FIELD OF THE INVENTION

This invention is related to the field of viral control, more specifically to the control of viral replication, especially of chronic viruses and particularly hepatitis C virus (HCV) and hepatitis B (HBV).

BACKGROUND ART

Infecting viruses have been a plague for the host cells to which they are targeted from the very beginning of their existence. Although some endogenous mechanisms exist for the control of viral infection, such as the viral induced gene for gene defence mechanisms in plants, or the RNAi mechanisms in all higher eukaryotes, which is enhanced by increase in temperature (fever), and of course the immunological responses in vertebrates, natural resistance mechanisms have not prevented the widespread occurrence of viral infections.

Viruses, basically consisting of nucleic acid material (the genome) packaged in a protein and lipid shell, have in common that they need a host cell to reproduce, but the mechanisms of reproduction depend strongly on the type of virus. With respect to replication, two main types of viruses are discerned, ones with DNA as nucleic acid material and ones with RNA. Further differentiation exists in that they can be single-stranded or double-stranded. Especially with respect to replication a further group should be mentioned, the retroviruses, which replicate by forming DNA from their RNA.

The Baltimore classification of viruses (Baltimore, D., 1974 Harvey Lect. 70 Series: 57-74) is based on the mechanism of mRNA production. Viruses must generate mRNAs from their genomes to produce proteins and replicate themselves, but different mechanisms are used to achieve this in each virus family. Viral genomes may be single-stranded (ss) or double-stranded (ds), RNA or DNA, and may or may not use reverse transcriptase (RT). Additionally, ssRNA viruses may be either sense (+) or antisense (-). This classification places viruses into seven groups: • I: dsDNA viruses (e.g. Adenoviruses, Herpesviruses, Poxviruses) » II: ssDNA viruses (+)sense DNA (e.g. Parvoviruses)

» III: dsRNA viruses (e.g. Reoviruses)

» IV: (+)ssRNA viruses (+)sense RNA (e.g. Picornaviruses, Togaviruses)

• V: (-)ssRNA viruses (-)sense RNA (e.g. Orthomyxoviruses, Rhabdoviruses)

• VI: ssRNA-RT viruses (+)sense RNA with DNA intermediate in life-cycle (e.g. Retroviruses)

• VII: dsDNA-RT viruses (e.g. Hepadnaviruses)

Chronic viruses are viruses with long latency periods in infected individuals, before disease symptoms develop. Chronic viruses include human immunodeficiency virus (HIV), Karposi's sarcoma-associated herpesvirus (KSHV), Eppstein-barr virus (EBV, another herpesvirus), Hepatitis B virus (HBV) and Hepatitis C virus (HCV). Especially hepatitis viruses and HIV are a major global health concern.

HCV is the causal agent of chronic liver infections, which develop into lethal cirrhosis or liver cancer in a substantial percentage of infected individuals.

HCV belongs to the Flaviviridae. Virus particles consist of a plus stranded genomic RNA molecule wrapped up with C protein to nucleocapsids. Lipid envelopes with glycoprotein projections surround the nucleocapsids. The RNA molecule encodes a single large polyprotein, which is co- and post-translationally cleaved by viral and cellular proteases into 10 different mature viral proteins, with the structural proteins (C, El, E2 and p7) located in the amino-terminal part and the non-structural proteins (NS2, NS3, NS4a, NS4b, NS5a and NS5b) located at the carboxy-terminus. From a different reading frame than the polyprotein overlapping with the C-encoding region another non-structural protein (pi 6) is encoded. Virus particles have a tropism for hepatocytes and lymphoid cells. Upon entrance, the nucleocapsids are released in the cytoplasm and the genomic RNA molecules are translated directly. Virus replication takes place in the cytoplasm of infected cells. The majority of infected individuals becomes chronically infected and frequently shows chronic hepatitis. The history of chronic HCV infections can vary dramatically between individuals. Some have minimal liver disease and never develop complications. Others develop severe and often lethal cirrhosis and/or hepatocellular carcinoma's after many years of infection. Cirrhosis is momentarily the leading indication for liver transplantations. HCV is characterised by its presence at very low titres in the infected cells during the chronic phase and by its extremely high genomic variability. These characteristics significantly contribute to the lack of successful anti-HCV therapies.

HCV infections are currently treated with alpha-interferon alone or in combination with ribavirin, albeit with a low success rate. Additionally, the costs of both drugs are very high. Gene therapy has been considered as an attractive alternative novel therapy against other chronic viruses, such as HIV (Romano et al., Stem Cells 17: 191-202, 1999; Engel and Kohn, Front. Biosci. 4: 26-33, 1999; Buchsacher and Wong-Staal, Human Gene Therapy 12: 1013-1019, 2001). Gene therapy for HCV has been suggested in WO 03/087371. Previously studied targets for gene therapy and drug discovery include the NS3-NS4A protease and the NS5B polymerase. The protease domain of the NS3-NS4A protease includes the N-terminal third of NS3 and a short stretch of NS4A, which has been reported to function as a cofactor. A high-resolution structure of the protease has enabled the development of protease inhibitors which are either substrate analogs, inhibitors containing a serine trap, or product-mimicking inhibitors. NS3 also includes a helicase domain in the C- terminal 500 amino acids, the structure of which has enabled the development of small molecule inhibitors of helicase function. The NS5B polymerase has also been a target for high resolution structural studies and drug design. Inhibitors of viral polymerases include substrate (nucleoside) analogs, product (pyrophosphate) analogs, and nonnucleoside inhibitors.

HCV viral infection does not provide immune protection. This fact, together with the virus's high variability in antigenic structure recognized by the immune system, has hindered the development of an effective serum therapy and vaccines to protect individuals against HCV infection.

Hepatitis B virus (HBV) is a member of the Hepadnavirus family. The virus particle, (virion) consists of an outer lipid envelope and an icosahedral nucleocapsid core composed of protein. The nucleocapsid encloses the viral DNA and a DNA polymerase that has reverse transcriptase activity. The outer envelope contains embedded proteins which are involved in viral binding of, and entry into, susceptible cells. The virus is one of the smallest enveloped animal viruses with a virion diameter of 42nm, but pleomorphic forms exist, including filamentous and spherical bodies lacking a core. These particles are not infectious and are composed of the lipid and protein that forms part of the surface of the virion, which is called the surface antigen (HBsAg), and is produced in excess during the life cycle of the virus. The life cycle of Hepatitis B virus is complex. Hepatitis B is one of a few known non-retro viral viruses which use reverse transcription as a part of its replication process. The virus gains entry into the cell by binding to a receptor on the surface of the cell and enters it by endocytosis. Because the virus multiplies via RNA made by a host enzyme, the viral genomic DNA has to be transferred to the cell nucleus by host proteins called chaperones. The partially double stranded viral DNA is then made fully double stranded and transformed into closed circular supercoiled DNA (cccDNA) that serves as a template for transcription of four viral mRNAs. The largest mRNA, (which is longer than the viral genome), is used to make the new copies of the genome and to make the capsid core protein and the viral DNA polymerase. These four viral transcripts undergo additional processing and go on to form progeny virions which are released from the cell or returned to the nucleus and re-cycled to produce even more copies. The long mRNA is then transported back to the cytoplasm where the virion P protein synthesizes DNA via its reverse transcriptase activity.

Acute infection with hepatitis B virus is associated with acute viral hepatitis - an illness that begins with general ill-health, loss of appetite, nausea, vomiting, body aches, mild fever, dark urine, and then progresses to development of jaundice. It has been noted that itchy skin has been an indication as a possible symptom of all hepatitis virus types. The illness lasts for a few weeks and then gradually improves in most affected people. A few patients may have more severe liver disease (fulminant hepatic failure), and may die as a result of it. The infection may be entirely asymptomatic and may go unrecognized.

Chronic infection with Hepatitis B virus may be either asymptomatic or may be associated with a chronic inflammation of the liver (chronic hepatitis), leading to cirrhosis over a period of several years. This type of infection dramatically increases the incidence of hepatocellular carcinoma (liver cancer). Chronic carriers are encouraged to avoid consuming alcohol as it increases their risk for cirrhosis and liver cancer. The hepatitis B virus primarily interferes with the functions of the liver by replicating in hepatocytes. HBV virions (DANE particle) bind to the host cell via the preS domain of the viral surface antigen and are subsequently internalized by endocytosis. PreS and IgA receptors are accused for this interaction. HBV-preS specific receptors are primarily expressed on hepatocytes, however, viral DNA and proteins have also been detected in extrahepatic sites, suggesting that cellular receptors for HBV may also exist on extrahepatic cells. During HBV infection, the host immune response causes both hepatocellular damage and viral clearance. Both the innate immune response, particular dendritic cells and NK-cells, and the adaptive immune response, particularly virus-specific cytotoxic T lymphocytes (CTLs), contributes to most of the liver injury associated with HBV infection. By killing infected cells and by producing antiviral cytokines capable of purging HBV from viable hepatocytes, immune cells eliminate the virus.

Although none of the available drugs are very effective in eliminating the virus, they can stop viral replication and likely minimize liver damage such as cirrhosis and liver cancer. Currently, there are seven drugs licensed for treatment of hepatitis B infection in the United States. These medications consist of the antiviral drugs lamivudine (Epivir), adefovir (Hepsera), tenofovir (Viread), telbivudine (Tyzeka) and entecavir (Baraclude) and the two immune system modulators interferon alpha-2a and pegylated interferon alfa-2a (Pegasys). The use of interferon, which requires injections daily or thrice weekly, has been supplanted by long-acting pegylated interferon, which is injected only once weekly. However, some individuals are much more likely to respond than others and this might be because of the genotype of the infecting virus or the patient's heredity. The treatment works by reducing the viral load, (the amount of virus particles as measured in the blood), which in turn reduces viral replication in the liver.

Despite the numerous attempts to find inhibitors of the hepatitis viruses and for other viral infections, which for at least HBV have led to several drugs now being on the market, there is still need for alternative therapies. SUMMARY OF THE INVENTION

The invention now concerns a method for inhibition of a viral infection comprising exposing said virus to mesenchymal stem cells (MSCs), preferably said virus is a hepatitis virus, more preferably HCV or HBV.

In a preferred embodiment, the inhibition is the inhibition of viral replication in a host cell. In a further preferred embodiment the cells harbouring the virus are co-cultured with MSCs. Alternatively, the cells harbouring the virus are contacted with exudate of MSCs. In the latter case, it is preferred that the exudate is concentrated by removing small molecules, preferably smaller than 3 kD, more preferably smaller than 10 kD, even more preferably smaller than 50 kD, preferably by filtration. Also, it is preferred that the exudate is heat-treated, preferably by subjecting it to a temperature of at least 6O ⁰ C for at least 20 minutes, more preferably for at least 100 ⁰ C for at least 30 minutes.

In a further preferred embodiment, the antiviral active component produced by the MSCs is a protein with an amino acid sequence of more than approximately 400 amino acids.

The invention also comprises the use of mesenchymal stem cells (MSCs) for the inhibition of a viral infection, preferably a hepatitis virus, more preferably HCV or HBV. For said use, the MSCs are co-cultured with cells harbouring said virus.

The invention also comprises the use of exudate of MSCs for the inhibition of a viral infection, preferably a hepatitis virus, more preferably HCV or HBV. Preferably, said exudate is concentrated by removing small molecules, preferably smaller than 3 kD, more preferably smaller than 10 kD and even more preferably smaller than 50 kD, by filtration. In another embodiment, a preferred use is wherein said exudate is heat-treated, preferably by subjecting it to a temperature of at least 6O ⁰ C for at least 20 minutes, more preferably for at least 100 ⁰ C for at least 30 minutes. Further, preferably the exudate comprises a protein with an amino acid sequence of more than 80 amino acids.

Also part of the invention is a pharmaceutical composition for use in the treatment of viral infection, wherein said composition comprises MSCs or exudate from MSCs. LEGENDS TO THE FIGURES

Figure 1. Potent inhibition of HCV replication by co-culture MSC with Huh7 HCV replicon cells (ET). HCV replication was determined by measuring luciferase activity after 48 hours treatment. Co-cultured MSC with ET cells (30,000 cells) resulted in 90+3% inhibition of HCV replication at 1:1 ratio, 34+10% inhibition at 1:10 and 25+6% at 1 :100 ratio (P<0.01). Control groups of co-cultured ET cells with parental Huh7 cells or mouse fibroblast cell line (L-cells) did not result in significant inhibition of viral replication.

Figure 2. Inhibition of HCV replication by MSC conditional medium (MSC-CM). MSC-CM was harvested and pooled. Huh-7 HCV replicon (ET) cells were treated with MSC-CM at different concentrations. HCV replication was determined by measuring luciferase activity 2 days after treatment. 70+9% inhibition of viral replication was observed at 50% concentration and 48+8% inhibition at 10% concentration (P<0.01 ) .

Figure 3. Concentrated MSC-CM (C-MSC-CM) retains potent anti-viral activity. 25-fold concentrated MSC conditioned medium was prepared by using 3 kD cutoff centrifugal filter devices from pooled MSC-CM. HCV replication was determined by measuring luciferase activity 2 days after treatment. 87±5%, 76±5% and 24±11% inhibition of HCV replication were observed at 10%, 2% and 0.5% of C- MSC-CM treated groups, respectively (P<0.01). The leftover fraction of MSC-CM that passed the 3 kD filter lost its anti-viral activity.

Figure 4. Anti-viral effect of MSC is independent of cell viability. MSC- CM or C-MSC-CM has no significant effect on Huh7 ET cell viability, as determined by MTT assay. MTT assay was performed by determining the optical density at 490 nm (OD490).

Figure 5. MSC-CM retains its anti-viral activity after boiling. MSC-CM treated at 65°C or 100 ⁰ C for 30 min possess similar antiviral activity as the untreated MSC-CM.

Figure 6. MSC-CM does not contain selectable levels of IFNs. Different concentration of IFN-α can stimulate luciferase activity in a dose-dependent manner, while MSC-CM dose not stimulate luciferase activity. Figure 7. Suppression of HCV replication by MSC is not dependent on GTP depletion. Supplementation with 50 μmol/L guanosine slightly inhibited HCV replication, but did not overturn the inhibition by MSC-CM.

Figure 8. Suppression of HCV replication by serum-free MSC conditioned medium.

Figure 9. Concentrated MSC-CM prepared by 10 kD cutoff filter retains anti-viral activity. 25-fold concentrated MSC conditioned medium was prepared by using 10 kD cutoff centrifugal filter devices from pooled MSC-CM. HCV replication was determined by measuring luciferase activity 2 days after treatment. The leftover fraction of MSC-CM that passed the 10 kD filter lost its anti- viral activity.

Figure 10. 50 kD cutoff filter concentrated the anti-viral activity but also some anti-viral activity is retained in the leftover fraction.. 25-fold concentrated MSC conditioned medium was prepared by using 50 kD cutoff centrifugal filter devices from pooled MSC-CM. HCV replication was determined by measuring luciferase activity 2 days after treatment. More then 90% inhibition of HCV replication was observed at the 20% C-MSC-CM condition. The leftover fraction of MSC-CM that passed the 50 kD filter resulted in less then 40% inhibition of HCV replication, suggesting that some of the antiviral compounds have a smaller molecular size.

Figure 11. Suppression of HBV by MSC-CM. HBV mRNA level in the HBV cell line model, HepG2.2.15, was significantly suppressed (up to 67% reduction) by treated with 10% C-MSC-CM.

DETAILED DESCRIPTION

Mesenchymal stem cells or MSCs are multipotent stem cells that can differentiate into a variety of cell types. Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, adipocytes, hepatoctes, and beta-pancreatic islets cells. The term Multipotent Stromal Cell has been proposed as a better replacement because the cells, called MSCs by many labs today, do not have the capacity to reconstitute an entire organ, but can encompass multipotent cells derived not only from bone-marrow, but from other non- marrow tissues, such as adult adipose tissue, liver, lymphoid organs, muscle side- population cells or the Wharton's jelly present in the umbilical cord, as well as in the dental pulp of deciduous baby teeth. Unlike most other human adult stem cells, mesenchymal stem cells can be obtained in quantities appropriate for clinical applications, making them good candidates for use in tissue repair. Techniques for isolation and amplification of mesenchymal stem cells in culture have been established and the cells can be maintained and propagated in culture for long periods of time, without loosing their differentiation capacity to all the above cell types. Mesenchymal stem cells can also be frozen to preserve them, and when they are thawed they function apparently normally, thus allowing for future "off-the-shelf therapy approaches. Perhaps one of the important considerations for human applications is that mesenchymal stem cells can be derived from adipose tissue, liver perfusate, bone marrow or other tissue samples from a given patient, expanded in clinical grade cultures, and given back to the patient.

In a preferred embodiment of the present invention, the stem cells are cultured in serum-free medium. This prevents contamination of the exudate of MSCs by pathogenic components that are derived from the calf or cow serum that is normally used. Such pathogenic components can for instance be virus particles or prions. When it is contemplated of giving the MSCs as part of a pharmaceutical composition, it is of essence that the receiver of the treatment will not be exposed to pathogenic components.

In vivo, MSCs are able to repair damaged tissue from kidney, heart, liver, pancreas and gastrointestinal tract. Furthermore, they also have antiproliferative, immunomodulatory and anti-inflammatory effects and are low immunogenic. Although the mechanism underlying the immunosuppressive effects of MSCs has not been clearly defined, their immunosuppressive properties have already been exploited in the clinical setting. Therefore, MSCs might have implications for treatment of allograft rejection, graft- versus-host disease, rheumatoid arthritis, autoimmune inflammatory bowel disease and other disorders in which immunomodulation and tissue repair are required.

However, no effects of MSCs on viral infection and replication have been reported. It is known for hepatitis B virus, that MSC can take up virus particles, as detected by antibody staining, but do not support productive infection (Rong, Q. et al., 2008, J. Viral Hepatitis 15:607-614). Neither cccDNA nor pregenomic RNA could be detected in MSCs, consistent with their anti-(hepatitis B) viral activity. One study has shown that when MSCs are challenged with VSV or Sendai viruses in vitro, these cells will produce relevant quantities of interferon-gamma (IFN-γ) (Kang, et al., 2005 Exp. Hematol. 33: 796-803). In this same study it was reported that unstimulated MSCs produce only very low levels of IFN-γ, consistent with our own observations. Recent study showed that MSCs can be genetically modified using adeno-associated virus vectors to express interferon-alpha (IFN-α) as a therapeutic strategy for lung melanoma metastasis tested in a mouse model (Ren, et al., 2008 Stem Cells 26: 2332- 38). Though MSCs are potent inhibitors, recent evidence suggests that MSCs do not inhibit T cell responses directed against virus-specific antigens like EBV and CMV (Karlson, et al., 2008 Blood 112:532-541).

Further, MSCs have been proposed as viral packaging cell lines for transport of viruses and transduction into target tissues (Silva, F., Nardi, N., 2006, Med. Hypoth. 67:922-925; Hakkarainen, T. et al., 2007, Human Gene Therapy 18:627-641, Ricks, D.M., et al., 2008, Stem Cells Dev. 17:441-450).

In the experimental part it is clearly shown thatone or more factors that are excreted from MSCs is able to inhibit viral replication of HCV and HBV. At least one factor is larger than 50 kD, because filtration of the exudate through a 3 kD, 10 kD and/or through a 50 kD filter, in which all compounds less than the indicated size were filtered out of the exudate, resulted in a concentrated MSC preparation with an enrichment of antiviral activity. These results indicate that one or more large (>50 kD) compounds are responsible for this effect. Furthermore, at least one antiviral compound(s) seems to have a molecular mass between 10 and 50 kD (approx. 80-400 aa), equivalent to the mass of all interferons (180-200 aa) and most chemokines, cytokines and growth factors. The antiviral activity of this latter compound is less than of the >50kD compound(s). Heat treatment, for instance for sterilization of the exudate, did not decrease the antiviral activity, indicating that, if the compound is of a proteinaceous structure, the activity is mainly residing in the primary structure of the protein(s) and is independent of a specific folding of the protein.

In the search for the nature of the activity of the MSC exudate, it has been discovered that there is no interferon-like activity of the MSCs that would be responsible for the antiviral activity. An attempt to show that the activity is mediated through the IMPDH/GTP pathway, via which the known antiviral compounds ribavirin and mycophenolic acid cause their effects, also proved negative. These negative results indicate that the inhibitory nature of the exudate of MSCs on the replication of viruses seems to be mediated through a new pathway. One embodiment of the invention is a method for treating viral infections in a mammal by exposing the cells in which the virus is replicating to MSCs or to the exudate of MSCs. In order to obtain MSCs or the exudate, the MSCs are obtained from the human bone marrow, liver or spleen, though other sources for MSC could be considered. MSCs from bone marrow are commercially available, MSCs from liver and or spleen can be prepared as indicated in the examples. The exudate of the MSCs can, if necessary, be enriched by passing it through a 10 kD cut-off filter, discarding the filtrate and resuspending the retentate. Not only will the remaining exudate be enriched in antiviral activity, but also any toxic or pathogenic low molecular substance will be deleted from it by this filtration step. Further, resuspending the retentate enables using medium or solvent that is acceptable in pharmaceutical formulations.

Therapy according to the method of the invention can be accomplished by contacting the cells in which the virus is replicating with the MSCs or the exudate, both in vivo or ex vivo. For treatment of viral infections like hepatitis B and C, where the cells in which the virus is replicating are liver cells, this can for instance be effected by perfusing the liver of a diseased subject with a liquid harboring the MSCs or with exudate of the MSCs. It is also possible to administer the MSCs to the diseased subject by intravenous, intraportal or subcutaneous injections. A person skilled in the art will be well aware of the methods of administering stem cells, see e.g. Reiser, J. et al., 2005, Expert Opin. Biol. Ther. 5:1571-1584. MSC exudates or concentrated fractions thereof can be considered for oral administration, as well. The fact that the antiviral activity of MSC products is heat-insensitive, suggests that these compounds can retain activity after ingestion. Further, they can be provided with site- specific delivery vehicles for targeting the MSCs to those tissue in which the cells that harbour the replicating virus are residing (Caplan, A.I., 2005, Tissue Eng. 11 :1198- 1211). Finally, it would be possible to engraft both autologous and allogenous MSCs in organs of the diseased subject, such as the liver. In order to boost the antiviral effect of the MSCs, they can be ex vivo transfected with DNA sequences that encode for an antiviral peptide/protein or DNA-sequences that give rise to antiviral effects through RNA interference. In the latter case, a construct is provided to the MSCs enabling production of a double-stranded RNA molecule which is highly homologous to a nucleotide sequence of the virus. Techniques for designing such a construct and to transfect MSCs are well known in the art. Pharmaceutical compositions comprising MSCs or exudate of MSCs may further comprise standard ingredients for pharmaceutical compositions, such as pharmaceutically acceptable carriers, etc.

EXAMPLES

1. Isolation and culture of mesenchymal stem cells from liver perfusates, liver biopsy and spleen

Liver graft preservation fluid (perfusates) was collected from human liver grafts at the time of transplantation. During the backtable procedure, the grafts were perfused through the portal vein with 1-2 L of University of Wisconsin (UW) solution to remove residual blood from the vasculature, then the effluent perfusates were collected (perfusates I). Immediately before transplantation, the donor liver was perfused with 200 to 500 ml of human albumin solution under hydrostatic pressure, and the effluent perfusate was collected from the vena cava (perfusates II). Perfusate I and II were pooled and mononuclear cells were isolated by density gradient centrifugation using Ficoll Paque Plus.

End stage liver disease tissue samples were obtained from the explanted liver from liver transplantation recipients, and stored in UW solution at 4 ⁰ C. Within 24h, the liver biopsy was minced with a scalpel knife, incubated with sterile filtered 0.5 mg/ml collagenase type IV (Sigma, Zwijndrecht, The Netherlands) in RPMI for 30 min at 37°C under continuous stirring. After two washes in RPMI, the cells from the dissociated tissue were transferred to 24 or 12-well plates (Corning-Costar, NY, USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Lonza, Venders, Belgium) supplemented with 10% fetal calf serum (Hyclone, Logan, Utah), 100 IU/ml penicillin and 100 μg/ml streptomycin.

Isolation of MSC from spleen is similar to the method for liver tissue. Bone marrow MSC is commercially available. The medical ethical committee of the Erasmus MC approved the use of liver materials for this study.

2. Production of mesenchymal stem cells conditioned medium (MSC-CM)

MSC (1*10 ⁶ cells ) were cultured in a 75cm ² flask in 15ml DMEM (Lonza, Venders, Belgium) supplemented with 10% fetal calf serum (Hyclone, Logan, Utah), 100 IU/ml penicillin and 100 μg/nil streptomycin. When the cell density reached 70-90%, culture medium was refreshed and collected after 3 days. Control conditioned medium was produced from parental Huh-7 cells and mouse fibroblast cell line (L-cells) in indicated fashion as for MSC. 25-fold concentrated MSC-CM (C-MSC-CM) was prepared by using 3 KD cutoff centrifugal filter devices (Millipore Corporation, Billerica, MA, USA).

3. Heat-inactivation of MSC-CM

Collected MSC-CM was incubated at 65°C or 100°C in a water bath for 30min.

4. Culture of HCV replication model

Huh-7 cells containing a subgenomic HCV bicistronic replicon (I389/NS3- 3V/LucUbiNeo-ET, Huh-7 ET, kindly provided by prof dr RaIf Bartenschlager, University of Heidelberg, Germany) were maintained with DMEM supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 250 μg/ml G418 (Sigma, Zwijndrecht, The Netherlands).

5. Determination of HCV replication

HCV replication in Huh-7 ET was monitored by luciferase activity. Briefly, 30,000 cells were plated in 96-wells. 2 days later, 100 mM luciferin potassium salt (Sigma) was added to Huh-7 ET cells and incubate for 30 min at 37 ⁰ C. Luciferase activity was quantified using a LumiStar Optima luminescence counter (BMG LabTech, Offenburg, Germany).

6. Co-culture of MSC with Huh-7 ET

Co-culture of MSC and Huh-7 ET (30,000 cells) was performed in 96-wells at different ratio (MSC: ET: 1 : 1, 10 or 100), including parental Huh7 and fibroblast cells as control groups. HCV replication was determined by measuring luciferase activity 48h later.

7. Treatment of Huh-7 ET with MSC-CM and C-MSC-CM

30,000 Huh-7 ET cells were plated in 96-wells. Different concentration of MSC-CM, C-MSC-CM and heat-inactivated MSC-CM were treated. HCV replication was determined by measuring luciferase activity after 48h. 8. Interferon report assay

Interferons (IFNs) are crucial mediators in the innate immune response against viruses. To determine whether the anti- viral effect of MSC is dependent on the action of IFN, an interferon response reporter assay was performed. Some evidence suggests that MSC can produce for instance IFN-gamma under certain conditions. pIRES-Luc is a plasmid containing an IFN response element which drives luciferase expression. IFN response of 293 -T cells was determined by measuring luciferase activity after transduction with pIRES-Luc and treatment with different concentrations of IFN-α (Figure 6). A similar experiment was performed to test the presence of IFN in MSC- CM. Moreover, no IFN-α and IFN-γ was detectable in MSC-CM by ELISA detection (data not shown). Based on these data we conclude that MSC-CM does not contain detectable levels of IFN and therefore IFNs do not seem to account for the antiviral activity of MSC in this setting.

9. Guanosine supplementation ofhuh-7 ETcells

IMPDH inhibitors like ribavirin and mycophenolic acid can inhibit viral replication by reducing the cellular guanosine nucleotide (GTP) pool. Supplementation with exogenous guanosine can overcome this inhibitory effect. To investigate whether the inhibition of HCV replication by MSC-CM is dependent on IMPDH inhibition and/or GTP depletion, we investigated the effect of 50 μmol/L guanosine supplementation. Figure 7 shows that addition of 50 μmol/L guanosine slightly inhibited HCV but had no effect of inhibition by MSC-CM, suggesting that the IMPDH/GTP pathway is not involved in the antiviral action of MSC. Suppression of HCV replication by MSC is not dependent on GTP depletion.

10. Production of serum free mesenchymal stem cells conditioned medium (MSC-CM- SF)

0.5 x 106 MSC were cultured in 25cm2 flask in 5ml normal MSC culture medium. When cell density reached 90-95%, medium was replaced with serum free Iscove's Modified Dulbecco's Medium (IMDM) (Lonza, Venders, Belgium) supplemented with 10 ng/ml epidermal growth factor (EGF) and 10 ng/ml basic fibroblast growth factor (bFGF). Conditioned culture medium was collected after 3 days. 25-fold concentrated MSC-CM (C-MSC-CM-SF) was prepared by using 3 KD cutoff centrifugal filter devices (Millipore Corporation, Billerica, MA, USA). Inhibition of HCV replication by serum free MSC conditioned medium (MSC-CM- SF) was determined by measuring luciferase activity 2 days after treatment. Results are shown in Fig. 8. HCV viral replication was inhibited by 53±3% with 1:1 diluted MSC-CM-SF (50%). Concentrated MSC-CM-SF inhibited HCV replication at 20%, 5% and 2% concentration by 73+6%, 61±7% and 52±4%, respectively (P<0.01).

11. Production of concentrated mesenchymal stem cells conditioned medium by WKD cutofffllter

MSC-CM was collected and 25-fold concentrated MSC-CM (C-MSC-CM) was prepared by using 10 kD cutoff centrifugal filter devices (Millipore Corporation, Billerica, MA, USA). HCV replication was determined by measuring luciferase activity 2 days after treatment

Concentrated MSC-CM prepared by both 3 kD and 10 kD (Figure 9) cutoff filters resulted in an enrichment of antiviral activity. Inhibition of 84±4%, and 67±4% was observed at 10% and 2% concentration of C-MSC-CM, respectively (P<0.01). This result indicates that the antiviral compound(s) have a molecular mass greater then 10 kDalton, equivalent to a protein of approximately 80 amino acids in size.

12. Production of concentrated mesenchymal stem cells conditioned medium by 50 kD cutofffllter

MSC-CM was collected and 25-fold concentrated MSC-CM (C-MSC-CM) was prepared by using 50 kD cutoff centrifugal filter devices (Millipore Corporation, Billerica, MA, USA). HCV replication was determined by measuring luciferase activity 2 days after treatment.

50 kD cutofffllter separates anti-viral compounds into two fractions (Figure 10). Inhibition of 95±2%, and 74±5% was observed at 20% and 2% concentration of C- MSC-CM, respectively (p<0.01). Different from 3 and 10 kD filter, the leftover fraction of 50 kD filter retains 37±5% inhibition of HCV replication at 50% concentration (p<0.01). These results indicate that at least one of the antiviral compound(s) has a molecular mass greater then 50 kDalton, equivalent to a protein of approximately 400-450 amino acids in size. Furthermore, at least one antiviral compound(s) seems to have a molecular mass between 10 and 50 kD (approx. 80-400 aa), equivalent to the mass of all interferons (180-200 aa) and most chemokines, cytokines and growth factors. The antiviral activity of this latter compound is less than of the >50kD compound(s).

13. Culture at treatment with C-MSC-CM of HBV infection model, HepG2.2.15 HepG2.2.15 cell line is an established model (since 1987) for hepatitis B virus infection and replication, supporting a productive infection of HBV. Cells are grown in Williams' E medium (Gibco, Paisley, UK) supplemented with 5% fetal calf serum (FCS; Hyclone, Logan, UT). Cells were treated with 10% C-MSC-CM for 48h and lysed by Trizol for RNA isolation. cDNA was prepared using Promega's AMV reverse transcriptase following standard protocols. Real-time quantitative PCR was performed using primers to target HBs region of the HBV genome. Housekeeping gene GAPDH was included as reference gene.

10% C-MSC-CM significantly reduced HBV mRNA level by 67±4% (p<0.01) (Figure 11).