EP1497324A2 - CD4 sp + /sp T-LYMPHOCYTE-SPECIFIC HEPATITIS C VIRUS EPITOPES - Google Patents
CD4 sp + /sp T-LYMPHOCYTE-SPECIFIC HEPATITIS C VIRUS EPITOPESInfo
- Publication number
- EP1497324A2 EP1497324A2 EP03717293A EP03717293A EP1497324A2 EP 1497324 A2 EP1497324 A2 EP 1497324A2 EP 03717293 A EP03717293 A EP 03717293A EP 03717293 A EP03717293 A EP 03717293A EP 1497324 A2 EP1497324 A2 EP 1497324A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- epitopes
- hepatitis
- virus
- hcv
- lymphocyte
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the invention relates to hepatitis C virus epitopes that are specific to CD4 + T lymphocytes and to vaccines that contain these epitopes.
- HCV hepatitis C virus
- Flaviviridae family The hepatitis C virus, hereinafter referred to as HCV, was identified in 1989 and is an RNA virus from the Flaviviridae family. It ⁇ o. , consists of an RNA single strand of approx. 9400 nucleotides which encode a precursor polyprotein approx. 3000 amino acids long. This polyprotein is translated in an open reading frame and proteolytically cleaved post-translationally.
- the virus is highly variable, and there are various virus isolates known as genotypes
- hepatitis C is one of the most important chronic viral infections. There are currently 5 at least 180 million infected individuals. According to calculations by the Centers of Disease Control in the USA, there will also be an increase in hepatitis C-associated diseases by 2010 due to the long latency period after infection with HCV. 0 The HCV is predominantly transmitted parenterally and was the main cause of post-transfusion hepatitis NonA-NonB until it was discovered. Routine testing of all blood products with 2nd and 3rd generation HCV antibody tests has dramatically reduced the number of post-transfusion hepatitis. The so-called sporadic hepatitis C and iv drug abuse are now considered the main means of transmission of new HCV infections. No measures are currently known to effectively prevent new infections in this way.
- HCV causes chronic inflammation of the liver (hepatitis), which over the course of many years can lead to further complications, such as cirrhosis of the liver.
- hepatitis As part of cirrhosis of the liver that has existed for years, about 5% of all infected people develop hepatocellular carcinoma. In the western world, hepatitis C therefore ranks first as an indication for liver transplantation. The health care costs of these transplants are significant.
- ni is the sum of the reactions with 3 ⁇ Sl ⁇ 6
- n 2 is the sum of the reactions with SI> 6
- m is the number of tests against the respective peptide, where m> 15, and MW is the mean of all impact factors.
- the Impcat factor of the CD4 + T lymphocyte-specific HCV epitopes> MW and ⁇ MW + 1 * Sta, in particular> MW +1 * Sta and ⁇ MW + 2 * Sta, particularly preferably> MW + 2 * Sta is preferred.
- CD4 + T lymphocyte-specific HCV epitopes comprising one or more peptides selected from the group:
- HCV epitopes specific to CD4 + T lymphocytes mentioned below containing the sequence: EP001 GPRLGVRATRKTSER
- CD4 + T lymphocyte-specific HCV epitopes according to the invention are particularly preferred, selected from the group of epitopes EP001 to EP0017 with the above-mentioned sequence. These epitopes EP001 to EP017 according to the invention have an impact factor of> MW + 2 * Sta.
- HCV epitopes are also to be used for immunotherapy of chronic hepatitis C or a vaccine, further criteria are a high degree of conservation between different virus subtypes and a high degree of promiscuity in binding to different HLA class II molecules.
- the HCV epitopes identified and characterized in this way should be available for a vaccine for the prophylaxis and / or therapy of an HCV infection.
- a unique group of patients was identified, namely patients with acute hepatitis C who achieve permanent or at least temporary virus elimination in over 50% of cases.
- the entire virus was covered with overlapping synthetic peptides of 15 to 20 amino acids in length.
- a standardized lymphocyte proliferation assay was used as the test system.
- a formula was defined which is based on the frequency of detection of an epitope and the strength of the respective immune response.
- the invention is based on the selection of a particular patient collective, examinations with defined peptides and an algorithm for the identification of highly immunogenic CD4 + T cell epitopes which are suitable for the development of a prophylactic or therapeutic vaccine.
- the algorithm determines the "impact factor” (IF) of the respective epitope and is defined as follows:
- ni corresponds to the sum of the reactions with 3 ⁇ Sl ⁇ 6, n 2 to the sum of the reactions with Sl> 6 and m to the number of tests carried out against the respective peptide, this serves to normalize the values.
- the peptides we found were m> 15.
- the stimulation index (Sl) is usually calculated from the raw data of a proliferation assay and represents the multiplication factor of the measured sample compared to the control. An Sl of 3 is considered significant.
- the mean value was calculated from the impact factor of all peptides tested, each impact factor being determined according to Formula 1.
- our solution of the task is limited to the peptides whose IF are two standard deviations above the mean of all IF.
- HCV epitopes specific to CD4 + T lymphocytes means a defined region of a hepatitis C protein which, owing to its structure, “fits” into the complementary binding site of a CD4 + T lymphocyte receptor and thereby a highly specific reaction triggers.
- a "peptide screening" with approximately 450 selected different peptides (15-22mers) was carried out for a virus-specific CD4 + T cell response in the patient population described above.
- the peptides represent the entire virus protein, whereby we used 15mers with 5 amino acid overlapping areas or 20-22mers with 10 amino acids overlapping areas in order to record all relevant epitopes.
- Table 1 shows the respective positions in the HCV genome of the epitopes according to the invention, stating the respective virus isolate reference (Table 1 / column 4).
- the information on the amino acid position (Table 1 / Column 2) should only be understood as an approximation, because due to the high mutation rate of the virus in the different virus isolates can cause changes in position.
- the conserved epitopes are of particular importance for prophylactic and therapeutic vaccinations, which is shown by the constant sequence of the different virus isolates of an epitope (see also Table 1 / column 5).
- the epitopes according to the invention are listed in Table 1.
- Genotype 1 b AUTHORS Trowbridge.R. and Gowans.E.J. TITLE Molecular cloning of an Australian isolate of hepatitis C virus JOURNAL Arch. Virol. 143 (3), 501-511 (1998)
- Genotype 1b AUTHORS Takamizawa, A., Mori.C, Fuke.l., Manabe.S., Murakami.S., Fujita.J., Onishi.E., Andoh.T., Yoshida.l. and Okayama.H.
- TITLE Transcripts from a Single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee
- Genotype 1a AUTHORS CHOO.Q.-L, RICHMAN.K.H., HAN.J.H., BERGER.K., LEE.C,
- Genotype 1 b AUTHORS Tanaka.T., Kato.N., Nakagawa.M., Ootsuyama.Y., Cho.M.J.,
- TITLE Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis
- Genotype 1 b AUTHORS Chen.P.J., Lin.M.H., Tai.K.F., Liu.P.C, Lin.C.J. and
- Taiwanese hepatitis C virus genome sequence determination and mapping the 5 'termini of viral genomic and antigenomic RNA JOURNAL Virology 188 (1), 102-113 (1992)
- Genotype 1a AUTHORS Inchauspe.G., Zebedee.S., Lee.D.H., Sugitani.M., Nasoff.M. and Prince.A.M.
- Genotype 4 AUTHORS Chamberlain R, Adams N, Saeed AA, Simmonds P, Elliott RM
- TITLE Complete nucleotide sequence of a type 4 hepatitis C virus variant, the predominant genotype in the Middle East.
- TITLE The complete coding sequence of hepatitis C virus genotype 5a, the predominant genotype in South Africa.
- Genotype 6a AUTHORS Adams, N.J., Chamberlain, R.W., Taylor.LA, Davidson, F.,
- Genotype 1 b AUTHORS Honda, M., Kaneko.S., Unoura.M., Kobayashi.K. and
- Genotype 1 b AUTHORS Okamoto.H., Kojima.M., Okada.S., Yoshizawa.H., Lizuka.H.,
- Genotype 1a AUTHORS Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M.
- the so-called proliferation assay according to the following protocol was used as the test system.
- PBMC peripheral blood mononuclear cells
- RPMI1640 fetal calf serum
- 50 ⁇ l of this cell suspension concentration of 1x10 6 cells per ml
- the cells were stimulated by adding the peptides.
- the final concentration of the peptides was 10 ⁇ g / ml.
- the cell culture plates were cultivated for 5 days at 37 ° C. and 5% CO 2 , then 3 H-thymidine was added, and the incorporation of the radioactive 3 H was measured as a measure of the cell stimulation.
- an additional filter was defined and referred to below as an impact factor (IF).
- the impact factor (IF) is based on a point system that not only measures the frequency of relevant reactions, i.e. Stimulation index (Sl) greater than 3, as used conventionally, but also takes into account the strength of these reactions.
- Peptides with a high impact factor are not only characterized by a large stimulation index, i.e. strong specific reactivity but also by the repeated i.e. specific reaction found in different people.
- Epitopes that trigger strong HCV-specific CD4 + T cell responses measured in different patients are of great relevance for future vaccination approaches.
- the epitopes according to the invention are further distinguished by the fact that a clear specific CD4 + T cell activity on these peptides correlates with a decrease in the virus titer. These epitopes in particular are therefore likely to be ideal candidates for a vaccine.
- a specific vaccination reaction against these peptides could prevent the disease on the one hand and / or lead to their healing on the other hand, or at least have a favorable effect on the course of an HCV infection.
- the epitopes according to the invention are highly immunogenic, highly conserved sequences of the HCV, some of which are located in the immediate vicinity of known CD8 + T lymphocyte-specific HCV epitopes.
- HCV epitopes specific to CD4 + T lymphocytes they can mediate so-called T cell help for cytotoxic CD8 + T lymphocytes in addition to induction of CD4 + T lymphocytes.
- T cell help for cytotoxic CD8 + T lymphocytes are activated by the cytokines of stimulated CD4 + T lymphocytes.
- the peptides are characterized by frequent significant reactions in different patients with different MHC class II types
- the MHC Class II system is extremely polymorphic.
- the task of the MHC molecules is to bind peptide fragments derived from the body's own and pathogenic (e.g. hepatitis C virus) proteins and to express them on the cell surface for the detection and activation of specific CD4 + T lymphocytes. This system enables an effective and specific
- Class II types i.e. different people, the same peptide on their
- MHC class II molecule can express what in vitro by measurable in different people, directed against the same peptide
- CD4 + activity can be detected by one of these peptides
- Epitopes according to the invention have immunological relevance in different individuals.
- epitopes according to the invention are particularly suitable for both therapeutic and prophylactic peptide vaccination, which is directed against HCV.
- Another solution is a vaccine which contains a combination of the epitopes EP001 to EP017 according to the invention.
- the vaccine can particularly preferably contain a mixture of the epitopes EP001 to EP017 according to the invention.
- other HCV epitopes may also be present.
- the epitopes according to the invention can be used alone or with one or more auxiliary substances as medicaments, preferably as vaccines.
- the vaccine according to the invention contains at least one epitope according to the invention, preferably a mixture of epitopes according to the invention. However, other HCV epitopes may also be present.
- the excipients are preferably selected from the group consisting of fowl pox virus, modified vaccinia virus Ankara, virosomes, TransVax ® and other immune response enhancing substances.
- the vaccine according to the invention can be administered orally, parenterally, intramuscularly, intravenously, subcutaneously or intracutaneously.
- the epitopes according to the invention are epitopes that can be used as a T cell stimulating vaccine.
- a vaccine which contains the epitopes according to the invention, has a vaccination with the entire virus protein, which contains various epitopes for virus-specific T-lymphocytes and only B-lymphocytes and CD4 + T lymphocytes induce the advantage that it selectively induces specific T lymphocytes, CD4 + and / or CD8 + T lymphocytes. It also avoids antagonistic effects or the risk of iatrogenic autoimmune reactions that can occur when vaccinating with whole proteins.
- the epitopes according to the invention additionally have a higher immunogenicity compared to the entire virus protein, as a result of which a better vaccination result is achieved.
- the vaccine according to the invention thus enables the induction of an immune response in healthy people and therefore serves as a prophylactic vaccination.
- the vaccine according to the invention can also induce an immune response in chronically HCV-infected people and thus serve as a therapeutic vaccine.
- the coding cDNA of these epitopes can be used in a DNA vaccine, a special vaccination method.
- the DNA coding for the corresponding epitopes is cloned into a vector. This construct is in turn administered parenterally to the individual to be vaccinated (e.g. Immunology and Cell Biology, volume 75, pages 382 to 388).
- different DNA sequences can encode one of the epitopes according to the invention (see Current protocols, Wiley).
- the epitopes according to the invention can also be used in the diagnosis of the course of an HCV infection by monitoring the amount of CD4 + T lymphocytes which specifically recognize the epitope in question in the blood of the patient with a hepatitis C infection. This can be carried out, for example, using a diagnostic kit which comprises one or more of the epitopes according to the invention.
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Abstract
The invention relates to hepatitis C virus epitopes that are CD4<+> T-lymphocyte specific, in addition to vaccines that contain said epitopes.
Description
CD4+ T-Lymphozyten spezifische Hepatitis C Virus-Epitope CD4 + T lymphocyte specific hepatitis C virus epitopes
5 Die Erfindung betrifft Hepatitis C Virus-Epitope, die gegenüber CD4+ T- Lymphozyten spezifisch sind, sowie Impfstoffe, die diese Epitope enthalten.5 The invention relates to hepatitis C virus epitopes that are specific to CD4 + T lymphocytes and to vaccines that contain these epitopes.
Das Hepatitis C Virus, im nachstehenden HCV genannt, wurde 1989 identifiziert und ist ein RNA Virus aus der Familie der Flaviviridae. Es ιo., besteht aus einem RNA-Einzelstrang von ca. 9400 Nukleotiden, die ein ca. 3000 Aminosäuren langes Vorläuferpolyprotein kodieren. Dieses Polyprotein wird in einem offenen Leserahmen translatiert und posttranslationell proteolytisch gespalten. Das Virus ist hochvariabel, und es existieren verschiedene Virusisolate, die als Genotypen bezeichnetThe hepatitis C virus, hereinafter referred to as HCV, was identified in 1989 and is an RNA virus from the Flaviviridae family. It ιo. , consists of an RNA single strand of approx. 9400 nucleotides which encode a precursor polyprotein approx. 3000 amino acids long. This polyprotein is translated in an open reading frame and proteolytically cleaved post-translationally. The virus is highly variable, and there are various virus isolates known as genotypes
15 werden und deren geographische Verteilung sehr unterschiedlich ist. Mehr als sechs Genotypen werden heute weltweit unterschieden. Diese Genotypen wiederum werden in Subtypen unterteilt. Die genetische Variabilität besteht interindividuell und intraindividuell (innerhalb eines infizierten Individuums). Die intraindividuellen Subtypen sind die 0 sogenannten HCV Quasispezies, verwandte aber unterschiedliche Virussequenzen, die bei ungenauer Replikation entstehen.15 and their geographical distribution is very different. Today, more than six genotypes are differentiated worldwide. These genotypes are subdivided into subtypes. The genetic variability is inter-individual and intra-individual (within an infected individual). The intraindividual subtypes are the so-called HCV quasi-species, but they are related to different virus sequences that result from inaccurate replication.
Mit einer Prävalenz von ca. ein bis drei Prozent weltweit ist Hepatitis C eine der bedeutendsten chronischen Virusinfektionen. Man geht derzeit von 5 mindestens 180 Mio. infizierten Individuen aus. Nach Berechnungen der Centers of Disease Control in den USA wird es aufgrund der langen Latenzzeit nach der Infektion mit dem HCV außerdem noch zu einem Anstieg der Hepatitis C assoziierten Erkrankungen bis zum Jahre 2010 kommen. 0
Das HCV wird überwiegend parenteral übertragen und war bis zu seiner Entdeckung die Hauptursache für die Posttransfusionshepatitis NonA-NonB. Durch routinemäßiges Testen aller Blutprodukte mit HCV-Antikόrpertests der 2. und 3. Generation hat die Zahl der Posttransfusionshepatitiden drastisch abgenommen. Die sogenannte sporadische Hepatitis C sowie i.v.- Drogenmissbrauch gelten heute als Hauptübertragungswege neuer HCV Infektionen. Es sind derzeit keine Maßnahmen bekannt, um Neuinfektionen auf diesen Wegen wirksam zu verhindern.With a prevalence of around one to three percent worldwide, hepatitis C is one of the most important chronic viral infections. There are currently 5 at least 180 million infected individuals. According to calculations by the Centers of Disease Control in the USA, there will also be an increase in hepatitis C-associated diseases by 2010 due to the long latency period after infection with HCV. 0 The HCV is predominantly transmitted parenterally and was the main cause of post-transfusion hepatitis NonA-NonB until it was discovered. Routine testing of all blood products with 2nd and 3rd generation HCV antibody tests has dramatically reduced the number of post-transfusion hepatitis. The so-called sporadic hepatitis C and iv drug abuse are now considered the main means of transmission of new HCV infections. No measures are currently known to effectively prevent new infections in this way.
Das HCV verursacht eine chronische Leberentzündung (Hepatitis), die im langjährigem Verlauf zu weiteren Komplikationen, wie einer Leberzirrhose, führen kann. Im Rahmen einer jahrelang bestehenden Leberzirrhose kommt es bei ca. 5% aller Infizierten zur Entwicklung eines hepatozellulären Karzinoms. In der westlichen Welt nimmt die Hepatitis C deshalb den ersten Rang als Indikation für die Lebertransplantation ein. Die Kosten für das Gesundheitswesen durch diese Transplantationen sind erheblich.HCV causes chronic inflammation of the liver (hepatitis), which over the course of many years can lead to further complications, such as cirrhosis of the liver. As part of cirrhosis of the liver that has existed for years, about 5% of all infected people develop hepatocellular carcinoma. In the western world, hepatitis C therefore ranks first as an indication for liver transplantation. The health care costs of these transplants are significant.
Bei chronischer Hepatitis C sind zwar gegen fast alle Virusproteine Antikörper nachweisbar, es gibt im Gegensatz zur Hepatitis B jedoch keine Anti-HCV-Antikörperkonstellation, die eine Immunität gegenüber HCV oder eine Ausheilung anzeigt. Auch die Präsenz von Antikörpern gegen das HCV während einer chronischen HCV Infektion mildert den Verlauf nicht. Im Gegenteil scheint eine erfolgreiche Therapie mit einem Absinken der Antikörpertiter verbunden zu sein. Daher ist es nicht möglich, eine Infektion mit Hepatitis C durch eine konventionelle, prophylaktische Impfung mit Hüllprotein, wie sie bei der Hepatitis B erfolgreich durchgeführt wird, zu verhindern. Eine prophylaktische Impfung ist daher derzeit nicht verfügbar.In chronic hepatitis C, antibodies to almost all virus proteins are detectable, but in contrast to hepatitis B, there is no anti-HCV antibody constellation that indicates immunity to HCV or a cure. The presence of antibodies to the HCV during a chronic HCV infection does not reduce the course. On the contrary, successful therapy appears to be associated with a decrease in antibody titers. It is therefore not possible to prevent infection with hepatitis C by conventional prophylactic vaccination with coat protein, as has been successfully done in hepatitis B. Prophylactic vaccination is therefore currently not available.
Die einzige derzeit zugelassene Therapie der chronischen Hepatitis C ist eine Behandlung mit Interferon alpha allein oder in Kombination mitThe only currently approved therapy for chronic hepatitis C is treatment with interferon alpha alone or in combination with
Ribavirin für 6 bis 12 Monate. Diese Therapieform ist sehr kostenintensiv, mit erheblichen Nebenwirkungen belastet und führt nur in ca. 50% der Fälle
zu einer dauerhaften Viruselimination. Peptidepitope enthaltend T- Zellepitope sind bereits identifiziert worden (Diepolder et al. J. Virol. 1997, EP: 00 121 138.2 PCT: WO 02/26785A2). Diese Epitope waren an einem Patientenkollektiv durch Züchtung von virus-spezifischen CD4+ T-Zell Klonen identifiziert worden.Ribavirin for 6 to 12 months. This form of therapy is very cost-intensive, has considerable side effects and only works in about 50% of the cases for permanent virus elimination. Peptide epitopes containing T cell epitopes have already been identified (Diepolder et al. J. Virol. 1997, EP: 00 121 138.2 PCT: WO 02 / 26785A2). These epitopes had been identified in a patient population by culturing virus-specific CD4 + T cell clones.
Hinzu kommt, dass bisherige systematische Untersuchungen im wesentlichen auf Daten basieren, die an Patienten mit einer chronischen Hepatitis C Infektion gewonnen wurden, und eine spontane Elimination des Virus zu dem Zeitpunkt, an dem bereits eine chronische HCV Infektion vorliegt, äußerst selten vorkommt. Epitope, die an Patienten mit einer chronischen HCV Infektion gefunden wurden sind daher nicht mit der Ausheilung der Erkrankung assoziiert.In addition, previous systematic examinations are mainly based on data obtained from patients with chronic hepatitis C infection and spontaneous elimination of the virus at the time when there is already a chronic HCV infection is extremely rare. Epitopes found in patients with chronic HCV infection are therefore not associated with the healing of the disease.
Daher liegt der vorliegenden Erfindung die Aufgabe zugrunde, gegenüber CD4+ T-Lymphozyten spezifische HCV Epitope zu identifizieren, die mit Viruselimination oder Virussuppression assoziiert sind.It is therefore the object of the present invention to identify HCV epitopes specific to CD4 + T lymphocytes which are associated with virus elimination or virus suppression.
Die Lösung der Aufgabe sind CD4+ T-Lymphozyten spezifische HCV- Epitope mit einem Impaktfaktor (IF) > Mittelwert (MW)+2*StaThe solution to the problem are CD4 + T lymphocyte-specific HCV epitopes with an impact factor (IF)> mean (MW) + 2 * Sta
wobeiin which
IF = "1 *1 - "2 * 1 ,5 0Q (Formel l ) mIF = " 1 * 1 -" 2 * 1, 5 0Q (formula l) m
wobei ni der Summe der Reaktionen mit 3 < Sl < 6, n2 der Summe der Reaktionen mit SI > 6 und m der Anzahl der Tests gegen das jeweilige Peptid entspricht, wobei m >15 ist, und MW der Mittelwert aller Impaktfaktoren ist.
Bevorzugt ist der Impcat Faktor der erfindungsgemäßen CD4+ T- Lymphozyten spezifischen HCV-Epitope > MW und < MW +1*Sta, insbesondere > MW +1 *Sta und < MW +2*Sta, besonders bevorzugt > MW +2*Sta.where ni is the sum of the reactions with 3 <Sl <6, n 2 is the sum of the reactions with SI> 6 and m is the number of tests against the respective peptide, where m> 15, and MW is the mean of all impact factors. The Impcat factor of the CD4 + T lymphocyte-specific HCV epitopes> MW and <MW + 1 * Sta, in particular> MW +1 * Sta and <MW + 2 * Sta, particularly preferably> MW + 2 * Sta is preferred.
Eine bevorzugte Lösung der Aufgabe sind CD4+ T-Lymphozyten spezifische HCV-Epitope, umfassend ein oder mehrere Peptide, ausgewählt aus der Gruppe:A preferred solution to the problem are CD4 + T lymphocyte-specific HCV epitopes, comprising one or more peptides selected from the group:
(EP001 GPRLGVRATRKTSER, (EP002 ARSLTPCTCGSSDLY, (EP003 SSDLYLVTRHADVIP, (EP004 MWKCLIRLKPTLHGP, (EP005 VLVDILAGYGAGVAG, (EP006 THYVPESDAAARVTQILSSL, (EP007 TITQLLKRLHQWINEDCSTP, (EP008 CSGSWLRDVWDWICTVLTDF, (EP009 GAQITGHVKNGSMRIVGPKT, (EP010 EVTRVGDFHYVTGMTTDNVK, (EP011 CPCQVPAPEFFTEVDGVRLH, (EP012 FTEVDGVRLHRYAPACKPLL, (EP013 TSMLTDPSHITAETAKRRLA, (EP014 SSSASQLSAPSLKATCTTHH, (EP015 REVSVAAEILRKSRKFPPAM, (EP016 PLLESWKDPDYVPPWHGCP, und (EP017 DWCCSMSYTWTGALITPCA sowie Derivate hiervon mit gleicher oder ähnlicher Spezifität.(EP001 GPRLGVRATRKTSER, (EP002 ARSLTPCTCGSSDLY, (EP003 SSDLYLVTRHADVIP, (EP004 MWKCLIRLKPTLHGP, (EP005 VLVDILAGYGAGVAG, (EP006 THYVPESDAAARVTQILSSL, (EP007 TITQLLKRLHQWINEDCSTP, (EP008 CSGSWLRDVWDWICTVLTDF, (EP009 GAQITGHVKNGSMRIVGPKT, (EP010 EVTRVGDFHYVTGMTTDNVK, (EP011 CPCQVPAPEFFTEVDGVRLH, (EP012 FTEVDGVRLHRYAPACKPLL, (EP013 TSMLTDPSHITAETAKRRLA, (EP014 SSSASQLSAPSLKATCTTHH, (EP015 REVSVAAEILRKSRKFPPAM, (EP016 PLLESWKDPDYVPPWHGCP, and (EP017 DWCCSMSYTWTGALITPCA as well as derivatives thereof with the same or similar.
Eine weitere bevorzugte Lösung der Aufgabe sind die nachstehend genannten gegenüber CD4+ T-Lymphozyten spezifische HCV-Epitope, enthaltend die Sequenz:
EP001 GPRLGVRATRKTSERA further preferred solution to the problem are the HCV epitopes specific to CD4 + T lymphocytes mentioned below, containing the sequence: EP001 GPRLGVRATRKTSER
EP002 ARSLTPCTCGSSDLYEP002 ARSLTPCTCGSSDLY
EP003 SSDLYLVTRHADVIP EP004 MWKCLIRLKPTLHGPEP003 SSDLYLVTRHADVIP EP004 MWKCLIRLKPTLHGP
EP005 VLVDILAGYGAGVAGEP005 VLVDILAGYGAGVAG
EP006 THYVPESDAAARVTQILSSLEP006 THYVPESDAAARVTQILSSL
EP007 TITQLLKRLHQWINEDCSTPEP007 TITQLLKRLHQWINEDCSTP
EP008 CSGSWLRDVWDWICTVLTDF EP009 GAQITGHVKNGSMRIVGPKTEP008 CSGSWLRDVWDWICTVLTDF EP009 GAQITGHVKNGSMRIVGPKT
EP010 EVTRVGDFHYVTGMTTDNVKEP010 EVTRVGDFHYVTGMTTDNVK
EP011 CPCQVPAPEFFTEVDGVRLHEP011 CPCQVPAPEFFTEVDGVRLH
EP012 FTEVDGVRLHRYAPACKPLLEP012 FTEVDGVRLHRYAPACKPLL
EP013 TSMLTDPSHITAETAKRRLA EP014 SSSASQLSAPSLKATCTTHHEP013 TSMLTDPSHITAETAKRRLA EP014 SSSASQLSAPSLKATCTTHH
EP015 REVSVAAEILRKSRKFPPAMEP015 REVSVAAEILRKSRKFPPAM
EP016 PLLESWKDPDYVPPWHGCP und/oderEP016 PLLESWKDPDYVPPWHGCP and / or
EP017 DWCCSMSYTWTGALITPCA.EP017 DWCCSMSYTWTGALITPCA.
Insbesondere bevorzugt sind die erfindungsgemäßen CD4+ T-Lymphozyten spezifischen HCV-Epitope, ausgewählt aus der Gruppe der Epitope EP001 bis EP0017 mit der vorstehend genannten Sequenz. Diese erfindungsgemäßen Epitope EP001 bis EP017 haben einen Impact factor von > MW+2*Sta.The CD4 + T lymphocyte-specific HCV epitopes according to the invention are particularly preferred, selected from the group of epitopes EP001 to EP0017 with the above-mentioned sequence. These epitopes EP001 to EP017 according to the invention have an impact factor of> MW + 2 * Sta.
Da diese Epitope im weiteren für eine Immuntherapie der chronischen Hepatitis C bzw. einer Vakzine eingesetzt werden sollen, sind weitere Kriterien ein hoher Grad an Konservierung zwischen verschiedenen Virussubtypen sowie ein hoher Grad an Promiskuität der Bindung an verschiedene HLA-Klasse II Moleküle. Die so identifizierten und charakterisierten HCV-Epitope sollen für einen Impfstoff zur Prophylaxe und/oder Therapie einer HCV-Infektion zur Verfügung stehen.
Zur Lösung der Aufgabe wurde ein einzigartiges Patientenkollektiv identifiziert, nämlich Patienten mit akuter Hepatitis C, die in über 50% der Fälle eine dauerhafte oder zumindest vorübergehende Viruselimination erreichen. Um alle möglichen CD4+ T Zellepitope zu untersuchen wurde das ganze Virus mit überlappenden, synthetischen Peptiden von 15 bis 20 Aminsäuren Länge abgedeckt. Als Testsystem wurde ein standardisiertes Lymphozyten-Proliferationsassay verwendet. Um sowohl den Grad an Konservierung zwischen verschiedenen Virussubtypen als die Bindungspromiskuität hinsichtlich des jeweiligen genetischen Hintergrunds der Patienten zu erfassen, wurde eine Formel definiert, die auf der Häufigkeit der Erkennung eines Epitops und der Stärke der jeweiligen Immunreaktion beruht.Since these epitopes are also to be used for immunotherapy of chronic hepatitis C or a vaccine, further criteria are a high degree of conservation between different virus subtypes and a high degree of promiscuity in binding to different HLA class II molecules. The HCV epitopes identified and characterized in this way should be available for a vaccine for the prophylaxis and / or therapy of an HCV infection. To solve the problem, a unique group of patients was identified, namely patients with acute hepatitis C who achieve permanent or at least temporary virus elimination in over 50% of cases. In order to investigate all possible CD4 + T cell epitopes, the entire virus was covered with overlapping synthetic peptides of 15 to 20 amino acids in length. A standardized lymphocyte proliferation assay was used as the test system. In order to measure both the degree of preservation between different virus subtypes and the binding promiscuity with regard to the respective genetic background of the patient, a formula was defined which is based on the frequency of detection of an epitope and the strength of the respective immune response.
Die Erfindung basiert auf der Auswahl eines besonderen Patientenkollektives, Untersuchungen mit definierten Peptiden und einem Algorithmus zur Identifizierung hochimmunogener CD4+ T-Zellepitope, die für die Entwicklung einer prophylaktischen oder therapeutischen Vakzine geeignet sind.The invention is based on the selection of a particular patient collective, examinations with defined peptides and an algorithm for the identification of highly immunogenic CD4 + T cell epitopes which are suitable for the development of a prophylactic or therapeutic vaccine.
Der Algorithmus bestimmt den „Impaktfaktor" (IF) des jeweiligen Epitops und definiert sich folgendermaßen:The algorithm determines the "impact factor" (IF) of the respective epitope and is defined as follows:
n-i *1 + n2 * 1 ,5 „, „ ____.,__, ._ni * 1 + n 2 * 1, 5 "," ____., __, ._
IF = - - ! * 100 (Formel l) mIF = - -! * 100 (Formula 1) m
ni der Summe der Reaktionen mit 3 < Sl < 6, n2 der Summe der Reaktionen mit Sl > 6 und m der Anzahl der durchgeführten Tests gegen das jeweilige Peptid entspricht, dies dient zur Normierung der Werte. Bei den von uns gefundenen Peptiden war m >15 .
Der Stimulationsindex (Sl) wird üblicherweise aus den Rohdaten eines Proliferationsassays berechnet und stellt den Multiplikationsfaktor der gemessenen Probe gegenüber der Kontrolle dar. Ein Sl von 3 gilt als signifikant.ni corresponds to the sum of the reactions with 3 <Sl <6, n 2 to the sum of the reactions with Sl> 6 and m to the number of tests carried out against the respective peptide, this serves to normalize the values. The peptides we found were m> 15. The stimulation index (Sl) is usually calculated from the raw data of a proliferation assay and represents the multiplication factor of the measured sample compared to the control. An Sl of 3 is considered significant.
Des weiteren wurde der Mittelwert aus den Impaktfaktor aller getesteten Peptide berechnet, wobei jeder Impaktfaktor nach Formel 1 bestimmt wurde. Um relevante Epitope statistisch präzise zu bestimmen, beschränkt sich unsere Lösung der Aufgabe auf die Peptide, deren IF zwei Standardabweichungen über dem Mittelwert aller IF liegen.Furthermore, the mean value was calculated from the impact factor of all peptides tested, each impact factor being determined according to Formula 1. In order to determine relevant epitopes statistically precisely, our solution of the task is limited to the peptides whose IF are two standard deviations above the mean of all IF.
Im Sinne der vorliegenden Erfindung bedeutet "gegenüber CD4+ T- Lymphozyten spezifische HCV-Epitope" eine definierte Region eines Hepatitis C Proteins, die auf Grund ihrer Struktur in die komplementäre Bindungsstelle eines CD4+ T-Lymphozyten-Rezeptors "passt" und dadurch hochspezifisch eine Reaktion auslöst.For the purposes of the present invention, “HCV epitopes specific to CD4 + T lymphocytes” means a defined region of a hepatitis C protein which, owing to its structure, “fits” into the complementary binding site of a CD4 + T lymphocyte receptor and thereby a highly specific reaction triggers.
Da die primäre Aminosäurestruktur der HCV Proteine bekannt ist, wurden hier insgesamt über 450 synthetische Peptide (15-20mere) verwendet, die jeweils zwischen 5 und 10 Aminosäuren überlappen und die bekannten Struktur- und Nichtstruktur-Proteine umspannen.Since the primary amino acid structure of the HCV proteins is known, a total of over 450 synthetic peptides (15-20mers) were used, each overlapping between 5 and 10 amino acids and spanning the known structural and non-structural proteins.
Kollektiv:Collective:
Es wurde ein besonderes Patientenkollektiv gewählt und dieses auf für die Ausheilung der Erkrankung relevanten HCV spezifischen CD4+ T-Zell- epitope untersucht. Das Patientenkollektiv, nämlich Patienten mit akuter Hepatitis C, ist schwierig zu identifizieren, da einerseits ihr Vorkommen bei einer Häufigkeit von ca. 1 :100 000 in der deutschen Bevölkerung liegt und andererseits ausschließlich T-Lymphozyten in der akuten Phase der Erkrankung getestet wurden. Dies bedeutet eine erhebliche Einschränkung der Anzahl der verwendbaren Proben. Schließlich wurden innerhalb dieses
Patientenkollektiv nur Patienten berücksichtigt, die in der Lage waren das Virus spontan zu klären bzw. temporär zu kontrollieren (dies sind nur ca. 60% der Patienten mit akuter Hepatitis C), da nur hier das Immunsystem erfolgreich gegen das Virus vorgegangen ist. Hingegen stellt eine spontane Elimination des Virus zu einem späteren Zeitpunkt (chronische HCV) eine Rarität dar. Auch findet sich in der späten, chronischen Phase nur eine geringe oder nicht detektierbare, gegen das Virus gerichtete CD4+ T-Zellantwort. Von herausragender Wichtigkeit sind daher vor allem Epitope, die mit temporärer oder dauerhafter Viruskontrolle einher gehen. Diese Epitope bzw. die gemessene Reaktion auf diese Peptide sind also mittelbar oder unmittelbar mit der Ausheilung der HCV Infektion assoziiert und stellen somit ideale Kandidaten für zukünftige "Peptidimpfungen" dar. Dies trifft auf die erfindungsgemäßen Peptidsequenzen bzw. Peptide zu.A special group of patients was chosen and examined for CD4 + T cell epitopes that are specific for HCV and heal the disease. The patient population, namely patients with acute hepatitis C, is difficult to identify because on the one hand their occurrence is approximately 1: 100,000 in the German population and on the other hand only T-lymphocytes were tested in the acute phase of the disease. This means a considerable limitation of the number of samples that can be used. Finally, within this The patient group only considers patients who were able to clear the virus spontaneously or to control it temporarily (this is only approx. 60% of patients with acute hepatitis C), because only here the immune system has successfully acted against the virus. In contrast, spontaneous elimination of the virus at a later point in time (chronic HCV) is a rarity. Also in the late, chronic phase there is only a small or undetectable CD4 + T cell response directed against the virus. Epitopes associated with temporary or permanent virus control are therefore of particular importance. These epitopes or the measured response to these peptides are therefore indirectly or directly associated with the healing of the HCV infection and thus represent ideal candidates for future “peptide vaccinations”. This applies to the peptide sequences or peptides according to the invention.
Peptide:peptides:
Es wurde ein "Peptidscreening" mit ca. 450 ausgewählten unterschiedlichen Peptiden (15-22mere) hinsichtlich einer virusspezifischen CD4+ T- Zellantwort bei oben beschriebenen Patientenkollektiv durchgeführt.A "peptide screening" with approximately 450 selected different peptides (15-22mers) was carried out for a virus-specific CD4 + T cell response in the patient population described above.
Die Peptide repräsentieren das gesamte Virusprotein, wobei wir 15mere mit 5 Aminosäuren langen überlappenden Bereichen bzw. 20-22mere mit 10 Aminosäuren überlappenden Bereichen verwendeten um möglichst alle relevanten Epitope zu erfassen.The peptides represent the entire virus protein, whereby we used 15mers with 5 amino acid overlapping areas or 20-22mers with 10 amino acids overlapping areas in order to record all relevant epitopes.
Der Tabelle 1 sind die jeweiligen Positionen in HCV Genom der erfindungsgemäßen Epitope unter Angabe der jeweiligen Virusisolat Referenz zu entnehmen (Tabelle 1 / Spalte 4). Die Angaben zur Aminosäureposition (Tabelle 1 / Spalte 2) sind nur als Näherung zu verstehen, da aufgrund der hohen Mutationsrate des Virus bei den
verschiedenen Virusisolaten zu Änderungen der Position kommen kann. Besondere Bedeutung für prophylaktische und therapeutische Impfungen dürfte den konservierten Epitopen zu kommen, was sich in der gleichbleibenden Sequenz der unterschiedlichen Virusisolate eines Epitops zeigt (s.a. Tabelle 1 / Spalte 5).Table 1 shows the respective positions in the HCV genome of the epitopes according to the invention, stating the respective virus isolate reference (Table 1 / column 4). The information on the amino acid position (Table 1 / Column 2) should only be understood as an approximation, because due to the high mutation rate of the virus in the different virus isolates can cause changes in position. The conserved epitopes are of particular importance for prophylactic and therapeutic vaccinations, which is shown by the constant sequence of the different virus isolates of an epitope (see also Table 1 / column 5).
Aus früheren eigenen Untersuchungen ging hervor, dass bestimmten Bereichen des Virus besondere immunologische Bedeutung zukommt, deshalb wurden diese Bereiche des Virusgenoms (NS3-NS4) mit Hilfe von Peptiden (20mere mit 10 Aminosäuren langen überlappenden Bereichen) zusätzlich getestet.Previous own investigations showed that certain areas of the virus are of particular immunological importance, which is why these areas of the virus genome (NS3-NS4) were additionally tested with the help of peptides (20mers with overlapping areas with 10 amino acids).
In Tabelle 1 sind die erfindungsgemäßen Epitope aufgeführt.The epitopes according to the invention are listed in Table 1.
Tabelle 1 :Table 1 :
Liste 1 : Virusisolate List 1: Virus isolates
11
Genotype: 1 b AUTHORS Trowbridge.R. and Gowans.E.J. TITLE Molecular cloning of an Australian isolate of hepatitis C virus JOURNAL Arch. Virol. 143 (3), 501-511 (1998)Genotype: 1 b AUTHORS Trowbridge.R. and Gowans.E.J. TITLE Molecular cloning of an Australian isolate of hepatitis C virus JOURNAL Arch. Virol. 143 (3), 501-511 (1998)
22
Genotype: 1b AUTHORS Takamizawa,A., Mori.C, Fuke.l., Manabe.S., Murakami.S., Fujita.J., Onishi.E., Andoh.T., Yoshida.l. and Okayama.H.Genotype: 1b AUTHORS Takamizawa, A., Mori.C, Fuke.l., Manabe.S., Murakami.S., Fujita.J., Onishi.E., Andoh.T., Yoshida.l. and Okayama.H.
TITLE Structure and organization of the hepatitis C virus genome isolated from human carriersTITLE Structure and organization of the hepatitis C virus genome isolated from human carriers
JOURNAL J. Virol. 65 (3), 1105-1113 (1991)JOURNAL J. Virol. 65 (3), 1105-1113 (1991)
3 Genotype: 1 a AUTHORS Yanagi.M., Purcell.R.H., Emerson.S.U. and Bukh.J.3 Genotypes: 1 a AUTHORS Yanagi.M., Purcell.R.H., Emerson.S.U. and Bukh.J.
TITLE Transcripts from a Single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzeeTITLE Transcripts from a Single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee
JOURNAL Proc. Natl. Acad. Sei. U.S.A. 94 (16), 8738-8743 (1997)JOURNAL Proc. Natl. Acad. Be. U.S.A. 94 (16), 8738-8743 (1997)
4 Genotype:2a AUTHORS Okamoto.H., Okada.S., Sugiyama.Y, Kurai.K., lizuka.H.,4 Genotype: 2a AUTHORS Okamoto.H., Okada.S., Sugiyama.Y, Kurai.K., Lizuka.H.,
Machida.A., Miyakawa.Y. and Mayumi.M.Machida.A., Miyakawa.Y. and Mayumi.M.
TITLE Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human JOURNAL J. Gen. Virol. 72 (Pt 11), 2697-2704 (1991)TITLE Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human JOURNAL J. Gen. Virol. 72 (Pt 11), 2697-2704 (1991)
5 Genotype:2b AUTHORS Okamoto.H., Kurai.K., Okada.S., Yamamoto.K., Lizuka.H.,5 Genotype: 2b AUTHORS Okamoto.H., Kurai.K., Okada.S., Yamamoto.K., Lizuka.H.,
Tanaka.T., Fukuda.S., Tsuda.F. and Mishiro.S.Tanaka.T., Fukuda.S., Tsuda.F. and Mishiro.S.
TITLE Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates: comparative study of four distinet genotypesTITLE Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates: comparative study of four distinet genotypes
JOURNAL Virology 188 (1), 331-341 (1992) 6JOURNAL Virology 188 (1), 331-341 (1992) 6
Genotype: 1a AUTHORS CHOO.Q.-L, RICHMAN.K.H., HAN.J.H., BERGER.K., LEE.C,Genotype: 1a AUTHORS CHOO.Q.-L, RICHMAN.K.H., HAN.J.H., BERGER.K., LEE.C,
DONG.C, GALLEGOS.C, COIT.D., MEDINA-SELBY.A., BARR.P.J., WEINER.A.J.,DONG.C, GALLEGOS.C, COIT.D., MEDINA-SELBY.A., BARR.P.J., WEINER.A.J.,
BRADLEY.D.W., KUO.G. and HOUGHTON.M.BRADLEY.D.W., KUO.G. and HOUGHTON.M.
TITLE Genetic organization and diversity of the hepatitis C virus JOURNAL Proc. Natl. Acad. Sei. U.S.A. 88 (6), 2451-2455 (1991)TITLE Genetic organization and diversity of the hepatitis C virus JOURNAL Proc. Natl. Acad. Be. U.S.A. 88 (6), 2451-2455 (1991)
77
Genotype: 1 b AUTHORS Tanaka.T., Kato.N., Nakagawa.M., Ootsuyama.Y., Cho.M.J.,Genotype: 1 b AUTHORS Tanaka.T., Kato.N., Nakagawa.M., Ootsuyama.Y., Cho.M.J.,
Nakazawa.T., Hijikata.M., Ishimura.Y and Shimotohno.K.
TITLE Molecular cloning of hepatitis C virus genome from a Single Japanese earrier: sequence Variation within the same individual and among infected individualsNakazawa.T., Hijikata.M., Ishimura.Y and Shimotohno.K. TITLE Molecular cloning of hepatitis C virus genome from a Single Japanese earrier: sequence Variation within the same individual and among infected individuals
JOURNAL Virus Res. 23 (1-2), 39-53 (1992JOURNAL Virus Res. 23 (1-2), 39-53 (1992
8 Genotype:1 b AUTHORS Kato.N., Hijikata.M., Ootsuyama.Y, Nakagawa.M.,8 Genotype: 1 b AUTHORS Kato.N., Hijikata.M., Ootsuyama.Y, Nakagawa.M.,
Ohkoshi,S.,Sugimura,T. and Shimotohno.K.Ohkoshi, S., Sugimura T. and Shimotohno.K.
TITLE Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitisTITLE Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis
JOURNAL Proc. Natl. Acad. Sei. U.S.A. 87 (24), 9524-9528 (1990) 9JOURNAL Proc. Natl. Acad. Be. U.S.A. 87 (24), 9524-9528 (1990) 9
Genotype:1 b AUTHORS Chen.P.J., Lin.M.H., Tai.K.F., Liu.P.C, Lin.C.J. andGenotype: 1 b AUTHORS Chen.P.J., Lin.M.H., Tai.K.F., Liu.P.C, Lin.C.J. and
Chen.D.S.Chen.D.S.
TITLE The Taiwanese hepatitis C virus genome: sequence determination and mapping the 5' termini of viral genomic and antigenomic RNA JOURNAL Virology 188 (1), 102-113 (1992)TITLE The Taiwanese hepatitis C virus genome: sequence determination and mapping the 5 'termini of viral genomic and antigenomic RNA JOURNAL Virology 188 (1), 102-113 (1992)
1010
Genotype:1a AUTHORS Inchauspe.G., Zebedee.S., Lee.D.H., Sugitani.M., Nasoff.M. and Prince.A.M.Genotype: 1a AUTHORS Inchauspe.G., Zebedee.S., Lee.D.H., Sugitani.M., Nasoff.M. and Prince.A.M.
TITLE Genomic strueture of the human prototype strain H of hepatitis C virus: comparison with American and Japanese isolatesTITLE Genomic strueture of the human prototype strain H of hepatitis C virus: comparison with American and Japanese isolates
JOURNAL Proc. Natl. Acad. Sei. U.SA 88 (22), 10292-10296 (1991)JOURNAL Proc. Natl. Acad. Be. U.SA 88 (22), 10292-10296 (1991)
1111
Genotype:3b AUTHORS Chayama.K. Toranomon Hospital, Department ofGenotype: 3b AUTHORS Chayama.K. Toranomon Hospital, Department of
Gastroenteroloty; 2-2-2 Toranomon, Minato-ku, Tokyo 105, Japan TITLE Direct SubmissionGastroenteroloty; 2-2-2 Toranomon, Minato-ku, Tokyo 105, Japan TITLE Direct Submission
JOURNAL Submitted (18-FEB-1995) to the DDBJ/EMBUGenBank databases.JOURNAL Submitted (18-FEB-1995) to the DDBJ / EMBUGenBank databases.
COMMENT D26556:Submitted (20-Jan-1994) to DDBJ by: Kazuaki Chayama.COMMENT D26556: Submitted (20-Jan-1994) to DDBJ by: Kazuaki Chayama.
1212
Genotype:4 AUTHORS Chamberlain R , Adams N, Saeed AA, Simmonds P, Elliott RMGenotype: 4 AUTHORS Chamberlain R, Adams N, Saeed AA, Simmonds P, Elliott RM
TITLE : Complete nucleotide sequence of a type 4 hepatitis C virus variant, the predominant genotype in the Middle East.TITLE: Complete nucleotide sequence of a type 4 hepatitis C virus variant, the predominant genotype in the Middle East.
JOURNAL: J Gen Virol. 1997 Jun;78 ( Pt 6):1341-7.JOURNAL: J Gen Virol. 1997 Jun; 78 (Pt 6): 1341-7.
13 Genotype:5a AUTHORS Chamberlain RW, Adams NJ, Taylor LA, Simmonds P, Elliott13 Genotype: 5a AUTHORS Chamberlain RW, Adams NJ, Taylor LA, Simmonds P, Elliott
RM.RM.
TITLE The complete coding sequence of hepatitis C virus genotype 5a, the predominant genotype in South Africa.TITLE The complete coding sequence of hepatitis C virus genotype 5a, the predominant genotype in South Africa.
JOURNAL Biochem Biophys Res Commun. 1997 Jul 9;236(1):44-9.
14JOURNAL Biochem Biophys Res Commun. 1997 Jul 9; 236 (1): 44-9. 14
Genotype: 6a AUTHORS Adams, N.J., Chamberlain, R.W., Taylor.LA, Davidson, F.,Genotype: 6a AUTHORS Adams, N.J., Chamberlain, R.W., Taylor.LA, Davidson, F.,
Lin,C.K.,Elliott,R.M. and Simmonds.P.Lin, C. K., Elliott, R.M.. and Simmonds.P.
TITLE Complete coding sequence of hepatitis C virus genotype 6a JOURNAL Biochem. Biophys. Res. Commun. 234 (2), 393-396 (1997)TITLE Complete coding sequence of hepatitis C virus genotype 6a JOURNAL Biochem. Biophys. Res. Commun. 234 (2), 393-396 (1997)
1515
Genotype:1 b AUTHORS Honda, M., Kaneko.S., Unoura.M., Kobayashi.K. andGenotype: 1 b AUTHORS Honda, M., Kaneko.S., Unoura.M., Kobayashi.K. and
Murakami.S.Murakami.S.
TITLE Sequence comparisons for a hepatitis C virus genome RNA isolated from a patient with liver cirrhosisTITLE Sequence comparisons for a hepatitis C virus genome RNA isolated from a patient with liver cirrhosis
JOURNAL Gene 120 (2), 317-318 (1992)JOURNAL Gene 120 (2), 317-318 (1992)
1616
Genotype:1 b AUTHORS Okamoto.H., Kojima.M., Okada.S., Yoshizawa.H., lizuka.H.,Genotype: 1 b AUTHORS Okamoto.H., Kojima.M., Okada.S., Yoshizawa.H., Lizuka.H.,
Tanaka.T., Muchmore.E.E., Peterson.DA, Ito.Y. and Mishiro.S. TITLE Genetic drift of hepatitis C virus during an 8.2-year infection in a ehimpanzee: variability and stability JOURNAL Virology 190 (2), 894-899 (1992)Tanaka.T., Muchmore.E.E., Peterson.DA, Ito.Y. and Mishiro.S. TITLE Genetic drift of hepatitis C virus during an 8.2-year infection in a ehimpanzee: variability and stability JOURNAL Virology 190 (2), 894-899 (1992)
1717
Genotype:1a AUTHORS Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M.Genotype: 1a AUTHORS Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M.
Title: Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.Title: Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.
Journal: Science 1989 Apr 21 ;244(4902):359-62Journal: Science 1989 Apr 21; 244 (4902): 359-62
Genotype: 1bGenotype: 1b
Primary consensus sequence complete 1 b genomes available in EMBL database (January 2000)Primary consensus sequence complete 1 b genomes available in EMBL database (January 2000)
Testsystem:Test System:
Als Testsystem wurde der sogenannte Proliferationsassay nach folgendem Protokoll eingesetzt. Nach Dichtegradientenzentrifugation auf Ficollgradienten von heparinisiertem Blut wurden die frischen peripheren mononukleären Blutzellen (PBMC) isoliert und in einem Kulturmedium (RPMI1640, Gibco) suspendiert. 50 μl dieser Zellsuspension (Konzentration von 1x106 Zellen pro ml) wurden auf sterile 96 Loch-Kulturplatten verbracht.
Durch Zugabe der Peptide wurden die Zellen stimuliert. Die Endkonzentration der Peptide betrug 10μg/ml. Die Zellkulturplatten wurden über 5 Tage bei 37°C und 5%C02 kultiviert, anschließend mit 3H-Thymidin versetzt und als Maß für die Zellstimulation wurde der Einbau des radioaktiven 3H gemessen.The so-called proliferation assay according to the following protocol was used as the test system. After density gradient centrifugation on Ficoll gradients from heparinized blood, the fresh peripheral blood mononuclear cells (PBMC) were isolated and suspended in a culture medium (RPMI1640, Gibco). 50 μl of this cell suspension (concentration of 1x10 6 cells per ml) were placed on sterile 96-well culture plates. The cells were stimulated by adding the peptides. The final concentration of the peptides was 10μg / ml. The cell culture plates were cultivated for 5 days at 37 ° C. and 5% CO 2 , then 3 H-thymidine was added, and the incorporation of the radioactive 3 H was measured as a measure of the cell stimulation.
Auswertung:Evaluation:
Bei unseren Untersuchungen wurden nur Peptidreaktionen als signifikant angesehen, deren Stimulationsindex (Sl) größer 3 war (3x erhöht gegenüber den Kontrollen, bzw. den irrelevanten Peptiden).In our investigations, only peptide reactions were considered significant if their stimulation index (Sl) was greater than 3 (3x increased compared to the controls or the irrelevant peptides).
Um einerseits die Möglichkeit falsch positiver Reaktionen sowie irrelevanter Kreuzreaktionen zu minimieren und andererseits eine Hierarchie hinsichtlich der biologischen Wertigkeit relevanter Epitope zu erstellen, wurde ein zusätzlicher Filter definiert und im Folgenden als Impaktfaktor (IF) bezeichnet.In order to minimize the possibility of false positive reactions and irrelevant cross-reactions on the one hand and on the other hand to create a hierarchy with regard to the biological value of relevant epitopes, an additional filter was defined and referred to below as an impact factor (IF).
Dem Impaktfaktor (IF) liegt ein Punktesystem zugrunde, welches nicht nur die Häufigkeit relevanter Reaktionen, d.h. Stimulationsindex (Sl) größer 3, wie herkömmlicherweise, verwendet, sondern auch die Stärke dieser Reaktionen berücksichtigt.The impact factor (IF) is based on a point system that not only measures the frequency of relevant reactions, i.e. Stimulation index (Sl) greater than 3, as used conventionally, but also takes into account the strength of these reactions.
Dieses Bewertungssystem wurde für jedes untersuchte Peptid angewendet und ist nach folgender Formel definiert:This rating system was used for each peptide examined and is defined according to the following formula:
IF = "1 *1 + "2 * L5 , ,m (Formel l) mIF = " 1 * 1 + " 2 * L5,, m (formula l) m
wobei rti der Summe der Reaktionen mit 3 < Sl < 6, n2 der Summe der Reaktionen mit Sl > 6 und
m der Anzahl der Tests gegen das jeweilige Peptid entspricht, wobei m >15 war.where rti the sum of the reactions with 3 <Sl <6, n 2 the sum of the reactions with Sl> 6 and m corresponds to the number of tests against the respective peptide, where m was> 15.
Beispiel:Example:
Gegen EP007 wurden in 16 (m) unabhängigen Versuchen folgende spezifischen Sl's (Stimulationsindex) gemessen: 0,91 ; 1 ,07; 1 ,10; 1 ,24; 1 ,32; 1 ,33; 1 ,40; 1 ,46; 1 ,81 ; 1 ,84; 3,01 ; 3,25; 4,38; 5,32; 7,58 und 12,77.The following specific Sl 's (stimulation index) were measured in 16 (m) independent experiments against EP007: 0.91; 1, 07; 1, 10; 1, 24; 1.32; 1.33; 1.40; 1.46; 1.81; 1.84; 3.01; 3.25; 4.38; 5.32; 7.58 and 12.77.
Damit ergibt sich n1 = 4 (4 Werte >3 und <6) ; n2 = 2 (2 Werte >6), bzw. eingesetzt in die FormelThis results in n 1 = 4 (4 values> 3 and <6); n 2 = 2 (2 values> 6), or used in the formula
4 + 34 + 3
IF = * 100 (Formel l )IF = * 100 (Formula 1)
1616
ein Impaktfaktor von 43,75 für dieses Peptid. Da bei den von uns getesteten Peptiden der Mittelwert aller Impaktfaktoren 7,36 und die Standardabweichung 6,75 betrug, entspricht dieser Impaktfaktor von 43,75 einem Wert > MW +2*Sta. Für jedes untersuchte Peptid wurde der jeweilige Impaktfaktor berechnet. Aus allen Impaktfaktoren ließen sich dann Mittelwert und Standardabweichung errechnen.an impact factor of 43.75 for this peptide. Since the average of all impact factors for the peptides we tested was 7.36 and the standard deviation was 6.75, this impact factor of 43.75 corresponds to a value> MW +2 * Sta. The respective impact factor was calculated for each peptide examined. The mean and standard deviation could then be calculated from all impact factors.
Somit ergibt sich je nach Höhe des Impaktfaktors eine biologischimmunologisch bedeutsame Hierarchie. Peptide mit hohem Impaktfaktor zeichnen sich nicht nur durch einen großen Stimulationsindex, d.h. starke spezifische Reaktiviät, sondern auch durch die wiederholt d.h. bei unterschiedlichen Personen gefundene spezifische Reaktion aus.Depending on the level of the impact factor, this results in a hierarchy that is of biological importance. Peptides with a high impact factor are not only characterized by a large stimulation index, i.e. strong specific reactivity but also by the repeated i.e. specific reaction found in different people.
Diese hier getroffene Selektion soll der immunologischen Wertigkeit, der hier aufgeführten Peptide, besser gerecht werden. Epitope, die starke und bei unterschiedlichen Patienten gemessene HCV spezifisch CD4+ T-Zell Antworten auslösen, haben für künftige Impfansätze eine hohe Relevanz.
Die erfindungsgemäßen Epitope zeichnet weiterhin aus, dass eine deutliche spezifische CD4+ T-Zell Aktivität auf diese Peptide mit einer Abnahme des Virustiters korreliert. Gerade diese Epitope dürften somit ideale Kandidaten für eine Vakzine darstellen. Eine spezifisch Impfreaktion gegen diese Peptide, könnte die Erkrankung auf der einen Seite verhindern und/oder auf der anderen Seite zu deren Ausheilung führen, zumindest aber den Verlauf einer HCV Infektion günstig beeinflussen.This selection made here should better do justice to the immunological value of the peptides listed here. Epitopes that trigger strong HCV-specific CD4 + T cell responses measured in different patients are of great relevance for future vaccination approaches. The epitopes according to the invention are further distinguished by the fact that a clear specific CD4 + T cell activity on these peptides correlates with a decrease in the virus titer. These epitopes in particular are therefore likely to be ideal candidates for a vaccine. A specific vaccination reaction against these peptides could prevent the disease on the one hand and / or lead to their healing on the other hand, or at least have a favorable effect on the course of an HCV infection.
Die erfindungsgemäßen Epitope sind hochimmunogene, hochkonservierte und zum Teil in unmittelbarer Nachbarschaft zu bekannten CD8+ T- Lymphozyten spezifischen HCV-Epitopen gelegene Sequenzen des HCV. Als gegenüber CD4+ T-Lymphozyten spezifische HCV-Epitope können diese neben der Induktion von CD4+ T-Lymphozyten auch sogenannte T-Zell- Hilfe für zytotoxische CD8+ T-Lymphozyten vermitteln. Diese CD8+-T- Lymphozyten werden durch die Zytokine stimulierter CD4+ T-Lymphozyten aktiviert.The epitopes according to the invention are highly immunogenic, highly conserved sequences of the HCV, some of which are located in the immediate vicinity of known CD8 + T lymphocyte-specific HCV epitopes. As HCV epitopes specific to CD4 + T lymphocytes, they can mediate so-called T cell help for cytotoxic CD8 + T lymphocytes in addition to induction of CD4 + T lymphocytes. These CD8 + T lymphocytes are activated by the cytokines of stimulated CD4 + T lymphocytes.
Des weiteren zeichnen sich die Peptide durch häufige signifikante Reaktion bei verschiedenen Patienten mit unterschiedlichen MHC Klasse II TypenFurthermore, the peptides are characterized by frequent significant reactions in different patients with different MHC class II types
(major histocompability complex) aus. Das MHC Klasse II System ist ausgesprochen polymorph. Die Aufgabe der MHC Moleküle ist es von körpereigenen und pathogenen (z.B. Hepatitis C Virus) Proteinen stammende Peptidfragmente zu binden und sie zur Erkennung und Aktivierung von spezifischen CD4+T-Lymphozyten an der Zelloberfläche zu exprimieren. Dieses System ermöglicht eine wirkungsvolle und spezifische(major histocompability complex). The MHC Class II system is extremely polymorphic. The task of the MHC molecules is to bind peptide fragments derived from the body's own and pathogenic (e.g. hepatitis C virus) proteins and to express them on the cell surface for the detection and activation of specific CD4 + T lymphocytes. This system enables an effective and specific
Immunantwort gegen Pathogene wie z.B. HCV. Da nun verschiedene MHCImmune response to pathogens such as HCV. Since different MHC
Klasse II Typen, d.h. unterschiedliche Personen, dasselbe Peptid auf ihremClass II types, i.e. different people, the same peptide on their
MHC Klasse II Molekül exprimieren können, was in vitro durch bei unterschiedlichen Personen messbare, gegen dasselbe Peptid gerichteteMHC class II molecule can express what in vitro by measurable in different people, directed against the same peptide
CD4+ Aktivität wiederfindet, kann bei diesen Peptiden von einerCD4 + activity can be detected by one of these peptides
Promiskuität ausgegangen werden. Dies bedeutet, dass die
erfindungsgemäßen Epitope immunologische Relevanz bei verschiedenen Individuen besitzen.Promiscuity. This means that the Epitopes according to the invention have immunological relevance in different individuals.
Zusammenfassend eignen sich gerade die erfindungsgemäßen Epitope im hohen Maße sowohl für eine therapeutische als auch prophylaktische Peptidimpfung, die gegen das HCV gerichtet ist.In summary, the epitopes according to the invention are particularly suitable for both therapeutic and prophylactic peptide vaccination, which is directed against HCV.
Eine weitere Lösung ist ein Impfstoff, der eine Kombination der erfindungsgemäßen Epitope EP001 bis EP017 enthält. Der Impfstoff kann insbesondere bevorzugt ein Gemisch aus den erfindungsgemäßen Epitopen EP001 bis EP017 enthalten. Allerdings können auch weitere HCV Epitope anwesend sein.Another solution is a vaccine which contains a combination of the epitopes EP001 to EP017 according to the invention. The vaccine can particularly preferably contain a mixture of the epitopes EP001 to EP017 according to the invention. However, other HCV epitopes may also be present.
Die erfindungsgemäßen Epitope können allein oder mit einem oder mehreren Hilfsstoffen als Arzneimittel, vorzugsweise als Impfstoff, eingesetzt werden. Der erfindungsgemäße Impfstoff enthält mindestens ein erfindungsgemäßes Epitop, vorzugsweise ein Gemisch aus erfindungsgemäßen Epitopen. Allerdings können auch weitere HCV Epitope anwesend sein.The epitopes according to the invention can be used alone or with one or more auxiliary substances as medicaments, preferably as vaccines. The vaccine according to the invention contains at least one epitope according to the invention, preferably a mixture of epitopes according to the invention. However, other HCV epitopes may also be present.
Die Hilfsstoffe werden vorzugsweise ausgewählt aus der Gruppe, bestehend aus fowlpoxvirus, modifiziertes Vakziniavirus Ankara, Virosomen, Transvax® und anderen die Immunreaktion verstärkenden Stoffen.The excipients are preferably selected from the group consisting of fowl pox virus, modified vaccinia virus Ankara, virosomes, TransVax ® and other immune response enhancing substances.
Der erfindungsgemäße Impfstoff kann oral, parenteral, intramuskulär, intravenös, subcutan oder intracutan verabreicht werden. "The vaccine according to the invention can be administered orally, parenterally, intramuscularly, intravenously, subcutaneously or intracutaneously. "
Bei den erfindungsgemäßen Epitopen handelt es sich um Epitope, die als T- Zell-stimulierender Impfstoff eingesetzt werden können. Ein Impfstoff, der die erfindungsgemäßen Epitope enthält, hat gegenüber einer Impfung mit dem gesamten Virusprotein, das verschiedenste Epitope für virusspezifische T-Lymphozyten enthält und nur B-Lymphozyten und
CD4+T-Lymphozyten induziert, den Vorteil, dass es spezifische T- Lymphozyten, CD4+- und/oder CD8+-T-Lymphozyten selektiv induziert. Außerdem werden dadurch antagonistische Effekte bzw. die Gefahr von iatrogen erzeugten Autoimmunreaktionen, die bei Impfungen mit ganzen Proteinen auftreten können, vermieden. Die erfindungsgemäßen Epitope haben zusätzlich eine höhere Immunogenität im Vergleich zu dem gesamten Virusprotein, wodurch ein besseres Impfergebnis erreicht wird.The epitopes according to the invention are epitopes that can be used as a T cell stimulating vaccine. A vaccine, which contains the epitopes according to the invention, has a vaccination with the entire virus protein, which contains various epitopes for virus-specific T-lymphocytes and only B-lymphocytes and CD4 + T lymphocytes induce the advantage that it selectively induces specific T lymphocytes, CD4 + and / or CD8 + T lymphocytes. It also avoids antagonistic effects or the risk of iatrogenic autoimmune reactions that can occur when vaccinating with whole proteins. The epitopes according to the invention additionally have a higher immunogenicity compared to the entire virus protein, as a result of which a better vaccination result is achieved.
Der erfindungsgemäße Impfstoff ermöglicht somit im gesunden Menschen die Induktion einer Immunantwort und dient daher als prophylaktische Impfung. Auch bei chronisch HCV-infizierten Menschen kann der erfindungsgemäße Impfstoff eine Immunantwort induzieren und somit als therapeutischer Impfstoff dienen.The vaccine according to the invention thus enables the induction of an immune response in healthy people and therefore serves as a prophylactic vaccination. The vaccine according to the invention can also induce an immune response in chronically HCV-infected people and thus serve as a therapeutic vaccine.
Die kodierende cDNA dieser Epitope kann in einem DNA-Impfstoff, einer speziellen Impfmethode, eingesetzt werden. Dabei wird die für die entsprechenden Epitope codierende DNA in einen Vector Moniert. Dieses Konstrukt wird wiederum dem zu impfenden Individuum parenteral verabreicht (z.B. Immunology and Cell Biology, Band 75, Seite 382 bis 388). Entsprechend des degenerierten genetischen Codes können verschiedene DNA-Sequenzen eines der erfindungsgemäßen Epitope kodieren (siehe Current protocols, Wiley).The coding cDNA of these epitopes can be used in a DNA vaccine, a special vaccination method. The DNA coding for the corresponding epitopes is cloned into a vector. This construct is in turn administered parenterally to the individual to be vaccinated (e.g. Immunology and Cell Biology, volume 75, pages 382 to 388). According to the degenerate genetic code, different DNA sequences can encode one of the epitopes according to the invention (see Current protocols, Wiley).
Die erfindungsgemäßen Epitope können auch in der Diagnose des Verlaufs einer HCV-Infektion Verwendung finden, indem die Menge an CD4+ T- Lymphozyten, die spezifisch das betreffende Epitop erkennen, im Blut des Patienten mit einer Hepatitis C Infektion überwacht wird. Dies kann beispielsweise mit einem diagnostischen Kit durchgeführt werden, der eines oder mehrere der erfindungsgemäßen Epitope umfasst.
SequenzprotokollThe epitopes according to the invention can also be used in the diagnosis of the course of an HCV infection by monitoring the amount of CD4 + T lymphocytes which specifically recognize the epitope in question in the blood of the patient with a hepatitis C infection. This can be carried out, for example, using a diagnostic kit which comprises one or more of the epitopes according to the invention. sequence Listing
<110> Iπnuusystems GmbH<110> Iπnuusystems GmbH
<120> CD4+Lymphozyten spezifische HCV EPITOPE<120> CD4 + lymphocyte specific HCV EPITOPE
<130> PCT 1854-099 <150> EP 02 008 033.9<130> PCT 1854-099 <150> EP 02 008 033.9
<151> 2002-04-10<151> 2002-04-10
<160> 17 <170> Patentin ersion 3.1<160> 17 <170> Patent version 3.1
<210> 1<210> 1
<211> 15<211> 15
<212> PRT <213> Hepatitis C virus<212> PRT <213> Hepatitis C virus
<400> 1<400> 1
Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg 1 5 10 15Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg 1 5 10 15
<210> 2<210> 2
<211> 15<211> 15
<212> PRT<212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400> 2 Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr 1 5 10 15<400> 2 Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr 1 5 10 15
<210> 3 <211> 15<210> 3 <211> 15
<212> PRT<212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400><400>
Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro 1 5 10 15Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro 1 5 10 15
<210> 4<210> 4
<211> 15<211> 15
<212> PRT<212> PRT
<213> Hepatitis C virus <400> 4<213> Hepatitis C virus <400> 4
Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro 1 5 10 15Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro 1 5 10 15
<210>
<211> 15<210> <211> 15
<212> PRT<212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400> 5<400> 5
Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly 1 5 10 15Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly 1 5 10 15
<210> 6<210> 6
<211> 20<211> 20
<212> PRT<212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400> 6<400> 6
Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Gin Ile 1 5 10 15Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Gin Ile 1 5 10 15
Leu Ser Ser Leu 20Leu Ser Ser Leu 20
<210> 7<210> 7
<211> 20<211> 20
<212> PRT<212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400> 7<400> 7
Thr Ile Thr Gin Leu Leu Lys Arg Leu His Gin Trp Ile Asn Glu Asp 1 5 10 15Thr Ile Thr Gin Leu Leu Lys Arg Leu His Gin Trp Ile Asn Glu Asp 1 5 10 15
Cys Ser Thr Pro 20Cys Ser Thr Pro 20
<210> 8<210> 8
<211> 20<211> 20
<212> PRT<212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400> 8<400> 8
Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp Ile Cys Thr Val 1 5 10 15Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp Ile Cys Thr Val 1 5 10 15
Leu Thr Asp Phe 20Leu Thr Asp Phe 20
<210> 9<210> 9
<211> 20<211> 20
<212> PRT<212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400> 9
Gly Ala Gin Ile Thr Gly His Val Lys Asn Gly Ser Met Arg Ile Val 1 5 10 15<400> 9 Gly Ala Gin Ile Thr Gly His Val Lys Asn Gly Ser Met Arg Ile Val 1 5 10 15
Gly Pro Lys Thr 20Gly Pro Lys Thr 20
<210> 10 <211> 20<210> 10 <211> 20
<212> PRT<212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400> 10<400> 10
Glu Val Thr Arg Val Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr 1 5 10 15Glu Val Thr Arg Val Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr 1 5 10 15
Asp Asn Val Lys 20Asp Asn Val Lys 20
<210> 11 <211> 20<210> 11 <211> 20
<212> PRT<212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400> 11<400> 11
Cys Pro Cys Gin Val Pro Ala Pro Glu Phe Phe Thr Glu Val Asp Gly 1 5 10 15Cys Pro Cys Gin Val Pro Ala Pro Glu Phe Phe Thr Glu Val Asp Gly 1 5 10 15
Val Arg Leu His 20Val Arg Leu His 20
<210> 12 <211> 20<210> 12 <211> 20
<212> PRT<212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400> 12<400> 12
Phe Thr Glu Val Asp Gly Val Arg Leu His Arg Tyr Ala Pro Ala Cys 1 5 10 15Phe Thr Glu Val Asp Gly Val Arg Leu His Arg Tyr Ala Pro Ala Cys 1 5 10 15
Lys Pro Leu Leu 20Lys Pro Leu Leu 20
<210> 13 <211> 20<210> 13 <211> 20
<212> PRT<212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400> 13<400> 13
Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Thr Ala Lys 1 5 10 15
Arg Arg Leu Ala 20Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Thr Ala Lys 1 5 10 15 Arg Arg Leu Ala 20
<210> 14<210> 14
<211> 20<211> 20
<212> PRT <213> Hepatitis C virus<212> PRT <213> Hepatitis C virus
<400> 14<400> 14
Ser Ser Ser Ala Ser Gin Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys 1 5 10 15Ser Ser Ser Ala Ser Gin Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys 1 5 10 15
Thr Thr His His 20Thr Thr His His 20th
10> 1510> 15
11> 2011> 20
12> PRT12> PRT
13> Hepatitis C virus13> Hepatitis C virus
<400> 15<400> 15
Arg Glu Val Ser Val Ala Ala Glu Ile Leu Arg Lys Ser Arg Lys Phe 1 5 10 15Arg Glu Val Ser Val Ala Ala Glu Ile Leu Arg Lys Ser Arg Lys Phe 1 5 10 15
Pro Pro Ala Met 20Pro Pro Ala Met 20
<210> 16<210> 16
<211> 20<211> 20
<212> PRT <213> Hepatitis C virus<212> PRT <213> Hepatitis C virus
<400> 16<400> 16
Pro Leu Leu Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro Pro Val Val 1 5 10 15Pro Leu Leu Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro Pro Val Val 1 5 10 15
His Gly Cys Pro 20His Gly Cys Pro 20
<210> 17<210> 17
<211> 20 <212> PRT<211> 20 <212> PRT
<213> Hepatitis C virus<213> Hepatitis C virus
<400> 17 Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile 1 5 10 15
Thr Pro Cys Ala 20
<400> 17 Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile 1 5 10 15 Thr Pro Cys Ala 20
Claims
1. CD4+ T-Lymphozyten spezifische HCV-Epitope1. CD4 + T lymphocyte specific HCV epitopes
mit einem Impaktfaktor (IF) > Mittelwert (MW)+2*Stawith an impact factor (IF)> mean (MW) + 2 * Sta
wobeiin which
IF = (Formel l ) IF = (Formula 1)
wobei n-i der Summe der Reaktionen mit 3 < Sl < 6, n2 der Summe der Reaktionen mit Sl > 6 und m der Anzahl der Tests gegen das jeweilige Peptid entspricht, wobei m >15 ist, und MW der Mittelwert aller Impaktfaktoren ist.where ni is the sum of the reactions with 3 <Sl <6, n 2 is the sum of the reactions with Sl> 6 and m is the number of tests against the respective peptide, where m is> 15, and MW is the mean of all impact factors.
2. CD4+ T-Lymphozyten spezifische HCV-Epitope nach Anspruch 1 , umfassend ein oder mehrere Peptide, ausgewählt aus der Gruppe, bestehend aus:2. CD4 + T lymphocyte-specific HCV epitopes according to claim 1, comprising one or more peptides selected from the group consisting of:
(EP001 ) GPRLGVRATRKTSER,(EP001) GPRLGVRATRKTSER,
(EP002) ARSLTPCTCGSSDLY,(EP002) ARSLTPCTCGSSDLY,
(EP003) SSDLYLVTRHADVIP, (EP004) MWKCLIRLKPTLHGP,(EP003) SSDLYLVTRHADVIP, (EP004) MWKCLIRLKPTLHGP,
(EP005) VLVDILAGYGAGVAG,(EP005) VLVDILAGYGAGVAG,
(EP006) THYVPESDAAARVTQILSSL,(EP006) THYVPESDAAARVTQILSSL,
(EP007) TITQLLKRLHQWINEDCSTP,(EP007) TITQLLKRLHQWINEDCSTP,
(EP008) CSGSWLRDVWDWICTVLTDF, (EP009) GAQITGHVKNGSMRIVGPKT,(EP008) CSGSWLRDVWDWICTVLTDF, (EP009) GAQITGHVKNGSMRIVGPKT,
(EP010) EVTRVGDFHYVTGMTTDNVK, (EP011 ) CPCQVPAPEFFTEVDGVRLH,(EP010) EVTRVGDFHYVTGMTTDNVK, (EP011) CPCQVPAPEFFTEVDGVRLH,
(EP012) FTEVDGVRLHRYAPACKPLL,(EP012) FTEVDGVRLHRYAPACKPLL,
(EP013) TSMLTDPSHITAETAKRRLA,(EP013) TSMLTDPSHITAETAKRRLA,
(EP014) SSSASQLSAPSLKATCTTHH, (EP015) REVSVAAEILRKSRKFPPAM,(EP014) SSSASQLSAPSLKATCTTHH, (EP015) REVSVAAEILRKSRKFPPAM,
(EP016) PLLESWKDPDYVPPWHGCP, und(EP016) PLLESWKDPDYVPPWHGCP, and
(EP017) DWCCSMSYTWTGALITPCA sowie Derivate hiervon mit gleicher oder ähnlicher Spezifität.(EP017) DWCCSMSYTWTGALITPCA and derivatives thereof with the same or similar specificity.
3. CD4+ T-Lymphozyten spezifische HCV-Epitope mit einer oder mehreren im Anspruch 2 genannten Sequenzen.3. CD4 + T lymphocyte-specific HCV epitopes with one or more of the sequences mentioned in claim 2.
4. CD4+ T-Lymphozyten spezifische HCV-Epitope nach einem der vorstehenden Ansprüche mit einer der Sequenzen EP001 bis EP017.4. CD4 + T lymphocyte-specific HCV epitopes according to one of the preceding claims with one of the sequences EP001 to EP017.
5. Impfstoff, mindestens ein HCV-Epitop nach einem der vorstehenden Ansprüche umfassend.5. Vaccine comprising at least one HCV epitope according to any one of the preceding claims.
6. Impfstoff nach Anspruch 5, zusätzlich mindestens einen Hilfsstoff, ausgewählt aus der Gruppe, bestehend aus fowlpoxvirus, modifiziertem Vakziniavirus Ankara, Virosomen, Transvax, CPG und anderen die Immunreaktion verstärkenden Stoffen, umfassend.6. Vaccine according to claim 5, additionally comprising at least one excipient selected from the group consisting of fowlpoxvirus, modified vaccinia virus Ankara, virosomes, Transvax, CPG and other substances which enhance the immune response.
7. Impfstoff nach einem der Ansprüche 5 oder 6 in Form einer Injektionslösung.7. Vaccine according to one of claims 5 or 6 in the form of a solution for injection.
8. HCV-Epitop nach einem der Ansprüche 1 bis 4 als Arzneimittel.8. HCV epitope according to one of claims 1 to 4 as a medicament.
9. Verwendung mindestens eines HCV-Epitops nach einem der Ansprüche 1 bis 4 zur Herstellung einer Zusammensetzung zur9. Use of at least one HCV epitope according to one of claims 1 to 4 for the preparation of a composition for
Prophylaxe und/oder Therapie einer Hepatitis C Infektion. Prophylaxis and / or therapy for a hepatitis C infection.
10. Verwendung mindestens eines HCV-Epitop nach einem der Ansprüche 1 bis 4 in der Diagnose einer Hepatitis C Infektion.10. Use of at least one HCV epitope according to one of claims 1 to 4 in the diagnosis of a hepatitis C infection.
11. Diagnostischer Kit, mindestens eines der Epitope nach einem der Ansprüche 1 bis 4 umfassend.11. Diagnostic kit, comprising at least one of the epitopes according to one of claims 1 to 4.
12. cDNA, eines der HCV-Epitope nach einem der Ansprüche 1 bis 4 kodierend.12. cDNA encoding one of the HCV epitopes according to one of claims 1 to 4.
13. Impfstoff, mindestens eine cDNA nach einem der Ansprüche 1 bis 4 umfassend. 13. Vaccine comprising at least one cDNA according to one of claims 1 to 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP03717293A EP1497324A2 (en) | 2002-04-10 | 2003-04-10 | CD4 sp + /sp T-LYMPHOCYTE-SPECIFIC HEPATITIS C VIRUS EPITOPES |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP02008033A EP1357127A1 (en) | 2002-04-10 | 2002-04-10 | Hepatitis c virus epitopes specific for cd4+ t-cell lymphocytes |
| EP02008033 | 2002-04-10 | ||
| EP03717293A EP1497324A2 (en) | 2002-04-10 | 2003-04-10 | CD4 sp + /sp T-LYMPHOCYTE-SPECIFIC HEPATITIS C VIRUS EPITOPES |
| PCT/EP2003/003732 WO2003084988A2 (en) | 2002-04-10 | 2003-04-10 | Cd4+ t-lymphocyte-specific hepatitis c virus epitopes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1497324A2 true EP1497324A2 (en) | 2005-01-19 |
Family
ID=28685850
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP02008033A Withdrawn EP1357127A1 (en) | 2002-04-10 | 2002-04-10 | Hepatitis c virus epitopes specific for cd4+ t-cell lymphocytes |
| EP03717293A Withdrawn EP1497324A2 (en) | 2002-04-10 | 2003-04-10 | CD4 sp + /sp T-LYMPHOCYTE-SPECIFIC HEPATITIS C VIRUS EPITOPES |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP02008033A Withdrawn EP1357127A1 (en) | 2002-04-10 | 2002-04-10 | Hepatitis c virus epitopes specific for cd4+ t-cell lymphocytes |
Country Status (4)
| Country | Link |
|---|---|
| US (2) | US7270820B2 (en) |
| EP (2) | EP1357127A1 (en) |
| AU (1) | AU2003221569A1 (en) |
| WO (1) | WO2003084988A2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4342519B2 (en) * | 2003-09-22 | 2009-10-14 | 株式会社グリーンペプタイド | Hepatitis C virus-derived peptide |
| WO2013150450A1 (en) * | 2012-04-02 | 2013-10-10 | Universidade Do Porto | Hcv homolog fragments, cell-lines and applications thereof |
| SG10201912866RA (en) * | 2015-06-25 | 2020-02-27 | Univ Nanyang Tech | Broad-spectrum anti-infective peptides |
| WO2018055535A2 (en) | 2016-09-21 | 2018-03-29 | The Governors Of The University Of Alberta | Hepatitis c virus immunogenic compositions and methods of use thereof |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6027729A (en) * | 1989-04-20 | 2000-02-22 | Chiron Corporation | NANBV Diagnostics and vaccines |
| US6007982A (en) * | 1990-12-14 | 1999-12-28 | Innogenetics N.V. | Synthetic antigens for the detection of antibodies to hepatitis C virus |
| ES2066547T3 (en) * | 1991-03-01 | 1995-03-01 | Akzo Nobel Nv | PEPTIDES, IMMUNOCHEMICALLY REACTIVE WITH ANTIBODIES DIRECTED AGAINST HEPATITIS NO-A, NO-B VIRUSES. |
| CA2070952A1 (en) * | 1991-06-11 | 1992-12-12 | Makoto Seki | Gene of hepatitis c virus or fragment thereof, polypeptide encoded by the same |
| DE4209215A1 (en) * | 1991-07-04 | 1993-01-07 | Boehringer Mannheim Gmbh | HCV PEPTIDE ANTIGEN AND METHOD FOR DETERMINING HCV |
| US6689363B1 (en) * | 1992-01-29 | 2004-02-10 | Epimmune Inc. | Inducing cellular immune responses to hepatitis B virus using peptide and nucleic acid compositions |
| US5980899A (en) * | 1992-06-10 | 1999-11-09 | The United States Of America As Represented By The Department Of Health And Human Services | Identification of peptides that stimulate hepatitis C virus specific cytotoxic T cells |
| AU1818299A (en) * | 1997-12-10 | 1999-06-28 | Washington University | Anti-pathogen system and methods of use thereof |
| WO2001021189A1 (en) * | 1999-07-19 | 2001-03-29 | Epimmune Inc. | Inducing cellular immune responses to hepatitis c virus using peptide and nucleic acid compositions |
| SI1263936T1 (en) * | 2000-03-14 | 2006-06-30 | Bavarian Nordic As | Altered strain of the modified vaccinia virus ankara (mva) |
| AUPQ776100A0 (en) * | 2000-05-26 | 2000-06-15 | Australian National University, The | Synthetic molecules and uses therefor |
| AU2001272257A1 (en) * | 2000-07-07 | 2002-01-21 | Medmira Inc. | Hcv mosaic antigen composition |
| EP1195381A1 (en) * | 2000-09-28 | 2002-04-10 | Immusystems GmbH | Hepatitis c virus epitopes specific for cd4+ t-cell lymphocytes |
-
2002
- 2002-04-10 EP EP02008033A patent/EP1357127A1/en not_active Withdrawn
-
2003
- 2003-04-10 EP EP03717293A patent/EP1497324A2/en not_active Withdrawn
- 2003-04-10 WO PCT/EP2003/003732 patent/WO2003084988A2/en not_active Application Discontinuation
- 2003-04-10 AU AU2003221569A patent/AU2003221569A1/en not_active Abandoned
-
2004
- 2004-10-07 US US10/962,145 patent/US7270820B2/en not_active Expired - Fee Related
-
2007
- 2007-08-13 US US11/837,953 patent/US20080089903A1/en not_active Abandoned
Non-Patent Citations (1)
| Title |
|---|
| See references of WO03084988A2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2003084988A3 (en) | 2004-11-11 |
| AU2003221569A8 (en) | 2003-10-20 |
| AU2003221569A1 (en) | 2003-10-20 |
| US20050249754A1 (en) | 2005-11-10 |
| US7270820B2 (en) | 2007-09-18 |
| WO2003084988A2 (en) | 2003-10-16 |
| EP1357127A1 (en) | 2003-10-29 |
| US20080089903A1 (en) | 2008-04-17 |
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