DE102009044224A1 - Method for production of HCV virus-like particles - Google Patents

Abstract

The present invention describes a method for the production of HCV-based virus-like particles (VLPs) with recombinant yeast cells, in particular with fission yeasts.

Description

Field of the invention

The present invention describes a method for the production of virus-like particles (VLPs) with recombinant yeast cells, in particular with fission yeast cells (Schizosaccharomyces).

background

The invention of vaccine against infectious diseases is without question one of the biggest milestones in the history of medicine. At present, vaccinations prevent more than 3 million deaths each year. From an economic perspective, this means savings of more than a billion dollars a year.

However, the success of a genetically engineered vaccine always depends on safety considerations for possible mutational and recombination events. Moreover, especially for chronic diseases and cancers, there are often no effective therapies or even vaccines that can cure or prevent the disease. One reason for this could be that strong and effective induction of the immune response is necessary to control and cure infected or neoplastic cells. Another reason could be that even more specific selection of key antigens is needed, especially for labeling neoantigens on tumors.

For these reasons, the development of new vaccines and technology platforms as carriers of such vaccines or simply for the production of such vaccines is of great importance.

An interesting starting point is the use of virus-like particles (VLPs) as non-infectious particles but still immunogenic vaccines. VLPs consist of viral structural proteins that self-assemble into a virus-like host cell in a host cell To assemble particles without including nucleic acids in their interior: it has been shown that they are highly immunogenic and could bypass most of the safety aspects mentioned above ( Noad & Roy, 2003 ).

• i. ein mittlerer Durchmesser der VLPs (< 0,05 μm), der optimal ist für die Aufnahme der VLPs durch dentritische Zellen ( Fifis et al., 2004 )
• ii. eine effiziente Aktivierung der Antigen-präsentierenden Zellen (APCs) ( Lenz et al., 2003 );
• iii. eine Aktivierung von CD8 positiven cytotoxischen Killerzellen ( Ruedl et al., 2002 ) und
• iv. eine potente Stimulation der B-Zell vermittelten Immunantworten durch Aktivierung des B-Zellrezeptors auf der Oberfläche von B-Zellen.

Regarding the immunogenicity of VLPs, it has recently been shown that stimulation of the adaptive immune system by VLPs is dependent on several factors:

• i. a mean diameter of the VLPs (<0.05 μm) which is optimal for the uptake of VLPs by dendritic cells ( Fifis et al., 2004 )
• ii. an efficient activation of the antigen-presenting cells (APCs) ( Lenz et al., 2003 );
• iii. an activation of CD8-positive cytotoxic killer cells ( Ruedl et al., 2002 ) and
• iv. potent stimulation of B cell mediated immune responses by activation of the B cell receptor on the surface of B cells.

The latter observation was first demonstrated in studies using organic polymers whose surface was filled with haptens. It could be shown that 20-25 haptens arranged at a distance of 5-10 nm were sufficient to induce T cell independent B cell activation ( Dintzis et al., 1976 ; Moon et al., 1995 ).

At present, two VLP-based vaccines are approved for the prevention of hepatitis B infection and human papilomavirus infection. The success of these vaccines has raised new expectations in this area and demonstrated the importance of developing new vaccines, particularly the development of VLP-based vaccines against other viral pathogens.

The production of hepatitis C virus (HCV) VLPs takes place, for example, in mammalian cell cultures. Here, transiently transfected mammalian cells express the viral structural proteins that self-assemble into VLPs and then secreted into the cell medium. However, the use of mammalian cell cultures is a very expensive and time-consuming process and also carries the risk of contamination.

For these reasons, there is continuous research for the development of new, inexpensive and time-saving techniques for VLP production to avoid the above-mentioned disadvantages. As a result, it has been shown in recent years that unicellular organisms such as fungi and yeasts in principle to VLP production can be used. For example, the hepatitis B S protein forms particles when it is niger expressed in the yeast Aspergillus ( Plueddemann & Zyl, 2003 ). In addition, one could successful production of chimeric VLPs consisting of hepatitis B and hepatitis E virus proteins in the yeast Pichia pastoris ( Li et al., 2004 ). The production of HCV VLPs was also intensively tested with this yeast, but produced VLPs of this organism are only formed intracellularly and are not secreted into the culture medium ( Acosta-Rivero et al., 2001 ).

A major disadvantage of the described use of fungi and yeasts for VLP production is the lack of secretion of the VLPs. Accordingly, non-secreted VLPs must be obtained by several isolation steps from within the host cells. This has been described for both the yeast Saccharomyces cerevisiae and the yeast Schizosaccharomyces pombe. Saccharomyces cerevisiae and Schizosaccharomyces pombe are two very well-characterized yeasts that can efficiently produce but not release or secrete some VLPs. Sasagawa et al. (1995) For example, the production of HPV VLPs could be demonstrated with the yeast Schizosaccharomyces pombe, but they could not be detected in the culture medium of the yeasts. Consequently, the particles had to be laboriously isolated, which also poses a risk of contamination of the VLP fractions and always entails a loss of volume.

It is believed that the outer cell wall of yeast cells hinders particle formation id secretion. For this reason, a method has been developed in which spheroplasts that have no outer cell wall are produced. This method is also used by Sakuragi et al. (2002) used to secrete HIV particles from cells of the yeast Saccharomyces cerevisiae after the cell wall is removed. To remove the cell wall, enzymatic cell wall degradation must be performed, but this modifies both yeast cells and VLPs. Unfortunately, the lack of suitable expression rates is a well-known phenomenon in many established expression systems, such as, e.g. In the yeast Saccharomyces cerevisiae or Pichia pastoris. For reasons that are unexplainable, some proteins are expressed better than other proteins, which may not be expressible or even undetectable. There are a few possible reasons for this phenomenon: it is believed that specific microRNAs may be involved in a mis-expression or mis-translation of various proteins. Other scientists believe that different proteins or enzymes are involved in rapid degradation of the recombinant proteins. This has the consequence that the desired protein is no longer detectable.

For all these reasons it can be seen that the production of VLPs with yeast cells is difficult and that improved expression systems and suitable methods for VLP production are needed. This is particularly true for the production of functional HCV VLPs using recombinant microorganisms.

Subject of the invention

For the reasons given above, the invention described herein provides an alternative method and an alternative expression system for the production of VLPs, particularly HCV VLPs. This is a cost-effective, time-saving and simple process in which modification of the VLPs is avoided by complex isolation steps and yet can be produced competitive amounts of VLPs. It is a further object of the invention to use HCV VLPs for diagnostic purposes, as carrier molecules and as a vaccine.

Summary of the invention

The object of the present invention is achieved by the method according to claim 1, which comprises the use of recombinant yeast strains which can functionally express the structural genes needed for the self-assembly of HCV-based virus-like particles, and the culture this recombinant yeast under suitable culture conditions for the production of virus-like particles. Furthermore, this method involves the isolation of the virus-like particles from the supernatant from the cultured yeast cells.

The presented method of HCV VLP production is based on the use of recombinant yeast strains expressing the HCV structural proteins that self-assemble into VLPs intracellularly. The yeast cells are cultured under suitable conditions and then cells and cell fragments are separated from the culture medium. Optionally, the yeast cells can be treated with a detergent or sonicated and separated again from the cell supernatant. Subsequently, the isolation of the VLPs from the cell culture supernatants takes place.

For the purposes of this invention, sodium dodecyl sulfate is used as a detergent to treat the cells. Further, this invention encompasses a method in which the recombinant yeast strains were stably transformed with at least one vector encoding the genetic information for expression of the HCV structural proteins needed to form HCV VLPs. Furthermore, the invention encompasses a method in which the structural genes underlying the intracellular formation of HCV VLP originate from the group of structural genes of the HCV genome. This group consists of the following structural genes: HCV core (gene type 1a), HCV envelope (HCV E1, gene type 1a) and HCV envelope2 (HCV E2, gene type 1a). Furthermore, in this method, the structural genes as fusion genes, consisting of genes of the group of HCV structural genes and a heterologous gene. Furthermore, this method comprises recombinant yeast strains derived from at least one of the following parental strains: Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces japanicus and Schizosaccharomyces kambucha.

Furthermore, the invention encompasses, inter alia, the use of recombinant fission yeasts (Schizosaccharomyces) for the production and secretion of HCV VLPs. Furthermore, the use of fission yeasts transiently or stably transformed with at least one vector as described above. This vector encodes the cDNA needed for expression of the HCV structural proteins. The genetic information encoded by the vector comes from the group of structural genes of the HCV genome which are HCV core (gene type 1a), HCV E1E2 (gene type 1a), combinations of these genes or derivatives thereof which are needed to form HCV VLPs. In addition, the method comprises fission yeasts (Schizosaccharomyces) which are derived from at least one of the following parental strains: Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces japanicus and Schizosaccharomyces kambucha.

Furthermore, the invention encompasses isolated HCV VLPs which are produced by recombinant fission yeasts and secreted into the cell supernatant and which are subsequently isolated from the medium. HCV VLPs obtained by this method consist of HCV core proteins (gene type 1a), HCV core proteins (type 1a) and / or HCV E1 / E2 proteins (type 1a). One use of the HCV VLPs generated by this method is the development of a medicament for treating or preventing viral infections or as part of a kit.

Brief description of the pictures

shows the schematic representation of the workup of individual samples from the culture supernatant of Spalthefestämmen expressing the HCV proteins under different culture conditions.

shows core concentrations in culture supernatant of fission yeasts measured by HCV-Core ELISA. The samples studied were concentrated using a membrane that separates molecules to a molecular weight of 100 kDa.

shows core concentrations in the culture supernatant of spheroplasts measured by HCV-Core ELISA.

shows core concentrations measured after centrifugation using a 20% sucrose cushion in the sediments using HCV-Core ELISA.

shows core concentrations in the culture supernatant of spheroplasts, which were quantified after centrifugation with the aid of a 20 percent sucrose pad using HCV core ELISA.

shows core concentrations in the culture supernatant of spheroplasts quantitated by HCV-Core ELISA. The samples studied were concentrated using a membrane that separates molecules to a molecular weight of 100 kDa.

shows core concentrations in the filtrate of the spheroplast supernatant, which has passed through a membrane with a permeability to molecules <100 kDa.

shows core concentrations in the culture supernatant of spheroplasts treated with 1% SDS. The samples studied were concentrated using a membrane which separates molecules with a wipe> 100 kDa.

shows core concentrations in the filtrate of the spheroplast supernatant treated with 1% SDS. The filtrate has passed through a membrane with a permeability to molecules <100 kDa.

shows an electron micrograph of HCV VLPs in the culture supernatant of the untreated strain CAD100.

Detailed description of the invention

It has already been described in the literature that yeast-based VLPs are not detectable in the culture medium of the producing cells, but only after the yeast cell wall has been enzymatically degraded. This observation is not very surprising at first glance, since it is also known that secretory yeast proteins remain in the periplasmic space between the outer cell wall and the inner cell membrane ( Moreno et al., 1985 ; Schweingruber et al., 1986 ).

Another explanation for a lack of secretion is the size of the VLPs: Assuming that the core proteins with a molecular wipe of 22 kDa to VLPs, composed of about 200 proteins per particle together, resulting protein aggregates, the cell wall due to their Size can not happen. This would be especially true for enveloped VLPs whose diameter is even greater. Based on this assumption, secretion would only be expected by the production of spheroplasts, as has already been shown for the secretion of HIV-VLPs by the yeast Saccharomyces cerevisiae ( Sakuragi et al., 2002 ).

Contrary to all preconceptions, the present inventors present a novel and surprisingly efficient expression system, namely another recombinant yeast for the production of virus-like particles (VLPs) using the method presented here, the VLPs being isolated from the supernatant of the cell culture of the said recombinant yeasts can be.

The yeast used in this method belongs to the genus of fission yeasts, which is evolutionarily remote from other and the yeast Saccharomyces cerevisiae and therefore significantly different from this. In contrast to some papers published so far, the inventors were able to show that recombinant fission yeasts are able to express HCV structural proteins and allow or even actively secrete virus-like release of the particles.

In a preferred embodiment, the yeast species is Schizosaccharomyces for the method of producing HCV VLPs described in this invention. This comes from the group of the following strains: Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces japonicus and Schizosaccharomyces kambucha or combination thereof.

The term "virus-like particles" is based on the virus-like structure of the particles that are assembled from the viral structural proteins of the corresponding viruses. For some viruses, these proteins are embedded in a double lipid layer. Such virus-like particles are generally non-infective because they do not pack nucleic acids inside you.

The method described in this invention is based on the use of recombinant yeast strains expressing viral structural proteins encoded by structural genes. The expressed viral structural proteins are necessary for self assembly of the VLPs.

The term "viral structural gene" means and encompasses those genes which are responsible for the production of specific proteins or peptides necessary for the formation of the particle structure normally surrounding nucleic acids and regulatory elements in its interior. This structure is often referred to as capsid, core or capsid structure. The term "viral structural genes" also includes and includes modified structural genes derived from a desired virus but which include alterations, nucleic acid exchange, deletions or insertions. The term "viral structural genes" means and also includes fusion genes consisting of structural genes of a desired virus having at least one heterologous gene.

The term "self-assembly" of the virus particles in the context of this invention refers to those steps involved in the propagation of the virus for intracellular organization of the proteins for formation of complex three-dimensional virus particles. In general, structural proteins tend to self-assemble.

According to the present invention, recombinant yeast cells are cultured under suitable conditions. Suitable conditions include medium composition, pH, temperature, culture time, stirring rate, etc. Under suitable conditions, the yeast is able to secrete and / or export the produced and self-formed VLPs into the culture medium, from which they are determined by the method described in this invention be isolated.

Since yeast cells do not grow adherent, the intact yeast cells or Zellfrakmente must first be separated from the culture medium z. B by a filtration or centrifugation step. The use of suitable filters or the speed of centrifugation must be adjusted depending on the size of the VLPs produced in order to avoid simultaneous separation of the VLPs from the medium.

In addition, the efficiency of the present method can be increased by either treating the culture supernatant, retentate samples, filtrate samples, or the cell or spheroplast sediments after centrifugation with a detergent or sonicated, coherent or aggregated Particles or particle cell structures are isolated.

Subsequently, secreted or exported VLPs as described in this invention are isolated from the yeast culture medium, e.g. B. by a sucrose cushion centrifugation and / or ultracentrifugation according to the standard protocol.

According to one embodiment of the present invention, recombinant yeast cells are used which have been transformed with a vector which inhibits the expression of heterologous proteins, such as e.g. B. the viral structural proteins encoded.

The term "vector" describes linear or circular DNA transport vehicles, such as. As DNA fragments, plasmids, cosmids or artificial chromosomes, which may include in addition to the desired nucleic acid and regulatory sequences, selection marker genes and replicons (for autonomous propagation of the vector). Therefore, the vectors used in this invention can be propagated in a unicellular organism such as the yeast cell and also isolated from these organisms.

According to the present invention, for the production of recombinant yeast strains, the integrative vector pCAD1 ( Dragan et al., 2005 ) and the autosomal replicating vector pREP1 ( Maundrell, 1993 ). The vector pCAD1 integrates into the leu1 locus of chromosome II and is not lost even under non-selective conditions.

In the case of the autosomal replicating vector (pREP1), one or more copies of the vector are present in the cell. Due to the autosomal propagation of the vector in the cell, the recombinant yeast strains contain many copies of the vector and thus many copies of the expression cassette with the desired nucleic acid, here the genes for the expression of the viral structural proteins.

Alternatively, transformation may also be by integration of the vector or parts thereof into the yeast genome as a stable transformation. Typically, but not exclusively, integration occurs through recombination events between homologous sequences of the vector and the yeast genome. In these cases, only the genome-integrated expression cassette is present.

According to another embodiment of the present invention, the structural genes necessary for self-assembly of the VIPs are derived from hepatitis viruses. According to the present invention, the structural proteins are derived from hepatitis C virus (HCV), preferably from the following group of genes: HCV core protein (genotype 1a) and HCV E1 / E2 (gene type 1a).

As shown in detail in the examples, the method underlying this invention is for the production of VLPs with recombinant fission yeasts. While the amount of core proteins to be detected (by Elisa technology, described in the example) despite concentration in the cell supernatants of fission yeasts is very low ( ), so the amount can be reduced by at least a factor of 20, 40 or 80 or even by a factor of 100, by previously processing the cells into spheroplasts and processing the samples (sucrose cushion centrifugation, filtration), by pre-processing the samples with a suitable detergent ( ), be increased. Alternatively, the retentate fractions or sedimented cell fractions can be treated by sonication, preferably by ultrasound.

It appears that the fission yeast (Schizosaccharomyces) is surprisingly well suited for the method on which this invention is based. This is most likely due to the fact that fission yeast differs evolutionarily from other yeast genera. It is believed that the difference between the genus of yeast exists in the structure of the outer cell wall and that this difference contributes to the fission yeast being useful as a host organism for VLP production described in this invention.

As shown, fission yeasts are remarkably well-suited for efficient production and release of the VLPs into the cell culture supernatant, and this represents a significant improvement over conventional VLP production methods. This is particularly true for methods that also used to prepare spheroplasts becomes.

A comparison of the method underlying this invention - as also demonstrated in the examples - with standard methods gives the following:
Detection of HCV core proteins in the sediments ( ) and cell culture supernatants ( ) of the yeast strain CAD102, obtained after a sucrose-pad centrifugation, very clearly show the formation of VLPs on the basis of the theoretical distribution of complex virus particles. Before centrifugation, core proteins are detectable in the culture medium, while after centrifugation core proteins are detectable almost exclusively in the sediments. The same applies approximately to the examined samples of the yeast strain CAD103. The strain CAD100 produces only core proteins, which are mainly detected in the sediments after a sucrose cushion centrifugation ( ), while also a low concentration in the supernatant is detectable. Based on the theoretically calculated sedimentation time, it can be assumed that most of the core proteins form VLPs, otherwise sedimentation of the core proteins would not occur. The detection of core proteins in the supernatant can be explained as follows: It is possible that some core particles are enclosed by the host cell membrane and therefore have different sedimentation conditions or are single core proteins that do not form protein aggregates.

Filtration using a membrane permeable to molecules <100 kDa in molecular weight shows that core proteins are detectable only in the retentate samples of strains CAD102 and CAD103 (see signals from both strains in FIG and ). Since single core proteins with a molecular mass of 22 kDa would have passed through the membrane, this also provides an indication of the formation and secretion of VLPs with the yeast strains CAD102 and CAD103.

In samples of strain CAD100, a significant proportion of core proteins pass through the membrane. The results of the sucrose cushion centrifugation suggest that monomeric core proteins are present. This assumption can also be confirmed by the filtration result.

When retentate samples from the filtration are treated with 1% SDS and then filtered again, there is an astonishing increase in core concentration. This increase can be seen in all core-expressing strains and especially in the strain CAD102 ( ) to be watched. Signal strength measured in the MB163 control strain is not affected by SDS treatment. Moreover, the core signal strength within the fractions that has passed through the membrane is also not significantly altered by SDS treatment (cf. With ). If retentate samples from yeast cells with an intact cell wall are treated with SDS, a core signal can be detected for the first time using ELISA technology.

The results of treatment of retentate specimens of the spheroplast supernatant with SDS show that hardly any core proteins have passed through the membrane (cf. With ). SDS treatment may lead to the dissolution of virus envelopes and / or make the epitopes of core proteins more readily accessible for antibody detection without, however, disrupting their structure. This could explain why core proteins remain in the retentate after SDS treatment. An SDS treatment of the strain CAD100 shows, analogously to the experiments without SDS treatment ( ) that core proteins are allowed to pass through the membrane ( ). This also confirms our hypothesis that not all core proteins occur as VLPs but also in monomeric form.

Finally, electron micrographs of the culture medium of the strain CAD100 confirmed the secretion of VLPs ( ).

In summary, the method of this invention is particularly suitable for producing VLPs and for isolating significant amounts of VLP directly from the culture supernatant.

According to this invention, its underlying method is suitable for producing and isolating VLPs which consist of one or more structural proteins necessary for the assembly of VLPs. The VLPs can be derived from the group of flaviviruses, retroviruses, hepatitis viruses and in particular from the hepatitis C virus. For this method, yeast strains containing the structural genes needed for VLP production and derived from the group of flaviviruses, retroviruses and hepatitis viruses are used.

Alternatively, the yeast strains may contain fusion genes consisting of the structural genes of the above-mentioned selected group or combination of these genes or fusion genes of a structural protein of said group with at least one further heterologous gene.

The recombinant yeast strains used in this method can be from the group Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces japonicus and Schizosaccharomyces kambucha. The use of recombinant fission yeasts for the production of selected VLPs may be advantageous because of their evolutionary differences to the yeast Saccharomyces. In particular, the intracellular enzyme composition or the supply of microRNAs between both yeast species are believed to be very different so that the ability to functionally express various proteins is not comparable or predictable.

According to this invention, recombinant fission yeasts are transformed with at least one vector encoding the structural genes needed for the formation of VLPs (as described above). In particular, the recombinant fission yeasts are able to produce structural proteins of HCV, in particular the proteins HCV core (gene type 1a) and HCV E1 / E2 (gene type 1a).

The invention described provides a method for producing isolated VLPs which are produced by recombinant fission yeasts and secreted into the culture supernatant, from which they can subsequently be isolated. The VLPs isolated with the method described in this invention consist essentially of HCV core proteins (Gentype 1a) and / or HCV E1 / E2 proteins (Gentype 1a) or combinations thereof.

According to this invention, the isolated VLPs are useful for the preparation of a medicament for the treatment or prevention of viral infections. In the case of HCV VLPs, these are particularly suitable for developing a therapeutic or preventive vaccine.

A suitable drug comprises the isolated VLPs in a suitable buffer or solution, such as, e.g. As water or phosphate buffer. However, other suitable additives, carrier molecules, diluents or excipients for, for example, oral, intramuscular or intravenous applications may be used for the preparation of the medicament or vaccine.

Furthermore, the VLPs isolated according to the present invention are used as components of a kit containing the isolated VLPs and at least one vessel. Many applications of such a kit are conceivable and include, but are not limited to, diagnostic tests for human and veterinary medicine or the immunization of animals such as rabbits, mice, rats ... for the production of antibodies as well as for the immunization of humans. Furthermore, the VLPs isolated according to this invention are used as imaging methods in diagnostics. For these purposes, one of the structural proteins is fused to a heavy metal. Furthermore, VLPs can be loaded with contrast agents, for example.

Examples

EXAMPLE 1: PREPARATION OF HCV VLPs WITH RECOMBINANT COLLARS

RNA origin and RNA extraction

The cDNA encoding the expression of the core viral proteins E1 and E2 originates from a patient serum number 22057, from the serum bank of the Department of Internal Medicine II - Gastroenterology, Hepatology, Endocrinology, Diabetology and Nutritional Medicine, the University Hospital in Frankfurt. RNA extraction was carried out using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer

vector

The amplified cDNAs were cloned into the TOPO XL vector of the kit pCR TOPO XL cloning kit from Invitrogen (Carlsbad, CA). As expression vectors for the fission yeast, the integrative vector pCAD1 (FIG. Dragan et al., 2005 ) and the autosomal replicating vector pREP1 ( Maundrell, 1993 ) used.

Reverse transcription (RT-PCR) of the RNA

The cDNA was prepared by RT-PCR. For this, 3 μl of the extracted RNA (# 22057) were mixed with 1.5 μl random hexamers (Invitrogen, Carlsbad, CA, USA) and 0.5 μl RNAse free water and incubated for 10 minutes at 65 ° C and immediately cooled in an ice bath. In parallel, the following PCR components were mixed together: 7.5 μl RNAse-free water, 5 μl of the pretreated RNA mixture from previous step, 1.4 μl 25 mM MgCl ₂ , 4 μl 10 mM dNTPs (Invitrogen, Carlsbad, CA, USA) , 2.5 10 × PCR reaction buffer + MgCl ₂ (Roche, Basel, Switzerland), 1 μl RNaseOUT (recombinant ribonuclease inhibitor) 40 U μl-1 (Invitrogen), 1 μl Superscript II reverse transcriptase. This was followed by incubation at 42 ° C. for 60 minutes.

Amplification of the cDNA

Because fission yeast does not possess a signal peptide sequence, ( Weihofen et al., 2002 ), but required for correct processing of the HCV proteins, the following expression cassettes were prepared: NH ₂ -CORE export signal COOH (protein 1) and NH ₂ export signal E1-E2-COOH (protein 2).

HCV core and envelope proteins E1 / E2 were amplified using the Expand Long Template Kit (Roche). The components of the PCR were mixed together as follows: 27 μl of RNAse-free water, 6 μl of the RT-PCR-generated cDNA as template for amplification of the core gene, 1 μl of plasmid M289 + 22057 in the case of amplification of the envelope proteins, 5 μl of 10 mM dNTPs (Invitrogen), 5 μl of 10 × Expand PCR Buffer 3, 1.5 μl 25 mM MgCl ₂ , 1 μl DMSO, 1 μl Expand enzyme mix with 3.5 U μl-1.

For amplification of the core gene, the following primers were used:

For the amplification of genes E1 / E2 the following primers were used:

The first primer provides the core gene with a 5'-terminal NdeI and a 3'-terminal BamHI site. A stop codon was introduced by the second primer. The envelope proteins E1 and E2 were amplified with primers 3 and 4 as fusion genes with an ER localization sequence (derived from the core gene). Likewise, a 5'-terminal NdeI and a 3'-terminal BamHI site were introduced with the primers.

PCR program

• 1 T = 95 ° C, t = 2.0 min
• 2 T = 95 ° C, t = 10 s
• 3 T = 50 ° C, t = 30 s
• 4 T = 68 ° C, t = 2.5 min
• 5 GOTO 2, 35 times
• 6 T = 68 ° C, t = 10 min
• 7 T = 10 ° C, t =.

The correct size of the amplicon was confirmed by agarose gel electrophoresis. The amplified DNA was isolated from the gel and cloned into the pCR TOPO XL vector (Invitrogen).

In the following, the cDNA coding for the HCV core protein becomes core and the cDNA coding for the envelope proteins is called ENV.

sequencing

Sequencing of the amplified genes was performed using the BigDye Terminator v1.1 cycle sequencing kit and the ABI Prism 3100 Genetic Analyzer. The sequence PCR was prepared as follows: 7.5 μl of water, 2 μl of BigDye solution, 2 μl of pCR TOPO XL vector and 0.5 μl of sequencing primer (M13 primer, which are contained in the Topo XL cloning kit).

PCR program:

• 1 T = 95 ° C, t = 2.0 min
• 2 T = 95 ° C, t = 10 s
• 3 T = 45 ° C, t = 15 s
• 4 T = 60 ° C, t = 4 min
• 5 T = 72 ° C, t = 10 min
• 5 GOTO 2, 35 times
• 6 T = 72 ° C, t = 10 min
• 7 T = 10 ° C, for storage

For the sequencing of the E1 / E2 sequence further sequence-specific primers were used (E1-257-2s, HVR1-inner sense, E2-1b-6a).

Construction of the expression vectors

The pCR TOPO XL vector with the coding sequences for the expression of the core and envelope proteins E1 / E2 were digested with the enzymes NdeI / BamHI and the corresponding fragments were isolated from the agarose gel after electrophoretic separation. Subsequently, core and ENV sequences were cloned into both vectors pCAD1 and pREP1. The resulting expression vectors were named pCAD1-CORE, pCAD1-ENV, pREP1-CORE and pREP1-ENV.

Construction of the fission yeasts

The fission yeast strain NCYC2036 (MB163) with the gene type h-ura4.dl18 was used as a starting point for the production of fission yeast strains expressing HCV protein. Cryocompetent MB163 cells were prepared as described ( Suga and Hatakeyama, 2005 ) and transformed either with the vectors pCAD1-CORE or pCAD1-ENV, which led to the production of the fission yeast strains CAD100 and CAD101. The correct integration of the pCAD1 plasmid into the yeast cell genome was verified by growth control on EMM media containing phloxin B. The integration of the desired cDNAs was additionally confirmed by colony PCR using primers 1, 2, 3 and 4.

In a second step, the strain pCAD100 was transformed with the vector pREP1-ENV and the strain pCAD101 with the vector pREP1-CORE by means of the lithium acetate method ( Okazaki et al., 1990 ). The yeast strains produced are summarized in Table 2. Table 2: Splitting yeasts tribe origin expressed proteins Type of replication Expression cassette per cell CAD100 NCYC2036 CORE chromosomally 1 CAD101 NCYC2036 EPS chromosomally 1 CAD102 CAD100 CORE chromosomally 1 EPS autosomal lots CAD103 CAD101 EPS chromosomally 1 CORE autosomal lots

Production of HCV proteins by means of fission yeasts

Mildew strains MB163, CAD100, CAD101, CAD102 and CAD103 were seeded on EMM-containing media to which either 0.01% uracil (for MB163) or 0.01% leucine (for CAD100 and CAD101) was added. All plates contained thiamine at a concentration of 5 M to repress the expression of recombinant proteins during the growth phase at 30 ° C. After three days of growth time, 10 ml of precultures from the yeast strains were prepared with EMM and the corresponding amino acid additives and without the addition of thiamine and incubated for 24 h at 30 ° C. and a shaking speed of 150 rpm. Thereafter, biomass was produced in a 100 ml culture in the absence of thiamine and in the presence of the corresponding amino acid substitutions (30 ° C for 24 h, 150 rpm). The cells were centrifuged at 3000 g for 5 minutes and washed twice in ZymDig buffer (50 mM NaH 2 PO 4 / Na 2 HPO 4, pH = 8, 2% glucose, 16% sucrose) and finally resuspended in 4 ml of ZymDig buffer. Zymolyase 20T from ICN Biomedicals (Aurora, OH, USA) was added at a concentration of 20 mg mL-1 and this cell suspension was incubated at 30 ° C for 24 h with shaking (150 rpm). Zymolyase 20T is an enzyme mix derived from the organism Arthrobacter luteus. Its -1,3-glucan laminaripentaohydrolase activity is responsible for the degradation of the cell wall, resulting in the formation of spheroplasts. Subsequently, the cells were centrifuged (3000 g, 5 min) and the supernatant was discarded because of the high zymolyase concentration. After three washes with ZymDig buffer, the cells were resuspended again with 4 ml of ZymDig buffer and incubated overnight (30 ° C 150 rpm). Finally, the cell suspension was centrifuged (3000 g, 5 min) and the supernatant was stored at 4 ° C after addition of 1 mM PMSF and 1 mM DTE.

Preparation of the samples

shows a schematic overview of the preparation of the individual samples. The culture medium supernatant was designated M, while the samples treated with ZymDig buffer after the second treatment were designated S samples. The letters A to G refer to the position in the schematic drawing. Untreated samples of M and S were designated MA and SA samples and stored.

There were 1.5 ml of supernatant on a sucrose cushion (20%) and centrifuged (10 ⁵ g, 1 h). Sample B is a sample taken from the liquid above the sucrose cushion, while Sample C is the sediment resuspended with PBS. An additional 2 ml of the supernatant was filtered (10 ³ g, 1 h) using a membrane which is permeable to proteins with a molecular weight of <100 kDa. This resulted in the samples D (flow) and E (retentate). The retentate samples were further incubated with 1% SDS for 30 min and then filtered again under the above conditions. These samples were designated F (flow) and G (retentate).

Protein concentration determination

The amount of protein in the samples was measured using the Pierce BCA assay (Rockford, IL, USA) with few changes. The final volume of the measured solution was composed as follows: 100 μl of a 1:40 dilution consisting of Solution B and Solution A were mixed with either 50 μl BSA or a 1:10 dilution of the supernatants. The mixture was incubated for 1 hour at room temperature in a microtiter plate. The color reaction was then measured photometrically at a wavelength of = 595 nm in the Tecan Genios MTP reader (Geneva, Switzerland).

Detection of the HCV core

HCV core proteins in the supernatant of the fission yeasts were carried out by means of the HCV CORE antigen ELISA test system from Ortho-Clinical Diagnostics (Raritan, NJ, USA) according to the manufacturer's instructions. This kit is a qualitative diagnostic assay that recommends special quality features for reliable diagnostic results. Because we only used the ELISA kit to demonstrate successful CORE expression, the manufacturer's recommended quality control guidelines were not respected. The measured concentrations of the core protein were normalized based on the total protein amount of the respective samples. Furthermore, since no concentration-dependent calibration was performed, it is not known whether there is a linear relationship between the core concentration and the signal strengths. Consequently, no core signals were detected in samples D, E, F and G of strain CAD101, which expresses only the HCV envelope proteins. The signal strengths of these samples are comparable to those of the MB163 control strain.

Protein production with fission yeasts

The integration of the desired cDNAs CORE and / or ENV was confirmed for all constructed fission yeast strains (Table 2) by means of colony PCR. Two combinations were chosen for the construction of VLP-secreting fission yeast strains. The parent strain expresses either CORE or ENV using an integrative vector (with only one expression cassette per cell). In addition, ENV or CORE cDNAs were introduced into the cells using an autosomal replicating vector (many expression cassettes per cell) to allow formation of VLPS. This procedure was mainly used to find out what amounts of cDNA are suitable for VLP production. The strains CAD100 and CAD101 show a normal growth compared to the control strain MB163, while the strain CAD102 grows significantly slower. In the phase-contrast microscope, an untypical phenotype can be detected in strains Cad100 and CAD102, based on an increase in intracellular altered structures and a change in cell shape.

Core proteins are secreted into the culture medium

In the MA samples, a weak signal could be detected compared to the MB163 control strain. A sucrose cushion centrifugation failed to improve the signal strength. After filtration of the samples through a membrane which is permeable to proteins <100 kDa, differences between the control strain and the strains CAD100 and CAD102 could be measured ( ).

Core proteins are secreted by spheroplasts

After spheroplasts were prepared by enzymatic Zymolyase 20T digestion, they were incubated for a further 24 hours in ZymDig buffer. Supernatants of these samples (SA samples) were analyzed by core ELISA and a significant core concentration was measured in the samples of strains CAD100, CAD102 and CAD103 ( ). Only very low core concentrations (insignificant amounts) were to be measured in samples of the control strain and the ENV expressing strain CAD101. Compared to the core concentrations of intact cells, the core signals in the supernatants of spheroplasts were significantly stronger. Moreover, treatment of the supernatants with 1% SDS followed by filtration resulted in further enhancement of core signals in the samples of strains CAD100 and CAD102 compared to the control strain MB163.

Sucrose cushion centrifugation

The sucrose cushion centrifugation is a centrifugation in which the samples are layered over a sugar gradient (here consisting of 20% sucrose). This is a common one Method for the separation of VLPs from culture media. Both the resuspended sediments and the liquid above the sugar pad were analyzed by HCV Core ELISA. Since the sediment was resuspended with 0.5 ml of PBS, it can be assumed that the sample was approximately 3 times concentrated. In the sediments of the strains CAD100 and CAD102, a significantly higher core concentration was measurable compared to untreated supernatants ( ). In the samples of the strain CAD103 no improvement but a reduction of the concentration could be measured. Even with the control strains (MB163 and CAD101), which do not express core proteins, a non-significant increase in core concentration could be observed. This may be a consequence of the concentration of the samples. At the same time, a decrease in the core signals in the liquid above the sucrose cushion was measurable in all samples in which a gain of the core signals in the sediment samples was measured ( ).

Core proteins are retained by a membrane which is permeable to proteins with a molecular weight of <100 kDa

By filtration of the supernatants of spheroplasts, which were resuspended in ZymDig buffer, the core proteins were retained by the membrane (retentate samples) ( ). However, no clear concentration effect could be observed. For the strain CAD103, a reduction of the core concentration could also be detected. It is also interesting that only with samples of the strain CAD100, which overexpressed only the core protein, core signals were also measurable in the flow-through samples. This was not the case for samples of strains CAD102 and CAD103 ( ). If the core concentrations of MB163 in the flow-through samples (background signals) are subtracted from the measured values of strains CAD102 and CAD103, no core signal is present in these flow-through samples (cf. With ).

Core concentrations after SDS treatment

Each 0.3 ml of the retentate samples was treated with 1% SDS and then filtered again. This resulted in amazing increases in core concentration. The increase was recorded in all core-expressing strains and was particularly marked in samples of the strain CAD102 (FIG. ). For samples of the MB163 strain, this effect was undetectable. Also for the flow-through samples, SDS treatment did not increase the core concentration (cf. With ).

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This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited non-patent literature

• Noad & Roy, 2003 [0005]
• Fifis et al., 2004 [0006]
• Lenz et al., 2003 [0006]
• Ruedl et al., 2002 [0006]
• Dintzis et al., 1976 [0007]
• Moon et al., 1995 [0007]
• Plueddemann & Zyl, 2003 [0010]
• Li et al., 2004 [0010]
• Acosta-Rivero et al., 2001 [0010]
• Sasagawa et al. (1995) [0011]
• Sakuragi et al. (2002) [0012]
• Moreno et al., 1985 [0030]
• Schweingruber et al., 1986 [0030]
• Sakuragi et al., 2002 [0031]
• Dragan et al., 2005 [0045]
• Maundrell, 1993 [0045]
• Dragan et al., 2005 [0068]
• Maundrell, 1993 [0068]
• Weihofen et al., 2002 [0070]
• Suga and Hatakeyama, 2005 [0080]
• Okazaki et al., 1990 [0081]

Claims (15)

Method for producing HCV-based virus-like particles (VLPs) The use of a recombinant yeast cell which expresses the structural proteins needed for self-assembly of the virus-like particle, The culture of the recombinant yeast under suitable conditions, The separation of the yeast cells and cell fragments from the supernatant, Optionally, the treatment of the yeast cell fraction or the retentate fraction with a detergent or by sonication, and the separation of the treated yeast cell fraction or retentate fraction from the supernatant, and The separation of the virus-like particles from the supernatant of the recombinant yeast cell culture. A method according to claim 1, characterized in that the yeast strains are selected from at least one parental strain selected from the group consisting of Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces japonicus and Schizosaccharomyces kambucha. Method according to claim 1 or 2, characterized in that the detergent used is sodium dodecyl sulfate. A method according to any one of claims 1 to 3, characterized in that the recombinant yeast strain is transformed with at least one copy of a vector which encodes the genes necessary for the expression of the viral structural proteins and which is required for the intracellular formation of HCV-based virus-like particles become. A method according to any one of claims 1 to 3, characterized in that the recombinant yeast strain is stably transformed by the insertion of at least one copy of a vector encoding the expression of the viral structural proteins required to produce HCV-based virus-like particles , Method according to one of claims 1 to 5, characterized in that the structural genes, which are required for the self-assembly of the virus-like particles are selected from the group consisting of structural genes of the hepatitis C virus HCV. Method according to claim 6, characterized in that the structural genes required for the self-assembly of a virus-like particle are selected from a group consisting of the structural genes HCV core protein, HCV subtype 1a core protein (again gene type 1a ) and HCV E1E2 protein. Method according to one of claims 1 to 6, characterized in that the structural genes comprise and encode a fusion gene of a structural gene selected from the group consisting of structural genes of the hepatitis virus and a heterologous gene. Use of recombinant Schizosaccharomyces cells for the production of secreted HCV-based virus-like particles. Use according to claim 9, characterized in that the recombinant fission yeast strain is stably or transiently transformed with at least one copy of a vector comprising the structural genes or derivatives of the hepatitis C virus structural genes (HCV) selected from the group consisting of structural genes for the HCV core. Protein, HCV subtype 1a core protein (again, genotype 1a), HCV E1E2 protein, combinations thereof, and derivatives thereof, which are required for self-assembly of a virus-like particle Use according to any one of claims 9 to 10, characterized in that the Schizosaccharomyces is selected from at least one strain selected from the group consisting of Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces japonicus and Schizosaccharomyces kambucha. Isolated HCV-based virus-like particles produced by recombinant Schizosaccharomyces and secreted into the supernatant, which were isolated from the Schizosaccharomyces cell culture supernatant. An isolated HCV-based virus-like particle prepared according to the method of any one of claims 1 to 8, wherein the virus-like particles consist essentially of HCV core protein, HCV subtype 1a core protein and / or HCV E1E2 protein ( Gentype 1a) exist. Use of the isolated HCV-based virus-like particles according to claims 12 and 13 for the manufacture of a medicament for the treatment or prevention of viral infections or HCV infection. Use of the isolated HCV-based virus-like particles according to claims 12 and 13, in a kit containing the isolated wax-like particles and at least one container.

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