WO2000061811A2 - Method of predicting susceptibility to hiv infection or progression of hiv disease - Google Patents

Abstract

A method of predicting susceptibility to HIV infection, or progression to AIDS after seroconversion, by detecting a polymorphism in the IL-10 promoter gene. In particular, the detected polymorphism is IL10-5'-592A, in which there is a transition from C to A at the -592 position in the IL10 promoter, which eliminates an ETS binding motif. Subjects who are either homozygous or heterozygous for the allele have a greater incidence of HIV seroconversion, and more rapid progression to AIDS, particularly more than 5 years after seroconversion. The polymorphism of the present invention can be used in association with other alleles, such as CCR5-Δ32, CCR5-P1, CCR2-641, SDF1-3'A, MBL and HLA-A, -B, and -C, to determine an individual's susceptibility to HIV infection, and provide a prognosis for disease progression in those who have been infected. The presence of the allele can also assist in the selection of more aggressive prophylactic or therapeutic treatment for individuals who are at greater risk for infection or disease progression.

Description

METHOD OF PREDICTING SUSCEPTIBILITY TO HIV INFECTION OR PROGRESSION OF HIV DISEASE

FIELD This invention concerns diagnosis and treatment of HIV disease, and is particularly directed to detection of a genetic polymorphism associated with enhanced rates of HIV infection and progression to AIDS.

BACKGROUND It is known that the promoter region of IL-10 has several polymorphisms. Eskdale and Gallagher (Immunogenetics 42:444, 1995), and Eskdale et al., (Immunogenetics 45:82, 1996) have reported that there are polymorphic CA repeats over 1000 base pairs upstream of the proposed IL-10 TATA box. In addition, single nucleotide polymorphisms (SNPs) have been identified at position - 1082 (a G - A transition), and at -819 (a C - T transversion) and at position -592 (a C - A transversion), where all the positions are counted from the transcription start site. (Lazarus et al., J. Rheum. 24:2314, 1997). The -592 polymorphism is referred to as the "A allele" because of the nucleotide A at position -592. Alleles that have a C at position -592 are referred to as the "C allele." The -592 SNP has been studied in relation to its linkage to several diseases. For instance, the -592 SNP has been found to correlate with production of Ro antibodies and renal impairment in systemic lupus erythematosus (SLE) (Lazarus et al. 1997). Rosenwasser and Boorish. (Am. J. Respir. Crit. Care Med. 156:5152, 1997) disclosed an association between the A allele and asthma. This reference disclosed that subjects with the A form of the promoter allele demonstrate significantly less IL-10 synthesis than do others. Turner et al. described an association between the A allele and increased "graft rejection, and also stated that presence of the A allele predicts low IL-10 production. Allelic variations associated with certain genes (e.g., CCR5-Δ32, CCR5-P1, CCR2-641 ,

SDF1-3Α, MBL and HLA-A, -B, and -C) have been correlated with HIV infection and/or AIDS progression (Dean et al. , Science 273 : 1856, 1996; Smith et al. , Science 277:959, 1997; Winkler et al, Science 279:389, 1998; Martin et al, Science 282:1907, 1998; Garred et al, Lancet 349:236, 1997; Liu et al., Cell 86:367, 1996; Samson et al, Nature 382:722, 1996; Zimmerman et al., Mol. Med. 3:23, 1997; Michael et al, Nature Med. 3:338, 1997; Huang et al, Nat. Med. 2:1240, 1996; Kostrikis et al, Nat. Med. 3:350, 1998; Mummidi et al, Nat. Med. 4:786, 1998; McDermott et al., Lancet 352:866, 1998). The influence of CCR5-Δ32 and CCR5-Δ32 (and methods of their detection) is disclosed in Smith et al. (Science 277:959, 1997), which is incorporated herein by reference. Several references have also disclosed that enhanced IL-10 expression is a marker for progression of HIV-1 infection and onset of AIDS. Ichiki et al. (J. Alter. Clin. Immunol. 99:pS46, 1997), for example, states that serum IL-10 is higher in HIV-infected persons who rapidly progress to AIDS. This finding is attributed to the ability of IL-10 to inhibit the immunopotentiating capacity of IL-12. Immunol 99 pS50, 1997) also states that IL- 10 expression is increased m HI V-positive individuals, which is thought to lead to immune unresponsiveness

SUMMARY It has surprisingly been found that the A allele is associated with an mcreased susceptibility to

HIV infection and progression of HIV disease to AIDS and death This finding is particularly surprising in view of the prior disclosure in the art that the A allele is associated with decreased IL-10 production, while rapid progression to AIDS has been associated with mcreased IL-10 production, which was thought to lead to immune unresponsiveness Hence the association between the polymorphism in the A allele, and the increased susceptibility to HIV infection and progression, was unexpected

The invention therefore includes a method of predicting a susceptibility to HIV infection or HIV progression in a subject, by detecting a polymorphism m the human IL-10 promoter of the subject, and particularly in the negative regulatory region of the promoter, wherein presence of the polymorphism indicates a susceptibility to HIV infection or HIV progression In particular embodiments, the polymorphism is IL10-5'-592A, or an allele (such as IL10-R(-397S)-283), which is tightly linked to the IL10-5'-592A allele)

Another embodiment of the invention is a method of predicting susceptibility to HIV infection or HIV progression in a subject, by obtaining a test sample of DNA containing the IL-10 promoter sequence of the subject, and detecting a polymorphism in the IL-10 promoter that is associated with decreased expression of IL 10 The polymorphism may be detected using a variety of techniques, including restriction digestion, probe hybπdization. nucleic acid amplification, and nucleotide sequencing Nucleic acid amplification can include use of the polymerase chain reaction with pπmers such as those shown in SEQ ID NOs 39 and 40 Alternatively, the polymorphism can be detected with a nucleic acid probe to the human TZ.70-5 '-5 °24 polymorphism m the test sample, such as allele-specific ohgonucleotides that hybπdize with the human IL10-5'-592A and -592C alleles

When a probe is used to predict susceptibility to HIV infection or HIV progression in the subject, the method can include obtaining from the subject a test sample of DNA comprising the human IL- 10 promoter sequence The test sample is then contacted with at least one nucleic acid probe for a human IL-10 promoter sequence polymorphism that is associated with increased susceptibility to HIV infection or HIV progression in a subject The hybπdization sample is maintained under conditions sufficient for specific hybπdization of the human IL-10 promoter sequence with the nucleic acid probe, such that specific hybπdization can be detected to indicate increased susceptibility to HIV infection or HIV progression in the subject

Other embodiments of the invention include a kit for use in diagnosing an increased susceptibility to HIV infection or HIV progression m a subject The kit can include a probe that specifically hybπdizes to the human IL-10 promoter sequence polymorphism (such as IL10-5'-592A) that is associated with the mcreased susceptibility to HIV infection or HIV progression Alternatively, the kit can include PCR pπmers for amplifying the allele for subsequent detection

The availability of the predictive test of the present invention can help provide prognostic information to persons infected with the HIV virus, and can also assist in the selection of treatments for these people Subjects who have the polymorphism may, for example, require more aggressive treatment with anti -viral medications than other subjects who do have the polymorphism Presence of the polymorphism can also be used to help select treatments (such as administration of IL 10 or gene therapy with IL-10) for particular patients who may have an undesired low endogenous IL10 level Alternatively, the presence of the polymorphism could be used as a predictor that very aggressive treatment could substantially eradicate the virus from the infected person

The present invention also mcludes generation of normograms or other predictive algoπthms that can be used, in association with allele status, to prognose probable survival or years to development of AIDS following HIV seroconversion These normograms can be based not only on the presence of the IL10-5'-592A allele, but can also include other alleles that in combination with the IL10-5'-592A allele affect the course of HI V disease

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed descπption of a several embodiments, which proceeds with reference to the accompanying figures

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic map of the human IL- 10 gene on chromosome 1 q31 -32 Exons, the AUG start codon untranslated regions, SNP (single nucleotide polymorphism) and STR (short tandem repeat) polymorphisms are shown, along with putative transcπptional factor bindmg sites identified by sequence homology in relation to negative and positive regulatory elements The positive regulatory region of the promoter is labeled "pos reg '^" (corresponding to nucleotides -1100 to -900) and the negative regulatory region of the promoter is labeled "neg reg " (corresponding to nucleotides -800 to - 300) Transcnption factor binding motifs are also shown, in relation to the -1082 SNP and -592 SNP

FIG. 2 is a series of graphs illustrating the effect of STR and SNP vanants of IL10 in AIDS cohorts, as they are related to the development of AIDS in individuals, according to both the 1987 (AIDS-1987) and 1993 (AIDS-1993) clinical definitions of AIDS The number of patients and number of events (N), P value (P) and relative hazard (RH) are based on a Cox proportional hazards model FIG 2A shows data for progression to AIDS-1993 among Caucasian individuals homozygous for IL10- R(-3975)-283/283, compared to those beanng other STR allele genotypes in Caucasians FIG 2B shows Kaplan-Meier curves demonstrating time to AIDS-1987 m sublets, m combined Caucasian cohorts, based on IL10-5'-592A (abbreviated IL10-5Α) genotypes The genotypes shown are IL10-5'-592 C/C (IL10-+/+), IL10-+/5Α, and IL10-5Α/5Α FIG 2B shows time to onset in Caucasians of AIDS-1993 for IL10-5'-592 C/C (IL10-+/+) IL10-+/5Α and IL 10-5 'A/5 'A FIG 2D is similar to FIG 2C, but combmes data for all ethnic groups until onset of AIDS-1987 FIG 2E is similar to FIG 2D, but is limited to data for the MACS (high πsk factors for infection) cohort, for progression to AIDS-1993 FIG 2F is similar to FIG 2D, but is limited to the MHCS cohort progression to AIDS-1987

FIG. 3 is a bar graph illustrating the effect of IL10-+/5Α and 5Α/5Α genotype frequencies on four AIDS endpomts m partitioned time mtervals after HIV-1 infection in Caucasians The time intervals are 0-5 years, 5-10 years and greater than 10 years since seroconversion The endpomts are a CD4 count less than 200, progression to AIDS-1993, progression to AIDS-1987, and death The genotypic frequencies m each time category reflect only failing seroconverters within the first ten years After ten years, both seroprevalents and seroconverters are included Mantel-Haenszel Chi-Square tests of the three time categones are indicated for frequency differences The number of subjects for each time category, P values (P) and the population frequency of IL10-+/5A plus 5 'A/5 'A genotypes in Caucasians (arrow, 0 417) are also shown

FIG. 4 is a seπes of graphs showing Kaplan-Meier curves of the fraction of patients who are free of AIDS, at 0-14 years, as defined by 1987 clinical cπteπa (AIDS-1987) and 1993 clinical cπteπa (AIDS-1993) FIG 4A illustrates the fraction of AIDS-free subjects (using AIDS 1993 cπteπa) with the 5Α/5Α or +/5'A genotypes, compared to the -5'- C/C (+/+) genotype FIG 4B is similar to FIG 4A, but the fractions are calculated using AIDS 1987 cπteπa FIGS 4C and 4D show the fraction of AIDS free subjects (using AIDS 1993 and 1987 cntena) for CCR2-64I or CCR5-Δ32 beanng genotypes, compared to +/+ genot pes FIGS 4E and 4F respectively show the effect of both IL10-5Α and CCR2-64I or CCR5-Δ32 genotypes on development of AIDS-1993 and AIDS-1987 m combined Caucasian cohorts

FIG. 5 illustrates differential DNA-protein binding between synthesized o gonucleotide probes specific for IL10-5Α vs IL10-+ Both allele nucleotides resolve the faster migrating SP-1 complex, but only IL10-+ specifically binds the slower migrating complex (arrow) NS=non-stιmulated. Comp = cold competition with double stranded ohgonucleotides

SEQUENCE IDENTIFICATION LISTINGS SEQ ID NO 1-38 are STR pπmer sequences for the STR loci listed in Table 1 (where each pπmer sequence is labeled with its corresponding Sequence Identification Number)

SEQ ID NO 39 and 40 are forward and reverse pπmer sequences (respectively) used for detection of the SNP at the 311 bp IL10-SNP-592 site, as disclosed in Example 3

SEQ ID NO 41 and 42 are forward and reverse pπmer sequences (respectively) used for detection of the SNP at the 193 bp IL 10-SNP- 1082 site, as disclosed in Example 3

SEQ ID NO 43 is a sequence of a probe that may be used to detect a single base pair polymorphism at position -592 SEQ ID NO 44 is a sequence of a SP-1 bmdmg site in HIV-1 LTR. DETAILED DESCRIPTION Abbreviations

IL Interleukin

IL10-5Α IL10-5'-592A PBMC Peπpheral blood monocyte

+/+ IL10-5'-592 C/C

SNP Single nucleotide polymorphism

SP1 Stimulating protein 1

STR Short tandem repeats ALIVE AIDS Link to the Intravenous Expeπence

HGDS Hemophilia Growth and Development Study

MHCS Multicenter Hemophiliac Cohort

MACS Multicenter AIDS Cohort Study

SFCC San Francisco City Clinic Study

Definitions

"AIDS-1993" refers to the 1993 definition of AIDS provided by the Centers for Disease

Control (CDC), which included HIV-1 infection m the presence of an AIDS-defining illness (such as

Pneumocystts pneumonia) or a CD4 level of less than 200 "AIDS-1987" refers to the more stringent 1987 definition of AIDS provided by the Centers for

Disease Control (CDC), which included HIV-1 infection in the presence of an AIDS-defining illness

(such as Pneumocystts pneumonia)

"Amplification" of a nucleic acid molecule (e g , a DNA or RNA molecule) refers to use of a technique that increases the number of copies of a nucleic acid molecule in a specimen An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide pπmers, under conditions that allow for the hybπdization of the pπmers to nucleic acid template in the sample The pπmers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid The product of amplification may be characteπzed by electrophoresis, restπction endonuclease cleavage patterns, oligonucleotide hybndization or hgation, and/or nucleic acid sequencing using standard techniques Other examples of amplification include strand displacement amplification as disclosed in U S Patent No 5 744.311 , transcnption-free isothermal amplification, as disclosed in U S Patent No 6,033,881 , repair cham reaction amplification, as disclosed in WO

90/01069, ligase chain reaction amplification, as disclosed in EP-A-320 308, gap filling ligase chain reaction amplification, as disclosed in 5,427,930 and NASBA™ RNA transcnption-free amplification, as disclosed m U S Patent No 6,025,134 "CD4 level" refers to the population of CD4+ helper T-lymphocytes, usually expressed in CD4 cells per cubic millimeter of a blood sample

CCR2-64I refers to a mutation within the first transmembrane region of the CCR2 chemokine and HIV-1 receptor gene, that occurs at a frequency of 10-15% among Caucasians and African Amencans, and exerts a protective effect against progression to AIDS m HlV-mfected individuals The mutation is a G-to-A nucleotide substitution at position 190 (counting from the ATG start codon) that changes the ammo acid residue vahne at position 64 to lsoleucme, as descnbed in Smith et al , Science 277 959-965, 1997

CCR5-Δ32 refers to a mutation with a 32-nucleotιde deletion within the CCR5 gene, which has been descπbed in subjects who remained uninfected despite repeated exposures to HIV- 1 , as disclosed in Huang, et al , Nature Medicine 2 1240-1243, 1996

CCR5-P1 refers to a combination of 10 polymorphic nucleotide residues at positions 208(G), 612(A), 626(C), 627(C). 630(C), 647(C), 676(A), 684(T), 714(C) and 811(G) as disclosed m Martin et al (Scιence 282 1907-1911, 1998) "HIV disease" refers to a well recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by an HIV virus, as determined by antibody or western blot studies Laboratory findings associated with this disease include a progressive decline in T-helper cells

"lnterleukιn-10 promoter" is in the 5' flanking sequence of the IL-10 gene and the sequence of this region is provided m Eskdale et al (Immunogenetics 46 120- 128, 1997)

"Interleukιn-10 vanants" includes any IL10 protein, including proteins with substitutions, additions or deletions, which do not substantially diminish the activity of IL-10

"Marker" refers to a DNA sequence polymorphism flanked by conserved regions Conserved regions flanking the marker can be used for the design and construction of primers for amplifying the relevant DNA portions

A "tightly linked" allele is one that is linked in non-random chromosomal association (linkage disequilibnum) with another allele In particular embodiments, a tightly linked allele is one that is indicative of the presence of the polymorphism of interest (such as the IL10-5 'A polymorphism), for example one that can predict a neighboring site with an increased likelihood from 1-100%, for example an increased likelihood of at least 50% 75% or 98% Alternatively, the association is statistically significant to a p value of 0 5 or less for example 0 1 or less

A "genetic marker indicative of the IL10-5 'A polymorphism" refers to a genetic marker that is genetically linked with and co-segregates with the IL10-5 'A polymorphism, e g as DNA allehc variants of the genetic markers that are detectable in fragmented genomic DNA samples (or PCR regions) isolated from the population

"Co-segregation" means that a specific allele of one polymorphic locus (e g , a genetic marker) is heπted together with a specific allele of another locus (e g the 77,70-5 'A polymorphism) Co- segregation occurs when the two loci are on the same chromosome and are located physically close enough so that the rate of genetic recombination between the two loci is low or zero Two loci co- segregating m a statistically significant manner (typically determined by linkage analysis and by determining an LOD score in population studies, or by a determination of statistical significance m other studies) are located on the same chromosome, in the same contiguous stretch of DNA, physically close to one another, typically within 50 million base pairs or less

"Linkage" means that two loci (such as a genetic marker and the 77.70-5 'A polymorphism) are mhented together more often than would be expected by chance Genetic linkage is a reflection of the physical location of the loci on the same chromosome segment Unlinked loci have a 50% chance of being separated by a recombination event and assort independently at meiosis Loci that are close together are less likely to be separated by recombination, and are therefore more likely to be mhented together The distance between linked loci is measured in terms of the frequency of recombination occurring between them, and is expressed in centimorgans (cM)

A marker and the polymorphism are said to be linked to one another if the marker and the locus co-segregate in certain families or by genetic linkage analysis and statistical probability calculations

Statistically, linkage is the probability that two different markers are linked, and this probability may be expressed as the LOD score at a selected recombination fraction, or as a statistically significant association An LOD score of 3 or greater is statistically significant evidence of linkage between a pair of genetic markers An LOD score of less than -2 is considered statistically significant evidence that a pair of markers, or a marker and a disease phenotype, are not linked and are not co-segregating together An example of linked markers is a calculated LOD score of 3 0 at a recombinant fraction (RF) of 0 0, means that the subject marker has a statistical significance (p<0 05) of being linked closely (/ e 0 0) to the other marker at a 95% confidence level Methods of calculating LOD scores are discussed in U S Patent No 5,449,604, which is incorporated by reference "Linkage disequi bπum" occurs when a particular combination of alleles at two closely linked loci appears more frequently than would be expected by chance Linkage disequihbπum is often observed between a gene of interest (e g a mutant allele, such as IL10-5Α) and a flanking allele (such as IL10-R(-3795)-283) which can then also be used as a marker for the condition "SDF1 -3'A" refers to a polymorphic allele that is associated with protection against progression to death in patients with HIV disease See Van Rij et al (AIDS 12 F85, 1998)

"Transition" is a type of genetic point mutation characteπzed by substitution of one pynmidme (G/C) by the other pyπmidine, or of one puπne (A/T) for the other punne

"Transversion" is a type of genetic point mutation characteπzed by replacement of a punne with a pynmidme, or a pynmidme with a punne "African Amencan" refers to blacks Findings in the African American group can be generalized to persons of the same racial group who reside m other countries Association Between IL-10 Alleles and HIV Infection or Disease Progression

A genetic epidemiological scan of patients enrolled in AIDS cohorts for 19 candidate gene- nked short tandem repeat (STR) polymorphisms revealed significant genotype associations for HIV-1 infection and progression to AIDS with markers adjacent to and tracking (by linkage disequi bπum) common smgle nucleotide polymorphic (SNP) vanants in the IL 10 promoter region that differentially regulate IL10 production

Among AIDS cohorts (N=3337), study participants carrying the IL10-5'-592A (IL10-5Α) promoter allele were at increased πsk for HIV-1 infection, and once infected they progressed to AIDS more rapidly than homozygotes for the alternative IL10-5'-592 C/C (IL10-+/+) genotype, particularly m the later stages of HIV-1 infection (e g , five years after seroconversion) An estimated 25-30% of long term non-progressors (who avoid clinical AIDS for ten or more years after HIV-1 infection) can be attnbuted to their IL 10 (+/+) promoter genotype The alternative 77, 70-5 A and + IL 10 promoter alleles are functionally distinct in relative IL10 production, m retention of an ETS transcnption factor recognition sequence, and in binding to specific putative nuclear transcnption factors

Pπor genetic epidemiologic analysis of AIDS cohorts has implicated at least six human loci whose alleles assert differential influence on HIV infection and/or AIDS pafhogenesis among mfected individuals CCR5-A32, CCR5-P1, CCR2-64I, SDF1-3Α, MBL smd HLA-A, -B and -C To detect genetic effects of other polymorphic vanants, candidate loci were identified using closely linked short tandem repeat (STR) polymorphisms (also called microsatellites) Their allele and genotype frequencies among clinical subdivisions of AIDS cohorts were tested for association with HIV-1 infection and the rate of progression to AIDS STRs are typically (but not always) located outside the coding region of functional genes Because they are abundant (over 100,000 in the human genome) and substantially randomly distributed, they are frequently informative in linkage mapping and population association studies (Dib et al , Nature 380 152, 1996, Daniels et al ,Am J Med Genet 74 319, 1997, Schwab et al , Nat Genet 11 325, 1995)

Using these techniques, associations were found between alleles of two STR loci in the region of the IL10 gene on human chromosome 1 q31 -32, which were found to be related to differential HIV- 1 infection incidence and rate of AIDS progression A single nucleotide polymorphism (SNP) within the upstream regulatory region of IL 10 was demonstrated to be in non-random chromosomal association

(/ e , linkage disequihbπum) with the adjacent STR loci and to associate with differential HIV-1 infection and disease progression Alternative SNP alleles for this -592 promoter site have been shown previously to mediate two-fold differences in IL 10 concentrations (Rosenwasser and Bonsh, Am J Respir Crit Care Med 156 SI 52, 1997 ) The present invention demonstrates allele specific differential affinity to nuclear transcnption factors that recognize the IL 10 promoter gene sequence

These combined genetic, epidemiologic and functional results indicate that mcreased expression of the IL10 gene helps reduce HIV-1 infection and pathogenic progression The discovery that mcreased expression of IL10 helps reduce and limit progression of HIV infection also enables a vanety of new therapeutic interventions in the treatment of HIV disease The new appreciation of the role of IL10 in HIV infection permits treatment by exogenous administration of IL-10 (or vanants) m individuals who are heterozygous or homozygous for the IL10-5Α gene Dosages of IL-10 can be, for example, those shown US Patent No 5,665,345, to inhibit HIV replication, for example 1000 to 1,000,000 units, particularly 20,000 to 200,000 units administered to a subject

To perform a diagnostic test for the presence or absence of the polymorphism in an individual, a suitable genomic DNA-containing sample from a subject is obtained and the DNA extracted usmg conventional techniques However, the majonty of the present discussion is directed to the use of DNA from individuals because of its ease of isolation and the like

For example, DNA can be prepared by standard methods Most typically, a blood sample, a buccal swab, a hair follicle preparation or a nasal aspirate is used as a source of cells to provide the DNA The extracted DNA is then subjected to amplification, for example, using the polymerase chain reaction (PCR) according to standard procedures The allele of the single base-pair polymorphism is determined by conventional methods including manual and automated fluorescent DNA sequencmg, pπmer extension methods (Nύafoτov, et al , Nucl Acids Res 22 4167-4175, 1994), oligonucleotide hgation assay (OLA) (Nickerson et al , Proc Natl Acad Set USA 87 8923-8927, 1990), allele-specific PCR methods (Rust et al , Nucl Acids Res 6 3623-3629, 1993), RNase mismatch cleavage, single strand conformation polymorphism (SSCP), denatunng gradient gel electrophoresis (DGGE), Taq-Man, oligonucleotide hybndization, and the like

An example of a genotype associated with HIV infection and progression is the IL10-5A allele The absence of this genotype indicates a relative resistance to HIV infection and progression of HIV infection to AIDS In addition to the genotype descnbed above, other alleles associated with vanable HIV infection rates or HIV progression to AIDS can also be detected and used in combination with the IL10-5A allele to predict the probability that the subject will progress to AIDS within a given time period These other alleles include (without limitation) CCR5-Δ32, CCR5-P1, CCR2-641, SDF1-3Α, MBL stadHLA-A, -B, and -C

The marker of the present invention can be utilized for the detection of, and differentiation of, individuals who are homozygous and heterozygous for the IL 10-5 'A allele The value of identifying individuals who carry the A allele (/ e , individuals who are heterozygous or homozygous for the 77,70- 5'A allele) is that these individuals can then initiate or customize therapy (such as combination chemotherapy) to reduce viral load in the body, or undergo more aggressive treatment of the disease, and beneficially its course

As will be readily understood and appreciated, with respect to any sequences disclosed herem, complementary sequences reverse complementary sequences, and/or other similar sequence information are equivalent to the disclosed sequences and are therefore contemplated m accordance with the present mvention The following examples are mtended to illustrate but not to limit the invention

EXAMPLE 1

STR alleles for nineteen loci tracking seventeen AIDS candidate genes (Table 1) were screened for distortions in allele frequency, genotype frequency and Hardy- Wemberg equi bπum (the tendency for genotype frequencies to occur according to a polynomial distribution) in compaπsons of HIV-1 infected patients to those with documented high HIV-1 exposure who had not become infected In addition, all prospective recessive and dominant STR genotypes were tested for association with differential rates of progression to AIDS usmg a Cox proportional hazards model as descnbed previously (Cox Proportional Hazard Regression, SAS Release 6, 1 0, SAS Institute, Cary, NC JR Stat Soc 1972,B 34 187 Allison, Survival analyses using the SAS system A practical guide SAS Institute, Cary, NC, 155-197, 1995, Levin, Ada Umo Int Contra Cancrum 9, 531, 1953 )

One STR, IL10-G(-1140)(see FIG 1), a CA repeat 1 kb upstream of the IL10 gene (FIG 1), showed a weak association with sensitivity to HIV-1 infection (p=0 03, 10 df for 11 allele locus), while all other loci showed no significant associations with infection (Table 1 ) For the disease progression rate association tests, two loci revealed highly significant associations (IL10-R-3975, p=0 003, and GAAT12D11, an STR within 1 cM of CCR5 and CCR2 (Stephens et al , Am J Hum Genet 62 1507, 1998), p=0 0009) Following correction for multiple tests, both retained statistical significance (Table 1) A survival analysis illustrating the association of lL10-R(-3975)-283/283 homozygosity with relatively rapid progression to AIDS is presented in Fig 2A

Table 1

Candidate gene chromosome STR locus STR No. of STR primer sequences SEQ ID Infection tests AIDS-1993 Bonferroni position symbol distance alleles No. Progression corrected from gene tests P-value

P-value (df) P-value (allele)

Interleukin 10 lq31-32 77,70-G lkb 1 1 5'-CAACCCAACTGGCTCCC-3' 0.03 (10)* 0.17 (157)

5'- ATGGAGGCTGGATAGGAGGT -3'

IL10-R 4kb 6 5'-CCCTCCAAAATCTATTTGCATA-3' 3 0.43 (5) 0.003 (283)*** 0.02* 5'-CTCATCAAGAAGCCCAAAGC-3' 4

Interleukin 1 alpha 2ql3 ILIA 0.4kb 1 1 5'-TTCCATTCCTGACTTCCAGG-3' 5 0.65 (10) 0.14 (327) 5'-GATGAAGGTAAGTTGGAGACA-3' 6

Interleukin 1 2ql2 IL1RA NR 1 1 5'-GATCATTGGATGTTGCATGG-3' 7 0.39( 10) 0.15 (93) receptor antagonist 5'-GGACTTCAGGTAGACAGAG-3' 8

CCR2/CCR5 3p21 AFMB362WB9 lcM 5'-TGGGCAAAGGACTTAAATC-3' 9 0.07( 3) 0.05 (214)* 0.20

5'-C ACTTATTTTCTGGTGTGTGTATGT-3' 10

GAAT12DU 0.2cM 5'-CATTCCTGTGACTGTCCCTC-3' 11 0.06 (4) 0.0009 0.005" 5'-GAGGCTGATGTAGGGGATGA-3' 12 (197)***

STRL-33 3p21 D3S2354 0.5 cR 7 5'-GCTCTTGGCTGTGTTAACTG-3' 13 0.18 (6) 0.05 (137) 5'-AGCTTATTTGGATTCCATCAG-3' 14

Interleukin 5 3p26-24 IL5RA 0.4kb 1 1 5'-GCTGCCAATTGTAGCACTCA-3' 15 0.33 (10) 0.48 (251) receptor alpha 5'-GTGTATCCTGGATCAGGCCTC-3' 16

Interleukin 2 4q26-q27 1L2 <0.1kb 15 5'-AAAGAGACCTGCTAACAC-3' 17 0.44 (14) 0.1 1 (202) 5'-GATGCTTTATTTTCTTGAAC-3' 18

Group-specific 4ql2 GC <0.1kb 7 5'-GAATACTTCCGGAAGATGAGTCC-3' 19 0.36 (6) 0.14(268) component 5'-GGTGGGCGCCTATAATCC-3' 20

(Table 1 continued next page)

Table 1 (continued)

Candidate gene chromosome STR locus STR No of STR pnmer sequences SEQ ID Infection tests Progression tests Bonferroni position symbol distance alleles No AIDS-1993 corrected from gene P-value

P-value (df) P-value (allele)

Interleukin 9 5q31 1 IL9 0 5kb 12 5'-TCTCAGTGAGGTTAACTAATTTGCC-3' 21 0 97 (1 1) 0 21 (425) 5'-GTACCTGCCAAGAGCCAGAGT-3' 22

TATA box-binding 6q27 TBP in exon 14 5'-GACCCCACAGCCTATTCAGA-3' 23 0 07 (13) 0 17 (183) piotein 5'-TTGACTGCTGAACGGCTGCA-3' 24

Tu oi nectosis 6p21 3 TNFB <0 2kb 2 5'-GGT i TCTCTGACTGCATCTT-3' 25 0 28 (1) 0 19 (162) factoi beta 5'-TCATGGGGAGAACCTGCAGAG-3' 26

Interferon alpha 9p22 IFNA NR 7 5'-TGCGCGTTAAGTTAATTGGTT-3' 27 0 69 (6) 0 34 (145) 5'-GTAAGGTGGAAACCCCCACT-3' 28

T cell 12pl3 CD4 1 2 kb 9 5'-AATTGTTGGAGTCGCAAGCT-3' 29 0 27 (8) 0 10 (226) antιgen/T4/Leu3 5'-GTCTGAAAAAAGTGGGGCTG-3' 30

Interferon gamma 12q24 1 IFNG 0 5kb 9 5'-TTACAACACAAAATCAAATC-3' 31 0 82 (8) 0 19 (189) S'-ATACAAAAACAAAAAACAGCAAAGC-S' 32 v-eibA related gene 19pl3 1 ERBAL2 NR 12 5'-TCAGCCTGGGGGAGATCT-3' 33 0 38 (1 1) 0 13 (183) 5'-AGTTGGCCATCCAGCCTG-3' 34

Interleukin 2 22ql l 2-ql3 IL2RB O lkb 12 5'-CTAG AGGGACCTGCTTGTGT-3 ' 35 0.33 (1 1) 0 53 (285) receptor beta 5'-GCCTGAAACTCCTCAAGCAC-3' 36

Properdin Xpl l 3-pl l 23 PFC 15kb 13 5'-CCTGAGGATAGTGTCAGCGAT-3' 37 0 10 (12) 0 41 (240) 5'-CTTTCAGGGCTACTGGTCACT-3' 38

Infection and disease progression association with two STRs (AFMB362wB9 and GATT12D11) tightly linked to CCR5 was not unexpected, because CCR5-A32 had previously been associated with reduced HIV-1 infection and progression (O'Bπen and Dean, Sci. Amer. 277 44, 1997, Dean et al , Science 273 1856, 1996, Zimmerman et al , Mol Med 3 23, 1997, Michael et al , Nature Med. 3 338, 1997, Y Huang et al , Nat Med. 2 1240, 1996, Stephens et al ,Am J Hum Genet

62 1507, 1998) However, an ILIO effect was not anticipated The IL10 associated signal with linked STRs for both HIV-1 infection and the rate of AIDS progression among infected patients (Table 1 , FIG 2A) was studied further by analyzing previously identified single nucleotide polymorphisms (SNPs) within the upstream region of the ILIO gene (FIG 1) Two vanants within this region were examined for association with HIV-1 infection or disease progression The first vanant was a G-A transition at position -1082, and the second vanant was a C-A transversion at position -592 This second vanant was designated 77, 70-5 '-592 A, and abbreviated IL10-5Α

A third vanant within this region (a C to T at position -819) did not have to be analyzed separately from the -592 polymorphism because it was in 100% linkage disequilibπum with the -592 polymorphism For a descπption of the -819 polymorphism, see Turner et al , European J Immunogenetics 24 1-8, 1997

Alleles and genotypes for the IL10-SNP-1082 G-A vanant showed no significant allele or genotype association with infection (Table 2) or AIDS progression, however the position -592 vanant (IL10-5Α) was associated with both increased infection and progression The allele frequencies of 77.70- 5',4 in four ethnic groups were as follows Caucasians, 0 236 (n=2208), African Amencans, 0 400 (n=937). Hispanics, 0 327 (n=l 53) and Asians, 0 600 (n=47) The allele frequencies of the most common or wild type allele (IL10-5 -592C abbreviated 77,70-+) is equal to one minus the IL10-5Α frequencies for each ethnic group

The influence of the IL10-5Α on HIV-1 infection was examined in a categorical analysis of allele and genotype frequency distnbutions (termed a defined disease category analysis) among 575 HIV- 1 infected patients and 431 HIV- 1 -exposed uninfected Caucasians (Table 2) IL10-5Α allele or genotypic frequencies were equivalent in an initial companson of exposed (or at πsk) uninfected versus infected individuals in separate or combined cohort analysis (Table 2, p > 0 05) However, a group of 72 high-nsk exposed uninfected individuals from MACS (those with extremely high-πsk sexual practices) (Huang et al., Nat Med 2 1240, 1996. Detels et al , J Acquir Immune Defic Syndr 1 1263, 1994) showed a modest reduction in 77.70-5 'A alleles or IL 10-5 'A bearing genotypes relative to HIV- 1 infected individuals (31 0% among high-nsk uninfected individuals compared to 45 1 % among infected patients, FET p=003 Table 2) These results indicate that the IL10- 5 'A allele was associated with increased risk for infection (odds ratio = 1 75. FET, p=0 03) A role for IL10-5Α in disease progression among 769 HIV- 1 -infected seroconverters was demonstrated using a Cox proportional hazards model (Cox, Proportional Hazard Regression, SAS Release 6, 1 0, SAS Institute, Cary, NC JR Stat Soc 1972 B 34,187 Allison Survival analyses using the SAS system A practical guide SAS Institute, Cary, NC 1995,155-197, Levm, Ada Unio Int Contra Cancrum 9 531, 1953), and the results are shown FIGS 2B-F and Table 3 For combined and individual cohort analyses, the heterozygous IL10-+/5Α and homozygous IL10-5Α/5Α patients were mdistinguishable m the rates of progression to four AIDS endpomts (e g , FIG 2B) However, a highly significant acceleration to AIDS progression was apparent among HIV-infected Caucasians of genot pes IL10-5ΑI5Α oτIL10-+/5'A compared to 77,70-+/+ homozygotes for every AIDS outcome (P=0 0009- 0 05, Table 3) This susceptibility effect was also apparent when the protective effects of other AIDS restπction alleles, CCR2-64I and CCR5-A32, were considered as co-vanables in the adjusted Cox analyses (Table 3)

Table 2 IL10 promoter SNP allele and genotype frequencies influence on HIV-1 infection

IL10-SNP N Allele % Genotype %

A P(FET) GG GA A A P(FET)

IL10-SNP- HIV-positive 461 46 5 53 5 0 92 20 4 52 3 27 3 0 55 1082

HIV-negative 418 46 3 53 7 22 0 48 6 29 4

5',4 +/+ +/5'A S'A/5'A

ILlO-SNP-592 HIV-positive 575 75 6 24 4 0 27 57 2 36 9 5 9 0 79

HIV-negative 431 76 9 23 1 59 4 35 0 5 6

+ 5'A +Λ- +/5'A 5 'A/5 'A

ILlO-SNP-592 HIV-positive 377 74 3 25 7 0 04* 54 9 38 7 6 4 0 11

HREU* 72 81 3 18 8 68 1 26 4 5 6

+/+ +/5'A or

5Α/5Α

IL10-SNP-592 HIV-positive 377 54 9 45 1 0 03*

HREU* 72 68 1 31 0

*HREU-Hιgh πsk exposed uninfected based on high exposure in MACS through frequent sexual exposures (34)

P-values are from Fisher's exact test, *p<0 05 Table 3

Survival analysis tor progression to four AIDS outcomes among seroconverters as a function ofIL]0-5'A/5'A or +/5'A vs IL10-+/+ genotype all ethnic groups Caucasians African Americans outcome Interval

Years n/event RHu p-value AdiRH' p-value n/event RHu p-value AdjRH¹ p-value n/event RHu p-value AdjRH¹ p-value

CD4 all 758/381 1 14 0 23 1 24 0 06 505/276 1 27 0 05 1 30 0 03 218/83 0 86 0 53 1 13 0 67

--200 0-5 0 60 0 93 1 02 091 1 10 061 1 14 045 0 63 0 10 0 71 029

>5 1 43 0 02 1 51 0 009 1 44 0 03 1 46 0 02 1 93 0 19 3 41 0 05

AIDS- all 766/462 1 28 0 01 1 37 0002 511/336 1 44 00009 1 45 00008 220/102 092 0 69 1 22 1 42

1993 0-5 1 06 0 66 1 17 0 26 1 23 0 21 1 27 0 15 0 74 0 24 0 93 0 80

>5 1 54 0 002 1 58 0 0009 1 63 0 0009 1 61 0 001 1 42 0 37 1 93 0 13

AIDS- all 769/332 1 43 0 002 1 44 0 002 514/265 1 51 0 001 1 51 0 001 220/53 1 08 0 81 1 12 0 73

1987 0-5 1 26 0 24 1 25 0 27 1 21 0 42 1 26 0 33 1 48 0 37 1 31 0 57

>5 1 52 0 003 1 54 0 002 1 65 0 0007 1 63 0 001 0 77 0 54 0 96 0 93

L

Death all 769/252 1 32 0 03 1 32 0 03 514/213 1 40 0 02 1 41 0 02 220/31 1 01 0 81 1 07 0 87 I

0-6 1 04 0 86 1 05 0 82 1 05 0 85 1 09 0 75 1 02 0 96 0 96 0 94

>6 1 49 0 01 1 48 0 02 1 57 0 007 1 56 0 008 1 25 0 74 1 32 0 69

'Statistical Analysis Analyses were performed separately for each cohort, for Caucasians, tor African American and for combined ethnic groups Cox models (26,27) were used for calculating relative hazards and p values for separate and combined cohorts and racial groups Analysis were age stratified for those individuals <30, 30 to 40 or >40 vears old Kaplan-Meier survival analyses were used to visualize the effects of significant variables The time dependent effects were examined by analyses of two and three year windows of the tour outcomes Non-significant windows in the initial infection period were grouped together and later times considered separately in further Cox analyses

Time was not taken into account in the all models and was included as interaction term in the 0-5 and >5 year models (except death where 0-6 and >6 years was used) 'Relative hazards were shown as RHu from unadjusted models and as adjRH from adjusted models in which the effects ot the previously described CCR5 Δ32 and CCR2 641 genotypes were taken into account The effects of 1LI0 and CCR (CCR5 Δ32 &_ CCR2 641) were additive since, in the Cox analyses interaction terms were not significant (P 0 53-0 87) for the tour outcomes in Caucasians

'African Americans show a strong ILK⁾ 5 A AIDS acceleration effect in the >5 yr interval tor CD4 < 200 and AIDS- 1993 endpomts, particularly in relative hazards ad_justed tor the influence ot CCR5- +/Δ32 and CCR2 +/64I protective genotypes (RH 3 41 and 1 93, respectively)

The patterns of AIDS acceleration illustrated m FIG 2 show that the effect of the IL 10-5 'A allele is more substantial in later stages of HIV-1 infection (for example, 5 years following seroconversion) This effect is demonstrated for combined cohorts in Table 3, usmg a relative hazards model partitioned into rapid progressors (0-5 years after seroconversion) and late/slow and medium progressors (> 5 years after seroconversion) In Table 4, a partitioned time mterval approach with combined Caucasian, separate MACS and MHCS cohorts is presented These analyses show that acceleration to AIDS associated with 77.70-5'A bearing genotypes was more pronounced starting approximately 5 years after HIV-1 exposure, but the 1L10-5Α allele has no significant effect on AIDS progression m the initial five years after infection The time dependence of ILl 0-5'A promoter mediated disease acceleration contrasts to the CCR5-P1/P1 promoter allele acceleration which is strongest in the early stages (0-5 years) of HIV-1 infection (Martin et al , Science 282 1907, 1998)

Table 4

Survival analysis for progression to four AIDS outcomes among seroconverters as a function of ILl 0-5'A/ 5'A or +/5'A vs 77,70-+/+ in combined Caucasian, MACS and MHCS cohorts

AIDS Cohorts all (0-19 yrs ) 0-5 yrs * >5 yrs * outcomes n/event RHu p-value RHu p-value RHu p-value

CD4<200 Caucasians 505/276 1 27 0 05 1 10 0 61 1 44 0 03 MACS 345/183 1 42 0 02 1 21 0 32 1 83 0 01 MHCS 151/92 1 04 0 88 0 59 0 31 1 22 046

AIDS-1993 Caucasians 511/336 1 44 0 0009 1 23 0 21 1 63 0 0009 MACS 345/234 1 56 0 0008 1 28 0 17 1 96 0 0005 MHCS 157/100 1 34 0 17 0 97 0 95 1 48 0 11

AIDS-1987 Caucasians 514/265 1 51 0 001 1 21 0 42 1 65 0 0007 MACS 348/186 1 52 0 005 1 19 0 49 1 74 0 003 MHCS 157/78 1 49 0 10 1 15 0 83 1 56 009

Death Caucasians 514/213 1 40 0 02 1 05 0 85 1 57 0 007 MACS 348/147 1 34 0 08 1 05 0 87 1 55 0 04 MHCS 157/65 1 46 0 15 0 95 0 95 1 53 0 13

*For death a cutoff of 6 years was used

The tendency of IL10-5Α bearing genotypes to progress to AIDS rapidly was further demonstrated m a defined disease category analysis, and the results are demonstrated in FIG 3

Seroprevalent patients (those who enter the cohorts already HTV-1 positive) were placed in the slow/non- progressor category, because avoiding AIDS for longer penods (; e , > 10 years) is informative whether the interval of infection is precise or is greater than the time penod since enrollment The results (FIG 3) reveal a marked decrease in ILl 0-5'A beaπng genotypes in patients who avoid AIDS for 10 or more years after infection (compared to those who develop AIDS in less than 10 years p=0 0007-005), supporting the conclusion that patients carrying one or two ILl 0-5'A alleles progress relatively rapidly to AIDS The results of both the survival and defined disease category analyses (FIGS 2 and 3. Tables 3,4) affirm a strong dominant 77,70-5'A association with more rapid progression to different AIDS outcomes, particularly in the later stages of HIV-1 infection

The association of the STR 7770-R(-3975) with infection and disease progression is the result of a strong linkage disequi bnum between IL10-R(-3975) allele 283 (FIG 2A) and the ILl 0-5'A promoter allele (FIG 2B-F) Thus, a haplotype survey of 1698 human chromosomes (Caucasian) showed that 94% of IL10-5Α bearing chromosomes carry an ILl 0-R(-3975)-283 allele, a significant departure from random expectation for association of included alleles in that haplotype (68%, p<0 0001)

The AIDS accelerating effects for ILl 0-5'A were compared to the protective effects of CCR5- Δ32 and CCR2-64I in delaying AIDS (FIG 4) Because the protective effects of CCR2-64I and CCR5- Δ32 are dominant, genetically independent and equivalent in their influence on AIDS progression (Smith et al , Science 277 959, 1997), the CCR5 and CCR2 protective genotypes (CCR5-+/Δ32, CCR2-+/64I, and CCR2-64I/64I) were combined, and survival of patients determined in combined cohorts for 77,70- 5'A acceleration of AIDS (FIGS 4A and B) and for CCR2/5 protection (FIGS 4C and D) Separations of the genotype specific survival curves are gradual, and the strength of the 5 effects are equivalent (if not slightly greater), but in opposite directions of the CCR2/5 effects

Even more revealing is an analysis of CCR5, CCR2 and IL10 genotypes together (FIGS 4E and 4F) where patients protected by CCR5-A32 or CCR2-64I with the 7770-+/+ (also protective) genotype avoid AIDS considerably longer than CCR5-Δ32 or CCR2-64I protected patients carrying the ILl 0-5'A accelerating genotypes (red vs green lines RH=0 62, p=0 02, RH=0 65, p=0 05 for AIDS-1993 and AIDS-1987 respectively) Similarly ILl 0-5'A accelerating genotypes carrying wildtype (susceptible) CCR2-+ and CCR5-+ alleles (black lines, FIGS 4E and 4F) progress more rapidly than any genotypic group including IL 1075+/+ plus CCR5-+ or CCR2-+ (i e , CCR5/2 susceptible) genotypes (black vs blue lines RH=1 38, p=0 02 RH=1 47, p=0 01 for AIDS-1993 and AIDS-1987 respectively) These patterns demonstrate the cumulative effects in cohort populations of these genetic influences EXAMPLE 2

IL10-5Α Binding Sites The DNA sequence surrounding the ILl 0-5'A vanant (positions -604 to -581) was examined for homology to recognized bmdmg sites for known transcnption factors (Heinemeyer et al , Nucl Acids Res 26 362, 1998) and shown to include motifs specific for SP- 1 and ETS family binding sites (FIG 1 ) The 7770-+ sequence lacks the ETS motif while the ILl 0-5'A retains it posing a potential molecular mechanism for a reported two-fold mcrease m IL10 production in peπpheral blood mononuclear cells of IL 10- +/+ homozygotes compared to 7770-5 'A/5 'A homozygotes (Rosenwasser and Boπsh, Am J Respir Crit Care Med 156 SI 52, 1997)

To resolve potential allele distinctions in vitro, DNA sequence oligonucleotides representing 7770-+ and ILl 0-5'A (-604 to -581, extending approximately 11 bp on either side of SNP-592) were synthesized and tested for specific DNA binding protein recognition using the electrophoretic mobility shift assay (EMSA) with nuclear extracts from phytohaemagglutimn stimulated pnmary human penpheral blood T-lymphocytes (PBL) (Yu et al , J Immunol. 157 26, 1996) Two specific binding complexes were resolved usmg 7770-+ specific oligonucleotides, but only one of these bound to 77,70- 5' A specific oligonucleotides (FIG 5) Specificity for the binding was demonstrated by the fact that competitive binding of cold (100-fold excess) allele specific oligonucleotides eliminated complex formation completely (FIG 5) Cold synthetic SP-1 oligonucleotide effectively competed with and eliminated the faster migrating complex, implicating SP-1 as binding both ILIO promoter alleles A synthetic oligonucleotide designed from the ETS core consensus sequence (Hememeyer et al., Nucl Acids Res 26 362, 1998) did not compete for either complex formation indicating that nuclear proteins other than ETS family members may be involved in the formation of the 7770-+ binding complex Methods. Nuclear extracts were prepared as descnbed previously (Yu et al , J Immunol

157 126 1996) Cell pellets were resuspended in lysis buffer A (50 mM KCI, 25 mM Hepes, pH 7 8, 0 5% Nonidet P-40, 1 mM phenylmehylsulfonyl fluoπde, 10 μg/ml leupeptm. 20 μg/ml aprotmin, 100 μM dithiothreitol) and subsequently incubated on ice for 5 min Cellular suspensions were collected by centnfugation at 2000 rpm and the supernatant was decanted Nuclei were washed in buffer A without Nonidet P-40, and harvested by centnfugation at 2000 rpm Nuclear pellets were resuspended in extraction buffer B (500 mM KCI, 25 mM Hepes, pH 7 8, 10% glycerol, 1 mM phenylmefhylsulfonyl fluoride, 10 μg/ml leupeptm, 20 μg/ml aprotinin, 100 μM dithiothreitol), frozen in dry ice. thawed slowly on ice, and finally centnfuged at 14,000 rpm for 10 minutes The supernatant w as harvested and nuclear proteins quantified with the biomchonmic acid protein assay reagent (Pierce, Rockford, IL) Single-stranded oligonucleotides were commercially synthesized (Life Technologies, Rockville,

MD) to span -604 to -581 of the IL-10 promoter as follows

5'-GACCCCGCCTGTCCTGTAGGAAGC-3' (ILIO-5'C), and 5'-GACCCCGCCTGTACTGTAGGAAGC-3' (ILl 0-5'A) (both from SEQ ID No 43) SP-1 bindmg site in HIV-1 LTR was GGGAGGCGTGGCCTGGGCGGACTGGGGAGTGGCGA (SEQ ID NO 44) Complementary strands were annealed by combining 2 μg of each oligonucleotide and 6 μl of 10X annealing buffer (500 mM Tπs, 100 mM MgCL, and 50 mM dithiothreitol) in a 60 μl reaction, placing in a boiling water bath for 5 minutes and allowing to cool to room temperature The DNA- protein binding reaction was in a 20 μl reaction mixture consisting of 7 μg of nuclear protein extract, 1 μg poly (dl - dC) (Sigma), 4 μl of 5X binding buffer (60 mM Hepes, 7 5 mM MgCL, 300 mM KCI. 1 mM Ethylenediamine-tetraacetic acid, 2 5 mM dithiothreitol, 50% glycerol and [4 - (2 - Aminoethyl) - Benzenesulfonyl Fluoπde Hydrochlonde]) and 1 5 x 10⁴ cpm of 32P-labeled oligonucleotide probe In cold oligonucleotide competition expenments, 100-fold excess of unlabeled oligonucleotide probe was added 10 minutes prior to adding the radiolabeled oligonucleotide probe

Individuals with the ILl 0-5'A vanant have a vanant promoter sequence, that decreases IL10 expression following immune stimulation The results of this Example illustrate that differential transcnption may be due to altered recognition sites such as ETS, which consequently affects transcnptional activation and ILIO production, for example by decreasing activation and IL-10 production

EXAMPLE 3 Genotype Assessment Using PCR Detection of the SNP at the 311 bp ILlO-SNP-592 site was performed using the following pπmers

IL-10-SNP-592-A 5'-TACTCTTACCCACTTCCCCC-3' (forward) (SEQ ID NO 39), and IL10-SNP-592-Z 5'-TGAGAAATAATTGGGTCCCC-3' (reverse) (SEQ ED NO 40) Detection of the ILlO-SNP-1082 site (193 bp) was performed with IL10-SNP-1082-A 5'-CACTACTAAGGCTCCTTTGGG-3' (forward) (SEQ ID NO 41), and

IL10-SNP-1082-Z 5'-CCTGGATTAAATTGGCCTT-3' (reverse) (SEQ ID NO 42) Other pπmers are listed in Table 1

PCRs were earned out in a volume of 20 μl containing 50 ng of genomic DNA, 0 5 mM of each pnmer, 250 μM of each dNTP, 10 mM Tπs-HCI (pH 8 3) 50 mM KCI, 2 0 and 2 5 mM MgC12 and 0 72 units of AmpliTaq Gold (Perkm-Elmer, Norwalk, CT, USA) PCR amplifications were performed in 0 2 ml 96 well reaction plates m the GeneAmp PCR system 9600 (Perkin-Elmer) with a program consisting of 9 minutes denatunng at 94° C, followed by 35 cycles of 30 second denaturing at 94° C, 30 second annealing at 59° C - 53° C and 1 mmute extension at 72° C, followed by a final extension penod of 10 minutes at 72° C For the SNPs, amplified fragments were digested with 3 to 5 units of Rsal (7770-SNP-592) or EcoNI (7770-SNP- 1082) and separated in 3 5% TBE agarose gel (Amresco, Solon, OH, USA)

For microsatelhte genotyping, electrophoresis was earned out in an ABI PRISM 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA, USA) using 0 2 mm thick denatunng 5% Long- Ranger (FMC) gels containing 10% urea Microsatelhte PCR products (1 5 to 2 0 μl) were mixed with 0 3 μl of internal land standard (GENESCAN-500 TAMRA) and 2 μl of formamide loading buffer, denatured at 94° C for 5 minutes, cooled and then immediately loaded into the gel Sizing of DNA peaks and allele calling were performed using the GeneScan (ver 2 1) and ABI PRISM Genotyper (ver 2 0) software based on molecular weight as descnbed in the user's manual

The study group included 848 seroconverters 1863 seroprevalents and 627 sero-negatives for a total of 3337 (Caucasian 2208, Afncan Amencan 937 Hispanic 153, Asian 16) from five AIDS cohorts AIDS Link to the Intravenous Expenence (ALIVE) (Vlahov et al , NIDA Res Monogr 109 75,

1991), Hemophilia Growth and Development Study (HGDS) (Hilgartner et al ,Am J Pediatr Hematol Oncol 15 208, 1993),

Multicenter Hemophiliac Cohort (MHCS) (Goedert et al , N Engl J Med 321 1141, 1989), Multicenter AIDS Cohort Study (MACS) (Kaslow e/ α/ Am J Epidemiol 126 310 1987) and San Francisco City Clinic Study (SFCC) (Buchbmder, AIDS 8 1123, 1994)

Seroconversion date was estimated as the midpoint between the first positive HIV-1 antibody test date and the last negative antibody test HTV-1 infected SFCC patients (N=90) were considered as sero-prevalent because they had no previous HIV-1 antibody negative visit, and also because this cohort has a disproportionate number of slow/longer term progressors to AIDS (Buchbmder, AIDS 8 1123 , 1994, Donfield et al, Science 280 1819, 1998, Smith et al, Nat. Med. 3 1052, 1997 ) Four separate end points reflecting advancing AIDS morbidity were considered (I) CD4<200 cells/mm³, (u) AfDS- 1993, the CDC 1993 definition of AIDS (that is HIV-1 infection plus AIDS-defining illness or declme of CD4 T lymphocytes to < 200, see MMWR 1992,41 (no RR-17) or death, (in) the more stringent AIDS- 1987 definition (that is HIV-1 -infected plus AIDS defining illness, see MMWR 1987,36, suppl 1,1- 15S) or death and IV) death duπng follow-up for an HIV-1 -infected patient Time to endpomts was calculated from seroconversion date DNAs were extracted from immortal lymphoblastoid B cell lines established for each patient (O'Bnen and Dean, Sci. Amer. 277 44, 1997. Dean et al, Science 273 1856. 1996) EXAMPLE 4

Polymorphism Gene Probes and Markers Sequences surrounding and overlapping the single base-pair polymorphism of the present invention can be useful for a number of gene mapping, targetmg, and detection procedures For example, genetic probes can be readily prepared for hybπdization and detection of the ILl 0-5'A polymorphism As will be appreciated, probe sequences may be greater than about 16 or more oligonucleotides m length and possess sufficient complementanty to distinguish between the C (in the dominant allele) and A (m the ILl 0-5'A polymorphism) Similarly, sequences surrounding and overlapping the single base-pair polymorphism of the present invention can be utilized in allele specific hybπdization procedures

Sequence sunoundmg and overlapping the polymorphism of the present invention, or any portion or subset thereof that allows one to identify the polymorphism, are highly useful Thus, in accordance with another aspect of the present invention there is provided a genetic marker predictive of a the ILl 0-5'A polymorphism, compnsmg a partial sequence of the human genome including at least about 16 contiguous nucleotide residues including "X" in the following nucleotide sequence GACCCCGCCTGTXCTGTAGGAAGC (SEQ ID NO 43) and sequences complementary therewith, wherem "X" represents C or a single base-pair polymorphism of the C that is present at X in the dominant allele of the human population For example, the polymorphism is a C to A transition, but can also include a C to G or C to T transversion

EXAMPLE 5 Detecting SNPs The SNP at ILl 0-5'-A can be detected by a vanety of techniques These techniques mclude allele-specific oligonucleotide hybπdization (ASOH)(Stonekιng et al . Am J Hum Genet 48 370-382, 1991 ) which involves hybndization of probes to the sequence, stringent washing, and signal detection Other new methods mclude techniques that incorporate more robust scoring of hybndization Examples of these procedures include the hgation chain reaction (ASOH plus selective hgation and amplification), as disclosed in Wu and Wallace, Genomws 4 560-569, 1989, mini-sequencing (ASOH plus a single base extension) as discussed m Syvanen, Methods Mol Biol 98 291-298, 1998, and the use of DNA chips (miniaturized ASOH with multiple oligonucleotide arrays) as disclosed m Lipshutz et al.

(BioTechmques 19 442-447, 1995) Alternatively, ASOH with smgle- or dual- labeled probes can be merged with PCR, as m the 5'-exonuclease assay (Held et al , Genome Res 6 986-994, 1996), or with molecular beacons (as m Tyagi and Kramer, Nat. Biotechnol 14 303-308, 1996)

An even more recently introduced technique is dynamic allele-specific hybπdization (DASH), which involves dynamic heating and coincident monitormg of DNA denaturation, as disclosed by Howell et al (Nature Biotechnol 17 87-88, 1999) A target sequence is amplified by PCR in which one pπmer is biotinylated The biotmylated product strand is bound to a streptavidin-coated microtiter plate well, and the non-biotinylated strand is nnsed away with alkali wash solution An oligonucleotide probe, specific for one allele, is hybndized to the target at low temperature This probe forms a duplex DNA region that interacts with a double strand-specific intercalating dye When subsequently excited, the dye emits fluorescence proportional to the amount of double-stranded DNA (probe-target duplex) present The sample is then steadily heated while fluorescence is continually monitored A rapid fall m fluorescence indicates the denatunng temperature of the probe-target duplex Using this technique, a smgle-base mismatch between the probe and target results in a significant lowenng of melting temperature (T_m) that can be readily detected

A vanety of other techniques can be used to detect the polymorphism in DNA

EXAMPLE 6

Differentiation of Individuals who are Homozygous Versus Heterozygous for the Polymorphism As will be appreciated, the oligonucleotide hgation assay (OLA), as descnbed at Nickerson et al (Proc Nad Acad Set USA 87 8923-8927, 1990), allows the differentiation between individuals who are homozygous versus heterozygous for the ILl -5' -A polymorphism This feature allows one to rapidly and easily determine whether an individual is 7770-R-(-3975)-283/283 homozygous, which is linked to a relatively rapid progression to AIDS Alternatively, OLA can be used to determine whether a subj ect is homoz gous at the 7770-5 'A allele

As an example of the OLA assay, when earned out in microtiter plates, one well is used for the determination of the presence of the ILl 0-5'A allele and a second well is used for the determination of the presence of the ILl 0-5 'C allele Thus, the results for an individual who is heterozygous for the polymorphism will show a signal m each of the A and C wells, and an individual who is homozygous for the ILl 0-5'A polymorphism will show a signal in only the A well

Claims

We claim:

1 A method of predicting a susceptibility to HIV infection or HIV progression m a subject, comprising detecting a pol morphism in a human EL- 10 promoter of the subject, wherem presence of the polymorphism indicates the susceptibility to HIV infection or HIV progression

2 The method of claim 1 , wherem the polymorphism is 7770-5 '-592A

3 The method of claim 2, wherem detecting the presence of the polymorphism compnses providing DNA from the subject, and assessing the DNA for the presence of the ILl 0-5 '-592A polymorphism 4 The method of claim 3 , further compnsing determining whether the sub|ect is homozygous or heterozygous for the polymorphism

5 The method of claim 2, further compnsing detecting one or more other alleles associated with HIV infection or progression

6 The method of claim 5, wherein the one or more other alleles is one or more alleles selected from the group of CCR5-Δ32, CCR5-P1, CCR2-641, SDFl-3 'A, MBL and 77 4-.4, -B, and -C.

7 The method of claim 6, wherein the one or more other alleles is one or more alleles selected from the group of CCR5-Δ32 and CCR2-64]

8 The method of claim 2, wherein the polymorphism is detected by detecting an STR within 4 kb of the ILIO gene, which is in linkage disequihbπum with ILl 0-5 '-592A 9 The method of claim 8, wherein the STR is selected from one or more of the group of 7770-

G-7740 and 7770-R-3975

10 The method of claim 3. wherein the assessing step is performed by a process which compnses subjecting the DNA or RNA to amplification using oligonucleotide pnmers flanking the polymorphism 11 The method of claim 10, wherem the assessing step further compnses an oligonucleotide hgation assay

12 The method of claim 10, wherein the assessing step compnses polymerase chain reaction amplification of the DNA from the subject with oligonucleotide pπmers of SEQ ID NO 39 and SEQ ID NO 40 13 A method of predicting susceptibility to HIV infection or HIV progression in a subject, compnsing obtainmg a test sample of DNA containing a human IL-10 promoter sequence of the subject, and detecting the polymorphism in the IL-10 promoter, wherein the presence of the polymorphism indicates the susceptibility 14 The method of claim 13 , wherem detecting the polymoφhism compnses using at least one technique chosen from the group consisting of restriction digestion, probe hybndization, nucleic acid amplification, and nucleotide sequencmg

15 The method of claim 14, wherem detecting the polymoφhism compnses using nucleic acid amplification

16 The method of claim 15, wherein detecting the polymoφhism compnses using polymerase chain reaction nucleic acid amplification

17 The method of claim 16, wherem detecting the polymoφhism comprises performing the polymerase chain reaction amplification with pπmers selected from the group of SEQ ID NO 39 and SEQ ID NO 40

18 The method of claim 14, wherem detecting the polymoφhism comprises performing probe hybπdization with a nucleic acid probe to the human IL10-5'-592A polymoφhism m the test sample

19 The method of claim 14. wherein detecting the polymoφhism compnses detecting the polymoφhism by hybndization of an allele-specific oligonucleotide with the human ILl 0-5 '-592 A allele 20 The method of claim 13 , wherem the polymoφhism is IL 10-5 '-592 A

21 A method of predicting susceptibility to HIV infection or HIV progression in a subject, compnsing obtaimng from the subject a test sample of DNA comprising the human IL-10 promoter sequence, contacting the test sample with at least one nucleic acid probe for a human IL- 10 promoter sequence polymoφhism that is associated with increased susceptibility to HIV infection or

HIV progression in a subject, maintaining the hybndization sample under conditions sufficient for specific hybπdization of the human IL-10 promoter sequence with the nucleic acid probe, and detecting specific hybπdization of the human IL- 10 promoter sequence with the nucleic acid probe, wherein specific hybndization of the human IL-10 promoter sequence with the nucleic acid probe indicates increased susceptibility to HIV infection or HIV progression m the subject

22 The method of claim 21. wherem the polymoφhism is 7770-5 '-592 A

23 The method of claim 22, wherein the probe is present on a substrate 24 The method of claim 23, wherein the substrate is a cDNA aπay

25 A kit for use in diagnosing an increased susceptibility to HIV infection or HIV progression in a subject, compnsing a probe that specifically hybndizes to a human IL-10 promoter sequence polymoφhism that is associated with the increased susceptibility to HIV infection or HIV progression

26 The kit of claim 25, wherem the probe specifically hybridizes to a human IL10-5'-592A polymoφhism

27 A nucleic acid probe that specifically hybndizes to a human IL10-5'-592A polymoφhism