WO2002029098A2 - Genetic factors affecting the outcome of viral infections - Google Patents

Abstract

There is disclosed a method of predicting the outcome of a viral infection in a human subject, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the IL10RB gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the IL10RB gene. The method is particularly useful as a genetic screen to predict the outcome of diseases caused by hepatitis B virus, hepatitis C virus and HIV.

Description

Genetic factors affecting the outcome of viral infections

The present invention is concerned with the identification of genetic factors associated with the outcome of viral infections in human subjects. In particular, the invention provides genetic screens which can be used to predict the outcome of viral infection, for example infection with hepatitis B virus, hepatitis C virus and human immunodeficiency virus .

Background

The chronic hepatitis B virus (HBV) infection is a serious, genetically influenced disease for which no fully effective treatments are available. It is estimated that 300 million people worldwide are chronically infected with hepatitis B virus (HBV) . In most populations over 80% subjects rapidly clear the viral infection whereas the remainder become persistent carriers. A large proportion of these carriers, about one million each year, will die from the end stage sequelae of cirrhosis and hepatocellular carcinoma. Limited data from one twin study and some HLA association studies indicate a genetic component to variable outcome of HBV infection. The identification of the genetic factors determining the outcome of HBV infection could lead to innovative therapeutic interventions . It has previously been described that genetic variation in the IL-10 promoter region is associated with the clearance of HBV infection in two populations (WO 00/08215) . Interestingly, the IL-10 promoter allele associated with increased levels of gene expression was associated with clearance of the virus, suggesting that additional IL-10 may facilitate viral clearance rather than the converse.

In order to identify further genes associated with HBV infection the inventors performed a search through a genome-wide scan in 151 Gambian families with 203 affected sib-pairs where disease predisposing genes are likely to be segregating. Only one region of the entire genome, the long arm of chromosome 21, showed statistically significant evidence of linkage. The peak lod score in this region of linkage was at a microsatellite marker located in the intron of the i^'nterleukin 10 receptor B gene (IL-10RB) . Further family based association studies were carried out in the Gambian families used for the genome scan and a further set of Italian families including just over 60 sib-pairs with the cases of persistent HBV infection. In both populations the inventors found a highly significant (p < 0.001) association with a coding change in the IL-10RB gene. There was also association with variants in the flanking interferon- gamma receptor chain-B (IFNGR2) gene. The same ILIORB gene polymorphism was associated with HCV infection in a case control study of Europeans.

Interestingly, the inventors have found that the major gene identified as linked to and associated with HBV infection is also clearly associated with HCV infection. The estimated number of people who are suffering from chronic Hepatitis C virus (HCV) infection is similar to that for hepatitis B. Unlike hepatitis B the very large numbers of these people come from the developed world where blood transfusion and intravenous drug use are common. Up to 80% of subjects infected with HCV will establish a chronic infection and perhaps 20% of subjects with HCV infection will develop cirrhosis. Currently available alpha interferon therapy can be used to control viral replication in up to 40% of those chronically infected with HBV. Fewer patients treated for HCV infection with interferon achieve a sustained remission. There are therefore compelling clinical, public health and commercial reasons for improving the treatment of both hepatitis B and C virus infections and providing more accurate tests for determining prognosis. Identification of non-HLA genetic susceptibility factors provides an important new route to understanding the key biochemical pathways affecting these viral diseases. The inventors have further demonstrated an i'mmunogenetic association between polymorphic variation in the ILIORB gene and the outcome of infection with human immunodeficiency virus (HIV) .

The immunogenetic associations identified by the present inventors have utility in at least two areas. First, the genetic variants shown to be associated with viral disease have utility in predicting the outcome of infection and likely response to treatments. The variants described or flanking polymorphisms in linkage disequilibrium with these genes and variants may also be used to assess these risks and probabilities. Secondly, these genetic associations identify two new molecular targets, the IL-10 receptor and the interferon gamma receptor, for development of new drugs to modulate or prevent disease associated with HBV, HCV and HIV.

Therefore, in accordance with a first aspect the invention provides a method of predicting the outcome of a viral infection in a human subject, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the ILIORB gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the ILIORB gene.

In the context of this application, the term predicting the outcome of a viral infection' may encompass, non-exclusively, predicting susceptibility to development of a persistent infection, also predicting the likelihood that a (non-infected) subject will develop a persistent infection following exposure to a virus and also predicting survival time following infection.

The polymorphic variant of the ILIORB gene will preferably be one for which an association with an aspect of viral disease, such as for example, susceptibility to persistent infection, rate of viral clearance, length of survival following infection. Associations between genetic variation in the IL-10RB gene and aspects of viral disease may be established using family-based or population-based association studies, similar to those described in the experimental reports included herein. An example of a polymorphic variant which has been shown to be associated with an aspect of viral disease is the IL10RB-K47E polymorphism (Legare, 1997) . This polymorphism is a single nucleotide change A to G at nucleotide number 216435 in the genomic sequence deposited under GenBank accession number AP001716.

In one embodiment the method of the invention may be used to predict the outcome of infection with a hepatitis virus, in particular hepatitis B virus (HBV) or hepatitis C virus (HCV) . In a preferred embodiment, the method of the invention may comprise genotyping a human subject for the IL10RB-K47E polymorphism, wherein the presence of at least one A allele is taken as an indication of susceptibility to persistent hepatitis B infection whereas the presence of at least one G allele is taken as an indication of susceptibility to persistent hepatitis C infection. Thus, AA homozygotes are scored as susceptible to persistent HBV infection, GG homozygotes as susceptible to persistent HCV infection and GA heterozygotes as being more susceptible than GG homozygotes and AA homozygotes to persistent infection with HBV or HCV, respectively.

The invention also encompasses a method which comprises genotyping a non-infected subject for the IL10RB-K47E polymorphism, wherein the presence of at least one A allele is taken as an indication that infection with hepatitis B virus will more likely lead to persistent hepatitis B infection whereas the presence of one or more G alleles is taken as an indication that infection with hepatitis C virus will more likely lead to persistent hepatitis C infection.

A ^λnon-infected' subject is taken to mean a subject which shows no sign of infection according to clinically accepted criteria. In a further embodiment the method of the invention may be used to predict the outcome of infection with a human immunodeficiency virus.

The present inventors have observed an association between a polymorphic variant of the ILIORB gene and prolonged survival in HIV infected individuals. Therefore, in a preferred embodiment the. invention provides a method which comprises genotyping a human subject for the IL10RB-K47E polymorphism, wherein the presence of at least one G allele is taken as an indication of prolonged survival following HIV infection.

Also included within the scope of the invention is a method of predicting the likely course of a viral infection and resultant disease in a human subject, the method _^comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the ILIORB gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the ILIORB gene.

This method may, advantageously be used to predict the likely course of an infection with HBV or HCV. In the context of HBV or HCV infection the term ^predicting the likely course of an infection' encompasses, but is not necessarily limited to, predicting the likely rate of disease progression in a human subject infected with HCV or HBV, the rate of disease progression being defined by reference to fibrotic changes in the liver. The actual course of infection may be monitored by measurement of viral load. Fibrotic changes in the liver may be monitored by liver biopsy.

The polymorphic variant of the ILIORB gene is preferably one which is associated with susceptibility to persistent viral infection. Thus, in a preferred embodiment the invention provides a method of predicting the likely course of an infection with HBV or HCV which comprises genotyping the said subject for the IL10RB-K47E polymorphism, wherein the presence of at least one A allele is taken as an indication of susceptibility to persistent HBV infection whereas the presence of at least one G allele is taken as an indication of susceptibility to persistent HCV infection.

In a second aspect, the invention provides a method of predicting the outcome of a viral infection in a human subject, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the INFGR2 gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the INFGR2 gene. In this aspect the polymorphic variant in the IFNGR2 gene is preferably one which is associated with an aspect of viral disease such as, for example, susceptibility to persistent infection, disease progression, rate of viral clearance, length of survival following infection and rate of liver fibrosis in the case of hepatitis virus infections. An example of such polymorphic variants is the IFNGR2+2080 and IFNGR2+2087 polymorphisms. Associations between genetic variation in the IFNGR2 gene and aspects of viral disease may be established using family-based or population-based association studies, similar to those described in the experimental reports included herein.

In one embodiment of this second aspect of the invention the method may be used to predict the outcome of infection with hepatitis virus, in particular HBV or HCV. In preferred embodiments, the method may comprise genotyping the subject for the IFNGR2+2080 polymorphism, in which case the presence of at least one G allele is taken as an indication of susceptibility to persistent HBV infection or it may comprise genotyping the subject for the IFNGR2+2087 polymorphism, in which case the presence of at least one T allele is taken as an indication of susceptibility to persistent infection with HCV or HBV.

In particular embodiments, the above methods may comprise genotyping of a non-infected subject for the IFNGR2+2080 polymorphism, in which case the presence of at least one G allele is taken as an indication of that infection with HBV will more likely lead to persistent infection or genotyping a non-infected subject for the IFNGR2+2087 polymorphism, in which case the presence of at least one T allele is taken as an indication of that infection with either HCV or HBV will more likely lead to persistent infection.

This aspect of the invention also provides a method of predicting the likely course of a viral infection and resultant disease in a human subject, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the INFGR2 gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the INFGR2 gene.

This method is particularly useful for predicting the likely course of infection with HBV or HCV. As defined above, the term predicting the likely course of an infection' encompasses, but is not necessarily limited to, predicting the likely rate of disease progression or rate of liver fibrosis in a human subject infected with HCV or HBV. The polymorphic variant of the IFNGR2 gene is preferably one which is associated with susceptibility to persistent viral infection. Thus, in a preferred embodiment the invention provides a method of predicting the likely course of an infection with HBV or HCV which comprises genotyping the subject for the IFNGR2+2080 polymorphism, wherein the presence of at least one G allele is taken as an indication of susceptibility to persistent hepatitis B infection. In a further preferred embodiment the invention provides a method of predicting the likely course of an infection with HBV or HCV which comprises genotyping the subject for the IFNGR2+2087 polymorphism, wherein the presence of at least one T allele is taken as an indication of susceptibility to persistent hepatitis B virus infection and persistent hepatitis C virus infection.

Particular embodiments of the above-described methods according to the first and second aspects of invention provide genetic screens by which to test for susceptibility to persistent infection with HBV or HCV. Thus, the methods may be used to identify individuals who are ^Λat risk' of developing persistent infection because of their genetic make-up. This may in turn allow early intervention with treatment regimes aimed at either preventing infection or preventing the establishment of persistent infection in infected individuals. For example, testing of uninfected individuals may help to identify those who would benefit from vaccination because they are at increased risk of developing persistent disease if they become infected. Similarly, the methods may be used to test individuals in which infection with HBV and/or HCV has been confirmed in order to identify those who would benefit from particular regimes of treatment because they are at increased risk of developing a persistent infection. In a further aspect, the invention provides a method of predicting response to therapy for a human subject infected with a virus, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the ILIORB gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the ILIORB gene.

This method may advantageously be used to predict response to therapy for human subjects infected with HBV, HCV or HIV. In a preferred embodiment, the method comprises genotyping the subject for the IL10RB-K47E polymorphism.

The invention still further provides a method of predicting response to therapy for a human subject infected with a virus, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the INFGR2 gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the INFGR2 gene.

This method may again be used to predict response to therapy for human subjects infected with HBV or HCV. In preferred embodiments, the method may comprise genotyping the subject for the IFNGR2+2080 polymorphism and/or genotyping the subject for the IFNGR2+2087 polymorphism.

The above-described methods based on screening for polymorphic variation in the ILIORB or IFNGR2 genes may be used to predict the need for or utility of vaccines designed to prevent or modulate HBV or HCV infection, particularly in uninfected individuals who would be genetically ^λat risk' of developing a persistent infection if they were to become infected.

These methods may also be used to predict response to therapeutic treatment of persistent hepatitis infections with therapeutic interferon or interferon derivatives or other anti-viral agents.

In addition to screening for polymorphic variants in the ILIORB gene or the IFNGR2 gene, for all aspects of the invention it is also contemplated to screen for the presence or absence of genetic variants in linkage disequilibrium with polymorphic loci in these genes and also methods which involve screening for genetic variants in close physical proximity to polymorphic loci in the ILIORB or IFNGR2 genes.

As would be readily apparent to persons skilled in the art of human genetics, "linkage disequilibrium" occurs between a marker polymorphism (e.g. a DNA polymorphism which is ^xsilent' ) and a functional polymorphism (i.e. genetic variation which affects phenotype or which contributes to a genetically determined trait) if the marker is situated in close proximity to the functional polymorphism. Due to the close physical proximity, many generations may be required for alleles of the marker polymorphism and the functional polymorphism to be separated by recombination. As a result they will be present together on the same haplotype at higher frequency than expected, even in very distantly related people. As used herein the term "close physical proximity" means that the two markers/loci in question are close enough for linkage disequilibrium to be likely to arise .

In accordance with the invention, the step of determining the genotype of an individual at a given polymorphic locus, commonly known as ^genotyping' may advantageously comprise screening for the presence or absence in the genome of the subject of both the common allele and the variant allele or may comprise screening for the presence or absence of either individual allele, it generally being possible to draw conclusions about the genotype of an individual at a polymorphic locus having two alternative allelic forms just by screening for one or other of the specific alleles. For example, in order to determine the genotype of an individual for the IL10RB-K47E polymorphism one would preferably screen for both the A allele and the G allele but might conceivably screen for either individual allele.

In accordance with the invention, genotyping can be carried out using any suitable methodology known in the art and it is to be understood that the invention is in no way limited by the precise technique used to perform such genotyping. Known techniques for scoring polymorphic variants are listed below:

Polymorphic variants may be scored by sequencing regions of the genome. Typically, a region of the genomic DNA including the polymorphic locus will first be amplified, for example using PCR, in order to provide a template for sequencing.

Polymorphic variants by also be scored by sequence-specific oligonucleotide hybridisation. In this technique, a DNA fragment spanning the polymorphic locus is amplified using PCR. The PCR products are then blotted onto a suitable membrane and hybridised with labelled allele-specific oligonucleotide probes.

A further technique is the ligase detection reaction or LDR, which is based on the theory that ligation between two oligonucleotides will only take place if bases on either side of the junction are complementary to the opposite strand. This technique is preferred for genotyping the IL10RB-K47E polymorphism.

In addition to the above, SNPs are commonly scored using PCR-based techniques, such as the amplification refractory mutation system (ARMS) described by Newton et al . , 1989. This method generally involves performing DNA amplification reactions using genomic DNA as the template and two different primer pairs, the first primer pair comprising an allele-specific primer which under appropriate conditions is capable of hybridising selectively to the wild type allele and a non allele- specific primer which binds to a complementary sequence elsewhere within the gene in question, the second primer pair comprising an allele-specific primer which under appropriate conditions is capable of hybridising selectively to the variant allele and the same non allele-specific primer. This method may sometimes be referred to as PCR-SSP rather than ARMS (see Bunce et al . , 1995).

If the SNP results in the abolition or creation of a restriction site then genotyping can be carrying out by performing PCR using non-allele specific primers spanning the polymorphic site and digesting the resultant PCR product using the appropriate restriction enzyme (also known as PCR-RFLP) . Restriction fragment length polymorphisms, including those resulting from the presence of a single nucleotide polymorphism, may also be scored by digesting genomic DNA with an appropriate enzyme then performing a Southern blot using a labelled probe corresponding to the polymorphic region (see Sambrook et al . , 1989) . Further known techniques for the scoring of single nucleotide polymorphisms (see review by Schafer and Hawkins, 1998) include mass spectrometry, particularly matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, Roskey et al. 1996), single nucleotide primer extension (Shumaker et al. 1996; Pastinen et al. 1997) and DNA chips or microarrays (Underhill et al. 1996; Gilles et al. 1999). The use of DNA chips or microarrays could enable simultaneous genotyping at many different polymorphic loci in a single individual or the simultaneous genotyping of a single polymorphic locus in multiple individuals.

The known techniques for scoring polymorphisms are of general applicability and it would therefore be readily apparent to persons skilled in the art that known techniques could be adapted for the scoring of the ILIORB and IFNGR2 polymorphism described herein. As would be readily apparent to those skilled in the art, genotyping is generally carried out on genomic DNA prepared from a suitable tissue sample obtained from the subject under test. Most commonly, genomic DNA is prepared from a sample of whole blood, according to standard procedures which are well known in the art.

The genetic associations observed by the present inventors identify the IL-10 receptor and the interferon gamma receptor as novel molecular targets for therapeutic intervention in the treatment and/or prevention of hepatitis virus infection. In the case of the IL-10 receptor and as set out more fully in the example the present inventors have demonstrated that the ILI0RB-K47E polymorphism has a direct effect on the function of the receptor.

Thus, in a further aspect the invention contemplates use of the IL-10 receptor in the identification of chemical compounds which are capable of preventing or modulating infection with hepatitis virus or HIV and also use of the interferon gamma receptor in the identification of chemical compounds which are capable of preventing or modulating infection with hepatitis virus. In particular, the invention contemplates methods of screening for compounds which block or enhance activation of the IL- 10/IL-lO receptor signalling pathway or the IFNγ/IFNγ receptor signalling pathway.

A variety of methods may be used to identify compounds which either block or enhance activation of the IL-lO/IL-10 receptor signalling pathway. In a particular embodiment the invention provides a cell- based screening method for identifying compounds having potential pharmacological activity in the prevention or modulation of infection with hepatitis virus or HIV. The method comprises exposing a cell expressing the IL-10 receptor to a test compound and measuring an indicator of IL-10 receptor signalling in the presence and absence of the test compound. An increase in IL-10 receptor signalling in the presence of the test compound is an indication that the test compound has potential pharmacological activity in the prevention or modulation of infection with hepatitis virus or HIV.

Indicators of IL-10 receptor signalling include activation of signalling molecules such as the Stat transcription factors (Statlα, Stat 3 and possibly

Stat5) and Tyk2. Methods for the measurement of these indicators are known in the art and described, for example, by Kotenko et al., EMBO J. 16: 5894-5903 (1997). Kotenko et al. measure Stat activation by EMSA using a suitable target DNA sequence and an anti- STATS antibody.

The cell expressing the IL-10 receptor can be any cell which expresses a functional IL-10 receptor on the surface thereof, a functional receptor being one capable of intracellular signalling in the presence of IL-10. This definition encompasses variant IL-10 receptor proteins which exhibit functional receptor signalling in the presence of IL-10. Most preferably the cell expressing the IL-10 receptor will be a host cell, for example a COS cell, transfected with DNA encoding the two chains of the IL-10 receptor. Kotenko et al ( ibid) have previously shown that such cells become responsive to exogenously applied IL-10, indicating that the IL-lO/IL-10 receptor signalling pathway is active in such cells. Measurement of indicators of IL-10 receptor signalling may require disruption of the cell, as described by Kotenko et al, ibid.

The above methodology may be used to screen for compounds which affect IL-10 receptor signalling by acting directly on the 11-10 receptor. The invention also provides a related screen in which the cell expressing the IL-10 receptor is exposed to IL-10 and then measuring an indicator of IL-10 receptor signalling in the presence and absence of the test compound. This screen may be used to identify compounds which modulate IL-lO/IL-10 receptor signalling.

Although initially it was believed that only IL- 10 was a ligand for the IL10 receptor it is now clear that IL-22 (also known as IL-TIF) is a further ligand for the IL-10 receptor (Kotenko et al. J Biol Chem. 276, 2725-32 (2001)). The invention can also therefore be used to identify pathways simulated through the IL10 receptor by IL-22 as affecting the risk of hepatitis infection outcome. Thus the invention also provides a related screen in which the cell expressing the IL-10 receptor is exposed to IL-22 and then measuring an indicator of IL-10 receptor signalling in the presence and absence of the test compound. The recognition that IL-22 is a ligand for the IL-10 receptor identified in this invention also teaches the possibility of using this recently identified cytokine to stimulate the IL10 receptor pathway in patients with hepatitis B or other viral infections .

A similar screen to that described above may be performed to screen for compounds which modulate

IFNγ/IFNγ receptor signalling. Thus the invention further contemplates a screening method for identifying compounds having potential pharmacological activity in the prevention or modulation of infection with hepatitis virus. The method comprises exposing a cell expressing the IFNγ receptor to a test compound and measuring an indicator of IFNγ receptor signalling in the presence and absence of the test compound. An increase in IFNγ receptor signalling in the presence of the test compound is an indication that the test compound has potential pharmacological activity in the prevention or modulation of infection with hepatitis virus .

Indicators of IFNγ receptor signalling include cell surface expression of MHC class I molecules and activation of signalling molecules such as the Stat transcription factor Static.. Methods for the measurement of these indicators are known in the art and described, for example, by Kotenko et al., EMBO J. 16: 5894-5903 (1997) .

The cell expressing the IFNγ receptor can be any cell which expresses a functional IFNγ receptor on the surface thereof, a functional receptor being one capable of intracellular signalling in the presence of IL-10. This definition encompasses variant IFNγ receptor proteins which exhibit functional receptor signalling in the presence of IFNγ. Most preferably the cell expressing the IFNγ receptor will be a host cell, for example a COS cell, transfected with DNA encoding the two chains of the IFNγ receptor, Hu-IFN- γRl and Hu-IFN-γR2.

The invention also provides the related screen in which the cell expressing the IFNγ receptor is exposed to IFNγ and then measuring an indicator of IFNγ receptor signalling in the presence and absence of the test compound. This screen may be used to identify compounds which modulate IFNγ/IFNγ receptor signalling.

There is no limitation on the types of candidate compounds to be tested in the screening methods of the invention. Test compounds may include compounds having a known pharmacological or biochemical activity, compounds having no such identified activity and completely new molecules or libraries of molecules such as might be generated by combinatorial chemistry. Compounds which are DNA, RNA, PNA, polypeptides or proteins are not excluded.

Also within the scope of the invention are compounds identified as having potential pharmacological activity in the treatment of hepatitis virus and/or HIV infection using the screening methods of the invention. Such compounds may be useful as lead compounds in the development of pharmaceutical agents for the treatment of hepatitis virus and/or HIV infection.

In a still further aspect the invention provides a method of treating a persistent hepatitis virus infection which method comprises administering to a patient in need thereof a therapeutically effective amount of a medicament comprising interleukin-10 or IL-22 and a pharmaceutically acceptable carrier, diluent or excipient therefor.

The invention further provides for use of IL-10 or IL-22 for the manufacture of a medicament for the treatment of persistent hepatitis virus infection.

In a preferred embodiment, the invention provides for use of IL-10 or IL-22 in the treatment of chronic hepatitis B virus infection.

The invention also contemplates use of either IL- 10 or IL-22 and an interferon and combinations thereof including IL-10 and interferon IL-22 and interferon or any combination of two or more for the treatment of hepatitis virus infection, particularly chronic HBV infection. In this embodiment, the IL-10/ IL- 22/interferon combination (s) are preferably administered in pegylated form, i.e. complexed with polyethylene glycol. The interferon may be a type I or type II interferon. The invention further contemplates the use of IL- ΪO/interferon or IL-22/interferon chimeras in the treatment of hepatitis virus infection, particularly chronic HBV infection.

The present invention will be further understood with reference to the following experimental report.

Experimental 1-Hepatitis Virus

Families and Subjects Multicase families with two or more siblings who are chronic HBV carriers were identified from the databases of previous studies throughout The Gambia. Healthy carriers were also recruited for the case control study by screening of local blood donors. Ethical approval for this Gambian study was granted by the local Scientific Co-ordinating Committee and the Joint Gambian Government/Medical Research Council Ethical Committee. All siblings older than 10 years (not vaccinated against Hepatitis B) and parents of the previously known chronic HBsAg carriers were invited to participate in the study and give a blood sample. A questionnaire was completed to document the family status and vaccine history. Written informed consent was obtained from all participants. Individuals tested for HBsAg positive and anti Core IgM negative were defined as HBV chronic carriers. HBV surface antigen was detected with reserve passive agglutination (Hepto-test, Wellcome Diagnostics) . All negative samples were further re-tested for hepatitis B surface antigen with an immunoradiometric assay (Sorin Biomedical) to achieve highest possible sensitivity. An HBeAg/anti-HBe immunoradiometric assay kit (DiaSorin) was used according to manufacturers instructions for detection of HBeAg, Anti-HBe, total Anti-Core, and Anti-Core IgM.

The main ethnic groups represented in the study were Mandinka (65%) Wollof (8%), Fula (5%), Jola (7%), Sarahule (7%) and Serere (8%). 1180 people from different areas and ethnic groups of The Gambia were bled during the study. 138 Gambian families with 200 affected sib-pairs were selected from the genotyping.

Genotyping and Analysis

Genomic DNA was extracted from peripheral blood lymphocytes from 138 families with two or more siblings who are chronic HBV carriers. All parental DNA samples were genotyped when available: 55 families had both parents available, 68 families had one parent available, and 15 families had neither parent available. The genome-wide scan with an average intermarker spacing of 15-20 cM was performed by use of over 300 highly polymorphic microsatellite markers from the ABI PRISM™ linkage mapping set version 2 (PE Biosystems) . These loci were amplified with the fluorescently labelled primers in separate PCRs, and the products were then multiplexed into panels by being pooled before electrophoresis on ABI 373A sequencers (PE Biosystems) which use a laser to detect fluorescence-labelled PCR product. DNA fragment sizing was performed using the GENESCAN™ 672 AND Genotyper™ software programs (PE Biosystems) . Single point lod scores and probabilities were calculated using the SIBPAIR-ANALYZE software package. 90 affected sib-pairs from 62 families were initially used for the 1^st round genome scan. 28 markers with suggestive linkage (LOD score > 1.0) were genotyped for the 2^nd genome scan with 82 affected sib-pairs of 56 families from the same population. The region of LOD score > 2 on chromosome 21 were further investigated with additional microsatellite markers. The higher-density mapping was performed on the original samples and on additional 20 families consisted of 28 affected sib-pairs.

Results

In an attempt to define genetic factors that determine the outcome of hepatitis B (and possibly also C) virus infection, a search was made throughout the entire genome for genetic linkage to chronic HBV infection with over 300 polymorphic microsatellite markers in West African families. Through a major two year field effort it was possible to recruit two hundred and three affected sibling pairs and many of their parents in The Gambia, extract DNA from blood samples and transport these to Oxford. A genome wide linkage analysis was performed and single point lod scores and probabilities calculated using the SIBPAIR- ANALYZE software package. Only one region of the entire genome, on the long arm of chromosome 21 showed a lod score in excess of 2. The peak lod score in this region of linkage was at a microsatellite marker located in the intron of IL-10 receptor B (also termed IL-10R2) gene. This gene codes for what was formerly known as an orphan type II cytokine receptor but is now known to be a necessary component of IL-10 receptor. The gene is about 30 Kb in size. Significant linkage (LOD score = 3.55, p-value = 0.000026) was found for this marker within the interleukin-10 receptor B (IL/10R2/I110RB) gene on chromosome 21q22.1 (Table 1). The region of linkage contains four interesting positional candidate genes in a 180 KB segment of DNA. These are the two components of the interferon α/β- receptor (IFNAR1 and IFNAR2), the second component of the interferon γ-receptor (IFNGR2) and, as stated above, the second chain of the IL-10 receptor (ILIORB) . These four genes belong to the class II cytokine receptor (CRII) family that is a group of structurally, genetically and functionally related proteins sharing D200 domains of 200 amino acids and a similar intron/exon structure encoding the intracellular domain. The four genes span about 180kb with a gene order of IFNAR2-IL10R2-IFNAR1-IFNGR2 from centromere to telomere. Although this cluster has been fully sequenced no other expressed sequence has been identified in this region. The gene cluster is polymorphic and sequence variation has been characterised and reported in the literature. The inventors chose to genotype these published polymorphisms listed in Table 2 to search for association with these genes. Table 3 summarises the findings from family-based association studies with these polymorphisms. Both the Gambian families used for the genome scan and a further set of Italian families with cases of persistent HBV were studied. The Italian families included just over 60 affected sibling pairs and parents. In both populations there is one highly significant (P <0.001) association with a coding change in the IL-10RB gene. This change is a substitution of A to G in exon 2 of the ILIORB gene resulting in an amino acid change from lysine to glutamate at position 47 of the chain (denoted I110RB- K47E) . This variant is in the extracellular domain of the receptor. Less strong association was observed for two non-coding changes in the flanking interferon gamma receptor-2 gene and IFNGR2+2080 and +2087, : these two single nucleotide substitutions are in the 3' UTR of IFNGR2 gene (Table 3). Analysis of linkage disequilibrium between the IL-10RB and the IFNGR2 markers found some weak but non-significant allelic association in each population. These polymorphisms were examined further in case-control samples from Gambians over 10 years of age who were persistent carriers of HBsAg or who had cleared the virus. The IL-10RB gene variant showed again a significant association with HBsAg carriage (Table 4), providing further evidence that the G a lele of this gene is associated with viral clearance .

Association with HCV infection No family or twin studies have been conducted in HCV infection and to date no family clustering has been reported. Taken with the low rates of intra- familial transmission, these figures suggest that sib- pair analysis of sufficient power to establish a genetic component will be impracticable. Therefore, the polymorphisms identified by the present inventors from the genomic screen in HBV were tested in case- control studies of HCV infection.

The polymorphisms listed in Table 2 were examined also in case-control studies of HCV infection in Europeans. The IL10RB-K47E variant showed an association with susceptibility to persistent HCV infection. Other flanking polymorphisms shown in the table failed to show an association, supporting the relevance of the association with the IL-10RB gene (Table 5) in both types of chronic viral hepatitis. Notably the allelic associations are converse in HBV and HCV infection, with the A allele predisposing to HBV persistence and the G allele predisposing to HCV persistence.

Discussion The genome wide linkage study has identified a single peak of significant linkage on the long arm of chromosome 21. Interestingly, genes on chromosome 21 were speculated to be relevant to HBV persistence over 30 years ago following the observation that cases of trisomy 21 had a high rate of viral carriage. The inventors have observed strong allelic association with one of the first positional candidate genes studied in this region - the second chain of the IL-10 receptor. In retrospect this is a particularly plausible gene for persistent HBV infection given the inventor' s earlier finding of an association of HBV persistence with promoter variants of the IL-10 gene. Association with sequence changes in the flanking IFNGR2 gene were also observed. The functional relevance of the IL-10RB gene is further supported by the nature of the variant studied, a radical change from a negatively to a positively charged amino acid. Further support for the role of this gene is provided by the findings in persistent hepatitis C virus infection. The same substitution is also associated with this other major form of persistent hepatitis, but intriguingly with a converse allelic association. This finding adds further weight to the evidence presented that the IL-10RB gene is a major locus for susceptibility to persistent HBV. Similar levels of allelic association are observed in the Gambian and Italian families indicating that this appears to be an important locus in Europeans as well as Africans. Table. 1 Linkage analysis of Gambian families at Chromosome 21q22 (SibPair-Analyze programme output single point analysis)

Table. 2 Polymorphisms at Interferon/Interleukin-10 Receptor Gene Cluster (21q22.1)

Table. 3a Family Association (TDT) in African and European HBV

Table. 3b Transmission data in the TDT in African and European HBV families (T/NT = no. of times transmitted/no. non-transmitted)

Table. 4 IL10RB-K47E and HBV infection in Gambians: case-control study data

Overall genotype χ²=7.89 p=0.019 (2df)

AG genotype χ²=7.0 p=0.008 OR=0.56 (0.34<OR<0.93)

Table. 5a Case-control Studies in European HCV

Table. 5b IL10RB-K47E and HCV infection status

Overall genotype χ²=13.44 p=0.001 (2df)

AG genotype 42 p=0.0004 0R=2.36(1.42<0R<3.9)

G allele χ²=9.8 p=0.0017 OR=1.88(1.24<OR<2.86)

Experimental 2-HIV

Patient cohorts

In collaboration with the MRC programme on AIDS in Uganda two cohorts of Ugandans with HIV infection have been followed for a period of 0-10 years. One is a rural cohort in the Masaka district and the other a peri-urban cohort from in or near Entebbe. Both cohorts have been described previously (Nunn et al. BMJ 1997, 315: 767-71; French et al. Lancet 2000, 355: 2106-11) .

Experimental approach

HIV serostatus was determined by ELISA tests and 806 HIV infected individuals followed in Entebbe and 160 HIV infected individuals followed in Masaka district. All individuals were genotyped for the IL10RB-K47E variant and survival to death measured and analysed using Kaplan-Meier statistical techniques.

Results

In a survival analysis of the Masaka cohort individuals of genotype AG survived longer than those of genotype AA (P=0.057 using the Tarone-Ware test of significance) . GG homozygotes were too rare for risk to be adequately defined. In the study in Entebbe, individuals with genotype GA also survived longer that individuals of genotype AA (logrank test, one tailed P=0.027). No statistically significant association of IL10RB-K47E genotype was found with susceptibility to HIV infection. However, the consistent observation of prolonged survival of GA heterozygotes compared to AA homozygotes in these two Ugandan studies provided a method of predicting outcome of HIV infection using such genotyping. Experiment 3

In the case of the IL-10 receptor we demonstrated that the IL10RB-K47E polymophism has a direct effect on the function of the receptor.

Experiments were performed to elucidate the mechanism underlying the genetic association of the IL-10R K47E polymorphism and persistent hepatitis infection. The primary objective was to ■ investigate the effect of the different genotypes on IL-10 signal transduction and function. Lipopolysaccharide (LPS) is know to stimulate macrophages to induce TNF-alpha release, that can be measured in supernatants, and this process is inhibited by IL10 acting through the IL10 receptor.

Blood (10ml) was taken from healthy volunteers and DNA isolated and genotyped for the ILIORB K47E change, a A-G nucleotide change. Monocyte-derived macrophages were isolated and cultured from the same individuals by standard methods. The numbers of viable cells were then counted with trypan blue and adjusted to a concentration of 5x10^ per ml. lOOμl of these cells was added per well, on a 96 well plate. The isolated macrophage had IL-10 added at a variety of concentrations and the plate was then incubated for one hour. This was to allow the IL-10 to effect the macrophages. After this time LPS (50μg) was added to each well. The wells were then made to a constant total volume of 200μl per well with complete medium. This was then incubated for 24 hours and the amount of TNFα produced assayed by a sandwich ELISA. Varying amounts of IL-10 were used as shown in the table, 0-0.5 ng/ml . The experiment was performed with cells from 22 healthy subjects of different genotypes, see table. The percentage inhibitions of TNFα in response to LPS of each subject (including genotype) is shown for 7 individuals of genotype AA, 9 of genotype AG and 6 of genotype GG (table 6) . Table . 6

These results show that IL-10 is a potent inhibitor of TNFα production, as expected, but importantly there is significantly more inhibition in those with the GG, than the GA and the AA genotype of the ILIORB gene (Mann-Whitney test, P < 0.05). Thus, the glutamate variant at position 47 of the ILIORB chain, corresponding to the nucleotide G, is associated with more IL10 receptor signalling activity. This is the variant associated with resistance to persistent hepatitis B virus infection- implying that more IL-10, or more IL-22, and in particular more activity of the IL-10 receptor signalling pathway is protective against persistent hepatitis B virus infection. This information is clearly of value in determining whether to use IL10 receptor pathway stimulators or blockers in the management of persistent hepatitis B virus infection, and in persistent hepatitis C virus and in other viral infections.

References

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Claims

CLAIMS :

1. A method of predicting the outcome of a viral infection in a human subject, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the ILIORB gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the ILIORB gene.

2. A method as claimed in claim 1 wherein the viral infection is a hepatitis virus infection.

3. A method as claimed in claim 2 wherein the viral infection is a hepatitis B virus infection or a hepatitis C virus infection.

4. A method as claimed in claim 3 wherein the polymorphic variant of the ILIORB gene is one which is associated with susceptibility to persistent viral infection.

5. A method as claimed in claim 4 which comprises genotyping the said subject for the

IL10RB-K47E polymorphism, wherein the presence of at least one A allele is taken as an indication of susceptibility to persistent hepatitis B infection whereas the presence of at least one G allele is taken as an indication of susceptibility to persistent hepatitis C infection.

6. A method as claimed in claim 4 which comprises genotyping a non-infected subject for the IL10RB-K47E polymorphism, wherein the presence of at least one A allele is taken as an indication that infection with hepatitis B virus will more likely lead to persistent hepatitis B infection whereas the presence of at least one G allele is taken as an indication that infection with hepatitis C virus will more likely lead to persistent hepatitis C infection .

7. A method as claimed in claim 1 wherein the viral infection is a human immunodeficiency virus infection.

8. A method as claimed in claim 7 wherein the^" polymorphic variant in the ILIORB gene is one which is associated with prolonged survival of HIV infected individuals.

9. A method as claimed in claim 8 which comprises genotyping the said subject for the IL10RB-K47E polymorphism, wherein the presence of at least one G allele is taken of an indication of prolonged survival following HIV infection.

10. A method of predicting the likely course of a viral infection and resultant disease in a human subject, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the ILIORB gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the ILIORB gene.

11. A method as claimed in claim 10 wherein the viral infection is a hepatitis virus infection.

12. A method as claimed in claim 11 wherein the viral infection is a hepatitis B virus infection or a hepatitis C virus infection.

13. A method as claimed in claim 12 wherein the polymorphic variant of the ILIORB gene is one which is associated with susceptibility to persistent viral infection.

14. A method as claimed in claim 13 which comprises genotyping the said subject for the

IL10RB-K47E polymorphism, wherein the presence of at least one A allele is taken as an indication of susceptibility to persistent hepatitis B infection whereas the presence of at least one G allele is taken as an indication of susceptibility to persistent hepatitis C infection.

15. A method of predicting the outcome of a viral infection in a human subject, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the INFGR2 gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the INFGR2 gene.

16. A method as claimed in claim 15 wherein the viral infection is a hepatitis virus infection.

17. A method as claimed in claim 16 wherein the viral infection is a hepatitis B virus infection or a hepatitis C virus infection.

18. A method as claimed in claim 17 wherein the polymorphic variant of the IFNGR2 gene is one which is associated with susceptibility to persistent viral infection.

19. A method as claimed in claim 18 which comprises genotyping the said subject for the

IFNGR2+2080 polymorphism, wherein the presence of at least one G allele is taken as an indication of susceptibility to persistent hepatitis B infection.

20. A method as claimed in claim 18 which comprises genotyping the subject for the IFNGR2+2087 polymorphism, wherein the presence of at least one T allele is taken as an indication of susceptibility to persistent hepatitis B virus infection and persistent hepatitis C virus infection.

21. A method as claimed in claim 18 which comprises genotyping a non-infected subject for the IFNGR2+2087 polymorphism, wherein the presence of at^' least one T allele is taken as an indication that infection with either hepatitis B virus or hepatitis C virus will more likely lead to persistent infection.

22. A method as claimed in claim 18 which comprises genotyping a non-infected subject for the IFNGR2+2080 polymorphism, wherein the presence of at least one G allele is taken as an indication that infection with hepatitis B virus will more likely lead to persistent hepatitis B infection.

23. A method of predicting the likely course of a viral infection and resultant disease in a human subject, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the INFGR2 gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the INFGR2 gene.

24. A method as claimed in claim 23 wherein the viral infection is a hepatitis virus infection.

25. A method as claimed in claim 24 wherein the viral infection is a hepatitis B virus infection or a hepatitis C virus infection.

26. A method as claimed in claim 25 wherein the polymorphic variant of the IFNGR2 gene is one which is associated with susceptibility to persistent viral infection.

27. A method as claimed in claim 26 which comprises genotyping the said subject for the IFNGR2+2080 polymorphism, wherein the presence of at least one G allele is taken as an indication of susceptibility to persistent hepatitis B infection.

28. A method as claimed in claim 26 which comprises genotyping the subject for the IFNGR2+2087 polymorphism, wherein the presence of at least one T allele is taken as an indication of susceptibility to persistent hepatitis B virus infection and persistent hepatitis C virus infection.

29. A method of predicting response to therapy for a human subject infected with a virus, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the ILIORB gene or of one or more polymorphic variants in linkage disequilibrium with or in close physical proximity to a polymorphic locus in the ILIORB gene.

30. A method as claimed in claim 29 wherein the viral infection is a hepatitis virus infection.

31. A method as claimed in claim 30 wherein the viral infection is a hepatitis B virus infection or a hepatitis C virus infection.

32. A method as claimed in claim 29 wherein the viral infection is a human immunodeficiency virus infection.

33. A method as claimed in any one of claims 29 to 32 which comprises genotyping the said subject for the IL10RB-K47E polymorphism.

34. A method of predicting response to therapy for a human subject infected with a virus, the method comprising screening for the presence or absence in the genome of said subject of one or more polymorphic variants of the INFGR2 gene or of one or more polymorphic variants in linkage disequilibrium With or in close physical proximity to a polymorphic locus in the INFGR2 gene.

35. A method as claimed in claim 34 wherein the viral infection is a hepatitis virus infection.

36. A method as claimed in claim 35 wherein the viral infection is a hepatitis B virus infection or a hepatitis C virus infection.

37. A method as claimed in any one of claims 34 to 36 which comprises genotyping the said subject for the IFNGR2+2080 polymorphism.

38. A method as claimed in any one of claims

34 to 37 which comprises genotyping the said subject for the IFNGR2+2087 polymorphism.

39. A method as claimed in claim 30 or claim 36 for use in predicting the need for or utility of vaccines designed to prevent or modulate hepatitis B or hepatitis C virus.

40. A method as claimed in claim 30 or claim 36 for use in predicting response to therapeutic interferon or interferon derivatives in persistent hepatitis virus infections.

41. A method of identifying a compound having potential pharmacological activity in the prevention or modulation of infection with hepatitis virus or HIV, which method comprises: providing a cell expressing the IL-10 receptor; and measuring an indicator of IL-lO/IL-10 receptor signalling in the presence or absence of a test compound, wherein an increase in IL-lO/IL-10 receptor signalling in the presence of the test

Compound is an indication that the test compound has potential pharmacological activity in the prevention or modulation of infection with hepatitis virus or HIV.

42. A method as claimed in claim 41 wherein the step of measuring an indicator of IL-lO/IL-10 receptor signalling in the presence or absence of a test compound is carried out in the presence of exogenous IL-10 or IL-22.

43. A method as claimed in claim 41 or claim 42 wherein the indicator of IL-10 receptor signalling is activation of a Stat transcription factor or activation of Tyk2.

44. A method of identifying a compound having potential pharmacological activity in the prevention or modulation of infection with hepatitis virus, which method comprises: providing a cell expressing the IFNγ receptor; and measuring an indicator of IFNγ/IFNγ receptor signalling in the presence or absence of a test compound, wherein an increase in IFNγ/IFNγ receptor signalling in the presence of the test compound is an indication that the test compound has potential pharmacological activity in the prevention or modulation of infection with hepatitis virus.

45. A method as claimed in claim 44 wherein the step of measuring an indicator of IFNγ/IFNγ receptor signalling in the presence or absence of a test compound is carried out in the presence of IFNγ.

46. A method as claimed in claim 44 or claim 45 wherein the indicator of IFNγ/IFNγ receptor signalling is cell-surface expression of MHC class I molecules or activation of Stat lα.

47. A method of treating a persistent hepatitis virus infection which method comprises administering to a patient in need thereof a therapeutically effective amount of a medicament comprising IL-10 or IL-22 and a pharmaceutically acceptable carrier, diluent or excipient therefor.

48. A method as claimed in claim 47 wherein the medicament comprises two or more of any of IL- 10, interferon and IL-22.

49. A method as claimed in claim 48 wherein the interferon is a type I interferon.

50. A method as claimed in claim 48 wherein the interferon is a type II interferon.

51. A method as claimed in any one of claims 47 to 50 for the treatment of chronic hepatitis B virus infection.

52. Use of IL-10 or IL-22 for the manufacture of a medicament for the treatment of a persistent hepatitis virus infection.

53. Use as claimed in claim 51 wherein the medicament comprises two or more of any of IL-10 and interferon and IL-22.

54. A method as claimed in claim 53 wherein the interferon is a type I interferon.

55. A method as claimed in claim 53 wherein the interferon is a type II interferon.

56. Use as claimed in any one of claims 51 to 55 for the manufacture of a medicament for the treatment of chronic hepatitis B virus infection.