectromelia virus, Cowpox virus, Infectious bovine rhinotracheitis virus, Equine rhinopneumonitis (equine abortion) virus, Malignant catarrh virus of cattle, Feline rhinotracheitis virus, Canine herpes virus, Epstein-Barr virus (associated with infectious mononucleosis and Burkitt lymphoma), Marek's disease virus, Sheep pulmonary adenomatosis (Jaagziekte) virus, Cytomegaloviruses, Adenovirus group, Human papilloma virus, Feline panleucopaenia virus, Mink enteritis virus, African horse sickness virus (9 serotypes), Blue tongue virus (12 serotypes), Infectious pancreatic necrosis virus of trout, Fowl sarcoma virus (various strains), Avian leukosis virus (visceral, erythroblastic and myeloblastic), Osteopetrosis virus, Newcastle disease virus, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza 4, Mumps virus, Turkey virus, CANADA/58, Canine distemper virus, Measles virus, Respiratory syncytial virus, Myxovirus, Type A viruses such as Human influenza viruses, e.g., Ao/PR8/34, A1/CAM/46, and A2/Singapore/1/57; Fowl plaque virus; Type B influenza viruses, e.g., B/Lee/40; Rabies virus; Eastern equinine encephalitis virus; Venezuelan equine encephalitis virus; Western equine encephalitis virus; Yellow fever virus, Dengue type 1 virus (=type 6), Dengue type 2 virus (=type 5); Dengue type 3 virus; Dengue type 4 virus; Japanese encephalitis virus, Kyasanur Forest virus; Louping ill virus; Murray Valley encephalitis virus; Omsk haemorrhagic fever virus (types I and II); St. Louis encephalitis virus; Human rhinoviruses, Foot-and-mouth disease virus; Poliovirus type 1; Enterovirus Polio 2; Enterovirus Polio 3; Avian infectious bronchitis virus; Human respiratory virus; Transmissible gastro-enteritis virus of swine; Lymphocytic choriomeningitis virus; Lassa virus; Machupo virus; Pichinde virus; Tacaribe virus; Papillomavirus; Simian virus; Sindbis virus, and the like. The lipid vesicle synthetic carriers of the present invention can be used to bind synthetic peptide analogues of bacteria, for example, leprosy, tuberculosis, syphilis and gonorrhea. The lipid vesicle synthetic carriers of the present invention can also be used to bind synthetic peptide analogues of the following parasites: organisms carrying malaria (P. Falciparum, P. Ovace, etc.), Schistosomiasis, Onchocerca Volvulus and other filiarial parasites, Trypanosomes, Leishmania, Chagas disease, amoebiasis, hookworm, and the like. The lipid vesicle carriers of the present invention can be used to bind the peptides corresponding to amino acid sequences in the pre-S region of HBsAg. The lipid vesicle carriers of the present invention can also be used to bind amino acid sequences in the S region, as well as other amino acid sequences for other virus, etc. Amino acid sequences (corresponding to amino acids in the S region) which contains an antigenic determinant for hepatitis B surface antigen can be linked to the lipid vesicle carrier of the present invention. The immunogenicity of such peptides is enhanced by covalent linking to novel lipid vesicle (liposome) carriers also discovered by applicants. Example Synthesizing and Testing S(135-155) Derivatives Each of the conjugates ((1) to (26)) of S(135-155) listed in Table 1, except conjugate 3, was mixed 1:1 with complete Freund's adjuvant and injected into two New Zealand White rabbits (65 to 160 µg of peptide per rabbit). The rabbits were further injected at biweekly intervals with equal doses of conjugates in incomplete Freund's adjuvant (not used for conjugate 3). Blood specimens were taken two weeks after each injection. To prepare conjugates 1, and 4-8 (Table 1), 1 mg quantities of peptide 309-329 of the env gene product (S(135-155)) were activated with a two times molar excess of N-ethyl-N'(dimethyl-aminopropyl) carbodiimide (EDAC) and N-hydroxy-benzotriazole (NHBTA) and subsequently linked to equimolar quantities of poly-D-lysine and diaminoalkanes (from Fluka AG, Buchs, Switzerland), respectively, as described (Arnon, R., Sela, M., Parant, M. and Chedid., L., "Antiviral Response Elicited By A Completely Synthetic Antigen With Built-In Adjuvanticity", Proceedings of The National Academy of Science USA , 77,6769-6772, (1980)). To prepare conjugates 2 and 3, 1 mg quantities of each EDAC-activated S(135-155) and MDP (Calbiochem, San Diego, California) were linked to 10 mg of poly-D-lysine. Peptide 309-329 of the env gene product (800µg) was oxidized with ferricyanide (Dreesman et al, 1982 supra ), activated with EDAC as above and linked to 4 mg of LPH. Chromatography on Sephadex G-25 indicated complete linking of the peptide to LPH (conjugate 9). The oxidized, EDAC-activated peptide (1 mg) was also conjugated to 1 mg of polyvaline in a suspension of 2.5 ml of 1 M NaHCO 3 , pH 8.5, and 10 ml of CHCl 3 . The interphase and aqueous phase after centrifugation was used for immunization (conjugate 10). Liposomes were prepared by the method of Oku, N. Scheerer, J.F., and MacDonald, R.C., "Preparation of Giant Liposomes", Biochimica et Biophysica Acta , 692, 384-388 (1982). Stearylamine, dilauroyl lecithin and cholesterol were dissolved in glucose-saturated ethanol at final concentrations of 10, 23 and 1.43 mg/ml, respectively. For some liposome preparations, the concentration of dilauroyllecithin was decreased to 17.5 mg/ml and sphingomyelin was added (10 mg/ml). Other preparations contained as an additional component lipid A (420µg/ml; Calbiochem). The solutions were dialyzed against 0.1 M NaCH0 3 , pH 8.5, in dialysis bags with a molecular weight cut-off of 10 3 daltons for at least sixteen hours. The liposomes were treated for approximately six hours with glutaraldehyde (final concentration 30 mg/ml), mixed with 0.5 volumes of 33.9% (w/w) sodium diatrizoate, floated four times into 1 M NaCHO 3 by centrifugation for ten minutes at 10,000 rpm, and reacted with 0.84 to 1 mg of peptide 309-329 of the env gene product per 10 mg stearylamine overnight at 20°C. The linking of peptide 309-329 of the env gene product to liposomes under these conditions was complete. Some preparations were reacted additionally with 7.5 mg of RAT (Biosearch, San Rafael, California) per 10 mg of stearylamine for six hours at 20°C. The liposomes were floated three times into 0.14 M NaCl, 0.01 Tris-HCl-0.02% NaN 3 (TS) and dialyzed against TS-10 -4 M oxidized glutathione for at least sixteen hours. In some cases (20) and (21) the stearylamine-containing liposomes were not derivatized with GA but instead directly reacted with EDAC-activated peptide 309-329 of env gene product. Alternately, (18) and (19), the activated peptide 309-329 of env gene product was linked to glutaraldehyde-treated liposomes further derivatized by reaction with 0.2 M ethylene diamine at pH 8.5 overnight at 20°C followed by floating two times into 0.1 M NaHCO 3 , pH 8.5, reduction with 10uM sodium dithionite for one hour at 20°C and repeated floating into the same buffer. An aliquot of these liposomes was additionally reacted with EDAC-activated RAT. The liposomes were finally dialyzed against TS-10 -4 M oxidized glutathione. In one preparation (22), stearic acid was used instead of stearylamine for the preparation of liposomes. These were dialyzed against 0.01 M NaCl, activated with EDAC (50 mg/ml for two hours plus additional 25 mg/ml for one hour) at pH 5.5 and 20°C, floated two times into 0.01 M NaCl and reacted with the peptide 308-329 of the env gene product in 1 M NaHCO 3 ,pH 8.5, overnight. Polyglutaraldehyde microspheres were prepared as described by Margel, S., Zisblatt, S. and Rembaum, A. "Polyglutaraldehyde: A New Reagent For Coupling Proteins To Microspheres And For Labeling Cell-Surface Receptors. II. Simplified Labeling Method By Means Of Non-Magnetic And Magnetic Polyglutaraldehyde Microspheres", Journal of Immunological Methods , 28, 341-353 (1979), using Polysurf 10-36 B (Bartig Industries Inc., New Canaan, Conn., Margel & Offarim, (1983)). One mg of the peptide 309-329 of the env gene product was linked to approximately 50 mg of microspheres under conditions similar to those described for glutaralde-hyde treated liposomes. Conjugate 25 was prepared by treating the microspheres with 5 ml of 0.1 Mε-amino caproic acid at pH 8.5 overnight. After centrifugation, the microspheres were suspended in dimethylformamide (2ml) and reacted with 2 mg EDAC plus 670 ug NHBTA for one hour at 20°C. After centrifugation, the microspheres were resuspended in 2 ml of 0.1 M NaHCO 3 , pH 8.5, containing 1 mg of peptide 309-329 of the env gene product. All reagents listed above were of analytical grade and obtained from Sigma, St. Louis, Missouri, unless indicated otherwise. Free peptide 309-329 of the env gene product (mol. weight = 2,664 daltons) containing five cysteine residues was in a predominantly monomeric form, since it was eluted after molecular exclusion chromatography in about the same fractions as insulin A chain. Linking to diaminobutane and to other diamino-alkanes (data not shown) resulted in formation of S(135-155) polymers which were immunogenic and induced both antipeptide and anti-HBs antibodies. Preparations (4), (5) and (7) also induced anti-HBs, while polymers with diaminooctane or dodecane linkers (6) and (8) failed to do so (Fig. 8) for reasons not known. Oxidation of the peptide 309-329 of the env gene product resulted in polymerization (data not shown). The polymer linked to LPH (conjugate 9) induced high levels of anti-S(135-155) but no anti-HBs, unlike S(135-155) linked to KLH or LPH in its reduced form (Neurath et al., 1982, supra ). This finding again emphasizes the role of peptide conformation in inducing antibodies to the native protein. Linking of the oxidized peptide to highly hydrophobic poly-L-valine resulted in a conjugate (10) of low immunogenicity. S(135-155) linked to poly-D-lysine administered with Freund's adjuvant (1) or having covalently linked MDP and given without adjuvant (3) induced both anti-S(135-155) and anti-HBs. The latter conjugate administered with Freund's adjuvant (2) appeared poorly immunogenic. S(135-155) linked to glutaraldehyde treated liposomes containing stearylamine (conjugate 11) induced levels of anti-HBs comparable to those elicited by those elicited by conjugates with KLH or LPH (Neurath et al., 1982, supra ). Incorporation of sphingomyelin and.or lipid A, components reported to enhance the antigenicity of haptens inserted into liposomal membranes (Yasuda, T., Dancey, G.F. and Kinsky, S.C., "Immunogenicity Of Liposomal Model Membranes In Mice: Dependence On Phospholipid Composition", Proceedings Of The National Academy Of Sciences , 74, 1234-1236 (1977)), into the liposomes (conjugates 13, 15a, 16) failed to enhance anti-HBs, responses. Conjugates (18 and 19) prepared by linking S(135-155) to glutaraldehyde-treated liposomes through an ethylenediamine bridge rather than directly, had the capacity to induce anti-HBs but a considerable variability in response between individual rabbits was observed. S(135-155) before or after oxidation and subsequently linked to stearyl-amine-containing liposomes (not fixed with glutaraldehyde; preparations 20 and 21) or to stearic acid-containing liposomes (22) induced low levels of anti-S-135-155 and no measurable anti-HBs. S(135-155) linked directly to microspheres of polyglutaraldehyde (preparations 23 and 24) induced a primary anti-HBs response. However, the level of anti-HBs decreased in the course of immunization. Anti-HBs was undetectable in sera collected two weeks after the third immunization. S(135-155) linked to these microspheres through ε-amino-caproic acid (25) and l-cysteine (26) bridges, respectively, either failed (25) or was marginally efficient (26) in eliciting anti-HBs. S(135-155)-KLH or LPH conjugates elicited a primary anti-HBs response but the level of anti-HBs failed to increase in sera of rabbits after additional antigen doses (Neurath et al., 1982 supra ). With the conjugates described above, generally, a decrease of anti-HBs levels was observed four or six weeks after primary immunization (Fig. 9B), but exceptions were observed in a minority of rabbits (panel 5, Fig. 9A). This declining trend was uniformly reversed when RAT was inserted into liposomal membranes together with S(135-155) (for example Fig. 9C and Fig. 9D). The immunogenicity of haptens inserted into liposomal membranes depends on the phospholipid composition of the liposomes and seemed to be inversely related to the fluidity of these membranes (Yasuda et al., 1977 supra ; Dancey, G.F., Yasuda, T. and Kinsky, S.C. ,"Effect Of Liposomal Model Membrane Composition On Immunogenicity", The Journal Of Immunology , 120, 1109-1113 (1978)). Treatment of stearylamine-containing liposomes with glutaraldehyde was found to provide reactive groups suitable for linking of synthetic peptides and at the same time increases the rigidity of the lipid membranes. Such liposomes, especially when containing carrier function enhancing RAT sites (Alkan, S.S., Nitecki, D.E. and Goodman, J.W., "Antigen Recognition And the Immune Response: The Capacity of 1-Tryosine-Azobenzenearsonate To Serve As A Carrier For A Macromolecular Hapten", The Journal Of Immunology , 107, 353-358, (1971), and Alkan, S.S., Williams, E.B., Nitecki D.E. and Goodman, J.W.. "Antigen Recognition And the Immune Response. Hurmoral And Cellular Immune Responses To Small Mono- And Bifunctional Antigen Molecules", The Journal Of Experimental Medicine , 135, 1228-1246, (1972)), are a promising tool for preparing fully synthetic immunogens for eliciting anti-viral antibodies. List of cross-linkers and carriers used for the preparation of S(135-155) conjugates (1) Poly-D-lysine (mol. weight 3-7 x 10 4 ) [comparative ex.] (2) 1 + N-Acetylmuramyl-L-alanyl-D-isoglutamine (MDP) [comparative ex.] (3) = 2 [comp. ex.] (4) 1,4-diaminobutane (5) 1,6-diaminohexane (6) 1,8-diaminooctane (7) 1,10-diaminodecane (8) 1,12-diaminododecane (9) Oxidized S(135-155) linked to LPH [comp. ex] (10) Oxidized S(135-155) linked to poly-L-valine [comp. ex.] (11) Liposomes containing stearylamine, and treated with glutaraldehyde (12) = 11 = L-tyrosine-azobenzene-p-arsonate (RAT) (13) = 11 + Sphingomyelin (from bovine brain) (14) = 13 + RAT (15a) = 11 + Lipid A (15) = 15a + RAT (16) = 13 + Lipid A (17) = 16 + RAT (18) = 11 treated with ethylenediamine (19) = 18 + RAT (20) = Liposomes containing stearylamine reacted with oxidized S(135-155) (see 9) (21) = 20 except S(135-155) was oxidized after attachment to liposomes (22) Stearic acid containing liposomes (23) Polyglutaraldehyde micropheres [comp. ex.] (24) = 23 + RAT [comp. ex.] (25) = 23 treated with ε-aminocaproic acid [comp. ex.] (26) = 23 treated with L-cysteine [comp. ex.]

Claims: Non protein lipid vesicle carrier for a peptide, said lipid vesicle carrier having active sites on the outer surface thereof consisting of -COOH, -CHO, -NH 2 and -SH active sites, said lipid vesicle carrier comprising a lipid compound selected from the group consisting of a) aminoalkanes, diaminoalkanes, aminoalkenes and diaminoalkenes, each having 10 to 18 carbon atoms in the alkane or alkene part, b) fatty acids having 12 to 18 carbon atoms, c) fatty acid aldehydes and/or d) fatty acid mercaptans, said active sites being provided by said lipid compound, and thereby enhancing immunogenicity of such peptides by covalent linking to said lipid vesicle carriers, which themselves do not provoke an antibody response and said lipid vesicle carrier being stabilized by cross-linking. Non-protein lipid vesicle carrier according to claim 1, wherein aminoalkanes, diaminoalkanes, aminoalkenes or diaminoalkenes are treated with a polyaldehyde. Non-protein lipid vesicle carrier according to claims 1 and 2, wherein the lipid vesicle is further treated with L-or D- or LD-cysteine. Non-protein lipid vesicle carrier according to claim 1 wherein said lipid vesicle contains fatty acids having 12 to 18 carbon atoms and said lipid vesicle is stabilized with a carbodiimide. Non-protein lipid vesicle carrier according to claim 4 wherein said carbodiimide is N-ethyl-N'-(dimethylaminopropyl)carbodiimide. Non-protein lipid vesicle carrier according to claim 1, wherein said lipid involves a fatty acid aldehyde and is treated with glutaraldehyde. Non-protein lipid vesicle carrier according to claim 1, wherein said lipid vesicle comprises a fatty acid mercaptan and is further reacted with a dimaleide. Non-protein lipid vesicle carrier according to claim 4, wherein said dimaleimide is N,N'-phenylenedimaleimide. Non-protein lipid vesicle carrier according to claims 1 and 3, wherein said lipid compound is stearylamine. Non-protein lipid vesicle carrier according to claims 1, 4 and 5, wherein the fatty acid lipid compound is stearic acid. Non-protein lipid vesicle carrier according to claims 1 and 7, wherein said fatty acid mercaptan is octadecanethiol. Use of the non protein lipid vesicle carrier according to claims 1 to 11 as carrier for the synthetic peptide analog of hepatitis B surface antigen containing aminoacid sequences corresponding to aminoacid sequences in pre-S-gene region of the HBV. Use according to claim 12 containing stearyl amine as carrier for the peptide 309-329 of the env gene product.

Inventors: KENT STEPHEN B H, NEURATH ALEXANDER ROBERT, KENT, STEPHEN B.H., NEURATH, ALEXANDER ROBERT
Applicants: CALIFORNIA INST OF TECHN, NEW YORK BLOOD CENTER INC, CALIFORNIA INSTITUTE OF TECHNOLOGY, NEW YORK BLOOD CENTER, INC.
Application year: 1985
Main classification: A61K39
IPC codes: A61K9, C07K16, A61K39, G01N33, A61K47