WO2011042551A1

WO2011042551A1 - Method of generating hcv-derived virus-like particles

Info

Publication number: WO2011042551A1
Application number: PCT/EP2010/065127
Authority: WO
Inventors: Matthias Bureik; Calin-Aurel Dragan; Anette Dragan
Original assignee: Pombiotech Gmbh
Priority date: 2009-10-09
Filing date: 2010-10-08
Publication date: 2011-04-14
Also published as: DE102009044224A1

Abstract

The present invention refers to a method of producing HCV derived virus-like particles (VLPs) from recombinant yeast cells, particularly fission yeast cells.

Description

METHOD OF GENERATING HCV-DERIVED VIRUS-LIKE PARTICLES

FIELD OF THE INVENTION

The present invention refers to a method of producing virus-like particles (VLPs) from recombinant yeast cells, particularly fission yeast cells.

BACKGROUND OF THE INVENTION

The prevention of diseases by vaccination is without question one of the most significant medical achievements of mankind. Vaccines currently prevent more than 3 million deaths per year, and the positive economic impact is in excess of a billion dollars per year.

Nevertheless, the success of recombinant vaccines is always accompanied by safety issues due to possible reversion, recombination or mutation. Additionally, very often there are efficiency issues, such as that either recombinant but also traditional approaches have not always been effective in the treatment or prevention of disorders like chronic infections or cancer. One reason may be that the treatment of said disorders may require the induction of potent cellular immune responses to facilitate clearance of infected or neoplastic cells. Another reason may be that a still more specific selection of key antigens is needed, particularly for targeting neoantigens on tumors.

Therefore, there is a need for new development of vaccines and also novel platform technologies as carriers for such vaccines or simply for the production of such vaccines.

One interesting strategy is the use of virus-like particles (VLPs) as non-infectious particles but still immunogenic vaccines. VLPs are composed of viral structural proteins that retain the ability to self- assemble without requiring the presence of the viral genome: they have been shown to be highly immunogenic and may avoid most of the above-mentioned safety issues (Noad & Roy, 2003) .

It has been previously shown that the ability of VLPs to stimulate an adaptative immune response depends on several properties:

(i) the average diameter of VLPs (< 0.05 μπι), which is optimal for uptake of the VLPs by dendritic cells (Fifis et l, 2004);

(ii) efficient activation of the antigen presenting cells (APCs) (Lenz etal, 2003);

(iii) induction ofCD8⁺ activation by a cross-priming mechanism (Ruedl etal., 2002); and

(iv) potent stimulation of a B-cell mediated response by direct cross-linking of the BCR on B cells. This latter property was first demonstrated in studies using organic polymers decorated with haptens, which proved that 20-25 haptens spaced by 5—10 nm were enough for T-cell- independent B- cell activation (Dintzis etal, 1976; ond etal., 1995).

To date, two VLP vaccines, against Hepatitis B Virus (HBV) infection and Human Papilloma Virus (HPV) infection, have been shown to efficiently function in humans. This success causes a new expectation and demonstrates the need for still new developments of vaccines, particularly VLP- based vaccines to other viral pathogens.

For example, the production of Hepatitis C Virus (HCV)-VLPs is generally accomplished by using mammalian cell culture methods. In this case, transiently transfected mammalian cells are producing the proteins that subsequently form VLPs by self-assembly which then bud into the medium. However, the use of mammalian cell culture methods bears the risk of contamination and is quite cumbersome.

Accordingly, there is continuous research for the development of new efficient, easy-to handle and cost-saving production systems for VLPs, which do overcome the above mentioned drawbacks. As a result, during the last couple of years it could be shown that unicellular organisms like fungus and yeast can, in principle, be used for the production of VLPs. For instance, the HBV-S protein was found to self-assembly into VLPs when expressed in Aspergillus niger (Plueddemann& Zyl, 2003). Furthermore, successful chimeric HBV/HEVVLP formation could also be observed in the Pichia pastoris (Li etal, 2004). The same yeast was extensively used for studying the expression of HCV proteins, however only intracellular assembly and no budding could be observed (Acosta-Rivero et l, 2001).

A major drawback of the described uses of funguses and yeasts for the production of VLPs is that in the used expression systems the VLPs are not secreted from these unicellular organisms. Accordingly, a method to produce VLPs from yeast requires several labour-intensive steps for breaking the cells as well as isolating and purifying the VLPs from the protein extract. This has also been reported likewise for Saccharomyces cerevisiae cells but also Schizosaccharomyces pombe cells. Saccharomyces cerevisiae and Schizosaccharomyces pombe are two well-characterized and efficiently growing genus of yeast, which were found to efficiently produce some virus-like particles, but which did not release or secrete the particles. For example, Sasagawa etal. ( 995) reported an experimental set up to produce HPV virus-like particles using S. pombe. While they were able to demonstrate expression of the virus proteins by S. pombe, no export of particles into the supernatant was described. Accordingly, VLPs needed to be purified from the whole protein extraction, which still bears the burden and risk of contaminating remains in the purified particle extraction and also particle destruction.

It has been suggested that the outer cell wall of yeast cells interferes with particle formation and secretion. Thus, there was a development of establishing methods, which use sphaeroblasts without an outer cell wall. This is supported by Sakuragi et al. (2002) describing the budding of HIV- 1 gag particles from sphaeroblasts of Saccharomyces cerevisiae after the removal of the cell walls. The procedural step of removing the cell wall, however, involves enzymatic treatment and, thus, modifies not only the yeast cells but also the virus-like particles.

Unfortunately, the lack of suitable expression is a well-known phenomenon in many well- established expression system such as e.g. S. cerevui or Picbia pastoris. Without clear explanation it is often found that some proteins are better expressed than others, which might not be expressable or even detectable at all. Reasons for this are multiple and without being bound by the theory, it is believed that particularly the presence of cell specific microRNAs might be involved in the failure to successfully express or translate certain proteins. Other scientist favour the theory that the presence of cell specific proteins or enzymes might be involved in a rapid degradation of the protein of interest, having the consequence that no expressable protein can be detected.

It follows from the above that particularly the production of functional HCV VLPs seems difficult and consequently, there is still a need to provide improved expression systems and methods suitable for the production of HVC virus-like particles.

OBJECT OF THE INVENTION

In the light of the above it is an object of the invention to provide an alternative method and an alternative expression system for the production of VLPs, especially HCV-derived VLPs, which avoids modification of the virus-like particles due to purification steps and at the same time is cost effective, easy to handle and provides competitive yields of VLPs. It is a further object of the invention to provide HCV-derived VLPs for the use as diagnostical tools, as carrier molecules or as vaccines.

SUMMARY OF THE INVENTION

The object of the invention is achieved by the method according to claim 1, which comprises the use of recombinant yeast, which is capable of functionally expressing the structural genes needed for the self-assembly of a HCV-derived virus-like particle and which recombinant yeast is cultured under suitable conditions for the production of virus-like particles, wherein the method comprises the isolation of the virus-like particles from the supernatant of the cultured yeast cells.

For this the method of production of HCV derived virus-like particles (VLPs) comprises the steps of using of a recombinant yeast cells, which is capable of expressing the structural genes needed for the self-assembly of a virus-like particle,

culturing of the recombinant yeast under suitable conditions,

separating the yeast cells and cellular fragments from the supernatant, then

optionally, treating the yeast cells fraction or retentate with a detergence or sonication and again separating the treated yeast cell fraction or retentate from the supernatant, and

isolating of the virus-like particle from the supernatant of the recombinant yeast culture.

The invention comprises - iter alia— a method as above wherein the detergence is sodium dodecyl sulfate; further, a method as any of the above wherein the recombinant yeast is transformed with at least one copy of a vector encoding and capable of expressing the structural genes needed for the self-assembly of HCV derived virus-like particle; furthermore, a method as any of the above wherein the recombinant yeast is stably transformed by the insertion of at least one copy of a vector encoding and capable of expressing the structural genes needed for the self-assembly of HCV derived virus-like particle; furthermore, a method as any of the above wherein the structural genes needed for the self-assembly of a virus-like particle are selected from the group consisting of structural genes of hepatitis C virus (HCV); furthermore, a method as any of the above wherein the structural genes needed for the self-assembly of a virus-like particle are selected from the group consisting of structural genes HCV-core protein, HCV-subtype la-core protein (again la genotype) and HCV E1E2 protein; furthermore, a method as any of the above wherein the structural genes comprise and encode a fusion gene between a structural gene selected from the group consisting of structural genes hepatitis viruses and an heterologous gene; furthermore, a method as any of the above wherein the recombinant yeast is selected from at least one of the parent strains of the group consisting of Schizosaccharomyces ombe, Schizosaccharomyces octosporus, Schizosaccharomyces japonicus, Schizosac- charomyces kambucha, and Schizosaccharomyces cryophilus.

Furthermore, the the invention comprises— iter alia— the use of recombinant Schizosaccharomyces cells for the production of secreted HCV-derived virus-like particles; further the use as above wherein the recombinant Schizosaccharomyces is stably or transiently transformed with at least one copy of a vector encoding and capable of expressing the nucleic acid sequence encoding the structural genes or derivatives of the structural genes ofjiepatitis C virus (HCV) selected from the group containing structural genes for HCV-core protein, HCV-subtype la-core protein (again la genotype), HCV E1E2 protein, combinations thereof and derivatives thereof, which are needed for the self-assembly of a virus-like particle; furthermore, the use as any of the above wherein the Schizosac- charomyces is selected from at least one of the strains of the group consisting of Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces japonicus and Schizosaccharomyces kambu- cha.

Additionally, the invention comprises isolated HCV-derived virus-like particles, which have been expressed by and secreted of recombinant Schizosaccharomyces into the supernatant and which have been isolated from the supernatant of said Schizosaccharomyces cell culture; isolated HCV-derived virus-like particles obtained by the method as above, said virus-like particles essentially consisting of HCV-core, HCV-subtype la-core and/or HCV E1E2 protein; the use of the isolated HCV-derived virus-like particles as any of the above in the manufacture of a medicament for the treatment or prophylaxis of a virus infection or an HCV infection; the use of the isolated HCV-derived virus-like particle as any of the above in a kit comprising the isolated virus-like particles and at least one container.

SHORT DESCRIPTION OF THE FIGURES

FIGURE 1 shows the processing scheme for the different sample preparation of supernatants from fission yeast strains expressing HCV proteins at different incubation conditions.

FIGURE 2 shows CORE signals in the retentate of medium supernatant of fission yeast cells after ultrafiltration through a 100 kDa cutoff membrane detected by the CORE ELISA

FIGURE 3 shows CORE signals in the supernatant of fission yeast sphaeroblasts detected by the CORE ELISA. Sphaeroblasts were generated as described under Example 1.

FIGURE 4 shows CORE signals in the pellet fraction of fission yeast sphaeroblast supernatants after centrifugation through 20% sucrose cushions detected by the anti-CORE ELISA

FIGURE 5 shows CORE signals in the top 1 ml volume of fission yeast sphaeroblast supernatants after sucrose cushion centrifugation.

FIGURE 6 shows CORE signals detected in the retentate of sphaeroblast supernatant ultrafiltration through a 100 kDa cutoff membrane by centrifugation.

FIGURE 7 shows CORE signals detected in the flow- thro ugh of sphaeroblast supernatants gained by ultrafiltration through a 100 kDa cutoff membrane. FIGURE 8 shows CORE signals detected in the 1% SDS incubated retentate of sphaeroblast supernatants gained by ultrafiltration through a 100 kDa cutoff membrane.

FIGURE 9 shows CORE signals detected in the flow-through of 1% SDS incubated sphaeroblast supernatants gained by ultrafiltration through a 100 kDa cutoff membrane.

FIGURE 10 shows HCV VLPs from the supernatant of CAD100 monitored by electron microscopy.

DETAILED DESCRIPTION OF THE INVENTION

It has been reported that VLP signals were completely missing in the supernatants of recombinant yeast cells, while they were readily detectable in the supernatant of yeast sphaeroblast. The difference might appear not quite surprising at the first glance, since, it is known that yeast secretory proteins tend to get stuck in the "periplasmic space" (Moreno et al., 1985; Schweingruber et al., 1986).

Another explanation is that if the yeast expresses viral proteins with a theoretical mass of, for example, about 22 kDa, which is the size of the HCV Core protein, and assembles the expressed proteins into VLPs with an assumed composition of about 200 protein molecules per particle then, such an assembly would be difficult to secrete through the yeast cell wall. This would particularly hold true for enveloped VLPs since their diameter would be even greater. Under this assumption, a facilitated secretion would accordingly only be expected in the case of sphaeroblasts, as shown e.g. for the secretion of HrV-gag VLPs from S. cerevisiae sphaeroblasts (Sakuragi et al., 2002).

However, and against previous prejudice is now the achievement of the inventors to provide a new and surprisingly efficient expression system, namely a different a recombinant yeast for the method for producing virus-like particles (VLP), wherein the VLPs can be isolated from the supernatant of the cell culture of said recombinant yeast.

The "yeast cells" according to the method of the invention is selected from the fission yeast genus, from which it is known that they are evolutionary very distant and thus distinct from S. cerevisiae. Although contradictory reports in the literature, the inventors could show that recombinant fission yeast is actually capable of expressing HCV structural proteins and allows virus-like budding of the VLPs or even actively secrets VLPs.

According to a preferred embodiment the parent strains for generation of the recombinant yeast used in the method of the invention is Schizosaccharomyces. Further preferred the Schizosacckaromyces is selected from the group containing the strains of Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces japonicus, Schizosaccharomyces kambucha, and Schizosaccharomyces cryophilus or combinations thereof.

The term "virus-like particles" herein means a virus like structure consisting of viral protein(s) derived from the structural genes i.e. proteins of said virus. In some cases these proteins are embedded within a lipid bilayer. Such particles resemble the virus from which they were derived but lack viral nucleic acid, meaning that they are not infectious.

The method of the invention uses recombinant yeast cells, which are capable of expressing structural genes of a virus, which are needed for the self-assembly of VLPs.

The term "structural genes of a virus" herein means and includes genes that control the production of a specific protein or peptide, which is needed to built the stabilising or supporting structure of a virus, i.e. needed for the self-assembly of the virus particle, which packages— under normal conditions - the nucleic acid material of the virus as well as regulatory factors or proteins, which are necessary for proper virus propagation. This stabilising and supporting structure is often also referred to as the capsule, the CORE or the capsid structure of a virus. The term "structural genes" herein means and includes also modified structural genes, which originate from a selected virus, but which comprise nucleic acid rearrangements, exchanges, deletions or insertions. Furthermore, the term includes fusion genes between the structural genes of a selected virus and at least one heterologous gene.

The term "self-assembly" herein means and refers to the fundamental principle, which generates structural organization in virus propagation. Generally, the structural genes expressed according to the invention have a tendency to self assemble.

According to the method of the invention the recombinant yeast is cultivated under suitable conditions, which in general are known to the skilled person. In brief, suitable conditions for the cultur- ing of the recombinant yeast comprise medium composition, pH, temperature, incubation period, agitation speed, etc. Under said suitable conditions the recombinant yeast of the invention is capable to secrete and/or export the self-assembled VLPs to the supernatant, from where they will be isolated according the method of the invention.

Since yeast cell in culture are normally non adhesive, it is advisable for isolating the VLPs from the supernatant of the recombinant yeast culture to separate in a first step the intact yeast cells and also any yeast cell fragments from the culture medium e.g. by established technologies such as filtration or centrifugation. The application of filters or filter membranes as well as the applicable speed for a centrifugation step must be adjusted to the expected particle size, in order to avoid also separating the virus-like particles from the supernatant.

Additionally, to increase the efficiency the yeast cell culture, the retentante (and analogously: permeate or flow-through) of the filtration or the pellet of the centrifugation is treated with either a detergence or treated by sonication, preferably ultrasonication, to allow and enforce disintegration of clotted particles or particles clotted to cellular structures.

Subsequently, the virus-like particle, which have been segregated and/or released into the supernatant of the yeast cell culture, are isolated according to the method of the invention e.g. by sucrose cushion centrifugation and ultracentrifugation of the supernatant according to standard protocols.

According to one embodiment the method of the invention uses recombinant yeast cells, which have been transformed with a vector carrying and capable of expressing the heterologous genes, such as the structural genes of a virus.

The term "vector" herein comprises DNA vehicles of circular or linear structure, such as DNA fragments, plasmids, cosmids or artificial chromosomes, which in addition to the desired nucleic acid sequence may contain regulatory sequences, selective marker genes and replicons enabling the autonomous replication of the vector. Hence, the vector according to the present invention can easily be amplified in a unicellular host organism, such as yeast, but can also be isolated from said unicellular host organism.

According to a further embodiment the vector used for generating recombinant yeast useful in the method of the invention is pCADl, an integrative vector (Dragan et i, 2005), or pREPl, an auto- somally replicating vector (Maundrell, 1993).

According to the invention the vector is used to deliver the desired structural genes, which are needed for the self-assembly of VLPs, into a yeast cell. The transformation of the yeast cells are stable under selective conditions. The transformation can be stable due to an integration of the vector or parts of the vector including the heterologous genes into the yeast genome. Typically, but not limiting, integration, i.e. insertion of the vector, occurs due to recombination between homologous sequences of the vector and the yeast genome. In this case only one expression cassette per cell is present. - Si -

According to one embodiment of the invention the vector pCADl is used, which integrates into the leul locus of chromosome II and cannot be lost even under non-selective conditions. Without being bound to the theory, it is believed that the chromosome II is a particular suitable locus for integration of structural virus genes, as the transcription activity of chromosome II is very high. It further seems that the integration of the recombinant genes into the Leul locus positively influences and promotes the secretion of the VLPs. It is unclear whether this effect is due to the high transcription rate of the chromosome II in the recombinant yeast organism and/or a metabolic change, due to the interruption of the Leul locus in the recombinant yeast organism. In any case the increased expression of the recombinant protein allows secretion into the medium of yeast cells.

In case of the autosomal replicating vector (pREPl) one or more copies of the vector are present in the cytoplasm of the recombinant yeast cell. Typically, the vectors used have the capacity to auto- somally replicate, which leads to recombinant yeast strains, which do carry many vector molecules and thus many expression cassettes for the desired nucleic acid sequence, namely the heterologous genes such as the structural genes of a virus. It is believed that as in yeast cells, which are recombinant by said autosomal vector and therefore show a high expression rate of the structural viral genes, due to this high expression also secretion into the medium can be found. Accordingly, it is believed — without being bound by the theory - that an increased amount of recombinantly expressed protein in the recombinant cell is helpful for the method of the present invention and thus the secretion of VLPs into the medium.

According to a further embodiment of the present invention the structural genes needed for the self- assembly of VLPs are selected from the structural genes of viruses of the family of hepatitis viruses. According to still a further embodiment the structural genes are selected from the genes of Hepatitis C Virus (HCV), preferably from the group consisting of the genes encoding HCV-CORE protein, HCV-subtype la-CORE protein and HCV E1E2 protein.

As will be demonstrated in more details in the examples, the method of the invention allows the production of VLPs with recombinant fission yeast cells. While without the step of treating the retentate or pellet fraction of the fission yeast cell culture according to the invention the detectable amount of VLPs (ELISA Assay as described in the Example) in the supernatant is rather low (Fig. 2), the yield of the treated retentate or fraction can be increased by at least a factor 20, preferably a factor 40, more preferably a factor 80, more preferably a factor 100 by treating the retentate or pellet fraction of the fission yeast cell culture with a suitable detergent (Fig 8) . Alternatively, the retentate or pellet fraction of the fission yeast cell culture can also be treated by sonication, preferable ultrasonication. It seems that Schizosaccharomyces is surprisingly well suited for the method of the invention. This is most probably due to the evolutionary distance of the genus Schizosaccharomyces from other yeast genus. It can be speculated that the structure of the outer cell wall is different and due to this difference particularly well suited for the method of the invention.

As demonstrated, the isolated recombinant Schizosaccharomyces of the invention and the method according the invention are surprisingly efficient in producing and releasing VLPs into the supernatant of yeast cell culture and thus provide an advantageous improvement in the production methods for VLPs. This holds true particularly if comparing the method of the invention to previously known methods working with sphaeroblasts.

A comparison of the method of the invention - as illustrated also in the examples - to standard methods reveals the following: The ELISA analysis carried out on the pellet and the supernatant fractions of fission yeast cell culture expressing HCV structural proteins according to the example shows for instance, that the pellet fraction (Fig. 4) and the supernatant fraction (Fig. 5) of strain CAD 102 after sucrose centrifugation clearly reflect the theoretical distribution of fully assembled VLPs. Before the centrifugation there was a CORE signal present in the medium while after centrifugation the CORE signal appears nearly solely in the pellet fraction. Although not so accentuated, the same happens to CORE signals from CAD 103. Strain CAD 100 produces CORE protein that, according to the sucrose cushion centrifugation results, was mainly concentrated in the pellet fraction (Fig. 4) but shows a small fraction still present in the supernatant. From time requirements in theory it can be judged that most of the CORE particles are probably arranged in greater aggregates otherwise no precipitation could have been observed. The remaining CORE signal in the pellet fraction of CADIOO can be explained in two different ways. Either due to the longer pellet time of enveloped CORE VLPs, some of the particles could have remained suspended in the supernatant or some of the CORE subunits did not form the VLP structure and, therefore, were not completely removed from the supernatant.

Clearly, a 100 kDa membrane filtration retained all of the CORE signals from CAD 102 and CAD103 samples (compare CAD102 and CAD103 signals in Fig. 6 to signals in Fig. 7). Since the CORE protein with a molecular mass of about 22 kDa should have easily passed the membrane, it is highly probable that it was kept in high mass aggregates which is just another hint towards the particle nature of CORE secretion in strains CAD102 and CAD103.

In samples of strain CADIOO a significant portion of CORE signals remained in the flow-through (Fig. 7). It was hypothesized in the upper section whether CORE could have been monomeric in order to explain signals still existing in the supernatant after sucrose cushion centrifugation. Regarding the here mentioned CORE signals in the flow-through fraction of the 100 kDa membrane rather point towards monomeric CORE.

When retentate from the ultrafiltration was incubated in 1% SDS and centrifuged again through a 100 kDa membrane an astonishing signal increase could be detected in the ELISA system. The increase was present in samples from all CORE expressing strains but was especially dramatic for strain CAD 102 (Fig. 8). Thereby, signals from the processed retentate from parental strain MB 163 remained at the same level as shown by the retentate of the sphaeroblast supernatant ultrafiltration. Moreover, when the flow-through of the retentate fractions was analyzed, the signal intensities were nearly exactly the same as for the supernatant flow-through of the ultrafiltration carried out in absence of 1% SDS (compare Fig. 6 with Fig. 9). Applying a 1% SDS treatment to retentate samples from yeast cells with intact cell walls rendered the CORE signals for the first time detectable by the ELISA system. However, the signals were not strong compared to cells lacking the wall (data not shown) .

The 100 kDa retention results with SDS treated samples show a complete depletion of CORE signals in the flow-through to background level (compare depletion in Fig. 7 to Fig. 9). Possibly, treatment of fission yeast made HCV or even native HCV particles by SDS sheds off the envelope assembly and/or makes CORE epitopes more accessible without disrupting the inner icosahedrons. This could explain why CORE signals are still retained by the membrane after SDS treatment. Similar to the flow-through analysis of CAD 100 samples not treated with SDS (Fig. 7) CORE can still be detected after treatment with SDS in the flow-through (Fig. 9) again backing our hypothesis of incomplete VLP formation. Finally, when retentate medium samples were analyzed an increase of CORE signal could be detected, thereby, demonstrating direct VLP secretion by intact fission yeast cells into the medium.

Summarizing the above method according to the invention is particularly useful in producing VLP s and allows isolating a considerably increased yield of VLPs directly from the supernatant of a yeast cell culture.

According to one embodiment the method of the invention is used to produce and isolate VLPs consisting of one or more of the structural proteins needed for the self-assembly of a virus-like particle deriving from the group of viruses comprising flaviviruses, retroviruses and hepatitis viruses.

For this the method of the invention uses a recombinant yeast which comprises and encodes the structural genes needed for the self-assembly of a virus-like particle deriving from the group of vi- ruses comprising flaviviruses, retroviruses and hepatitis viruses.

According to a further alternative embodiment the recombinant yeast comprises and encodes a fusion gene between a structural gene deriving from the selected from the group of viruses comprising flaviviruses, retroviruses, hepatitis viruses as well as combinations thereof fused - preferably in frame - to at least one additional heterologous gene. This heterologous gene may encode any antigen of interest, or any immunogenic protein or peptide, which one would like to present in the context of

According to a preferred embodiment the selected structural genes derive from the structural genes of hepatitis C virus (HCV) and are preferably selected from the group consisting of the structural genes encoding the HCV-core protein, HCV-subtype la-core protein (again the l a genotype) and HCV E1E2 protein.

According to a still preferred embodiment the recombinant yeast used in the method of the invention is selected from strains of the group consisting of Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces japonicus, Schizosaccharomyces kambucha, and Schizosaccharomyces cryophilus. The use of recombinant Schizosaccharomyces cells for the production of secreted virus-like particles is particularly advantageously due to the evolutionary based differences in comparison the Saccharomyces strains. Without being bound to the argument, it is believed that particularly the intracellular enzymatic composition or microRNA reservoir is quite distinct between Schizosaccharomyces and Saccharomyces, and thus, the capability to functionally express certain proteins can not be compared or predicted.

According to one embodiment the recombinant Schizosaccharomyces is transformed with a least one copy of a vector encoding and capable of expressing the structural genes needed for the self- assembly of a virus-like particle as described above. Particularly, according to a further embodiment the recombinant Schizosaccharomyces is capable of expressing the structural genes of hepatitis C virus (HCV) , preferably the structural genes encoding the HCV-core protein, HCV-subtype la-core protein (again la genotype) and HCV E1E2 protein.

According to still another embodiment the invention provides isolated virus-like particles (VLPs) , which have been expressed and secreted into the supernatant by the recombinant Schizosaccharomyces and which have been isolated from the supernatant of said Schizosaccharomyces cell culture.

The isolated virus-like particles obtained by the method according to one embodiment of the invention are virus-like particles essentially consisting of HCV-core, HCV-subtype la-core and/or HCV E1 E2 protein or combinations thereof.

Interestingly it was shown that the VLPs produced and isolated from the supernatant and/or pellet according to the present invention were immunologically detected by heterologous patient serum. These results prove that the VLPs according to the invention are useful to detect and therefore also to induce HCV specific antibodies. Accordingly the isolated virus-like particles according to the invention are Useful in the manufacture of a drug for the treatment or prophylaxis of a virus infection. In case of HCV VLPs these are particularly useful in the manufacture of a drug for the treatment or prophylaxis of an HCV infection. Thus the VLPs according the invention could be used as vaccine for the treatment and/or prophylaxis of an HCV infection.

A suitable medicament comprising the isolated VLPs of the invention can be formulated by containing only the VLPs in a pharmaceutically acceptable buffer or solution such as water or e.g. phosphate buffered saline. However, also every other to the skilled person known and suitable additive, carrier, diluent or excipient for either e.g. oral, intramuscular or intravenous application can be used for the formulation of this medicament or a corresponding vaccine.

Furthermore, the isolated virus-like particles according to the invention are provided in a kit comprising the isolated virus-like particles and at least one container. Such kit is useful for many applications including but not limited to clinical diagnostics, veterinarian diagnostics and the immunization of animals such as rabbits or mice or camelidae for the production of antibodies, but also humans for vaccination purposes. Additionally, the isolated virus-like particles according to the invention are useful for diagnostic imaging. For this purpose, one of the structural proteins may be fused in frame to a heavy metal chelating protein structure like phytochelatins or iso-peptides. Moreover, in vitro quantum dot fusions or dotting VLPs with adequate contrasting substances may be a viable way.

EXAMPLES

EXAMPLE 1: GENERATION OF HCV VIRUS-LIKE PARTICLES USING FISSION YEAST

CORE RNA source and purification

The source for the amplified cDNAs of the CORE and the Ε1Ε2 fusion protein of HCV la was the human serum #22057 from the patient serum bank of the Klinik fur Innere Medizin II - Gastroen- terologie, Hepatologie, Endokrinologie, Diabetologie und Ernahrungsmedizin. The extraction of HCV la RNA was done using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.

Vectors For cloning of amplified viral coding sequences, the pCR TOPO XL vector from Invitrogen (Carlsbad, CA, USA) was used. For expression in fission yeast the integrative vector pCAD 1 (Dragan et al., 2005) and the autosomally replicating vector pREPl (Maundrell, 1 93) were chosen.

RT-PCR of CORE

The amplification of HCV cDNA was preceded by a RT-PCR. The mixture for first RT-PCR step was 3 pi HCV RNA from the above RNA extraction, 1.5 μΐ random hexamer primers (Invitrogen, Carlsbad, CA, USA) and 0.5 ul RNAse free water. The mixture was incubated for 10 min at 65°C and immediately transferred on ice. The second step mixture was 7.5 pi RNAse free water, 5 pi template from RT step 1, 4 μΐ 25 mM MgCl₂, 4 μΐ 10 mM dNTPs (Invitrogen, Carlsbad, CA, USA), 2.5 lOx PCR reaction buffer + MgCl₂ (Roche, Basel, Switzerland), 1 μΐ RNaseOUT recombinant ribonuclease inhibitor 40 U ul-1 (Invitrogen), 1 μΐ Superscript II reverse transcriptase. The mixture was incubates at 42°C for 60 min and subsequently transferred on ice.

Amplification of cDNAs and cloning

Since fission yeast does not seem to possess a signal peptide peptidase (Weihofen et al., 2002), it was intended to express HCV components not as a part of the native poly-protein but from distinct expression cassettes. Therefore, the HCV protein will be synthesized as NH₂-CORE-ExportSignal- COOH (protein 1) and for the envelope proteins without p7 as NH₂-ExportSignal-El-E2-COOH (protein 2) .

The reverse transcribed cDNA were subjected to a PCR reaction using a modified Expand Long Template Kit (Roche). The PCR mixture was composed as follows: 27 μΐ RNAse free water, 6 μΐ from the second RT step as template in case of CORE amplification, 1 μΐ plasmid M289+22057 in case of the E1E2 polyprotein, 5 μΐ 10 mM dNTPs (Invitrogen), 5 μΐ of lOx Expand PCR buffer, 1.5 ul 25 mM MgCl₂, 1 μΐ DMSO, 1 μΐ Expand enzyme mix with 3.5 U ul-1. The primers used in case of CORE were:

5'- CAT ATG AGC ACG AAT CCT AAA CCT CAA -3' (primer 1; SEQ ID NO.: 1)

5'- GGA TCC TCA GGC TGA CGC GGG CAC AGT CAG -3' (primer 2; SEQ ID NO.: 2)

The primers used in case of E1E2 were:

5'- CAT ATG GAC CTC ATG GGG TAC ATA CCG CTC -3' (primer 3; SEQ ID NO.: 3) 5'- GGA TCC TCA CTC CGC TTG GGA TAT GAG TTG -3' (primer 4; SEQ ID NO.: 4) The first primer pair amplifies the HCV CORE with a 5 '-terminal Ndel and a 3'-terminal BamHI restriction site. An additional terminator codon was inserted by primer 2 after the ER localization sequence. Envelope proteins El and E2 were amplified by using primer 3 and 4 as a fusion construct with leading CORE ER localization sequence, 5'-terminal Ndel and 3'-terminal BamHI sites. The PCR thermal program was

The correct size of the PCR product was determined by agarose gel electrophoresis. The DNA was purified from the gel and cloned into the pCR TOPO XL vector (Invitrogen). The CORE containing cDNA sequence was designated as CORE while the E1E2 containing sequence as ENV for envelope proteins.

Sequencing of HCV cDNAs

Sequencing of the HCV CORE cDNA and HCV-pre-seq.-El/E2 cDNA was carried out using the ABI Prism 3100 Genetic Analyzer and the BigDye Terminator vl.l Cycle Sequencing Kit. The sequencing PCR mixture composition was 7.5 ul water, 2 pi BigDye solution, 2 μΐ pCR TOPO XL vector and 0.5 ul primer. The thermal program was:

The primers in case of CORE were standard M13 forward and reverse while in case of E1E2 additional internal primers were used (El-257-2s, HVRl-inner sense, E2-lb-6a). Expression plasmid construction

The pCR TOPO XL vectors bearing the CORE and ENV sequences were subjected to

Ndel/BamHI double restriction and separated from the vector by gel electrophoresis. After DNA isolation from agarose gels, the CORE and ENV sequences were cloned in both, pCADl and pREPl, yielding the expression plasmids pCADl-CORE, pCADl-ENV, pREPl-CORE and pREPl-ENV.

Fission yeast strain construction

The fission yeast strain NCYC2036 (MB163) with genotype h- ura4. ll8was used as the starting point for the generation of HCV protein expressing strains. Cryocompetent MB163 cells were prepared as described (Suga and Hatakeyama, 2005) and were transformed with either pCADl-CORE or pCADl-ENV generating the fission yeast strains CADIOO and CADIOI. Testing for correct chromosomal integration of the pCADl plasmids was performed by plating colonies on EMM medium Petri dishes containing phloxine B. The presence of the desired cDNAs was additionally checked by colony PCR using primers 1, 2, 3 and 4.

The second strain generation step intended to add the additional HCV components in order to coexpressing all crucial VLP proteins. Strain CADIOO was, therefore, transformed with pREPl- ENV while strain CADIOI was transformed with pREPl-CORE using the lithium acetate method (Okazaki et al., 1990) in both cases. The constructed fission yeast strains are listed in Table 2.

Table 2: Generated fission yeast strains expressed expression cassettes per strain parent replication type

protein cell

CAD 10

NCYC2036 CORE chromosomal 1

0

CAD 10

NCYC2036 ENV chromosomal 1

1

CAD 10

CAD I OO CORE chromosomal 1

2

ENV autosomal many

CAD 10

CAD I O I ENV chromosomal 1

3

CORE autosomal many Production of HCV proteins by fission yeast

Fission yeast strains MB163, CADIOO, CADlOl, CAD102 and CAD103 were spread on EMM plates containing either 0.01% uracil for MB163 or 0.01% leucine for CADIOO and CADlOl. Thiamine was present in all EMM agar media at a concentration of 5 μΜ in order to keep HCV expression repressed during initial colony growth at 30°C. A 10 ml EMM preculture with amino acid supplements but no thiamine was then made after three days of colony growth on plates and incubated at 30°C for 24 h and 150 rpm. Finally, bio mass was produced in a 100 ml EMM culture (30°C 24 h, 150 rpm) in absence of thiamine and in presence of the required amino acids.

The cells were then spun at 3000^ for 5 min and washed twice with ZymDig buffer (50 niM

NaH2P04/Na2HP04, pH = 8, 2% glucose, 16% sucrose) and resuspended in 4 ml ZymDig. Zymolyase 20T purchased from ICN Biomedicals (Aurora, OH, USA) was added at 20 mg mL-1 and the cell suspension was incubated at 30°C for 24 h at 150 rpm. Zymolyase 20T is an enzyme mixture from Arthrobacter luteus with the essential activity a-l,3-glucan laminaripentaohydrolase which is responsible for cell wall degradation and sphaeroblast formation. The growth medium from the biomass production was stored at 4°C with 1 mM PMSF and 1 mM DTE added.

Afterwards, the cells were centrifuged (3000^, 5 min) and the supernatant was discarded due to the high concentration of Zymolyase 20T. Three washing steps with ZymDig were performed resus- pending the cells again in 4 ml ZymDig and incubated (30°C 150 rpm) overnight. Finally, the cell suspension was centrifuged (3000^, 5 min) and the supernatant was stored at 4°C with added 1 mM PMSF and 1 mM DTE.

Sample preparation

The processing scheme for the different supernatants gained is shown in Fig. 1. The growth medium supernatants were designated as "M" samples while the samples stemming from the second incubation in ZymDig buffer were named "S" samples. The second letter indicates the location in the processing scheme.

Unprocessed samples from M and S samples for all strains were stored as such (samples MA or SA, see Fig. 1). An amount of 1.5 ml supernatant was centrifuged at 10⁵gfor 1 h through a 10 ml 20% sucrose cushion. Sample "B" denotes a sample taken from the top of the liquid after centriftigation while sample "C" is the PBS resuspended pellet. Another 2 ml of supernatant were centrifuged through a 100 kDa cutoff ultrafiltration membrane at 10 g for 1 h yielding the flow-through (sample "D") and the retentate (sample "Ε")· The retentate itself was subjected to an incubation with 1% SDS for 30 min and again passed through a 100 kDa membrane under the same conditions as above. Samples from this last step were designated as "F" and "G" for flow-through and retentate, respectively.

Protein concentration measurement

For the determination of total protein the BCAassay from Pierce (Rockford, IL, USA) was used with minor changes. Briefly, the total volume of the measured solution was 200 μΐ composed as follows: 100 ul 1:40 dilution of solution B into A and either 50 μΐ from a bovine serum albumin calibration row or a 1:10 diluted supernatant sample processed as described. The mixture was incubated for 1 h at room temperature in a microtiter plate (MTP) and then measured at λ = 595 nm in a Tecan Genios MTP reader (Geneva, Switzerland) .

Detection of HCV CORE

The presence of HCV CORE in supernatants from fission yeast incubations was analyzed with the antibody to HCV CORE antigen ELISA test system from Ortho-Clinical Diagnostics (Raritan, NJ, USA) according to the manufacturers recommendations. The ELISA detection kit was a qualitative, diagnostic test, where special restraints were recommended in order to assure reliable and clinically relevant results. Since we used the system to detect successfully the presence of CORE, although we used the absorption values without the quality assurance recommendations. Nevertheless, normalization of the data was done on the basis of total protein present in the samples in order to detect specific CORE signals. Furthermore, no concentration dependent calibration could be carried out, therefore, it is unknown whether there is a linear relationship between CORE concentration and ELISA signal. Samples D, E, F and G from strain CADI 01 expressing only envelope proteins were omitted from analysis due to ELISA signals ranging in the background of the parental strain MB163.

Fission yeast strains expressing HCV proteins

All constructed fission yeast strains (Tab.2) were subjected to a colony PCR analysis showing the presence of the cDNAs of either CORE or ENV or both. For the construction of VLP secreting strains, two genetic combinations were chosen. The parent strains were either expressing CORE or ENV from an integrative vector (only one expression cassette per cell) and had either ENV or CORE cDNAs added on autosomally replicating vectors (many expression cassettes per cell) in order to genetically complete the VLP structural component information within one strain. This was mainly done to analyze which ratio of cDNAs is suited for a significant VLP production. Strains CAD 100 and CADI 01 showed normal growth compared to wildtype MB 163 while CAD 102 showed a significantly slower growth. The phase-contrast microscopic phenotype of fully induced strains showed, irregularities of strains CAD 100 and CAD 102 consisting of an increase in intracellular bodies and altered cell shapes.

CORE is secreted in the medium of recombinant fission yeast

Looking for CORE signals in the MA samples revealed weak but detectable signals related to the parent strain MB 163. Sucrose cushion centrifugation did not improve the signal strength. However, when concentration with 100 kDa membranes was carried out, a difference could be detected between the parent strain and HCV CORE expressing strains CADIOO and CAD102 (Fig. 2).

CORE is secreted by recombinant fission yeast sphaeroblasts

After sphaeroblast generation by Zymolyase 20T, the cells were incubated for further 24 h in Zym- Dig buffer. Supernatants from these incubations (SA samples) were directly analyzed by CORE ELIS A revealing a significant signal in the supernatants of strains CADIOO, CAD 102 and CAD 103 (Fig. 3). Only an insignificant signal could be detected in the parental strain MB 163 and in the ENV expressing strain CADlOl. ELISA signals of the CORE overexpression are much stronger for sphaeroblasts supernatants than found in medium supernatants of cells. However, treatment of medium supernatants with 1% SDS after retention by 100 kDa cutoff membranes and recapture reveals strong signals in the CORE expressing strains CADIOO and CAD 102 compared to the parent strain MB 163 (see Fig. 10).

Sucrose cushion centrifugation

Centrifugation through a 20% sucrose cushion is a widespread technique for the concentration of VLPs from supernatants of producing cell culture systems. We analyzed the pellet fraction and, additionally, the top 1 ml volume after centrifugation in order to look for HCV CORE. The pellet fraction was resuspended in 0.5 ml PBS, therefore, a roughly 3 times concentrated solution could be expected.

First, the pellet fraction ELISA signals clearly improved for strain CADIOO and CAD102 compared to non-processed supernatant (Fig. 3) reflecting the concentration tendency in the pellet fractions (Fig. 4). For strain CAD103, no improvement but rather a loss of signal could be detected. Absence of CORE expression (parent strain MB163 and strain CADlOl) led to a slight increase but to overall insignificant results, possibly due to the concentration of the background agent acting in the ELISA system. Where significant signals were present in the SA samples, a clear drop in signal intensity could be observed in the top volume after centrifugation indicating the depletion of CORE (Fig. 5). The CORE signal is retained by 100 kDa molecular sieves

When supernatants from sphaeroblast incubations in ZymDig buffer were passed through a 100 kDa ultrafiltration membrane by centrifugation, CORE ELISA signals were retained by the membrane (Fig. 7). However, a concentration effect could not be reliably detected. For strain CAD 103, again, a slight loss in signal intensity was perceived. Equally interesting, only in the case of CAD 100 (overexpresses only CORE), the ELISA system detected its presence also in the flow-through where for all other CORE expressing strains (CAD102 and CAD103) this was not the case (Fig. 6). Setting the ELISA signal from the parental strain MB163 as background underlines the fact that the flow- through was completely devoid of HCV COREs in samples from strains CAD102 and CAD103 (compare signals in Fig. 6 to signals in Fig. 7) .

Signal increase after incubation in 1% SDS

Only 0.3 ml of retentate from the ultrafiltration were incubated in 1% SDS and centrifuged again through a 100 kDa membrane. An astonishing signal increase could then be detected in the ELISA system. The increase was present in samples from all CORE expressing strains but was especially dramatic for strain CAD 102 (Fig. 8). Thereby, signals from the processed retentate from parental strain MB 163 remained at the same level as shown by the retentate of the sphaeroblast supernatant ultrafiltration. Moreover, when the flow-through of the retentate fractions was analyzed, the signal intensities were nearly exactly the same as for the supernatant flow-through of the precedent ultrafiltration (compare Fig. 6 with Fig. 9). Increased signals could also be detected in the medium supernatant samples from intact fission yeast cells by processing in the same manner (see Fig. 10) .

REFERENCES

Acosta-Rivero, N., J. C. Aguilar, A. Musacchio, V. Falcn, A Via, M. C. de la Rosa, and J. Morales (2001, Sep). Characterization of the hcv core virus-like particles produced in the methylo- trophic yeast pichia pastoris. Biochem Biophys Res Commun 287(1), 122—125.

Dintzis, H.M., Dintzis, R.Z. & Vogelstein, B. Molecular determinants of immunogenicity: the immunon model of immune response. Proc Natl Acad Sci USA. 73, 3671-3675. (1 76).

Dragan, C.-A., S. Zearo, F. Hannemann, R. Bernhardt, and M. Bureik (2005, Apr). Efficient conversion of 11-deoxycortisol to Cortisol (hydrocortisone) by recombinant fission yeast schizosaccharomyces pombe. FEMS Yeast Res 5(6-7) , 621-625.

Fifis, T. et al. Size-dependent immunogenicity: therapeutic and protective properties of nano- vaccines against tumors. / Immunol. 173, 3148-3154. (2004). Lenz, P. etal. Interaction of papillomavirus virus-like particles with human myeloid antigen- presenting cells. Clin Immunol. 106, 231-237. (2003).

Li, H.-Z., H.-Y. Gang, Q.-M. Sun, X. Liu, Y.-B. Ma, M.-S. Sun, and C.-B. Dai (2004, Jun). Production in pichia pastoris and characterization of genetic engineered chimeric hbv/hev viruslike particles. Chin Med SciJ 19(2), 78-83.

Maundrell, K. (1993, Jan). Thiamine-repressible expression vectors prep and prip for fission yeast.

Gene 123(1), 127-130.

Milich, D.R. & McLachlan, A The nucleocapsid of hepatitis B virus is both a T-cell- independent and a T-cell-dependent antigen. Science. 234, 1398-1401. (1986).

Mond, J.J., Lees, A & Snapper, C.M. T cell-independent antigens type 2. Annu Rev Immunol. 13, ,655-692. (1995).

Moreno, S., T. Ruiz, Y. Sanchez, J. R. Villanueva, and L. Rodriguez (1985, Sep). Subcellular localization and glycoprotein nature of the invertase from the fission yeast schizosaccharomyces pombe. Arch Microbiol 142(A), 370-37 '4.

Noad, R. & Roy, P. Virus-like particles as immunogens. Trends Microbiol. 11, 438-444. (2003).

Okazaki, K., N. Okazaki, K. Kume, S. Jinno, K. Tanaka, and H. Okayama (1990, Nov). High- frequency transformation method and library transducing vectors for cloning mammalian cdnas by trans-complementation of schizosaccharomyces pombe. Nucleic Acids Res 18(22), 6485-6489.

Plueddemann, A and W. H. V. Zyl (2003, Sep). Evaluation of aspergillus niger as host for virus-like particle production, using the hepatitis b surface antigen as a model. Curr Genet 43(6), 439- 446.

Ruedl, G, Storni, T., Lechner, F., Bachi, T. & Bachmann, M.F. Cross-presentation of virus-like particles by skin-derived CD8(-) dendritic cells: a dispensable role for TAP. Eur J Immunol. 32, 818-825. (2002).

Sakuragi, S., T. Goto, K. Sano, and Y. Morikawa (2002, Jun). Hiv type 1 gag virus-like particle budding from sphaeroplasts of saccharomyces cerevisiae. Proc Natl Acad Sci USA 99(12), 7956-7961.

Sasagawa, T., P. Pushko, G. Steers, S. E. Gschmeissner, M. A Hajibagheri, J. Finch, L. Crawford, and M. Tommasino (1995, Jan). Synthesis and assembly of virus-like particles of human papillomaviruses type 6 and type 16 in fission yeast schizosaccharomyces pombe. Virology 206(1), 126-135.

Schweingruber, A M., F. Schoenholzer, L. Keller, R. Schwaninger, H. Trachsel, and M. E.

Schweingruber (1986, Jul). Glycosyiation and secretion of acid phosphatase in schizosaccharomyces pombe. Eur J Bio ch em 158(1), 133—140.

Suga, M. and T. Hatakeyama (2005, Jul). A rapid and simple procedure for high-efficiency lithium acetate transformation of cryopreserved schizosaccharomyces pombe cells. Yeast 22(10), 799— 804.

Claims

C L A I M S

Method for the production of HCV derived virus-like particles (VLPs) comprising

- the use of a recombinant fission yeast cell, which is capable of expressing the structural genes needed for the self-assembly of a virus-like particle,

- the culturing of the recombinant yeast under suitable conditions,

- the separation the yeast cells and cellular fragments from the supernatant,

- optionally, the treatment of the yeast cells fraction or retentate with a detergence or soni- cation, and the separation of the treated yeast cell fraction or retentate from the supernatant, and

- the isolation of the virus-like particle from the supernatant of the recombinant yeast culture.

Method according to claiml , wherein the recombinant yeast is selected from at least one of the parent strains of the group consisting of Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces japonicus, Schizosaccharomyces kambucha, and Schizosaccharomyces cryophilus.

Method according to claim 1 or 2, wherein the detergence is sodium dodecyl sulfate.

Method according to any of the claims 1 to 3, wherein the recombinant yeast is transiently transformed with at least one copy of an expression vector encoding and capable of expressing the structural genes needed for the self-assembly of HCV derived virus-like particle.

Method according to any of the claims 1 to 3, wherein the recombinant yeast is stably transformed by the insertion of at least one copy of an expression vector encoding and being capable of expressing the structural genes needed for the self-assembly of HCV derived virus-like particle.

Method according to claim 5, wherein the recombinant yeast is stably transformed by the insertion of at least one copy of an expression vector into chromosome II and/or the leul locus.

Method according to any of the claims 1 to 6, wherein the structural genes needed for the self-assembly of a virus-like particle are selected from the group consisting of structural genes of hepatitis C virus (HCV), HCV-core protein, HCV-subtype la-core protein (again la genotype) and HCV E1E2 protein.

8. Method according to any of the claims 1 to 7, wherein the structural genes comprise and encode a fusion gene between a structural gene selected from the group consisting of structural genes of HCV and an heterologous gene.

9. Use of recombinant Schizosaccharomyces cells for the production of secreted HCV-derived virus-like particles.

10. Use according to claim 9, wherein the recombinant Schizosaccharomyces is stably or transiently transformed with at least one copy of a vector encoding and capable of expressing the nucleic acid sequence encoding the structural genes or derivatives of the structural genes of hepatitis C virus (HCV) selected from the group containing structural genes for HCV-core protein, HCV-subtype la-core protein (again la genotype), HCV E1E2 protein, combinations thereof and derivatives thereof, which are needed for the self-assembly of a virus-like particle.

1 1. Use according to any of the claims 9 to 10, wherein the Schizosaccharomyces is selected from at least one of the strains of the group consisting of Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces japonicus, Schizosaccharomyces kambucha, and Schizosaccharomyces cryophilus.

12. Isolated HCV-derived virus-like particles, which have been expressed and secreted of recombinant Schizosaccharomyces into the supernatant and which have been isolated from the supernatant of said Schizosaccharomyces cell culture.

13. Isolated HCV-derived virus-like particles obtained by the method according to any of the claims 1 to 8, said virus-like particles essentially consisting of HCV-core, HCV-subtype la- core and/or HCV E1E2 protein.

14. Isolated HCV-derived virus-like particles according to the claims 12 and 13 for use in the treatment and/or prophylaxis of a hepatitis virus infection or an HCV infection.

15. Use of the isolated HCV-derived virus-like particle according to the claims 12 and 13 in a kit comprising the isolated virus-like particles and at least one additional container.