EP1385875A2

EP1385875A2 - Epitopes of virus hepatitis c specifically cd4+ t-lymphocytes

Info

Publication number: EP1385875A2
Application number: EP01985703A
Authority: EP
Inventors: Jörn Tilman GERLACH; Helmut Diepolder
Original assignee: Immusystems GmbH
Current assignee: Immusystems GmbH
Priority date: 2000-09-28
Filing date: 2001-09-28
Publication date: 2004-02-04
Also published as: AU2002223569A1; WO2002026785A3; US7108856B2; US20070048333A1; EP1195381A1; WO2002026785A2; US20030186224A1

Abstract

The invention relates to hepatitis C virus epitopes which are specific in relation to CD4<+> T lymphocytes, in addition to vaccinations which contain said epitopes.

Description

CD4 ⁺ T lymphocyte specific hepatitis C virus epitopes

The invention relates to hepatitis C virus epitopes which are specific for CD4 ⁺ T lymphocytes and to vaccines which contain these epitopes.

The hepatitis C virus, hereinafter referred to as HCV, was identified in 1989 and is an RNA virus from the Flaviviridae family. It consists of an RNA single strand of approximately 9400 nucleotides that encode a precursor polyprotein of approximately 3000 amino acids. 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, whose 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 intra-individual subtypes are the so-called HCV quasi-species, but they are related to different virus sequences that result from inaccurate replication.

With a prevalence of around one to three percent worldwide, hepatitis C is one of the most important chronic viral infections. At least 180 million infected individuals are currently expected. According to calculations by the Center 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. 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 transmission pathways for 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. 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 the cause of a liver transplant. The health care costs of these transplants are significant.

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. A therapeutic vaccination is therefore currently not available.

The only therapy currently approved is treatment with interferon alpha alone or in combination with ribavirin for 3 to 12 months. This form of therapy is very expensive, often has side effects and only leads to permanent virus elimination in approx. 40% of cases. From Journal of Virology, Volume 71, pages 6011 to 6019 and Hepatology, Volume 30, No. 4, 1999, pages 1088 to 1098 it is known that direct peripheral blood T cell stimulation and specificity analysis of HCV-specific T cell lines, Highly immunogenic T cell epitopes can be identified within the Gore, NS3 and NS4 regions of the hepatitis C virus. T-lymphocytes are isolated from peripheral blood and the HCV-specific CD4 + T-lymphocytes contained therein are enriched by repeated stimulation with the corresponding virus protein in vitro to produce so-called specific T cell lines. By analyzing the growth behavior (incorporation of radioactively labeled nucleotides) of these specific T cell lines after stimulation with HCV protein and / or the smaller subunits, the peptides, the sequence of the T cell epitope can be narrowed. In particular, the analysis of T cell lines, in comparison to T cell clones, gives inaccurate results due to the cell mixture with regard to the sequence specifically recognized by the CD4 + T lymphocytes.

It is therefore the object of the present invention to isolate HCV epitopes specific to CD4 ⁺ T lymphocytes and to make available the HCV epitopes identified by the CD4 + T lymphocytes for a vaccine for the prophylaxis and / or therapy of an HCV infection ,

The solution to the problem are the HCV epitopes which are specific to CD4 ⁺ T lymphocytes and which contain the sequence:

1) YLVAYQATVC;

2) WTSTWVLVGGVLAALAAYCL; 3) QYLAGLSTLPG;

4) IASLMAFTA;

5) FNILGGWVA; and or 6) SPVFTDNSSPPAVPQSFQVA

and derivatives thereof with comparable specificity.

The following derivatives of the HCV epitopes which contain the HCV epitopes 1) to 5) are particularly preferred:

7) GENLPYLVAYQATVCARAQA

8) EWTSTWVLVGGVLAALAA 9) FISGIQYLAGLSTLPGNPAIA

10) PGNPAIASLMAFTAAVTSP and / or

11) SQTLLFNILGGWVAAQLAA and derivatives thereof with comparable specificity.

Another solution to the problem are the following HCV epitopes, which are specific to CD4 ⁺ T lymphocytes and contain the sequence:

12) TSVRLRAYLNTPGLPVCQDH

13) STEDLVNLLPAILSPGALW 14) KLVALGINAVAYYRGLDVSVIPTSGDVW

15) SGKPAIIPDREVLYREFDEM

16) LGIGTVLDQAETAGA

17) ETAGARLWLATATP

18) CVTQTVDFSLDPTFT 19) RPSGMFDSSVLCECY

20) VFPDLGVRWCEKMAL and / or

21) KLGVPPLRVWRHRAR and their derivatives with comparable specificity.

Another solution is a vaccine which contains at least one of the epitopes 1) to 21) according to the invention. The vaccine preferably contains the epitope with the layer eel 773-1783, namely 3) QYLAGLSTLPG. The vaccine can particularly preferably contain a mixture of the epitopes 1) to 21) according to the invention. However, other HCV epitopes may also be present.

The invention is illustrated by means of the figures. Show it

Figure 1 shows the epitope mapping of the epitope at position aa1765-1784.

2 shows the mapping of the epitope at position aa1773-1783 using N- or C-terminally truncated peptides;

3 shows the epitope mapping of the epitope at position aa1785-1804;

4 the mapping of the epitope at position aa1787-1795 using N- or C-terminally truncated peptides;

5 shows the epitope mapping of the epitope at position aa1655-1674; and

Fig. 6 mapping the epitope at position aa1585-1594 using N- or C-terminally truncated peptides.

7 shows the epitope mapping of the epitope at position eal 535-1554 with 20mer peptides,

8 the epitope mapping of the epitope at the position eal 1875-1894 with 20mer peptides,

9 the mapping of the epitope at position aa1689-1708 using N- or C-terminally truncated peptides. The epitopes according to the invention are specifically recognized by CD4 ⁺ T lymphocytes which are formed in the patient in the case of a self-limited HCV infection.

The position of the epitopes according to the invention on the HCV protein is in each case for 1) aa1585-1594, 2) aa1655-1675, 3) aa1773-1783, 4) aa1787-1795, 5) aa1809-1817 and 6) aa1207-1226.7 ) aa1580 - 1599, 8) eel 654 - 1673, 9) eel 768 - 1788, 10) eel 782 - 1800, 11) eel 804 - 1822, 12) aa1535 - 1554, 13) aa1875 - 1894, 14) aa1406 - 1434 , 15) aa1689 - 1708, 16) aa1327 - 1341, 17) 1337 - 1352, 18) aa1457 - 1472, 19) aa1507 - 1522, 20) aa2581 - 2595 and 21) aa2911 - 2930 where the specified positions refer to, for example Publication CHOO, Q.-L. et al. Relate to PNAS 1991.

The epitopes according to the invention, containing the sequences 1) to 21) mentioned above, are preferably the sequences 1) to 21) themselves. In this embodiment of the invention, the epitopes according to the invention are selected from the group consisting of 1) YLVAYQATVC, 2) WTSTWVLVGGVLAALAAYCL, 3) QYLAGLSTLPG, 4) IASLMAFTA, 5) FNILGGWVA, 6) SPVFTDNSSPPAVPQSFQVA, 7) GENLPYLVAYQATVCARAQA, 8) EWTSTWVLVGGVLAALAA, 9) FISGIQYLAGLSTLPGNPAIA, 10) PGNPAIASLMAFTAAVTSP, 11) SQTLLFNILGGWVAAQLAA, 12) TSVRLRAYLNTPGLPVCQDH, 13) STEDLVNLLPAILSPGALW, 14) KLVALGINAVAYYRGLDVSVIPTSGDVW, 15) SGKPAIIPDREVLYREFDEM, 16) LGIGTVLDQAETAGA, 17) ETAGARLWLATATP, 18) CVTQTVDFSLDPTFT, 19) RPSGMFDSSVLCECY, 20) VFPDLGVRWCEKMAL and 21) KLGVPPLRVWRHRAR.

To identify these HCV epitopes specific to CD4 ⁺ T lymphocytes, infection was found in patients with acute hepatitis C who had a self-limited course of the disease and in whom At the same time, a strong HCV-specific T-lymphocyte activity could be detected in vitro, T-cell clones of these virus-specific T-lymphocytes isolated and cloned from T-cell lines using the classic limiting dilution method. This method allows multiplication of a single cell by many powers of ten and enables the characterization of antigen specificity with a clonal cell population. The HCV-specific T cell clones were used to precisely characterize the epitope recognized in each case by means of N-terminally and C-terminally truncated peptides, as shown in FIGS. 2, 4 and 6.

The derivatives according to the invention of epitopes 1) to 21) with comparable specificity can be found in the same way as these epitopes using the HCV-specific T cell clones by means of N- and C-terminal truncated peptides or by exchanging single or several amino acids in the sequences 1) to 21) and checking the specificity of these changed sequences.

The epitopes according to the invention are highly immunogenic, highly conserved and in the immediate vicinity of known HCV epitopes which are specific to CD8 + T lymphocytes.

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. In this context, the immediate proximity of the HCV epitopes found here to known CD8 + epitopes is particularly important.

Epitopes 1) to 16) were recognized by fresh peripheral mononuclear cells (PBMC) from 18 patients with acute HCV, namely for 1) of 6 patients, for 2) of 4 patients, for 3) of 6 patients, for 4) of 3 Patients for 5) of 4 patients, for 7) from one patient, for 8) from two different clones of a patient, and for 9) and 10) from one patient. Epitopes 11) to 16) were determined with the help of HCV-specific T cell clones from patients after liver transplantation, in whom virus elimination had taken place after the transplantation despite immunosuppression.

The epitopes according to the invention not only stimulated the T lymphocytes of the patients from whom the T cell clones originate, but also the fresh peripheral mononuclear cells (PBMC) from different patients with acute hepatitis C and a self-limited course. The HLA (class I and II) of these persons with a positive reaction to these epitopes, which is important for the antigen presentation, was different, so that a certain promiscuity of the peptides (presentation on different HLA receptors) can be assumed. In the case of a protective T cell vaccination, they are therefore ideally suited for vaccinating healthy people or hepatitis C patients with different HLA characteristics.

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, in particular the epitope at position aa1773-1783, to which further epitopes or epitopes according to the invention can optionally be added at other positions.

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.

In addition, this causes antagonistic effects or the risk of iatrogenic autoimmune reactions that occur with vaccinations with whole

Proteins can be avoided. 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 c-DNA 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 (eg 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). 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.

example 1

Heparinized blood was drawn from patients with a self-limited course of acute hepatitis C in the first 6 months after the onset of the disease. The fresh peripheral mononuclear blood cells (PBMC) were isolated by density gradient centrifugation on Ficoll gradients and suspended in a culture medium (RPMI1640, Gibco). 50 μl of this cell suspension (concentration of 1x10 ⁶ cells per ml) was placed on sterile 96-well culture plates. Ten samples of this cell mixture were stimulated by adding a recombinant HCV protein. The final concentration of the protein was 1 μg / ml. The cell culture plates were cultivated for 5 days at 37 ° C. and 5% CO ₂ . On day 6, culture IL-2 was added. The final concentration of IL-2 was 15U / ml and the cells were cultivated for a further three days at 37 ° C. and 5% CO ₂ . Those holes in the culture plate that appeared to be strongly stimulated under microscopic control on day ten were collected and distributed to a further culture plate in serial dilution stages. A hole was selected microscopically, which contained approximately 150 cells. These 150 cells were diluted with medium and spread over 300 holes so that statistically half a cell was contained in each hole. IL-2 was then added to a final concentration of 15U / ml. In addition, 3x10 ⁴ autologous and 3000 Rad irradiated PBMC and phythema agglutinin (PHA) were added to each hole as growth factor. The clones were expanded and then expanded into one Proliferation assay with HCV proteins tested for antigen specificity. The antigen specificity was first determined by stimulation with proteins and then, in the case of a positive result, with 20 amino acid long peptides (20mer peptides), as shown in FIG. 1.

For this purpose, T cell clones were stimulated with proteins, as shown in FIG. 1 on the left, and antigen-presenting cells, and the stimulation was measured as incorporation of radioactively labeled ³ H, as shown in FIG. 1 on the right. To further narrow the specifically recognized epitope, stimulation was carried out with 20 mer peptides corresponding to the protein sequence, each overlapping by 10 amino acids, and the incorporation of ³ H was measured again. The corresponding epitope was reduced to 20 amino acids.

The epitope, the smallest still stimulating sequence within the 20mer peptide, was analyzed with truncated peptides, i.e. the original 20mer peptide was truncated in the ascending order from the N-terminal as shown in Figure 2. The same procedure was used with peptides truncated from the C-terminal. A proliferation assay showed the amino acid to which the peptide could be truncated without having to accept any significant loss of stimulation.

The epitope QYLAGLSTLPG is obtained, as shown in FIG. 2. By comparing the characterized epitopes with sequences of the known genotypes of entire HCV proteins, a high degree of conservation of the epitopes was found despite the known high genetic variability of the HCV.

Since peptides are highly sensitive to degradation and the antigen-specific recognition of the peptide-HLA complex by the T-

Cell receptor can be subject to minor fluctuations in individual cases or can be fermented by loss of a single amino acid, as well the synthesis length of the peptides can be subject to fluctuations, the epitopes 1 to 5, when used in a vaccine, should each carry five amino acids N-terminal and C-terminal as protection against degradation or to optimize the synthesis and ensure the antigen presentation, these extended epitopes for example, epitopes 7 through 11 can be.

Example 2

Clones were obtained according to the procedure of Example 1. The antigen specificity of these clones was then determined as shown in Figure 3. The corresponding epitope with 20 amino acids is obtained. The epitope within this 20mer peptide was then determined as shown in Figure 4. The epitope IASLMAFTA is obtained.

Example 3

Clones were obtained according to the procedure of Example 1. The antigen specificity of these clones was then determined as shown in Figure 5. The corresponding epitope with 20 amino acids, namely WTSTWVLVGGVLAALAAYCL, is obtained according to Example 1.

Example 4

Clones were obtained according to the procedure of Example 1. The antigen specificity was also determined as described in Example 1 and the corresponding epitope with 20 amino acids was obtained. Then, as shown in Figure 6, the epitope was determined, namely YLVAYQATVC. Example 5

Following the procedure of Example 1, clones from patients after liver transplantation were made from routinely drawn blood. In patients after liver transplantation, the specificity of the T cell clones was analyzed using the ELISPOT assay. A detection antibody, in this case against interferon gamma, is attached to a nitrocellulose-coated microtiter plate and adheres to the nitrocellulose.

In the second step, the specific T cell clones are added together with antigen presenting cells and the antigen to be tested. After adding the specific antigen, the clonal T lymphocytes secrete Interferon gamma. This cytokine is "captured" by the detection antibodies over 48 hours. After removal of the cell mixture, a second specific antibody is added which recognizes and labels the fixed cytokine-antibody complex. The second antibody can then be visualized by a dye reaction so that the antigen-specific impress secreted cytokines as spots, which can finally be counted under the microscope and represent a measure of the antigen specificity in relation to the control.In analogy to the previously described proliferation test, epitopes can also be mapped with the ELISPOT assay.

Claims

claims

1. CD4 ⁺ T lymphocyte-specific HCV epitopes, containing the sequence:

1) YLVAYQATVC;

2) WTSTWVLVGGVLAALAAYCL; 3). QYLAGLSTLPG;

4) IASLMAFTA; 5) FNILGGWVA, and / or

6) SPVFTDNSSPPAVPQSFQVA and derivatives thereof with comparable specificity.

2. HCV epitope according to claim 1, with the following sequence:

1) YLVAYQATVC;

2) WTSTWVLVGGVLAALAAYCL;

3) QYLAGLSTLPG;

4) IASLMAFTA; 5) FNILGGWVA, or

6) SPVFTDNSSPPAVPQSFQVA.

3. Derivatives of an HCV epitope according to one of claims 1 or 2, containing the sequence: 7) GENLPYLVAYQATVCARAQA

8) EWTSTWVLVGGVLAALAA

9) FISGIQYLAGLSTLPGNPAIA

10) PGNPAIASLMAFTAAVTSP and / or

1 1) SQTLLFNILGGWVAAQLAA and derivatives thereof with comparable specificity.

4. derivative of an HCV epitope according to claim 3, having the following sequences:

7) GENLPYLVAYQATVCARAQA

8) EWTSTWVLVGGVLAALAA 9) FISGIQYLAGLSTLPGNPAIA

10) PGNPAIASLMAFTAAVTSP or

11) SQTLLFNILGGWVAAQLAA.

5. CD4 ⁺ T lymphocyte-specific HCV epitopes, containing the sequence:

12) TSVRLRAYLNTPGLPVCQDH

13) STEDLVNLLPAILSPGALW

14) KLVALGINAVAYYRGLDVSVIPTSGDVW

15) SGKPAIIPDREVLYREFDEM 16) LGIGTVLDQAETAGA

17) ETAGARLWLATATP

18) CVTQTVDFSLDPTFT

19) RPSGMFDSSVLCECY

20) VFPDLGVRWCEKMAL and / or 21) KLGVPPLRVWRHRAR and their derivatives with comparable specificity.

6. HCV epitope according to claim 5, with the following sequences: 12) TSVRLRAYLNTPGLPVCQDH 13) STEDLVNLLPAILSPGALW

14) KLVALGINAVAYYRGLDVSVIPTSGDVW

15) SGKPAIIPDREVLYREFDEM

16) LGIGTVLDQAETAGA

17) ETAGARLWLATATP 18) CVTQTVDFSLDPTFT

19) RPSGMFDSSVLCECY

20) VFPDLGVRWCEKMAL or 21) KLGVPPLRVWRHRAR.

7. Vaccine comprising at least one HCV epitope according to any one of the preceding claims.

8. Vaccine according to claim 7, additionally comprising at least one excipient selected from the group consisting of fowlpoxvirus, modified vaccinia virus Ankara, virosomes, Transvax and other substances which enhance the immune response.

9. Vaccine according to one of claims 7 or 8, in the form of a solution for injection.

10. HCV epitope according to one of claims 1 to 6 as a medicament.

11. Use of at least one HCV epitope according to one of claims 1 to 6 for the manufacture of a composition for the prophylaxis and / or therapy of a hepatitis C infection.

12. Use of at least one HCV epitope according to one of claims 1 to 6 in the diagnosis of a hepatitis C infection.

13. Diagnostic kit, comprising at least one of the epitopes according to one of claims 1 to 6.

14. c-DNA encoding one of the HCV epitopes according to one of claims 1 to 6.

15. Vaccine comprising at least one c-DNA according to claim 14.