CA2283633A1 - Peptide vaccine to prevent development of several herpes virus infections and/or atherosclerotic plaque - Google Patents
Peptide vaccine to prevent development of several herpes virus infections and/or atherosclerotic plaque Download PDFInfo
- Publication number
- CA2283633A1 CA2283633A1 CA 2283633 CA2283633A CA2283633A1 CA 2283633 A1 CA2283633 A1 CA 2283633A1 CA 2283633 CA2283633 CA 2283633 CA 2283633 A CA2283633 A CA 2283633A CA 2283633 A1 CA2283633 A1 CA 2283633A1
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- Prior art keywords
- pro
- gly
- arg
- ala
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- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
- C07K16/088—Varicella-zoster virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/245—Herpetoviridae, e.g. herpes simplex virus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
- C07K16/087—Herpes simplex virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
- C07K16/089—Cytomegalovirus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
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- A61K2039/70—Multivalent vaccine
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
- C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
- C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
- C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
A vaccine is disclosed for the prophylaxis against pathogenic development of atherosclerotic plaque in a mammalian subject susceptible thereto which comprises: 10 to 30 % by weight of the compound of SEQ ID 2; 10 to 30 % by weight of the compound of SEQ ID 4; 10 to 30 % by weight of the compound of SEQ ID 6; 10 to 30 % by weight of the compound of SEQ ID 8 in combination with a pharmaceutically acceptable innert carrier.
Description
PEPTIDE VACCINE TO PREVENT DEVELOPMENT OF SEVERAL HEI?PES VIRUS INfECTIONS
IWD/OR ATNEROSCLEROTIC PLAQUE
SPECIFICATION
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of US patent application 08/281,702 filed 27 July 1994.
FIELD OF THE INVENTION
This invention relates to a vaccine against herpes virus for _ treatment and prevention of the development of several herpes virus infections and/or atherosclerotic plaques. More particularly the invention relates to a herpes vaccine containing peptides encoded by parts of herpes virus DNA with homology to alpha-subunits of human G proteins and that acts as a prophylaxis against pathogenic development of several herpes infections and/or atherosclerotic plaque in a mammalian subjected susceptible thereto.
BACKGROUND OF THE ~ UENTION
It is generally accepted that atherogenesis is triggered by primary injury to the endothelial lining of the arterial walls. This injury is believed to be the result of exposure of the underlying smooth muscle cells to several factors of non-infectious origin (hormones, low density lipoproteins, growth factors, among others). The prevailing view is that human atherosclerosis (AS) is a pleiotropic process with various causes. See Ross, R., The Pathogenesis of Atherosclerosis: An Update, New England J. Med.,314, 488 to 500 (1986).
IWD/OR ATNEROSCLEROTIC PLAQUE
SPECIFICATION
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of US patent application 08/281,702 filed 27 July 1994.
FIELD OF THE INVENTION
This invention relates to a vaccine against herpes virus for _ treatment and prevention of the development of several herpes virus infections and/or atherosclerotic plaques. More particularly the invention relates to a herpes vaccine containing peptides encoded by parts of herpes virus DNA with homology to alpha-subunits of human G proteins and that acts as a prophylaxis against pathogenic development of several herpes infections and/or atherosclerotic plaque in a mammalian subjected susceptible thereto.
BACKGROUND OF THE ~ UENTION
It is generally accepted that atherogenesis is triggered by primary injury to the endothelial lining of the arterial walls. This injury is believed to be the result of exposure of the underlying smooth muscle cells to several factors of non-infectious origin (hormones, low density lipoproteins, growth factors, among others). The prevailing view is that human atherosclerosis (AS) is a pleiotropic process with various causes. See Ross, R., The Pathogenesis of Atherosclerosis: An Update, New England J. Med.,314, 488 to 500 (1986).
A fundamentally new etiological factor: herpes virus infection was reported by Fabricant et al, who demonstrated that chickens infected with Marek Disease Yirus (MDV) have an unusually high incidence of atherosclerotic plaque {ASP) in the arteries. See Fabricant, C.G. et al, Yirus-Induced Cholesterol Crystals, Science , 181, 566 to 567 (1973); and Fabricant, C.G. et al, Virus-Induced Atherosclerosis, J. Exp. Med., 148, 335 to 340 (1978). Since that time data have been accumulated suggesting herpes virus in AS in humans. It was shown that different herpes viruses can alter smooth muscle cells lipid metabolism and induce cholesterol and cholesterol ester accumulation in these cells. See Fabricant, C.G. et al, Herpes Virus Infection Enhances Cholesterol and Cholesterol Ester - Accumulation in Cultured Arterial Smooth Muscle Cells, Am. J. Pathol, 105, 176 to 184 (1981); Fabricant, C.G. et al, Herpes Virus-Induced Atherosclerosis in Chickens, Fed. Proc., 42, 2476 to 2479 (1983); Melnick, J.L. et al, Cytomegalovirus Antigen within Human Arterial Smooth Muscle Cells, anc , ii, 644 to 647 (1983); Gyorkey, F. et al, Herpesviridae in the Endothelial and Smooth Muscle Cells of Proximal Aorta in Atherosclerotic Patients, Exp. Mol. Pathol, 40, 328 to 339 (1984); Hajjar.
et al, Virus-Induced Atherosclerosis: Herpes Virus Infection Alters Aortic Cholesterol Metabolism and Accumulation, Am. J. Pathol., 122, 62 to 70 (1986); Adam et al, High Levels of Cytomegalovirus Antibody in Patients Requiring Vascular Surgery for Atheroscierosis, ancet, 2, 291 to 293 (1987); Petrie, Association of Herpesvirus/Cytomegalovirus Infections with Human Atherosclerosis, Pro4. Med. Virol., 35, 21 to 42 (1988); Grattan, M.T. et al, Cytomegalovirus Infection is Associated with Cardiac Allograft Rejection and Atherosclerosis, J. A. Med. Assoc.. 261, 3561 to 3566 (1989);
Mc Donald, K. et al, Association of Coronary Artery Disease in Cardiac Transplant Recipients with Cytomegalovirus Infection, Am. J. Cardiol., 64, 359 to 362 (1989); Visser et al, Granulocyte-Mediated Injury in Herpes Simplex Virus-Infected Human Endothelium, j<,~s Invest., 60, 296 to 304 (1989); Melnick, J.L. et al, Possible Role of Cytomegalovirus in Atherogenesis, J. Am. Assoc.,, 263, 2204 to 2207 (1990); Bruggeman, C.A. et al, The Possible Role of Cytomegalovirus in Atherogenesis, Proa Med.
Tirol., 38, 1 to 26 (1991); Melnick, J.L. et al, Accelerated Graft Atherosclerosis Following Cardiac Transplantation; Do Viruses Play a Role?, ~]in. Cardiol., 14 {Supp. II), 21 to 26 (1991); and Ha~~ar , D.P., Viral Pathogenesis of Atherosclerosis, Am. J. Pathols, 133, 1195 to 1211 (1991).
In addition the DNA of various herpesviruses showed positive hybridization with ASP DNA; see Benditt, E.P. et al, Viruses in the Etiology of Atherosclerosis, Proc. Natl. Acad. Sci., 80, 6386 to 6389 (1983); Pyrzak, R. et al, Detection of Specific DNA Segments of Marek's Disease Herpes Virus in Japanese Quail Susceptible to Atherosclerosis, Athero~clerosis, 68, 77 to 85 (1987); Petrie, B.L. et al, Nucleic Acid Sequences of Cytomegalovirus in Cultured Human Arterial Tissue, J.J. Inf.
155, 158 to 159 (1987); Yamashiroya, H.M. et al, Herpesviridae in Coronary Arteries and Aorta of Young Trauma Victims, Am. J. Pathol, 130, 71 to 79 (I988); and Hendrix, M.G.R. et al, The Presence of Cytomegalovirus Nucleic Acids in Arterial Walls of Patients Suffering From Grade III
Atherosclerosis, Am. J. Pathol., 134, 1151 to 1157 (1989).
No systematic attempts to demonstrate a viral presence in ASP
by direct isolation of infectious HSY from ASP and by detection of viral replication in ASP by Electron Microscopy have been reported. A viral presence in ASP would explain the presence of HSY-like DNA in ASP, and redirect research to determine the molecular mechanisms of viral involvement in etiology of atherosclerosis. In such a case, the possibility of a contamination of ASP in the blood vessels by HSV also has to be excluded.
et al, Virus-Induced Atherosclerosis: Herpes Virus Infection Alters Aortic Cholesterol Metabolism and Accumulation, Am. J. Pathol., 122, 62 to 70 (1986); Adam et al, High Levels of Cytomegalovirus Antibody in Patients Requiring Vascular Surgery for Atheroscierosis, ancet, 2, 291 to 293 (1987); Petrie, Association of Herpesvirus/Cytomegalovirus Infections with Human Atherosclerosis, Pro4. Med. Virol., 35, 21 to 42 (1988); Grattan, M.T. et al, Cytomegalovirus Infection is Associated with Cardiac Allograft Rejection and Atherosclerosis, J. A. Med. Assoc.. 261, 3561 to 3566 (1989);
Mc Donald, K. et al, Association of Coronary Artery Disease in Cardiac Transplant Recipients with Cytomegalovirus Infection, Am. J. Cardiol., 64, 359 to 362 (1989); Visser et al, Granulocyte-Mediated Injury in Herpes Simplex Virus-Infected Human Endothelium, j<,~s Invest., 60, 296 to 304 (1989); Melnick, J.L. et al, Possible Role of Cytomegalovirus in Atherogenesis, J. Am. Assoc.,, 263, 2204 to 2207 (1990); Bruggeman, C.A. et al, The Possible Role of Cytomegalovirus in Atherogenesis, Proa Med.
Tirol., 38, 1 to 26 (1991); Melnick, J.L. et al, Accelerated Graft Atherosclerosis Following Cardiac Transplantation; Do Viruses Play a Role?, ~]in. Cardiol., 14 {Supp. II), 21 to 26 (1991); and Ha~~ar , D.P., Viral Pathogenesis of Atherosclerosis, Am. J. Pathols, 133, 1195 to 1211 (1991).
In addition the DNA of various herpesviruses showed positive hybridization with ASP DNA; see Benditt, E.P. et al, Viruses in the Etiology of Atherosclerosis, Proc. Natl. Acad. Sci., 80, 6386 to 6389 (1983); Pyrzak, R. et al, Detection of Specific DNA Segments of Marek's Disease Herpes Virus in Japanese Quail Susceptible to Atherosclerosis, Athero~clerosis, 68, 77 to 85 (1987); Petrie, B.L. et al, Nucleic Acid Sequences of Cytomegalovirus in Cultured Human Arterial Tissue, J.J. Inf.
155, 158 to 159 (1987); Yamashiroya, H.M. et al, Herpesviridae in Coronary Arteries and Aorta of Young Trauma Victims, Am. J. Pathol, 130, 71 to 79 (I988); and Hendrix, M.G.R. et al, The Presence of Cytomegalovirus Nucleic Acids in Arterial Walls of Patients Suffering From Grade III
Atherosclerosis, Am. J. Pathol., 134, 1151 to 1157 (1989).
No systematic attempts to demonstrate a viral presence in ASP
by direct isolation of infectious HSY from ASP and by detection of viral replication in ASP by Electron Microscopy have been reported. A viral presence in ASP would explain the presence of HSY-like DNA in ASP, and redirect research to determine the molecular mechanisms of viral involvement in etiology of atherosclerosis. In such a case, the possibility of a contamination of ASP in the blood vessels by HSV also has to be excluded.
None of the above references deals with the preparation of a vaccine against any form of the herpes virus. The following reference deals with the preparation of a herpes vaccine against Marek's Disease Herpes-Virus in chickens: Fabricant, J. et al, Vaccination Prevents Atherosclerosis Induced by Marek's disease Herpesvirus, College of Veterinary Medicine and Medicine, Cornell University, Ithaca and New York, N.Y. The reference appeared as an abstract in the Federation of American Societies for Experimental Biology, 65th Annual Meeting, Atlanta (1981).
The vaccine employed against Marek's Disease Herpesvirus in chickens was derived from Turkey herpesvirus (HVT). There is no indication that a vaccine against atherosclerosis caused by human herpes virus could be prepared. There is certainly no suggestion to employ a herpes vaccine containing homologous peptide sequences to those of the viral DNA found in strains of the herpes virus that effect humans.
U.S. Patent 4,038,381 discloses a vaccine for the prevention and treatment of vascular conditions, comprising a combination of a tuberculosis antigen with an antiherpetic vaccine. There is no suggestion to employ the four polypeptides of the present invention as the active ingredients in the vaccine. The reference also states that the individual tuberculosis antigen and antiherpetic vaccine had no known per se ability in the prevention or treatment of vascular disease.
OBJECT Of THE INVENTION
It is the object of the invention to provide a universal vaccine as a prophylaxis against pathogenic development of several herpes infections and/or atherosclerotic plaque in a mammalian subject susceptible thereto.
Y.. ...
SUMMARY OF THF~~NVENTION
We have found such a vaccine that is effective as a prophylaxis against pathogenic development of several herpes infections and/or atherosclerotic plaque in mammalian subjects, including humans. The vaccine contains four new peptides as described herein below in the indicated proportions:
(a) 10 to 30x by weight of the compound Ala Pro leu Pro Ala Pro Ata Pro Pro Ser Thr Pro Pro Gly Pro Glu Pro Ala Pro Ala Gln Pro Ala Ala Pro Arg Ala Ala (Seq ID 2);
(b) 10 to 3076 by weight of the compound Ala Pro Pro Glu Ala Asp Ala Arg Thr Leu Arg Arg Pro Gly Pro Pro Leu Pro Leu Pro Pro Ser Leu Leu Pro (Seq ID 4);
(c) 10 to 30X by weight of the compound Gly Thr Asp Gly Pro Ala Arg Gly Gly Gly Ser Gly Gly Gly Arg Gly Pro Gly Gly Gly Arg Gly Gly Pro Arg Gly (Seq ID 6); and (d) 10 to 30% by weight of the compound Gly Trp Ala Ala Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Arg Gln Arg Arg Ala Ala Arg Arg Arg Arg (Seq ID 8);
in combination with a pharmaceutically acceptable inert vaccine carrier such as normal saline or a physiological oil (e. g. corn oil, sunflower oil).
Preferably each of the four polypeptides is present in the compositions in equal proportions by weight: that is the compositions preferably contain 25% of each of the four polypeptides.
The compositions are prepared by incorporating each of the four polypeptides in the pharmaceutically acceptable inert vaccine carrier such as normal saline or a physiological oil in an adequate concentration of said polypeptides. Preferably there is present 1.0 to 100 ~g of each polypeptide per ml of pharmaceutical composition. Fore preferably one dose of vaccine (1 ml) contains equal parts (20 beg) of each of the 4 polypeptides. Thus the preferred total amount of polypeptides in one dose of vaccine is 80 fig.
Since each of the polypeptides is itself a new compound, each of them, individually, as well as collectively, is considered to be part of the invention as well.
Also contemplated to be within the scope of the invention is a method of prophylaxis of pathogenic development of several herpes virus infections and/or atherosclerotic plaques in a mammalian subject susceptible thereto which comprises the step of administering to said mammalian subject, a therapeutically effective amount of the pharmaceutical composition containing the four polypeptide sequences as described hereinabove. The herpes infections whose development can be prevented include Herpes Simplex I, Herpes Simplex II, Cytomegalovins, Epstein-Barr Virus, Herpes Zoster and Kaposi~s Sarcoma.
The compositions may preferably be administered to a mammalian subject parenterally, such as by injection. More preferably the compositions are administered by subcutaneous, intramuscular, intra-arterial, intravenous or intradermal injection. A preferred dosage of the compositions is 1 ml every 20 days administered in a series of 6 intramuscular injections. The full cycle of treatment may consist of 2 or 3 such courses with 3 month intervals in between.
Use of an adjuvant, for instance inorganic gels such as alum, aluminum hydroxide or aluminum phosphate that increase antigenic response, is optional in the compositions.
REF DESCRIPTION OF THE DRAW) NGS
The above and other objects, features and advantages will become more readily apparent from the following description, reference being made to the accompanying drawings in which:
Fig. 1 is a map of the expression vector plasmid pRSET (prior art).
Figs. 2 and 3 are series of bar graphs showing the results of an enzyme-linked immunosorbent assay (ELISA) on rabbit sera #76 and #77 obtained from rabbits before and after immunization with the present peptide-containing vaccine using the peptide having Sequence ID #2 to bind the antibodies in the sera to determine antibody formation.
Figs. 4 and 5 are series of bar graphs showing the results of an enzyme-linked immunosorbent assay (ELISA) on rabbit sera #76 and #77 obtained from rabbits before and after immunization with the present peptide-containing vaccine using the peptide having Sequence ID #4 to bind the antibodies in the sera to determine antibody formation.
Figs. 6 and 7 are series of bar graphs showing the results of an enzyme-linked immunosorbent assay (ELISA) on rabbit sera #76 and #77 obtained from rabbits before and after immunization with the present peptide-containing vaccine using the peptide having Sequence ID #6 to bind the antibodies in the sera to determine antibody formation.
Figs. 8 and 9 are series of bar graphs showing the results of an enzyme-linked immunosorbent assay (ELISA) on rabbit sera #76 and #77 obtained from rabbits before and after immunization with the present peptide-containing vaccine using the peptide having Sequence ID #8 to bind the antibodies in the sera to determine antibody formation.
Fig. 10 is a comparative electrophoretic motility study (in 4%
acrylamide gel) of the "DNA-GGC-sites-fragments" per se, with alpha-proteins of nuclei from human blood vessel endothelium (with and without ASP) and with rabbit sera (with and without antibodies against a mixture of several herpes virus peptides).
Lane a "DNA-GGC-sites-fragment" (25,000 cpm, 2.5 ng of DNA) in the absence of added protein extract and any sera;
Lanes b through e: "DNA-GGC-sites-fragments" in the same concentration +
aliquot of the extracts of purified nuclei from human blood vessel endothelium cells with ASP (b, c) + aliquot of the normal (pre-immune) rabbit sera N76(d) and N77(e) in dilution 1:2.
Lanes f and g: "DNA-GGC-sites-fragments" in the same concentration +
aliquot of the extracts of purified nuclei from human blood vessel endothelium cells without ASP.
Lane h: "DNA-GGC-sites-fragments" in the same concentration + k~xture of aliquots of the extract of purified nuclei from endothelial blood vessel cells with ASP + aliquot of the mixture of the post-immune rabbit sera N76 and N77 in dilution 1:100.
lanes i through 1: the same situation as in Lane "h~, but the mixtures of irtmune sera were used in a decreased dilution of 1:50(i), 1:30 0), 1:20(k), and 1:10(1).
Fig. 11 shows the results of another technique of gel retardation experiments in 5% PAAG. The immunosera number 77(N4) and Number 76(N6) (with dilution 1:20) had decreased the formation (amount of DNA protein complex between DNA-GGC-fragment labelled by 32P and alpha-protein nuclei from human blood vessels with ASP - in comparison with corresponding preimmune sera (N5 and N7). On the other side both of inmune sera (N1) and (N2), as well as the preinmune sera number 76(N3) had no influence on the formation of such complexes, if sera were used in dilution of 1:100 Preparation of the Vaccine against Atherosclerosis The vaccine may be prepared by recombinant DNA techniques by chemical synthesis or by automated solid phase synthesis. When preparing the vaccine by recombinant DNA techniques the following steps are employed:
Recombinant DNA Synthesis 1. Accumulation of Virus Particles For isolation of the fragments of DNA that encode peptides having Seq ID 2 and Seq ID 6, it is necessary to accumulate Herpes Simplex Virus Type 1, and for isolation of the fragments of DNA that encode peptides having Seq ID 4 and Seq ID 8, it is necessary to accumulate Human Cytomegalovirus.
Herpes Simplex Yirus 1 and Human Cytomegalovirus are each cultivated in diploid human embryonic lung cells (HECL).
Tissue Cultures For isolation of Herpes Simplex Yirus 1 and Human Cytomegalovirus, it is necessary to use diploid human embryonic lung cells (e. g. semi-continuous cells). These cells are derived from embryonic lung tissue and following initial dispersal, they can be redispersed and regrown many times (30 to 50 times). Human embryonic lung tissue, which can be obtained from embryos of 10 to 12 weeks, provide a most valuable source for harvesting a number of different herpes viruses, including Herpes Simplex Viruses and Human Cytomegalovirus.
Semi-continuous cells have a normal chromosome count (diploid) and show the phenomenon of contact inhibition. An inoculum of each virus listed above is placed on the monolayer and allowed to absorb for 1 hour.
It is then removed and fresh medium is added. Cultures are incubated at 37'C and they are inspected regularly by microscopy for evidence of virus growth. The culture medium is normally changed on the day after inoculation to minimize the effect of toxins that may persist in the inoculum, and is then replaced periodically to replenish the supply of nutrients for the cells. Cultures are incubated for various lengths of time depending on the virus. While the cytopathic effects of a concentrated inoculum of herpes virus may appear overnight, a low level of cytomegalovirus may take 3 to 4 weeks to appear.
The cells infected by herpes viruses may be cultivated in suspension also.
For inoculation of the tissue cultures to prepare the peptide vaccines, the following viruses may be used:
Herpes Simplex Type 1 (Human herpesvirus 1, Herpesvirus homines type 1) ATCC YR-539, Strain MacIntyre.
Cytomegalovirus ATCC YR-538 Strain: AD-1fi9.
2. isolation of the following four nucleotide Sequences from the Viral DNA that Code for the Four peptides indicated above:
Ala Pro Leu Pro Ala Pro Ala Pro Pro Ser Thr Pro Pro Gly Pro Glu CCC GCC CCC GCC CAG CCC GCG GCG CCC CGG GCC GCC 84 (Seq ID 1);
Pro Ala Pro Ala Gln Pro Ala Ala Pro Arg Ala Ala Ala Pro Pro Glu Ala Asp Ala Arg Thr Leu Arg Arg Pro Gly Pro Pro CTG CCG CTG CCG CCT TCC CTT CTC CCG 75 (Seq ID 3);
Leu Pro Leu Pro Pro Ser Leu Leu Pro Gly Thr Asp Gly Pro Ala Arg Gly Gly Gly Ser Gly Gly Gly Arg Gly CCC GGT GGC GGA AGA GGT GGC CCC CGC GGG 78 (Seq ID 5); and Pro Gly Gly Gly Arg Gly Gly Pro Arg Gly Gly Trp Ala Ala Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Gly Arg CGT CGC CGC CAG CGG CGA GCG GCA CGG AGA CGG AGG 84 (Seq ID 7) Arg Arg Arg Gln Arg Arg Ala Ala Arg Arg Arg Arg .
Note the DNA sequences assigned Seq ID numbers 1, 3, 5, and 7 are the gene sequences containing the codons to obtain the peptides having Seq ID Nos. 2, 4, 6, and 8. The DNA fragments having Seq ID Nos 1,3,5, and 7 are regarded as novel intermediate compounds that constitute part of the present invention.
The Origin and Utility of these Four Oligonucleotides The Oligonucieotide of Seq ID 1 is the part of the Herpes Simplex Virus Type 1 immediate early (IE) gene 3 for the transcriptional activator IE 175 (= ICP 4). Its 84 nucleotides are located from by 3760 up to 3844 according to the known gene nucleotide sequence Herpes Simplex Virus Type 1, Viridae; DS-DNA Enveloped Viruses; Herpes Viridae, Alpha-Herpes Virinae. This same oligonucleotide may also be found in the complete short unique region 2 with partial terminal and inverted repeats in DNA, HSV 1, Strain 17.
The oligonucleotide of Seq ID 3 is the part of the Human Cytomegalovirus (Strain AD 169) complete genome (from base pair 70001 up to base 80100). Its 75 nucleotides are located from by 2342 up to 2416 in this region of the Human Cytomegalovirus gene Strain AD=169 according to Human Cytomegalovirus, Viridae, DS-DNA Enveloped Viruses, Herpesviridae, Betaherpesvirinae.
The oligonucleotide of Seq ID 3 is in the Human Cytomegalovirus F fragment DNA encoding DNA Polymeraseycoprotein B also and has homology with DNA from the following viruses:
(a) Epstein-Barr Virus, artifactual joining of B95-8 complete gene and the sequences from raga of the large deletion found in B95-8 (from base pair 70001 to 80100) about 70X.
{b) HSV1 (strain 17) complete short unique region with inverted repeat DNA, (from by 10001 to 20100) about 65X.
(c) HSV2 ORF1, ORF2, and ORF 3 (LAT) gene about 65X.
The oligonucleotide with Seq ID5 is the part of Herpes Simplex virus type one (NSY 1) latency associated transcript (LAT).
Its 78 nucleotides are localized from 2255 up to 2332 positions of LAT gene according to Herpes Simpiex Virus Type 1, Yiridae, DS-DNA
Enveloped Viruses; Herpes Viridae, Alpha-Herpesvirinae.
This oligonucleotide has homology:
with Herpes Simplex virus type 1 Bam H1 fragment 8 DNA sequence - about 97.5%:
with Herpes Simplex virus type 1 gene encoding two latency -related proteins - about 97.5%;
with Pseudorabies virus immediate - early gene -about 70%;
with Herpes Simplex virus type 2 ORF1, ORF2, and ORF3 (LAT) gene- about 70%;
with Epstein-Barr virus, artifactual joining of B95 - 8 complete genome and the sequences from ra3i of the large deletion found in B95 - 8 (from base 70001 to 80100 - about 65.5X;
with Bovine Herpesvirus type 1 early - intermediate transcription control protein_(BICP4) gene - about 70%;
with Human Cytomegalovirus UL56 gene - 67.5%;
The oligonucleotide with Seq ID 7 is part of Human Cytomegalovirus (HCMV) short unique region, short repeats, and part of long repeat (from base 1 to base 10100).
Its 84 nucleotides are localized from 5340 up to 5424 positions in this part of genome according to Human Cytomegalovirus, Viridae; DS-DNA Enveloped Virules; Herpes Viridae;
Betaherpesvirinae This oligonucleotide has homology:
with Equine Herpesvirus 4 (EHV4) genome, thymidine kinase (TK) and glycoprotein H (GH) genes - about 71%
with Herpes Simplex virus type 2 immediate - early (IE5) protein mRNA, 5' end - about 65%;
with Herpes Simplex virus type 1 complete genome from base 70001 to base 80100 - about 65.5%.
Isolation of the oligonucleotides sequences from the viral DNA
that code for the four peptides indicated above.
The viral DNA is isolated from corresponding viral suspension obtained from HELL infected by Herpes Simplex 1 or Human Cytomegalovirus (see above about cell cultures and virus strains), purified by agarose gel electrophoresis (AGE) according to Myers, R.M. et al, "Detection and Localization of Single Base Changes by Denaturing Gradient Gel Electrophoresis; Methods Enzymol. 155:501 to 527 (1987) and Myers et al, "In Genome Analysis: A Practical Approach" (Ed. K. Oavies) p. 95 IRL Press, Oxford (1988), treated by restriction enzymes and subjected by polyacrylamide gel electrophoresis.
The isolation of oligonucleotide with Seq ID 1 from Herpes Simplex virus type I immediate early (IE) gene 3 for transcriptional activator IE 175 (= ICP4) is produced after treating DNA by restriction enzyme Ncol with recognition sequence CGATGG. This procedure according to PAAG, EF gives the polynucleotide with 2308 pair of bases (from 2637 up to 4935 position) with the actual fragment located from 3760 up to 3844 position.
The isolation of oligonucleotide with Seq ID 3 from HCMV
(strain AD169) complete genome is produced after treating of DNA by restriction enzyme Bst E III with recognition sequence GATC. This procedure after PAAG, EF gives the polynucleotide with 2182 pair of base -from 715 to 2898 position - with the actual fragment from 2342 up to 2416 position.
The isolation of oligonucleotide with Seq. ID5 from HSVI (LAT) is produced after treating of DNA by restriction enzyme Nco 1 with recognition sequence CCATGG. This procedure after PAAG, EF gives the polynucletide with 1737 pair of base -from 659 up to 2396 position - with actual fragment from 2255 up to 2332 position.
The isolation of oligonucleotide with Seq IO 7 from HCMV -short unique region, short repeats, and part of long repeat is produced after treating of DNA by restriction enzyme Nco 1 also. This procedure after PAAG, EF gives the polynucleotide with 4807 pair of base with actual fragment from 5340 up to 5424.
The structures of the Herpes Simplex Virus Type I Immediate Early (IE) Gene 3 for Transcriptional Activator IE 175; HCMY (Strain AD
169); HSVI (LAT); and HCMY - Short Unique Region are known in the art and may be found in EMBL-37.
The construction of recanbinant plasmid DNA for expression of the four peptides, with Seq Nos. 2, 4, 6 and 8.
All 4 oligonucleotides are introduced into a polylinker of plasmid pRSET with sites of restriction, including Nco 1 and Bst E III.
This plasmid may be used to transform E.Coli (AB 109 strain). For this aim bacteria are treated with CaCl2 which makes their membranes slightly permeable (competent bacteria). The transformed bacteria are then selected by growing them on a medium containing ampicillin. pRSET is a commercial multicopy expressing vector with a high level of protein expression.
Chemical Synthesis The four sequences having the formulae Seq. Nos. 2,4,6 and 8 may also be prepared by direct peptide synthesis that is well known in the art according to the syntheses employed in The Pegtides, Schroeder and Luebke, Yol. I, Methods of Peptide Synthesis, Academic Press (1965).
Preferably each of the four polypeptides having Seq Nos. 2,4,6 and 8 are synthesized starting from the amino terminal acid and forming the peptide bond between the carboxy terminal of the given amino acid and the amino terminal of the next given amino acids. This procedure is carried out until all of the amino acids needed to make each of the four peptides are formed into whole chains.
Where it is necessary to employ an amino-protecting group to protect an N-terminal amino substituent to carry out the synthesis of one or more of the four above-mentioned peptides, the approaches of pages 3 through 51 of The Pegtides may be employed. Where it is necessary to employ a carboxy-protecting group to protect either a C-terminal carboxy group or a carboxy group forming part of a side chain (i.e. Glu, Asp) to carry out the synthesis of one or more of the four above-mentioned polypeptides, the methods of page 52 through 75 of the reference are employed.
Glycine and alanine are relatively simple amino acids common to the presently claimed peptides. Where it is necessary to block the amino terminal, carbobenzoxy groups are employed. Where it is necessary to block the carboxy terminal, a benzyl ester is formed. See pages 137 and 138 of The Pe tip ides.
In fact the information regarding blocking the C- and N-terminals of simple amino acids such as glycine and alanine without highly reactive side chains is still highly relevant to the blocking of all amino acids involved in the synthesis of the peptides of the present invention.
One amino acid common to all four peptides of the Seq Nos 2, 4, 6 and 8 is arginine. Arginine has a guanido group on its side chain and sometimes this group may be responsible for undesired side reactions.
Pages 167 through 174 of The Pe~.tides discusses peptide synthesis using a number of different blocking groups to protect the guanido side chain.
Pages 175 and 176 discuss peptide synthesis involving arginine where the guanido side chain need not be blocked.
Another amino acid that is well represented among the four peptides of this invention is proline. Proline is a heterocyclic amino acid with an amino functional group. Where it is necessary to block the amino group, pages 146 through 148 of The Peptides provides details.
Serine is an amino acid present in three of the four new peptides. Serine contains a side chain that includes a hydroxy group. In some situations the hydroxy group may undergo undesired side reactions.
The Peptides on pages 207 through 214 describes peptide synthesis using serine with and without protecting groups for the hydroxy group on the side chain.
Threonine is another amino acid present in three of the four new polypeptides that also contains a side chain having a hydroxy substituent. In some situations the hydroxy group may undergo undesired side reactions. The Peptides on pages 214 through 216 describes peptide synthesis using threonine with and without protecting groups for the hydroxy group on the side chain.
Tryptophan is an amino acid present in the new peptide of Seq.
ID No. 8. Tryptophan is an indole and thus contains an indole nitrogen that can undergo undesired side reactions. Pages 148 through 150 of The Peptides describes peptide synthesis using tryptophan.
Glycine and alanine are relatively simple amino acids common to the presently claimed peptides. Where it is necessary to block the amino terminal, carbobenzoxy groups are employed. Where it is necessary to block the carboxy terminal, a benzyl ester is formed Where it is necessary during peptide synthesis to facilitate the reaction of the C-terminal of a given amino acid or peptide, the activated ester technique as described in The Peetides on pages 97 to 108 may be employed.
Solid Phase Synthesis of the Four Peptides The synthesis of each of the four peptides with the Sequence ID Nos. 2, 4, 6 and 8 according to the instant patent application was carried out. Each of the four peptides was produced by the Automation of Solid Phase Synthesis with the following High Performance Liquid Chromatography (HPLC). See AminoTech. 1991. Biochemical and Reagents for Peptide Synthesis. AminoTech Catalogue, AminoTech, Nepean, Ontario.
The amount of the first HPLC-peptide (product 9410-147, Seq. ID
No. 2) produced equalled 15 mg. The amino acid analysis of this peptide is presented in Table 1.
The amount of the second HPLC-peptide (product 9410-148, Seq.
ID No. 2) produced equalled 20 mg. The amino acid analysis of this peptide is presented in Table 2.
The amount of the third HPLC-peptide {product 9410-149 Seq. ID
No. 6) produced equalled 15 mg. The amino acid analysis of this peptide is presented in Table 3.
The amount of the fourth HPLC-peptide (product 9410-151 Seq.
ID No. 8) produced equalled 50 mg. The amino acid analysis of this peptide - is presented in Table 4.
FINAL REPORT OF AMINO
ACID ANALYSIS FOR
SYNTHETIC PEPTIDE
HAVING SEQ ID NO. 2 Date: 10-3-93 Sample: Peptide ~:
RESIDUES EXPECTED COMPOSITION DETECTED COMPOSITION
Asp/Asn Thr 1 1.05 Ser 1 0.96 Glu/gln 2 2.08 Pro 12 12.48 Gly 1 1.05 A1a 9 9.4 Cys Ilal Met Ile Leu 1 0.95 Tyr Phe His Lys Arg 1 1. 04 Trp the peptide was hydrolyzed for one hour with 6N HCl containing 0.1% phenol at 160°C.
** The composition of the peptide was analyzed on a reverse-phase HPLC column.
FINAL REPORT OF AMINO
ACID ANALYSIS FOR
SYNTHETIC PEPTIDE
HAVING SEQ ID N0.
Date: 10-3-94 Sample: Peptide ~:
RESIDUES EXPECTED COMPOSITION DETECTED COMPOSITION
Asp/Asn 1 0.95 Thr 1 1.04 Ser 1 1.05 Glu/gln 1 0.96 Pro 9 9.38 Gly 1 0.96 Ala 3 3.12 Cys Yal Met Ile Leu 5 4.81 Tyr Phe His Lys Arg 3 3.13 Tr i I I
* The peptide was hydro~yzed for one nour wiin tin nm conia~n~ng 0.1% phenol at 160°C.
** The composition of the peptide was analyzed on a reverse-phase HPLC column.
FINAL REPORT OF AMINO
ACID ANALYSIS FOR
SYNTHETIC PEPTIDE
HAVING SEQ ID N0.
Date: 10-3-94 Sample: Peptide ~:
RESIDUES EXPECTED COMPOSITION DETECTED COMPOSITION
Asp/Asn 1 1.05 Thr 1 0.96 Ser 1 1.05 Glu/gln Pro 3 2.88 Gly 15 15.42 Ala 3 1.03 Cys Yal Met Ile Leu Tyr Phe His Lys Arg 4 4.16 Tr The peptide was hydrolyzed for one hour with 6N HCl containing 0.1% phenol at 160°C.
** The composition of the peptide was analyzed on a reverse-phase HPLC column.
FINAL REPORT OF AMINO
ACID ANALYSIS FOR
SYNTHETIC PEPTIDE
HAVING SEQ ID N0.
Date: 10-3-94 Sample: Peptide ~:
RESIDUES EXPECTED COMPOSITION DETECTED COMPOSITION
Asp/Asn Thr Ser Glu/gln 1 1.04 Pro ' Gly 4 4.18 Ala 4 3.84 Cys Va1 Met Ile Leu Tyr Phe His Lys Arg 18 18.66 Tr 1 1.05 * The peptide was hydroiyzea ror one noun w~Ln an nm coniain~mg O.I% phenol at 160°C.
** The composition of the peptide was analyzed on a reverse-phase HPLC column.
Determination of Immunogenic Activity of the Peptide Vaccine 1. Coupling Peptides to Protein Carriers Ilith 6lutaraldehyde Glutaraldehyde is a bifunctional coupling agent that couples amino groups on the peptide to amino groups on the protein carrier Keyhole Limpet Hemocyanin (KLH). For preparing each complex of peptide-KLH with glutaraldehyde, it was necessary to carry out the following procedures:
(1) 20 mM glutaraldehyde were prepared;
(2) KLH was dissolved in water;
{3) Each of the four peptides was added to the water individually;
{4) The glutaraldehyde was added dropwise with stirring to the water over the course of 5 minutes at room temperature.
Stirring of the solution was continued for another 30 minutes. The solution became yellow.
(5j Glycine was then added to the solution to block any unreacted glutaraldehyde and allowed to remain for 30 minutes;
(6) Excess peptide and reagent were then removed by either exhaustive dialysis in phosphate-buffered saline (see Kagan & Glick 1979.
"Oxyitocin", Methods of Hormone Radioimmunoassay. B.B. Jaffe & H.R. Behrman, eds. pp 328 to 329, Academic Press, NYj.
2. Immunization of Rabbits on the Basis of a Special Schedule of Infections by a Mixture of Equal Amounts of A11 Peptides For the study to determine the inmunogenic activity of the four peptides, a mixture of equal amounts of all four of the peptides with Seq.
ID numbers 2, 4, 6 and 8 with KLH was used. Immunization of two rabbits (designated R76 and R77) was carried out according to the following immunization schedule:
(a) first bleeding;
(b) day 1, first injection;
(c) day 8, second injection;
(d) day 24, third injection;
(e) day 40, fourth injection;
(f) day 55, fifth injection;
(g) from 70th day, second and final bleeding The immunization dose was 0.5 mg per injection.
3. ELISA Titration of Rabbit's Iapmunosera with Synthetic Peptides In the present case for the measurement of antibodies in rabbit sera, each of the four peptide reagents (which are the same four peptides that are the active ingredients in the vaccine) was fixed to a specific plastic microplate, incubated with each -test serum (from Rabbit R76 or R77) (obtained both before and after administering the vaccine) at dilutions of 1:30,000, 1:10,000: 1:3,000 and 1:1,000), washed, and then reinoculated with an anti-immunoglobulin labelled with an enzyme, namely, horseradish peroxidase.
The enzyme activities were measured by adding the substrate for the enzyme and estimating the color reaction in a spectrophotometer. The amount of antibody bound to the absorbed peptide reagents is proportional to the enzyme activity. Once the substrate for the enzyme is added the enzymatic activity is determined and the amount of unknown antibody is determined as a function of the measured enzyme activity.
The ELISA was carried out using the following steps:
1. A 96-well plate was coated with 20 ug/ml of free peptide in 0.01 M sodium phosphate buffer, pH 7.2 containing 0.1 M NaCI (PBS) (50 ul /
well, 4 overnight), 2. The plate was washed twice with PBS and block the wells with TANA'sR blocking solution for one hour at 37° C, 3. The wells were incubated with diluted serum (using l.Ox BSA
/ PBS for dilution, 37° C for 2 - 4 hours), 4. Each well was washed four times with PBS, and then incubated with 1 : 3,000 diluted goat antirabbit IgG-horseradish peroxidase conjugate (TANA Lab., using 1.09 8SA / PBS for dilution) for 1 hour.
The vaccine employed against Marek's Disease Herpesvirus in chickens was derived from Turkey herpesvirus (HVT). There is no indication that a vaccine against atherosclerosis caused by human herpes virus could be prepared. There is certainly no suggestion to employ a herpes vaccine containing homologous peptide sequences to those of the viral DNA found in strains of the herpes virus that effect humans.
U.S. Patent 4,038,381 discloses a vaccine for the prevention and treatment of vascular conditions, comprising a combination of a tuberculosis antigen with an antiherpetic vaccine. There is no suggestion to employ the four polypeptides of the present invention as the active ingredients in the vaccine. The reference also states that the individual tuberculosis antigen and antiherpetic vaccine had no known per se ability in the prevention or treatment of vascular disease.
OBJECT Of THE INVENTION
It is the object of the invention to provide a universal vaccine as a prophylaxis against pathogenic development of several herpes infections and/or atherosclerotic plaque in a mammalian subject susceptible thereto.
Y.. ...
SUMMARY OF THF~~NVENTION
We have found such a vaccine that is effective as a prophylaxis against pathogenic development of several herpes infections and/or atherosclerotic plaque in mammalian subjects, including humans. The vaccine contains four new peptides as described herein below in the indicated proportions:
(a) 10 to 30x by weight of the compound Ala Pro leu Pro Ala Pro Ata Pro Pro Ser Thr Pro Pro Gly Pro Glu Pro Ala Pro Ala Gln Pro Ala Ala Pro Arg Ala Ala (Seq ID 2);
(b) 10 to 3076 by weight of the compound Ala Pro Pro Glu Ala Asp Ala Arg Thr Leu Arg Arg Pro Gly Pro Pro Leu Pro Leu Pro Pro Ser Leu Leu Pro (Seq ID 4);
(c) 10 to 30X by weight of the compound Gly Thr Asp Gly Pro Ala Arg Gly Gly Gly Ser Gly Gly Gly Arg Gly Pro Gly Gly Gly Arg Gly Gly Pro Arg Gly (Seq ID 6); and (d) 10 to 30% by weight of the compound Gly Trp Ala Ala Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Arg Gln Arg Arg Ala Ala Arg Arg Arg Arg (Seq ID 8);
in combination with a pharmaceutically acceptable inert vaccine carrier such as normal saline or a physiological oil (e. g. corn oil, sunflower oil).
Preferably each of the four polypeptides is present in the compositions in equal proportions by weight: that is the compositions preferably contain 25% of each of the four polypeptides.
The compositions are prepared by incorporating each of the four polypeptides in the pharmaceutically acceptable inert vaccine carrier such as normal saline or a physiological oil in an adequate concentration of said polypeptides. Preferably there is present 1.0 to 100 ~g of each polypeptide per ml of pharmaceutical composition. Fore preferably one dose of vaccine (1 ml) contains equal parts (20 beg) of each of the 4 polypeptides. Thus the preferred total amount of polypeptides in one dose of vaccine is 80 fig.
Since each of the polypeptides is itself a new compound, each of them, individually, as well as collectively, is considered to be part of the invention as well.
Also contemplated to be within the scope of the invention is a method of prophylaxis of pathogenic development of several herpes virus infections and/or atherosclerotic plaques in a mammalian subject susceptible thereto which comprises the step of administering to said mammalian subject, a therapeutically effective amount of the pharmaceutical composition containing the four polypeptide sequences as described hereinabove. The herpes infections whose development can be prevented include Herpes Simplex I, Herpes Simplex II, Cytomegalovins, Epstein-Barr Virus, Herpes Zoster and Kaposi~s Sarcoma.
The compositions may preferably be administered to a mammalian subject parenterally, such as by injection. More preferably the compositions are administered by subcutaneous, intramuscular, intra-arterial, intravenous or intradermal injection. A preferred dosage of the compositions is 1 ml every 20 days administered in a series of 6 intramuscular injections. The full cycle of treatment may consist of 2 or 3 such courses with 3 month intervals in between.
Use of an adjuvant, for instance inorganic gels such as alum, aluminum hydroxide or aluminum phosphate that increase antigenic response, is optional in the compositions.
REF DESCRIPTION OF THE DRAW) NGS
The above and other objects, features and advantages will become more readily apparent from the following description, reference being made to the accompanying drawings in which:
Fig. 1 is a map of the expression vector plasmid pRSET (prior art).
Figs. 2 and 3 are series of bar graphs showing the results of an enzyme-linked immunosorbent assay (ELISA) on rabbit sera #76 and #77 obtained from rabbits before and after immunization with the present peptide-containing vaccine using the peptide having Sequence ID #2 to bind the antibodies in the sera to determine antibody formation.
Figs. 4 and 5 are series of bar graphs showing the results of an enzyme-linked immunosorbent assay (ELISA) on rabbit sera #76 and #77 obtained from rabbits before and after immunization with the present peptide-containing vaccine using the peptide having Sequence ID #4 to bind the antibodies in the sera to determine antibody formation.
Figs. 6 and 7 are series of bar graphs showing the results of an enzyme-linked immunosorbent assay (ELISA) on rabbit sera #76 and #77 obtained from rabbits before and after immunization with the present peptide-containing vaccine using the peptide having Sequence ID #6 to bind the antibodies in the sera to determine antibody formation.
Figs. 8 and 9 are series of bar graphs showing the results of an enzyme-linked immunosorbent assay (ELISA) on rabbit sera #76 and #77 obtained from rabbits before and after immunization with the present peptide-containing vaccine using the peptide having Sequence ID #8 to bind the antibodies in the sera to determine antibody formation.
Fig. 10 is a comparative electrophoretic motility study (in 4%
acrylamide gel) of the "DNA-GGC-sites-fragments" per se, with alpha-proteins of nuclei from human blood vessel endothelium (with and without ASP) and with rabbit sera (with and without antibodies against a mixture of several herpes virus peptides).
Lane a "DNA-GGC-sites-fragment" (25,000 cpm, 2.5 ng of DNA) in the absence of added protein extract and any sera;
Lanes b through e: "DNA-GGC-sites-fragments" in the same concentration +
aliquot of the extracts of purified nuclei from human blood vessel endothelium cells with ASP (b, c) + aliquot of the normal (pre-immune) rabbit sera N76(d) and N77(e) in dilution 1:2.
Lanes f and g: "DNA-GGC-sites-fragments" in the same concentration +
aliquot of the extracts of purified nuclei from human blood vessel endothelium cells without ASP.
Lane h: "DNA-GGC-sites-fragments" in the same concentration + k~xture of aliquots of the extract of purified nuclei from endothelial blood vessel cells with ASP + aliquot of the mixture of the post-immune rabbit sera N76 and N77 in dilution 1:100.
lanes i through 1: the same situation as in Lane "h~, but the mixtures of irtmune sera were used in a decreased dilution of 1:50(i), 1:30 0), 1:20(k), and 1:10(1).
Fig. 11 shows the results of another technique of gel retardation experiments in 5% PAAG. The immunosera number 77(N4) and Number 76(N6) (with dilution 1:20) had decreased the formation (amount of DNA protein complex between DNA-GGC-fragment labelled by 32P and alpha-protein nuclei from human blood vessels with ASP - in comparison with corresponding preimmune sera (N5 and N7). On the other side both of inmune sera (N1) and (N2), as well as the preinmune sera number 76(N3) had no influence on the formation of such complexes, if sera were used in dilution of 1:100 Preparation of the Vaccine against Atherosclerosis The vaccine may be prepared by recombinant DNA techniques by chemical synthesis or by automated solid phase synthesis. When preparing the vaccine by recombinant DNA techniques the following steps are employed:
Recombinant DNA Synthesis 1. Accumulation of Virus Particles For isolation of the fragments of DNA that encode peptides having Seq ID 2 and Seq ID 6, it is necessary to accumulate Herpes Simplex Virus Type 1, and for isolation of the fragments of DNA that encode peptides having Seq ID 4 and Seq ID 8, it is necessary to accumulate Human Cytomegalovirus.
Herpes Simplex Yirus 1 and Human Cytomegalovirus are each cultivated in diploid human embryonic lung cells (HECL).
Tissue Cultures For isolation of Herpes Simplex Yirus 1 and Human Cytomegalovirus, it is necessary to use diploid human embryonic lung cells (e. g. semi-continuous cells). These cells are derived from embryonic lung tissue and following initial dispersal, they can be redispersed and regrown many times (30 to 50 times). Human embryonic lung tissue, which can be obtained from embryos of 10 to 12 weeks, provide a most valuable source for harvesting a number of different herpes viruses, including Herpes Simplex Viruses and Human Cytomegalovirus.
Semi-continuous cells have a normal chromosome count (diploid) and show the phenomenon of contact inhibition. An inoculum of each virus listed above is placed on the monolayer and allowed to absorb for 1 hour.
It is then removed and fresh medium is added. Cultures are incubated at 37'C and they are inspected regularly by microscopy for evidence of virus growth. The culture medium is normally changed on the day after inoculation to minimize the effect of toxins that may persist in the inoculum, and is then replaced periodically to replenish the supply of nutrients for the cells. Cultures are incubated for various lengths of time depending on the virus. While the cytopathic effects of a concentrated inoculum of herpes virus may appear overnight, a low level of cytomegalovirus may take 3 to 4 weeks to appear.
The cells infected by herpes viruses may be cultivated in suspension also.
For inoculation of the tissue cultures to prepare the peptide vaccines, the following viruses may be used:
Herpes Simplex Type 1 (Human herpesvirus 1, Herpesvirus homines type 1) ATCC YR-539, Strain MacIntyre.
Cytomegalovirus ATCC YR-538 Strain: AD-1fi9.
2. isolation of the following four nucleotide Sequences from the Viral DNA that Code for the Four peptides indicated above:
Ala Pro Leu Pro Ala Pro Ala Pro Pro Ser Thr Pro Pro Gly Pro Glu CCC GCC CCC GCC CAG CCC GCG GCG CCC CGG GCC GCC 84 (Seq ID 1);
Pro Ala Pro Ala Gln Pro Ala Ala Pro Arg Ala Ala Ala Pro Pro Glu Ala Asp Ala Arg Thr Leu Arg Arg Pro Gly Pro Pro CTG CCG CTG CCG CCT TCC CTT CTC CCG 75 (Seq ID 3);
Leu Pro Leu Pro Pro Ser Leu Leu Pro Gly Thr Asp Gly Pro Ala Arg Gly Gly Gly Ser Gly Gly Gly Arg Gly CCC GGT GGC GGA AGA GGT GGC CCC CGC GGG 78 (Seq ID 5); and Pro Gly Gly Gly Arg Gly Gly Pro Arg Gly Gly Trp Ala Ala Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Gly Arg CGT CGC CGC CAG CGG CGA GCG GCA CGG AGA CGG AGG 84 (Seq ID 7) Arg Arg Arg Gln Arg Arg Ala Ala Arg Arg Arg Arg .
Note the DNA sequences assigned Seq ID numbers 1, 3, 5, and 7 are the gene sequences containing the codons to obtain the peptides having Seq ID Nos. 2, 4, 6, and 8. The DNA fragments having Seq ID Nos 1,3,5, and 7 are regarded as novel intermediate compounds that constitute part of the present invention.
The Origin and Utility of these Four Oligonucleotides The Oligonucieotide of Seq ID 1 is the part of the Herpes Simplex Virus Type 1 immediate early (IE) gene 3 for the transcriptional activator IE 175 (= ICP 4). Its 84 nucleotides are located from by 3760 up to 3844 according to the known gene nucleotide sequence Herpes Simplex Virus Type 1, Viridae; DS-DNA Enveloped Viruses; Herpes Viridae, Alpha-Herpes Virinae. This same oligonucleotide may also be found in the complete short unique region 2 with partial terminal and inverted repeats in DNA, HSV 1, Strain 17.
The oligonucleotide of Seq ID 3 is the part of the Human Cytomegalovirus (Strain AD 169) complete genome (from base pair 70001 up to base 80100). Its 75 nucleotides are located from by 2342 up to 2416 in this region of the Human Cytomegalovirus gene Strain AD=169 according to Human Cytomegalovirus, Viridae, DS-DNA Enveloped Viruses, Herpesviridae, Betaherpesvirinae.
The oligonucleotide of Seq ID 3 is in the Human Cytomegalovirus F fragment DNA encoding DNA Polymeraseycoprotein B also and has homology with DNA from the following viruses:
(a) Epstein-Barr Virus, artifactual joining of B95-8 complete gene and the sequences from raga of the large deletion found in B95-8 (from base pair 70001 to 80100) about 70X.
{b) HSV1 (strain 17) complete short unique region with inverted repeat DNA, (from by 10001 to 20100) about 65X.
(c) HSV2 ORF1, ORF2, and ORF 3 (LAT) gene about 65X.
The oligonucleotide with Seq ID5 is the part of Herpes Simplex virus type one (NSY 1) latency associated transcript (LAT).
Its 78 nucleotides are localized from 2255 up to 2332 positions of LAT gene according to Herpes Simpiex Virus Type 1, Yiridae, DS-DNA
Enveloped Viruses; Herpes Viridae, Alpha-Herpesvirinae.
This oligonucleotide has homology:
with Herpes Simplex virus type 1 Bam H1 fragment 8 DNA sequence - about 97.5%:
with Herpes Simplex virus type 1 gene encoding two latency -related proteins - about 97.5%;
with Pseudorabies virus immediate - early gene -about 70%;
with Herpes Simplex virus type 2 ORF1, ORF2, and ORF3 (LAT) gene- about 70%;
with Epstein-Barr virus, artifactual joining of B95 - 8 complete genome and the sequences from ra3i of the large deletion found in B95 - 8 (from base 70001 to 80100 - about 65.5X;
with Bovine Herpesvirus type 1 early - intermediate transcription control protein_(BICP4) gene - about 70%;
with Human Cytomegalovirus UL56 gene - 67.5%;
The oligonucleotide with Seq ID 7 is part of Human Cytomegalovirus (HCMV) short unique region, short repeats, and part of long repeat (from base 1 to base 10100).
Its 84 nucleotides are localized from 5340 up to 5424 positions in this part of genome according to Human Cytomegalovirus, Viridae; DS-DNA Enveloped Virules; Herpes Viridae;
Betaherpesvirinae This oligonucleotide has homology:
with Equine Herpesvirus 4 (EHV4) genome, thymidine kinase (TK) and glycoprotein H (GH) genes - about 71%
with Herpes Simplex virus type 2 immediate - early (IE5) protein mRNA, 5' end - about 65%;
with Herpes Simplex virus type 1 complete genome from base 70001 to base 80100 - about 65.5%.
Isolation of the oligonucleotides sequences from the viral DNA
that code for the four peptides indicated above.
The viral DNA is isolated from corresponding viral suspension obtained from HELL infected by Herpes Simplex 1 or Human Cytomegalovirus (see above about cell cultures and virus strains), purified by agarose gel electrophoresis (AGE) according to Myers, R.M. et al, "Detection and Localization of Single Base Changes by Denaturing Gradient Gel Electrophoresis; Methods Enzymol. 155:501 to 527 (1987) and Myers et al, "In Genome Analysis: A Practical Approach" (Ed. K. Oavies) p. 95 IRL Press, Oxford (1988), treated by restriction enzymes and subjected by polyacrylamide gel electrophoresis.
The isolation of oligonucleotide with Seq ID 1 from Herpes Simplex virus type I immediate early (IE) gene 3 for transcriptional activator IE 175 (= ICP4) is produced after treating DNA by restriction enzyme Ncol with recognition sequence CGATGG. This procedure according to PAAG, EF gives the polynucleotide with 2308 pair of bases (from 2637 up to 4935 position) with the actual fragment located from 3760 up to 3844 position.
The isolation of oligonucleotide with Seq ID 3 from HCMV
(strain AD169) complete genome is produced after treating of DNA by restriction enzyme Bst E III with recognition sequence GATC. This procedure after PAAG, EF gives the polynucleotide with 2182 pair of base -from 715 to 2898 position - with the actual fragment from 2342 up to 2416 position.
The isolation of oligonucleotide with Seq. ID5 from HSVI (LAT) is produced after treating of DNA by restriction enzyme Nco 1 with recognition sequence CCATGG. This procedure after PAAG, EF gives the polynucletide with 1737 pair of base -from 659 up to 2396 position - with actual fragment from 2255 up to 2332 position.
The isolation of oligonucleotide with Seq IO 7 from HCMV -short unique region, short repeats, and part of long repeat is produced after treating of DNA by restriction enzyme Nco 1 also. This procedure after PAAG, EF gives the polynucleotide with 4807 pair of base with actual fragment from 5340 up to 5424.
The structures of the Herpes Simplex Virus Type I Immediate Early (IE) Gene 3 for Transcriptional Activator IE 175; HCMY (Strain AD
169); HSVI (LAT); and HCMY - Short Unique Region are known in the art and may be found in EMBL-37.
The construction of recanbinant plasmid DNA for expression of the four peptides, with Seq Nos. 2, 4, 6 and 8.
All 4 oligonucleotides are introduced into a polylinker of plasmid pRSET with sites of restriction, including Nco 1 and Bst E III.
This plasmid may be used to transform E.Coli (AB 109 strain). For this aim bacteria are treated with CaCl2 which makes their membranes slightly permeable (competent bacteria). The transformed bacteria are then selected by growing them on a medium containing ampicillin. pRSET is a commercial multicopy expressing vector with a high level of protein expression.
Chemical Synthesis The four sequences having the formulae Seq. Nos. 2,4,6 and 8 may also be prepared by direct peptide synthesis that is well known in the art according to the syntheses employed in The Pegtides, Schroeder and Luebke, Yol. I, Methods of Peptide Synthesis, Academic Press (1965).
Preferably each of the four polypeptides having Seq Nos. 2,4,6 and 8 are synthesized starting from the amino terminal acid and forming the peptide bond between the carboxy terminal of the given amino acid and the amino terminal of the next given amino acids. This procedure is carried out until all of the amino acids needed to make each of the four peptides are formed into whole chains.
Where it is necessary to employ an amino-protecting group to protect an N-terminal amino substituent to carry out the synthesis of one or more of the four above-mentioned peptides, the approaches of pages 3 through 51 of The Pegtides may be employed. Where it is necessary to employ a carboxy-protecting group to protect either a C-terminal carboxy group or a carboxy group forming part of a side chain (i.e. Glu, Asp) to carry out the synthesis of one or more of the four above-mentioned polypeptides, the methods of page 52 through 75 of the reference are employed.
Glycine and alanine are relatively simple amino acids common to the presently claimed peptides. Where it is necessary to block the amino terminal, carbobenzoxy groups are employed. Where it is necessary to block the carboxy terminal, a benzyl ester is formed. See pages 137 and 138 of The Pe tip ides.
In fact the information regarding blocking the C- and N-terminals of simple amino acids such as glycine and alanine without highly reactive side chains is still highly relevant to the blocking of all amino acids involved in the synthesis of the peptides of the present invention.
One amino acid common to all four peptides of the Seq Nos 2, 4, 6 and 8 is arginine. Arginine has a guanido group on its side chain and sometimes this group may be responsible for undesired side reactions.
Pages 167 through 174 of The Pe~.tides discusses peptide synthesis using a number of different blocking groups to protect the guanido side chain.
Pages 175 and 176 discuss peptide synthesis involving arginine where the guanido side chain need not be blocked.
Another amino acid that is well represented among the four peptides of this invention is proline. Proline is a heterocyclic amino acid with an amino functional group. Where it is necessary to block the amino group, pages 146 through 148 of The Peptides provides details.
Serine is an amino acid present in three of the four new peptides. Serine contains a side chain that includes a hydroxy group. In some situations the hydroxy group may undergo undesired side reactions.
The Peptides on pages 207 through 214 describes peptide synthesis using serine with and without protecting groups for the hydroxy group on the side chain.
Threonine is another amino acid present in three of the four new polypeptides that also contains a side chain having a hydroxy substituent. In some situations the hydroxy group may undergo undesired side reactions. The Peptides on pages 214 through 216 describes peptide synthesis using threonine with and without protecting groups for the hydroxy group on the side chain.
Tryptophan is an amino acid present in the new peptide of Seq.
ID No. 8. Tryptophan is an indole and thus contains an indole nitrogen that can undergo undesired side reactions. Pages 148 through 150 of The Peptides describes peptide synthesis using tryptophan.
Glycine and alanine are relatively simple amino acids common to the presently claimed peptides. Where it is necessary to block the amino terminal, carbobenzoxy groups are employed. Where it is necessary to block the carboxy terminal, a benzyl ester is formed Where it is necessary during peptide synthesis to facilitate the reaction of the C-terminal of a given amino acid or peptide, the activated ester technique as described in The Peetides on pages 97 to 108 may be employed.
Solid Phase Synthesis of the Four Peptides The synthesis of each of the four peptides with the Sequence ID Nos. 2, 4, 6 and 8 according to the instant patent application was carried out. Each of the four peptides was produced by the Automation of Solid Phase Synthesis with the following High Performance Liquid Chromatography (HPLC). See AminoTech. 1991. Biochemical and Reagents for Peptide Synthesis. AminoTech Catalogue, AminoTech, Nepean, Ontario.
The amount of the first HPLC-peptide (product 9410-147, Seq. ID
No. 2) produced equalled 15 mg. The amino acid analysis of this peptide is presented in Table 1.
The amount of the second HPLC-peptide (product 9410-148, Seq.
ID No. 2) produced equalled 20 mg. The amino acid analysis of this peptide is presented in Table 2.
The amount of the third HPLC-peptide {product 9410-149 Seq. ID
No. 6) produced equalled 15 mg. The amino acid analysis of this peptide is presented in Table 3.
The amount of the fourth HPLC-peptide (product 9410-151 Seq.
ID No. 8) produced equalled 50 mg. The amino acid analysis of this peptide - is presented in Table 4.
FINAL REPORT OF AMINO
ACID ANALYSIS FOR
SYNTHETIC PEPTIDE
HAVING SEQ ID NO. 2 Date: 10-3-93 Sample: Peptide ~:
RESIDUES EXPECTED COMPOSITION DETECTED COMPOSITION
Asp/Asn Thr 1 1.05 Ser 1 0.96 Glu/gln 2 2.08 Pro 12 12.48 Gly 1 1.05 A1a 9 9.4 Cys Ilal Met Ile Leu 1 0.95 Tyr Phe His Lys Arg 1 1. 04 Trp the peptide was hydrolyzed for one hour with 6N HCl containing 0.1% phenol at 160°C.
** The composition of the peptide was analyzed on a reverse-phase HPLC column.
FINAL REPORT OF AMINO
ACID ANALYSIS FOR
SYNTHETIC PEPTIDE
HAVING SEQ ID N0.
Date: 10-3-94 Sample: Peptide ~:
RESIDUES EXPECTED COMPOSITION DETECTED COMPOSITION
Asp/Asn 1 0.95 Thr 1 1.04 Ser 1 1.05 Glu/gln 1 0.96 Pro 9 9.38 Gly 1 0.96 Ala 3 3.12 Cys Yal Met Ile Leu 5 4.81 Tyr Phe His Lys Arg 3 3.13 Tr i I I
* The peptide was hydro~yzed for one nour wiin tin nm conia~n~ng 0.1% phenol at 160°C.
** The composition of the peptide was analyzed on a reverse-phase HPLC column.
FINAL REPORT OF AMINO
ACID ANALYSIS FOR
SYNTHETIC PEPTIDE
HAVING SEQ ID N0.
Date: 10-3-94 Sample: Peptide ~:
RESIDUES EXPECTED COMPOSITION DETECTED COMPOSITION
Asp/Asn 1 1.05 Thr 1 0.96 Ser 1 1.05 Glu/gln Pro 3 2.88 Gly 15 15.42 Ala 3 1.03 Cys Yal Met Ile Leu Tyr Phe His Lys Arg 4 4.16 Tr The peptide was hydrolyzed for one hour with 6N HCl containing 0.1% phenol at 160°C.
** The composition of the peptide was analyzed on a reverse-phase HPLC column.
FINAL REPORT OF AMINO
ACID ANALYSIS FOR
SYNTHETIC PEPTIDE
HAVING SEQ ID N0.
Date: 10-3-94 Sample: Peptide ~:
RESIDUES EXPECTED COMPOSITION DETECTED COMPOSITION
Asp/Asn Thr Ser Glu/gln 1 1.04 Pro ' Gly 4 4.18 Ala 4 3.84 Cys Va1 Met Ile Leu Tyr Phe His Lys Arg 18 18.66 Tr 1 1.05 * The peptide was hydroiyzea ror one noun w~Ln an nm coniain~mg O.I% phenol at 160°C.
** The composition of the peptide was analyzed on a reverse-phase HPLC column.
Determination of Immunogenic Activity of the Peptide Vaccine 1. Coupling Peptides to Protein Carriers Ilith 6lutaraldehyde Glutaraldehyde is a bifunctional coupling agent that couples amino groups on the peptide to amino groups on the protein carrier Keyhole Limpet Hemocyanin (KLH). For preparing each complex of peptide-KLH with glutaraldehyde, it was necessary to carry out the following procedures:
(1) 20 mM glutaraldehyde were prepared;
(2) KLH was dissolved in water;
{3) Each of the four peptides was added to the water individually;
{4) The glutaraldehyde was added dropwise with stirring to the water over the course of 5 minutes at room temperature.
Stirring of the solution was continued for another 30 minutes. The solution became yellow.
(5j Glycine was then added to the solution to block any unreacted glutaraldehyde and allowed to remain for 30 minutes;
(6) Excess peptide and reagent were then removed by either exhaustive dialysis in phosphate-buffered saline (see Kagan & Glick 1979.
"Oxyitocin", Methods of Hormone Radioimmunoassay. B.B. Jaffe & H.R. Behrman, eds. pp 328 to 329, Academic Press, NYj.
2. Immunization of Rabbits on the Basis of a Special Schedule of Infections by a Mixture of Equal Amounts of A11 Peptides For the study to determine the inmunogenic activity of the four peptides, a mixture of equal amounts of all four of the peptides with Seq.
ID numbers 2, 4, 6 and 8 with KLH was used. Immunization of two rabbits (designated R76 and R77) was carried out according to the following immunization schedule:
(a) first bleeding;
(b) day 1, first injection;
(c) day 8, second injection;
(d) day 24, third injection;
(e) day 40, fourth injection;
(f) day 55, fifth injection;
(g) from 70th day, second and final bleeding The immunization dose was 0.5 mg per injection.
3. ELISA Titration of Rabbit's Iapmunosera with Synthetic Peptides In the present case for the measurement of antibodies in rabbit sera, each of the four peptide reagents (which are the same four peptides that are the active ingredients in the vaccine) was fixed to a specific plastic microplate, incubated with each -test serum (from Rabbit R76 or R77) (obtained both before and after administering the vaccine) at dilutions of 1:30,000, 1:10,000: 1:3,000 and 1:1,000), washed, and then reinoculated with an anti-immunoglobulin labelled with an enzyme, namely, horseradish peroxidase.
The enzyme activities were measured by adding the substrate for the enzyme and estimating the color reaction in a spectrophotometer. The amount of antibody bound to the absorbed peptide reagents is proportional to the enzyme activity. Once the substrate for the enzyme is added the enzymatic activity is determined and the amount of unknown antibody is determined as a function of the measured enzyme activity.
The ELISA was carried out using the following steps:
1. A 96-well plate was coated with 20 ug/ml of free peptide in 0.01 M sodium phosphate buffer, pH 7.2 containing 0.1 M NaCI (PBS) (50 ul /
well, 4 overnight), 2. The plate was washed twice with PBS and block the wells with TANA'sR blocking solution for one hour at 37° C, 3. The wells were incubated with diluted serum (using l.Ox BSA
/ PBS for dilution, 37° C for 2 - 4 hours), 4. Each well was washed four times with PBS, and then incubated with 1 : 3,000 diluted goat antirabbit IgG-horseradish peroxidase conjugate (TANA Lab., using 1.09 8SA / PBS for dilution) for 1 hour.
5. Each well was washed four times with PBS, and then incubated with TANA'sR calorimetric ELISA substrate (tetra methylbenzene /
H202 solution), 6. The enzyme reaction was stopped with TANA'sR ELISA-stopping buffer {diluted phosphoric acid), 7. The plates were read using 450 nm.
On the base of this technique these data were obtained:
FIG. 2. Binding of rabbit #76 serum to first peptide with Seq.
ID No. 2.
FIG. 3. Binding of rabbit #77 serum to first peptide with Seq.
ID No. 2.
FIG. 4. Binding of rabbit #76 serum to second peptide with Seq. ID No. 4.
FIG. 5. Binding of rabbit #77 serum to second peptide with Seq. ID No. 4.
FIG. 6. Binding of rabbit #76 serum to third peptide with Seq.
ID No. 6. -FIG. 7. Binding of rabbit I77 serum to third peptide with Seq.
ID No. fi.
FIG. 8. Binding of rabbit X76 serum to fourth peptide with Seq. ID No. 8.
FIG. 9. Binding of rabbit X77 serum to fourth peptide with Seq. ID No. 8.
In both rabbit sera (R76 and R77) the high levels of antibodies against each of our four peptides were determined in contrast with these sera before immunization.
On the basis of ELISA titrations, it was found that two rabbits produced high titer antibodies against each of the four tested peptide reagents with Seq. ID No. 2, 4, 6 and 8. The results are presented in Figures 2 through 9. Note the large difference in the inmunogenic activity in the rabbit blood before and after the peptide-containing vaccine was administered to the rabbits.
In two of such sera from iamunized rabbit N76 (R76) and immunized rabbit N77 (R77) and in pre-immune (normal) sera (R78) in the Laboratory of Microbiological Associates, Inc. (MA) the level (titers) of antibodies were determined against:
Herpes Simplex Virus 1 (HSY1) - on the basis of MA'sELISA
Herpes Simplex Virus 2 (HSV2) - on the basis of Whittaker kit, ELISA, Human Cytomegalovirus (HCMY) - on the basis of Whittaker kit, ELISA, Yaricella Zoster Virus (YZY) on the basis of MA's IFA, Epstein - Bar Virus (EBV) - on the basis of MA's. IFA.
Titers were determined by comparison with the mean + 99%
confidence interval for control (normal, pre-immune) serum R78 (See: Tables through 8).
As one can see, strong differenti-al reactivity was obtained for:
R76 - against HSY-1 + HSY-2 and HCMV, R77 - against HSY-1 + HSV-2 and HCMY.
Neither antibody showed reactivity against EBV (YCA) antigen.
Although reactivity against VZV was relatively strong for R76 and R77, the R78 titer was also elevated and, therefore, did not allow low titered differential results.
Tables 5, 6, 7 and 8 showing the 95x and 99X confidence intervals for R78 titration in each assay follow hereinbelow:.
-- CONFIDENCE INTERVALS (POOLED) --Var MEAN STD LOWER UPPER LOWER UPPER
Name ERR 95% 95% 99% 99%
0.156 0.073 -0.022 0.333 -0.114 0.425 0.224 0.073 0.046 0.401 -0.046 0.493 40 0.166 0.073 -0.012 0.343 -0.104 0.435 80 0.15 0.073 -0.028 0.328 -0.119 0.419 160 0.193 0.073 0.015 0.37 -0.077 0.462 320 O.I1 0.073 -0.068 0.287 -0.16 0.379 640 0.104 0.073 -0.074 0.282 -0.165 0.373 1280 0.128 0.073 -0.05 0.306 -0.141 0.397 -- CONFIDENCE INTERVALS (POOLED) --VAR MEAN STD LOWER UPPER LOWER UPPER
NAME ERR 95% 95% 99% 99%
10 1.153 0.041 1.053 1.252 1.001 1.304 20 0.657 0.041 0.557 0.756 0.505 0.808 0.534 0.041 0.434 0.634 0.383 0.685 80 0.304 0.041 0.204 0.404 0.153 0.455 160 0.226 0.041 0.126 0.326 0.075 0.377 320 0.181 0.041 4.081 0.28 0.029 0.332 640 0.15 0.041 0.05 0.249 -0.002 0.301 1280 0.13 0.041 0.03 0.23 0.021 0.281 -- CONFIDENCE INTERVALS (POOLED) --VAR MEAN STD LOWER UPPER LOWER UPPER
NAME ERR 95% 95% 99% 99%
0.929 0.049 0.809 1.048 0.747 1.11 0.561 0.049 0.441 0.68 0.379 0.742 40 0.368 0.049 0.248 0.487 0.186 0.549 80 0.261 0.049 0.141 0.381 0.08 0.442 160 0.171 0.049 0.051 0.29 -0.011 0.352 320 0.124 0.049 0.004 0.244 -0.057 0.305 640 0.102 0.049 -0.018 0.221 -0.08 0.283 1280 0.104 0.049 -0.015 0.224 -0.077 0.286 STUDY - VZV
-- CONFIDENCE INTERVALS (POOLED) --VAR MEAN STD LOWER UPPER LOWER UPPER
NAME ERR 95x 95% 99% 99X
1.364 0.155 0.966 1.762 0.74 1.988 1.077 0.155 0.679 1.474 0.453 1.7 40 1.069 0.155 0.671 1.467 0.445 1.693 80 0.632 0.155 0.234 1.029 0.008 1.255 160 0.44 0.155 0.042 0.837 -0.184 1.063 320 0.454 O.I55 0.056 0.851 -0.17 1.077 640 0.145 0.155 -0.253 0.543 -0.479 0.769 1280 0.529 0.219 -0.033 1.091 -0.353 1.411 The immunogenisity of each herpetic peptide and its mixtures as a result of ELISA-titration of irtmune rabbit sera with several members of Herpesvirus family.
Ser/Yir HSV1 HSY2 HSY1+2 HCMV EBV VZV MDY
Anti-P1 3000 3000 0 10000 10000 10000 3000 Anti-P2 0 0 0 1000 0 3000 3000 Anti-P3 3000 3000 1000 3000 10000 3000 3000 Anti-P4 3000 3000 1000 3000 10000 3000 10000 Anti-P5 0 0 0 0 3000 0 0 Anti-4P 3000 3000 0 1000 10000 3000 10000 Anti-5P 0 0 0 1000 0 0 3000 Contr.ser 0 0 0 0 0 0 0 At present we are providing some additional data concerning the antibody titers in sera of rabbits who have been immunized with the polypeptide vaccine according to the present invention.
After immunization of the rabbits by the polypeptide vaccine containing the four polypeptides we have obtained the next level of specific antibodies against the different herpes viruses in an ELISA test;
against Herpes Simplex Virus I 1:3000 against Herpes Simplex Yirus II 1:3000 against Human Cytomegalovirus 1:1000 against Epstein-Barr Yirus 1:10,000 against Yaricella-Zoster Virus 1:3,000 against Marek~s Disease Virus 1:10,000.
In these experiments we have used the homogenates of cell cultures infected by several herpes viruses (from American Cell Culture Collection) as antigens for ELISA titration.
Thus, the data demonstrate that "antibody titers increase not only over control after multiple infections of the pharmaceutical composition..." but also possess the real protective activity of these sera against all actual members of the Herpes Viridae family.
The data presented above show that the new vaccine according to the present invention displays a real immunogenic activity, and a protective activity and this activity shows the effectiveness of the new peptide -- containing vaccine to stimulate herpes antibody production against all herpes viruses. The vaccine may be designated as a polyvalent or universal herpes vaccine EFFICACY OF THE PEPTIDE-CONTAINING VACCINE TO PREVENT THE .
DEVELOPMENT OF ATHEROSCLEROTIC PLAQUE
Background Information It is known in the art that an increase of G proteins in the blood (Gs proteins) that stimulate the production of cyclic AMP prevents the development of atherosclerotic plaque. The new vaccine containing the polypeptides with Seq. ID Nos. 2, 4, 6 and 8 prevents the development of atherosclerotic plaque by stimulating the production of Gs proteins.
Atherogenesis is a consequence of persistent herpes virus infection development in blood vessel walls. In the course of this development, synthesis of a considerable number of virus-specified regulator proteins (transcriptional factors) takes place in the vessel -walls. Some of these regulator proteins repress viral DNA replication and transcription, thus preventing a chronic herpes viral infection from becoming an acute infection. Owing to the homology between certain sections of the alpha subunit of the Gs protein gene and those of the genes of a number of herpes viruses, individual virus-specific transcription factors bind with GGC-GGC-GGC sections in the alpha subunit of the 6s protein gene and suppress the transcription of this gene. This in turn, decreases translation and synthesis of the Gs protein. A decrease in Gs protein production reduces cyclic AMP formation. This causes a reduction of the synthesis of cholesterol ether hydrolase, which in turn increases - atherogenesis.
The antibodies generated in the rabbit in response to the in~ect9on of our polypeptide vaccine bind with one of the transcription factors described above, this factor being related to the GGC-GGC-GGC sites in the Gs protein gene. These antibodies prevent the emergence of a complex between the Gs protein gene site and the abovementioned transcription factor. The antibodies prevent the inhibitory influence of the persistent herpes virus infection on the synthesis of Gs proteins, thus mitigating the extent of atherogenesis.
Experimental Section The prevention of the formation of the DNA-protein complex containing DNA from the Gs protein gene and protein from the herpes transcription factor by means of the antibodies generated by administration of our peptide vaccine to rabbits is illustrated with the results of the model experiments described below.
The study of DNA-protein interaction has been conducted by the shift-mobility assay method.
Shift Mobility Assay A1. Extraction of Alpha-Protein-from Nuclei Purified nuclei from normal blood vessel endothelium and blood vessel endothelium with atherosclerotic plaques (ASP) were extracted with 0.35 M NaCI. Purified nuclei were pelleted by low-speed centrifugation and re-suspended by vortexing to a final DNA concentration of 0.4 mg/ml in 0.35 M NaCI, 5 mM Na-EDTA, 10 mM 2-mercaptoethanol, 10 mM Tris-HC1 (pH 7.5) containing the five proteinase inhibitors (Phenylmethylsulfonyl fluoride - (PMSF), antipain, leupeptin, chymostatin, and pepstatin A) at the concentration used in the nuclei isolation. After 30 minutes at 30'C with occasional vortexing, the suspension was centrifuged at 10,000 x g for 15 minutes. The supernatant containing the alpha-protein was used either immediately or after storage at -70°C in the presence of 15% glycerol.
These alpha-proteins are new factors of herpes virus transcription and are different from the heretofore known factors of herpes virus transcription such as Spll, Ergl, Wtl and others. The new transcriptional factors have the ability to make the stable DNA-protein complex in vitro in the experimental model system (as described hereinbelow) and in the human organism as well as with a specific site on the alpha-subunit of the Gs proteins.
A2. Preparation of DNA-Fragments with GGC-Sites from the Promoter Region of Mouse Ribosome Protein Gene Fragments containing GGC sites were isolated from the promoter region of mouse ribosome protein gene following complete digestion of purified mouse DNA with either Hind III or Mbo II restriction enzymes. The fragments were purified separately by preparative electrophoresis in 6%
polyacrylamide eluted from the gels, and thereafter separately end-labeled with 32P by T4 polynucleotide kinase and gamma-32P-ATP, following a treatment with bacterial alkaline phosphatase (Maxam and Gilbert, 1980).
The labelled DNA was purified by extractions with chloroform:isopropanol (24:1 by weight) in the presence of 1% SDS, 1M NaCI and ethanol-precipitated in the presence of 10 mg/ml of linear polyacrylamide as a carrier. Equal amounts of the 32P-counts of the Hind III and MboII
produced fragments of DNA were then mixed together to yield the final DNA
sample with the GGC-GGC-GGC sites.
A3. Detection of the DNA-Protein Interactions by the Shift Mobility Assay The "DNA-Protein" interactions were investigated by the Shift-Mobility Assay on a low ionic strength 4% polyacrylamdide gel (See FIG.
10).
As it is shown, pure DNA-fragment DNA (see A2) in the absence of any addition of protein extract, migrates in the gel in a discrete band.
See FIG. 10, Lane "a", while in the presence of protein extract (extracted from the purified nuclei of blood vessel endothelium with ASP), most of the DNA failed to enter the gel. See FIG. 10, Lanes "b" and "c". The same result was shown with a mixture of "DNA" plus "ASP" plus rabbit sera where the rabbit sera did not contain the antibodies against the herpes peptides generated by the rabbit upon administration of the polypaptide vaccine.
See FIG. l0, Lanes "d" and "e".
In the presence of an aliquot of extracts of purified nuclei of human blood endothelum without ASP (normal endothelium "NE") the "DNA"
migrated to an intermediate extent. See Lanes "f" and "g" of FIG. 10.
In the presence of "ASP" and rabbit immune sera ("IS") in dilution 1:50 to 1:10 "DNA" oigrates as a discrete band as pure DNA (see FIG. 10, Lanes "i" through "1" and compare with the results in Lane "a", which shows pure "DNA" without protein or immune sera). In the case of the addition to the complex of "DNA" plus "ASP" the "IS" in dilution 1:100 and 1:50, the "DNA" migrates to an intermediate extent. See FIG. 10, Lane "h"
Further analysis of the shift mobility assay as shown in Fig.
is as follows:
Fig. 10 shows the electrophoretic mobility in 4& polyacrylamide gel of the "DNA-GGC sites fragments", with alpha proteins of nuclei from human blood vessel endothelium (with and without ASP) and with rabbit sera (with and without antibodies generated by administration of the present polypeptide vaccine to rabbits).
Lane "a" "DMA-GGC fragment" (25,000 cpm, 2.5 ng of DNA) in the absence of added protein extract and any sera.
Lanes "b" through "e" "DNA-GGC site fragments" in the same concentration + aliquot of the extracts of purified nuclei from human blood vessel endothelium cells with ASP (b, c) and + aliquot of the normal (pre-immune) rabbit sera - N76{d) and N77(e) in dilution 1:2.
Lanes "f" and "g" "DNA-GGC- sites fragments" in the same concentration + aliquot of the extracts of purified nuclei from normal human blood vessel endothelial cells without ASP.
Lane "h" "DNA-GGC sites fragment" in the same concentration + a mixture of aliquots of the extract of purified nuclei from endothelial -blood vessel cells with ASP + aliquot of the mixture of the post-immune rabbit sera N76 and N77 in a dilution of 1:100.
Lanes "I" through ~1" are the same situation as in Lane "h", but the mixture of immune sera was used in a dilution of 1:50(I); 1:30 0 );
1:20(k); and 1:10(1) These data show that the usage of immune rabbit sera against several herpesvirus peptides can prevent formation of the complex between DNA GGC-GGC-GGC binding sites on the mouse ribosomal protein gene which shares this binding site in common with the genes expressing the Gs proteins and the protein extract from the nuclei of blood vessel endothelium with ASP" and consequently prevents the development of ASP".
In the experiment presented above the antibodies in the immune rabbit sera decrease the formation of the DNA-protein complex by more than 90% according to the data in Figure 10 and that such a decrease in the formation of the complex will facilitate the in vivo expression of the Gs protein genes to produce Gs proteins which in turn leads to increased cyclic AMP production and less formation of atherosclerotic plaque (ASP);
We performed an additional experiment using gel retardation to analyze the mobility of the "DNA-GGC sites fragment" in 5% PAAG. The results are shown in Fig. 11.
According to Fig. 11, the irtununosera number 77 (N4) and number 76 (N6) (with dilution 1:20) had decreased the formation (amount) of DNA-protein complex between DNA-GGC-fragment labelled by 32P and the alpha-protein nuclei from human blood vessel cells with ASP in comparison with the amount of the DNA-protein complex when the corresponding pre-immune sera N5 and N7 were employed instead.
WO 97/34630 . PCT/US97/05282 Both of the immune sera N1 and N2 as well as the preimnune sera 76 (N3) had no influence on the formation (amount) of the DNA-protein complex, when such sera were used in a dilution of 1:100.
These additional data confirm that the use of inmune rabbit sera containing antibodies generated by administration of the present peptide vaccine, can prevent the formation of a DNA-protein complex between DNA-GGC site fragments characteristic of the gene that expresses the Gs proteins and protein extract the nuclei of blood vessel endothelium with ASP, characteristic of the herpes transcriptional factor, and consequently the polypeptide vaccine can prevent the development of ASP.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
{i) APPLICANT: 6olubev, Daniel B. ET AL
(ii) TITLE OF INVENTION: PEPTIDE VACCINE TO PREVENT DEVELOPMENT OF
SEVERAL iiERPES INFECTIONS AND/OR ATHEROSCLEROTIC PLAQUE
ATHEROSCLEROSIS
(iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Karl F. Ross, PC
(B) STREET: 5676 Riverdale Ave.
(C) CITY: Bronx (D) STATE: NY
(E) COUNTRY: USA
(F) ZIP: 10471-0900 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release X1.0, Version X1.25 (vi) CURRENT APPLICATION DATA: -(A) APPLICATION NUMBER:
(B) FILING DATE:
{C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Myers, Jonathan E.
(B) REGISTRATION NUMBER: 26,963 (C) REFERENCE/DOCKET NUMBER: 19236N
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (718) 884-6600 (B) TELEFAX: (718) 601-1099 (C) TELEX: 620428 (2) INFORMATION FOR SEQ ID N0:1: -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
{A) NAME/KEY: CDS
(B) LOCATION: 1..84 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
GCC CCC CTC CCC GCG CCC GCG CCC CCC TCC ACG CCC CCG GGG CCC GAG
Ala Pro Leu Pro Ala Pro Ala Pro Pro Ser Thr Pro Pro Gly Pro Glu CCC GCC CCC GCC CAG CCC GCG GCG CCC CGG GCC GCC
Pro Ala Pro Ala Gln Pro Ala Ala Pro Arg Ala Ala (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Ala Pro Leu Pro Ala Pro Ala Pro Pro Ser Thr Pro Pro Gly Pro Glu Pro Ala Pro Ala Gln Pro Ala Ala Pro Arg Ala Ala (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
WO 97/34b30 PCT/US97/05282 (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..75 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GCT CCT CCA GAG GCC GAC GCG CGG ACC CTC CGA CGT CCT GGC CCG CCG
Ala Pro Pro Glu Ala Asp Ala Arg Thr Leu Arg Arg Pro Gly Pro Pro CTG CCG CTG CCG CCT TCC CTT CTC CCG
Leu Pro Leu Pro Pro Ser Leu Leu Pro (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Ala Pro Pro Glu Ala Asp Ala Arg Thr Leu Arg Arg Pro Gly Pro Pro Leu Pro Leu Pro Pro Ser Leu Leu Pro (2) INFORMATION FOR SEQ ID N0:5:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single -(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.78 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GGC ACC GAC GGC CCC GCC CGA GGA GGC GGA AGC GGA GGA GGA CGC GGC
Gly Thr Asp Gly Pro Ala Arg Gly Gly Gly Ser Gly Gly Gly Arg Gly CCC GGT GGC GGA AGA GGT GGC CCC CGC GGG
Pro Gly Gly Gly Arg Gly Gly Pro Arg Gly (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Gly Thr Asp Gly Pro Ala Arg Gly Gly Gly Ser Gly Gly Gly Arg Gly Pro Gly Gly Gly Arg Gly Gly Pro Arg Gly {2) INFORMATION FOR SEQ ID N0:7:
(i} SEQUENCE CHARACTERISTICS:
(A} LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear {ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.78 (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
{iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..84 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
GGC TGG GCT GCG CGG CGG GGC CGG CGA CGG GGA CGG CGG CGG GGA CGA
Gly Trp Ala Ala Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Gly Arg CGT CGC CGC CAG CGG CGA GCG GCA CGG AGA CGG AGG
Arg Arg Arg Gln Arg Arg Ala Ala Arg Arg Arg Arg (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (8) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Gly Trp Ala Ala Arg Arg 61y Arg Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Arg Gln Arg Arg Ala Ala Arg Arg Arg Arg
H202 solution), 6. The enzyme reaction was stopped with TANA'sR ELISA-stopping buffer {diluted phosphoric acid), 7. The plates were read using 450 nm.
On the base of this technique these data were obtained:
FIG. 2. Binding of rabbit #76 serum to first peptide with Seq.
ID No. 2.
FIG. 3. Binding of rabbit #77 serum to first peptide with Seq.
ID No. 2.
FIG. 4. Binding of rabbit #76 serum to second peptide with Seq. ID No. 4.
FIG. 5. Binding of rabbit #77 serum to second peptide with Seq. ID No. 4.
FIG. 6. Binding of rabbit #76 serum to third peptide with Seq.
ID No. 6. -FIG. 7. Binding of rabbit I77 serum to third peptide with Seq.
ID No. fi.
FIG. 8. Binding of rabbit X76 serum to fourth peptide with Seq. ID No. 8.
FIG. 9. Binding of rabbit X77 serum to fourth peptide with Seq. ID No. 8.
In both rabbit sera (R76 and R77) the high levels of antibodies against each of our four peptides were determined in contrast with these sera before immunization.
On the basis of ELISA titrations, it was found that two rabbits produced high titer antibodies against each of the four tested peptide reagents with Seq. ID No. 2, 4, 6 and 8. The results are presented in Figures 2 through 9. Note the large difference in the inmunogenic activity in the rabbit blood before and after the peptide-containing vaccine was administered to the rabbits.
In two of such sera from iamunized rabbit N76 (R76) and immunized rabbit N77 (R77) and in pre-immune (normal) sera (R78) in the Laboratory of Microbiological Associates, Inc. (MA) the level (titers) of antibodies were determined against:
Herpes Simplex Virus 1 (HSY1) - on the basis of MA'sELISA
Herpes Simplex Virus 2 (HSV2) - on the basis of Whittaker kit, ELISA, Human Cytomegalovirus (HCMY) - on the basis of Whittaker kit, ELISA, Yaricella Zoster Virus (YZY) on the basis of MA's IFA, Epstein - Bar Virus (EBV) - on the basis of MA's. IFA.
Titers were determined by comparison with the mean + 99%
confidence interval for control (normal, pre-immune) serum R78 (See: Tables through 8).
As one can see, strong differenti-al reactivity was obtained for:
R76 - against HSY-1 + HSY-2 and HCMV, R77 - against HSY-1 + HSV-2 and HCMY.
Neither antibody showed reactivity against EBV (YCA) antigen.
Although reactivity against VZV was relatively strong for R76 and R77, the R78 titer was also elevated and, therefore, did not allow low titered differential results.
Tables 5, 6, 7 and 8 showing the 95x and 99X confidence intervals for R78 titration in each assay follow hereinbelow:.
-- CONFIDENCE INTERVALS (POOLED) --Var MEAN STD LOWER UPPER LOWER UPPER
Name ERR 95% 95% 99% 99%
0.156 0.073 -0.022 0.333 -0.114 0.425 0.224 0.073 0.046 0.401 -0.046 0.493 40 0.166 0.073 -0.012 0.343 -0.104 0.435 80 0.15 0.073 -0.028 0.328 -0.119 0.419 160 0.193 0.073 0.015 0.37 -0.077 0.462 320 O.I1 0.073 -0.068 0.287 -0.16 0.379 640 0.104 0.073 -0.074 0.282 -0.165 0.373 1280 0.128 0.073 -0.05 0.306 -0.141 0.397 -- CONFIDENCE INTERVALS (POOLED) --VAR MEAN STD LOWER UPPER LOWER UPPER
NAME ERR 95% 95% 99% 99%
10 1.153 0.041 1.053 1.252 1.001 1.304 20 0.657 0.041 0.557 0.756 0.505 0.808 0.534 0.041 0.434 0.634 0.383 0.685 80 0.304 0.041 0.204 0.404 0.153 0.455 160 0.226 0.041 0.126 0.326 0.075 0.377 320 0.181 0.041 4.081 0.28 0.029 0.332 640 0.15 0.041 0.05 0.249 -0.002 0.301 1280 0.13 0.041 0.03 0.23 0.021 0.281 -- CONFIDENCE INTERVALS (POOLED) --VAR MEAN STD LOWER UPPER LOWER UPPER
NAME ERR 95% 95% 99% 99%
0.929 0.049 0.809 1.048 0.747 1.11 0.561 0.049 0.441 0.68 0.379 0.742 40 0.368 0.049 0.248 0.487 0.186 0.549 80 0.261 0.049 0.141 0.381 0.08 0.442 160 0.171 0.049 0.051 0.29 -0.011 0.352 320 0.124 0.049 0.004 0.244 -0.057 0.305 640 0.102 0.049 -0.018 0.221 -0.08 0.283 1280 0.104 0.049 -0.015 0.224 -0.077 0.286 STUDY - VZV
-- CONFIDENCE INTERVALS (POOLED) --VAR MEAN STD LOWER UPPER LOWER UPPER
NAME ERR 95x 95% 99% 99X
1.364 0.155 0.966 1.762 0.74 1.988 1.077 0.155 0.679 1.474 0.453 1.7 40 1.069 0.155 0.671 1.467 0.445 1.693 80 0.632 0.155 0.234 1.029 0.008 1.255 160 0.44 0.155 0.042 0.837 -0.184 1.063 320 0.454 O.I55 0.056 0.851 -0.17 1.077 640 0.145 0.155 -0.253 0.543 -0.479 0.769 1280 0.529 0.219 -0.033 1.091 -0.353 1.411 The immunogenisity of each herpetic peptide and its mixtures as a result of ELISA-titration of irtmune rabbit sera with several members of Herpesvirus family.
Ser/Yir HSV1 HSY2 HSY1+2 HCMV EBV VZV MDY
Anti-P1 3000 3000 0 10000 10000 10000 3000 Anti-P2 0 0 0 1000 0 3000 3000 Anti-P3 3000 3000 1000 3000 10000 3000 3000 Anti-P4 3000 3000 1000 3000 10000 3000 10000 Anti-P5 0 0 0 0 3000 0 0 Anti-4P 3000 3000 0 1000 10000 3000 10000 Anti-5P 0 0 0 1000 0 0 3000 Contr.ser 0 0 0 0 0 0 0 At present we are providing some additional data concerning the antibody titers in sera of rabbits who have been immunized with the polypeptide vaccine according to the present invention.
After immunization of the rabbits by the polypeptide vaccine containing the four polypeptides we have obtained the next level of specific antibodies against the different herpes viruses in an ELISA test;
against Herpes Simplex Virus I 1:3000 against Herpes Simplex Yirus II 1:3000 against Human Cytomegalovirus 1:1000 against Epstein-Barr Yirus 1:10,000 against Yaricella-Zoster Virus 1:3,000 against Marek~s Disease Virus 1:10,000.
In these experiments we have used the homogenates of cell cultures infected by several herpes viruses (from American Cell Culture Collection) as antigens for ELISA titration.
Thus, the data demonstrate that "antibody titers increase not only over control after multiple infections of the pharmaceutical composition..." but also possess the real protective activity of these sera against all actual members of the Herpes Viridae family.
The data presented above show that the new vaccine according to the present invention displays a real immunogenic activity, and a protective activity and this activity shows the effectiveness of the new peptide -- containing vaccine to stimulate herpes antibody production against all herpes viruses. The vaccine may be designated as a polyvalent or universal herpes vaccine EFFICACY OF THE PEPTIDE-CONTAINING VACCINE TO PREVENT THE .
DEVELOPMENT OF ATHEROSCLEROTIC PLAQUE
Background Information It is known in the art that an increase of G proteins in the blood (Gs proteins) that stimulate the production of cyclic AMP prevents the development of atherosclerotic plaque. The new vaccine containing the polypeptides with Seq. ID Nos. 2, 4, 6 and 8 prevents the development of atherosclerotic plaque by stimulating the production of Gs proteins.
Atherogenesis is a consequence of persistent herpes virus infection development in blood vessel walls. In the course of this development, synthesis of a considerable number of virus-specified regulator proteins (transcriptional factors) takes place in the vessel -walls. Some of these regulator proteins repress viral DNA replication and transcription, thus preventing a chronic herpes viral infection from becoming an acute infection. Owing to the homology between certain sections of the alpha subunit of the Gs protein gene and those of the genes of a number of herpes viruses, individual virus-specific transcription factors bind with GGC-GGC-GGC sections in the alpha subunit of the 6s protein gene and suppress the transcription of this gene. This in turn, decreases translation and synthesis of the Gs protein. A decrease in Gs protein production reduces cyclic AMP formation. This causes a reduction of the synthesis of cholesterol ether hydrolase, which in turn increases - atherogenesis.
The antibodies generated in the rabbit in response to the in~ect9on of our polypeptide vaccine bind with one of the transcription factors described above, this factor being related to the GGC-GGC-GGC sites in the Gs protein gene. These antibodies prevent the emergence of a complex between the Gs protein gene site and the abovementioned transcription factor. The antibodies prevent the inhibitory influence of the persistent herpes virus infection on the synthesis of Gs proteins, thus mitigating the extent of atherogenesis.
Experimental Section The prevention of the formation of the DNA-protein complex containing DNA from the Gs protein gene and protein from the herpes transcription factor by means of the antibodies generated by administration of our peptide vaccine to rabbits is illustrated with the results of the model experiments described below.
The study of DNA-protein interaction has been conducted by the shift-mobility assay method.
Shift Mobility Assay A1. Extraction of Alpha-Protein-from Nuclei Purified nuclei from normal blood vessel endothelium and blood vessel endothelium with atherosclerotic plaques (ASP) were extracted with 0.35 M NaCI. Purified nuclei were pelleted by low-speed centrifugation and re-suspended by vortexing to a final DNA concentration of 0.4 mg/ml in 0.35 M NaCI, 5 mM Na-EDTA, 10 mM 2-mercaptoethanol, 10 mM Tris-HC1 (pH 7.5) containing the five proteinase inhibitors (Phenylmethylsulfonyl fluoride - (PMSF), antipain, leupeptin, chymostatin, and pepstatin A) at the concentration used in the nuclei isolation. After 30 minutes at 30'C with occasional vortexing, the suspension was centrifuged at 10,000 x g for 15 minutes. The supernatant containing the alpha-protein was used either immediately or after storage at -70°C in the presence of 15% glycerol.
These alpha-proteins are new factors of herpes virus transcription and are different from the heretofore known factors of herpes virus transcription such as Spll, Ergl, Wtl and others. The new transcriptional factors have the ability to make the stable DNA-protein complex in vitro in the experimental model system (as described hereinbelow) and in the human organism as well as with a specific site on the alpha-subunit of the Gs proteins.
A2. Preparation of DNA-Fragments with GGC-Sites from the Promoter Region of Mouse Ribosome Protein Gene Fragments containing GGC sites were isolated from the promoter region of mouse ribosome protein gene following complete digestion of purified mouse DNA with either Hind III or Mbo II restriction enzymes. The fragments were purified separately by preparative electrophoresis in 6%
polyacrylamide eluted from the gels, and thereafter separately end-labeled with 32P by T4 polynucleotide kinase and gamma-32P-ATP, following a treatment with bacterial alkaline phosphatase (Maxam and Gilbert, 1980).
The labelled DNA was purified by extractions with chloroform:isopropanol (24:1 by weight) in the presence of 1% SDS, 1M NaCI and ethanol-precipitated in the presence of 10 mg/ml of linear polyacrylamide as a carrier. Equal amounts of the 32P-counts of the Hind III and MboII
produced fragments of DNA were then mixed together to yield the final DNA
sample with the GGC-GGC-GGC sites.
A3. Detection of the DNA-Protein Interactions by the Shift Mobility Assay The "DNA-Protein" interactions were investigated by the Shift-Mobility Assay on a low ionic strength 4% polyacrylamdide gel (See FIG.
10).
As it is shown, pure DNA-fragment DNA (see A2) in the absence of any addition of protein extract, migrates in the gel in a discrete band.
See FIG. 10, Lane "a", while in the presence of protein extract (extracted from the purified nuclei of blood vessel endothelium with ASP), most of the DNA failed to enter the gel. See FIG. 10, Lanes "b" and "c". The same result was shown with a mixture of "DNA" plus "ASP" plus rabbit sera where the rabbit sera did not contain the antibodies against the herpes peptides generated by the rabbit upon administration of the polypaptide vaccine.
See FIG. l0, Lanes "d" and "e".
In the presence of an aliquot of extracts of purified nuclei of human blood endothelum without ASP (normal endothelium "NE") the "DNA"
migrated to an intermediate extent. See Lanes "f" and "g" of FIG. 10.
In the presence of "ASP" and rabbit immune sera ("IS") in dilution 1:50 to 1:10 "DNA" oigrates as a discrete band as pure DNA (see FIG. 10, Lanes "i" through "1" and compare with the results in Lane "a", which shows pure "DNA" without protein or immune sera). In the case of the addition to the complex of "DNA" plus "ASP" the "IS" in dilution 1:100 and 1:50, the "DNA" migrates to an intermediate extent. See FIG. 10, Lane "h"
Further analysis of the shift mobility assay as shown in Fig.
is as follows:
Fig. 10 shows the electrophoretic mobility in 4& polyacrylamide gel of the "DNA-GGC sites fragments", with alpha proteins of nuclei from human blood vessel endothelium (with and without ASP) and with rabbit sera (with and without antibodies generated by administration of the present polypeptide vaccine to rabbits).
Lane "a" "DMA-GGC fragment" (25,000 cpm, 2.5 ng of DNA) in the absence of added protein extract and any sera.
Lanes "b" through "e" "DNA-GGC site fragments" in the same concentration + aliquot of the extracts of purified nuclei from human blood vessel endothelium cells with ASP (b, c) and + aliquot of the normal (pre-immune) rabbit sera - N76{d) and N77(e) in dilution 1:2.
Lanes "f" and "g" "DNA-GGC- sites fragments" in the same concentration + aliquot of the extracts of purified nuclei from normal human blood vessel endothelial cells without ASP.
Lane "h" "DNA-GGC sites fragment" in the same concentration + a mixture of aliquots of the extract of purified nuclei from endothelial -blood vessel cells with ASP + aliquot of the mixture of the post-immune rabbit sera N76 and N77 in a dilution of 1:100.
Lanes "I" through ~1" are the same situation as in Lane "h", but the mixture of immune sera was used in a dilution of 1:50(I); 1:30 0 );
1:20(k); and 1:10(1) These data show that the usage of immune rabbit sera against several herpesvirus peptides can prevent formation of the complex between DNA GGC-GGC-GGC binding sites on the mouse ribosomal protein gene which shares this binding site in common with the genes expressing the Gs proteins and the protein extract from the nuclei of blood vessel endothelium with ASP" and consequently prevents the development of ASP".
In the experiment presented above the antibodies in the immune rabbit sera decrease the formation of the DNA-protein complex by more than 90% according to the data in Figure 10 and that such a decrease in the formation of the complex will facilitate the in vivo expression of the Gs protein genes to produce Gs proteins which in turn leads to increased cyclic AMP production and less formation of atherosclerotic plaque (ASP);
We performed an additional experiment using gel retardation to analyze the mobility of the "DNA-GGC sites fragment" in 5% PAAG. The results are shown in Fig. 11.
According to Fig. 11, the irtununosera number 77 (N4) and number 76 (N6) (with dilution 1:20) had decreased the formation (amount) of DNA-protein complex between DNA-GGC-fragment labelled by 32P and the alpha-protein nuclei from human blood vessel cells with ASP in comparison with the amount of the DNA-protein complex when the corresponding pre-immune sera N5 and N7 were employed instead.
WO 97/34630 . PCT/US97/05282 Both of the immune sera N1 and N2 as well as the preimnune sera 76 (N3) had no influence on the formation (amount) of the DNA-protein complex, when such sera were used in a dilution of 1:100.
These additional data confirm that the use of inmune rabbit sera containing antibodies generated by administration of the present peptide vaccine, can prevent the formation of a DNA-protein complex between DNA-GGC site fragments characteristic of the gene that expresses the Gs proteins and protein extract the nuclei of blood vessel endothelium with ASP, characteristic of the herpes transcriptional factor, and consequently the polypeptide vaccine can prevent the development of ASP.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
{i) APPLICANT: 6olubev, Daniel B. ET AL
(ii) TITLE OF INVENTION: PEPTIDE VACCINE TO PREVENT DEVELOPMENT OF
SEVERAL iiERPES INFECTIONS AND/OR ATHEROSCLEROTIC PLAQUE
ATHEROSCLEROSIS
(iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Karl F. Ross, PC
(B) STREET: 5676 Riverdale Ave.
(C) CITY: Bronx (D) STATE: NY
(E) COUNTRY: USA
(F) ZIP: 10471-0900 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release X1.0, Version X1.25 (vi) CURRENT APPLICATION DATA: -(A) APPLICATION NUMBER:
(B) FILING DATE:
{C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Myers, Jonathan E.
(B) REGISTRATION NUMBER: 26,963 (C) REFERENCE/DOCKET NUMBER: 19236N
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (718) 884-6600 (B) TELEFAX: (718) 601-1099 (C) TELEX: 620428 (2) INFORMATION FOR SEQ ID N0:1: -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
{A) NAME/KEY: CDS
(B) LOCATION: 1..84 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
GCC CCC CTC CCC GCG CCC GCG CCC CCC TCC ACG CCC CCG GGG CCC GAG
Ala Pro Leu Pro Ala Pro Ala Pro Pro Ser Thr Pro Pro Gly Pro Glu CCC GCC CCC GCC CAG CCC GCG GCG CCC CGG GCC GCC
Pro Ala Pro Ala Gln Pro Ala Ala Pro Arg Ala Ala (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Ala Pro Leu Pro Ala Pro Ala Pro Pro Ser Thr Pro Pro Gly Pro Glu Pro Ala Pro Ala Gln Pro Ala Ala Pro Arg Ala Ala (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
WO 97/34b30 PCT/US97/05282 (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..75 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GCT CCT CCA GAG GCC GAC GCG CGG ACC CTC CGA CGT CCT GGC CCG CCG
Ala Pro Pro Glu Ala Asp Ala Arg Thr Leu Arg Arg Pro Gly Pro Pro CTG CCG CTG CCG CCT TCC CTT CTC CCG
Leu Pro Leu Pro Pro Ser Leu Leu Pro (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Ala Pro Pro Glu Ala Asp Ala Arg Thr Leu Arg Arg Pro Gly Pro Pro Leu Pro Leu Pro Pro Ser Leu Leu Pro (2) INFORMATION FOR SEQ ID N0:5:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single -(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.78 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GGC ACC GAC GGC CCC GCC CGA GGA GGC GGA AGC GGA GGA GGA CGC GGC
Gly Thr Asp Gly Pro Ala Arg Gly Gly Gly Ser Gly Gly Gly Arg Gly CCC GGT GGC GGA AGA GGT GGC CCC CGC GGG
Pro Gly Gly Gly Arg Gly Gly Pro Arg Gly (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Gly Thr Asp Gly Pro Ala Arg Gly Gly Gly Ser Gly Gly Gly Arg Gly Pro Gly Gly Gly Arg Gly Gly Pro Arg Gly {2) INFORMATION FOR SEQ ID N0:7:
(i} SEQUENCE CHARACTERISTICS:
(A} LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear {ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.78 (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
{iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..84 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
GGC TGG GCT GCG CGG CGG GGC CGG CGA CGG GGA CGG CGG CGG GGA CGA
Gly Trp Ala Ala Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Gly Arg CGT CGC CGC CAG CGG CGA GCG GCA CGG AGA CGG AGG
Arg Arg Arg Gln Arg Arg Ala Ala Arg Arg Arg Arg (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (8) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Gly Trp Ala Ala Arg Arg 61y Arg Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Arg Gln Arg Arg Ala Ala Arg Arg Arg Arg
Claims (3)
1. A pharmaceutical composition for preventing pathogenic development of several herpes infections and/or atherosclerotic plaques, which consists essentially of:
(a) 10 to 30% by weight of a polypeptide having Sequence ID 2:
Ala Pro Leu Pro Ala Pro Ala Pro Pro Ser Thr Pro Pro Gly Pro Glu Pro Ala Pro Ala Gln Pro Ala Ala Pro Arg Ala Ala 20 25 ;
(b) 10 to 30% by weight of a polypeptide having Sequence ID 4:
Ala Pro Pro Glu Ala Asp Ala Arg Thr Leu Arg Arg Pro Gly Pro Pro Leu Pro Leu Pro Pro Ser Leu Leu Pro 20 25;
(c) 10 to 30% by weight of a polypeptide having Sequence ID 6:
Gly Thr Asp Gly Pro Ala Arg Gly Gly Gly Ser Gly Gly Gly Arg Gly Pro Gly Gly Gly Arg Gly Gly Pro Arg Gly 20 25 ; and (d) 10 to 30% by weight of a polypeptide having Sequence ID 8:
Gly Trp Ala Ala Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Arg Gln Arg Arg Ala Ala Arg Arg Arg Arg
(a) 10 to 30% by weight of a polypeptide having Sequence ID 2:
Ala Pro Leu Pro Ala Pro Ala Pro Pro Ser Thr Pro Pro Gly Pro Glu Pro Ala Pro Ala Gln Pro Ala Ala Pro Arg Ala Ala 20 25 ;
(b) 10 to 30% by weight of a polypeptide having Sequence ID 4:
Ala Pro Pro Glu Ala Asp Ala Arg Thr Leu Arg Arg Pro Gly Pro Pro Leu Pro Leu Pro Pro Ser Leu Leu Pro 20 25;
(c) 10 to 30% by weight of a polypeptide having Sequence ID 6:
Gly Thr Asp Gly Pro Ala Arg Gly Gly Gly Ser Gly Gly Gly Arg Gly Pro Gly Gly Gly Arg Gly Gly Pro Arg Gly 20 25 ; and (d) 10 to 30% by weight of a polypeptide having Sequence ID 8:
Gly Trp Ala Ala Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Gly Arg Arg Arg Arg Gln Arg Arg Ala Ala Arg Arg Arg Arg
2. A method of preventing pathogenic development of several herpes virus infections and/or atherosclerotic plaques in a mammalian subject subjectable thereto, which comprises the step of administering to said mammalian subject, a therapeutically effective amount of the pharmaceutical composition defined in claim 1.
3. The method of preventing pathogenic development of several herpes infections and/or atherosclerotic plaques in a mammalian subject defined in claim 2 wherein the pharmaceutical composition is administered by injection.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/618,917 US5837262A (en) | 1994-07-27 | 1996-03-20 | Pharmaceutical compositions against several herpes virus infections and/or atherosclerotic plaque |
US08/618,917 | 1996-03-20 | ||
PCT/US1997/005282 WO1997034630A1 (en) | 1996-03-20 | 1997-03-19 | Peptide vaccine to prevent development of several herpes virus infections and/or atherosclerotic plaque |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2283633A1 true CA2283633A1 (en) | 1997-09-25 |
Family
ID=24479675
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA 2283633 Abandoned CA2283633A1 (en) | 1996-03-20 | 1997-03-19 | Peptide vaccine to prevent development of several herpes virus infections and/or atherosclerotic plaque |
Country Status (5)
Country | Link |
---|---|
US (1) | US5837262A (en) |
EP (1) | EP0967992A4 (en) |
AU (1) | AU2722097A (en) |
CA (1) | CA2283633A1 (en) |
WO (1) | WO1997034630A1 (en) |
Families Citing this family (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6183752B1 (en) * | 1997-02-05 | 2001-02-06 | Pasteur Merieux Serums Et Vaccins | Restenosis/atherosclerosis diagnosis, prophylaxis and therapy |
CA2406807C (en) | 2000-04-21 | 2010-04-06 | Amgen Inc. | Methods and compositions for the prevention and treatment of anemia |
JP4004952B2 (en) | 2000-11-28 | 2007-11-07 | ザ ユニヴァーシティ オヴ シカゴ | Genetically engineered herpesvirus for the treatment of cardiovascular disease |
US8562583B2 (en) | 2002-03-26 | 2013-10-22 | Carmel Pharma Ab | Method and assembly for fluid transfer and drug containment in an infusion system |
US7867215B2 (en) | 2002-04-17 | 2011-01-11 | Carmel Pharma Ab | Method and device for fluid transfer in an infusion system |
SE523001C2 (en) | 2002-07-09 | 2004-03-23 | Carmel Pharma Ab | Coupling component for transmitting medical substances, comprises connecting mechanism for releasable connection to second coupling component having further channel for creating coupling, where connecting mechanism is thread |
WO2004064903A1 (en) | 2003-01-21 | 2004-08-05 | Carmel Pharma Ab | A needle for penetrating a membrane |
US7942860B2 (en) | 2007-03-16 | 2011-05-17 | Carmel Pharma Ab | Piercing member protection device |
US7975733B2 (en) | 2007-05-08 | 2011-07-12 | Carmel Pharma Ab | Fluid transfer device |
US8657803B2 (en) | 2007-06-13 | 2014-02-25 | Carmel Pharma Ab | Device for providing fluid to a receptacle |
US8029747B2 (en) | 2007-06-13 | 2011-10-04 | Carmel Pharma Ab | Pressure equalizing device, receptacle and method |
US8622985B2 (en) | 2007-06-13 | 2014-01-07 | Carmel Pharma Ab | Arrangement for use with a medical device |
US10398834B2 (en) | 2007-08-30 | 2019-09-03 | Carmel Pharma Ab | Device, sealing member and fluid container |
US8287513B2 (en) | 2007-09-11 | 2012-10-16 | Carmel Pharma Ab | Piercing member protection device |
US8075550B2 (en) | 2008-07-01 | 2011-12-13 | Carmel Pharma Ab | Piercing member protection device |
US8523838B2 (en) | 2008-12-15 | 2013-09-03 | Carmel Pharma Ab | Connector device |
US8790330B2 (en) | 2008-12-15 | 2014-07-29 | Carmel Pharma Ab | Connection arrangement and method for connecting a medical device to the improved connection arrangement |
US8480646B2 (en) | 2009-11-20 | 2013-07-09 | Carmel Pharma Ab | Medical device connector |
USD637713S1 (en) | 2009-11-20 | 2011-05-10 | Carmel Pharma Ab | Medical device adaptor |
US9168203B2 (en) | 2010-05-21 | 2015-10-27 | Carmel Pharma Ab | Connectors for fluid containers |
US8162013B2 (en) | 2010-05-21 | 2012-04-24 | Tobias Rosenquist | Connectors for fluid containers |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3122669A1 (en) * | 1980-06-12 | 1982-02-11 | Asta-Werke Ag, Chemische Fabrik, 4800 Bielefeld | "METHOD FOR PRODUCING NEW MUTANTS OF HERPES SIMPLEX VIRUS TYPE 1 AND TYPE 2" |
US4693981A (en) * | 1983-12-20 | 1987-09-15 | Advanced Genetics Research Institute | Preparation of inactivated viral vaccines |
US5534258A (en) * | 1994-07-27 | 1996-07-09 | Bio-Virus Research Incorporated | Polypeptides to prevent atherosclerotic plaque |
-
1996
- 1996-03-20 US US08/618,917 patent/US5837262A/en not_active Expired - Fee Related
-
1997
- 1997-03-19 EP EP97921084A patent/EP0967992A4/en not_active Withdrawn
- 1997-03-19 CA CA 2283633 patent/CA2283633A1/en not_active Abandoned
- 1997-03-19 WO PCT/US1997/005282 patent/WO1997034630A1/en not_active Application Discontinuation
- 1997-03-19 AU AU27220/97A patent/AU2722097A/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
EP0967992A1 (en) | 2000-01-05 |
AU2722097A (en) | 1997-10-10 |
US5837262A (en) | 1998-11-17 |
EP0967992A4 (en) | 2004-06-30 |
WO1997034630A1 (en) | 1997-09-25 |
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Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Discontinued |