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WO2009035170A1 - Method for diagnosis and induction of resistance to virus - Google Patents

Method for diagnosis and induction of resistance to virus Download PDF

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Publication number
WO2009035170A1
WO2009035170A1 PCT/JP2008/067213 JP2008067213W WO2009035170A1 WO 2009035170 A1 WO2009035170 A1 WO 2009035170A1 JP 2008067213 W JP2008067213 W JP 2008067213W WO 2009035170 A1 WO2009035170 A1 WO 2009035170A1
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Prior art keywords
virus
snp
rac2
hiv
subject
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French (fr)
Inventor
Yasuyoshi Kanari
Masaaki Miyazawa
Shinji Irie
Mario S. Clerici
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ImmunoClin Ltd
Osaka Industrial Promotion Organization
Toppan Inc
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ImmunoClin Ltd
Osaka Industrial Promotion Organization
Toppan Printing Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention is related generally to the analytical testing of the samples obtained from a subject, and more particularly the method for detecting genetic predisposition of a subject to resistance to virus.
  • the invention also provides a chip and kit for detecting such genetic predisposition.
  • the invention further provides an application of identified genetic predisposition to induce resistance to virus infection.
  • biomarkers such as single nucleotide polymorphisms (SNPs), nucleotide insertion mutations, nucleotide deletion mutations, microsatellite markers or other DNA sequence variations.
  • SNPs single nucleotide polymorphisms
  • nucleotide insertion mutations nucleotide insertion mutations
  • nucleotide deletion mutations microsatellite markers or other DNA sequence variations.
  • inherited polymorphisms and somatic mutations in oncogenes or tumor suppressor genes are widely regarded as being indicative of a susceptibility to certain cancers, especially in view of the associations between mutated oncogenes or deleted tumor suppressor genes and certain cancers.
  • some individuals are highly susceptible or resistant to infection, especially viral infection.
  • Prediction of disease susceptibility is beneficial for those possessing predisposing genes in order to avoid unnecessary contacts with known etiological agents, chemicals, or viruses, and to take known and developing preventative means. It is also useful in the design of a vaccine against viral disease or for gene therapy. In addition, prediction of the speed of disease progression may allow an opportunity for individualized, more efficient management or therapeutic intervention.
  • HIV human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • HIV- 1 human immunodeficiency virus type 1
  • HIV-I seroconversion Reference 1, O'Brien et al., "Polygenic and multifactorial disease gene association in man", Annu. Rev. Genet., 2000, vol.34, pp.563-591; Reference 2, O'Brien et al., "Human genes that limit AIDS", Nat. Genet., 2004, vol.36, pp.565-574).
  • MHC major histocompatibility complex
  • AIDS restriction genes can explain only up to 15 % of the host determinants that control HIV I/AIDS (Reference 2, O'Brien et al., "Human genes that limit AIDS", 2004, Nat. Genet., vol.36, pp.565-574; Reference 10, Fellay, J. et al., "A whole-genome association study of major determinants for host control of HIVl", Science, Published online July 19 2007, 10.1126/science.1143767), and the presence of other resistance genes has been predicted (Reference 2, O'Brien et al., "Human genes that limit AIDS", 2004, Nat. Genet., vol.36, pp.565-574).
  • the inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423 (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024; Reference 18, WO2004/035825, International publication of PCT application, 29 April 2004).
  • the inventors of the present invention genotyped 74 HIV-exposed but uninfected and 77 HIV-infected individuals enrolled from the same geographical region.
  • SNPs are significantly associated with ESN individuals under a dominant gene hypothesis at the CardlO, CDC42EP1 and GRAP2 loci (Reference 19, WO2006/067506, International publication of PCT application, 29 June 2006).
  • exact molecular genetic mechanisms that explain the observed resistance of ESN individuals to HIVl acquisition had not been provided.
  • the inventors of the present invention performed expression analyses of all the genes and open reading frames located in the candidate segment of human chromosome 22 and extensive genome sequencing, and identified previously unforeseen association between certain sequence polymorphisms in the Rac2 gene locus and the ESN (HIV- 1-exposed but uninfected or exposed seronegative) status.
  • the sequence polymorphism associated with ESN confers a higher expression levels of this gene, and is associated with poor replication of CCR5-tropic HIV in peripheral blood mononuclear cells.
  • SNPs and specific haplotypes of them are representative of the above sequence polymorphism and thus predictive of a resistance to HIVl infection, and these are designated by the following SNP ID Nos: rs9610683, rs9610682, rs2284037, rs739042, rs2284036, rs739041, ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss7340
  • these SNPs and haplotypes have an effect on the Rac2 gene expression level and HIV replication.
  • these SNPs and haplotypes are useful for detecting genetic predisposition to resistance to virus infection, and also to provide preventative and therapeutic means to confer resistance to virus infection.
  • an aspect of the present invention provides- (l) An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, including: a) genotyping a sample of the subject at a site of at least one SNP selected from the group consisting of SNPs designated by following SNP ID Nos ⁇ (i) rs739041, (ii) rs739042, and (iii) rs2284037; and b) assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP is a resistance allele, wherein the resistance allele is as follows (SNP ID No. : allele) : G) rs739041 : T; (ii) rs739042 : T; (iii) and rs2284037 : T.
  • An ex vivo method for detecting genetic predisposition of a subject to resistance to virus including: a) genotyping a sample of the subject at sites of SNPs designated by following SNP ID Nos: (i) rs2284037, ( ⁇ ) rs739042 and (iii) rs73904l; and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (j) rs2284037 : T; (ii) rs739042 : T; (iii) rs739041 : T.
  • An ex vivo method for detecting genetic predisposition of a subject to resistance to virus including: a) genotyping a sample of the subject at a site of at least one SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, and (iii) rs2284036; and b) assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP or nucleotide polymorphism is a resistance allele, wherein the resistance allele is as follows (SNP ID No. : allele) : (i) rs9610683 : C; (ii) rs9610682 : A; and (iii) rs2284036 : T.
  • [20] (6) A method for inducing or enhancing resistance to virus in a subject, including enhancing the Rac2 gene expression in the subject.
  • the Rac2 gene expression may be enhanced by using a DNA-binding protein, peptide, oligonucleotide, or nucleotide analogue that binds to the Rac2 repressor protein binding site located in the Rac2 gene region 1 (SEQ ID No:32).
  • the Rac2 gene expression may be enhanced by inhibiting the expression of an Rac2 repressor protein by RNAi technique.
  • nucleic acid exhibiting a Rac2 repressor protein binding activity, wherein the nucleic acid is selected from the following (A) and (B)-
  • (B) a nucleic acid having more than 80% identity with the nucleotide sequence of SEQ ID No:32.
  • nucleic acid in the nucleic acid according to the above (l ⁇ ), the nucleic acid may have the following alleles at the SNP sites in the nucleotide sequence of SEQ
  • a method for screening a candidate therapeutic agent against virus including: a) contacting an agent with a nucleic acid fragment having a partial or entire nucleotide sequence of the nucleic acid according to the above (l ⁇ ) or (ll); b) measuring an interaction between the agent and the nucleic acid fragment; and c) selecting the agent having the interaction with the nucleic acid fragment as a candidate therapeutic agent against virus.
  • a method for screening a candidate therapeutic agent against virus including: a) administering an agent to a biological material; b) measuring an expression level of the Rac2 gene in the biological material; and c) selecting the agent enhancing expression level of the Rac2 gene in the biological material as a candidate therapeutic agent against virus.
  • [30] A method for performing clinical trial for prevention, reduction, prophylaxis or treatment against virus, including: a) detecting genetic predisposition of subjects by the method according to any one of the above methods (l) - (5); b) stratifying the subject by the result of the detected genetic predisposition; c) giving a treatment to the stratified subject; and d) assessing the effect of the prevention, reduction, prophylaxis or treatment to the stratified subject.
  • the virus may be one selected from the group consisting of HIV, HCV and
  • the virus may be any virus.
  • HIV HIV or HCV.
  • the virus may be any virus.
  • the virus may be one selected from the group consisting of HIV, HCV and HPV.
  • the virus may be HIV or
  • the virus may be HIV.
  • the virus may be one selected from the group consisting of HIV, HCV and
  • the virus may be any virus.
  • HIV HIV or HCV.
  • the virus may be any virus.
  • the genotyping may be performed by the Invader assay.
  • a chip used in the method according to the above method (2) including a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs2284037, (ii) rs739042 and (iii) rs739041, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:3, 4 and 6.
  • a chip used in the method according to the above method (3) including a base plate and probe sets detecting SNPs and nucleotide polymorphisms designated by the SNP ID Nos (i) ss73405466, (ii) rs9798725, (iii) ss73405467, (iv) rs5995400, (v) rs6000619, (vi) rs5756570, (vii) rs36110509, (viii) rs2899284, Gx) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix
  • a chip used in the method according to the above method (4) including a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs9610683 and (ii) rs9610682, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:i and 2.
  • a chip used in the method according to the above method (5) including a base plate and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, and (iii) rs2284036, wherein the probe sets comprises at least one probe comprises more than 7 bases of partial sequence or partial complementary sequence of the nucleotide sequence selected from the group consisting of SEQ ID NO:l, 2, and 5.
  • a kit used in the method according to the above method (5) including a reagent and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, and (iii) rs2284036, wherein the probe sets comprises at least one probe comprises more than 7 bases of partial sequence or partial complementary sequence of the nucleotide sequence selected from the group consisting of SEQ ID NO:i, 2, and 5.
  • a method for screening a candidate therapeutic agent against virus including: a) administering an agent to a biological material; b) measuring an intensity of a binding between a Rac2 repressor protein and a binding site thereof, in the biological material; and c) selecting the agent inhibiting the binding in the biological material as a candidate therapeutic agent against virus.
  • Figure 1 is an illustration of the distribution of SNPs and nucleotide polymorphisms in the Rac2 locus.
  • Figure 2 shows a result of DNA microarray analysis comparing the changes in gene expression levels during the HIV-I antigenic stimulation in peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • Figure 3 is a coefficient map of linkage disequilibrium (LD) between each pair of SNP alleles.
  • Figure 4 is a graph showing luciferase activities induced by the Rac2 promoter and the polymorphic region 1 indicated in Figure 1.
  • Figure 5 is a graph showing changes in the expression levels of the Rac2 gene in PBMC stimulated with the HIV-I peptides.
  • Figure 6 is a graph showing HIVl p24 concentrations measured at 3 and 5 days after virus inoculation grouped by the Rac2 region 1 genotypes.
  • Figure 7 is an illustration of locations and spans of known genes and open reading frames in the candidate region in human chromosome 22.
  • An aspect of the present invention provides an ex vivo method, a chip and a kit detecting genetic predisposition of a subject to resistance to virus. Further, another aspect of the present invention provides a method, biopharmaceutical and nucleic acid for inducing or enhancing resistance to virus in a subject.
  • virus examples include, but are not limited to, Human immunodeficiency virus (HIV), Human papilloma virus (HPV), Hepatatis C virus (HCV), Human herpes virus (HHV), Cytomegalovirus (CMV), Small round structured virus (SRSV) and Influenza (Flu) virus.
  • HIV Human immunodeficiency virus
  • HPV Human papilloma virus
  • HCV Hepatatis C virus
  • HHV Human herpes virus
  • CMV Human herpes virus
  • CMV Cytomegalovirus
  • SRSV Small round structured virus
  • Influenza virus Influenza virus
  • the virus is HIV, HPV or HCV. More preferably, the virus is HIV. HIV includes HIVl and HIV-2.
  • the term “genetic predisposition” refers to genetic factors which influence the phenotypes of an organism. However, the effect of the genetic predisposition can be modified by environmental conditions.
  • the term "subject”, as used herein, refers to an object for the method, for detecting genetic predisposition.
  • the subject is a primate such as human, monkey and ape. More preferably, the subject is human. Most preferably, the human is Caucasian.
  • the term "resistance to virus”, as used herein, refers to an act or power to prevent virus infection or virus-induced disease progression.
  • the present invention is based on the research demonstrating that particular SNPs and haplotypes located in the Rac2 gene region are significantly associated with ESN (HIV- 1-exposed but uninfected or exposed seronegative) individuals. Summary of the research is described below.
  • the inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423.
  • the inventors of the present invention genotyped 74 ESN and 77 HIV-infected individuals enrolled from the same area of Italy at multiple loci of known SNP (see Figure 7 for their chromosomal locations and Table 1 for complete listing of the observed genotypes).
  • Table 1 is a table showing allele distribution at known SNP loci in chromosome 22 among the ESN and HlVl-infected individuals.
  • Expression levels of all the genes located in the above candidate region were compared between the ESN and HIV-infected individuals by using DNA microarrays. Among all the chromosome 22 genes tested, only a small number were constitutively expressed at high levels or induced upon the stimulation with HIV-I antigens ( Figure 2). One of the induced genes is the Rac2, which encodes a Rho-subfamily small GTPase that is selectively expressed in and involved in the activation of T- helper type-1 (ThI) cells.
  • Rac2 encodes a Rho-subfamily small GTPase that is selectively expressed in and involved in the activation of T- helper type-1 (ThI) cells.
  • the Myh9 and Lgalsl were also induced upon the antigenic stimulation.
  • the chromosomal segments spanning the Myh9, I12rb, Rac2-Pscd4, CardlO, and Lgalsl loci were sequenced (see Figure 7 for the location of the sequenced segments), and the frequencies of ESN and HIV-I -infected individuals possessing a particular allele at each of the observed polymorphic loci were compared. Notable accumulation of sequence polymorphisms with significantly different allele frequencies between the two groups was observed in a segment between the CardlO and Rac2 ( Figure 3 and Table 2).
  • SNPs and nucleotide polymorphisms located in the regions 1 and 2 as shown in Figure 1 are listed in the SEQUENCE LISTING with SEQ ID Nos 1 through 31.
  • the SNP ID numbers for each SEQ ID Nos are: (1) rs9610683, (2) rs9610682, (3) rs2284037, (4) rs739042, (5) rs2284036, (6) rs739041, (7) ss73405466, (8) rs9798725, (9) ss73405467, (l ⁇ ) rs5995400, (11) rs6000619, (12) rs5756570, (13) rs36110509, (14) rs2899284, (15) rs6000618, (16) rs6000617, (17) rs9610677, (18) rs6000616, (19) rs9610676, (20) rs9610675, (21) rs9622582
  • SNPs significantly accumulated in ESN individuals are listed in Table 3.
  • detected alleles are as follows, respectively: C/G, A/G, T/C, T/C, T/C, and C/T.
  • SNPs accumulated in ESN individuals are as follows, respectively: C, A, T, T, T, and T. [Table 3]
  • one aspect of the present invention provides use of the above listed SNPs and nucleotide sequence polymorphisms for diagnosis of resistance to HIV infection.
  • rs739041 SEQ ID No. 6
  • rs739042 SEQ ID No. 4
  • rs2284037 SEQ ID No. 3
  • rs9610683 SEQ ID No. l
  • rs9610682 SEQ ID No. 2
  • rs2284036 SEQ ID No. 5
  • the SNP ID Nos are identification numbers used in dbSNP which is world's largest database for nucleotide variations and is part of the US National Center for Biotechnology Information (NCBI).
  • the "rs” ID number is an identification tag assigned by NCBI to a group of SNPs that map to an identical location. The rs ID number is assigned after submission.
  • the "ss” ID number is simply a unique identifier assigned by NCBI when SNP is submitted to dbSNP.
  • Anyone can download the information about SNPs having SNP ID Nos from the following site! http 7/www.ncbi.nlm.nih. gov/projects/SNP/index.html
  • Haplotypes of the central block consist of SNPs designated by SNP ID Nos. rs2284037, rs739042 and rs739041.
  • Hapotypes of the right block consist of SNPs and nucleotide polymorphisms designated by SNP ID Nos.
  • ss73405466 rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73405482, ss73405484 and ss73405485.
  • the haplotype TTT in the central block were significantly accumulated in ESN individuals.
  • the above polymorphic regions 1 and 2 in the Rac2 locus colocalized with the chromosomal segments that showed >50% sequence homology between humans and mice (HHl and HH2 in Figure l) and one of the intragenic regions previously reported to exhibit elevated cytosine methylation in hematopoietic cells (Reference 20, Ladd, RD.
  • the inventors detected no sequence polymorphisms in the Rac2 promoter. Consistent with the above notions, transfection of the luc constructs harboring the Rac2 promoter resulted in high levels of luciferase expression both in the T- lineage Jurkat and macrophage-lineage THP-I cells (Figure 4). Further, coexistence of the region 1 fragment possessing the CCC haplotype strongly suppressed the luc expression in both types of cells, indicating the presence of a repressor protein binding site within region 1. Importantly, when the region 1 fragment possessing the TTT haplotype coexisted, significantly higher luciferase activities were induced in comparison with those induced with the promoter alone.
  • the Rac2 region 1, but not region 2, haplotypes were also associated with in vivo expression levels of the Rac2 in HIV-I antigen-stimulated PBMC.
  • the levels of Rac2 expression were examined quantitatively by real-time PCR in cultured PBMC, a 2-fold increase of the Rac2 message was observed after 6 hours of stimulation with the HIV-I peptides in the individuals homozygously possessing the TTT haplotype in region 1 ( Figure 5).
  • the levels of Rac2 expression decreased after the antigenic stimulation in cells homozygous for the CCC haplotype.
  • haplotypic differences in region 2 did not affect the Rac2 induction after the antigenic stimulation, again indicating that the polymorphic region 1, but not region 2, harbors a functional regulatory element of the Rac2 expression.
  • the regulatory Rac2 intxon haplotype TTT which is significantly more accumulated among ESN individuals, is associated with a restricted replication of CCR5-tropic HIV-I.
  • the possible gene-dose effect in the induction of Rac2 after the antigenic stimulation ( Figure 5) might explain the apparent differences in median p24 levels between the individuals homozygous and heterozygous for the .TTThaplotype.
  • Rac2 is involved in chemotaxis and phagocytic function of neutrophils and macrohages (Reference 21, Ambruso, D.R. et al., "Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation", Proc Natl Acad Sci U S A, 2000, vol.
  • Rh2 is required for the migration of Bl lymphocytes, a specific subpopulation of B-lymphocytes, from the peritoneal cavity to gut-associated lymphoid tissues (Reference 26, Croker, B.A. et al., "The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-Ia and marginal zone B lymphocytes", J.Immunol., 2002, vol.168, pp.3376-3386), and Bl-derived plasma cells are the predominant sources of pathogen-reactive mucosal IgA (Reference 27, Fagarasan, S.
  • this virus resistance mechanism mediated by the Rac2 gene expression also has effect against all other viruses infected via mucosal route such as Human papilloma virus (HPV), Hepatatis C virus (HCV), Human herpes virus (HHV), Cytomegalovirus (CMV), Small round structured virus (SRSV), Influenza (Flu) virus and the like.
  • HPV Human papilloma virus
  • HCV Hepatatis C virus
  • HHV Human herpes virus
  • CMV Cytomegalovirus
  • SRSV Small round structured virus
  • Influenza Influenza virus and the like.
  • SNP associated with differences in the course of HCV infection is located in the region close to the Rac2 gene (Reference 29, Saito, T., et al., "Genetic variations in humans associated with differences in the course of hepatitis C", Biochemical and Biophysical Research Communication (BBRC), 2004, vol.317, pp.335-341).
  • one aspect of the present invention provides an ex vivo method, a chip and a kit for detecting genetic predisposition of a subject to resistance to virus.
  • one aspect of the present invention provides a method and a biopharmaceutical (Rac2 protein, inhibitor of Rac2 repressor protein, or the Rac2 gene inducer) for inducing or enhancing resistance to virus in a subject by utilizing virus resistance mechanism mediated by Rac2.
  • a biopharmaceutical Rost2 protein, inhibitor of Rac2 repressor protein, or the Rac2 gene inducer
  • one aspect of the present invention provides a method for screening a candidate therapeutic agent against virus by utilizing Rac2 or the Rac2 regulatory region as a target molecule.
  • another aspect of the present invention provides use of the Rac2 repressor protein binding site for a gene therapy to induce resistance to virus in a subject.
  • the ex vivo method for detecting genetic predisposition of a subject to resistance to virus comprises following (a) and (b): (a) is genotyping a sample of the subject at a site of at least one SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos rs9610683, rs9610682, rs2284037, rs739042, rs2284036, and rs73904l; [67] And (b) is assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP or nucleotide polymorphism is a resistance allele, wherein the resistance allele is as follows (SNP ID No.
  • rs9610683 C
  • rs9610682 A
  • rs2284037 T
  • rs739042 T
  • rs2284036 T
  • rs739041 C.
  • rs739041 (i) rs739042, and (iii) rs2284037 are more significant for detecting genetic predisposition to resistance to virus.
  • the term "genotyping” refers to a process of determining the genetic constitutions such as nucleotide sequence of an individual with a biological assay.
  • sample refers to a biological sample or material obtained from a subject, for example a cell, tissue, organ, blood, hair or the like.
  • site refers to a position or location of specific nucleotide or polynucleotide in the genome.
  • nucleotide polymorphism refers to difference of nucleotide sequence between members of a species (or between paired chromosomes in an individual).
  • SNP single nucleotide polymorphism
  • allele refers to a particular form of a gene or DNA sequence at a specific chromosomal location (locus).
  • the ex vivo method of the present invention comprises (a) genotypying a sample of the subject at sites of SNPs and nucleotide polymorphism constituting a haplotypel and (b) assessing the subject as having genetic predisposition of resistance to virus if the haplotype which is significantly accumulated in ESN individuals is present.
  • haplotype refers to a set of closely linked genetic markers present on one chromosome which tends to be inherited together.
  • the haplotypes significantly accumulated in ESN individuals are following three haplotypes:
  • a haplotype including: (SNP ID No. : allele) (i) rs9610683 : Cl and (ii) rs9610682 : Al
  • a haplotype including: (SNP ID No. : allele) (i) rs2284037 : T; (ii) rs739042 : T; and (iii) rs739041 : T; and
  • a haplotype including: (SNP ID No. : allele) (i) rs9610683 : C; (ii) rs9610682 : Al and (iii) rs2284036 : T.
  • any techniques publicly known in the art can be used, including single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis by denaturing high-performance liquid chromatography (DHPLC), direct DNA sequencing, Invader assay, and TaqMan assay.
  • SSCP single-strand conformation polymorphism
  • DPLC denaturing high-performance liquid chromatography
  • direct DNA sequencing Invader assay
  • TaqMan assay TaqMan assay
  • SNP or nucleotide polymorphism can be detected by the single-strand conformation polymorphism (SSCP) analysis in which electrophoretic separation of single -stranded nucleic acids based on subtle differences in sequence (often a single base pair) results in a different secondary structure and a measurable difference in mobility through a gel (Reference 30, Orita, M. et al., "Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction", Genomics, 1989, vol.5(4), pp.874-879).
  • SSCP single-strand conformation polymorphism
  • nucleotide polymorphism can be detected by the heteroduplex analysis by denaturing high-performance liquid chromatography (DHPLC) that is based on DNA heteroduplex formation and separation of heteroduplex from homoduplex molecular species by means of ion-pair reverse phase HPLC (Reference 31, Oefner, P.J. et al., "DNA mutation detection using denaturing high performance liquid chromatography", Current protocols in human genetics, 1998, p.7 -10:l-7 -10 -12).
  • DPLC denaturing high-performance liquid chromatography
  • SNP or nucleotide polymorphism can be detected by direct DNA sequencing, such as the chain termination method (Reference 32, Sanger, F. et al., "DNA sequencing with chain-terminating inhibitors", Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1977, vol.74(12), pp.5463-5467), which are well known in the art.
  • chain termination method reference 32, Sanger, F. et al., "DNA sequencing with chain-terminating inhibitors", Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1977, vol.74(12), pp.5463-5467
  • a subsequence of the Rac2 gene region encompassing the SNPs and nucleotide polymorphisms is amplified and either cloned into a suitable plasmid and then sequenced, or sequenced directly.
  • SNP can be detected by Invader assay.
  • Invader assay three single -stranded DNA chains form a ternary complex with one base-pair overlap.
  • This complex is composed of a DNA target oligonucleotide, which contains the SNP sequence of interest, and two other oligonucleotides.
  • the upstream oligonucleotide is designated as invader oligo and downstream oligonucleotide is designated as a probe.
  • These three oligonucleotide strands hybridize to one another, forming a one base-pair junction causing the 5' end of the probe to form a unhybridized 5' flap.
  • This 5' flap is then cleaved by a Cleavase enzyme, resulting in the release of the 5' flap of the probe.
  • FRET cassette is labeled with a fluorophore (F) and a quencher (Q) so cleavage between them generates a fluorescence signal.
  • FRET cassette is self hybridized at 5' portion, and 5' flap is hybridized with 3' portion of the FRET cassette so as to form a ternary complex with one base -pair overlap.
  • Cleavase enzyme cleavage of 5' end of FRET cassette by Cleavase enzyme causes fluorescence signal (Reference 33, U.S. Patent No. 5,846,717; Reference 34, Lyamichev, V. et al., "Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes", Nature Biotechnology, 1999, vol.l7(3), pp.292-296.).
  • SNP can be detected by TaqMan assay which uses two types (wild type and mutant type) of probes having fluorescence dye and quencher and utilizes 5' to 3' nuclease activity of Taq polymerase.
  • DNA fragment harboring SNP site is amplified by PCR primer.
  • TaqMan probe anneals to the DNA.
  • the PCR primer extends to 5 ' side of the TaqMan probe and there is no mismatch between the TaqMan probe and DNA
  • the TaqMan probe is degraded by the 5' to 3' nuclease activity of the Taq polymerase. With the degradation of the TaqMan probe, fluorescence dye is separated by quencher and fluorescence is generated.
  • the present invention provides a method for inducing or enhancing resistance to virus in a subject. This method comprises enhancing the Rac2 gene expression in the subject.
  • the step of enhancing the Rac2 gene expression can be performed by any technique known in the art, for example, modulation of the Rac2 repressor protein binding site by using a DNA-binding protein, peptide, oligonucleotide, nucleotide analogue or chemical compound.
  • RNAi technique may be used for inhibiting the expression of a Rac2 repressor protein in order to enhance the Rac2 gene expression.
  • the present invention provides use of Rac2 protein in the preparation of a medicament for a treatment or prophylaxis of virus-induced disease.
  • the embodiment of the present invention provides a method for treatment or prophylaxis of virus-induced disease including administering Rac2 protein to a subject.
  • Rh2 protein refers to a full length polypeptide of Rac2 protein, its homologue or functional fragment thereof.
  • the amino acid sequence and coding sequence of Rac2 is registered in Genbank under accession number NM_002872.
  • the present invention provides a nucleic acid exhibiting the Rac2 repressor protein binding activity (the polymorphic region 1) which is selected from the following nucleic acids : (A) a nucleic acid having the nucleotide sequence of SEQ ID No:32; and (B) a nucleic acid having more than 80%, 90% or 95% identity with the nucleotide sequence of SEQ ID No:32.
  • one aspect of the present invention provides a nucleic acid exhibiting reduced Rac2 repressor protein binding activity, which has specific alleles at the sites of SNPs in the nucleotide sequence of SEQ ID No.32 as follows (base number in the SEQ ID No.32 : allele): (i) 132 : C; ( ⁇ ) 144 : A; (iii) 1276 : T; (iv) 1959 : T; (v) 2301 :T, and (vi) 2379 : T.
  • the term "identity”, as used herein, refers to a percentage of identical nucleic acid bases among two nucleotide sequences which are aligned in high order match. The percentage of the identity can be calculated by BLAST program under default setting (Reference 36, Altschul, S. F. et al, "Gapped BLAST and PSI-BLAST: A new generation of protein database search programs", Nucleic Acids Res., 1997, vol.25, pp.3389-3402).
  • the nucleic acid may comprise DNA, RNA or nucleic acid analogs such as uncharged nucleic acid analogs including, but not limited to, peptide nucleic acids (PNAs) which are disclosed in International publication WO 92/20702 (Reference 37).
  • PNAs peptide nucleic acids
  • Such sequences can routinely be synthesized using variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry.
  • the above identified nucleic acid with SNP alleles can have modulated Rac2 repressor protein binding activity.
  • Rac2 repressor protein binding activity can be controlled, thereby affecting the Rac2 expression.
  • the nucleic acid of the present invention can be used as a Rac2 repressor protein binding sequence and can be utilized for a gene therapy in order to enhance the Rac2 gene expression and induce resistance to virus in a subject.
  • the present invention provides a method for screening a candidate therapeutic agent against virus by utilizing the Rac2 repressor protein binding site as a target molecule.
  • This method comprises following: (a) contacting an agent with a nucleic acid fragment having the partial or entire nucleotide of the Rac2 region 1 nucleic acid of the present invention; (b) measuring an interaction between the agent and the nucleic acid fragment; and (c) selecting the agent having the interaction with the nucleic acid fragment as a candidate therapeutic agent against virus.
  • a candidate therapeutic agent refers to a chemical compound, lead compound or biological molecule which has a potential activity to induce resistance to virus in a subject.
  • the present invention provides a method for screening a candidate therapeutic agent against virus. This method comprises the following: (a) administering an agent to a biological material; (b) measuring an expression level of the Rac2 gene in the biological material; and (c) selecting the agent enhancing expression level of the Rac2 gene in the biological material as a candidate therapeutic agent against virus.
  • biological material refers to the material which possesses a fragment of or the entire Rac2 gene or the Rac2 gene regulatory element, for example, DNA fragment, a cell, a tissue, an organ, an organism and individual.
  • the present invention provides a method for performing clinical trial for prevention, reduction, prophylaxis or treatment against virus. This method comprises following: (a) detecting genetic predisposition of subjects by the detecting method of the present invention;
  • the term "clinical trial” refers to any research studies designed to collect clinical data on response to a particular treatment, and includes, but not limited to, phase I, phase II, and phase III clinical trials.
  • stratifying the subject refers to arrangement of each individual in a clinical trial population in a hierarchical order according to any status such as resistance level to virus infection or virus disease progression.
  • the present invention provides a chip or a kit for detecting genetic predisposition of a subject for resistance to virus.
  • the chip comprises base plate and at least one probe detecting SNP or nucleotide polymorphism.
  • the kit comprises reagent and at least one probe detecting SNP or nucleotide polymorphism.
  • the SNP or nucleotide polymorphism is selected from the group consisting of SNPs and nucleotide polymorphisms designated by the SNP ID Nos rs9610683, rs9610682, rs2284037, rs739042, rs2284036, and rs739041.
  • the SNP or nucleotide polymorphism may also include ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73405482, ss73405484 and ss73405485.
  • the probes detecting each SNP or nucleotide polymorphism designated by each SNP ID Nos comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO: l (for rs9610683), 2 (for rs9610682), 3 (for rs2284037), 4 (for rs739042), 5 (for rs2284036), and 6 (for rs73904l).
  • the probes detecting each SNP or nucleotide polymorphism designated by each SNP ID Nos may also comprises more than 7 bases of partial sequence or partial complementary sequence of 7 (for ss73405466), 8 (for rs9798725), 9 (for ss73405467), 10 (for rs5995400), 11 (for rs6000619), 12 (for rs5756570), 13 (for rs36110509), 14 (for rs2899284), 15 (for rs6000618), 16 (for rs6000617), 17 (for rs9610677), 18 (for rs6000616), 19 (for rs9610676), 20 (for rs9610675), 21 (for rs9622582), 22 (for ss73405476), 23 (for ss73405477), 24 (for ss73405479), 25 (for rs5756568), 26 (for rs933223), 27 (for
  • chip refers to a tool or instrument for biological analysis having a portable size.
  • the scope of the "chip” includes a plate or array used in biological experiments.
  • base plate refers to a material holding a probe or probe sets.
  • reagent refers to any chemical compound, solution or protein for a reaction detecting SNP or nucleotide polymorphism.
  • examples of the reagent include, but are not limited to, polymerase, Clevase enzyme, restriction enzyme, ligase, fluorescent dye, quencher, reaction buffer, hybridization buffer or etc.
  • the probe sequence may comprise DNA, RNA or nucleic acid analogs such as uncharged nucleic acid analogs including, but not limited to, peptide nucleic acids (PNAs) which are disclosed in International publication WO 92/20702 (Reference 37).
  • PNAs peptide nucleic acids
  • Such sequences can routinely be synthesized using variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry. Once synthesized, oligonucleotide probes may be labeled by any well-known methods.
  • the probe comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID Nos 1 - 31.
  • the probe has 7 to 100 bases, more preferably, 10 to 50 bases, and most preferably, 15 to 30 bases.
  • the probe can comprise any sequence other than partial sequence or partial complementary sequence of SEQ ID Nos 1 - 31.
  • the probe may comprise linker sequence fixed to the base plate or 5' flap sequence for invader reaction.
  • the probe is used for detecting SNP or nucleotide polymorphism in any form.
  • the probe can be used as "invader oligo" or “probe” in the invader assay described above.
  • the probe can be used as "TaqMan probe”.
  • ESN ESN, their HIV-I -infected partner, and unexposed control individuals were enrolled with written informed consents as described previously (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024), and genetic analyses were performed with an approval from the ethical committee of the Kinki University School of Medicine. PBMC were stimulated with a mixture of HIV-I Gag and Env peptides as described (Reference 38, Clerici, M.
  • Genomic DNA was amplified with primers (enlisted in the SEQUENCE LISTING under SEQ ID Nos: 33 - 72) and sequenced. Case-control analyses of the observed genotypes, calculation of the coefficients of LD, and extraction of haplotype compositions were performed by using the SNPAlyze ver. 6.0 (DYNACOM Co., Ltd., Yokohama, Japan). Putative Rac2 regulatory regions were cloned into the pGL3 plasmid (Promega Corporation, Madison, U.S.A.), transfected into human Jurkat cells, and the Dual-Luciferase Reporter Assays (Promega) were performed.
  • the inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423 (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HlVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024; Reference 18, WO2004/035825, International publication of PCT application, 29 April 2004).
  • the inventors of the present invention genotyped 74 ESN and 77 HIV-infected individuals enrolled from the same area of Italy at multiple loci of known SNP (see Figure 7 for their chromosomal locations and Table 1 for complete listing of the observed genotypes).
  • PBMC peripheral blood mononuclear cells
  • the Rac2 and Lgalsl loci are located close to the two SNPs at which the allele frequencies were different between the ESN and HIV-infected groups ( Figure 2).
  • the augmented expression of the Rac2 after the antigenic stimulation was further confirmed by real-time PCR ( Figure 5).
  • Total RNA was prepared from antigen-stimulated PBMC as described (Reference 39, Biasin, M. et al., "Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G ⁇ a possible role in the resistance to HIV of HIV-exposed seronegative individuals", J.Infec.Dis., 2007, vol.195, pp.960-964), and cDNA was produced in the presence of an RNase inhibitor by using the T7-oligo(dT)24 primer. The resultant cDNA was purified, and biotinylated cRNA was prepared by using Biotin-16UTP and MEGAscript transcription kit (Ambion, Inc., Austin, U.S.A.).
  • Two to 10 oligodeoxynucleoti.de probes were designed for each of the genes enlisted in Figure 7 by using the TargetSpecifier (CombiMatrix Corporation, Mukilteo, U.S.A.) and synthesized on microarray chips. After prehybridization, denatured biotin-conjugated cRNA samples were hybridized at 45 0 C, overnight. After washing and blocking, microarray chips were incubated with Cy 3- conjugated streptavidin and washed vigorously. A fluorescence image of each microarray was scanned by a GenePix (Molecular Devices Corporation, Union City, U.S.A.), and analyzed by using the Microarray Imager (CombiMatrix Corporation).
  • TargetSpecifier CombiMatrix Corporation, Mukilteo, U.S.A.
  • LD between SNP was analyzed by likelihood ratio test as described (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024), and exact P values were obtained.
  • the inventors sequenced the chromosomal segments spanning the I12rb, Rac2-Pscd4, Mnfg, CardlO, and Lgalsl loci and compared the obtained nucleotide sequences between the ESN and HIV-1-infected individuals for the possible presence of polymorphisms with skewed genotype frequencies between the groups.
  • the Myh9 locus located close to the D22S277 marker, the expression of which was apparently increased in some examined individuals upon the antigenic stimulation (Figure 2), was also sequenced.
  • the inventors detected no sequence polymorphisms with a significant case-control difference at the Myh9, I12rb, and Lgalsl loci.
  • the SNP genotypes were highly linked to each other within the above Rac2 region and across the CardlO-Rac2 segment (Figure 3).
  • the SNP within the Rac2 region 1 constitute a small number of haplotypes with skewed frequencies between the groups ⁇ the region 1 haplotype TTT was significantly more accumulated among the ESN individuals, while the haplotype CCC was more frequently found in the HIV-infected group.
  • 34 of the 36 ESN individuals sequenced (94%) possessed the TTT haplotype on at least one chromosome, while 47 of the 65 HIV-I -infected individuals possessed the CCC haplotype, and 18 (28%) lacked the TTT.
  • Figure 3 shows distribution of observed SNP with significant case-control differences by adopting either a dominant (red lines) or a recessive (blue lines) hypothesis. P values shown are those calculated by ⁇ 2 (chi-square) test. See Figure 9 for odds ratios and P values calculated by Fisher's exact test. A representative value is shown for each cluster of SNP with significant case-control differences in the Rac2 locus, with the upper and lower brackets corresponding respectively to the regions 1 and 2 in Figure 1. Coefficients of LD between each pair of SNP alleles are shown here in lvalues and colour-scaled by using SNPAlyze ver. 6.0.
  • Figure 1 shows distribution of sequence polymorphisms (arrows) in the Rac2 locus with those showing significant case-control differences between ESN and HIV-infected groups shown in red. Longer arrows indicate SNP with case-control differences of P ⁇ 0.05. Chromosomal regions showing >50 % sequence homologies between humans and mice are indicated with horizontal lines labelled HH for high homology. In the polymorphic region 2, there were also gaps (small filled squares in the diagram) and base deletions (-): ⁇ l, a 28-bp deletion (— ) relative to the database-reported genome sequence (+); ⁇ 2, an 82-bp insertion (+) relative to the reported genome sequence (— )>" and ⁇ 3, a 34-bp insertion (+).
  • Haplotypes in each of the three blocks are those identified by the Four Gamete method using SNPAlyze ver. 6.0, and all observed linkages between each haplotype across the blocks are shown. The numbers of individuals possessing a particular haplotype are compared between the two groups by Fisher's exact test.
  • the genomic fragments that harbored the polymorphic region 1 isolated from representative healthy control individuals who homozygously possessed either the TTT or CCC haplotype were cloned and inserted into the downstream of the luc gene along with the known Rac2 core promoter (Reference 20, Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333) placed in the upstream.
  • the Rac2 promoter is known to be strong and promiscuous, and tissue -specific expression of this gene is regulated by the presence of putative repressor protein (Reference 20, Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic" specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333). In the present study, the inventors detected no sequence polymorphisms in the Rac2 promoter.
  • the Rac2 region 1, but not region 2 haplotypes were also associated with in vivo expression levels of the Rac2 in HIV-I antigen-stimulated PBMC.
  • the levels of Rac2 expression were examined quantitatively by real-time PCR in cultured PBMC, a 2-fold increase of the Rac2 message was observed after 6 hours of stimulation with the HIV-I peptides in the individuals homozygously possessing the TTT haplotype in region 1 ( Figure 5).
  • the levels of Rac2 expression decreased after the antigenic stimulation in cells homozygous for the CCC haplotype.
  • haplotypic differences in region 2 did not affect the Rac2 induction after the antigenic stimulation, again indicating that the polymorphic region 1, but not region 2, harbors a functional regulatory element of the Rac2 expression.
  • FIG. 5 shows changes in the expression levels of the Rac2 gene in PBMC stimulated with the HIV-I peptides.
  • Real-time PCR analyses were performed with samples obtained from 22 ESN and 8 HIV-1-infected individuals, which were prepared as described for Figure 2, and the results were re-grouped based on the separately determined region 1 and region 2 genotypes. Data are shown here as ratios of the Rac2 expression between 1 and 6 hours after the antigenic stimulation calculated as 2" MCT , and bars indicate S.E.M.
  • Statistical analyses were done by one-way ANOVA with Dunn's post-tests for multiple comparisons, and a significant group -wise difference was observed between the homozygous TTT and CCC groups.
  • the numbers of individuals possessing each identified genotype were: 10 TTT/TTT, 14 TTT/CCC, and 6 CCC/CCCin region 1, and 10 T/T, 14 T/G, and 6 G/G in region 2.
  • FIG. 6 shows replication of HIV- 1 in cultured PBMC.
  • Uninfected healthy control individuals were genotyped with written informed consent for the Rac2 region 1 and region 2 haplotypes, and their PBMC were infected with each of the 4 different HIV-I clones or isolates, or with the dual-tropic isolate 89.6. None of the individuals included here possessed the CCR5 ⁇ 32 allele.
  • Data shown here are p24 concentrations measured at 3 and 5 days after virus inoculation grouped by the region 1 genotypes, with horizontal bars indicating median values.
  • Statistical comparisons were done by two-way ANOVA for repeated measures with Dunn's post-tests. When group -wise differences were indicated, individual levels of significance were determined by two-tailed t-tests.
  • the dual-tropic 89.6 replicated efficiently in PMBC of all three genotypes, and no significant differences were observed in p24 concentrations between the CCC/CCC and individuals at day 5 (not shown).
  • the 2504-bp region 1 and 8904-bp region 2 genomic fragments were amplified by PCR using the following oligonucleotide primers and LA-Taq polymerase (TAKARABIO, Inc., Ohtsu, Japan):
  • Rh2RlL ATCCTCGAGCACCAAGCTGGACCTGCGGGACGACAAG
  • Rac2RlR ATCGTCGACGAGAGGATGTCACTCGCTCTGAGTCACATG
  • Rac2R2L ATCCTCGAGCTCCCACCTAGATGGGTCTGATCCTCCAG and Rac2R2R ATCCTCGAGTTTTGATGTAGCATAGCTCCCAGTAACTTTCAG
  • the cloned Rac2 genomic fragments were inserted to the SaK restriction site located downstream of the luc gene within the pGL3 plasmid (Promega Corporation, Madison, U.S.A.) harbouring the previously described Rac2 cove promoter (-260 to +130 bp) (Reference 20, Ladd, P.D.
  • PBMC peripheral blood mononuclear cells
  • HIV-I p24 Absolute levels of HIV-I p24 were measured using the Alliance HIV-I p24 ELISA kit (PerkinElmer Inc., Waltham, U.S.A.). HIV-I BaL and IIIB were provided by Drs. S. Gartner, M. Popovic, and R. Gallo (Courtesy of the NIH AIDS Research and Reference Reagent Program). HIV-I primary isolates were kind gifts from Prof. C. -R Perno, University of Roma, Tor Vergata, Italy.

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Abstract

The present invention provides an ex vivo method, a chip and a kit detecting genetic predisposition of a subject for resistance to virus. The present method, chip and kit utilize specific SNPs and nucleotide polymorphisms accumulated in ESN (HIV- 1-exposed but uninfected) individuals as markers of resistance to virus. Further, the present invention provides preventative and therapeutic means to confer resistance to virus infection.

Description

DESCRIPTION
METHOD FOR DIAGNOSIS AND INDUCTION OF RESISTANCE TO
VIRUS
FIELD OF THE INVENTION
[Ol] The present invention is related generally to the analytical testing of the samples obtained from a subject, and more particularly the method for detecting genetic predisposition of a subject to resistance to virus. The invention also provides a chip and kit for detecting such genetic predisposition. The invention further provides an application of identified genetic predisposition to induce resistance to virus infection.
Priority is claimed on PCT/JP2007/068591, filed September 12, 2007, the contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[02] It is known in the art that it is possible to diagnose a predisposition to certain diseases with the use of biomarkers such as single nucleotide polymorphisms (SNPs), nucleotide insertion mutations, nucleotide deletion mutations, microsatellite markers or other DNA sequence variations. For example, inherited polymorphisms and somatic mutations in oncogenes or tumor suppressor genes are widely regarded as being indicative of a susceptibility to certain cancers, especially in view of the associations between mutated oncogenes or deleted tumor suppressor genes and certain cancers. It is also known that some individuals are highly susceptible or resistant to infection, especially viral infection. Prediction of disease susceptibility is beneficial for those possessing predisposing genes in order to avoid unnecessary contacts with known etiological agents, chemicals, or viruses, and to take known and developing preventative means. It is also useful in the design of a vaccine against viral disease or for gene therapy. In addition, prediction of the speed of disease progression may allow an opportunity for individualized, more efficient management or therapeutic intervention.
[03] Additionally, the development of an effective vaccine or therapeutic agent against major viral diseases such as human immunodeficiency virus (HIV) infection is a pressing matter with global socio-economic ramifications. HIV is the causative agent of acquired immunodeficiency syndrome (AIDS). One of the keys to the development of such a vaccine is the understanding of the mechanisms of natural resistance against HIV infection. And in the development of such a therapeutic agent, especially in the clinical trial, it is necessary to assess genetic predisposition of the subject to resistance or susceptibility to HIV infection and its disease progression, because the measured effects of the candidate agent in question can be significantly biased by the presence of genetically determined susceptibility or resistance in the subject.
[04] Several human genes are associated with resistance against human immunodeficiency virus type 1 (HIV- 1) infection or delayed development of AIDS after HIV-I seroconversion (Reference 1, O'Brien et al., "Polygenic and multifactorial disease gene association in man", Annu. Rev. Genet., 2000, vol.34, pp.563-591; Reference 2, O'Brien et al., "Human genes that limit AIDS", Nat. Genet., 2004, vol.36, pp.565-574). These include genes encoding chemokine receptors and their ligands, cytokines, receptors expressed on antigen-presenting or natural killer cells, and those mapped within the major histocompatibility complex (MHC) (Reference 3, Dean, M. et al., "Genetic restriction of HIV-I infection and progression to AIDS by a deletion allele of the CCR5 structural gene", Science, 1996, vol.273, pp.1856-1862; Reference 4, Liu, R. et al., "Homozygous defect in HIVl coreceptor accounts for resistance of some multiply-exposed individuals to HIV-I infection", Cell, 1996, vol.86, pp.367-377; Reference 5, Samson, M. et al., "Resistance to HIV-I infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene", Nature, 1996, vol.382, pp.722-725; Reference 6, Winkler, C. et al., "Genetic restriction of AIDS pathogenesis by an SDF-I chemokine gene variant", Science, 1998, vol.279, pp.389-393," Reference 7, Gonzalez, E. et al., "The influence of CCL3L1 gene-containing segmental duplications on HIV- I/AIDS susceptibility", Science, vol.307, pp.1434- 1440; Reference 8, Carrington, M. et al., "HLA and HIV Heterozygote advantage and B*35-Cw*04 disadvantage", Science, vol.283, pp.1748-17525 Reference 9, Martin, M.P. et al., "Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS", Nature Genet., 2002, vol.31, pp.429-434; Reference 10, Fellay, J. et al., "A whole- genome association study of major determinants for host control of HIV-I", Science, Published online July 19 2007, 10.1126/science.1143767; Reference 11, Beyrer, C. et al., "Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand", J.Infect.Dis., 1999, vol.79, pp.59-68; Reference 12, Liu H. et al., "Analysis of genetic polymorphisms in CCR5, CCR2, stromal cell-derived factor- 1, RANTES, and dendritic cell-specific intercellular adhesion molecule- 3- grabbing nonintegrin in seronegative individuals repeatedly exposed to HIV-I", J. Infec. Dis., 2004, vol.190, pp.1055-1058). [05] However, the known AIDS restriction genes (ARG) can explain only up to 15 % of the host determinants that control HIV I/AIDS (Reference 2, O'Brien et al., "Human genes that limit AIDS", 2004, Nat. Genet., vol.36, pp.565-574; Reference 10, Fellay, J. et al., "A whole-genome association study of major determinants for host control of HIVl", Science, Published online July 19 2007, 10.1126/science.1143767), and the presence of other resistance genes has been predicted (Reference 2, O'Brien et al., "Human genes that limit AIDS", 2004, Nat. Genet., vol.36, pp.565-574). [06] Among existing human clusters showing natural resistance to HIVl acquisition, there is a distinct group known as HIVl-exposed but uninfected or exposed seronegative (ESN) individuals who have evidence of multiple and repeated exposures to HIVl through unprotected sexual contacts, but nevertheless possess no serum IgG reactive to the viral antigens (Reference 11, Beyrer, C. et al., "Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand", J.Infect.Dis., 1999, vol.79, pp.59-68; Reference 12, Liu H. et al., "Analysis of genetic polymorphisms in CCR5, CCR2, stromal cell-derived factor- 1, RANTES, and dendritic cell-specific intercellular adhesion molecule- 3-grabbing nonintegrin in seronegative individuals repeatedly exposed to HIV-I", J. Infec. Dis., 2004, vol.190, pp.1055-1058; Reference 13, Mazzoli S. et al., "HIV-specific mucosal and cellular immunity in HIV- seronegative partners of HIV-seropositive individuals", Nature Med., 1997, vol.3, pp.1250-1257; Reference 14, Biasin, M. et al., "Mucosal and systemic immune activation is present in human immunodeficiency virus-exposed seronegative women", J. Infect. Dis., 2000, vol.182, pp.1365-1374; Reference 15, Belec, L. et al., "Cervicovaginal secretory antibodies to HIV type 1 that block viral transcytosis through epithelial barriers in highly exposed HIVl "seronegative African women", J. Infect. Dis., 2001, vol.184, pp.1412- 1422; Reference 16, Locaputo, S. et al., "Mucosal and systemic HIVspecific immunity in HlVexposed but uninfected heterosexual males", AIDS, 2003, vol.17, pp.351-358; Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2"13 are associated with HlVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024).
[07] The detection of HIVspecific T- lymphocyte responses and HIVreactive IgA antibodies in mucosal secretions (Reference 13, Mazzoli S. et al., "HIVspecific mucosal and cellular immunity in HIV seronegative partners of HIV-seropositive individuals", Nature Med., 1997, vol.3, pp.1250-1257; Reference 14, Biasin, M. et al., "Mucosal and systemic immune activation is present in human immunodeficiency virus-exposed seronegative women", J. Infect. Dis., 2000, vol.182, pp.1365-1374; Reference 15, Belec, L. et al., "Cervicovaginal secretory antibodies to HIV type 1 that block viral transcytosis through epithelial barriers in highly exposed HIV-1-seronegative African women", J. Infect. Dis., 2001, vol.184, pp.1412- 1422," Reference 16, Locaputo, S. et al., "Mucosal and systemic HIV-specific immunity in HIV-exposed but uninfected heterosexual males", AIDS, 2003, vol.17, pp.351-358; Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HlVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024) indicates that ESN individuals have been exposed to HIV-I and have mounted HIV-specific immune responses, but the exposures have not resulted in productive infection.
[08] Attempts to associate the ESN status with previously described ARG have so far been unsuccessful (Reference 11, Beyrer, C. et al., "Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand", J.Infect.Dis., 1999, vol.79, pp.59-68; Reference 12, Liu H. et al., "Analysis of genetic polymorphisms in CCR5, CCR2, stromal cell-derived factor- 1, RANTES, and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin in seronegative individuals repeatedly exposed to HIV-I", J. Infec. Dis., 2004, vol.190, pp.1055-1058; Reference 13, Mazzoli S. et al., "HIV-specific mucosal and cellular immunity in HIV- seronegative partners of HIV-seropositive individuals", Nature Med., 1997, vol.3, pp.1250-1257; Reference 14, Biasin, M. et al., "Mucosal and systemic immune activation is present in human immunodeficiency virus-exposed seronegative women", J. Infect. Dis., 2000, vol.182, pp.1365-1374; Reference 15, Belec, L. et al., "Cervico vaginal secretory antibodies to HIV type 1 that block viral transcytosis through epithelial barriers in highly exposed HIV-1-seronegative African women", J. Infect. Dis., 2001, vol.184, pp.1412-14225 Reference 16, Locaputo, S. et al., "Mucosal and systemic HIV-specific immunity in HlVexposed but uninfected heterosexual males", AIDS, 2003, vol.17, pp.351-358; Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024).
[09] The inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423 (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024; Reference 18, WO2004/035825, International publication of PCT application, 29 April 2004). To further narrow down the chromosomal region where the putative immune resistance gene is located, the inventors of the present invention genotyped 74 HIV-exposed but uninfected and 77 HIV-infected individuals enrolled from the same geographical region. Several SNPs are significantly associated with ESN individuals under a dominant gene hypothesis at the CardlO, CDC42EP1 and GRAP2 loci (Reference 19, WO2006/067506, International publication of PCT application, 29 June 2006). However, exact molecular genetic mechanisms that explain the observed resistance of ESN individuals to HIVl acquisition had not been provided. [lθ] References
(1) O'Brien et al., "Polygenic and multifactorial disease gene association in man", Annu. Rev. Genet., 2000, vol.34, pp.563-591.
(2) O'Brien et al., "Human genes that limit AIDS", Nat. Genet., 2004, vol.36, pp.565-574.
(3) Dean, M. et al., "Genetic restriction of HIVl infection and progression to AIDS by a deletion allele of the CCR5 structural gene", Science, 1996, vol.273, pp.1856-1862.
(4) Liu, R. et al., "Homozygous defect in HIVl coreceptor accounts for resistance of some multiply-exposed individuals to HIVl infection", Cell, 1996, vol.86, pp.367-377. (5) Samson, M. et al., "Resistance to HIV-I infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene", Nature, 1996, vol.382, pp.722-725.
(6) Winkler, C. et al., "Genetic restriction of AIDS pathogenesis by an SDF-I chemokine gene variant", Science, 1998, vol.279, pp.389-393.
(7) Gonzalez, E. et al., "The influence of CCL3L1 gene-containing segmental duplications on HIV" I/AIDS susceptibility", Science, vol.307, pp.1434- 1440.
(8) Carrington, M. et al., "HLA and HIV: Heterozygote advantage and B*35-Cw*04 disadvantage", Science, vol.283, pp.1748- 1752.
(9) Martin, M.P. et al., "Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS", Nature Genet., 2002, vol.31, pp.429-434.
(10) Fellay, J. et al., "A whole-genome association study of major determinants for host control of HIVl", Science, Published online July 19 2007, 10.1126/science.1143767.
[11]
(11) Beyrer, C. et al., "Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand", J.Infect.Dis., 1999, vol.79, pp.59-68.
(12) Liu H. et al., "Analysis of genetic polymorphisms in CCR5, CCR2, stromal cell-derived factor- 1, RANTES, and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin in seronegative individuals repeatedly exposed to HIVl", J. Infec. Dis., 2004, vol.190, pp.1055-1058.
(13) Mazzoli S. et al., "HIV-specific mucosal and cellular immunity in HIV seronegative partners of HlVseropositive individuals", Nature Med., 1997, vol.3, pp.1250-1257.
(14) Biasin, M. et al., "Mucosal and systemic immune activation is present in human immunodeficiency virus-exposed seronegative women", J. Infect. Dis., 2000, vol.182, pp.1365-1374. (15) Belec, L. et al., "Cervicovaginal secretory antibodies to HIV type 1 that block viral transcytosis through epithelial barriers in highly exposed HIV-1-seronegative African women", J. Infect. Dis., 2001, vol.184, pp.1412-1422.
(16) Locaputo, S. et al., "Mucosal and systemic HIV- specific immunity in HIV-exposed but uninfected heterosexual males", AIDS, 2003, vol.17, ρp.351-358.
(17) Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HlVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024.
(18) WO2004/035825, International publication of PCT application, published on 29 April 2004.
(19) WO2006/067506, International publication of PCT application, published on 29 June 2006.
(20) Ladd, PD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333.
(21) Ambruso DR, Knall C, Abell AN, Panepinto J, Kurkchubasche A, Thurman G, Gonzalez-Aller C, Hiester A, deBoer M, Harbeck RJ, Oyer R, Johnson GL, Roos D. Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation. Proc Natl Acad Sci U S A. 2000 97:4654-4659.
(22) Williams DA, Tao W, Yang F, Kim C, Gu Y, Mansfield P, Levine JE, Petryniak B, Derrow CW, Harris C, Jia B, Zheng Y, Ambruso DR, Lowe JB, Atkinson SJ, Dinauer MC, Boxer L. Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency. Blood 2000 96:1646-1654.
(23) Yamauchi A, Kim C, Li S, Marchal CC, Towe J, Atkinson SJ, Dinauer MC. Rac2"deficient murine macrophages have selective defects in superoxide production and phagocytosis of opsonized particles. J Immunol. 2004 173:5971-5979.
(24) Diebold BA, Bokoch GM. Molecular basis for Rac2 regulation of phagocyte NADPH oxidase. Nat Immunol. 2001 2:211-215.
(25) Bokoch GM, Diebold BA. Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood 2002 100:2692-2696.
[12]
(26) Croker, B.A. et al., "The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-Ia and marginal zone B lymphocytes", J.Immunol., 2002, vol.168, pp.3376-3386.
(27) Fagarasan, S. et al., "T-independent immune response: new aspects of B cell biology", Science, 2000, vol.290, pp.89-92.
(28) Li, B. et al., "Role of the guanosine triphosphatase Rac2 in T helper 1 cell differentiation", Science, 2000, vol.288, pp.2219-2222.
(29) Saito, T., et al., "Genetic variations in humans associated with differences in the course of hepatitis C", Biochemical and Biophysical Research Communication (BBRC), 2004, vol.317, pp.335-341.
(30) Orita, M. et al., "Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction", Genomics, 1989, vol.5(4), pp.874-879.
(31) Oefner, P.J. et al., "DNA mutation detection using denaturing high performance liquid chromatography", Current protocols in human genetics, 1998, p.7 -10:1-710 -12.
(32) Sanger, F. et al., "DNA sequencing with chain-terminating inhibitors", Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1977, vol.74(l2), pp.5463-5467.
(33) U.S. Patent No. 5,846,717
(34) Lyamichev, V. et al., "Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes", Nature Biotechnology, 1999, vol.17(3), pp.292-296. (35) Holland, P.M. et al., "Detection of specific polymerase chain reaction product by utilizing the 5'-—3' exonuclease activity of Thermus aquaticus DNA polymerase", Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1991, vol.88(l6), pp.7276-7280.
(36) Altschul, S.F. et al, "Gapped BLAST and PSI-BLAST: A new generation of protein database search programs", Nucleic Acids Res., 1997, vol.25, pp.3389-3402.
(37) WO 92/20702, International publication of PCT application, published on 26 November 1992
[13]
(38) Clerici, M. et al., "Interleukin-2 production used to detect antigenic peptide recognition by T-lymphocytes from asymptomatic HIV- seropositive individuals", Nature, 1989, vol.339, pp.383-385.
(39) Biasin, M. et al., "Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G^ a possible role in the resistance to HIV of HIV-exposed seronegative individuals", J.Infec.Dis., 2007, vol.195, pp.960-964.
(40) Meskauskas, A. & Dinman, J.D., "Ribosomal protein L3: Gatekeeper to the A site", MoI. Cell., 2007, vol.23, pp.877-888.
(41) Asano, K. et al., "Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits", J.Biol. Chem., 1997, vol.272, pp.27042-27052.
(42) Yu, H., Leitenberg et al., "Deficiency of small GTPase Rac2 affects T cell activation", J.Exp.Med., 2001, vol.194, pp.915-925.
(43) Jacobelli, J. et al., "A single class II myosin modulates T cell motility and stopping, but not synapse formation", Nat.Immunol., 2004, vol.5, pp.531-538.
(44) Ouellet, M. et al., "Galectin-1 acts as a soluble host factor that promotes HIV-I infectivity through stabilization of virus attachment to host cells", J.Immunol., 2005, vol.174, pp.4120-4126.
SUMMARY OF THE INVENTION [14] The inventors of the present invention performed expression analyses of all the genes and open reading frames located in the candidate segment of human chromosome 22 and extensive genome sequencing, and identified previously unforeseen association between certain sequence polymorphisms in the Rac2 gene locus and the ESN (HIV- 1-exposed but uninfected or exposed seronegative) status. The sequence polymorphism associated with ESN confers a higher expression levels of this gene, and is associated with poor replication of CCR5-tropic HIV in peripheral blood mononuclear cells.
More specifically, some SNPs and specific haplotypes of them are representative of the above sequence polymorphism and thus predictive of a resistance to HIVl infection, and these are designated by the following SNP ID Nos: rs9610683, rs9610682, rs2284037, rs739042, rs2284036, rs739041, ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73405482, ss73405484 and ss73405485.
Further, the inventors of the present invention found that these SNPs and haplotypes have an effect on the Rac2 gene expression level and HIV replication. Thus these SNPs and haplotypes are useful for detecting genetic predisposition to resistance to virus infection, and also to provide preventative and therapeutic means to confer resistance to virus infection. [15] Accordingly, an aspect of the present invention provides- (l) An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, including: a) genotyping a sample of the subject at a site of at least one SNP selected from the group consisting of SNPs designated by following SNP ID Nos^ (i) rs739041, (ii) rs739042, and (iii) rs2284037; and b) assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP is a resistance allele, wherein the resistance allele is as follows (SNP ID No. : allele) : G) rs739041 : T; (ii) rs739042 : T; (iii) and rs2284037 : T.
[16] (2) An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, including: a) genotyping a sample of the subject at sites of SNPs designated by following SNP ID Nos: (i) rs2284037, (ϋ) rs739042 and (iii) rs73904l; and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (j) rs2284037 : T; (ii) rs739042 : T; (iii) rs739041 : T.
[17] (3) An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, including: a) genotyping a sample of the subject at sites of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) ss73405466, (ii) rs9798725, (iii) ss73405467, (iv) rs5995400, (v) rs6000619, (vi) rs5756570, (vii) rs36110509, (viii) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xvϋi) ss73405479, (xix) rs5756568, (xx) rs933223, (xxi) rs933222, (xxii) rs933321, (xxiii) ss73405482, (xxiv) ss73405484 and (xxv) ss73405485l and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (i) Ss73405466 : G; (ii) rs9798725 : T; (iii) ss73405467 : GGCCTCATCCTTCCAAGTTCAAGTTCAG; (iv) rs5995400 : C; (v) rs6000619 : A; (vi) rs5756570 : C; (vii) rs36110509 : gap; (viii) rs2899284 : T; (ix) rs6000618 : T; (x) rs6000617 : Tl (xi) rs9610677 : Al (xii) rs6000616 : C; (xiii) rs9610676 : Al (xiv) rs9610675 : Tl (xv) rs9622582 : Cl (xvi) ss73405476 : Tl (xvii) ss73405477 : Tl (xviii) ss73405479 : Tl (xix) rs5756568 : Gl (xx) rs933223 : Cl (xxi) rs933222 : Al (xxii) rs933321 : Al (xxiii) ss73405482 : Al (xxiv) ss73405484 : gapl and (xxv) ss73405485 : TCCCATAATCCAGGTGGGAGGAACCCAGTGGGAGGTAATTGAA [18] (4) An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, including: a) genotyping a sample of the subject at sites of SNPs designated by following SNP ID Nos: (i) rs9610683 and (ii) rs9610682; and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (i) rs9610683 : C; and (ii) rs9610682 : A.
[19] (5) An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, including: a) genotyping a sample of the subject at a site of at least one SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, and (iii) rs2284036; and b) assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP or nucleotide polymorphism is a resistance allele, wherein the resistance allele is as follows (SNP ID No. : allele) : (i) rs9610683 : C; (ii) rs9610682 : A; and (iii) rs2284036 : T.
[20] (6) A method for inducing or enhancing resistance to virus in a subject, including enhancing the Rac2 gene expression in the subject.
[21] (7) In the method according to the above method (6), the Rac2 gene expression may be enhanced by using a DNA-binding protein, peptide, oligonucleotide, or nucleotide analogue that binds to the Rac2 repressor protein binding site located in the Rac2 gene region 1 (SEQ ID No:32).
[22] (8) In the method according to the above method (6), the Rac2 gene expression may be enhanced by inhibiting the expression of an Rac2 repressor protein by RNAi technique.
[23] (9) Use of Rac2 protein in the preparation of a medicament for the treatment or prophylaxis of virus-induced disease.
[24] (lθ) A nucleic acid exhibiting a Rac2 repressor protein binding activity, wherein the nucleic acid is selected from the following (A) and (B)-
(A) a nucleic acid having the nucleotide sequence of SEQ ID No:32>' and
(B) a nucleic acid having more than 80% identity with the nucleotide sequence of SEQ ID No:32.
[25] (ll) In the nucleic acid according to the above (lθ), the nucleic acid may have the following alleles at the SNP sites in the nucleotide sequence of SEQ
ID No:32 (base number in the SEQ ID No:32 : allele):
(i) 132 : Cl (ϋ) 144 : A; (iii) 1276 : T; (iv) 1959 : T; (v) 2301 : T; and
(vi) 2379 : T.
[26] (12) Use of the nucleic acid according to the above (10) or (ll) as a Rac2 repressor protein inhibitor.
[27] (13) Use of the nucleic acid according to the above (lθ) or (ll) for a gene therapy to induce resistance to virus in a subject.
[28] (14) A method for screening a candidate therapeutic agent against virus, including: a) contacting an agent with a nucleic acid fragment having a partial or entire nucleotide sequence of the nucleic acid according to the above (lθ) or (ll); b) measuring an interaction between the agent and the nucleic acid fragment; and c) selecting the agent having the interaction with the nucleic acid fragment as a candidate therapeutic agent against virus.
[29] (15) A method for screening a candidate therapeutic agent against virus, including: a) administering an agent to a biological material; b) measuring an expression level of the Rac2 gene in the biological material; and c) selecting the agent enhancing expression level of the Rac2 gene in the biological material as a candidate therapeutic agent against virus.
[30] (16) A method for performing clinical trial for prevention, reduction, prophylaxis or treatment against virus, including: a) detecting genetic predisposition of subjects by the method according to any one of the above methods (l) - (5); b) stratifying the subject by the result of the detected genetic predisposition; c) giving a treatment to the stratified subject; and d) assessing the effect of the prevention, reduction, prophylaxis or treatment to the stratified subject.
[31] (17) In the method according to any one of the above methods (l) ■ (8), the virus may be one selected from the group consisting of HIV, HCV and
HPV
[32] (18) In the method according to the above method (17), the virus may be
HIV or HCV.
[33] (19) In the method according to the above method (18), the virus may be
HIV.
[34] (20) In the use according to the above use (9) or (13), the virus may be one selected from the group consisting of HIV, HCV and HPV.
[35] (21) In the use according to the above use (20), the virus may be HIV or
HCV.
[36] (22) In the use according to the above use (21), the virus may be HIV.
[37] (23) In the method according to any one of the above methods (14) - (16), the virus may be one selected from the group consisting of HIV, HCV and
HPV.
[38] (24) In the method according to the above method (23), the virus may be
HIV or HCV.
[39] (25) In the method according to the above method (24), the virus may be
HIV.
[40] (26) In the method according to any one of the above methods (l) - (5), the genotyping may be performed by the Invader assay.
[41] (27) A chip used in the method according to the above method (l), including a base plate and at least one probe detecting SNP selected from the group consisting of SNPs designated by the SNP ID Nos (i) rs739041, (H) rs739042, (iii) and rs2284037, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO: 6, 4, and 3.
[42] (28) A chip used in the method according to the above method (2), including a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs2284037, (ii) rs739042 and (iii) rs739041, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:3, 4 and 6.
[43] (29) A chip used in the method according to the above method (3), including a base plate and probe sets detecting SNPs and nucleotide polymorphisms designated by the SNP ID Nos (i) ss73405466, (ii) rs9798725, (iii) ss73405467, (iv) rs5995400, (v) rs6000619, (vi) rs5756570, (vii) rs36110509, (viii) rs2899284, Gx) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix) rs5756568, (xx) rs933223, (xxi) rs933222, (xxii) rs933321, (xxiii) ss73405482, (xxiv) ss73405484 and (xxv) ss73405485, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31.
[44] (30) A chip used in the method according to the above method (4), including a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs9610683 and (ii) rs9610682, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:i and 2.
[45] (31) A chip used in the method according to the above method (5), including a base plate and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, and (iii) rs2284036, wherein the probe sets comprises at least one probe comprises more than 7 bases of partial sequence or partial complementary sequence of the nucleotide sequence selected from the group consisting of SEQ ID NO:l, 2, and 5.
[46] (32) A kit used in the method according to the above method (l), including a reagent and at least one probe detecting SNP selected from the group consisting of SNPs designated by the SNP ID Nos (i) rs739041, (ii) rs739042, (iii) and rs2284037, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO: 6, 4, and 3.
[47] (33) A kit used in the method according to the above method (5), including a reagent and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, and (iii) rs2284036, wherein the probe sets comprises at least one probe comprises more than 7 bases of partial sequence or partial complementary sequence of the nucleotide sequence selected from the group consisting of SEQ ID NO:i, 2, and 5.
(34) A method for screening a candidate therapeutic agent against virus, including: a) administering an agent to a biological material; b) measuring an intensity of a binding between a Rac2 repressor protein and a binding site thereof, in the biological material; and c) selecting the agent inhibiting the binding in the biological material as a candidate therapeutic agent against virus.
BRIEF DESCRIPTION OF THE DRAWINGS [48]
Figure 1 is an illustration of the distribution of SNPs and nucleotide polymorphisms in the Rac2 locus.
Figure 2 shows a result of DNA microarray analysis comparing the changes in gene expression levels during the HIV-I antigenic stimulation in peripheral blood mononuclear cells (PBMC).
Figure 3 is a coefficient map of linkage disequilibrium (LD) between each pair of SNP alleles. [49]
Figure 4 is a graph showing luciferase activities induced by the Rac2 promoter and the polymorphic region 1 indicated in Figure 1.
Figure 5 is a graph showing changes in the expression levels of the Rac2 gene in PBMC stimulated with the HIV-I peptides.
Figure 6 is a graph showing HIVl p24 concentrations measured at 3 and 5 days after virus inoculation grouped by the Rac2 region 1 genotypes.
Figure 7 is an illustration of locations and spans of known genes and open reading frames in the candidate region in human chromosome 22.
DETAILED DESCRIPTION OF THE INVENTION
[50] An aspect of the present invention provides an ex vivo method, a chip and a kit detecting genetic predisposition of a subject to resistance to virus. Further, another aspect of the present invention provides a method, biopharmaceutical and nucleic acid for inducing or enhancing resistance to virus in a subject.
Examples of the virus include, but are not limited to, Human immunodeficiency virus (HIV), Human papilloma virus (HPV), Hepatatis C virus (HCV), Human herpes virus (HHV), Cytomegalovirus (CMV), Small round structured virus (SRSV) and Influenza (Flu) virus. Preferably, the virus is HIV, HPV or HCV. More preferably, the virus is HIV. HIV includes HIVl and HIV-2.
[51] In the present specification and claims, the term "genetic predisposition" refers to genetic factors which influence the phenotypes of an organism. However, the effect of the genetic predisposition can be modified by environmental conditions. The term "subject", as used herein, refers to an object for the method, for detecting genetic predisposition. Preferably, the subject is a primate such as human, monkey and ape. More preferably, the subject is human. Most preferably, the human is Caucasian.
The term "resistance to virus", as used herein, refers to an act or power to prevent virus infection or virus-induced disease progression. [52] The present invention is based on the research demonstrating that particular SNPs and haplotypes located in the Rac2 gene region are significantly associated with ESN (HIV- 1-exposed but uninfected or exposed seronegative) individuals. Summary of the research is described below. [53] The inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423.
To further narrow down the location of the putative HIV-I resistance gene, the inventors of the present invention genotyped 74 ESN and 77 HIV-infected individuals enrolled from the same area of Italy at multiple loci of known SNP (see Figure 7 for their chromosomal locations and Table 1 for complete listing of the observed genotypes).
[Table 1]
Difference in genotype frequencies
Genotype distribution among the between the groups by a dominant gene
Genotype distribution among the HIV-exposβd but uninfected model HIV-infected individuals individuals
,-.1 , Odds ratio 95X CI (Fisher)
N Tota Tota
SNP ID (rs)* Linked locus 1 1/2 2 1 1/2 2 ND D I
132618 APOL3 17 34 22 1 74 16 40 21 0 77
7078 MYH9 40 26 6 2 74 51 21 4 1 77
762906 RABL4 2 16 55 1 74 1 24 52 0 77
713829 C22orf33 10 42 20 2 74 7 38 32 0 77
C22oιi33
9610624 0 52 18 4 74 0 58 18 1 77 (axon 1 coding)
228942 IL2RB 49 19 5 1 74 52 23 2 0 77
229526 C1QTNF6 5 26 41 2 74 3 24 49 1 77
2284036 RAO2 45 25 2 2 74 43 31 1 2 77
HCV2236051 RAC2 74 0 0 0 74 77 0 0 0 77
2015524 CARDtO 42 15 0 3 60 45 10 0 5 60
9619680 CARDW 74 0 0 0 74 77 0 0 0 77
9610775 CARDIO 46 21 2 5 74 41 21 10 5 77 0.0313 s 0.185 0.039 - 0.878
738298 CARDtO 44 11 0 5 60 41 13 2 4 60
2235336 CDC42EP1 34 34 4 2 74 30 30 17 0 77 0.0043 s 0.208 0.066 - 0.651
2076087 CDC42EP1 12 28 14 6 60 17 27 12 4 60
2281099 LGALS2 0 0 54 0 54 0 0 58 1 59
139883 SOXtO 13 35 25 1 74 16 36 24 1 77
2267374 MAFF 13 30 29 2 74 20 31 24 2~ 77
2267393 GTPBPl 9 29 36 0 74 9 30 36 2 77
APOBEC3G
8177832 68 6 0 0 74 68 7 0 2 77 Cexon 4 coding)
APOBEC3G
17496046 67 7 0 0 74 70 6 0 1 (axon 6 coding) 77
738331 RPL3 30 33 10 1 74 26 38 11 2 77
GRAP2
137959 36 32 2 4 74 33 36 7 1 (promoter) 77
GRAP2
137988 10 20 24 1 55 6 20 (intron 1) 6 1 33
QRAP2
2267420 3 12 58 1 74 2 19 (intron 2) 54 2 77
GRAP2
137997 1 19 49 5 (intron 3) 74 1 23 50 3 77
GRAP2
138000 19 40 14 1 74 20 37 20 0 (intron 3) 77
GRAP2
12759 74 0 0 0 (exon 8 coding) 74 76 0 0 1 77
GRAP2
138011 52 20 2 0 74 48 26 (31 intron) 1 2 77
5757899 TNRCSB 28 36 8 2 74 30 33 13 1 77
138354 XPNPEP3 21 42 10 1 74 25 41 10 1 77
202642 TOB2 2 27 45 0 74 4 22 51 0 77
139562 CCDC134 1 20 49 4 74 2 21 53 1 77
7290134 BAFF-R 37 28 7 2 74 38 26 11 2 77
6002555 CENPM 6 28 37 3 74 8 28 40 1 77
5751231 TCF20 3 27 43 1 74 4 29 43 1 77
137086 POLD1P3 4 28 41 1 74 6 42 28 1 77 0.0218 s 2.196 1.139 - 4.235
2024643 A4GALT 26 20 4 0 50 26 26 6 0 58 Table 1 is a table showing allele distribution at known SNP loci in chromosome 22 among the ESN and HlVl-infected individuals. [54] Expression levels of all the genes located in the above candidate region were compared between the ESN and HIV-infected individuals by using DNA microarrays. Among all the chromosome 22 genes tested, only a small number were constitutively expressed at high levels or induced upon the stimulation with HIV-I antigens (Figure 2). One of the induced genes is the Rac2, which encodes a Rho-subfamily small GTPase that is selectively expressed in and involved in the activation of T- helper type-1 (ThI) cells. The Myh9 and Lgalsl were also induced upon the antigenic stimulation. [55] The chromosomal segments spanning the Myh9, I12rb, Rac2-Pscd4, CardlO, and Lgalsl loci were sequenced (see Figure 7 for the location of the sequenced segments), and the frequencies of ESN and HIV-I -infected individuals possessing a particular allele at each of the observed polymorphic loci were compared. Notable accumulation of sequence polymorphisms with significantly different allele frequencies between the two groups was observed in a segment between the CardlO and Rac2 (Figure 3 and Table 2).
[Table 2]
Dominant model Recessive model
Gene Woolfs 95% Cl WoolFs 95% Cl oNr iInU locus Uvo sided two-
Chi-square P-value Fisher's Odds ratio Low High Chi-square P-value sided Odds ratiG Low High exact test Fisher's rs8142629 - _ - - 00529 0818 1 12 02536 56783 rs9610778 22192 01363 02885 01954 001S8 20307 15873 02077 02275 04565 01333 15639 rs7291745 _ _ - 04689 04935 06345 22222 02149 229753
SS73405559 _ - - - 04689 04935 06345 22222 02149 229753 rs60007B1 00669 07959 1 06897 00407 116841 02349 06279 07706 13333 0416 42737 r6519065 09967 03181 04161 04444 0088 22451 00566 0812 1 08684 02715 27776 rs9610776 _ - - 0 1 1 1 0243 41145 rs4080481 00115 09145 1 09333 02648 32895 13766 02407 03592 20455 06139 68148
RS932327 04571 0499 05204 06384 01708 23706 01959 0658 0771 07692 02405 24605 rs932326 17812 0182 02489 03556 00748 16907 0028 08671 1 11 03603 33581 hCV26000412 - - - - 25664 01092 02151 52174 0578 470974 rs2235334 10566 0304 04116 04327 00851 22002 02025 06527 07622 13241 03893 45033
SS73405565 06465 04214 1 - - 27585 00967 01476 - rs5756708 0007935 0929 1 09375 02266 38792 01329 07154 07671 07972 02355 26989 rs5750446 01395 07088 1 06786 00878 52435 02923 05887 07745 13636 04424 42028 rs7285572 0714 03981 1 - 30383 00813 01336 - rs1997674 0003168 09551 1 10556 01606 69386 02923 05887 07745 13636 04424 42028 rs738304 01812 06704 1 06429 0083 49811 01232 07256 07759 08148 02595 2559 rs9610774 02471 06191 1 05619 00563 56131 01098 07404 08179 11647 04724 28715 ts3817806 16842 01944 0375- - - 18063 0179 02385 0405 01049 15633 rs3817805 _ _ _ 06094 0435 04769 14457 05717 36561 rs3817804 - - - - 01203 07287 07968 11948 04368 32685
§ hCV25994982 20225 0155 03333 - - 19363 01641 0216 29231 06098 140108 rs3817803 _ 14249 02326 03289 176 06917 44784 rs3817802 - - - - - 07759 03784 04846 04874 00957 24814
O rs3817801 _ - 10114 03146 03428 16029 06366 40356 rs3817800 _ 10114 03146 03428 16029 06366 40356
- - - - 10114 03146 03428 16029 06366 40356
SS73405568 17679 01836 03636 - 39286 00475 00689 4 09349 171135 rs12627982 - - - - - 12277 02679 03313 16923 0664 4313 rs5756705 08424 03587 05333 05323 01362 20805 57016 00169 002 27922 11879 55633
IS2235333 23855 01225 02088 03068 00639 1473 31798 00746 00915 21429 09212 49847 rs2235332 - - - 03822 05364 07095 0596 01138 31221
SS73405569 05439 04608 1 - _ _ 00007788 09777 1 10157 03407 30278 rs2235331 20147 01558 02064 03333 00688 16157 03049 05808 06722 12632 05509 28962
SS74804654 _ _ 12629 02611 03643 20119 05898 6863 rs5756701 _ _ 09717 03243 03709 18571 0539 63987 rs5750442 14571 02274 04118- 07071 04004 05681 1625 05224 5055 rs2295155 _ _ _ 00121 09123 1 10714 0314 36555 rs5756700 _ 05208 04705 0494 06 01487 24213 rs5756699 - . - - 12432 02649 03109 04667 01203 18096 rs5756698 _ - - - 05208 04705 0494 06 01487 24213 rs5756697 _ 08102 03681 05216 05652 01621 19709
SS73405574 _ _ _ 02841 0594 06723 06296 01137 34865
SS73405577 - - 00399 08417 1 12857 01085 152376 rs12169694 - - - - 05208 04705 0494 06 01487 24213 rs5750411 15306 0216 04- - - 08102 03681 05216 05652 01621 19709 ss73405583 15306 0216 04- - - 01812 06704 1 06429 0083 49811 rs3761441 _ _ _ 0002455 09605 1 096 01909 48265
SS74804657 _ _ 02102 06466 1 152 02515 91879 rs11089844 - - - - - 01992 06554 07102 07083 01551 32349 rs2071839 _ - - 02618 06089 07256 06957 01725 28047 rs2071840 - - - - - 03284 05666 07232 06667 01658 26811 rs6000748 00269 08697 1 08854 02068 37906 01232 07256 07759 08148 02595 2559 rs5756692 _ - - - 03284 05666 07232 06667 01658 26811
Cn SS74804658 _ _ 03284 05666 07232 06667 01658 26811 i rs2284053 - - 03284 05666 07232 06667 01658 26811 rs2284052 _ _ _ - 00903 07638 1 08 01864 3434
SS74804659 _ _ - 08268 03632 063S9 27692 02854 268652
SS74804660 - - 22668 01322 02331 03111 00647 14957 rs9607464 13889 02386 05102 - _ _ 27972 00944 0163 38571 0736 202151 rs9610767 - 01727 06777 1 14615 02422 88201 rs2071841 _ - 0003168 09551 1 10556 01606 69386
SS74804661 14571 02274 04118- _ 01395 07088 1 06786 00878 52435 rs3788527 - - - - 08539 03554 04267 04722 00939 2375 rs739166 0106 07448 07442 08 02085 30702 00601 08063 1 08636 02674 27894
SS73405555 12628 02611 03146 17333 06602 45511 0149 06995 08119 08333 033 21042
SS73405554 05525 04573 1 - - 00114 0915 1 09091 0158 52302
06671 08115 08163 03236 20594
04348 06071 01988 18539
04909 06267 072 02822 18369
07647 0824 0875 03649 20933
06048 06519 07917 03265 19193
04799 05068 07308 03057 1747
04508 19032 04453 81342
04799 05068 07308 03057 1747
04799 05068 07308 03057 1747
Figure imgf000024_0001
04861 05079 07343 03075 17536 hCV25627052 - - - - - 16441 01998 05493 -. -
SS73405550 05542 04566 1- _ - 08115 03677 04479 19333 04515 82778
SS73405549 _ _ _ _
SS73405548 - - - - - 66736 000979 00182 108621 12136 97216
HCV25760891 - - - - - - 14509 02284 02486 25376 05339 120621 hCV256276S1 05704 04501 1 - _ _ 07323 03921 04538 1871 04376 79994
SS73405547 - - - - - 70675 000785 00156 114815 12797 103013
SS73405543 19018 01679 03469 - _ _ 02217 06377 06908 14516 03055 68975
155756604 0682 04089 04632 14881 05776 38335 22875 01304 01418 04825 01858 12528 rs2413460 0443 05057 06872 18 03134 103397 00547 0815 1 08718 02761 27525
SS73405539 - - 0002455 09605 1 096 01909 48265 rs6000657 0714 03981 1 - - 0009462 09225 1 09231 01839 46323 is8138201 - - - - - 08109 03678 0637 04286 00651 28218 rs2284050 06415 04232 04949 18261 0413 80745 0 1 1 1 03064 32641 rs53830145 06415 04232 04949 18261 0413 80745 03643 05461 07635 06909 02074 23014 rs6000655 03384 05607 07163 15652 03434 7135 08672 03517 03883 05667 01705 18833
SS73405534 - - - - 30383 00813 01336 _
SS73405530 - - - 20202 01552 02017 02857 0047 17374
SS73405529 _ 20202 01552 02017 02857 0047 17374
SS73405528 - - - - - 18241 01768 02144 03036 00501 18387 rs3831702 00669 07959 1 06897 00407 116841 01301 07183 07681 125 03714 42066 rs1122084 0714 03981 1 - _ 42095 00402 00515 025 00634 09863
SS73405526 - 10263 0311 03908 30769 03187 297102
3 SS73405523 - - 16865 01941 03806 4 04318 370559 tJ ts2006178 00173 08953 1 10909 02985 39863 20963 01477 01969 03529 00834 14944
(£ _ _ 18241 01768 02144 03036 00501 18387
SS73405519 _ _ 18241 01768 02144 03036 00501 18387 rs733271 03985 05278 07391 15455 03979 60028 3107 0078 01134 02879 00689 12032 rs5756596 11194 02901 04905 21818 05037 94501 3107 0078 01134 02879 00689 12032 rs5756S95 11194 02901 04905 21818 05037 94501 3107 0078 01134 02879 00689 12032
|-s2899289 2303 01291 01666 34545 06525 182902 05182 04716 05659 06538 02051 20849
SS73405515 - - - - 08109 03678 0637 04286 00651 28218 rs5756592 2303 01291 01666 34545 06525 182902 05182 04716 05659 06538 02051 20849 rs4239887 05322 04657 06852 19 03318 108791 30879 00789 00938 035 01067 1148 rs4239886 14571 02274 05059 - 05397 04626 05431 16 04544 56341 rs763082 16953 01929 03154 25714 06029 10967 3107 0078 01134 02879 00689 12032
[54325860 12659 02605 0346 2125 05633 80157 3107 0078 01134 02879 00689 12032 rs4565465 07802 03771 0518 18214 04768 69576 3107 0078 01134 02879 00689 12032 rs5756591 14803 02237 03168 24286 0567 104025 27943 00946 01183 03048 00726 12795 rs2284047 05208 04705 07199 17246 03884 75581 02377 06259 07635 07403 02206 24845 rs2284046 14803 02237 03168 24286 0567 104025 15873 02077 03451 04318 01149 16223
[S2284045 14803 02237 03168 24286 0567 104025 15873 02077 03451 04318 01149 16223 ts1018808 14803 02237 03168 24286 0567 104025 27943 00946 01183 03048 00726 12795
SS73405510 10771 02994 04877 21587 04949 94168 30498 00807 01122 02888 00685 12169 rs5756590 06085 04354 04959 1803 0405 80266 01126 07372 07768 08205 02583 26065 rs7288236 28986 00887 014- - - 19704 01604 02428 23333 07066 77046 rs7288504 28986 00887 014- _ 19704 01604 02428 23333 07066 77046
SS73405509 28986 00887 014- 19704 01604 02428 23333 07066 77046
SS73405506 06803 04095 1 - - - 0 1 1 1 01517 6593
SS73405505 - - - - 0 1 1 1 01517 6593 rs13058636 06803 04095 1 - _ 00777 07804 1 12174 0305 48599 rs4821626 37037 00543 O 0746 - _ 34353 00638 00858 30513 09205 101144 rs2284043 00277 08679 1 08718 0173 4392 06933 04051 05532 16364 05115 5235
SS73405500 - - . _ - O 1 1 1 01517 6593 rs2284042 01812 06704 1 06429 0083 49811 01202 07288 0779 12222 03928 38027
SS73405499 - - - _ - 21277 01447 02653 - rs2284040 01812 06704 1 06429 0083 49811 04831 0487 05691 15 0477 47168 rs1041895 01812 06704 1 06429 0083 49811 04831 0487 05691 15 0477 47168
IS5750405 22113 0137 01825 03714 00983 1403 23911 0122 01476 0381 01102 13171 rs5750404 22113 0137 01825 03714 00983 1403 23911 0122 01476 0381 01102 13171 rs1041894 22425 01343 02192 475 05258 42909 3 00833 01482 27857 08613 90099 rs5995411 00183 08925 1 0913 02441 34151 22059 01375 02165 0375 01005 13993
IS5995410 00183 08925 1 0913 02441 34151 22059 01375 02165 0375 01005 13993 rs3213557 00183 08925 1 0913 02441 34151 22059 01375 02165 0375 01005 13993 rs4821615 00242 08763 1 11304 02414 52931 00123 09118 1 09333 02753 31646 ss73405422 05643 04525 1 - - - 01906 06624 1 05357 00312 91868 ss73405423 01527 0696 1 05714 00334 97717 02693 06038 07068 06771 01544 29683
SS73405424 01527 0696 1 05714 00334 97717 02693 06038 07068 06771 01544 29683 rs9610689 12159 02702 05453 02679 00224 3202 09962 03182 04893 21 04807 91737
TB6000632 05039 04778 06552 05385 00957 30288 09826 03216 03686 05432 01616 18259
SS73405425 16845 01943 03778 - - - 30252 0082 01097 35 08174 149861
SS73405426 15126 02187 02652 04 00905 17682 0365 05457 07224 15278 03845 60704 rs2213430 01458 07025 1 07143 01264 40374 01383 071 0754 12727 03567 45417 rs2213431 - - - - - 01197 07294 1 15455 01291 185016 rs2285109 10844 02977 04069 04239 00819 21946 0002085 09636 1 10303 0286 37113 rs2339773 23205 01277 02837 01867 00177 19689 21139 0146 02055 03951 01115 14 rs2239775 05992 04389 1 - - - 06588 0417 05327 16941 04717 6084
IS22239774 00728 07873 1 06786 00399 115265 00338 08541 1 08889 02531 31215 rs6000622 - - - - 01585 06906 1 144 02375 87316
(S9607434 16118 02042 03878 - 00137 0907 1 09286 02679 32191
SS73405428 16118 02042 03878 - - 00007347 09784 1 10182 02767 37468
I-S929023 00582 08094 1 125 02034 7683 0262 06088 07574 13737 04066 46414 rs4821614 14859 02229 02975 04333 01109 16934 13352 02479 03447 04606 01219 17409 ts2284038 05731 0449 04873 05833 01433 23749 10022 03168 03541 05067 01323 19402 rs11704316 15341 02155 04- 01555 06933 0753 13 03524 4796 rs9610686 1004 03163 04511 04727 01073 20826 1293 02555 03327 04571 01169 17873 rs5756573 1478 02241 04091 - _ 02375 0626 0676 06522 01159 36694 rs12166336 0133 07153 1 05926 00346 101426 05672 04514 05372 0625 01833 21311 rs12170S12 17768 01825 02977 - 00188 0891 1 09396 03852 22918 hCV2493926 - - - - - 03899 05323 0765 0675 01955 23308 ts9607433 05682 0451 1 - - - 0273 06013 06577 07875 03212 19308 rs1476002 1148 0284 05345 - - - 15722 02099 024 17857 07175 44444
SS73405427 11301 02878 O 5366 - 27775 00956 01225 04571 01802 116 rs9607432 05594 04545 1 - 03942 05301 06563 07517 03081 18345
[-9610685 05594 04545 1- 02235 06364 08221 08059 03291 19735 rs9607431 1148 0284 05345- _ - 0046 08303 08273 11 04601 26297 rs12484612 10872 02971 04059 1746 06076 50171 14734 02248 02735 05911 0252 13863
IS9610684 11301 02878 05366 - - 0002728 09583 1 09773 04125 23156 rs2285110 12346 02665 03226 04118 00826 20539 00103 0919 1 09579 04179 21954 rs33917324 31833 00744 01229 03529 01087 11457 3146 00761 00974 04423 01777 11011 rs9610683 23653 01241 01602 03636 00963 13736 16045 02053 02719 05783 02468 13547 rs9010682 2886 00894 01038 03312 00883 12417 16045 02053 02719 05783 02468 13547 rs2284037 57468 00165 00162 01801 00389 0835 20567 01515 01858 05362 02276 12631
IS4140870 05431 04611 06534 04412 00474 41085 11339 02869 03935 06297 02681 14792 rs739042 49433 00262 00362 0246 00666 09083 09036 03418 037 06538 02716 15742 rs5845330 - - _ 12305 02673 02967 17667 06417 48642
IB11089831 11664 02801 05325 - 21418 01433 02519 02286 0027 19378 rs2284036 2976 00845 01558- 17549 01853 02035 05565 02328 13304 rs739041 48555 00276 0036 02482 00671 09183 05032 04781 04968 07241 02963 17696 ss73405460 02471 06191 1 05619 00563 56131 1319 02508 0291 06071 02583 14271 rs4820276 11852 02763 04051 03143 00352 2804 16457 01995 0285 05714 02421 13487
SS73405463 - - - - - 12277 02679 02986 36471 03189 417037 rs9610680 06093 0435 06502 04214 00453 39227 09836 03213 04024 06569 02857 15102
SS73405466 00873 07676 1 085 0289 24999 12084 02717 02886 0625 02697 14486 rs12166968 00154 09012 1 08571 0075 9799 02173 06411 07414 07143 01727 29542 rs9798725 04795 04886 05185 15591 04403 55209 14856 02229 0297 16728 07292 38373 ss73405467 0442 05062 05221 15323 04325 54282 13052 02533 02982 16221 07056 37292 ts5995400 03699 05431 05303 14785 04169 52429 06867 04073 05261 14241 06163 32908
IB13054143 00154 09012 1 08571 0075 9799 00681 07942 1 08438 02352 30268
IS6000619 0442 05062 05221 15323 04325 54282 13052 02533 02982 16221 07056 37292 rs9610679 17679 01836 05517- 19141 01665 02067 055 02348 12884 rs9610678 17679 01836 05517 - 19141 01665 02067 055 02348 12884
IS5756570 0442 05062 05221 15323 04325 54282 15225 02172 02894 17059 07278 39986
IS36110509 15105 02191 0282 23387 05853 93449 14805 02237 02943 16762 07273 38631 rs2899284 12817 02576 03361 2 05927 67489 06497 04202 05272 14091 06112 32486 rs6000618 0005525 09407 1 10507 02852 38709 06497 04202 05272 14091 06112 32486 rs13058180 06566 04178 04483 15 05606 40134 00136 09071 1 10556 04255 26184 rs6000617 00891 07653 07414 12258 03214 46751 07679 03809 04057 14531 06289 33574 rs9B10677 00891 07653 07414 12258 03214 46751 07679 03809 04057 14531 06289 33574 rs6000616 00891 07653 07414 12258 03214 46751 07679 03809 04057 14531 06289 33574 rs961067β 00891 07653 07414 12258 03214 46751 07679 03809 04057 14531 06289 33574 rs9610675 00636 08009 1 11875 03118 45224 09836 03213 04024 15223 06622 34998 rs9622582 00137 09068 1 10833 02838 41357 11049 02932 03946 15714 0675 36581
SS73405476 00137 09068 1 10833 02838 41357 11049 02932 03946 15714 0675 36581
SS73405477 00137 09068 1 10833 02838 41357 11049 02932 03946 15714 0675 36581
SS73405478 _ - _
SS73405479 00137 09068 1 10833 02838 41357 11049 02932 03946 15714 0675 36581 rs5756568 0055 08146 1 12222 0228 65528 10726 03004 03319 16765 06288 44699
SS74804655 - - _ 20329 01539 02076 0479 01729 13267
SS74804656 2475 01157 02028 - - 05704 04501 0587 25 02154 290114 rs933223 00511 08212 1 11667 03062 44451 08305 03621 04047 14732 06392 33953 ιt>933222 00636 08009 1 11875 03118 45224 07071 04004 05286 14286 06212 32852 rs6000614 17476 01862 05511 - 25839 0108 01321 04857 01999 11801 rs933321 13192 02507 05148 - _ 37251 00536 00692 34909 09559 127491
SS73405482 12708 02596 05192 - _ 32683 00706 01226 32 08862 115555 ss73405483 - - - - r-4820274 32923 00696 01454 - 09652 03259 03774 05556 01711 1804
SS73405485 - - - - - - - rs4821605 01107 07394 1 06207 00365 105554 03851 05349 05587 06875 02101 225
1S4820273 29738 00846 01647 - 10576 03038 03863 055 01751 17273
R54820272 45536 00329 00639 - - 10576 03038 03863 055 01751 17273
SS73405486 04835 04868 04964 06087 01491 24843 01743 06763 07702 12821 0399 41197 rs9607428 19666 01608 02044 02885 00471 1767 00314 08594 1 09 02804 28885
SS73405487 21458 0143 01822 03727 00973 14279 00856 07699 1 08392 02591 27178
SS73405488 0049 08249 1 08667 02439 30802 04343 05099 05624 0675 02093 2177 ss73405489 21701 01407 02886 01975 00189 20633 27729 00959 01304 036 01065 12168
SS73405491 05733 0449 0718 17778 03964 79737 15242 0217 03393 21818 06254 76112
SS73405492 00903 07638 1 08 01864 3434 02377 06259 07635 07403 02206 24845
SS73405493 15306 0216 04- _
SS73405495 - - - - - 25888 01076 01314 0372 01097 12618 rs3218250 _ _ - - 03482 05552 1 2 01926 207724 rs3218251 31854 00743 01516 - - - 25738 01086 01438 26667 07928 89695 rs228979 01391 07091 1 05862 00344 99911 01391 07091 07716 125 03867 40407 rs3218252 03032 05819 07222 15217 03393 68256 00582 08094 1 11667 03332 40846 ts3218253 29217 00874 01684 - - - 06269 04285 05555 16095 04938 52458 rs2284035 02776 05983 07197 15 03298 6822 00121 09123 1 09333 02736 31844 t.3218255 - - - - - - - rs228978 29738 00846 01647 - 19957 01578 02527 22857 0719 72665 rs3218258 29738 00846 01647 - _ 09067 0341 03966 175 05505 55634 rs228976 01142 07354 1 07037 0091 54444 02266 0634 07734 13203 04201 41495 rs3218261 14091 02352 042- - - 03284 05666 07232 06667 01658 26811 rs2284034 01395 07088 1 06786 00878 52435 0001142 0973 1 10196 03306 31446 Table 2 shows genotypes at all detected polymorphic loci between the CardlO and Il2rb loci and the results of case-control analyses between the ESN and HIV-1-infected individuals.
The observed case-control differences apparently increased toward the downstream (centromeric) portion of the Rac2 locus, and there was one region, between the 5th and 6th Rac2 exons (region l), where multiple polymorphic loci with statistically significant case-control differences between the ESN and HIV-infected groups were accumulated (Figure 1 and Figure 3).
[56] Thirtyone SNPs and nucleotide polymorphisms located in the regions 1 and 2 as shown in Figure 1 are listed in the SEQUENCE LISTING with SEQ ID Nos 1 through 31. The SNP ID numbers for each SEQ ID Nos are: (1) rs9610683, (2) rs9610682, (3) rs2284037, (4) rs739042, (5) rs2284036, (6) rs739041, (7) ss73405466, (8) rs9798725, (9) ss73405467, (lθ) rs5995400, (11) rs6000619, (12) rs5756570, (13) rs36110509, (14) rs2899284, (15) rs6000618, (16) rs6000617, (17) rs9610677, (18) rs6000616, (19) rs9610676, (20) rs9610675, (21) rs9622582, (22) ss73405476, (23) ss73405477, (24) ss73405479, (25) rs5756568, (26) rs933223, (27) rs933222, (28) rs933321, (29) ss73405482, (30) ss73405484 and (31) ss73405485. Among these, the SNPs significantly accumulated in ESN individuals are listed in Table 3. For each of SEQ ID Nos 1 to 6 in region 1, detected alleles are as follows, respectively: C/G, A/G, T/C, T/C, T/C, and C/T. Moreover, for each of SEQ ID Nos. 1 to 6 in region 1, SNPs accumulated in ESN individuals are as follows, respectively: C, A, T, T, T, and T. [Table 3]
Table 3. Case-control associations between chromosome 22 genotypes and the
ESN status.
Linked Locus/location Allele/haplotype Exact P Odds 95% CI gene (model)* (Fisher) ratio
CardlO ss73405568 T (res) 0.069 4.00 0.935-17.1 rs5756705 C (res) 0.020 2.79 1.188-6.56
Pscd4 ss73405548 T (res) 0.018 10.86 1.214-97.2 ss73405547 T (res) 0.016 11.48 1.280-103.0
Rac2 rs2284037 T (dom) 0.016 0.180 0.0399-0.835 rs739042 T (dom) 0.036 0.246 0.066-0.908 rs739041 T (dom) 0.036 0.248 0.067-0.918
Rac2 Intron 5 TTT (dom) 0.0084 0.154 0.033-0.704 haplotype
* res, recessive; dom, dominant.
[57] Based on the research described above, that is the significant accumulation of a particular allele at the indicated loci in ESN individuals who show resistance to HIV acquisition despite repeated sexual exposures to HIV-I, one aspect of the present invention provides use of the above listed SNPs and nucleotide sequence polymorphisms for diagnosis of resistance to HIV infection.
Among these SNPs and nucleotide polymorphisms, (i) rs739041 (SEQ ID No. 6), (ii) rs739042 (SEQ ID No. 4), and (iii) rs2284037 (SEQ ID No. 3) are most significant for detecting genetic predisposition to resistance to virus.
Moreover, (iv) rs9610683 (SEQ ID No. l), (v) rs9610682 (SEQ ID No. 2), and (vi) rs2284036 (SEQ ID No. 5) are also significant for detecting genetic predisposition to resistance to virus.
The SNP ID Nos are identification numbers used in dbSNP which is world's largest database for nucleotide variations and is part of the US National Center for Biotechnology Information (NCBI). The "rs" ID number is an identification tag assigned by NCBI to a group of SNPs that map to an identical location. The rs ID number is assigned after submission. The "ss" ID number is simply a unique identifier assigned by NCBI when SNP is submitted to dbSNP. Anyone can download the information about SNPs having SNP ID Nos from the following site! http 7/www.ncbi.nlm.nih. gov/projects/SNP/index.html
[58] The above described SNP genotypes are highly linked to each other within the above Rac2 region, with the levels of LD being higher among the ESN than in HIV-infected individuals in the region 2 (Figure 3). Thus, the polymorphic loci within the regions 1 and 2 constitute small numbers of haplotypes. In particular, the region 1 downstream haplotype TTTis highly accumulated among the ESN individuals: 34 of the 36 ESN individuals whose genomic DNA sequenced (94%) possessed the TYThaplotype, while 47 of the 65 HlVl-infected individuals possessed the CCC haplotype (P - 0.0084). [59] Figure 1 illustrates the observed haplotypes divided in three blocks. Haplotypes of the left block consist of SNPs designated by SNP ID Nos. rs9610683 and rs9610682. Haplotypes of the central block consist of SNPs designated by SNP ID Nos. rs2284037, rs739042 and rs739041. Hapotypes of the right block consist of SNPs and nucleotide polymorphisms designated by SNP ID Nos. ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73405482, ss73405484 and ss73405485.
The haplotype TTT in the central block were significantly accumulated in ESN individuals. Thus, one aspect of the present invention provides use of the above three haplotypes for diagnosis of resistance to HIV=infection. [60] The above polymorphic regions 1 and 2 in the Rac2 locus colocalized with the chromosomal segments that showed >50% sequence homology between humans and mice (HHl and HH2 in Figure l) and one of the intragenic regions previously reported to exhibit elevated cytosine methylation in hematopoietic cells (Reference 20, Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333), indicating that these regions may contain conserved regulatory elements. To examine this directly, the genomic fragments that harbored the polymorphic region 1 isolated from representative healthy control individuals who homozygously possessed either the TTT or CCC haplotype were cloned and inserted into the downstream of the luc gene along with the known Rac2 core promoter (Reference 20, Ladd, PD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333) placed in the upstream. The Rac2 promoter is known to be strong and promiscuous, and tissue-specific expression of this gene is regulated by the presence of putative repressor protein (Reference 20, Ladd, P.D. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333). In the present study, the inventors detected no sequence polymorphisms in the Rac2 promoter. Consistent with the above notions, transfection of the luc constructs harboring the Rac2 promoter resulted in high levels of luciferase expression both in the T- lineage Jurkat and macrophage-lineage THP-I cells (Figure 4). Further, coexistence of the region 1 fragment possessing the CCC haplotype strongly suppressed the luc expression in both types of cells, indicating the presence of a repressor protein binding site within region 1. Importantly, when the region 1 fragment possessing the TTT haplotype coexisted, significantly higher luciferase activities were induced in comparison with those induced with the promoter alone. Interestingly, however, this effect was observed only in the T-lineage Jurkat cells, not in THP-1 cells. These results clearly indicate a functional difference of the region 1 fragments possessing the TTT or CCChaplotype"- the Rac2 intvon haplotype TTT that is significantly more accumulated in the ESN individuals lacks the repressor activity and functions instead as an enhancer of gene expression in T-lineage cells, while the haplotype CCC remains repressive. When tested in Jurkat cells, the Rac2 region 2 fragments apparently reduced the expression of the luc gene regardless of the haplotype, indicating a specific importance of region 1 where the genotypes were skewed between the two phenotypic groups. The Rac2 region 1, but not region 2, haplotypes were also associated with in vivo expression levels of the Rac2 in HIV-I antigen-stimulated PBMC. Thus, when the levels of Rac2 expression were examined quantitatively by real-time PCR in cultured PBMC, a 2-fold increase of the Rac2 message was observed after 6 hours of stimulation with the HIV-I peptides in the individuals homozygously possessing the TTT haplotype in region 1 (Figure 5). On the other hand, the levels of Rac2 expression decreased after the antigenic stimulation in cells homozygous for the CCC haplotype. Importantly, haplotypic differences in region 2 did not affect the Rac2 induction after the antigenic stimulation, again indicating that the polymorphic region 1, but not region 2, harbors a functional regulatory element of the Rac2 expression.
[61] Finally, the Rac2 region 1 haplotypes did affect HIV" 1 replication in vitro. Thus, when PBMC from uninfected healthy control individuals possessing the homozygous CCC haplotype or harboring the TTT haplotype in region 1 were infected in vitro with HIV-I, different kinetics in the increase of p24 concentrations were observed. In supernatants of PBMC possessing the homozygous CCC haplotype, p24 concentrations were significantly higher as compared to what was observed in supernatants of cells harboring the TTT haplotype both upon infection with the CCR5-tropic BaL strain of HIV-I or with a primary CCR5"tropic HIV-I isolate, v.6 (Figure 6). Median p24 levels in cultures of the cells homozygous for the TTT haplotype at 5 days after infection with either one of the two CCR5-tropic viruses were <5% of those observed with the cells of the haplotype CCC. On the other hand, p24 concentrations did not differ significantly when PBMC of different region 1 haplotypes were inoculated with the primary CXCR4-tropic HIV-I isolate vl7, and p24 concentrations at 5 days after infection even became higher than those in the cells of the CCC haplotype when PBMC with the heterozygous haplotype were infected with the CXCR4-tropic laboratory strain IIIB. None of the individuals whose PBMC were used for the above in vitro infection experiments possessed the CCR5Δ32 allele. Thus, the regulatory Rac2 intxon haplotype TTT, which is significantly more accumulated among ESN individuals, is associated with a restricted replication of CCR5-tropic HIV-I. The possible gene-dose effect in the induction of Rac2 after the antigenic stimulation (Figure 5) might explain the apparent differences in median p24 levels between the individuals homozygous and heterozygous for the .TTThaplotype. [62] As Rac2 is involved in chemotaxis and phagocytic function of neutrophils and macrohages (Reference 21, Ambruso, D.R. et al., "Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation", Proc Natl Acad Sci U S A, 2000, vol. 97, pp4654-4659l Reference 22, Williams D.A. et al., "Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency", Blood, 2000, vol. 96, pp 1646" 16541 Reference 23, Yamauchi A et al., "Rac2- deficient murine macrophages have selective defects in superoxide production and phagocytosis of opsonized particles", J Immunol, 2004, vol. 173, pp5971"5979.) and is the predominant regulator of NADPH oxidase involved in superoxide production (Reference 24, Diebold BAet al., "Molecular basis for Rac2 regulation of phagocyte NADPH oxidase", Nat Immunol., 2001, vol. 2, pp211-215; Reference 25, Bokoch GM et al, "Current molecular models for NADPH oxidase regulation by Rac GTPase", Blood, 2002, vol. 100, pp2692-2696), the observed in vitro effects of the Rac2 region 1 haplotypes on HIV-I replication in peripheral blood mononuclear cells (Figure 6) may conceivably be explained by the possible influence of the different Rac2 expression levels on innate immune functions that affect HIV replication.
[63] It is also known that Rac2 is required for the migration of Bl lymphocytes, a specific subpopulation of B-lymphocytes, from the peritoneal cavity to gut-associated lymphoid tissues (Reference 26, Croker, B.A. et al., "The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-Ia and marginal zone B lymphocytes", J.Immunol., 2002, vol.168, pp.3376-3386), and Bl-derived plasma cells are the predominant sources of pathogen-reactive mucosal IgA (Reference 27, Fagarasan, S. et al., "Tindependent immune response: new aspects of B cell biology", Science, 2000, vol.290, pp.89"92). Therefore, the observed association between the high expression of the Rac2 gene and the ESN status might also explain the presence of HIV-1-reactive IgA in mucosal secretions of ESN individuals. Further, since Rac2 is pivotal in inducing the differentiation of CD4-positive effctor T cells toward ThI cells (Reference 28, Li, B. et al., "Role of the guanosine triphosphatase Rac2 in T helper 1 cell differentiation", Science, 2000, vol.288, pp.2219-2222), the observed association between the functionally active Rac2 regulatory polymorphism and ESN status also explains the higher production of interferon (lFN)-γ upon stimulation with HIV-I antigens in PBMC from ESN than those from HIV-I -infected individuals (Reference 13, Mazzoli S. et al., "HlVspecific mucosal and cellular immunity in HlVseronegative partners of HIV seropositive individuals", Nature Med., 1997, vol.3, pp.1250-1257; and Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HlVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024).
[64] Therefore, this virus resistance mechanism mediated by the Rac2 gene expression also has effect against all other viruses infected via mucosal route such as Human papilloma virus (HPV), Hepatatis C virus (HCV), Human herpes virus (HHV), Cytomegalovirus (CMV), Small round structured virus (SRSV), Influenza (Flu) virus and the like. It is noteworthy that a SNP associated with differences in the course of HCV infection is located in the region close to the Rac2 gene (Reference 29, Saito, T., et al., "Genetic variations in humans associated with differences in the course of hepatitis C", Biochemical and Biophysical Research Communication (BBRC), 2004, vol.317, pp.335-341).
[65] Accordingly, based on the above described research, one aspect of the present invention provides an ex vivo method, a chip and a kit for detecting genetic predisposition of a subject to resistance to virus.
In addition, one aspect of the present invention provides a method and a biopharmaceutical (Rac2 protein, inhibitor of Rac2 repressor protein, or the Rac2 gene inducer) for inducing or enhancing resistance to virus in a subject by utilizing virus resistance mechanism mediated by Rac2.
Further, one aspect of the present invention provides a method for screening a candidate therapeutic agent against virus by utilizing Rac2 or the Rac2 regulatory region as a target molecule. And another aspect of the present invention provides use of the Rac2 repressor protein binding site for a gene therapy to induce resistance to virus in a subject. [66] In one embodiment, the ex vivo method for detecting genetic predisposition of a subject to resistance to virus comprises following (a) and (b): (a) is genotyping a sample of the subject at a site of at least one SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos rs9610683, rs9610682, rs2284037, rs739042, rs2284036, and rs73904l; [67] And (b) is assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP or nucleotide polymorphism is a resistance allele, wherein the resistance allele is as follows (SNP ID No. : allele) : rs9610683 : C; rs9610682 : A; rs2284037 : T; rs739042 : T; rs2284036 : T; and rs739041 : C.
Among these SNPs and nucleotide polymorphisms, (i) rs739041, (ii) rs739042, and (iii) rs2284037 are more significant for detecting genetic predisposition to resistance to virus.
[68] In the present specification and claims, the term "genotyping" refers to a process of determining the genetic constitutions such as nucleotide sequence of an individual with a biological assay.
The term "sample", as used herein, refers to a biological sample or material obtained from a subject, for example a cell, tissue, organ, blood, hair or the like.
The term "site", as used herein, refers to a position or location of specific nucleotide or polynucleotide in the genome.
The term "nucleotide polymorphism", as used herein, refers to difference of nucleotide sequence between members of a species (or between paired chromosomes in an individual).
[69] The term "SNP" or "single nucleotide polymorphism", as used herein, refers to DNA sequence variation occurring when a single nucleotide A, T, C or G in the genome differs between members of a species (or between paired chromosomes in an idividuals).
The term "allele", as used herein, refers to a particular form of a gene or DNA sequence at a specific chromosomal location (locus).
The term "resistant allele", used herein for convenience to claim the invention, refers to the alleles accumulated in ESN individuals. [70] In another embodiment, the ex vivo method of the present invention comprises (a) genotypying a sample of the subject at sites of SNPs and nucleotide polymorphism constituting a haplotypel and (b) assessing the subject as having genetic predisposition of resistance to virus if the haplotype which is significantly accumulated in ESN individuals is present.
In the present specification and claims, the term "haplotype" refers to a set of closely linked genetic markers present on one chromosome which tends to be inherited together.
[71] In one embodiment, the haplotypes significantly accumulated in ESN individuals are following three haplotypes:
(1) A haplotype including: (SNP ID No. : allele) (i) rs9610683 : Cl and (ii) rs9610682 : Al
(2) A haplotype including: (SNP ID No. : allele) (i) rs2284037 : T; (ii) rs739042 : T; and (iii) rs739041 : T; and
(3) A haplotype including: (SNP ID No. : allele) (i) rs9610683 : C; (ii) rs9610682 : Al and (iii) rs2284036 : T.
[72] For genotyping a sample of the subject at sites of SNPs and nucleotide polymorphisms, any techniques publicly known in the art can be used, including single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis by denaturing high-performance liquid chromatography (DHPLC), direct DNA sequencing, Invader assay, and TaqMan assay.
In one embodiment, SNP or nucleotide polymorphism can be detected by the single-strand conformation polymorphism (SSCP) analysis in which electrophoretic separation of single -stranded nucleic acids based on subtle differences in sequence (often a single base pair) results in a different secondary structure and a measurable difference in mobility through a gel (Reference 30, Orita, M. et al., "Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction", Genomics, 1989, vol.5(4), pp.874-879).
[73] In one embodiment, nucleotide polymorphism can be detected by the heteroduplex analysis by denaturing high-performance liquid chromatography (DHPLC) that is based on DNA heteroduplex formation and separation of heteroduplex from homoduplex molecular species by means of ion-pair reverse phase HPLC (Reference 31, Oefner, P.J. et al., "DNA mutation detection using denaturing high performance liquid chromatography", Current protocols in human genetics, 1998, p.7 -10:l-7 -10 -12).
[74] In one embodiment, SNP or nucleotide polymorphism can be detected by direct DNA sequencing, such as the chain termination method (Reference 32, Sanger, F. et al., "DNA sequencing with chain-terminating inhibitors", Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1977, vol.74(12), pp.5463-5467), which are well known in the art.
In one embodiment of direct DNA sequence, a subsequence of the Rac2 gene region encompassing the SNPs and nucleotide polymorphisms is amplified and either cloned into a suitable plasmid and then sequenced, or sequenced directly.
[75] In one embodiment, SNP can be detected by Invader assay. In this assay, three single -stranded DNA chains form a ternary complex with one base-pair overlap. This complex is composed of a DNA target oligonucleotide, which contains the SNP sequence of interest, and two other oligonucleotides. The upstream oligonucleotide is designated as invader oligo and downstream oligonucleotide is designated as a probe. These three oligonucleotide strands hybridize to one another, forming a one base-pair junction causing the 5' end of the probe to form a unhybridized 5' flap. This 5' flap is then cleaved by a Cleavase enzyme, resulting in the release of the 5' flap of the probe.
Secondary reaction generates quantifiable signals as follows: FRET cassette is labeled with a fluorophore (F) and a quencher (Q) so cleavage between them generates a fluorescence signal. FRET cassette is self hybridized at 5' portion, and 5' flap is hybridized with 3' portion of the FRET cassette so as to form a ternary complex with one base -pair overlap. And then, cleavage of 5' end of FRET cassette by Cleavase enzyme causes fluorescence signal (Reference 33, U.S. Patent No. 5,846,717; Reference 34, Lyamichev, V. et al., "Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes", Nature Biotechnology, 1999, vol.l7(3), pp.292-296.).
[76] In one embodiment, SNP can be detected by TaqMan assay which uses two types (wild type and mutant type) of probes having fluorescence dye and quencher and utilizes 5' to 3' nuclease activity of Taq polymerase. DNA fragment harboring SNP site is amplified by PCR primer. On the process of the amplification, TaqMan probe anneals to the DNA. Where the PCR primer extends to 5 ' side of the TaqMan probe and there is no mismatch between the TaqMan probe and DNA, the TaqMan probe is degraded by the 5' to 3' nuclease activity of the Taq polymerase. With the degradation of the TaqMan probe, fluorescence dye is separated by quencher and fluorescence is generated. Where there is a mismatch between the Taqman probe and DNA, the probe is not degraded, and fluorescence does not occur. SNP can be detected by measurement of this fluorescence (Reference 35, Holland, P.M. et al., "Detection of specific polymerase chain reaction product by utilizing the 5'— 3' exonuclease activity of Thermus aquaticus DNA polymerase", Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1991, vol.88(16), pp.7276-7280).
[77] In one embodiment, the present invention provides a method for inducing or enhancing resistance to virus in a subject. This method comprises enhancing the Rac2 gene expression in the subject.
The step of enhancing the Rac2 gene expression can be performed by any technique known in the art, for example, modulation of the Rac2 repressor protein binding site by using a DNA-binding protein, peptide, oligonucleotide, nucleotide analogue or chemical compound. And RNAi technique may be used for inhibiting the expression of a Rac2 repressor protein in order to enhance the Rac2 gene expression.
[78] In one embodiment, the present invention provides use of Rac2 protein in the preparation of a medicament for a treatment or prophylaxis of virus-induced disease. In other words, the embodiment of the present invention provides a method for treatment or prophylaxis of virus-induced disease including administering Rac2 protein to a subject.
The term "Rac2 protein", as used herein, refers to a full length polypeptide of Rac2 protein, its homologue or functional fragment thereof. The amino acid sequence and coding sequence of Rac2 is registered in Genbank under accession number NM_002872.
[79] In one embodiment, the present invention provides a nucleic acid exhibiting the Rac2 repressor protein binding activity (the polymorphic region 1) which is selected from the following nucleic acids : (A) a nucleic acid having the nucleotide sequence of SEQ ID No:32; and (B) a nucleic acid having more than 80%, 90% or 95% identity with the nucleotide sequence of SEQ ID No:32.
Preferably, one aspect of the present invention provides a nucleic acid exhibiting reduced Rac2 repressor protein binding activity, which has specific alleles at the sites of SNPs in the nucleotide sequence of SEQ ID No.32 as follows (base number in the SEQ ID No.32 : allele): (i) 132 : C; (ϋ) 144 : A; (iii) 1276 : T; (iv) 1959 : T; (v) 2301 :T, and (vi) 2379 : T.
The term "identity", as used herein, refers to a percentage of identical nucleic acid bases among two nucleotide sequences which are aligned in high order match. The percentage of the identity can be calculated by BLAST program under default setting (Reference 36, Altschul, S. F. et al, "Gapped BLAST and PSI-BLAST: A new generation of protein database search programs", Nucleic Acids Res., 1997, vol.25, pp.3389-3402). [80] The nucleic acid may comprise DNA, RNA or nucleic acid analogs such as uncharged nucleic acid analogs including, but not limited to, peptide nucleic acids (PNAs) which are disclosed in International publication WO 92/20702 (Reference 37). Such sequences can routinely be synthesized using variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry.
The above identified nucleic acid with SNP alleles can have modulated Rac2 repressor protein binding activity. By using the above identified nucleic acid with SNP alleles, Rac2 repressor protein binding activity can be controlled, thereby affecting the Rac2 expression.
The nucleic acid of the present invention can be used as a Rac2 repressor protein binding sequence and can be utilized for a gene therapy in order to enhance the Rac2 gene expression and induce resistance to virus in a subject.
For example, one or more copies of Rac2 repressor protein binding sequence with or without the SNP alleles can be introduced into the target cell, thereby affecting the distribution of the Rac2 repressor protein and its binding to the Rac2 region 1, enhancing the Rac2 expression. [81] In another embodiment, the present invention provides a method for screening a candidate therapeutic agent against virus by utilizing the Rac2 repressor protein binding site as a target molecule. This method comprises following: (a) contacting an agent with a nucleic acid fragment having the partial or entire nucleotide of the Rac2 region 1 nucleic acid of the present invention; (b) measuring an interaction between the agent and the nucleic acid fragment; and (c) selecting the agent having the interaction with the nucleic acid fragment as a candidate therapeutic agent against virus.
In the present specification and Claims, the term "a candidate therapeutic agent" refers to a chemical compound, lead compound or biological molecule which has a potential activity to induce resistance to virus in a subject. [82] In one embodiment, the present invention provides a method for screening a candidate therapeutic agent against virus. This method comprises the following: (a) administering an agent to a biological material; (b) measuring an expression level of the Rac2 gene in the biological material; and (c) selecting the agent enhancing expression level of the Rac2 gene in the biological material as a candidate therapeutic agent against virus.
The term "biological material", as used herein, refers to the material which possesses a fragment of or the entire Rac2 gene or the Rac2 gene regulatory element, for example, DNA fragment, a cell, a tissue, an organ, an organism and individual.
The measurement of the expression level of the Rac2 gene can be performed by using Luciferase or other reporter gene which is functionally linked to the Rac2 promoter, or quantitative real-time polymerase chain reactions (PCR) with primers and probes specific for the Rac2 sequence. [83] In one embodiment, the present invention provides a method for performing clinical trial for prevention, reduction, prophylaxis or treatment against virus. This method comprises following: (a) detecting genetic predisposition of subjects by the detecting method of the present invention;
(b) stratifying the subject by the result of the detected genetic predisposition;
(c) giving a prevention, reduction, prophylaxis or treatment to the stratified subject; and (d) assessing the effect of the prevention, reduction, prophylaxis or treatment to the stratified subject. [84] In the present specification and claims, the term "clinical trial" refers to any research studies designed to collect clinical data on response to a particular treatment, and includes, but not limited to, phase I, phase II, and phase III clinical trials.
The term "stratifying the subject", as used herein, refers to arrangement of each individual in a clinical trial population in a hierarchical order according to any status such as resistance level to virus infection or virus disease progression.
Examples of the "prevention, reduction, prophylaxis" and "treatment", as used herein, include administering therapeutic agent, surgery and the like. [85] In another embodiment, the present invention provides a chip or a kit for detecting genetic predisposition of a subject for resistance to virus. The chip comprises base plate and at least one probe detecting SNP or nucleotide polymorphism. The kit comprises reagent and at least one probe detecting SNP or nucleotide polymorphism. The SNP or nucleotide polymorphism is selected from the group consisting of SNPs and nucleotide polymorphisms designated by the SNP ID Nos rs9610683, rs9610682, rs2284037, rs739042, rs2284036, and rs739041. The SNP or nucleotide polymorphism may also include ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73405482, ss73405484 and ss73405485.
[86] The probes detecting each SNP or nucleotide polymorphism designated by each SNP ID Nos comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO: l (for rs9610683), 2 (for rs9610682), 3 (for rs2284037), 4 (for rs739042), 5 (for rs2284036), and 6 (for rs73904l). The probes detecting each SNP or nucleotide polymorphism designated by each SNP ID Nos may also comprises more than 7 bases of partial sequence or partial complementary sequence of 7 (for ss73405466), 8 (for rs9798725), 9 (for ss73405467), 10 (for rs5995400), 11 (for rs6000619), 12 (for rs5756570), 13 (for rs36110509), 14 (for rs2899284), 15 (for rs6000618), 16 (for rs6000617), 17 (for rs9610677), 18 (for rs6000616), 19 (for rs9610676), 20 (for rs9610675), 21 (for rs9622582), 22 (for ss73405476), 23 (for ss73405477), 24 (for ss73405479), 25 (for rs5756568), 26 (for rs933223), 27 (for rs933222), 28 (for rs93332l), 29 (for ss73405482), 30 (for ss73405484) and 31 (for ss73405485). The sequences designated by SEQ ID Nos 1 - 31 represent flanking region sequences harboring each SNP or nucleotide polymorphism designated by each SNP ID Nos.
[87] In the present specification and claims, the term "chip" refers to a tool or instrument for biological analysis having a portable size. The scope of the "chip" includes a plate or array used in biological experiments.
The term "base plate", as used herein, refers to a material holding a probe or probe sets.
The term "reagent", as used herein, refers to any chemical compound, solution or protein for a reaction detecting SNP or nucleotide polymorphism. Examples of the reagent include, but are not limited to, polymerase, Clevase enzyme, restriction enzyme, ligase, fluorescent dye, quencher, reaction buffer, hybridization buffer or etc.
[88] The probe sequence may comprise DNA, RNA or nucleic acid analogs such as uncharged nucleic acid analogs including, but not limited to, peptide nucleic acids (PNAs) which are disclosed in International publication WO 92/20702 (Reference 37). Such sequences can routinely be synthesized using variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry. Once synthesized, oligonucleotide probes may be labeled by any well-known methods.
[89] The probe comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID Nos 1 - 31. Preferably, the probe has 7 to 100 bases, more preferably, 10 to 50 bases, and most preferably, 15 to 30 bases.
The probe can comprise any sequence other than partial sequence or partial complementary sequence of SEQ ID Nos 1 - 31. For example, the probe may comprise linker sequence fixed to the base plate or 5' flap sequence for invader reaction.
[90] The probe is used for detecting SNP or nucleotide polymorphism in any form. For example, the probe can be used as "invader oligo" or "probe" in the invader assay described above. In the above described TaqMan assay, the probe can be used as "TaqMan probe".
EXAMPLES
[91] The following EXAMPLES are presented in^ order to more fully describe the base of the present invention. This EXAMPLES should in no way be construed as limiting the scope of the invention as defined by the Claims. [92] Summary of Methods in EXMAPLEs
ESN, their HIV-I -infected partner, and unexposed control individuals were enrolled with written informed consents as described previously (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024), and genetic analyses were performed with an approval from the ethical committee of the Kinki University School of Medicine. PBMC were stimulated with a mixture of HIV-I Gag and Env peptides as described (Reference 38, Clerici, M. et al., "Interleukin-2 production used to detect antigenic peptide recognition by T-lymphocytes from asymptomatic HIV-seropositive individuals", Nature, 1989, vol.339, pp.383-385), and total RNA extracted for both microarray analyses and real-time PCR quantification of the Rac2 expression. The microarray analyses were performed by using 12K chips (CombiMatrix Corporation,Mukilteo, U.S.A) harbouring 2 to 10 probes for each of the genes enlisted in Figure 7. Genotyping of known SNP was performed by TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, U.S.A.). Genomic DNA was amplified with primers (enlisted in the SEQUENCE LISTING under SEQ ID Nos: 33 - 72) and sequenced. Case-control analyses of the observed genotypes, calculation of the coefficients of LD, and extraction of haplotype compositions were performed by using the SNPAlyze ver. 6.0 (DYNACOM Co., Ltd., Yokohama, Japan). Putative Rac2 regulatory regions were cloned into the pGL3 plasmid (Promega Corporation, Madison, U.S.A.), transfected into human Jurkat cells, and the Dual-Luciferase Reporter Assays (Promega) were performed. The in vitro infection of PBMC with HIV-I and measurements of p24 were performed as described elsewhere (Reference 39, Biasin, M. et al., J. Infec. Dis., 2007, vol.195, pp.960-964).
EXAMPLE 1
[93] COMPARISONS OF THE CHANGES IN GENE EXPRESSION
LEVELS DURING THE HIV-I ANTIGENIC STIMULATION
The inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423 (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HlVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024; Reference 18, WO2004/035825, International publication of PCT application, 29 April 2004). To further narrow down the location of the putative HIVl resistance gene, the inventors of the present invention genotyped 74 ESN and 77 HIV-infected individuals enrolled from the same area of Italy at multiple loci of known SNP (see Figure 7 for their chromosomal locations and Table 1 for complete listing of the observed genotypes).
[94] Consistent with our previous observations (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2"13 are associated with HlVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015- 1024), the number of enrollees possessing the allele 229 at the D22S423 locus was significantly higher among the ESN than among the HIV-infected individuals (P = 0.0009, Fisher's exact test). As shown in Figure 2 and Table 1 for the details, the numbers of individuals possessing a particular allele were different between the ESN and HIV-infected individuals at the SNP loci linked to the CardlO and GRAP2, the latter located closely to the D22S423 locus. Analyses of linkage disequilibrium (LD) revealed a strong linkage (P < 10~5) between the rs202642 and rsl39562 loci in the HIV-1-infected individuals, but the lack of such linkage among the ESN individuals, in consistency with the previously reported disruption of LD at the D22S276 locus (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2"13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024).
[95] Expression levels of all the genes located in the above candidate region were compared between the ESN and HIV-infected individuals by using DNA microarrays. Probes for the representative human cytokines, chemokine ligands, and transcription factors were also included, along with the plant and phage genes as negative controls. To selectively detect the possible HIV-reactive changes in the host gene expression without being biased by non- genetic factors, the inventors of the present invention stimulated peripheral blood mononuclear cells (PBMC) prepared from each examined individual with a pool of 6 and 5 synthetic peptides representing the promiscuous HIV-I Gag and gpl60 envelope epitopes (Reference 38, Clerici, M. et al., "Interleukin-2 production used to detect antigenic peptide recognition by T-lymphocytes from asymptomatic HlVseropositive individuals", Nature, 1989, vol.339, pp.383-385), respectively, and prepared RNA before and at various time-points after the antigenic stimulation. Data obtained with multiple and redundantly placed probes for each gene were normalized for the entire array between individuals and between different time-points by using the Lowess equation.
[96] The overall expression levels of the tested genes were not different between the cells prepared before and at 1 hour after the antigenic stimulation; however, drastic changes in the expression of several host genes were observed between 1 and 6 hours after the antigenic stimulation. Therefore, the inventors of the present invention focused their comparisons on changes in host gene expressions between 1 and 6 hours after the antigenic stimulation. The induction of multiple cytokine and chemokine genes including those encoding ILr6, TNF-α, and CCL3L1 was observed upon antigenic stimulation, and the expression levels of these genes were comparable in HIV-infected and in ESN individuals at 6 hours after the antigenic stimulation (Figure 2). This indicates that differences in gene expression between the tested groups, if any, were not due to possible HIV-induced changes in T-cell subset compositions.
[97] Among all the chromosome 22 genes tested, only a small number were constitutively expressed at high levels or induced upon the stimulation with HIV-I antigens (Figure 2). Of these, constitutive expression of the RPL3 and apparent induction of the EIF3S7 were expected because ribosomal protein L3 is an indispensable component of the peptidyltransferase center (Reference 40, Meskauskas, A. & Dinman, J.D., "Ribosomal protein L3'- Gatekeeper to the A site", MoI. Cell., 2007, vol.23, pp.877-888), and eIF3 is the largest eukaryotic initiation factor that plays a central role in protein synthesis (Reference 41, Asano, K. et al., "Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits", J.Biol.Chem., 1997, vol.272, pp.27042-27052).
[98] Three genes of particular interest in possible relation to the resistance to HIV infection were nevertheless preferentially expressed in some ESN individuals after the antigenic stimulation: the Rac2 encoding a Rho- subfamily small GTPase that is selectively expressed in and involved in the activation of T-helper type-1 (Thl) cells (Reference 28, Li, B. et al., "Role of the guanosine triphosphatase Rac2 in T helper 1 cell differentiation"-, Science, 2000, vol.288, pp.2219-2222," Reference 42, Yu, H., Leitenberg et al., "Deficiency of small GTPase Rac2 affects T cell activation", J.Exp.Med., 2001, vol.194, pp.915-925); the Myh9 encoding the only non-muscle myosin heavy chain expressed in T cells, which regulates T-cell motility (Reference 43, Jacobelli, J. et al., "A single class II myosin modulates T cell motility and stopping, but not synapse formation", Nat.Immunol., 2004, vol.5, pp.531-538); and the Lgalsl encoding a galactoside -binding lectin which may facilitate the attachment of HIV-I to the cell surface (Reference 44, Ouellet, M. et al., "Galectin-1 acts as a soluble host factor that promotes HIV-I infectivity through stabilization of virus attachment to host cells", J.Immunol., 2005, vol.174, pp.4120-4126). The Rac2 and Lgalsl loci are located close to the two SNPs at which the allele frequencies were different between the ESN and HIV-infected groups (Figure 2). The augmented expression of the Rac2 after the antigenic stimulation was further confirmed by real-time PCR (Figure 5).
[99] Detailed Method of DNA Microarray Analysis Study Population
Heterosexual couples discordant for HIV-I serostatus and healthy uninfected donors were enrolled from the Santa Maria Annunziata Hospital, Firenze, and the Luigi Sacco Hospital, Milano as described previously (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024). Written informed consent was obtained from all the enrolees. All the enrolees are Caucasians from Toscany region. The criteria for HIVl infection and the ESN status have been described (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HlVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024). The ethics committees of the above hospitals have approved the research protocols. [100] DNA Microarray analysis
Total RNA was prepared from antigen-stimulated PBMC as described (Reference 39, Biasin, M. et al., "Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G^ a possible role in the resistance to HIV of HIV-exposed seronegative individuals", J.Infec.Dis., 2007, vol.195, pp.960-964), and cDNA was produced in the presence of an RNase inhibitor by using the T7-oligo(dT)24 primer. The resultant cDNA was purified, and biotinylated cRNA was prepared by using Biotin-16UTP and MEGAscript transcription kit (Ambion, Inc., Austin, U.S.A.). Two to 10 oligodeoxynucleoti.de probes were designed for each of the genes enlisted in Figure 7 by using the TargetSpecifier (CombiMatrix Corporation, Mukilteo, U.S.A.) and synthesized on microarray chips. After prehybridization, denatured biotin-conjugated cRNA samples were hybridized at 450C, overnight. After washing and blocking, microarray chips were incubated with Cy 3- conjugated streptavidin and washed vigorously. A fluorescence image of each microarray was scanned by a GenePix (Molecular Devices Corporation, Union City, U.S.A.), and analyzed by using the Microarray Imager (CombiMatrix Corporation).
[101] Two to 10 oligodeoxynucleoti.de probes were designed for each of the genes enlisted in Figure 7, and detected fluorescence intensities were Lowess normalized and colour-scaled in Figure 2 with each horizontal line representing one probe. The sample columns are arranged to combine expression levels in each individual of all the tested genes at 1 and 6 hours after the antigenic stimulation. The data obtained with PBMC from 5 representative ESN and 4 representative HIV-1-infected individuals are shown. The right half of Figure 2 shows the summary of the results of SNP genotyping, the details of which are shown in Table 1. Corresponding chromosomal and microarray locations of representative genes are also indicated. Numbers of individuals possessing a particular allele are compared between the groups by adopting a dominant gene hypothesis using Fisher's exact test. LD between SNP was analyzed by likelihood ratio test as described (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024), and exact P values were obtained.
EXAMPLE 2
[102] DNA SEQUENCE POLYMORPHISMS ACCUMULATED IN ESN
INDIVIDUALS
Based on the above results of the exploratory experiments, the inventors sequenced the chromosomal segments spanning the I12rb, Rac2-Pscd4, Mnfg, CardlO, and Lgalsl loci and compared the obtained nucleotide sequences between the ESN and HIV-1-infected individuals for the possible presence of polymorphisms with skewed genotype frequencies between the groups. The Myh9 locus located close to the D22S277 marker, the expression of which was apparently increased in some examined individuals upon the antigenic stimulation (Figure 2), was also sequenced. The inventors detected no sequence polymorphisms with a significant case-control difference at the Myh9, I12rb, and Lgalsl loci. However, notable accumulation of sequence polymorphisms with significantly different genotype frequencies between the two groups was observed in a segment between the CardlO and Rac2 (Figure 3, and 9Al to 9D4). The observed odds ratios were high for the two SNP within the Pscd4 locus, but the number of individuals possessing the skewed allele was very low. There were three SNP located close to each other within the 5th intron of the Rac2 gene (region l) at which significant case-control differences between the ESN and HIV-infected groups were observed by adopting a dominant gene hypothesis.
[103] Importantly, the SNP genotypes were highly linked to each other within the above Rac2 region and across the CardlO-Rac2 segment (Figure 3). Thus, the SNP within the Rac2 region 1 constitute a small number of haplotypes with skewed frequencies between the groups^ the region 1 haplotype TTT was significantly more accumulated among the ESN individuals, while the haplotype CCC was more frequently found in the HIV-infected group. It is notable that 34 of the 36 ESN individuals sequenced (94%) possessed the TTT haplotype on at least one chromosome, while 47 of the 65 HIV-I -infected individuals possessed the CCC haplotype, and 18 (28%) lacked the TTT. In some individuals, the above Rae2 haplotype TTT was linked with the highly skewed Pscd4 haplotype TT as well as the CardlO haplotype TC; this particular CardlO-Rac2 haplotype was almost exclusively found in the ESN group. In the segment of Rac2 locus downstream of the exon 7 (region 2), there were multiple SNP and a few gaps which also constituted a few haplotypes (Figure 1). Although the distributions of these region 2 haplotypes did not differ significantly between the groups, coefficients of LD between the region 2 SNP were higher in the ESN than in the HIV-I infected group (Figure 3).
[104] Detailed Method of Identifying DNA Polymorphism Accumulated in ESN Individuals
Partially duplicated 71 genomic DNA samples from 30 ESN and 21 HIV-I -infected individuals were sequenced through the segments shown in Figure 7, and frequencies of each observed sequence polymorphism enlisted in Figure 9 were compared by case-control methods. Known AIDS restriction genes (ARG) were also genotyped with the above samples, and only 1 individual in each phenotypic group possessed the CCR5Δ32 allele heterozygously. No one possessed the CCR2-64I, and DCSIGN -336 SNP frequencies were not different between the groups.
[105] Figure 3 shows distribution of observed SNP with significant case-control differences by adopting either a dominant (red lines) or a recessive (blue lines) hypothesis. P values shown are those calculated by χ2 (chi-square) test. See Figure 9 for odds ratios and P values calculated by Fisher's exact test. A representative value is shown for each cluster of SNP with significant case-control differences in the Rac2 locus, with the upper and lower brackets corresponding respectively to the regions 1 and 2 in Figure 1. Coefficients of LD between each pair of SNP alleles are shown here in lvalues and colour-scaled by using SNPAlyze ver. 6.0. [106] Figure 1 shows distribution of sequence polymorphisms (arrows) in the Rac2 locus with those showing significant case-control differences between ESN and HIV-infected groups shown in red. Longer arrows indicate SNP with case-control differences of P < 0.05. Chromosomal regions showing >50 % sequence homologies between humans and mice are indicated with horizontal lines labelled HH for high homology. In the polymorphic region 2, there were also gaps (small filled squares in the diagram) and base deletions (-): Δl, a 28-bp deletion (— ) relative to the database-reported genome sequence (+); Δ2, an 82-bp insertion (+) relative to the reported genome sequence (— )>" and Δ3, a 34-bp insertion (+). Haplotypes in each of the three blocks are those identified by the Four Gamete method using SNPAlyze ver. 6.0, and all observed linkages between each haplotype across the blocks are shown. The numbers of individuals possessing a particular haplotype are compared between the two groups by Fisher's exact test.
EXAMPLE 3
[107] FUNCTIONAL ANALYSIS OF THE POLYMORPHIC REGIONS 1
AND 2
The above polymorphic regions 1 and 2 in the Rac2 locus colocalized with the chromosomal segments that showed >50% sequence homology between humans and mice (Figure l) and one of the intragenic regions previously reported to exhibit elevated cytosine methylation in hematopoietic cells (Reference 20, Ladd, P.D. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333), indicating that these regions may contain conserved regulatory elements. To examine this directly, the genomic fragments that harbored the polymorphic region 1 isolated from representative healthy control individuals who homozygously possessed either the TTT or CCC haplotype were cloned and inserted into the downstream of the luc gene along with the known Rac2 core promoter (Reference 20, Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333) placed in the upstream. The Rac2 promoter is known to be strong and promiscuous, and tissue -specific expression of this gene is regulated by the presence of putative repressor protein (Reference 20, Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic" specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333). In the present study, the inventors detected no sequence polymorphisms in the Rac2 promoter. Consistent with the above notions, transfection of the luc constructs harboring the Rac2 promoter resulted in high levels of luciferase expression both in the T- lineage Jurkat and macrophage-lineage THP-1 cells (Figure 4). Further, coexistence of the region 1 fragment possessing the CCC haplotype strongly suppressed the luc expression in both types of cells, indicating the presence of a Rac2 repressor protein binding site within region 1. Importantly, when the region 1 fragment possessing the TTT haplotype coexisted, significantly higher luciferase activities were induced in comparison with those induced with the promoter alone. Interestingly, however, this effect was observed only in the T-lineage Jurkat cells, not in THP" 1 cells. These results clearly indicate a functional difference of the region 1 fragments possessing the TTT ox CCC haplotype: the iføci? intron haplotype TTT that is significantly more accumulated in the ESN individuals lacks the repressor activity and functions instead as an enhancer of gene expression in T-lineage cells, while the haplotype CCC remains repressive. When tested in Jurkat cells, the Rac2 region 2 fragments apparently reduced the expression of the luc gene regardless of the haplotype, indicating a specific importance of region 1 where the genotypes were skewed between the two phenotypic groups.
[108] The Rac2 region 1, but not region 2, haplotypes were also associated with in vivo expression levels of the Rac2 in HIV-I antigen-stimulated PBMC. Thus, when the levels of Rac2 expression were examined quantitatively by real-time PCR in cultured PBMC, a 2-fold increase of the Rac2 message was observed after 6 hours of stimulation with the HIV-I peptides in the individuals homozygously possessing the TTT haplotype in region 1 (Figure 5). On the other hand, the levels of Rac2 expression decreased after the antigenic stimulation in cells homozygous for the CCC haplotype. Importantly, haplotypic differences in region 2 did not affect the Rac2 induction after the antigenic stimulation, again indicating that the polymorphic region 1, but not region 2, harbors a functional regulatory element of the Rac2 expression.
[109] Finally, the Rac2 region 1 haplotypes did affect HIV-I replication in vitro. Thus, when PBMC from uninfected healthy control individuals possessing the homozygous CCC haplotype or harboring the TTT haplotype in region 1 were infected in vitro with HIVl, different kinetics in the increase of p24 concentrations were observed. In supernatants of PBMC possessing the homozygous CCC haplotype, p24 concentrations were significantly higher as compared to what was observed in supernatants of cells harboring the TT-Thaplotype both upon infection with the CCR5-tropic BaL strain of HIVl or with a primary CCR5-tropic HIV-I isolate, v.6 (Figure 6). Median p24 levels in cultures of the cells homozygous for the TTT haplotype at 5 days after infection with either one of the two CCR5-tropic viruses were <5% of those observed with the cells of the haplotype CCC. On the other hand, p24 concentrations did not differ significantly when PBMC of different region 1 haplotypes were inoculated with the primary CXCR4-tropic HIV-I isolate vl7, and p24 concentrations at 5 days after infection even became higher than those in the cells of the CCC haplotype when PBMC with the heterozygous haplotype were infected with the CXCR4"tropic laboratory strain IIIB. None of the individuals whose PBMC were used for the above in vitro infection experiments possessed the CCR5Δ32 allele. Thus, the regulatory Rac2 intron haplotype TTT, which is significantly more accumulated among ESN individuals, is associated with a restricted replication of CCR5"tropic HIV-I. The possible gene-dose effect in the induction of Rac2 after the antigenic stimulation (Figure 5) might explain the apparent differences in median p24 levels between the individuals homozygous and heterozygous for the 7T_Thaplotype. [110] Detailed Method of Analysis of Polymorphic Regions 1 and 2
In Figure 4, Human Jurkat line T cells were trasfected with a luc plasmid harbouring the Rac2 promoter either with or without a genomic fragment of the putative regulatory regions. Data shown here are ratios of luciferase activities relative to those obtained by transfecting the luc gene without the promoter. The Dual-Luciferase reporter assays were performed at 18, 21, and 24 hours after the plasmid transfection, and averages (mean ± S.E.M) from 3 and 5 repeated experiments performed at 18 and 21 hours after transfection, respectively, are shown. Similar results were obtained at 24 hours after transfection. Statistical analyses were done by two-tailed paired zHest.
[ill] Figure 5 shows changes in the expression levels of the Rac2 gene in PBMC stimulated with the HIV-I peptides. Real-time PCR analyses were performed with samples obtained from 22 ESN and 8 HIV-1-infected individuals, which were prepared as described for Figure 2, and the results were re-grouped based on the separately determined region 1 and region 2 genotypes. Data are shown here as ratios of the Rac2 expression between 1 and 6 hours after the antigenic stimulation calculated as 2"MCT, and bars indicate S.E.M. Statistical analyses were done by one-way ANOVA with Dunn's post-tests for multiple comparisons, and a significant group -wise difference was observed between the homozygous TTT and CCC groups. Two-tailed t-test was then performed to evaluate the individual level of significance. The numbers of individuals possessing each identified genotype were: 10 TTT/TTT, 14 TTT/CCC, and 6 CCC/CCCin region 1, and 10 T/T, 14 T/G, and 6 G/G in region 2.
[112] Figure 6 shows replication of HIV- 1 in cultured PBMC. Uninfected healthy control individuals were genotyped with written informed consent for the Rac2 region 1 and region 2 haplotypes, and their PBMC were infected with each of the 4 different HIV-I clones or isolates, or with the dual-tropic isolate 89.6. None of the individuals included here possessed the CCR5Δ32 allele. Data shown here are p24 concentrations measured at 3 and 5 days after virus inoculation grouped by the region 1 genotypes, with horizontal bars indicating median values. Statistical comparisons were done by two-way ANOVA for repeated measures with Dunn's post-tests. When group -wise differences were indicated, individual levels of significance were determined by two-tailed t-tests. The dual-tropic 89.6 replicated efficiently in PMBC of all three genotypes, and no significant differences were observed in p24 concentrations between the CCC/CCC and
Figure imgf000056_0001
individuals at day 5 (not shown). [113] Method of Luciferase Assays
For functional analyses of the putative Rac2 regulatory regions, the 2504-bp region 1 and 8904-bp region 2 genomic fragments were amplified by PCR using the following oligonucleotide primers and LA-Taq polymerase (TAKARABIO, Inc., Ohtsu, Japan):
Rac2RlL ATCCTCGAGCACCAAGCTGGACCTGCGGGACGACAAG Rac2RlR ATCGTCGACGAGAGGATGTCACTCGCTCTGAGTCACATG Rac2R2L ATCCTCGAGCTCCCACCTAGATGGGTCTGATCCTCCAG and Rac2R2R ATCCTCGAGTTTTGATGTAGCATAGCTCCCAGTAACTTTCAG The cloned Rac2 genomic fragments were inserted to the SaK restriction site located downstream of the luc gene within the pGL3 plasmid (Promega Corporation, Madison, U.S.A.) harbouring the previously described Rac2 cove promoter (-260 to +130 bp) (Reference 20, Ladd, P.D. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333). Each of the resultant reporter constructs along with the pRL-TK were cotransfected into 1.0 to 1.2 x 106 human Jurkat cells using an amaxa Nucleofector (amaxa AG, Cologne, Germany) with the solution V and condition C- 17 according to the manufacturer's recommendations. After 18, 21 and 24 hours, cells were lysed and expressed luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega) according to the manufacturer's protocol. [114] Method of RNA Extraction and Real-Time PCR Analyses
Total RNA was extracted from antigen- stimulated PBMC as described (Reference 39, Biasin, M. et al., "Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G: a possible role in the resistance to HIV of HIV-exposed seronegative individuals", J.Infec.Dis., 2007, vol.195, pp.960-964), and cDNA was generated by using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer's protocol. Quantitative real-time PCR assays were performed by using an ABI 7900HT Real-Time PCR System (Applied Biosystems). Reactions were preformed using the TaqMan probes for the human Rac2 and GAPDH (Applied Biosystems) with Platinum Quantitative PCR SuperMix-UDG (Invitrogen Corporation, Carlsbad, U.S.A.). The relative expression levels of the Rac2 were calculated and normalized to those of GAPDH using the software provided with the ABI 7900HT PCR Systems. [115] Method of In Vitro HIV- 1 Infection and Measurement of p24
PBMC was stimulated for 2 days in complete medium containing 20 % foetal bovine serum, 5 mg/ml phytohemagglutinin, and 10 ng/ml recombinant human interleukin (IL) -2 at a concentration of 2 x 106 cells/ml. After a viability assessment, 3 x 106 cells were resuspended in medium containing 0.05 ng p24 equivalent per 106 cells of HIV-I, and incubated for 3 hours at 37°C. Infected cells were then washed and resuspended in 3 ml of complete medium containing IL-2, and were plated in 3 wells at 1 x 106 cells/well. Cultures were re-fed on day 3 and supernatants were collected on days 2, 3, and 5. Absolute levels of HIV-I p24 were measured using the Alliance HIV-I p24 ELISA kit (PerkinElmer Inc., Waltham, U.S.A.). HIV-I BaL and IIIB were provided by Drs. S. Gartner, M. Popovic, and R. Gallo (Courtesy of the NIH AIDS Research and Reference Reagent Program). HIV-I primary isolates were kind gifts from Prof. C. -R Perno, University of Roma, Tor Vergata, Italy.

Claims

1. An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at a site of at least one SNP selected from the group consisting of SNPs designated by following SNP ID Nos: (i) rs739041, (ϋ) rs739042, and (iii) rs2284037; and b) assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotype d SNP is a resistance allele, wherein the resistance allele is as follows (SNP ID No. : allele) : (i) rs739041 : T; (ii) rs739042 : T; and (iii) rs2284037 : T.
2. An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at sites of SNPs designated by following SNP ID Nos: (j) rs9610683 and (ii) rs9610682; and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (i) rs9610683 : C; and (ii) rs9610682 : A.
3. An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at a site of at least one SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, (iii) and rs2284036; and b) assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP or nucleotide polymorphism is a resistance allele, wherein the resistance allele is as follows (SNP ID No. '■ allele) : (i) rs9610683 : C; (ii) rs9610682 : A; and (iϋ) rs2284036 : T.
4. A method for inducing or enhancing resistance to virus in a subject, comprising enhancing the Rac2 gene expression in the subject.
5. The method according to Claim 4, wherein the Rac2 gene expression is enhanced by using a DNA-binding protein, peptide, oligonucleotide, or nucleotide analogue that binds to the sequence interacting with Rac2 gene repressor in the region 1.
6. The method according to Claim 4, wherein the Rac2 gene expression is enhanced by inhibiting an expression of the Rac2 repressor protein by RNAi technique.
7. Use of Rac2 protein in the preparation of a medicament for the treatment or prophylaxis of virus-induced disease.
8. A nucleic acid exhibiting a Rac2 repressor protein binding activity, wherein the nucleic acid is selected from the following (A) and (B):
(A) a nucleic acid having the nucleotide sequence of SEQ ID No:32>" and
(B) a nucleic acid having more than 80% identity with the nucleotide sequence of SEQ ID No:32.
9. The nucleic acid according to Claim 8, wherein the nucleic acid has the following alleles at the SNP sites in the nucleotide sequence of SEQ ID No:32 (base number in the SEQ ID No.32 : allele):
(i) 132 : C; (ϋ) 144 : A; (iii) 1276 : T; (iv) 1959 : T; (v) 2301 : T; and (vi) 2379 : T.
10. Use of the nucleic acid according to Claim 8 or 9 as a Rac2 repressor protein inhibitor.
11. Use of the nucleic acid according to Claim 8 or 9 for a gene therapy to induce resistance to virus in a subject.
12. A method for screening a candidate therapeutic agent against virus, comprising: a) contacting an agent with a nucleic acid fragment having the partial or entire nucleotide sequence of the nucleic acid according to Claim 8 or 91 b) measuring an interaction between the agent and the nucleic acid fragment! and c) selecting the agent having the interaction with the nucleic acid fragment as a candidate therapeutic agent against virus.
13. A method for screening a candidate therapeutic agent against virus, comprising: a) administering an agent to a biological material; b) measuring an expression level of Rac2 gene in the biological material; and c) selecting the agent enhancing expression level of Rac2 gene in the biological material as a candidate therapeutic agent against virus.
14. A method for performing clinical trial for prevention, reduction, prophylaxis or treatment against virus, comprising: a) detecting genetic predisposition of subjects by the method according to any one of Claims 1 - 3; b) stratifying the subject by the result of the detected genetic predisposition! c) giving a prevention, reduction, prophylaxis or treatment to the stratified subject; and d) assessing the effect of the prevention, reduction, prophylaxis or treatment to the stratified subject.
15. The method according to any one of Claims 1 ■ 6, wherein the virus is one selected from the group consisting of HIV, HCV and HPV.
16. The method according to Claim 15, wherein the virus is HIV or HCV.
17. The method according to Claim 16, wherein the virus is HIV.
18. The use according to Claims 7 or 11, wherein the virus is one selected from the group consisting of HIV, HCV and HPV.
19. The use according to Claim 20, wherein the virus is HIV or HCV.
20. The use according to Claim 21, wherein the virus is HIV.
21. The method according to any one of Claim 12 or 14, wherein the virus is one selected from the group consisting of HIV, HCV and HPV.
22. The method according to Claim 21, wherein the virus is HIV or HCV.
23. The method according to Claim 22, wherein the virus is HIV.
24. The method according to any one of Claims 1 or 3, wherein the genotyping is performed by the Invader assay.
25. A chip used in the method according to Claim 1, comprising a base plate and at least one probe detecting SNP selected from the group consisting of SNPs designated by the SNP ID Nos (i) rs739041, (ii) rs739042, and (iii) rs2284037, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:6, 4, and 3.
26. A chip used in the method according to Claim 1, comprising a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs2284037, (ii) rs739042 and (iii) rs739041, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:3, 4 and 6.
27. A chip used in the method according to Claim 2, comprising a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs9610683 and (ii) rs9610682, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:l and 2.
28. A chip used in the method according to Claim 3, comprising a base plate and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, (iii) rs2284036, wherein the probe sets comprises at least one probe comprises more than 7 bases of partial sequence or partial complementary sequence of the nucleotide sequence selected from the group consisting of SEQ ID NO^l, 2, or 5.
29. A kit used in the method according to Claim 1, comprising a reagent and at least one probe detecting SNP selected from the group consisting of SNPs designated by the SNP ID Nos (i) rs739041, (ii) rs739042, and (ϋi) rs2284037, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:6, 4, and 3.
30. A kit used in the method according to Claim 3, comprising a reagent and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, (iii) rs2284036, wherein the probe sets comprises at least one probe comprises more than 7 bases of partial sequence or partial complementary sequence of the nucleotide sequence selected from the group consisting of SEQ ID NO^l, 2, or 5.
31. A method for screening a candidate therapeutic agent against virus, comprising: a) administering an agent to a biological material! b) measuring an intensity of a binding between a Rac2 repressor protein and a binding site thereof, in the biological material; and c) selecting the agent inhibiting the binding in the biological material as a candidate therapeutic agent against virus.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Title
JANARDHAN A ET AL: "HIV-1 Nef Binds the DOCK2-ELMO1 Complex to Activate Rac and Inhibit Lymphocyte Chemotaxis", PLOS BIOLOGY, vol. 2, 2004, pages 65 - 76 *

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