ES2354694T3 - VACCINES AGAINST HCV. - Google Patents

Abstract

Hepatitis C virus (HCV) vaccine comprising at least the following three peptides: a) AYAAQGYKVLVLNPSVAAT or a part thereof containing at least one epitope and b) GEVQVVSTATQSFLATCINGVCWTV or a part thereof containing at least one epitope and c) HMWNFISGIQYLAGLSTLPGNPA or a part thereof that contains at least one epitope.

Description

The present invention relates to vaccines against HCV.

The immune system is a complex network of interrelated cell types and molecules, which has been developed to protect multicellular organisms from infectious microorganisms. It can be divided into innate (or natural) immunity in evolutionary older people and adaptive (or acquired) immunity. The innate immune system 5 recognizes patterns, which are normally common and essential for pathogens. For this limited number of molecular structures, germline encoded receptors have been developed. In comparison, adaptive immune system cells - B and T lymphocytes - can recognize a huge variety of antigenic structures. The receptors, named according to the types of cells that express them, B-lymphocyte receptor (BCR, their soluble versions are called antibodies) and T-cell receptor (TCR, only 10 cell-associated forms), 27, are generated by recombination somatic and show a clonal distribution. Thus, initially there is only a small number of cells with a certain specificity. When the antigen meets these cells it begins to divide (clonal expansion) to generate an effector population capable of coping with the antigen. After antigen removal, a specialized subpopulation of cells that specifically recognizes this antigen remains an immunological memory. Taken together the adaptive immune system 15 is delayed (compared to innate immunity), however it is specific and improves with repeated exposure to a given pathogen / antigen.

T lymphocytes have a central role in adaptive immunity. Its receptors (TCRs) recognize "major histocompatibility complex" (MHC or HLA): peptide complexes on the surface of cells. These peptides are called T-cell epitopes and represent antigen degradation products. There are two main classes of T lymphocytes: CD8 positive cytotoxic T lymphocytes (CTL) are restricted to MHC class I. CD4 positive helper T lymphocytes (HTL) are restricted to MHC class II. HTLs are essential for many adaptive immunity characteristics: activation of the so-called "cells presenting professional antigen" (APCs), immunoglobulin class switching (Ig), germ center reaction and maturation of Ig affinity, activation of CTL, immune memory, immune response regulation and others. 25

MHC molecules collect peptides within the cell and present them on the surface of the cells in the TCRs of T lymphocytes. There are two main classes of MHC, class I recognized by CTL positive CD8 and class II recognized by HTL positive CD4 .

MHC class I molecules consist of an alpha chain anchored in the 45 kDa membrane and the b2-immunoglobulin bound by 12 kDa non-covalent bonds (b2m). The resolution of the three-dimensional structure 30 by X-ray crystallography (Stern and Wiley 1994) reveals that the alpha chain has a slit, which is closed at both ends and accommodates peptides 8 to 11 amino acids in length. Class I molecules are expressed ubiquitously and the peptides they present originate from cytoplasmic proteins. These are degraded by the proteasome and the resulting peptides are actively transported to the endoplasmic reticulum (ER). There, with the help of various carbines, MHC: peptide complexes are formed and transported to the surface of the cells (Heemels 1995). Thus, MHC class I faces the proteome of a cell on its surface and allows T lymphocytes to recognize intracellular pathogens or malignant cells.

 MHC class II molecules consist of two membrane-anchored proteins (alpha and beta chain) of 35 kDa and 30 kDa, respectively. These together form a slit, open at both ends, which can accommodate peptides of variable length, usually 12 to 25 amino acids. Despite these differences, the 40 class I and II molecules have in common a striking structural similarity (Stern and Wiley 1994). Class II molecules are only expressed in dendritic cells (DC) including professional APC, B lymphocytes and macrophages / monocytes. These cells are specialized in absorbing and treating antigens in the endosomal pathway. Immediately after their biosynthesis, class II molecules are complexed by the so-called invariant chain (li), which prevents the binding of peptides in the ER. When vesicles containing class II: li 45 complexes are fused with degradation products containing exogenous antigen endosomes, li degrades until the cleft that binds MHC is only complexed by the so-called CLIP peptide. The latter is with the help of carbines such as HLA-DM exchanged for antigenic peptides (Villadangos 2,000). Finally, the MHC: peptide complexes occur again on the surface of the APCs, which interact in numerous ways with HTL.

Being both polygenic and extremely polymorphic, the MHC system is very complex. For the alpha 50 class I chain in humans there are three loci of the gene called HLA-A, -B and -C. Similarly, there are three class II alpha chain locus (DRA, DQA, DPA); for class II beta chain locus the situation is even more complex since there are four different DR beta chains (DRB1,2,3,5) plus DQB and DPB. Except for monomorphic DR alpha chain DRA, each gene locus is present in many different alleles (dozens to hundreds) in the population (Klein 1986). The different alleles have largely different binding specificities for 55 peptides. The alleles are designated, for example, HLA-A * 0201 or HLA-DRB1 * 0401 or HLA-DPA * 0101 / DPB * 0401.

T-cell epitopes have been identified by a variety of proposals (Van den Eynde 1997). For example, T lymphocyte lines and clones have been used to screen cDNA expression libraries for example in the context of COS cells transfected with the appropriate HLA molecule. Alternatively, biochemical proposals have been pursued. The latter involved the elution of natural ligands from MHC molecules on the surface of target cells, the separation of these peptides by various chromatographic stages, analysis of their reactivity with lymphocytes in epitope reconstitution assays and mass spectrometry sequencing. (Wölfel et al., 1994, Cox et al. 1994).

Recently, the arrival of highly sensitive cytokine detection assays such as the IFN-gamma ELIspot made it possible to use lymphocytes directly ex vivo for the screening of overlapping synthetic peptides (Maecker 2.001, Kern 2000, Tobery 2.001). Mainly, Kern et al. (1999 and 2000) used series of superimposed 10-9 peptide sets to map epitopes of CD8 + T lymphocytes in vitro. Later, Tobery et al. 2,001 modified this proposal and demonstrated that sets containing as many as 64 peptides can be used to screen T-cell epitopes both CD8 + and CD4 + in mice. These two procedures were based on the control of the specific response of the antigen by measurement of IFN-gamma production or by intracellular staining (Kern et al 2000) or in the ELIspot assay (Tobery et al. 2001). By using mixtures of 15-mer 15 the responses of CD4 + T cells are approximately equal to those detected when complete soluble protein was used as an antigen, while - not surprisingly - the responses of CD8 + T cells are significantly greater than Frequently insignificant responses detected with stimulation of soluble proteins. In addition, the responses of CD8 + T lymphocytes for a mixture of 15 amino acid peptides are similar to those obtained with a mixture of 8-12 amino acid peptides, selected to represent known minimum MHC class I epitopes. Most likely, the peptidases associated with the cell membrane are responsible for "cutting" the peptides to the optimum length in these circumstances (Maecker et al. 2001).

An interesting alternative is to screen combinatorial libraries of synthetic peptides with specific lymphocytes. For example, a decapeptide library consisting of 200 mixtures arranged in a positional scanning format has been used successfully for the identification of peptide ligands that stimulate clonotypic populations of T lymphocytes (Wilson, et al., J. Immunol. , 1999, 163: 6.424-6.434).

Many T-cell epitopes have been identified by the so-called "Reverse immunological approaches" Rammensee 1999). In this case the protein that gives rise to a potential T lymphocyte epitope is known and HLA binding residues are swept in its main sequence. Typically, dozens to hundreds of candidate peptides are synthesized or even a complete series of overlapping peptides are synthesized and their binding to HLA molecules is tested. Normally, the best binders are selected for further characterization with respect to their reactivity with T lymphocytes. This can be done for example by priming T lymphocytes in vitro or in vivo with the help of HLA transgenic mice.

Hepatitis C Virus (HCV) is a member of the flaviviridiae family, which chronically infects approximately 170 million people in the world. There are at least 6 HCV genotypes and more than 50 subtypes have been described. In America, Europe and Japan genotypes 1, 2 and 3 are the most common. The geographic distribution of HCV genotypes varies greatly, with genotype 1a being predominant in the USA and parts of Western Europe, while 1b predominates in Southern and Central Europe (Bellentani 2,000).

HCV is transmitted by the parenteral or percutaneous route and replicates in hepatocytes. Approximately 15% 40 of the patients suffer from acute self-limited hepatitis associated with viral elimination and recovery. Approximately 80% of infected people become chronic carriers. The infection persists asymptomatically frequently with delayed evolution over years, however HCV is ultimately a leading cause of cirrhosis, terminal liver disease and liver cancer (Liang, 2,000). The concentration and quality of the responses of both HTL and CTL determine whether patients recover (spontaneously or as a result of treatment) or develop chronic infection (Liang, 2,000).

The usual treatment of HCV comprises an association of pegylated interferon-alpha and antiviral ribavirin. Virological responses are achieved, depending on the genotype, in approximately 50% of HCV patients. The low tolerability and the considerable side effects of this treatment clearly require new therapeutic intervention including therapeutic vaccines (Comberg 2.002). However, at the present time 50 there is no detailed understanding of which epitopes in which combination of MHC lead to successful immune responses (Ward 2.002). Therefore, an extensive analysis of the T lymphocyte response against complete HCV is required for the development of vaccines based on therapeutic epitopes.

The HCV virion contains a 9.5 kilobase positive sense single stranded RNA genome that encodes a large single polyprotein of approximately 3,000 amino acids. The latter is treated with at least 10 55 proteins for both host and HCV-encoded proteolytic activities (Liang, 2,000). To a large degree,

HCV RNA-dependent RNA polymerase tends to error leading to the development of viral quasi-species and contributing to immunoscape variants (Farci 2,000).

WO 01 / 21189A discloses mechanisms by which an antigen is recognized by T lymphocytes to identify and prepare HCV epitopes to develop vaccines based on HCV-directed epitopes. US 5,683,864 A discloses combinations of HCV antigens consisting of an HCV polyprotein C 5 domain antigen and at least one additional antigen of any of the NS3 domain, the NS4 domain, the S domain or the domain NS5

Although many proposals have been made in the art over the past 15 years and many promising antigen candidates have been proposed during this time, there is still no HCV vaccine on the market (not a satisfactory therapeutic treatment; the only treatment available, such as a combination of alpha interferon and ribavirin was previously mentioned, it is only effective in a minority of patients). At the beginning after the publication of the HCV sequences, a myriad of epitopes and antigens were suggested as effective means of fighting HCV disease (eg, Koziel et al., J. Virol. 67 (12), 7,522-7,532; Shirai et al., J. Virol. 68 (5) (1994), 3,334-3,342; Leroux-Roels et al., Hepatology 23 (1) (1996), 8-16; Hoffmann et al., Hepatology 21 (3 ) (1995), 632-638; Sarobe et al., J. Clin. Invest. 102 (6) (1998), 1,239-1,248; Battegay et al., J. Virol. 69 (4) (1,995), 15 2,462-2,470; Wentworth et al., Int. Imunol, 8 (5) (1996), 651-659; Rehermann et al., J. Virol. 70 (10) (1996), 7.092-7.102; Diepolder et al. , J. Virol. 71 (8) (1997), 6.011-6.019; Lamonaca et al., Hepatology 30 (4) (1999), 1.088-1.098; WO 00/11186, WO 95/12766, WO 95 / 27733, document WO 94/20127, document WO 95/25122, document WO 95/22317, document WO 94/25601, document WO 92/03458 and document WO 93/00365 Additionally, important studies have been undertaken God of neutralization ex vivo 20 proofs of principle with chimpanzees, the other species other than homo sapiens that is susceptible to chronic HCV infection. Of course, the study of immune responses induced acute and chronic infections in individuals and chimpanzees, has revealed only so far an emerging image of potential protective immune responses following the establishment of active viral infection. However, the correlation of prophylactic vaccine protection after parenteral immunization is expected to be largely extra-hepatic prior to HCV exposure. Only a limited number of chimpanzee vaccine efficacy studies have been performed and these studies showed - at most - a protection of 5 out of 7 animals from infection after a low dose homologous stimulation using exactly the same clone for immunization and stimulation In addition, HCV has a high degree of variability that provides HCV with the ability to get rid of immune responses induced by vaccines or infection. In consideration of this problem, a combination of different large protein fragments has also been suggested as a DNA vaccine as an alternative vaccination strategy (Rollier et al., J. Virol. 78 (1) (2004), 187-196; Dunas-Carrera et al., Biotech. Appl. Biochem. Imm. Publ. (September 29, 2003; BA20030112)).

Despite all the progress that has been made in the last 15, especially in the last 10 years, there is still a pressing need to develop new treatments and vaccination strategies against HCV (Heile et al., 35 J. Virol. 74 ( 15) (2,000), 6,885-6,892).

 It is therefore an objective of the present invention to provide such effective treatments and vaccination strategies against HCV.

The present invention therefore provides a vaccine against the Hepatitis C virus (HCV) as claimed in claim 1. Preferred embodiments of this HCV vaccine are claimed in claims 2 to 17. The vaccine of the The present invention can be used for the preparation of a medicament for the prevention and treatment of an HCV infection. A process for the preparation of this vaccine is claimed in claim 19.

 In international patent application WO 04/024182, filed on August 27, 2003 (claiming priority A 1367/2002 of September 13, 2002), a new method for isolating 45 HCV peptides useful in vaccination is disclosed , especially HCV T lymphocyte epitopes. In this application, the concept of "HCV hot spot epitopes" was described for the first time: a "hot spot" of T-cell epitopes is defined as a short peptide sequence comprising at least more than one T-cell epitope. For example, two or more epitopes may be placed shortly one after the other (briefly defined as less than 5-10 amino acids) or directly one after the other or partially or even totally overlapping. The 50 hot spots may contain only class I or class II epitopes or a combination of the two. Epitopes on hot spots may have different HLA restrictions.

Due to the highly complex and selective routes of treatment of class I and class II antigens, referred to in the introduction, epitopes of T lymphocytes in the sequence of a polypeptide cannot be easily predicted. Although widely used, computer algorithms for predicting T lymphocyte epitopes have a high rate of both false negatives and false positives.

Thus, since even the epitopes of individual T lymphocytes are not obvious within the sequence of a polypeptide, the same is even more the case for hot spots. Various radically different experimental proposals are combined in accordance with the present invention for identification of T lymphocyte epitopes, including epitope capture, transgenic HLA animals and in vitro stimulation of human mononuclear cells. The three proposals are systematically applied to overlapping peptides that encompass the antigen of interest, allowing extensive identification of epitopes. In such extensive analysis, not limited to a particular HLA allele but rather by unraveling all possibly target epitopes in a population, epitope hot spots may become apparent. In an antigen, only some sequences, if any, show characteristics of hot spots. Thus the identification of a hot spot is always an amazing process. 10

 Hot spots of T lymphocyte epitopes offer important advantages: Hot spots can be activated and can be recognized by different clones of an individual's T lymphocytes. Hot spots (when they comprise epitopes with different HLA restriction) can interact with T lymphocytes of different non-HLA correspondence individuals.

Epitope-based vaccines have so far been directed to selected frequent HLA-alleles, for example 15 HLA-A2, which is expressed in approximately half of Caucasians. Since other alleles are less frequent, epitope-based vaccines with a large global population will have to comprise many different epitopes. The number of chemical entities (eg peptides) of a vaccine is limited by restrictions that originate from the manufacture, formulation and stability of the product.

The hot spots allow such epitope-based vaccines with a large global population extension, since they provide a potentially high number of epitopes for a limited number of peptides. Of course, the prior art (see the references mentioned about the epitopes discussed in the art and the fact that a vaccine against HCV has not yet been developed) as it is currently found, has clearly demonstrated that vaccine development Against HCV it has been considered problematic and that it has been a general failure to develop vaccines against HCV and treatments despite the abundance of references during the last 25 10 years, which explains the effectiveness of HCV antigens in obtaining humoral immune response and cell in various infection models. The treatment and prevention of HCV infection involve complexity and serious unpredictability.

The concept of the present invention by providing HCV vaccines based on the characterization of HCV hot spot epitopes now allows for the first time an HCV vaccination system that allows both adequate and specific prophylactic vaccination and an effective treatment regimen for patients. HCV infected, especially chronically infected patients.

It could be demonstrated by the inventors of the present application that the use of one or two specific epitopes only in the HCV vaccine is not sufficient to give satisfactory results in vaccination. Also the concept of providing a mixture of HCV proteins (Core, E1 and E2 as a DNA vaccine; Duenas-Carrera et 35 al. (2003)) or a mixture of large fragments of such HCV proteins (Core, E1 and E2 and NS3 as DNA vaccines; Rollier et al., (2004) did not lead to the desired results.

The HCV vaccines according to the present invention contain epitopes containing short polypeptides of the epitopes of the hot spots as indicated above. The present vaccines do not depend on the risks and uncertainties regarding DNA vaccines (absence of long-lasting immune response; largely extrahepatic action; mode of action, location, etc.). On the other hand, the present invention also does not face the various manufacturing problems with the production of complete proteins or long chain protein fragments.

The hot spot epitopes described herein have been identified by functional assays using mainly a Th1 / Tc1 type reaction (i.e., interferon gamma) as an output reading. It is known that antigens (complete or at least large fragments) not only contain such agonist epitopes, but can also encode antagonist ligands. Such antagonists potentially present in complete antigens, but absent in hot spots may impede the immunogenicity and efficacy of the vaccine. On the other hand, complete antigens will almost certainly induce a humoral immune response. Such antibody response has little effect per se on HCV residing in the cells, but can seriously compromise the desired T lymphocyte response. Epitopes that come from hot spots according to the present invention, due to their restricted length, have a much lower probability of inducing unwanted antibody responses. Finally, only relatively small parts of HCV antigens between different genotypes are conserved. Thus, the complete antigens used in a vaccine can induce responses against unconserved regions or epitopes, irrelevant to most patients. On the contrary, the hot spots 55 described herein come from conserved regions> 80% between the main genotypes 1, 2 and 3

HCV

 The super-HLA types (as disclosed for example in WO 01/21189) disclose the fact that in some cases a given HLA class I T cell epitope is not exclusively restricted to one and only one HLA allele it can also be recognized by T lymphocytes in the context of another HLA allele (example: a peptide can bind HLA-B * 0702 and -B * 3501). Such peptides by nature have a broader extension of HLA 5 in the population and are therefore interesting candidates for vaccine.

On the contrary, a hot spot of T lymphocyte epitopes according to the present invention differs significantly from such super-type HLA, because a hot spot according to the present invention is a peptide region in a protein that shows a number unusually high of different adjacent and even overlapping epitopes. The hot spot supplies a high number of epitopes in relatively small regions, while at the same time the appropriate treatment of the epitopes in the hot spots is guaranteed.

Of course, the hot spots according to the present invention show a remarkably low variation between different species of HCV that makes them even more favorable for vaccine proposals, because heterologous stimulation is also possible even if only small polypeptides are used. Preferably, the length of the epitopes according to the present invention is at least 6, 7, 8, 9, 10, 11 or 12 amino acids.

In accordance with the present invention the epitopes for a given vaccine can be selected from the hot spot regions as defined above. Of course, any region of complete hot spots in the vaccine or only parts thereof may be provided (provided that the part contains at least one epitope). The epitopes to be used in accordance with the present invention may also be variants of sequences with deletions (preferably 1, 2, 3, 4, 5, 6 or 7 amino acids) in the "standard" sequence of the dots hot as defined above. Preferably, the deletions within the sequence of hot spots do not modify the epitopes; Of course at least one epitope in the hot spot region has to be preserved despite the deletion.

Epitopes can be provided as polypeptides without modifications; modification of the chain of polypeptide with certain chemical or biological moieties may be appropriate, especially with respect to solubilization or pharmaceutical formulation considerations (for example, as disclosed in WO 01/78767). The epitopes used in the present HCV vaccine may comprise either the entire hot spot region or a contiguous fragment thereof (containing at least one of the epitopes contained in the hot spot) or comprising a non-contiguous portion of the hot spot (however, also containing at least one of the epitopes contained in the hot spot; in this case one or more connectors (eg, amino acid or peptide connectors) can be provided between the non-contiguous parts of this hot spot).

According to a preferred embodiment, the HCV vaccine according to the present invention comprises at least three, especially at least four epitopes, each of a different hot spot epitope.

Even more preferred, the HCV vaccine comprises at least five epitopes, each of a different hot spot epitope. Although the greater the number of epitopes in such an epitope-based vaccine, the greater the problems with respect to the formulation, a number of 4, 5 or 6 epitopes is the optimal balance between wide range of protective capabilities and formulation disadvantages. , especially with respect to production on an industrial scale.

In certain cases therefore also an HCV vaccine comprising at least six epitopes, each of a different hot spot epitope may be a preferred embodiment of the present invention. The optimal number of hot spot epitopes also depends on the relative interaction of the epitopes selected from each other which has been shown to be the main obstacle to certain combinations of short-length polypeptides in a vaccine.

Preferred HCV vaccines according to the present invention are characterized in that - in addition to the epitopes a), b) and c) of claim 1 - the additional epitopes are selected from the following groups:

In general, only one epitope should be selected from a group of epitopes to achieve good results. Of course, not all hot spot epitopes listed above are equally potent and efficacy may vary between different population groups. Preferably, a strategy of 5 individual vaccination can be applied for each group that is based primarily on extension for HLA. Therefore, a preferred HCV vaccine according to the present invention is characterized in that it comprises at least the following hot spot epitopes:

As an alternative to the precise antigens and epitopes listed herein, 10 antigen / epitope variants can be used in vaccines according to the present invention. Preferred variants are designed according to homologous epitopes (which contain one or more exchanges present in homologous HCV strains) or heteroclícticos. Among the preferred variants are those that vary from a sequence described herein by conservative amino acid substitutions. Such substitutions are those that replace a given amino acid in a polypeptide with another amino acid of similar characteristics. Typically, see conservative substitutions for substitutions one for another, between the aliphatic amino acids Ala, Val, Leu and Ile; exchange of the Ser and Thr hydroxyl moieties, exchange of the Asp and Glu acid moieties, substitution between the Asn and Gln amide moieties, exchange of the Lys and Arg basic moieties and substitutions between the Phe and Tyr aromatic moieties.

Particularly preferred in this regard are variants that have the amino acid sequence of any polypeptide described herein, in which residues of 1, 2, 3, 4 or 5 amino acids are substituted, removed or added, in any combination Among these, silent substitutions, additions and deletions are preferred, which do not modify the properties and activities of the polypeptide of the present invention. They are

Especially preferred in this regard conservative substitutions. Specifically, suitable amino acid substitutions are those that are contained in homologs for the sequences described herein. A suitable sequence variant of an antigen or epitope as disclosed herein therefore includes one or more variations that are present in one or more strains or HCV serotypes (preferably exchanges of 1, 2, 3, 4 or 5 amino acids that are based on such homologous variations). 5 Such antigens comprise sequences that may be naturally occurring sequences or newly created artificial sequences. These preferred antigen variants are based on such variations of sequences found in nature, for example forming a "mixed sequence" for the antigenic regions of the polypeptides according to the present invention. For example, a given HCV sequence as disclosed herein can be modified including one or more such variations, thereby creating an artificial variant (i.e., not found in nature) of this sequence of given antigens or epitopes (found in nature).

It is evident that also the epitopes or peptides from the present epitopes or peptides by improvement of amino acid exchanges, by conservation or at least not significantly preventing the ability of T lymphocyte activation of the epitopes to be covered by the epitopes or peptides according with the present invention. Therefore, the present epitopes or peptides also cover epitopes or peptides, which do not contain the original sequence as derived from a specific strain of HCV, but rather trigger the same T lymphocyte response or preferably an improved response. These epitopes are referred to as "heteroclitic." These include any epitope that can trigger the same T lymphocytes as the original epitope and preferably has a more potent activation capacity of T lymphocytes preferably in vivo or also in vitro. The respective homologous epitopes of other strains of HCV are also included in the present invention.

Heterocytic epitopes can be obtained by rational design, that is, taking into account the contribution of individual residues to the binding to MCH / HLA as disclosed for example by Ramensee et al. 1999 (Immunogenetics 50: 213-219) or Sturniolo et al. 1999 (Nature Biotechnology 17: 555-562), together with a systematic exchange of residues that potentially interact with the TCR and testing the resulting sequences with T lymphocytes directed against the original epitope. Such a design is possible for an expert in the field without much experimentation.

Another possibility includes screening peptide libraries with T lymphocytes against the original epitope. A preferred way is the positional scanning of synthetic peptide libraries. Such proposals have been described in detail for example by Blake et al. 1996 () and Hemmer et al. 1999 () and the references given therein.

As an alternative to epitopes represented by the amino acid sequence from HCV of similar origin or heterocytic epitopes, substances that mimic these epitopes can also be applied, for example, "peptidomimetics" or "retro-reverse-peptides".

Additional chemical groups, especially amino acid residues, linkers (and that bind to carriers), groups that facilitate transport, etc., can be added to the N- and / or C-terminal epitopes in the HCV vaccine according with the present invention. Amino acid junctions are preferred as disclosed in WO 35 01/78767.

Another aspect of the design of improved hot spot epitopes is their formulation or modification with substances that increase their ability to stimulate T lymphocytes. These include epitopes of helper, lipid or liposome T lymphocytes (or preferred modifications as disclosed in WO 01 / 78767).

Another way to increase the capacity of epitope T lymphocyte stimulation is its formulation with 40 immune stimulation substances such as antibodies, cytokines or chemokines such as interleukin-2, -7, -12, -18, type I and II interferons ( IFN), especially IFN-alpha, GM-CSF, TNF-alpha, flt3-ligand and others.

In accordance with one more aspect, the present invention relates to the use of an HCV epitope or HCV peptide according to the present invention for the preparation of a restricted HLA vaccine to treat or prevent hepatitis C virus infections. (HCV). Four. Five

Another preferred HCV vaccine according to the present invention comprises the following epitopes:

The present HCV vaccine preferably comprises at least one A2 epitope and at least one DR1 epitope.

The present HCV vaccine preferably comprises at least one DR7 epitope. fifty

The following combination of epitopes is considered as specifically powerful (at least one of the groups (1) to (5)):

It is preferred to provide auxiliary substances specifically suitable for the present HCV vaccines. Therefore, certain types of immunostimulatory substances have proven advantageous for the present invention.

Therefore, a preferred HCV vaccine also contains a peptide comprising an R1-XZXZNXZX-R2 sequence, in which N is an integer between 3 and 7, preferably 5, X is a natural amino acid residue and / or unnaturally positively charged, Z is an amino acid residue selected from the group consisting of L, V, I, F and / or W and R1 and R2 are independently selected from each other from the group consisting of -H, -NH2, -COCH3 , -COH, a peptide with up to 20 amino acid residues or a peptide reactive group or a peptide linker with or without peptide; X-R2 may be an amide, ester or thioester of the C-terminal amino acid residue of the peptide ("Peptide A").

Another preferred embodiment of the HCV vaccine further contains an immunostimulatory oligodeoxynucleic acid (ODN) molecule with the structure according to formula (I).

 fifteen

in which:

R1 is selected from hypoxanthine and uracil, any X is O or S,

any NMP is a monophosphate or -monothiophosphate 2 'deoxynucleoside, selected from the group consisting of monophosphate or monophosphate monothiophosphate or -monothiophosphate deoxyadenosine, deoxyguanosine, deoxyinosine, deoxycytosine, deoxyuridine, deoxythymidine, 2-methyl-deoxyinosine, 5-methyl-deoxycytosine-, 20 desoxipseudouridina , deoxyribosapurin, 2-amino-deoxyribosapurin, 6-S-deoxyguanine, 2-dimethyl-deoxyguanosine or N-isopentenyl-deoxyadenosine, NUC is a 2'-deoxynucleoside, selected from the group consisting of deoxyadenosine, deoxyguanosine, deoxyinosine, deoxycytosine, deoxycytosine, deoxycytosine, deoxycytosine, deoxycytosin , 2-methyl-deoxyuridine, 5-methyl-deoxycytosine, deoxipseudouridine, deoxyribosapurine, 2-amino-deoxyribosapurin, 6-

S-deoxyguanine, 2-dimethyl-deoxyguanosine or N-isopentenyl-deoxyadenosine,

a and b are whole numbers from 0 to 100 with the proviso that a + b is between 4 and 150 and

B and E are common groups for the 5 'or 3' ends of nucleic acid molecules ("I- / U-ODN").

An HCV vaccine composition according to the present invention may further comprise a polycationic compound, preferably a polycationic polymer, more preferably a polycationic peptide, especially polyarginine, polylysine or an antimicrobial peptide.

The polycationic compound or compounds to be used in accordance with the present invention can be any polycationic compound that shows the characteristic effect in accordance with WO 97/30721. Preferred polycationic compounds are selected from basic polypeptides, organic polycations, basic polyamino acids or mixtures thereof. These polyamino acids should have a chain length of at least 4 amino acid residues. Especially preferred are substances that contain peptide bonds such as polylysine, polyarginine and polypeptides that contain more than 20%, especially more than 50% of basic amino acids in a range greater than 8, especially greater than 20, amino acid residues or mixtures thereof. . Other preferred polycations and their pharmaceutical compositions are disclosed in WO 97/30721 (e.g., polyethyleneimine) and WO 99/38528. Preferably, these 15 polypeptides contain between 20 and 500 amino acid residues, especially between 30 and 200 residues. These polycationic compounds can be produced chemically or recombinantly or they can come from natural sources.

The cationic (poly) peptides can also be polycationic antibacterial microbial peptides. These (poly) peptides can be of prokaryotic or animal or plant origin or can be produced chemically or recombinantly. Peptides can also belong to the class of defensins. Such host defense peptides or defensins are also a preferred form of the polycationic polymer according to the present invention. In general, a compound is used that allows as an end product the activation (or regulation by decrease) of the adaptive immune system, preferably mediated by APCs (including dendritic cells), as polycationic polymer.

The polycationic substances used in the process according to the present invention are antimicrobial peptides from cathelicidin or derivatives thereof (WO 02/13857 A), especially antimicrobial peptides from mammalian cathelicidins, preferably human, bovid or mouse or neuroactive compounds such as growth hormone (human) (as disclosed for example in WO 01/24822).

Polycationic compounds from natural sources include HIV-REV or HIV-TAT (cationic peptides derived, antenapedia peptides, chitosan or other chitin derivatives) or other peptides derived from these peptides or proteins by biochemical or recombinant production. Other preferred polycationic compounds are catelin or related substances or derivatives of catelin, especially mouse, bovid or catechins and / or especially human cathelicidins. Related or derived cathine substances contain the total or parts of the cathine sequence with at least 15-20 amino acid residues. The derivations may include the substitution or modification of natural amino acids by amino acids that are not among the 20 classic amino acids. On the other hand, additional cationic moieties can be introduced into said cathine molecules. It is preferred that these choline molecules be combined with the HCV peptide composition according to the present invention. However, these catelin molecules have proved surprisingly effective as an adjuvant for an antigen without the addition of more adjuvants. It is therefore possible to use said 40 molecules of catelin as effective adjuvants in vaccine formulations with or without more immunoactivating substances. Preferably, the present HCV vaccine also contains an Al (OH) 3 adjuvant and / or a polycationic peptide.

According to a specifically preferred embodiment, the Peptide A mentioned above is KLKL5KLK and / or the I- / U-ODN mentioned above is oligo d (IC) 13. Four. Five

In certain embodiments, it may be preferred that the HCV vaccine further contains an oligodeoxynucleotide containing a CpG motif.

Preferably, the HCV vaccine according to the present invention is lyophilized in a form that is reconstitutable in 15 min at 37 ° C. Depending on the individual epitopes in the HCV vaccine, this can be a difficult task. Therefore, the present invention also provides effective means to formulate such an HCV vaccine.

The HCV vaccine according to the present invention preferably contains between 10 µg and 10 mg of each epitope (per dose).

Depending on the application, the number of individual peptides and especially their concentration in the mixture varies. In a preferred embodiment of the present invention, the concentration of the individual peptides in the HCV vaccine to be delivered to the patient ranges from 5 µg / ml to 5 mg / ml, preferably 50 µg / ml to 4 mg / ml, more preferably from 100 /g / ml to 3 mg / ml.

The lyophilized HCV vaccine according to the present invention preferably contains traces of acetic acid due to the lyophilization process according to the present invention.

In accordance with another aspect, the present invention also relates to the use of the present HCV vaccine for the preparation of a medicament for the prevention and treatment of an HCV infection, that is, by administering an effective immunizing dose of a vaccine. against HCV according to the present invention to an individual, especially a patient with HCV or an individual at risk of acquiring an HCV infection (for example, hospital staff, clinical research staff, blood donation staff, etc. .).

Solubilizing mixtures of peptides such as HCV vaccines according to the present invention can be difficult. It is therefore an object of the present invention to provide methods for solubilization of HCV peptide mixtures according to the present invention, that is, mixtures of peptides that are composed of two or more peptides, especially those consisting of hydrophilic peptides. thus 15 as hydrophobic. Another object is to make available a process for the manufacture of sterile and optionally lyophilized pharmaceutical formulations containing peptide mixtures.

Therefore, the present invention provides according to another aspect, a process for preparing a pharmaceutical preparation comprising the solubilization of a mixture of HCV peptides according to the present invention, characterized in that the mixture of peptides is solubilized by a solution aqueous containing at least one organic acid selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid and halogenated or hydroxylated forms thereof. To extend the durability time, the solubilized peptide mixture can also be sterilized (by filtration, irradiation, heat treatment, chemical sterilization or other procedures) and lyophilized.

In a preferred embodiment, the peptide mixture contains hydrophilic as well as hydrophobic peptides. Of course, the present process can also be used for mixtures of peptides containing a single type of peptides (specifically for mixtures containing for example two or more hydrophilic peptides).

In another preferred embodiment, the peptides that are dissolved by an aqueous solution according to the present invention contain at least 6, preferably at least 8, more preferably at least 10, especially at least 12 amino acids. Peptides as well as polypeptides containing a maximum number of 30, 40 or 50 amino acids (or even up to 100, although long epitopes are less preferred due to the disadvantages of longer antigen fragments) can be dissolved according to the present invention .

For this aspect of the present invention the peptides are characterized by their solubility. Peptides that are soluble in an aqueous solution of less than 100 µg / ml are considered hydrophobic. On the other hand, hydrophilic peptides dissolve in a buffered aqueous solution in concentration greater than 100 µg / ml. Hydrophilic peptides can also be divided into cationic peptides (> 20% basic amino acids including lysine, arginine, histidine), anionic (> 20% acidic amino acids including aspartic acid, glutamic acid) and hydroxyl-rich (> 30% amino acids containing –OH such as serine, threonine, tyrosine).

In accordance with another aspect, the present invention therefore relates to a process for the preparation of the present HCV vaccine characterized by the following steps:

- chemically synthesize at least two epitopes as defined above,

- solubilize these epitopes by an aqueous solution containing at least one organic acid selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid and halogenated or hydroxylated forms thereof,

- mix the solubilized epitopes and

- optionally lyophilize the mixed epitopes.

The HCV peptide mixture can be frozen (if lyophilization stage is not performed) for storage reasons.

The peptides of the peptide mixture according to the present invention are synthesized by classical chemical methods (eg, solid phase synthesis according to Merrifield). Of course the peptides are 50

they can also be obtained by chemical or enzymatic fragmentation of recombinantly produced proteins or natural isolates. In another embodiment, HCV peptides are isolated directly from eukaryotic and prokaryotic cells. In all three cases, additional purification of the peptide or polypeptide of interest will be required in some cases before it is lyophilized and mixed together.

It is a basic requirement for this specific procedure that the organic acids used in aqueous solutions for the solubilization of peptides are pharmaceutically acceptable. In a preferred embodiment of the present invention, the organic acids are acetic acid and / or formic acid optionally containing acetonitrile.

Experiments revealed, depending on the composition of the peptide mixture, that the concentration of the organic acid in the aqueous solution has to be at least 10%, preferably at least 20%, preferably at least 10%, preferably at minus 40%, preferably at least 50%, preferably at least 60%.

In some cases, the addition of organic acid derivatives (for example acetonitrile) to the aqueous solution may be useful for solubilizing mixtures of peptides containing a larger amount of hydrophobic peptides. Also organic solvents such as DMSO and DMF contribute to an improved dissolution of peptide mixtures containing an increased concentration of hydrophobic peptides. However, aqueous mixtures of peptides containing DMSO and / or DMF cannot be lyophilized.

In a preferred embodiment, volumetric agents are added to the peptide mixture when it is desired that the product be lyophilized. Especially at low concentrations of peptides the formation of a good freeze-drying filter cake is not guaranteed. Therefore, swelling agents are added to low amounts of peptides. Preferred swelling agents are sorbitol, mannitol, polyvinylpyrrolidone (PVP) and mixtures thereof.

In another preferred embodiment the solubilized peptide mixture is sterilized by filtration. To achieve improved product recovery and allow filtration of large quantities of the peptide mixture, the peptides it contains must be completely solubilized and no gel can be formed.

The solubilized and optionally sterilized peptide mixture can be lyophilized directly or after filling vials. Lyophilization is a useful and effective procedure for prolonging the durability time of peptide mixtures, as described above. With the solubilized peptides, a lyophilized preparation of a mixture of peptides can be obtained, containing a maximum of 5% residual solvent (water in aqueous systems) and traces of the organic acid, which is reconstitutable in a buffered aqueous solution containing NaCl and / or 10-minute sorbitol of> 95%, preferably 98%, especially 99%, resulting in a cloudy suspension or a clear solution (depending on the composition of the peptide mixture). Such rapid reconstitution is specifically necessary in cases of emergency, in the event that a ready-to-use solution must be present within a few minutes with an almost complete resolution of the total dose of a lyophilized vial.

The invention will be described in more detail by the following examples and figures, but the invention is of course not limited thereto.

Figure 1 shows that KLK / O-d (IC) 13 induces specific type 1 cellular immune responses of HCV peptides;

EXAMPLES

Example I. Binding of peptides from HCV to HLA class II molecules. 40

According to WO 04/024182, several sequences were synthesized that incorporate new overlapping reactive HCV peptide peptides or that avoid critical moieties such as cysteine. In these, their affinities for soluble class II HLA molecules were retested and the results were compared with those obtained with the original (Table 1).

Table 1. Binding of selected HCV peptides and their 15-mer counterparts for soluble HLA class II molecules ("+++" strong affinity, "++" intermediate affinity, "+" weak affinity, "-" without affinity, "nh" not done; the core binding remains are underlined).

The suppressed affinities for DRB1 * 0101 and DRB1 * 0401 molecules in the case of peptide 1,798 compared to their shorter counterparts (B84-B96) are probably due to the long sequence (26 amino acids) that a secondary structure can avoid that prevents Union. The 1,798 peptide in proteolytic cleavage, in vivo, must be expected to give rise to two shorter class II epitopes. The cysteine residues (C) removed in peptides 1,827 and 1,829 (derived from peptides C114 and 1,604, respectively) seem crucial for binding to 5 molecules of DRB1 * 0401 but do not essentially change the affinities for other DR subtypes tested.

Example II Identification and characterization of HCV epitope hot spots

As indicated in general terms, a hot spot of T-cell epitopes (a "hot spot") is defined as a short peptide sequence at least comprising more than one T-cell epitope. For example, two or more epitopes are they can be placed one shortly after another (briefly defined as less than 5-10 amino acids) or directly one after the other or partially or even completely overlapping. Hot spots may contain only class I or class II epitopes or a combination of both. Epitopes in hot spots may have different HLA restrictions.

Due to the highly complex and selective routes of treatment of class I and class II antigens, referred to in the introduction, T-cell epitopes cannot be easily predicted in the sequence of a polypeptide. 15 Although widely used, computer algorithms for predicting T-cell epitopes have a high rate of both false negatives and false positives.

Thus, since even the epitopes of individual T lymphocytes are not obvious in the sequence of a polypeptide, the same is even more the case for hot spots. Several radically different experimental proposals are combined in accordance with the present invention for identification of T lymphocyte epitopes, including epitope capture, transgenic HLA animals and in vitro stimulation of human mononuclear cells. The three proposals are systematically applied to overlapping peptides that extend over the antigen of interest, allowing extensive identification of epitopes. In such extensive analysis, not limited to a particular HLA allele, but rather by unraveling all possibly target epitopes in a population, the hot spots of epitopes may become apparent. In an antigen, only some sequences, if any, show characteristics of hot spots. Thus the identification of a hot spot is always a surprising fact.

The hot spots of the T cell epitopes offer important advantages: The hot spots can be activated and can be recognized by different clones of an individual's T lymphocytes. Hot spots (when they comprise epitopes with different HLA restriction) may interact with T lymphocytes of different individuals not corresponding to HLA.

Epitope-based vaccines have so far been targeted in selected frequent HLA alleles, for example HLA-A2, which is expressed in approximately half of Caucasians. Since other alleles are less frequent, epitope-based vaccines with a large global population will have to comprise many different epitopes. The number of chemical entities (eg peptides) of a vaccine is limited by restrictions that originate from the manufacture, formulation and stability of the product.

The hot spots allow such epitope-based vaccines with a large global population extension, since they provide a potentially high number of epitopes for a limited number of peptides.

Table 2. Hot spots of T-cell epitopes in conserved regions of HCV.

Hot spots (incl. Some variations) are shown in bold, the epitopes contained in hot spots 40 in normal source. The number of peptides and the sequence are given, as well as the HLA class I and class II extension. Source data refers to references in the literature, if not provided by WO 04/024182.

Example III Hot spot Ipep 1,426 of HCV epitopes contains at least HLA-A * 0201 and various promising class II T cell epitopes.

The main objective of this experiment was to compare the immunogenicity of the “hot spot” Ipep 1.426, which contains at least one epitope of HLA-A * 0201 (Ipep 1,334) and 2 promiscuous class II epitopes (Ipeps 1,006 and 1,425) 5 for individual epitopes For this purpose, peripheral blood mononuclear cells (PBMC) from various healthy HLA blood donors were stimulated in vitro or with a mixture of 1,426 or a mixture of 1,334, 1,006, 1,425. These stimulation rounds were performed resulting in oligoclonal T lymphocyte lines. Then, the responses against the four peptides were evaluated by interferon-gamma ELIspot analysis (IFN-). 10

Peptide 1,426 induces T lymphocyte responses similarly well as individual epitopes comprised in its sequence. In particular, CD8 positive T lymphocytes directed against the 1,334 restricted epitope of HLA-A * 0201 were successfully generated.

Table 3: IFN- secretion induced by peptides of oligoclonal T lymphocyte lines. Lines of two healthy HLA type individuals were generated by 3 rounds of primer in vitro or with peptide 1,426 or a mixture of 15 peptides 1,006 + 1,425 * 1,334. The reactivity of CD4 and CD8 positive T cells in these lines was assessed by ELIspot IFN- (“+++” very strong, “++” strong, “+” significant, “-“ without IFN-gamma secretion).

(keep going)

Example IV The potent HCV-specific type 1 cellular responses are induced by the combined injection of five different peptides from HCV, the KLK antimicrobial peptide and the synthetic oligodeoxynucleotide o-d (IC) 13. 5

HLA-A * 0201 transgenic mice of mice (HHD1)

 Peptides

 Peptides p83, p84, p87, p89, p1,426 were used for vaccination. p83: HCV-derived peptide, (KPFGGGQIVGGVYLLPRRGPRL) p84: HCV-derived peptide,

 (GYKVLVLNPSVAAT) p87: HCV-derived peptide, (DLMGYIPAV) p89: HCV-derived peptide, (CINGVCWTV) p1.426: HCV-derived peptide, (HMWNFISGIQYLAGLSTLPGNPA) (p1.274 used for frequent stimulation (HGT-peptide) A * 0201 restricted) All peptides were synthesized by classical solid phase F-moc synthesis, purified by HPLC and analyzed by mass spectroscopy for purity dose: 20 porg per peptide / mouse

 KLK KLKLLLLLKLK-COOH

 It was synthesized by MPS (Multiple Peptide System, USA) dose: 10 nmoles / mouse

 oligo-d (IC) 13 (= ODN1a) ODN

 5’ICI CIC ICI CIC ICI CIC ICI CIC IC3 ’was synthesized by Purimex Nucleic Acid Technology, Göttingen

 Formulation

 10 nM Tris / 135nM NaCl; pH  7

Experimental assembly (5 mice / group):

1. HCV peptides

2. HCV + KLK + o-d (IC) peptides 13 10

On days 0, 14 and 28, s.c. was injected into HHD.1 mice on the two hind legs a total volume of 100 µl / leg) containing the compounds listed above. On day 35, CD4 + T lymphocytes as well as CD8 + magnetic separation (MACS, Miltenyi) were isolated from single cell suspensions of spleen cells (7 days after the last vaccination). T lymphocytes were incubated with medium (background control) or re-stimulated with irradiated spleen cells of natural HHD.1 mice such as APC in the presence of or the different peptides used for vaccination or the frequent peptide p1.274. After overnight incubation, IFN- production was analyzed using an ELIspot assay.

Figure 1 shows that in the joint injection of the five peptides from HVC with KLK / o-d (IC) 13,

induced high amounts of IFN- produced by CD4 + T lymphocytes against p84, p87, p89, p1.426. In addition, a strong IFN-producción production was detectable by CD8 + T lymphocytes against p87, p89.

Example V: Solubilization of peptide mixtures (HCV peptides plus poly-L-arginine):

The peptide mixture contains peptides 83, 84, 87, 89, 1,426 and pR as an immunizer (see Table 4), in which peptides 83 and 84 are soluble in water and DMSO, peptides 87 and 89 are poorly soluble. in water but dissolve easily in DMSO and peptide 1,426 is only soluble in DMSO. For the manufacture of a suspension formulation it is first necessary to dissolve peptides 83 and 84 in an aqueous buffer solution and peptides 87, 89 and 1,426 in DMSO. After sterile filtration the solutions can be combined to form the final suspension.

Water / acetonitrile mixtures as well as DMSO and DMF did not function as solvents and resulted in suspensions that could not be sterile filtered due to filter blockage. Surprisingly, it was found that a 50% acetic acid / water mixture dissolved all components. The obtained peptide solution containing the aforementioned peptides could easily be sterile filtered without blocking the filter.

Table 4 Peptide Mix I

 ID peptide

 Peptide sequence

 83

 KFPGGGQIVGGVYLLPRRGPRL

 84

 GYKVLVLNPSVAAT

 87

 DLMGYIPAV

 89

 CINGVCWTV

 1,426

 HMWNFISGIQYLAGLSTLPGNPA

 fifteen

The following examples (examples V.1 to V.12, tables 5 to 16) illustrate that mixtures of peptides containing at least two peptides with variable length and amino acid composition are suitable for solubilization in accordance with the present invention. In addition, the tables show the known affinities of the peptides for HLA class I and class II molecules.

Example V.1: 20

Example V.2:

Example V.3:

Example V.4: 5

Example V.5:

Example V.6:

Example V.7:

Example V.8: 5

Example V.9:

Example V.10:

Example V.11:

 5

Example V.12:

Example VI Background in design of additional HCV vaccine variants in accordance with the present invention

The preferred combination of hot spots I consists of 12 peptides from conserved regions (> 80% in 1,2,3 genotypes) of 5 different HCV antigens. In total, at least 8 major HLA class I alleles (1-8 times each) are extended, providing a population extension of 83-91% in Europe, 89-91% in the US. in Caucasians and 87-89% in Japanese people (estimated on numbers of HLA frequency studies in Gjertson and Terasaki, eds., HLA 1998; American Society of Histocompatibility and Immunogenetics, Lenexa, 5 kasas, USA). Similarly, the various important class II alleles are spread, mostly by so-called promiscuous peptides (binding to more than one HLA class II allele). Frequent, particularly individual alleles (DR1, 4, 7, 11) extend independently 6-8 times. To a large extent, DR11 extends 7 times. The presence of this allele has joined the spontaneous recovery of HCV infection (Thursz et al., 1999 Lancet: 354 (9.196): 2.19-24). The high number of epitopes contained in this combination of hot spots ensures not only a large population extension and therefore applicability of the vaccine, but also a broad immune response in individuals treated with said combination. This is a prerequisite for efficacy since spontaneous recovery from HCV infection has been linked to a broad immune response typically directed against 10 or more epitopes (Dav et al., 2.002 J. Virol. 76 (24): 12.584-95 ).

A T lymphocyte response against multiple epitopes also reduces the risk of developing variants of HCV epitope escape (HCV viruses, which do not contain the target epitope because of the immune response) because when more target epitopes are present harder time for the virus to change them simultaneously. Chimpanzee experiments have established that such HCV escape variants are an important mechanism for maintaining chronic infection (Weiner et al., 1995 Proc Natl Acad Sci USA: 92 (7): 2,755-9).

Finally, targeting most HCV antigens (Core, E2, NS3, 4,5) instead of for example only one also provides significant advantages: different antigens can be recognized differently by T lymphocytes, for example due to differences in the treatment of the antigen or dictated by the particular HLA haplotype of the person). Also, different antigens have different functional importance during the HCV life cycle.

The combination of hot spots I also contains individual epitopes from hot spots 25 (Ipep87 from Ipep87EX and Ipep89 from Ipep89EX). This presents the advantage of a deliberate approach to the desired response in a particular epitope. Using for example Ipep89 ensures that a restricted HLA-A2 CTL response is generated while using 89EX can lead to a HLA-A2 restricted CTL response, which can be compromised by a concomitant DR4 or DR7 restricted auxiliary response. . 30

Using hot spot combinations has a number of advantages over using full length antigens, either in the form of recombinant proteins or as DNA vaccines. As detailed in the preceding paragraph in certain cases using individual epitopes may be advantageous over using the full antigen hot spot. Second, the hot spots described herein have been identified by functional assays using mainly a Th1 / Tc1 type reaction (i.e., interferon gamma) as the output reading. It is known that antigens not only contain such agonist epitopes, but they can also encode antagonist ligands. Such antagonists potentially present in complete antigens, but absent in hot spots may impede the immunogenicity and efficacy of the vaccine. Next, the complete antigens will almost certainly induce a humoral immune response. Said antibody response has little effect per se on HCV residing in the cells, but can seriously compromise the desired response of T lymphocytes. Hot spots due to their restricted length have a much lower probability of inducing responses of unwanted antibodies. Finally, only relatively small parts of HCV antigens are conserved between different genotypes. Thus, the complete antigens used in a vaccine can induce responses against unconserved regions or epitopes, not frequent for most patients. In contrast, the hot spots described herein come from conserved regions> 80% among the 45 major HCV genotypes 1, 2 and 3.

The preferred combination of hot spots II consists of 7 peptides from conserved regions (> 80% in genotypes 1, 2, 3) of 5 different HCV antigens. In total at least 7 major HLA Class I alleles (1-5 times) are extended, providing a population extension of 81-90% in Europe, 89-91% in 5 US Caucasians. and 85-88% in Japanese people (estimated in numbers of HLA frequency studies in Gjertson and Terasaki eds., HLA 1998. American Society of Histocompatibility and Immunogenetics, Lenexa, Kansas, USA). Similarly, several important HLA class II alleles are spread, mostly by peptides called promiscuous (binding to more than one HLA class II allele). Particularly frequent individual alleles (DR1, 4, 7, 11) are extended independently 5-6 times. In large part, it extends DR11 5 times. The presence of this allele has been linked to the spontaneous recovery of HCV infection (Thursz et al., 1999 Lancet: 354 (9.196): 2.119-24).

By comparison with combination I, combination II has a smaller number of different peptides (7 versus 12), which has potential advantages in the manufacture, formulation and competitive production of said product. Most importantly, there is only one peptide with a cysteine side chain (1846). Therefore, only 1,846 homodimers could be formed, but not heterodimers or oligomers in this combination.

At the same time, combination II extends the same 5 HCV antigens and a similar number of alleles of both HLA class I and II as combination I. Therefore, the arguments discussed in the previous section also remain true for the combination. II.

From a formulation development point of view, combination II has several advantages over combination I (only 7 instead of 12 components). Most importantly, only one peptide (1846) supports a free cysteine side chain. Thus, only 1846 homodimers could be formed, but not hetero- or oligomers.

Example VII: results of immunogenicity of T lymphocytes from an optimization test of 102 doses of HCV vaccine

During the course of a clinical study a series of peptide / polyarginine mixtures was applied as a vaccine to healthy volunteers. Each group consisted of 12 individuals, positive for HLA-A2. The individuals received 4 vaccinations at monthly intervals (in 3 to 6 visits). Blood for immunological analysis was drawn at 1 to 6 visits one month after the last vaccination (visit 7) and 3 months after the last vaccination (visit 8). To control clinical studies, Intercell has established T-lymphocyte assays of the prior art to determine immunological criteria under adherence to GLP / GCP treatment: Interferon-gamma ELIspot test T-cell proliferation test, HLA / FACS tetramer assay . These assays allow reliable measurements of epitope-specific T lymphocyte responses induced by the therapeutic HCV vaccine. Immune responses of T-lymphocytes induced by the vaccine serve as substitution parameters for efficacy (Keilholz et al., J Immunother, March-April 2,002; 25 (2): 97-138). 10

As primary criteria, the immunogenicity of T lymphocytes was determined by a T lymphocyte proliferation assay:

The proliferation assay allows the detection of specific T lymphocytes of the peptides in biological samples such as human blood. The basis of the assay is that in stimulation with a peptide specifically recognized by its T-cell receptor it reacts by cytokine secretion and subsequent proliferation. Proliferation of cells 15 can be measured by a variety of means; Among the most sensitive proposals is the incorporation of radiolabeled thymidine into DNA previously synthesized in the cell division. This reaction can be performed in a 96-well plate. Each well contains a fixed number of cells, which are cultured in the presence of antigen / peptide for a couple of days. For the last 16-20 hours thymidine labeled with tritium (3H-thymidine) is added. The cells are then collected on a filter plate: the medium containing free radioactivity is washed away while the DNA is glued to the filter. The incorporated radioactivity can be quantified by means of a beta scintillation counter. Normal output is given as counts per minute (cpm).

As primary secondary criteria, immunogenicity of T lymphocytes was determined by gamma interferon ELIspot

ELIspot allows the quantification of functional T lymphocytes (i.e., cytokine secretion), specific to the peptide in biological samples such as human blood. The basis of the assay is that T lymphocytes in stimulation with a peptide specifically recognized by the T lymphocyte receptor react by cytokine secretion such as IFN-gamma. This reaction can be performed in a 96-well plate. The filter wells of that plate are coated with a specific Mab for IFN-range. As a consequence, each cell that secretes IFN-gamma leaves an IFN-gamma spot that can be visualized with a subsequent color reaction. Stains can be counted using 30 automated plate readers. The numbers obtained are a measure of the frequency of T lymphocytes that secrete IFN-gamma-specific peptides in the sample.

As an additional secondary main criterion, HLA / FACS tetramer analysis was performed.

HLA class I tetramers, soluble recombinant forms of an HLA molecule complex and antigenic peptide, bind to an antigen-specific T lymphocyte receptor used for recognition of T lymphocytes. 35 Through the use of flow cytometry with fluorescent tetramers. Antigen-specific CD8 + T lymphocytes can be enumerated and characterized reliably. The assay uses iTag tetramers custom made from HLA-A * 0201 produced by Beckman Coulter Immunomics complexed with HCV vaccine class I epitopes.

Individuals were classified as responders if they showed significant T lymphocyte responses in any of visits 4 to 8 and did not present a pre-treatment response.

Maximum Responding Indices were achieved (against any peptide, in any of visits 4 to 8, in any of the three T-lymphocyte assays of up to 92% versus 25% in a control group of 25 individuals who received saline.

Responding indices by peptides Ipep89, Ipep84 and Ipep1426: 45

As an example, the responses to peptides of hot sites of T lymphocyte epitopes Ipep89 (T-cell epitope of class I), Ipep84 and Ipep1426 are shown here. These responses were obtained after a cycle of 4 monthly vaccinations. For group B the vaccine contained poly-L-Arginine only. For Group C the vaccine contained a mixture of 5 peptides, including Ipep89, Ipep84 and Ipep1426. For Group G the vaccine contained a mixture of 5 peptides as in Group C plus poly-L-Arginine as a completely synthetic T lymphocyte adjuvant. Table 19 shows that in the control group B no responders are observed in the ELIspot and tetramer-HLA / FACS analyzes and 3 responders with, in total, only 3 positive visits. The latter can be interpreted as the index of false positive responders obtained in the proliferation test.

In Group C containing only but not poly-L-Arginine peptides, 1 responder was observed against Ipep84 in ELIspot, 5 and 4 responders against Ipep84 and Ipep1426, respectively, in proliferation and 4 responders against Ipep89 in tetramer analysis -HLA / FACS. Finally, the G group (same peptides as in C plus poly-L-arginine) showed responders in all three trials. To a large extent, especially consistent (all three peptides) and prolonged (see the number of total positive visits) ELIspot responses that give an indication 5 of functional T lymphocytes were dependent on poly-L-arginine as a synthetic T lymphocyte adjuvant.

image 1

 5

 10

 fifteen

 twenty

 25

 30

Example of an individual treatment (ELIspot): individual 5 of group C

Table 20 shows the results of interferon-gamma ELIspot obtained from a series of blood extractions from a particular individual of group G (5 peptides + poly-L-arginine). At visit 5, one month after the second vaccination, Ipep84, Ipep1426 class II epitopes already showed responses. After the third vaccination a maximum was reached in all responses and also the Ipep89 class I epitope showed a response. On the other hand, Ipep1426 showed a prolonged response even 3 months after the last vaccination at visit 8.

Table 20: ELIspot values of individual 5 of group G) for Ipep89, 84, 1426 over time

 (stains per 1 million PBMC, subtracted fund; non-significant values were set to zero))

 Visit 1 Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Visit 8

 Ipep89

 0 0 0 0 22 0 0

 Ipep84

 0 0 0 18 45 0 0

 Ipep1426

 0 0 0 22 92 16 16

 10

References:

Aichinger G et al., 1997, J. Biol. Chem. 272: 29.127-29.136,

Ausubel FM et al., (Authors), 2001. Current Protocols in Molecular Biology, Volumes 1-4, John Wiley and Sons (editorial).

Bellentani S et al., Microbes Infect. November 2,000; 2 (14) 1,757-63. fifteen

Bonifacino JS et al., (Authors), 2001. Current Protocols in Cell Biology, Volumes 1-2, John Wiley and Sons (editorial).

Cxomberg M et al., Curr Gastroenterol Rep. February 2,002; 4 (1): 23-30. Cox AL et al., 1994. Science 264: 716-719

Coligan JE et al., (Authors), 2001. Current Protocols in Immunology, Volumes 1-4, John Wiley and sons 20 (editorial).

Farci P et al., Semin Liver Dis. 2,000; 20 (1): 103-26 Gavin MA et al., 1993. J. Immunol. 151: 3,971-80

Gorga, JC et al., 1987. J. Biol .. Chem. 262: 16.087-16.094. Heemels, MT et al., 1995. Annu Rev. Biochem. 64: 463-491 Kern F et al., 2,000. Eur. J Immun 30: 1,676-1,682

Kern F et al., 1999. J. Virol. 73 (10): 8,179-8,184 25

Klein, J. 1986. Natural History of the MHC, John Wiley and Sons (editorial).

Kronenberg M et al., 1999. Immunol Today 20: 515-21.

Kwok WW et al., 2001. Trends Immunol. 22 (11): 583-588.

Lalvani A et al., 1997. J. Exp. Med. 186 (6): 859-865.

Liang TJ et al., Ann Intern Med. February 15, 2,000; 132 (4): 296-305. 30

Maecker HT et al., 2001. J Immunol Methods 255: 27-40

Nijman HW et al., 1993. Eur. J. Immunol., 6, 1,215-9

Novak N et al., 2001. J. Immunol. 166: 6,665-6,670.

Parker, KC et al., 1994. J. Immunol. 152: 163.

Rammensee, HG et al., 1999. Immunogenetics 50: 213-219

Stern LJ et al., 1994. Structure 2: 245-251

Sturniolo T et al., 1999. Nature Biotechnology 17: 555-562.

Tobery WT et al., 2001 J. Immunol. Methods 254: 59-66. 5

Valli A et al., 1993, J. Clin. Invest. 91: 616-628

Van der Erynde BJ, et al., 1997. Curr Opin Immunol. 5: 684-93

Villadangos JA et al., 2,000. Immunity 12: 233-239

Ward S et al., Clin Exp Immunol. May 2,002; 128 (2): 195-203.

Wilson DB et al., 1999. J. Immunol. 163: 6.424-6.434. 10

Claims (19)

1. Hepatitis C virus (HCV) vaccine comprising at least the following three peptides:

a) AYAAQGYKVLVLNPSVAAT or a part thereof containing at least one epitope and

b) GEVQVVSTATQSFLATCINGVCWTV or a part thereof containing at least one epitope and

c) HMWNFISGIQYLAGLSTLPGNPA or a part thereof containing at least one epitope. 5

2. Vaccine against HCV according to claim 1, characterized in that it comprises the following peptides:

GYKVLVLNPSVAAT, DLMGYIPAV, CINGVCWTV and HMWNFISGIQYLAGLSTLPGNPA 10

3. Vaccine against HCV according to claim 1 or 2, characterized in that it comprises the peptide KFPGGGQIVGGVYLLPRRGPRLGVRATRK.

4. Vaccine against HCV according to claim 1, characterized in that it comprises the DLMGYIPAV peptide.

5. Vaccine against HCV according to claim 1, characterized in that it comprises the CINGVCWTV peptide as an epitope containing part of GEVQVVSTATQSFLATCINGVCWTV. fifteen

6. Vaccine against HCV according to claim 1, characterized in that it comprises the peptide HMWNFISGIQYLAGLSTLPGNPA.

7. Vaccine against HCV according to claim 1, characterized in that it comprises the peptide GYKVLVLNPSVAAT as an epitope containing part of AYAAQGYKVLVLNPSVAAT.

8. Vaccine against HCV according to any one of claims 1 to 7, characterized in that it also contains an immunostimulatory peptide comprising a sequence R1-XZXZNXZX-R2, in which N is an integer between 3 and 7, preferably 5 , X is a positively charged natural and / or unnatural amino acid residue, Z is an amino acid residue selected from the group consisting of L, V, I, F and / or W and R1 and R2 are independently selected from each other in the group consisting of -H, -NH2, -COCH3, -COH, a peptide with up to 20 amino acid residues or a peptide reactive group or a peptide linker with or without peptide; X-R2 may be an amide, ester or thioester of the C-terminal amino acid residue of the peptide ("Peptide A").

9. Vaccine against HCV according to any one of claims 1 to 8, characterized in that it also contains an immunostimulatory oligodeoxynucleic acid (ODN) molecule with the structure according to formula (I).

 30 in which: R1 is selected from hypoxanthine and uracil, any X is O or S, any NMP is a 2 'deoxynucleoside monophosphate or monothiophosphate, selected from the group consisting of deoxyadenosine monophosphate or monothiophosphate, deoxyguanosine, deoxyinosine, deoxycytosine, deoxyuridine, deoxythymidine, 2-methyl-deoxyinosine, 5-methyloxidine, 5-methoxy deoxydosin, 5-methyloxidine, deoxydosin, 5-methyloxidine, deoxydosin, 5-methyloxidine, deoxydosin, 5-methyloxidine deoxydosin, 5-methyloxydine, desoxydosin, 5-methyloxydosine 2-amino-deoxyribosapurin, 6-S-deoxyguanine, 2-dimethyl-deoxyguanosine or N-isopentenyl-deoxyadenosine, NUC is a 2 'deoxynucleoside, selected from the group consisting of deoxyadenosine, deoxyguanosine, deoxyinosine, deoxycytosine, deoxyinosine, deoxythymidine, 2-methyl-deoxyuridine, 5-methyl-deoxycytosine, deoxypseudouridine, deoxyribosaporeir, 2-amino-deoxidine, 6-amino-deoxidine deoxyguanine, 2-dimethyl-deoxyguanosine or N-isopentenyl-deoxyadenosine, 10 a and b are whole numbers from 0 to 100 with the proviso that a + b is between 4 and 150 and B and E are common groups for the 5 'or 3' ends of nucleic acid molecules ("I- / U-ODN").

10. Vaccine against HCV according to any one of claims 1 to 9, characterized in that it also contains an Al (OH) 3 adjuvant.

11. Vaccine against HCV according to any one of claims 1 to 10, characterized in that it also contains an immunostimulatory polycationic peptide.

12. Vaccine against HCV according to any one of claims 8 to 11, characterized in that said Peptide A is KLKL5KLK.

13. Vaccine against HCV according to any one of claims 9 to 12, characterized in that said I- / U-ODN is oligo d (IC) 13. twenty

14. Vaccine against HCV according to any one of claims 1 to 13, characterized in that it also contains an immunostimulatory oligodeoxynucleotide containing a CpG motif.

15. Vaccine against HCV according to any one of claims 1 to 14, characterized in that it is lyophilized in a form that is reconstitutable in 15 min at 37 ° C.

16. Vaccine against HCV according to any one of claims 1 to 15, characterized in that it contains between 20 µg and 10 mg of each peptide.

17. Vaccine against HCV according to any one of claims 1 to 16, characterized in that it is lyophilized and contains traces of acetic acid.

18. Use of a vaccine according to any one of claims 1 to 17, for the preparation of a medicament for the prevention and treatment of an HCV infection. 30

19. Method for preparing a vaccine according to any one of claims 1 to 17, characterized by the following steps:

- chemically synthesizing the at least three peptides as defined in claims 1 to 17,

- solubilize these peptides by an aqueous solution containing at least one organic acid selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid and halogenated or hydroxylated forms thereof,

- mix the solubilized peptides and

- optionally lyophilize the mixed peptides.