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WO2011032078A1 - Détection de souches x4 du vih-1 par test de suivi des hétéroduplexes - Google Patents

Détection de souches x4 du vih-1 par test de suivi des hétéroduplexes Download PDF

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WO2011032078A1
WO2011032078A1 PCT/US2010/048604 US2010048604W WO2011032078A1 WO 2011032078 A1 WO2011032078 A1 WO 2011032078A1 US 2010048604 W US2010048604 W US 2010048604W WO 2011032078 A1 WO2011032078 A1 WO 2011032078A1
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hiv
coreceptor
qxr
patient
ccr5
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Binshan Shi
Barbara Weiser
Harold Burger
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Health Research Inc
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Health Research Inc
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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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the present invention relates to analysis of heteroduplexes using a heteroduplex duplex tracking assay using a denaturing separation method and its use for diagnostic methods, such as methods to monitor coreceptor use in treatment of human
  • HIV immunodeficiency virus
  • HIV uses a receptor-mediated pathway in the infection of host cells. HIV-1 requires contact with two cell-surface receptors to gain entry into cells and initiate infection; CD4 is the primary receptor. CXCR4 and CCR5, members of the chemokine receptor family of proteins, serve as secondary coreceptors for HIV- 1 isolates that are tropic for T-cell lines or macrophages, respectively (Deng et al (1996) Nature 381:661-6; Doranz et al (1996) Cell 86: 1149-59; and Berger et al (1998) Nature 391:240; Feng et al (1996) Science 272:872- 877; Samson et al (1996) Nature 382:722-725).
  • CXCR4 or CCR5 in conjunction with CD4, form a functional cellular receptor for entry of certain strains of HIV into cells.
  • Coreceptor use therefore plays a critical role in viral tropism, pathogenesis, and disease progression.
  • HIV-1 strains transmitted in vivo generally use CCR5 (R5 viruses), whether by sexual, parenteral, or mother-to-child transmission (Fenyo et al. (1998) Nature 391 :240; Samson et al. (1996) Nature 382:722-5; Shankarappa et al. (1999) J. Virol.
  • X4 strains not only infect an expanded spectrum of crucial target cells as compared to R5 viruses, but they also exhibit increased cytopathicity and mediate bystander killing of uninfected cells (Blaak et al. (2000) Proc. Natl. Acad. Sci. USA 97: 1269-74;
  • Envelope variants selectively interact with either CXCR4 or CCR5.
  • All of the known genetic determinates of coreceptor usage are found in the envelope gene (env), with the key determinates being found in the region of the env gene encoding the third variable (V3) domain of the gpl20 glycoprotein.
  • HIV-1 coreceptor utilization had been predicted according to the sequence of the V3 portion of the env gene (Hung CS et al. (1999); and Briggs DR et al. (2000)).
  • an accumulation of positively charged amino acid located in the V3 domain i.e., at positions 11 and 25 of the V3 domain and is a common feature of X4 viruses (Fouchier RA et al. (1992); Milich L.
  • V3 region of CXCR4-specific viruses also can exhibit greater sequence variation than their R5 -specific counterparts, in particular respect with common laboratory HIV isolates at HTLV-IIIB/LAV and JR-CSF (Milich L. et al. (1997)).
  • the difference in cell tropism correlates with disease progression.
  • viral populations are usually characterized by molecular heterogeneity. Strains isolated from individuals early in the course of their infection are usually M-tropic (Shankarappa et al. (1999); and Glushakova et al. (1999) J. Clin. Invest. 104:R7-R1 1).
  • M-tropic Shankarappa et al. (1999); and Glushakova et al. (1999) J. Clin. Invest. 104:R7-R1 1).
  • the X4 and R5 strains coexist to some extent in the viral swarm or population.
  • viruses isolated from approximately 50% of individuals with advanced immunodeficiency include viruses that are M- and T-tropic.
  • the emergence of X4 variants is associated with depletion of CD4 cells and acceleration of clinical disease.
  • cART may affect either the expression of CCR5 over CXCR4 or, alternatively, it may be influencing the kind of viral variant that predominates, such as CCR5-specific versus CXCR4-specific viruses.
  • CXCR4-specific strains There is a correlation between the emergence of CXCR4-specific strains and rapid HIV disease progression.
  • a diagnostic method would be useful to monitor the presence (or absence) of CXCR4-specific strains and/or CCR5-specific strains and shifts in coreceptor use over time.
  • a diagnostic method for use in monitoring shifts in coreceptor use may thereby be beneficial for measuring the therapeutic efficacy of various HIV treatment regimes, such as cART.
  • the effect of cART on coreceptor use by populations of virus has not heretofore been
  • CXCR4-specific strains The correlation between CXCR4-specific strains and rapid disease progression also indicates that a diagnostic method would be useful to monitor the presence of CXCR4- specific strains, shifts in coreceptor use associated with HIV disease progression, and to monitor the presence of CXCR4-specific strains and shifts in coreceptor use in patients undergoing antiretroviral therapy.
  • diagnostic methods for use in detecting CXCR4 isolates and/or monitoring shifts in coreceptor use would be beneficial for predicting disease progression over time or in response to treatment.
  • cell-based and molecular-based methods to monitor, measure, evaluate, detect, etc. HIV coreceptor use which are reliable, accurate, and easy to use as well as being qualitative and/or quantitative in their approach would be a welcomed advance to the art.
  • diagnostic methods e.g. cell-based and/or molecular-based methods, for measuring, monitoring, evaluating, detecting, etc. patient-derived HIV samples for coreceptor usage would be beneficial for evaluating HIV disease progression in the face of various anti HIV treatment and therapies.
  • diagnostic assays which result in significant improvement in the ability of the technique to detect X4 strains and distinguish them from R5 strains of HIV- 1 would be beneficial to the art.
  • Heteroduplex Tracking Assay can be carried out substantially in accordance with the guidance of Delwart et al (J.Virol. (1994) 68:6672-6683), Delwart et al (Science (1993) 262: 1257-1261), Nelson et al (J. Virol. (1997) 71:8850-8, Delwart et al (PCR Methods and Applications 4:S202-S216 (19950 Cold Springs Harbor), and U.S. Patent 5,851,759 (Weiner).
  • heteroduplex DNA is formed after hybridization of an exogenous nucleic acid probe with the target nucleic acid followed by electrophoresis on a native polyacrylamide gel.
  • the HTA can be used to detect discrete sequence variations, such as point mutations, insertions, and deletions.
  • the HTA assay has been used extensively to determine CCR5 and CXCR4 coreceptor usage by human immunodeficiency virus (HIV) as disclosed in U.S. application serial no. 12/174,141, filed July 16, 2008, U.S. application serial no. 1 1/654,897, filed January 17, 2007, U.S. patent no. 7,344,830, U.S. patent no., 7,294,458, and U.S. patent no. 6,727,060.
  • HAV human immunodeficiency virus
  • DGGE denaturing gradient gel electrophoresis
  • Non- electrophoretic nucleic acid separation methods that operate under denaturing conditions include denaturing high performance liquid chromatography (dHPLC).
  • the method includes aHTA comprising: (a) contacting a target nucleic acid with at least one exogenous probe nucleic acid to form a mixture under conditions that allow one or more heteroduplex species to form; (b) subjecting said mixture of heteroduplex species to a denaturing separation method, wherein said denaturing separation method can distinguish heteroduplex from homoduplex species by differential mobility in the separation medium; and (c) identifying the heteroduplex species present after separation.
  • the probe and/or target nucleic acids can be produced by amplification.
  • the amplification method can be, for example, PCR or molecular cloningasymmetric PCR.
  • the probe can be labeled, optionally prior to heteroduplex formation, and the label can comprise a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a chemiluminescent moiety, or an enzymatic moiety.
  • the separation method can include, but is not limited to, CDGE, CDCE, DGGE, TGGE, TGCE, TTGE, and dHPLC.
  • the probe can comprise at least one modified nucleotide residue that increases hybrid Tm.
  • the at least one modified residue can include, for example, one or more of LNA.
  • the exogenous nucleic acid probe can lack a GC clamp.
  • the method can include nucleic acid target in excess of exogenous nucleic acid probe.
  • the target nucleic acid can include a sequence of the V3 portion of the HIV-1 env gene which can be derived from a single isolate from an HIV-infected patient.
  • the exogenous nucleic acid probe can include a sequence of the V3 portion of the HIV- 1 env gene which can be derived from a HIV-1 CCR5 clone or a HIV-1 CXCR4 clone.
  • the present invention also relates to diagnostic methods and components thereof for determining the viral load of a population of acquired immunodeficiency virus using the CXCR4 coreceptor (X4-specific viral load) in a patient-derived biological sample.
  • This invention further relates to a method of determining when to initiate antiretroviral therapy in a patient.
  • the present invention also relates to a method of monitoring the efficacy of antiretroviral therapy in a patient.
  • the present invention encompasses a diagnostic method which may comprise determining the viral load of a population of acquired immunodeficiency (AIDS) virus using the CXCR4 coreceptor (X4-specific viral load) in a patient-derived biological sample comprising the steps of: (a) screening individual molecular clones of patient-derived acquired immunodeficiency primary isolate with a V3 loop sequencing assay to determine CCR5 coreceptor usage and CXCR4 coreceptor usage of each individual molecular clone; (b) determining the proportion of HIV using the CCR5 coreceptor (R5) versus the CXCR4 coreceptor (X4) wherein the proportion is expressed as a variable called the Quantity of X4 and R5 (QXR), which represents the fraction of virus in a specimen using the R5 coreceptor; (c) determining coreceptor specific viral loads of the patient-derived acquired acquired AIDS
  • the screening of individual molecular clones of patient-derived acquired immunodeficiency primary isolate to determine CCR5 coreceptor usage and CXCR4 coreceptor usage of each individual molecular clone is conducted with a V3 loop sequencing assay.
  • the present invention further encompasses a diagnostic method which may comprise determining the viral load of a population of acquired immunodeficiency virus using the CXCR4 coreceptor (X4-specific viral load) in a patient-derived biological sample.
  • the patient-derived biological sample is any bodily fluid or tissue.
  • the biological sample may be a bodily fluid which may be selected from the group consisting of blood, plasma, and spinal fluid.
  • the individual molecular clones may each comprise a DNA sequence corresponding to a portion of the HIV genome, the DNA sequence comprising at least a portion of the genetic determinates of coreceptor usage.
  • the genetic determinates may be derived from the env gene.
  • the molecular clones each may be derived from RNA of the patient-derived HIV and correspond to the HIV genome or a portion thereof and which comprise the genetic determinates of coreceptor usage or a portion thereof.
  • the molecular clones may be prepared by reverse transcription PCR (RT-PCR) of the RNA of the patient-derived HIV and at least one set of oligonucleotide primers.
  • RT-PCR reverse transcription PCR
  • at least one set of oligonucleotide primers may consist of the first set of primers in Table 3.
  • at least one set of oligonucleotide primers may include a second set of oligonucleotide primers, consisting of the second set of primers in Table 3.
  • the number of individual molecular clones may be at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 10, 000, at least 100,000, or at least 1,000,000. .
  • the heteroduplex tracking assay of the method may comprise the steps of: (a) amplifying the individual molecular clone or a portion thereof by PCR to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using a separation means; (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals coreceptor usage.
  • the labeled probe may be derived from a HIV-1 CCR5 clone or from a HIV-l bCXCR4 clone.
  • the labeled probe may comprise a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a
  • chemiluminescent moiety or an enzymatic moiety.
  • the method may be used (a) to assess or predict the degree of HIV progression, (b) to determine when to start or change antiretroviral treatment, or (c) to monitor the efficacy of antiretroviral treatment.
  • the viruses in a population are a mixture of those that use the R5 and X4 coreceptors.
  • the patient-derived biological sample may be any bodily fluid or tissue.
  • the biological sample may be a bodily fluid which may be selected from the group consisting of blood, plasma, and spinal fluid.
  • the individual molecular clones may each comprise a DNA sequence corresponding to a portion of the HIV genome, the DNA sequence comprising at least a portion of the genetic determinates of coreceptor usage.
  • the genetic determinates may be derived from the env gene.
  • the molecular clones each may be derived from RNA of the patient-derived HIV and correspond to the HIV genome or a portion thereof and which comprise the genetic determinates of coreceptor usage or a portion thereof.
  • the molecular clones may be prepared by RT-PCR of the RNA of the patient-derived HIV and at least one set of oligonucleotide primers.
  • at least one set of oligonucleotide primers may consist of the first set of primers in Table 3.
  • at least one set of oligonucleotide primers may include a second set of oligonucleotide primers, the second set may consist of the second set of primers in Table 3.
  • the number of individual molecular clones may be at least 20.
  • the heteroduplex tracking assay of the method may comprise the steps of: (a) amplifying the individual molecular clone or a portion thereof by PCR to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using a separation means; (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals coreceptor usage.
  • the labeled probe may be derived from a HIV-1 CCR5 clone or from a HIV-1 CXCR4 clone.
  • the labeled probe may comprise a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a
  • chemiluminescent moiety or an enzymatic moiety.
  • the antiretroviral therapy of the method may be any suitable antiretroviral treatment regimen. More preferably, the antiretroviral therapy may be selected from the group consisting of combination antiretroviral therapy (cART), protease inhibitors, fusion inhibitors, integrase inhibitors, coreceptor specific agents, nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • the nucleoside analoque reverse transcriptase inhibitor may be 3TC or AZT.
  • the nonnucleoside analogue reverse transcriptase inhibitor may be nevirapine.
  • a the viruses in a population are a mixture of those that use the R5 and X4 coreceptors.
  • the patient-derived biological sample may be any bodily fluid or tissue.
  • the biological sample may be a bodily fluid which may be selected from the group consisting of blood, plasma, and spinal fluid.
  • the individual molecular clones may each comprise a DNA sequence corresponding to a portion of the HIV genome, the DNA sequence comprising at least a portion of the genetic determinates of coreceptor usage.
  • the genetic determinates may be derived from the env gene.
  • the molecular clones each may be derived from RNA of the patient-derived HIV and correspond to the HIV genome or a portion thereof and which comprise the genetic determinates of coreceptor usage or a portion thereof.
  • the molecular clones may be prepared by RT-PCR of the RNA of the patient-derived HIV and at least one set of oligonucleotide primers.
  • at least one set of oligonucleotide primers may consist of the first set of primers in Table 3.
  • at least one set of oligonucleotide primers may include a second set of oligonucleotide primers, the second set consisting of the second set of primers in Table 3.
  • the number of individual molecular clones may be at least 20.
  • the heteroduplex tracking assay of the method may comprise the steps of: (a) amplifying the individual molecular clone or a portion thereof by PCPv to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using a separation means; (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals coreceptor usage.
  • the labeled probe may be derived from a HIV-1 CCR5 clone or from a HIV-1 CXCR4 clone.
  • the labeled probe may comprise a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a
  • chemiluminescent moiety or an enzymatic moiety.
  • the antiretroviral therapy of the method may be any suitable antiretroviral treatment regimen. More preferably, the antiretroviral therapy may be selected from the group consisting of combination antiretroviral therapy (cART), protease inhibitors, fusion inhibitors, integrase inhibitors, coreceptor specific agents, nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • the nucleoside analoque reverse transcriptase inhibitor may be 3TC or AZT.
  • the nonnucleoside analogue reverse transcriptase inhibitor may be nevirapine.
  • Various aspects of the HTA procedure, described heretofore, may be modified to accomplish this objective.
  • the modifications include novel use of sophisticated methods in nucleic acid research and gel electrophoresis that result in significant improvement in the ability of the technique to detect X4 strains and distinguish them from R5 strains of HIV- 1.
  • the new method is as feasible for use in a clinical lab as the previous HTA.
  • the new method focuses on methods to slow the electrophoretic mobility of HIV- 1 strains using the X4 coreceptor, relative to those that use R5, in order to amplify the separation of coreceptor- specific strains and facilitate their identification.
  • the method used is constant denaturing gel electrophoresis (CDGE).
  • CDGE constant denaturing gel electrophoresis
  • the present invention further encompasses a diagnostic method comprising determining the viral load of a population of acquired immunodeficiency (AIDS) virus using the CXCR4 coreceptor (X4-specific viral load) in a patient-derived biological sample comprising the steps of: (a) screening individual molecular clones of patient-derived acquired immunodeficiency primary isolate with a heteroduplex tracking assay using CDGE to determine CCR5 coreceptor usage and CXCR4 coreceptor usage of each individual molecular clone; (b) determining the proportion of HIV using the CCR5 coreceptor (R5) versus the CXCR4 coreceptor (X4) wherein the proportion is expressed as a variable called the Quantity of X4 and R5 (QXR), which represents the fraction of virus in a specimen using the R5 coreceptor; (c) determining coreceptor specific viral loads of the patient-
  • the biological sample is any bodily fluid or tissue.
  • the biological sample is a bodily fluid selected from the group consisting of blood, plasma, and spinal fluid.
  • the individual molecular clones each comprise a DNA sequence corresponding to a portion of the HIV genome, the DNA sequence comprising at least a portion of the genetic determinates of coreceptor usage.
  • the molecular clones are derived from R A of the patient-derived HIV and correspond to the HIV genome or a portion thereof and which comprise the genetic determinates of coreceptor usage or a portion thereof. Even more preferably, the genetic determinates are derived from the env gene.
  • the molecular clones are prepared by RT-PCR of the RNA of the patient-derived HIV and at least one set of oligonucleotide primers.
  • the at least one set of oligonucleotide primers consists of the first set of primers in Table 3.
  • the at least one set of oligonucleotide primers includes a second set of oligonucleotide primers, the second set consisting of the second set of primers in Table 3. It is also preferable that the number of individual molecular clones is at least 20.
  • the heteroduplex tracking assay comprises the steps of: (a) amplifying the individual molecular clone or a portion thereof by PCR to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using CDGE; (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals coreceptor usage.
  • the labeled probe is derived from a HIV- 1 CCR5 clone. Even more preferably, the labeled probe is derived from the sequences of the V3 portion of the HIV- 1 env gene.
  • the labeled probe may incorporate one or more locked nucleic acids (LNAs).
  • LNAs locked nucleic acids
  • the labeled probe preferably comprises a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a chemiluminescent moiety, or an enzymatic moiety.
  • the CDGE assay involves using a gel solution that enhances the detection of mutations.
  • the method is used (a) to assess or predict the degree of HIV progression, (b) to determine when to start or change antiretroviral treatment, or (c) to monitor the efficacy of antiretroviral treatment.
  • FIGURE 1 depicts the effect of combination antiretroviral therapy on HIV- 1 coreceptor use over time in representative study subjects.
  • Patients 1, 2, 6, 8, and 10 received new, combination therapy and Patient 13 remained untreated.
  • Drugs are abbreviated as follows: AZT, zidovudine; 3TC, lamivudine; Rit, ritonavir; Ind, indinavir; Saq, saquinavir; d4T, stavudine; Nel, nelfinavir; ddl, didanosine; ddC, zalcitabine; and Nev, nevirapine.
  • FIGURE 2 depicts the dynamics of the shift in coreceptor utilization immediately following initiation of HAART.
  • FIGURE 3 depicts an example of a template set-up for a PE2400 PCR tray -retainer.
  • FIGURE 4 depicts an example of a pattern produced by gel analysis based on an original RT layout, for use in selecting samples to be cloned/sequenced.
  • FIGURE 5 provides a graphical illustration of the various steps of the heteroduplex tracking assay (HTA) of the invention which provides for both qualitative and quantitative analysis of HIV coreceptor usage.
  • HTA heteroduplex tracking assay
  • FIGURE 6 provides a schematic representation of heteroduplex tracking assay (HTA) analysis of four different targets, including probe only, CCR5 -specific HIV V3 region only, CXCR4-specific HIV V3 region only, and a mixture or "quasispecies" of both CCR5- specific and CXCR4-specific HIV V3 regions.
  • HTA heteroduplex tracking assay
  • FIGURES 8-12 provide comparisons of HIV- 1 coreceptor utilization analysis method by improved HTA-CDGE and original HTA-QXR.
  • FIGURE 13 provides the clone map of an HIV-1 env V3 concensus sequence encoding CCR5 tropism.
  • the term "or (a) fragment(s) thereof as employed in the present invention and in context with polypeptides of the invention, comprises specific peptides, amino acid stretches of the polypeptides as disclosed herein. It is preferred that said "fragment(s) thereof is/are functional fragment(s).
  • the term "functional fragment” as used herein denotes a part of the above identified polypeptide of the invention which fullfils, at least in part, physiologically and/or structurally related activities of the polypeptide of the invention. It is also envisaged that the fragments, like the full-length polypeptides, may be distinguished between HIV strains in effecting binding.
  • the polypeptides of the present invention can be recombinant polypeptides expressed in eukaryotic cells, like mammalian cells.
  • nucleic acid hybridization may be used herein to refer to "molecular-based assays," and may include, for example, the heteroduplex binding assay of the invention.
  • present invention may also include methods that combine both cell-based and molecular based methods and should not be construed to be limited to either one or the other approach.
  • molecular clone may be used herein to refer to the cloning or PCR amplification of a portion of the HIV genome, such as a gene or a portion of a gene, which can then be analyzed in accordance with the molecular-based methods of the invention, especially the heteroduplex tracking assay.
  • genetic determinates may be used herein to refer to the molecular clones of portions of the env gene which allow a quantitative determination of the proportion of HIV specific for the CCR5 coreceptor and those specific for the CXCR4 coreceptor, for example the third variable (V3) region of the gpl20 glycoprotein.
  • PCR refers to the molecular biology technique known as polymerase chain reaction, disclosed by Mullis in U.S. Pat. Nos. 4,683,195 (Mullis et al) and 4,683,202, incorporated herein by reference. The following U.S.
  • Patents may also be referenced for information relating to PCR generally: 6,316,192; 6,309,837; 6,300,073; 6,300,072; 6,284,455; 6,270,977; 6,270,966; 6,268,143; 6,261,431; 6,251,607; 6,232,079; 6,225,093; 6,218,153; 6,207,425; 6,183,963; 6,180,372; 6,146,834; 6,087,097; 6,072,369; 6,068,974; 6,063,563; 6,046,039; 6,031,960; 6,017,699; 6,015,664; 6,015,534; 6,001,612; 5,972,602; 5,909,468; 5,905,732; 5,888,740; 5,883,924; 5,869,318; 5,853,991; 5,837,468; 5,827,657; 5,824,516; 5,824,479; 5,814,489; 5,7
  • denaturing separation method refers to laboratory techniques that can separate nucleic acid molecules under denaturing conditions.
  • the denaturing conditions can be chemical or thermal or both.
  • denatured separation method refers to a method for subjecting nucleic acid molecules to a two phase system under solution conditions that permit the partial melting of DNA duplex such that it that allows the operator to detect different species of molecules.
  • modified nucleotide residue refers to a non-naturally occurring chemical alteration of a nucleotide within a nucleic acid.
  • modified nucleotide residue include a Locked Nucleic Acid (LNA) or a 2'-methoxy-ribose.
  • LNA Locked Nucleic Acid
  • a nucleotide residue can incorporate more than one modification and a nucleic acid can have one or more modified nucleotide residues.
  • GC clamp refers to a 30-60 nucleotide stretch of oligonucleotide comprising guanine and cytosine residues typically incorporated into the 5'- end of an amplicon and used in denaturing separation methods such as CDGE, DGGE and dHPLC.
  • massively parallel sequencing refers to high-throughput DNA sequencing of clonally amplified or single DNA molecules that , typically, are spatially separated in a single flow cell generating hundreds of megabases to gigabases of nucleotide sequence output in a single run.
  • patient as used herein may be any animal, preferably a mammal, and even more preferably a human, infected with HIV.
  • infectious immunodeficiency virus refers to the infectious AIDS virus known to one of skill in the art and may be, but is not limited to, HIV-1 and/or HIV-2.
  • the term "genotype” may be used herein to refer to a strain of HIV at the genetic sequence level.
  • One of skill in the art appreciates that during the course of disease progression the pool of HIV in an infected individual may become a mixture of different strains which are different at the genetic level (i.e. have different "genotypes"). It is further understood by the skilled person that whether any particular strain of HIV from a population of virus in an infected individual is specific for CCR5 coreceptor or the CXCR4 coreceptor is dependent on the genetic determinates contained in that virus's genome, i.e. is reflected in that virus's genotype.
  • QXR is defined as the ratio of the number of clones identified as being specific to the CCR5 coreceptor compared to the total number of clones analyzed from a population of HIV contained in a sample of patient-derived HIV. It will be appreciated that during the course of disease progression that the pool of HIV in an infected individual can become a mixture of different strains which are different at the genetic level (i.e. have different "genotypes").
  • VL viral load
  • the "coreceptor specific viral load” is defined as the number of copies of virus RNA genome per unit volume that is specific for a certain coreceptor.
  • the R5- specific viral load is the number of copies of virusRNA genome per unit volume that is specific for CCR5 receptor and the X4-specific viral load is the number of copies of virus virus genome per unit volume that is specific for CXCR4 receptor.
  • HAART refers to any highly active antiretroviral therapy and is more recently referred to as combination antiretroviral therapy, or "cART", used interchangeably herein with “CART”. HAART and cART are also used herein
  • HAART may refer to three or more antiretroviral drugs in combination, and usually comprises one protease inhibitor and two or three reverse transcriptase inhibitors.
  • the heteroduplex tracking assay can be used to detect genetic variation. Using this method, one can establish genetic relationships between multiple viral DNA template molecules, such as the different genetic types (i.e. different genotypes) of HIV utilizing the different coreceptors.
  • the HTA is performed using the standard nondenaturing polyacrylamide gel electrophoresis for separation of the heteroduplex species and some of the heteroduplex species do not clearly resolve. Consequently, there is as a need to improve the resolution of heteroduplex species.
  • the HTA of the invention can include utilizing an exogenous nucleic acid probe which is mixed with an excess ("driver") of an nucleic acid target from a different source, i.e., the source for which typing or analysis of is desired, e.g.
  • the exogenous nucleic acid probes are then "driven” annealed completely into heteroduplexes with the driver target nucleic acid, and are separated using a denaturing separation method.
  • the exogenous nucleic acid probe can be labeled prior to mixing with the nucleic acid target.
  • the label can comprise a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a chemiluminescent moiety, or an enzymatic moiety.
  • the fluorescent moiety can be 6-FAM. Appropriate labels and their methods of preparation are well-known.
  • the nucleic acid probe or probes can be generated by amplification.
  • the nucleic acid target can be generated by amplification. Amplification can be performed by the PCR or asymmetric PCR.
  • the heteroduplexes can be detected by detecting the nucleic acid probe (as opposed to the target nucleic acid). The separation of heteroduplexes provides a visual display of the relationship between the two virus populations under study.
  • heteroduplex encompasses a doublestranded DNA molecule having
  • a heteroduplex can form by mixing together an exogenous labeled nucleic acid probe (e.g. a double-stranded DNA PCR product of a portion of the env gene of CCR5 -specific HIV) and a PCR product of a nucleic acid target sequence (e.g. a double-stranded DNA PCR product of the corresponding portion of the env gene of a CXCR4-specific HIV) such that
  • an exogenous labeled nucleic acid probe e.g. a double-stranded DNA PCR product of a portion of the env gene of CCR5 -specific HIV
  • a nucleic acid target sequence e.g. a double-stranded DNA PCR product of the corresponding portion of the env gene of a CXCR4-specific HIV
  • PCR product from the CXCR4-specific HIV will contain genetic determinates characteristic of CXCR4 type viruses, its nucleotide sequence will vary at specific locations with respect to the probe PCR product (which is derived from a CCR5-specific env sequence). These differences in sequence result in a heteroduplex which has altered mobility during separation with respect to homoduplexes.
  • a "homoduplex" can be formed between complementary strand pairs derived from an exogenous nucleic acid probe PCR product and a nucleic acid target PCR product such that their nucleotide sequences are the same.
  • the heteroduplex tracking assay can comprise the steps of (a) amplifying an individual molecular clone or a portion thereof by PCR to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using a denaturing separation method: and (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals coreceptor usage.
  • the exogenous nucleic acid probe can be derived from a HIV- 1 CCR5 clone. In another embodiment, the exogenous nucleic acid probe can be derived from a HIV- 1 CXCR4 clone.
  • the exogenous nucleic acid probe can by made by any of the nucleic acid chemical synthetic methods known in the art. Either or both the exogenous nucleic acid probe or the nucleic acid target can be amplified by any of the nucleic acid amplification methods known in the art.
  • the term "amplification” or “amplify” as used herein means one or more methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear.
  • a target nucleic acid may be either DNA or RNA. The sequences amplified in this manner form an "amplicon.” PCR (polymerase chain reaction) is disclosed by Mullis in U.S. Pat. Nos.
  • enzymatic replication and amplification methods include isothermal methods, rolling circle methods, Hot-start PCR, real-time PCR, Allele-specific PCR, Assembly PCR or Polymerase Cycling Assembly (PCA), Asymmetric PCR, RNA linear amplification, DNA linear amplification, Colony PCR, Emulsion PCR, Fast PCR, Real-Time PCR, nucleic acid ligation, Gap Ligation Chain Reaction (Gap LCR), Ligation-mediated PCR, Multiplex Ligation-dependent Probe Amplification, (MLPA), Gap Extension Ligation PCR (GEXL- PCR), quantitative PCR (Q-PCR), Quantitative real-time PCR (QRT-PCR), multiplex PCR, Helicase-dependent amplification, Intersequence-specific (ISSR) PCR, In
  • CDGE constant denaturing gel electrophoresis
  • CDGE separation principle is based on the melting behavior of DNA molecules.
  • a denaturing acrylamide gel double-stranded DNA is subjected to conditions that will melt the DNA in discrete segments called melting domains. The melting
  • Tm temperature of each domain
  • the Tm usually depends upon a specific nucleotide sequence composition. Mismatches, insertions, and deletions, however, will result in large decrements in Tm.
  • Tm of the lowest melting domain When the Tm of the lowest melting domain is reached, the DNA will become partially melted, creating branched molecules. Partial melting of the DNA reduces its mobility in a polyacrylamide gel. Since the Tm of a particular melting domain is sequence-specific, the presence of a mutation will alter the melting profile of that DNA when compared to the wild type. Thus, different DNA sequences containing different mutations will exhibit different mobilities compared to the wild-type DNA.
  • a 30 to 50bp GC rich sequence or so called GC clamp, is added to the 5 'end of one of the PCR primers, co-amplified, and thus introduced into the amplified target DNA fragments.
  • the GC-rich sequence acts as a high- melting domain which not only prevents the two DNA strands from complete dissociation into single strands but also increases the number of melting domains to be analyzed (Glavac and Dean, 1995).
  • the application of the GC clamp greatly increases the detection sensitivity and resolution; the disadvantage of the GC clamp, however, is that it can decrease the efficiency and specificity of amplification.
  • the addition of a GC clamp can present added difficulties for the detection of relatively short amplified DNA fragments. GC clamps have not been used for HTA wherein an exogenous nucleic acid probe is utilized.
  • denaturing electrophoresis is denaturing gradient gel
  • DGGE denaturing gradient gel electrophoresis
  • electrophoresis under denaturing conditions include, but are not limited to, constant denaturing (or denaturant) gel electrophoresis (CDGE), constant denaturing capillary electrophoresis (CDCE), thermal gradient gel electrophoresis (TGGE), thermal gradient capillary electrophoresis (TGCE), and temporal thermal gradient electrophoresis (TTGE).
  • CDGE constant denaturing gel electrophoresis
  • DPE constant denaturing capillary electrophoresis
  • TGGE thermal gradient gel electrophoresis
  • TGCE thermal gradient capillary electrophoresis
  • TTGE temporal thermal gradient electrophoresis
  • Methods other than electrophoresis are available, such as denaturing high performance liquid chromatography (dHPLC).
  • dHPLC denaturing high performance liquid chromatography
  • the exogenous nucleic acid probe can include at least one modified nucleotide that increases hybrid stability when annealed to the target nucleic acid.
  • denaturing separation methods typically incorporate a redesignGC clamp," typically 30-40 nucleotides long, into the PCR amplification primers so that the product has a resulting GC-clamp at the 5 ' end. This is performed in order to maximize the anomolous mobility of the interrogated sequence differences, such as for CDGE and DGGE.
  • the denaturing separation methods in the art form this GC- clamp by using a single 5 '-end primer in a PCR to yield duplexes with a single GC-clamp on the 5 'end.
  • the prior art methods would then subject the amplified duplexes to denaturation and reannealing to form heteroduplexes and subsequently subject the mixtures to the denaturation separation method.
  • the inventors recognized that because the HTA uses an exogenous nucleic acid probe, unlike the methods such as CDGE and DGGE, adding a GC- clamp is unwieldy at best. Consequently, the inventors arrived at the novel solution of incorporating modified nucleotides at the end of the probe in order to achieve an effect similar to the GC-clamp.
  • LNAs Locked Nucleic Acids
  • Such modifications can include bicyclic sugar moieties such as “Locked Nucleic Acids” (LNAs) in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'- C-oxymethylene linkage to form the bicyclic sugar moiety (reviewed in Elayadi et al, Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al, Chem. Biol, 2001, 8 1-7; and Orum et al, Curr. Opinion Mol.
  • LNAs Locked Nucleic Acids
  • ENATM is one non limiting example of an LNA.
  • LNAs are commercially available from Proligo (Paris, France and Boulder, Colo., USA) or Exiqon (Woburn,MA).
  • LNAs also form duplexes with complementary DNA, RNA or LNA with high thermal affinities.
  • Circular dichroism (CD) spectra show that duplexes involving fully modified LNA (esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex.
  • Nuclear magnetic resonance (NMR) examination of an LNA:DNA duplex confirmed the 3'-endo conformation of an LNA monomer. Recognition of double-stranded DNA has also been demonstrated suggesting strand invasion by LNA. Studies of mismatched sequences show that LNAs obey the Watson-Crick base pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands.
  • LNA monomers adenine, cytosine, guanine, 5- methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al, Tetrahedron, 1998, 54, 3607- 3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
  • TNA 3',2'-alpha- L-threose nucleic acid
  • TNA is capable of antiparallel Watson-Crick base pairing with complementary DNA, RNA and TNA oligonucleotides (Chaput et al, J. Am. Chem. Soc, 2003, 125, 856-857).
  • oligonucleotide mimetics have been prepared to include bicyclic and tricyclic nucleoside analogs (see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens et al, J. Am. Chem. Soc, 1999, 121, 3249-3255; Renneberg et al, J. Am. Chem. Soc, 2002, 124, 5993-6002; and Renneberg et al, Nucleic acids res., 2002, 30, 2751-2757).
  • modified nucleoside analogs have been oligomerized using the phosphoramidite approach and the resulting oligomeric compounds containing tricyclic nucleoside analogs have shown increased thermal stabilities (Tm's) when hybridized to DNA, RNA and itself. Oligomeric compounds containing bicyclic nucleoside analogs have shown thermal stabilities
  • Ribosyl and related sugar moieties are routinely modified at any reactive position not involved in linking.
  • a suitable position for a sugar substituent group is the 2'-position not usually used in the native 3' to 5'-internucleoside linkage.
  • Other suitable positions are the 3' and the 5'-termini.
  • 3'-sugar positions are open to modification when the linkage between two adjacent sugar units is a 2',5'-linkage.
  • Sugar substituent groups include, but are not limited to: OH; F; 0-, S-, or -alkyl; 0-, S-, or N-alkenyl; 0-, S- or -alkynyl; or O-alkyl-0- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C2 to Cio alkenyl and alkynyl. Particularly suitable are 0((CH2) n O) m CH 3 ,
  • n and m are from 1 to about 10.
  • One modification includes 2'-methoxyethoxy (2'-0— CH2CH2OCH 3 , also known as 2'- 0-(2-methoxyethyl) or 2'-MOE) (Martin et al, Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group.
  • 2'-dimethylaminooxyethoxy i.e., a 0(CH 2 ) 20N(CH 3 ) 2 group, also known as 2'-DMAOE, as described in examples hereinbelow
  • 2'-dimethylaminoethoxyethoxy also known in the art as 2'-0-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE
  • Sugar substituent groups include 0((CH 2 ) n O) m CH 3 , 0(CH 2 ) n OCH 3 , 0(CH 2 ) n NH 2 , 0(CH 2 ) n CH 3 , 0(CH 2 ) n ONH 2 , and 0(CH 2 ) n ON((CH 2 ) n CH 3 )) 2 , where n and m are from 1 to about 10.
  • nucleobase often referred to in the art simply as “base” or “heterocyclic base moiety” modifications or substitutions.
  • “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases also referred herein as heterocyclic base moieties include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
  • Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
  • Mutation Detection Enhancement (MDE®) Gel Solution is a commercially available (Lonza, Allendale,NJ) polyacrylamide-like matrix that has a high sensitivity to DNA conformational differences. This gel's unique structure causes DNA separation to occur on the basis of both size and conformation. MDE® Gel Solution's formulation enables the detection of more mutations by traditional heteroduplex and SSCP analysis than detection using standard polyacrylamide.
  • DNA sequencing can also be used for detecting sequence variation. Sequencing can be used to validate or augment the HTA. Methods for sequencing and/or identifying the V3 region may be any desired method, e.g., a method which is by or analogous to the methods cited in US Patent Nos.
  • the sequence variation of the V3 loop may be detected by performing any nucleic acid analysis techniques known to those of skill in the art.
  • suitable techniques include sequencing techniques (direct DNA sequencing which is also known as population-based sequencing (using either the dideoxy chain termination method or the Maxam-Gilbert method (see Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al, Recombinant DNA Laboratory Manual, (Acad. Press, 1988))., sequencing of single variants), pyrosequencing, gel electrophoresis
  • hybridization heteroduplex tracking assay, line probe assay, nucleic acid arrays (details on the use of nucleic acid arrays (DNA chips) for the detection of, for example, SNPs, see U.S. Pat. No. 6,300,063 issued to Lipshultz, et al, and U.S. Pat. No. 5,837,832 to Chee, et al), bead array).
  • Another sequencing method known in the art that can be used for detection of sequence variation of the V3 loop is known as massively parallel sequencing, next generation sequencing or deep sequencing (Voelkerding et al. (2009) Clin. Chem. 55(4):641-658; ten Bosch et al. (2008) J. Mol. Diag.
  • Genome Sequence FLX systems from 454 Corporation (a Roche Company), the Genome Analyzer from Illumina, and the SOLiD system from Life Technologies.
  • Single molecule massively parallel sequencers include those from, for example, Helicos Biosciences (the Heliscope) and Pacific Biosciences.
  • nucleic acid analysis techniques include restriction fragment length
  • polymorphism analysis cleavase fragment length polymorphism analysis as described in U.S. Pat. No. 5,843,669, random amplified polymorphic DNA (RAPD) analysis, arbitrary fragment length polymorphisms (AFLPs), differential sequencing with mass spectrometry, single based extension (SBE) of a fluorescently-labeled primer coupled with fluorescence resonance energy transfer (FRET) between the label of the added base and the label of the primer as described by Chen et al, (PNAS 94: 10756-61 (1997), single-strand conformation polymorphism analysis as described in Orita et al, Proc. Nat. Acad. Sci.
  • RAPD random amplified polymorphic DNA
  • AFLPs arbitrary fragment length polymorphisms
  • SBE single based extension
  • FRET fluorescence resonance energy transfer
  • high throughput analysis may be achieved by PCR multiplexing techniques well known in the art. (E.g., Z. Lin et al, Multiplex genotype determination at a large number of gene loci, Proc. Natl. Acad. Sci. USA 93(6):2582-87 [1996]).
  • additional methodologies may be achieved by combining existing nucleic acid analysis methodologies.
  • An example is ultradeep sequencing wherein a two stage PCR technique coupled with a novel pyrophosphate sequencing technique would allow the detection of sequence variants (SNP, indels and other DNA polymorphisms) in a rapid, reliable, and cost effective manner.
  • Conformation-sensitive gel electrophoresis of amplification products may also be used to analyze sequence variation of the V3 loop.
  • A. Markoff et al. Comparison of conformation-sensitive gel electrophoresis and single strand conformation polymorphism analysis for detection of mutations in the BRCA1 gene using optimized conformation analysis protocols, Eur. J. Genet. 6(2): 145-50 [1998]).
  • the sequence variation of the V3 loop may also be detected by performing immunological analysis techniques known to those of skill in the art such as ELISA and protein arrays.
  • the structure of the V3 loop helps to determine HIV coreceptor usage, and therefore methods that characterize V3 structure may also be used to determine whether a viral variant uses CCR5 or CXCR4 (T. Cardozo et al, Structural basis for coreceptor selectivity by the HIV-1 V3 loop. 2007 AIDS Res and Hum Retroviruses; 23:415-26).
  • sequence variation analysis assays encompasses the following non- limiting types of sequence variation analysis assays: PCR-free genotyping methods, single- step homogeneous methods, homogeneous detection with fluorescence polarization, "Tag" based DNA chip system, fluorescent dye chemistry, TaqMan genotype assays, Invader genotype assays, and microfluidic genotype assays, among others.
  • the authors of the present invention have surprisingly found that the viral load of acquired immunodeficiency virus in a patient-derived biological sample using the CXCR4 coreceptor (X4-specific viral load) is directly related to disease progression and clinical outcome.
  • the data presented herein strongly suggest that the X4-specific viral load determined by the methods provided herein is a powerful predictor in guiding clinical therapies including when to initiate antiretroviral therapy, the response to antiretroviral therapies, and clinical management.
  • the present invention relates to diagnostic methods and components thereof for determining the viral load of a population of acquired immunodeficiency virus using the CXCR4 coreceptor in a patient-derived biological sample.
  • the invention further relates to a method of determining when to initiate antiretroviral therapy in a patient.
  • the present invention also relates to a method of monitoring the efficacy of antiretroviral therapy in a patient.
  • the present invention encompasses a diagnostic method which may comprise determining the viral load of a population of acquired immunodeficiency virus using the CXCR4 coreceptor (X4-specific viral load) in a patient-derived biological sample.
  • the patient-derived biological sample is any bodily fluid or tissue.
  • the biological sample may be a bodily fluid which may be selected from the group consisting of blood, plasma, and spinal fluid.
  • the biological sample may be one which contains viral populations that are distinct from those in the readily obtained peripheral blood including the reservoirs of the genital tract and lymphoid tissue.
  • Patient-derived biological samples may be obtained by methods known to one of skill in the art. For instance, peripheral blood of HIV-infected individuals can be separated into plasma and cell components by methods known in the art. Primary viral isolates of HIV- 1 may also be obtained by co-culture with normal donor peripheral blood mononuclear cells (PBMCs). Titration of viral isolates in PBMCs can be carried out. These standard techniques are described throughout the literature; for example, see Fang et al (1995) Proc. Natl. Acad. Sci. USA 92: 12110-4.
  • the individual PCR products or molecular clones each comprise a DNA sequence corresponding to a portion of the HIV genome, the DNA sequence comprising at least a portion of the genetic determinates of coreceptor usage.
  • the genetic determinates are derived from the env gene.
  • the envelope protein may comprise gp 120, gp 160 or a portion thereof. Envelope sequences are predictive of coreceptor use on the basis of the overall charge of the V3 loop and the presence of basic or acidic residues at positions 275 and 287 of the env gene (Bhattacharya et al (1996) AIDS Res. Hum. Retrovir. 12:83-90; Hung et al (1999) J. Virol. 73:8216-26); and Cardozo et al (2007) AIDS Res. Hum. Retrov., 23:415-26.
  • Cloning strategies for isolating envelope genes of interest are well known to one of skill in the art. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning, A Laboratory Manual, 2 nd Ed., Cold Spring Harbor Laboratory Press, 1989.
  • the cloning methods used in the present invention will decrease the chance of sampling error or recombination.
  • high fidelity cloning of the samples above may be achieved by routine performance of multiple long RT-PCR reactions on limiting dilutions of RNA, followed by multiple PCR's on cDNAs obtained from each RT reaction.
  • performance of multiple PCR's on each cDNA preparation increases the likelihood of amplifying a different HIV-1 RNA species.
  • Short-term limited dilution techniques are also well known to one of skill in the art, see for example, Connor et al.
  • RNA is amplified to ⁇ 80 copies/ml.
  • the molecular clones each are derived from RNA of the patient-derived HIV and correspond to the HIV genome or a portion thereof and which comprise the genetic determinates of coreceptor usage or a portion thereof.
  • the molecular clones are prepared by RT-PCR of the RNA of the patient-derived HIV and at least one set of oligonucleotide primers.
  • at least one set of oligonucleotide primers consists of the first set of primers in Table 3.
  • at least one set of oligonucleotide primers includes a second set of oligonucleotide primers, the second set consisting of the second set of primers in Table 3.
  • the number of individual molecular clones is at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 10, 000, at least 100,000, or at least 1,000,000. .
  • the heteroduplex tracking assay of the method may comprise the steps of: (a) amplifying the individual molecular clone or a portion thereof by PCR to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using a separation means; (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals particular coreceptor usage.
  • the labeled probe may be derived from a known HIV-1 CCR5 clone or from a known HIV-1 CXCR4 clone.
  • the probe can be labeled and the label can be labeled probe comprises a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a chemiluminescent moiety, or an enzymatic moiety.
  • the heteroduplex tracking assay of the invention can be carried out substantially in accordance with the guidance of Delwart et al (J.Virol. (1994) 68:6672-6683), Delwart et al (Science (1993) 262: 1257-1261), Nelson et al (J. Virol. (1997) 71 :8850-8, Delwart et al (PCR Methods and Applications 4:S202-S216 (19950 Cold Springs Harbor), and U.S. Patent 5,851,759 (Weiner), each of which are incorporated in their entireties by reference.
  • the heteroduplex tracking assay can be used to analyze a portion of the HIV- 1 genome encompassing determinates of coreceptor utilization to understand, determine, monitor, or detect coreceptor usage. Genetic determinates of HIV- 1 coreceptor utilization can be found in the envelope gene (env), with key determinates being found in the third variable (V3) domain of the gpl20 glycoprotein.
  • env envelope gene
  • V3 variable domain of the gpl20 glycoprotein
  • the heteroduplex tracking assay of the invention can be carried out generally, while not being limited thereto, according to the basic steps of: (a) obtaining HIV viral RNA from the patient, (b) amplifying, e.g. reverse transcription (RT)-PCR, a portion of the viral genome containing genetic determinates of coreceptor usage, e.g. a genomic portion comprising the V3 domain of the gpl20 envelope glycoprotein, (c) forming heteroduplexes and/or homoduplexes with labeled nucleic acid-based probes prepared from a corresponding genomic region of a HIV strain, e.g.
  • RT reverse transcription
  • the same genomic portion comprising the V3 domain of gpl20, and (d) subjecting the heteroduplexes and homoduplexes to a separation system, e.g. electrophoresis through non-denaturing polyacrylamide gels, wherein the heteroduplexes and homoduplexes have differing and distinguishable mobilities that results in different mobility patterns, e.g. a electrophoretic pattern, such that the coreceptor usage can be determined.
  • a separation system e.g. electrophoresis through non-denaturing polyacrylamide gels
  • the presence of an electrophoretic pattern characteristic of X4- heteroduplexes can indicate the presence of CXCR4-specific viruses in the HIV sample.
  • the presence of an electrophoretic pattern characteristic of homoduplexes and R5 -heteroduplexes can indicate the presence of only CCR5-specific viruses.
  • a pattern characteristic of both homoduplexes and X4- and R5 -heteroduplexes can indicate that the HIV sample contains a mixed population of CCR5-specific and CXCR4-specific viruses.
  • the heteroduplex tracking assay can be performed at any point during disease progression or during, before, or after administering antiretroviral therapy. Further, the heteroduplex tracking assay can be carried out either to attain qualitative results or quantitative results.
  • RT-PCR reverse transcription PCR
  • heteroduplex tracking assay is based on the observation that when sequences were amplified by nested PCR from peripheral blood mononuclear cells of infected individuals, related DNA products coamplified from divergent templates could randomly reanneal to form heteroduplexes that migrate with reduced mobility in neutral polyacrylamide gels.
  • a first PCR product as a labeled probe, e.g. radioactive, or
  • nonradioactive which is mixed with an excess ("driver") of an unlabeled PCR product from a different source, i.e., the source for which typing or analysis of is desired, e.g. the PCR product defining the portion of the HIV genome with the coreceptor genetic determinates.
  • the probe sequences are then "driven” completely into heteroduplexes with the driver, and are separated, e.g. by gel electrophoresis, on the basis of size.
  • An autoradiogram or fluoroimage, for example, of the resulting polyacrylamide gel reveals these heteroduplexes and provides a visual display of the relationship between the two virus populations under study. The fact that heteroduplexes migrate with distinct mobilities indicates that the strand- specific composition of mismatched and unpaired nucleotides affects their mobility.
  • heteroduplex encompasses a doublestranded DNA molecule having
  • a heteroduplex can form by mixing together a labeled probe (e.g. a double-stranded DNA PCR product of a portion of the env gene of CCR5 -specific HIV) and a PCR product of a target sequence (e.g. a double-stranded DNA PCR product of the corresponding portion of the env gene of a CXCR4-specific HIV) such that
  • a labeled probe e.g. a double-stranded DNA PCR product of a portion of the env gene of CCR5 -specific HIV
  • a target sequence e.g. a double-stranded DNA PCR product of the corresponding portion of the env gene of a CXCR4-specific HIV
  • the PCR product from the CXCR4-specific HIV will contain genetic determinates characteristic of CXCR4 type viruses, its nucleotide sequence will vary at specific locations with respect to the probe PCR product (which is derived from a CCR5-specific env sequence). These differences in sequence result in a heteroduplex which has reduced mobility during electrophoresis with respect to homoduplexes oweing to a reduced level of base-pairing in the molecule.
  • the "homoduplex" may be formed between complementary strand pairs derived from a probe PCR product and a target PCR product such that their nucleotide sequences are the same.
  • the heteroduplex tracking assay can comprise the steps of (a) amplifying an individual molecular clone or a portion thereof by PCR to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using a separation means: and (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals coreceptor usage.
  • the labeled probe may be derived from a HIV- 1 CCR5 clone.
  • the labeled probe may be derived from a HIV- 1 CXCR4 clone.
  • the labeled probe can comprise a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a
  • chemiluminescent moiety or an enzymatic moiety.
  • Appropriate labels and their methods of preparation are well-known.
  • the diagnostic method may involve the use of microchips comprising nucleic acid molecules encoding a envelope protein, or a fragment thereof, preferably a V3 region fragment, especially including genetic determinates of coreceptor usage, on gene chips; or an envelope protein, or a fragment thereof, preferably a V3 region fragment, on protein-chips ⁇ See U.S. Patent Nos.
  • Diagnostic gene chips may comprise a collection of polypeptides that specifically detect a envelope protein, or fragments thereof, preferably V3 region fragments; or nucleic acid molecules that specifically detect a nucleic acid molecule encoding a envelope protein, or fragments thereof, preferably V3 region fragments; all of which may be used for the purposes of determining coreceptor use.
  • the envelope protein may be gpl60, gp 120, or a portion thereof.
  • the heteroduplex tracking assay of the invention can be used to provide both qualitative and quantitative information.
  • qualitative information can be derived using the HTA of the invention by analyzing the whole HIV population derived from an infected patient to determine whether the isolated population of HIV is CCR5 -specific, CXCR4-specific, or mixture of both types. It will be appreciated that qualitative information is based on the representative HIV population rather than individual clones therefrom.
  • quantitative information can be derived byusing the HTA of the present invention by analyzing individual HIV clones (e.g. portions of the HIV genome cloned into plasmidsthe HIV derived from a plurality of individual HIV viruses from the isolated whole population of HIV from the infected patient).
  • the data is analyzed with respect to their coreceptor usage and determining a ratio of CCR5-specific to CXCR4-specific clones.
  • the invention relates to determining the QXR ratio: the number of HIV clones that are identified as CCR5-specific compared to the total number of clones analyzed.
  • the HIV clone can refer to the amplified PCR product.
  • FIGURE 5 depicts a flow chart showing the qualitative and quantitative aspects of the HTA of the present invention.
  • HIV R A is extracted from the infected patient.
  • RT-PCR is carried out to obtain HIV cDNA, from which a PCR product (i.e. PCR amplicon) containing genetic determinates for coreceptor usage is amplied using PCR.
  • the PCR product is then gel purified.
  • the PCR product will be a mixed population of molecules - those genotypic for either CCR5 or CXCR4 coreceptors - whenever the isolated HIV sample contains both types of viruses.
  • the PCR product is analyzed by the HTA of the invention, which includes generally the steps of mixing together a labeled probe (e.g.
  • a PCR product corresponding to same region in a CCR5 strain as the amplified target PCR amplicon to be analyzed and the amplified target PCR amplicon to form homo- or heteroduplexes.
  • the molecules are then separated by gel electrophoresis, for example, on a 12% polyacrylamide gel. Electrophetic techniques are well known in the art. If the QXR ⁇ 1 on the qualitative test, then a quantitative test can be done. To perform the quantitative test, the V3 portion of the HIV envelope gene is molecularly cloned and each of 20 clones is analyzed by an individual HTA.
  • FIGURE 6 Exemplarly results are represented in FIGURE 6.
  • the figures shows four panels of schematic electropherograms.
  • the first panel is the negative control, i.e. labeled probe only.
  • the second panel shows the result of HTA of the V3 portion of the HIV envelope gene of a CCR5-tropic virus.
  • the third panel shows the result of HTA of the V3 portion of the HIV envelope gene of a CXCR4-tropic virus.
  • the fourth panel shows the result of HTA of a mixture of CCR5 and CXCR4 virus V3 regions.
  • Four different probes (each based on a CCR5 -specific control virus) were used to test each HIV sample.
  • the gels show
  • heteroduplex band patterns for those HIV samples containing CXCR4-specific and CCR5-specific viruses are heteroduplex band patterns for those HIV samples containing CXCR4-specific and CCR5- specific viruses.
  • the quantitative results of the heteroduplex tracking assay of the method of the present invention may be assessed by statistical methods well known to one of skill in the art.
  • QXR the proportion of plasma HIV-1 using CCR5
  • QXR 1 if all virus identified uses CCR5
  • the association between virologic responses and baseline QXR may be assessed by comparing the percentages of patients with undetectable HIV-1 RNA load across the different strata by using, for example, Fischer's exact test. Further, immunologic responses across two strata may be compared by Wilcoxon rank-sum tests. Kaplan-Meier curves and Cox proportional hazard regression models may be applied to quantify the association of baseline or follow-up QXR (equal 1 versus less than 1) with subsequent clinical progression, defined as a new clinical AIDS-defining event or death.
  • the quantitative results of the heteroduplex tracking assay of the method of the present invention may further be assessed by other statistical methods well known to one of skill in the art.
  • the concurrent log 2 transformed CD4 values and logio transformed HIV-1 loads in the univariable and multivariable Cox models may be included.
  • the inverse probability weights may be used to adjust for sampling bias.
  • STATA (Version 9.1, StataCorp, College Station, Texas) may be used for quantitative analyses.
  • the HTA method of the invention is used to assess or predict disease progression and/or mode of treatment.
  • the method is used (a) to assess or predict the degree of HIV progression, (b) to determine when to start or change antiretroviral treatment, or (c) to monitor the efficacy of antiretroviral treatment.
  • One of skill in the art e.g. a physician, preferably one specializing in the treatment of infectious disease
  • the frequency of application may vary, depending on various factors, for example, the age, sex, type of antiretroviral therapy administered to, or stage of disease progression in, a patient.
  • the frequency of application may vary, depending on various factors, for example, the age, sex, type of antiretroviral therapy administered to, or stage of disease progression in, a patient.
  • the viruses in a population are a mixture of those that use the R5 and X4 coreceptors.
  • the patient-derived biological sample is any bodily fluid or tissue.
  • the biological sample may be a bodily fluid which may be selected from the group consisting of blood, plasma, and spinal fluid.
  • the biological sample may be one which contains viral populations that are distinct from those in the readily obtained peripheral blood including the reservoirs of the genital tract and lymphoid tissue.
  • Patient-derived biological samples may be obtained by methods known to one of skill in the art. For instance, peripheral blood of HIV-infected individuals can be separated into plasma and cell components by methods known in the art. Primary viral isolates of HIV- 1 may also be obtained by co-culture with normal donor peripheral blood mononuclear cells (PBMCs). Titration of viral isolates in PBMCs can be carried out. These standard techniques are described throughout the literature; for example, see Fang et al (1995) Proc. Natl. Acad. Sci. USA 92: 12110-4.
  • the individual molecular clones each comprise a DNA sequence corresponding to a portion of the HIV genome, the DNA sequence comprising at least a portion of the genetic determinates of coreceptor usage.
  • the genetic determinates are derived from the env gene.
  • the envelope protein may comprise gp 120, gp 160 or a portion thereof. Envelope sequences are predictive of coreceptor use on the basis of the overall charge of the V3 loop and the presence of basic or acidic residues at positions 275 and 287 of the env gene (Bhattacharya et al (1996) AIDS Res. Hum. Retrovir. 12:83-90; Hung et al (1999) J. Virol. 73:8216-26; and Cardozo et al (2007) AIDS Res. Hum. Retrovir. 23:415-26).
  • the cloning methods used in the present invention will decrease the chance of sampling error or recombination.
  • high fidelity cloning of the samples above may be achieved by routine performance of multiple long RT-PCR reactions on limiting dilutions of RNA, followed by multiple PCR's on cDNAs obtained from each RT reaction.
  • performance of multiple PCR's on each cDNA preparation increases the likelihood of amplifying a different HIV-1 RNA species.
  • Short-term limited dilution techniques are also well known to one of skill in the art, see for example, Connor et al.
  • RNA is amplified to ⁇ 80 copies/ml.
  • the molecular clones each are derived from RNA of the patient-derived HIV and correspond to the HIV genome or a portion thereof and which comprise the genetic determinates of coreceptor usage or a portion thereof.
  • the molecular clones are prepared by RT-PCR of the RNA of the patient-derived HIV and at least one set of oligonucleotide primers.
  • at least one set of oligonucleotide primers consists of the first set of primers in Table 3.
  • at least one set of oligonucleotide primers includes a second set of oligonucleotide primers, the second set consisting of the second set of primers in Table 3.
  • the number of individual molecular clones is at least 20.
  • the heteroduplex tracking assay of the method may comprise the steps of: (a) amplifying the individual molecular clone or a portion thereof by PCPv to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using a separation means; (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals coreceptor usage.
  • the labeled probe may be derived from a HIV-1 CCR5 clone or from a HIV-1 CXCR4 clone.
  • the labeled probe comprises a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a chemiluminescent moiety, or an enzymatic moiety.
  • the heteroduplex tracking assay of the invention can be carried out substantially in accordance with the guidance of Delwart et al (J.Virol. (1994) 68:6672-6683), Delwart et al (Science (1993) 262: 1257-1261), Nelson et al (J.Virol. (1997) 71:8850-8; Delwart et al (PCR Methods and Applications 4:S202-S216 (19950 Cold Springs Harbor), and U.S. Patent 5,851,759 (Weiner), each of which are incorporated in their entireties by reference.
  • the heteroduplex tracking assay can be used to analyze a portion of the HIV- 1 genome encompassing determinates of coreceptor utilization to understand, determine, monitor, or detect coreceptor usage. Genetic determinates of HIV- 1 coreceptor utilization can be found in the envelope gene (env), with key determinates being found in the third variable (V3) domain of the gpl20 glycoprotein.
  • env envelope gene
  • V3 variable domain of the gpl20 glycoprotein
  • the heteroduplex tracking assay of the invention can be carried out generally, while not being limited thereto, according to the basic steps of: (a) obtaining HIV viral RNA from the patient, (b) amplifying, e.g. PCR and/or reverse transcription, a portion of the viral genome containing genetic determinates of coreceptor usage, e.g. a genomic portion comprising the V3 domain of the gpl20 envelope glycoprotein, (c) forming heteroduplexes and/or homoduplexes with labeled nucleic acid-based probes prepared from a corresponding genomic region of a HIV strain, e.g.
  • the same genomic portion comprising the V3 domain of gpl20, and (d) subjecting the heteroduplexes and homoduplexes to a separation system, e.g. electrophoresis through non-denaturing polyacrylamide gels, wherein the heteroduplexes and homoduplexes have differing and distinguishable mobilities that results in different mobility patterns, e.g. a electrophoretic pattern, such that the coreceptor usage can be determined.
  • a separation system e.g. electrophoresis through non-denaturing polyacrylamide gels
  • the presence of an electrophoretic pattern characteristic of X4- heteroduplexes can indicate the presence of CXCR4-specific viruses in the HIV sample.
  • the presence of an electrophoretic pattern characteristic of homoduplexes and R5-heteroduplexes can indicate the presence of only CCR5-specific viruses.
  • a pattern characteristic of both homoduplexes and X4- and R5-heteroduplexes can indicate that the HIV sample contains a mixed population of CCR5-specific and CXCR4-specific viruses.
  • the heteroduplex tracking assay can be performed at any point during disease progression or during, before, or after administering antiretroviral therapy. Further, the heteroduplex tracking assay can be carried out either to attain qualitative results or quantitative results.
  • RT-PCR reverse transcription PCR
  • heteroduplex tracking assay is based on the observation that when sequences were amplified by nested PCR from peripheral blood mononuclear cells of infected individuals, related DNA products coamplified from divergent templates could randomly reanneal to form heteroduplexes that migrate with reduced mobility in neutral polyacrylamide gels.
  • a first PCR product as a labeled probe, e.g. radioactive or
  • nonradioactive which is mixed with an excess ("driver") of an unlabeled PCR product from a different source, i.e., the source for which typing or analysis of is desired, e.g. the PCR product defining the portion of the HIV genome with the coreceptor genetic determinates.
  • the probe sequences are then "driven” completely into heteroduplexes with the driver, and are separated, e.g. by gel electrophoresis, on the basis of size.
  • An autoradiogram or fluoroimage, for example, of the resulting polyacrylamide gel reveals these heteroduplexes and provides a visual display of the relationship between the two virus populations under study.
  • the fact that heteroduplexes migrate with distinct mobilities indicates that the strand- specific composition of mismatched and unpaired nucleotides affects their mobility.
  • a "heteroduplex” encompasses a doublestranded DNA molecule having
  • a heteroduplex can form by mixing together a labeled probe (e.g. a double-stranded DNA PCR product of a portion of the e «vgene of CCR5 -specific HIV) and a PCR product of a target sequence (e.g. a double-stranded DNA PCR product of the corresponding portion of the env gene of a CXCR4-specific HIV) such that
  • a labeled probe e.g. a double-stranded DNA PCR product of a portion of the e «vgene of CCR5 -specific HIV
  • a target sequence e.g. a double-stranded DNA PCR product of the corresponding portion of the env gene of a CXCR4-specific HIV
  • the PCR product from the CXCR4-specific HIV will contain genetic determinates characteristic of CXCR4 type viruses, its nucleotide sequence will vary at specific locations with respect to the probe PCR product (which is derived from CCR5). These differences in sequence result in a heteroduplex which has reduced mobility during electrophoresis with respect to homoduplexes oweing to a reduced level of base-pairing in the molecule.
  • the "homoduplex" may be formed between complementary strand pairs derived from a probe PCR product and a target PCR product such that their nucleotide sequences are the same.
  • the heteroduplex tracking assay can comprise the steps of (a) amplifying an individual molecular clone or a portion thereof by PCR to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using a separation means: and (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals coreceptor usage.
  • the labeled probe may be derived from a HIV- 1 CCR5 clone.
  • the labeled probe may be derived from a HIV- 1 CXCR4 clone.
  • the labeled probe can comprise a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a
  • chemiluminescent moiety or an enzymatic moiety.
  • Appropriate labels and their methods of preparation are well-known.
  • the diagnostic method may involve the use of microchips comprising nucleic acid molecules encoding a envelope protein, or a fragment thereof, preferably a V3 region fragment, especially including genetic determinates of coreceptor usage, on gene chips; or an envelope protein, or a fragment thereof, preferably a V3 region fragment, on protein-chips ⁇ See U.S. Patent Nos.
  • Diagnostic gene chips may comprise a collection of polypeptides that specifically detect a envelope protein, or fragments thereof, preferably V3 region fragments; or nucleic acid molecules that specifically detect a nucleic acid molecule encoding a envelope protein, or fragments thereof, preferably V3 region fragments; all of which may be used for the purposes of determining coreceptor use.
  • the envelope protein may be gpl60, gp 120, or a portion thereof.
  • heteroduplex tracking assay of the invention can be used to provide both qualitative and quantitative information.
  • qualitative information can be derived using the HTA of the invention by analyzing the whole HIV population derived from an infected patient to determine whether the isolated population of HIV is CCR5-specific, CXCR5 -specific, or mixture of both types. It will be appreciated that qualitative information is based on the whole or substantially the whole HIV population rather than individual clones therefrom.
  • quantitative information can be derived using the HTA of the present invention by analyzing individual HIV clones (e.g.
  • the invention relates to determining the QXR ratio: the number of HIV clones that are identified as CCR5-specific compared to the total number of clones analyzed. It will be appreciated that the HIV clone refers to the cloned PCR product.
  • FIGURE 5 depicts a flow chart showing the qualitative and quantitative aspects of the HTA of the present invention.
  • HIV R A is extracted from the infected patient.
  • RT-PCR is carried out to obtain HIV cD A, from which a PCR product (i.e. PCR amplicon) containing genetic determinates for coreceptor usage is amplied using PCR.
  • the PCR product is then gel purified.
  • the PCR product will be a mixed population of molecules - those genotypic for either CCR5 or CXCR4 coreceptors - whenever the isolated HIV sample contains both types of viruses.
  • the PCR product is analyzed by the HTA of the invention, which includes generally the steps of mixing together a labeled probe (e.g.
  • a PCR product corresponding to same region in a CCR5 strain as the amplified target PCR amplicon to be analyzed and the amplified target PCR amplicon to form homo- or heteroduplexes.
  • the molecules are then separated by gel electrophoresis, for example, on a 12% polyacrylamide gel. Electrophetic techniques are well known in the art. If the QXR ⁇ 1 on the qualitative test, then a quantitative test can be done. To perform the quantitative test the V3 portion of the HIV envelope gene is molecularly cloned and each of 20 clones is analyzed by an individual HTA.
  • FIGURE 6 Exemplarly results are represented in FIGURE 6.
  • the figures shows four panels of schematic electropherograms.
  • the first panel is the negative control, i.e. labeled probe only.
  • the second panel shows the result of HTA of the V3 portion of the HIV envelope gene of a CCR5 virus.
  • the third panel shows the result of HTA of a CXCR4 virus.
  • the fourth panel shows the result of HTA of a mixture of CCR5 and CXCR4 virus V3 regions.
  • Four different probes (each based on a CCR5-specific control virus) were used to test each HIV sample.
  • the gels show heteroduplex band patterns for those HIV samples containing CXCR4-specific and CCR5-specific viruses.
  • the quantitative results of the heteroduplex tracking assay of the method of the present invention may be assessed by statistical methods well known to one of skill in the art.
  • QXR the proportion of plasma HIV-1 using CCR5
  • QXR 1 if all virus identified uses CCR5
  • the association between virologic responses and baseline QXR may be assessed by comparing the percentages of patients with undetectable HIV-1 RNA load across the different strata by using, for example, Fischer's exact test. Further, immunologic responses across two strata may be compared by Wilcoxon rank-sum tests. Kaplan-Meier curves and Cox proportional hazard regression models may be applied to quantify the association of baseline or follow-up QXR (equal 1 versus less than 1) with subsequent clinical progression, defined as a new clinical AIDS-defining event or death.
  • the quantitative results of the heteroduplex tracking assay of the method of the present invention may further be assessed by other statistical methods well known to one of skill in the art.
  • the concurrent log 2 transformed CD4 values and logio transformed HIV-1 loads in the univariable and multivariable Cox models may be included.
  • the inverse probability weights may be used to adjust for sampling bias.
  • STATA Very1, StataCorp, College Station, Texas
  • One of skill in the art e.g. a physician, preferably one specializing in the treatment of infectious disease
  • the frequency of application may vary, depending on various factors, for example, the age, sex, type of antiretroviral therapy administered to, or stage of disease progression in, a patient.
  • the antiretroviral therapy of the method is any suitable antiretroviral treatment regimen. More preferably, the antiretroviral therapy is selected from the group consisting of combination antiretroviral therapy (cART), protease inhibitors, fusion inhibitors, integrase inhibitors, coreceptor specific agents, nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • cART combination antiretroviral therapy
  • protease inhibitors fusion inhibitors, integrase inhibitors, coreceptor specific agents, nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • the nucleoside analogue reverse transcriptase inhibitor may be 3TC or AZT.
  • the nonnucleoside analogue reverse transcriptase inhibitor is nevirapine.
  • Antiretroviral therapy may include, but is not limited to, HAART, protease inhibitors, fusion inhibitors, integrase inhibitors, co-receptor specific agents, 3TC, AZT, nevirapine, non-nucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • HAART can be three or more antiretroviral drugs in combination.
  • the term "HAART" as used herein refers to an combination of highly active antiretroviral agents and usually comprises three drugs.
  • Typical reverse transcriptase inhibitors include nucleoside analogs, such as, but not limited to, (zidovudine, ( AZT, Retrovir), didanosine (ddl, Videx ) sanctionstavudine, (d4T, Zerit ), lamivudine, 3TC, Epivir), abacavir, (ABC, Ziagen), tenofovir, (TDF, Viread), combivir (CBV, combination of AZT and 3TC), and non-nucleoside reverse transcriptase inhibitors, e.g., nevirapine (NVP, Viramune), delavirdine (DLV, rescriptor), efavirenz, ( EFV, sustiva,).
  • nucleoside analogs such as, but not limited to, (zidovudine, ( AZT, Retrovir), didanosine (ddl, Videx ) contradictstavudine, (d4T, Zerit ), lam
  • Protease inhibitors include saquinavir, (SQV, Invirase), ritonavir (RTV, Norvir), indinavir, (IDV, Crixivan), nelfinavir, (NFV,Viracept ), fosamprenivir, FPV, Lexiva), kaletra (lopinavir and ritonavir) and fortovase (saquinavir in a soft gelatin form).
  • SQV Invirase
  • RTV Norvir
  • indinavir IDV, Crixivan
  • NFV nelfinavir
  • NFV nelfinavir
  • fosamprenivir FPV, Lexiva
  • kaletra lopinavir and ritonavir
  • fortovase saquinavir in a soft gelatin form.
  • HAART can also be "triple cocktail" therapy - a three drug regimen to combat HIV .
  • the patient-derived biological sample is any bodily fluid or tissue.
  • the biological sample may be a bodily fluid which may be selected from the group consisting of blood, plasma, and spinal fluid.
  • the biological sample may be one which contains viral populations that are distinct from those in the readily obtained peripheral blood including the reservoirs of the genital tract and lymphoid tissue.
  • Patient-derived biological samples may be obtained by methods known to one of skill in the art. For instance, peripheral blood of HIV-infected individuals can be separated into plasma and cell components by methods known in the art. Primary viral isolates of HIV- 1 may also be obtained by co-culture with normal donor peripheral blood mononuclear cells (PBMCs). Titration of viral isolates in PBMCs can be carried out. These standard techniques are described throughout the literature; for example, see Fang et al (1995) Proc. Natl. Acad. Sci. USA 92: 12110-4.
  • the individual molecular clones each comprise a DNA sequence corresponding to a portion of the HIV genome, the DNA sequence comprising at least a portion of the genetic determinates of coreceptor usage.
  • the genetic determinates are derived from the env gene.
  • the envelope protein may comprise gp 120, gp 160 or a portion thereof. Envelope sequences are predictive of coreceptor use on the basis of the overall charge of the V3 loop and the presence of basic or acidic residues at positions 275 and 287 of the env gene (Bhattacharya et al (1996) AIDS Res. Hum. Retrovir. 12:83-90; and Hung et al (1999) J. Virol. 73:8216-26; and; Cardozo et al (2007) AIDS Res. Hum. Retrovir. 23:415-26). Cloning strategies for isolating envelope genes of interest are well known to one of skill in the art. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning, A Laboratory Manual, 2 nd Ed., Cold Spring Harbor Laboratory Press, 1989.
  • the cloning methods used in the present invention will decrease the chance of sampling error or recombination.
  • high fidelity cloning of the samples above may be achieved by routine performance of multiple long RT-PCR reactions on limiting dilutions of RNA, followed by multiple PCR's on cDNAs obtained from each RT reaction.
  • performance of multiple PCR's on each cDNA preparation increases the likelihood of amplifying a different HIV-1 RNA species.
  • Short-term limited dilution techniques are also well known to one of skill in the art, see for example, Connor et al.
  • RNA is amplified to ⁇ 80 copies/ml.
  • the molecular clones each are derived from RNA of the patient-derived HIV and correspond to the HIV genome or a portion thereof and which comprise the genetic determinates of coreceptor usage or a portion thereof.
  • the molecular clones are prepared by PCR of the RNA of the patient- derived HIV and at least one set of oligonucleotide primers.
  • at least one set of oligonucleotide primers consists of the first set of primers in Table 3.
  • at least one set of oligonucleotide primers includes a second set of oligonucleotide primers, the second set consisting of the second set of primers in Table 3.
  • the number of individual molecular clones is at least 20.
  • the heteroduplex tracking assay of the method may comprise the steps of: (a) amplifying the individual molecular clone or a portion thereof by PCR to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using a separation means; (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals coreceptor usage.
  • the labeled probe may be derived from a HIV-1 CCR5 clone or from a HIV-1 CXCR4 clone.
  • the labeled probe comprises a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a chemiluminescent moiety, or an enzymatic moiety.
  • the heteroduplex tracking assay of the invention can be carried out substantially in accordance with the guidance of Delwart et al (J.Virol. (1994) 68:6672-6683), Delwart et al (Science (1993) 262: 1257-1261), Nelson et al (J.Virol. (1997) 71:8850-8; Delwart et al (PCR Methods and Applications 4:S202-S216 (19950 Cold Springs Harbor), and U.S. Patent 5,851,759 (Weiner), each of which are incorporated in their entireties by reference.
  • the heteroduplex tracking assay can be used to analyze a portion of the HIV- 1 genome encompassing determinates of coreceptor utilization to understand, determine, monitor, or detect coreceptor usage. Genetic determinates of HIV- 1 coreceptor utilization can be found in the envelope gene (env), with key determinates being found in the third variable (V3) domain of the gpl20 glycoprotein.
  • env envelope gene
  • V3 variable domain of the gpl20 glycoprotein
  • the heteroduplex tracking assay of the invention can be carried out generally, while not being limited thereto, according to the basic steps of: (a) obtaining HIV viral RNA from the patient, (b) amplifying, e.g. PCR and/or reverse transcription, a portion of the viral genome containing genetic determinates of coreceptor usage, e.g. a genomic portion comprising the V3 domain of the gpl20 envelope glycoprotein, (c) forming heteroduplexes and/or homoduplexes with labeled nucleic acid-based probes prepared from a corresponding genomic region of a HIV strain, e.g.
  • the same genomic portion comprising the V3 domain of gpl20, and (d) subjecting the heteroduplexes and homoduplexes to a separation system, e.g. electrophoresis through non-denaturing polyacrylamide gels, wherein the heteroduplexes and homoduplexes have differing and distinguishable mobilities that results in different mobility patterns, e.g. a electrophoretic pattern, such that the coreceptor usage can be determined.
  • a separation system e.g. electrophoresis through non-denaturing polyacrylamide gels
  • the presence of an electrophoretic pattern characteristic of X4- heteroduplexes can indicate the presence of CXCR4-specific viruses in the HIV sample.
  • the presence of an electrophoretic pattern characteristic of homoduplexes and R5 -heteroduplexes can indicate the presence of only CCR5-specific viruses.
  • a pattern characteristic of both homoduplexes and X4- and R5 -heteroduplexes can indicate that the HIV sample contains a mixed population of CCR5-specific and CXCR4-specific viruses.
  • the heteroduplex tracking assay can be performed at any point during disease progression or during, before, or after administering antiretroviral therapy. Further, the heteroduplex tracking assay can be carried out either to attain qualitative results or quantitative results.
  • RT-PCR reverse transcription PCR
  • heteroduplex tracking assay is based on the observation that when sequences were amplified by nested PCR from peripheral blood mononuclear cells of infected individuals, related DNA products coamplified from divergent templates could randomly reanneal to form heteroduplexes that migrate with reduced mobility in neutral polyacrylamide gels.
  • a first PCR product as a labeled probe, e.g. radioactive or
  • nonradioactive which is mixed with an excess ("driver") of an unlabeled PCR product from a different source, i.e., the source for which typing or analysis of is desired, e.g. the PCR product defining the portion of the HIV genome with the coreceptor genetic determinates.
  • the probe sequences are then "driven” completely into heteroduplexes with the driver, and are separated, e.g. by gel electrophoresis, on the basis of size.
  • An autoradiogram or fluoroimage, for example, of the resulting polyacrylamide gel reveals these heteroduplexes and provides a visual display of the relationship between the two virus populations under study. The fact that heteroduplexes migrate with distinct mobilities indicates that the strand- specific composition of mismatched and unpaired nucleotides affects their mobility.
  • heteroduplex encompasses a doublestranded DNA molecule having
  • a heteroduplex can form by mixing together a labeled probe (e.g. a double-stranded DNA PCR product of a portion of the e «vgene of CCR5 -specific HIV) and a PCR product of a target sequence (e.g. a double-stranded DNA PCR product of the corresponding portion of the env gene of a CXCR4-specific HIV) such that
  • a labeled probe e.g. a double-stranded DNA PCR product of a portion of the e «vgene of CCR5 -specific HIV
  • a target sequence e.g. a double-stranded DNA PCR product of the corresponding portion of the env gene of a CXCR4-specific HIV
  • the PCR product from the CXCR4-specific HIV will contain genetic determinates characteristic of CXCR4 type viruses, its nucleotide sequence will vary at specific locations with respect to the probe PCR product (which is derived from a CCR5-specific env sequence). These differences in sequence result in a heteroduplex which has reduced mobility during electrophoresis with respect to homoduplexes oweing to a reduced level of base-pairing in the molecule.
  • the "homoduplex" may be formed between complementary strand pairs derived from a probe PCR product and a target PCR product such that their nucleotide sequences are the same.
  • the heteroduplex tracking assay can comprise the steps of (a) amplifying an individual molecular clone or a portion thereof by PCR to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using a separation means: and (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals coreceptor usage.
  • the labeled probe may be derived from a HIV- 1 CCR5 clone.
  • the labeled probe may be derived from a HIV- 1 CXCR4 clone.
  • the labeled probe can comprise a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a
  • chemiluminescent moiety or an enzymatic moiety.
  • Appropriate labels and their methods of preparation are well-known.
  • the diagnostic method may involve the use of microchips comprising nucleic acid molecules encoding a envelope protein, or a fragment thereof, preferably a V3 region fragment, especially including genetic determinates of coreceptor usage, on gene chips; or an envelope protein, or a fragment thereof, preferably a V3 region fragment, on protein-chips ⁇ See U.S. Patent Nos.
  • Diagnostic gene chips may comprise a collection of polypeptides that specifically detect a envelope protein, or fragments thereof, preferably V3 region fragments; or nucleic acid molecules that specifically detect a nucleic acid molecule encoding a envelope protein, or fragments thereof, preferably V3 region fragments; all of which may be used for the purposes of determining coreceptor use.
  • the envelope protein may be gpl60, gp 120, or a portion thereof.
  • heteroduplex tracking assay of the invention can be used to provide both qualitative and quantitative information.
  • qualitative information can be derived using the HTA of the invention by analyzing the whole HIV population derived from an infected patient to determine whether the isolated population of HIV is CCR5 -specific, CXCR5 -specific, or mixture of both types. It will be appreciated that qualitative information is based on the whole or substantially the whole HIV population rather than individual clones therefrom.
  • quantitative information can be derived using the HTA of the present invention by analyzing individual HIV clones (e.g.
  • the invention relates to determining the QXR ratio: the number of HIV clones that are identified as CCR5-specific compared to the total number of clones analyzed. It will be appreciated that the HIV clone refers to the cloned PCR product.
  • FIGURE 5 depicts a flow chart showing the qualitative and quantitative aspects of the HTA of the present invention.
  • HIV RNA is extracted from the infected patient.
  • RT-PCR is carried out to obtain HIV cDNA, from which a PCR product (i.e. PCR amplicon) containing genetic determinates for coreceptor usage is amplied using PCR.
  • the PCR product is then gel purified.
  • the PCR product will be a mixed population of molecules - those genotypic for either CCR5 or CXCR4 coreceptors - whenever the isolated HIV sample contains both types of viruses.
  • the PCR product is analyzed by the HTA of the invention, which includes generally the steps of mixing together a labeled probe (e.g.
  • a PCR product corresponding to same region in a CCR5 strain as the amplified target PCR amplicon to be analyzed and the amplified target PCR amplicon to form homo- or heteroduplexes.
  • the molecules are then separated by gel electrophoresis, for example, on a 12% polyacrylamide gel. Electrophetic techniques are well known in the art. If the QXR ⁇ 1 on the qualitative test, then a quantitative test can be done. To perform the quantitative test the V3 portion of the HIV envelope gene is molecularly cloned and each of 20 clones is analyzed by an individual HTA.
  • FIGURE 6 Exemplarly results are represented in FIGURE 6.
  • the figures shows four panels of schematic electropherograms.
  • the first panel is the negative control, i.e. labeled probe only.
  • the second panel shows the result of HTA of the V3 region of the envelope gene of a CCR5 virus.
  • the third panel shows the result of HTA of the V3 region of the envelope gene of a CXCR4 virus.
  • the fourth panel shows the result of HTA of a mixture of CCR5 and CXCR4 virus V3 regions.
  • Four different probes (each based on a CCR5-specific control virus) were used to test each HIV sample.
  • the gels show heteroduplex band patterns for those HIV samples containing CXCR4-specific and CCR5-specific viruses.
  • the quantitative results of the heteroduplex tracking assay of the method of the present invention may be assessed by statistical methods well known to one of skill in the art.
  • QXR the proportion of plasma HIV-1 using CCR5
  • QXR 1 if all virus identified uses CCR5
  • the association between virologic responses and baseline QXR may be assessed by comparing the percentages of patients with undetectable HIV- 1 RNA load across the different strata by using, for example, Fischer's exact test. Further, immunologic responses across two strata may be compared by Wilcoxon rank-sum tests. Kaplan-Meier curves and Cox proportional hazard regression models may be applied to quantify the association of baseline or follow-up QXR (equal 1 versus less than 1) with subsequent clinical progression, defined as a new clinical AIDS-defining event or death.
  • the quantitative results of the heteroduplex tracking assay of the method of the present invention may further be assessed by other statistical methods well known to one of skill in the art.
  • the concurrent log 2 transformed CD4 values and logio transformed HIV-1 loads in the univariable and multivariable Cox models may be included.
  • the inverse probability weights may be used to adjust for sampling bias.
  • STATA (Version 9.1, StataCorp, College Station, Texas) may be used for quantitative analyses.
  • One of skill in the art e.g. a physician, preferably one specializing in the treatment of infectious disease
  • the frequency of application may vary, depending on various factors, for example, the age, sex, type of antiretroviral therapy administered to, or stage of disease progression in, a patient.
  • the antiretroviral therapy of the method is any suitable antiretroviral treatment regimen. More preferably, the antiretroviral therapy is selected from the group consisting of combination antiretroviral therapy (cART), protease inhibitors, fusion inhibitors, integrase inhibitors, coreceptor specific agents, nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • cART combination antiretroviral therapy
  • protease inhibitors fusion inhibitors, integrase inhibitors, coreceptor specific agents, nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • the nucleoside analogue reverse transcriptase inhibitor may be 3TC or AZT.
  • the nonnucleoside analogue reverse transcriptase inhibitor is nevirapine.
  • Antiretroviral therapy may include, but is not limited to, HAART, protease inhibitors, fusion inhibitors, integrase inhibitors, co-receptor specific agents, 3TC, AZT, FTC, efavirenz, nevirapine, non-nucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • HAART can be three or more antiretroviral drugs in combination.
  • the term "HAART" as used herein refers to a combination of highly active antiretroviral agents and usually comprises three drugs
  • Typical reverse transcriptase inhibitors include nucleoside analogs, such as, but not limited to, zidovudine, ( AZT, Retrovir), didanosine (ddl, Videx ) contradictstavudine, (d4T, Zerit ), lamivudine, 3TC, Epivir), abacavir, (ABC, Ziagen), tenofovir, (TDF, Viread), combivir (CBV, combination of AZT and 3TC), and non-nucleoside reverse transcriptase inhibitors, e.g., nevirapine (NVP, Viramune), delavirdine (DLV, rescriptor), efavirenz, ( EFV, sustiva,).
  • nucleoside analogs such as, but not limited to, zidovudine, ( AZT, Retrovir), didanosine (ddl, Videx ) contradictstavudine, (d4T, Zerit ), lamiv
  • Protease inhibitors include saquinavir, (SQV, Invirase), ritonavir (RTV, Norvir), indinavir, (IDV, Crixivan), nelfinavir, ( FV,Viracept ), fosamprenivir, FPV, Lexiva), kaletra (lopinavir and ritonavir) and fortovase (saquinavir in a soft gelatin form).
  • SQV Invirase
  • RTV Norvir
  • IDV Crixivan
  • nelfinavir FV,Viracept
  • fosamprenivir FPV, Lexiva
  • kaletra lopinavir and ritonavir
  • fortovase saquinavir in a soft gelatin form.
  • HAART can also be "triple cocktail" therapy - a three drug regimen to combat HIV.
  • the present invention further encompasses a diagnostic composition comprised of the methods of the present invention in the form of a kit.
  • the diagnostic composition may comprise the components as defined herein above wherein said components are bound to/attached to and/or linked to a solid support. It is furthermore envisaged, that the diagnostic composition may comprise nucleic acid sequences encoding an envelope protein, or a fragment thereof, preferably a V3 region fragment; or indicator cell lines of this invention; all of which may be contained on micro-chips identifiable with a suitable means for detection.
  • Solid supports are well known in the art and comprise, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubes etc.
  • Suitable methods for fixing/immobilizing cells, nucleic acid sequences, or polypeptides of the invention are well known and include, but are not limited to ionic, hydrophobic, covalent interactions and the like.
  • the diagnostic composition of the present invention may be used as a kit, inter alia, for carrying out the methods of the present invention, for example diagnostic kits or research tools. Additionally, the kit of the invention may contain suitable means for any other scientific, medical and/or diagnostic purposes.
  • Diagnostic compositions and kits of the present invention may be manufactured by standard procedures that are well known to one of skill in the art. Kits may advantageously include instructions for use and/or admixture of ingredients.
  • compositions and kits of the present invention are not limited to use with HIV, but may be used, based on the teachings herein and knowledge of one of skill in the art, to identify and quantitate analogous coreceptors of other lentiviruses, such as SIV and FIV. (See, for example, U.S. Patent Nos. 5,863,542 and 5,766,598).
  • Constant denaturing gel electrophoresis is a method that can separate heteroduplex DNA based on its melting behavior. It can separate heteroduplex DNA not only on the basis of mismatches, small insertions, or deletions, but also on the basis of sequence composition.
  • LNA locked nucleic acids
  • Applicants compared the mobility of each heteroduplex band in the gel relative to that of the single-stranded probe in the same gel lane, and defined Relative Mobility Percentage (RMP) as a gel measurement unit for HIV-1 tropism prediction based on data from HIV-1 strains of known tropism.
  • RMP Relative Mobility Percentage
  • the biological sample is any bodily fluid or tissue.
  • the biological sample is a bodily fluid selected from the group consisting of blood, plasma, and spinal fluid.
  • the individual DNA molecules each comprise a DNA sequence corresponding to a portion of the HIV genome, the DNA sequence comprising at least a portion of the genetic determinates of coreceptor usage.
  • the DNA molecules are derived from RNA of the patient- derived HIV and correspond to the HIV genome or a portion thereof and which comprise the genetic determinates of coreceptor usage or a portion thereof. Even more preferably, the genetic determinates are derived from the env gene.
  • the molecular clones are prepared by RT-PCR of the RNA of the patient-derived HIV and at least one set of oligonucleotide primers.
  • the at least one set of oligonucleotide primers consists of the first set of primers in Table 3.
  • the at least one set of oligonucleotide primers includes a second set of oligonucleotide primers, the second set consisting of the second set of primers in Table 3. It is also preferable that the number of individual molecular clones is at least 40.
  • the heteroduplex tracking assay comprises the steps of: (a) amplifying the individual molecular clone or a portion thereof by PCR to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof; (b) forming a population of heteroduplex molecules by contacting the amplified DNA with a labeled probe complementary to the amplified DNA under conditions sufficient to form heteroduplexes; (c) separating the population of heteroduplex molecules using CDGE; (d) detecting the presence or absence of heteroduplex molecules; wherein the presence or absence of heteroduplex molecules reveals coreceptor usage.
  • the labeled probe is derived from a HIV- 1 CCR5 clone. Even more preferably, the labeled probe is derived from the sequences of the V3 portion of the HIV- 1 env gene.
  • the labeled probe may incorporate one or more locked nucleic acids (LNAs). Locked nucleic acids are reported in the art (see Singh, S.K. et al. Chem. Commun. 4: 455- 456 (1998); Obika, S. et al. Tetrahedron Lett. 39: 5401-5404 (1998); and Koshkin et al, Tetrahedron 54: 3607-3630 (1998)).
  • Locked nucleic acid nucleosides are a class of nucleic acid analogues in which the ribose ring is "locked” by a methylene bridge connecting the 2'-0 atom and the 4'-C atom.
  • LNA nucleosides contain the common nucleobases (T, C, G, A, U) and are able to form base pairs according to standard Watson-Crick base pairing rules. However, by "locking" the molecule with the methylene bridge the LNA is constrained in the ideal conformation for Watson-Crick binding. When incorporated into a DNA oligonucleotide, LNA therefore makes the pairing with a complementary nucleotide strand more rapid and increases the stability of the resulting duplex.
  • the LNA modification confers the largest increase in base pairing stability of any commercially available DNA or RNA analogue: a positive AT m of up to 10 °C has been obtained upon substitution of one LNA nucleotide in DNA, and multiple incorporations give larger effects.
  • Other nucleic acid probe modifications sucgar and/or base
  • sugar and/or base that are known to one of skill in the art may be used in place of or in conjunction with LNAs.
  • the labeled probe preferably comprises a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a chemiluminescent moiety, or an enzymatic moiety. More preferably, the detectable moiety is a fluorescent dye, 6-FAM.
  • electrophoresis is performed using Constant Denaturing Gel Electrophoresis (CDGE).
  • CDGE assay involves using a gel solution that enhances the detection of mutations.
  • electrophoresis is performed using Denatruing Gradient Gel Electrophoresis (DGGE) or Temporal Temperature Gradient Gel Electrophoresis (TTGE).
  • DGGE Denatruing Gradient Gel Electrophoresis
  • TTGE Temporal Temperature Gradient Gel Electrophoresis
  • the method is used (a) to assess or predict the degree of HIV progression, (b) to determine when to start or change antiretroviral treatment, or (c) to monitor the efficacy of antiretroviral treatment.
  • HAART not only reduces the quantity of virus but also affects HIV-1 coreceptor use. Briefly, methods were devised for quantifying the proportion of viruses in patient-derived virus that used each coreceptor and monitoring the effect of combination antiretroviral therapy, particularly HAART, on coreceptor use. The Examples further show that QXR and X4-specific viral load are predictors of disease progression and clinical outcome.
  • HIV-1 RNA in plasma was quantitated by using NucliSens (Organon Teknika Corp., Durham, NC), with a lower limit of quantitation of ⁇ 80 copies/ml.
  • NucliSens Organon Teknika Corp., Durham, NC
  • the CCR5 genotype of each patient was determined as described (Samson et al. (1996)).
  • HIV- 1 Primary isolates of HIV- 1 were obtained by co-culture with normal donor PBMCs. Fang et al. (1995). Viral isolates were titrated in PBMCs (Fang et al. (1995)). Biological clones were derived from primary isolates by short-term limiting dilution cloning (Connor et al. (1997)).
  • HOS-CD4+ cell system Changes in coreceptor use of primary HIV-1 isolates and biological clones obtained from participants in the study over time were followed by using a HOS-CD4+ cell system.
  • the parental HOS-CD4+ line is a human osteogenic sarcoma cell line stably expressing high levels of CD4.
  • HOS-CD4+ cells transfected with genes encoding either CCR5 or CXCR4 in addition to CD4 (cell lines HOS-CD4.CCR5 and HOS-CD4.CXCR4 respectively) served as indicator lines for coreceptor use. Deng et al. (1996).
  • HOS- CD4.CCR5 and HOS-CD4.CXCR4 cells were seeded onto 12-well plates and, after 24 hours, inoculated with a standard quantity of titered virus; 10 2 TCID 50 of first passage primary viral isolates or biological clones were assayed in duplicate.
  • HIV JR-FL and LAV/HTLV-IIIB inoculated in parallel as CCR5- and CXCR4-specific positive control viruses, respectively, and uninoculated cells were used as negative controls.
  • parental HOS-CD4+ cells were also inoculated with duplicate primary and control isolates.
  • Results of the coreceptor use assay were then categorized in a semiquantitative manner according to p24 antigen level as follows: negative (p24 ⁇ 25 pg/ml), +/- (25 - 50 pg/ml), 1+ (50 - 250 pg/ml), 2+ (250 - 500 pg/ml), and 3+ (> 500 pg/ml).
  • SI syncytium- inducing
  • biologic clones which were derived from the patients' primary isolates by performing limiting dilution cultures, were isolated. Coreceptor use was then determined for 25 clones from each isolate by employing the HOS-CD4+ cell system. Biologic clones from these patients used either CCR5 or CXCR4; no dual tropic viruses were detected among the 525 clones by using Applicants' assay system.
  • the distribution of coreceptor use by the clones generally confirmed the semiquantitative results obtained for primary isolates; proportions of HIV-1 using each coreceptor appeared roughly similar whether the cloned virus or primary isolates were examined (Table 2 A, HIV-1 coreceptor use in primary viral isolates and biologic clones).
  • the MT2 assay to detect SI viruses in culture was also performed on primary isolates derived at each time point. These results confirmed the pattern of HIV- 1 coreceptor use described here. Thirteen of the fifteen patients were infected initially with SI virus. In all eleven of those who displayed SI virus and received new combination therapy, the phenotype changed at least transiently to non-syncytia inducing ( SI) after treatment (data not shown). Sequence Analyses of the HIV-1 V3 Loop
  • HIV-1 virions were isolated from plasma samples as described (Fang et al. (1996) J. AIDS Hum. Retro. 12:352-7). Reverse transcriptase polymerase chain reaction amplification produced a 920-bp amplicon spanning the V3 region of the env gene. Reaction conditions were controlled rigorously to minimize recombination and other artifacts (Fang et al. (1996)). Amplified products were cloned into a TOPOTM TA vector (Invitrogen, Carlsbad, CA), verified by restriction digestion, and sequenced. Alignment of the sequences was initially done using the PILEUP program in the GCG Suite (Genetics Computer Group, Madison, WI), then checked manually.
  • Envelope sequences were used to predict coreceptor use on the basis of the overall charge of the V3 loop and the presence of basic or acidic residues at positions 275 and 287 of the env gene (Bhattacharyya et al. (1996); and Hung et al. (1999)).
  • Table 2B Coreceptor use determined by cocultivation of PBMCs vs. sequence analysis of plasma HIV-1 RNA.
  • Table 2B shows a comparison of coreceptor use over time determined by two methods in representative study patients. At each time point, coreceptor use was assayed by co- cultivating PBMCs and determining the V3 loop sequence of virion-derived HIV-1 RNA.
  • the Wilcoxon Rank Sum Test was used to make comparisons between the magnitude of log viral level, CD4+ counts, and QXR values. Data for factors relating to changes in
  • QXR values were analyzed by multivariate Poisson regression. Variables included log HIV- 1 RNA levels, changes in viral levels, CD4+ cell counts, changes in CD4+ cell counts, and
  • D QXR(CCR5)+(1- QXR)(CXCR4); 0 ⁇ _QXR ⁇ 1, where D is the distribution of viral phenotypes. By design, it is a binomial population.
  • QXR values were constructed by relating data derived from the same patient sample by using three different analyses: biologic cloning, V3 sequencing of patient-derived molecular clones, and qualitative assays of primary isolates.
  • To construct QXR values inventors first calculated the proportion of biologic and, if available, molecular clones using CCR5 at each time point, then linked the proportion to the qualitative coreceptor use score (- to 3+) of primary isolates obtained simultaneously. Data that were not available were interpolated. The data were transformed to approximate a Poisson distribution. Poisson regression analysis was then performed to determine the factors associated with changes in QXR values.
  • QXR is a continuous, nonlinear variable between one and zero derived from the results presented here showing coreceptor use by biologically and molecularly cloned virus; it describes the mixed proportion of viruses using CCR5 and CXCR4.
  • a QXR value near one describes a population of viruses that almost all use CCR5; a value near zero describes a population that almost all use CXCR4.
  • Comparison of coreceptor usage in this patient was also performed using a recombinant assay that does not require culturable primary isolates.
  • the results of the recombinant assay were identical to the results obtained using virus derived from the patient's PBMC's.
  • Viral coreceptor usage was separately evaluated through the use of a Rapid Cell
  • This assay enables determination of coreceptor usage from cloned HIV env gene sequences obtained directly from patient samples (e.g. blood, mucosal tissue). This method allows for greater efficiency in determination of viral coreceptor usage, by circumventing the need for cultivation of primary isolates.
  • the Rapid Cell Fusion Assay can advantageously produce a result within one week after obtaining a patient sample.
  • the Rapid Cell Fusion Assay allows study of patient-derived virus obtained from sites other than the peripheral blood, particularly those sites from which cultured virus cannot be obtained. For example, while circulating macrophages and CD4 + T cells are the dominant reservoir of HIV-1, viral populations distinct from those in the peripheral blood exist in many reservoirs, including the genital tract. It is important to study these different reservoirs as HIV-1 viral populations in infected individuals demonstrate marked heterogeneity, with virus varying in the same compartment over time and in different compartments
  • the HL3T1 cell line was derived by stable transfection of parental HeLa cells with a chloramphenicol acetyltransferse (CAT) reporter construct containing a CAT gene is linked to an HIV-1 LTR promoter.
  • the HL3T1 cells produce CAT protein only upon introduction of an active HIV-1 Tat protein.
  • HL3T1 cells were transfected with a cloned env gene derived from a patient of interest. The cloned env gene product is expressed on the surface of the HL3T1 cells.
  • CAT chloramphenicol acetyltransferse
  • Indicator cell lines GHOST.CCR5 and GHOST.CXCR4 (respectively hereinafter "R5-tat” and “X4-tat") cells were transfected with pSV2tat72, a construct expressing high levels of HIV- 1 Tat under the control of the SV40 early promoter.
  • HL3T1 cells containing a cloned patient env gene were fused to R5-tat and X4-tat cells.
  • Cell surface envelope protein variants will selectively interact with either CCR5 or CXCR4. Fusion only occurs when an HL3T1 envelope protein interacts with an indicator cell expressing a compatible coreceptor. Therefore, HL3T1 cells will fuse with either R5-tat and X4-tat, depending on the patient's env gene specificity.
  • transfected HL3T1 and R5-tat or X4-tat cells were mixed in 6-well plates at 37°C and allowed to fuse for 48 hours. To quantitate fusion, the cells were lysed with 0.5% NP-40. Fusion of HL3T1 cells to R5-tat or X4-tat activated CAT gene expression. Aliquots of the cell lysates were monitored for CAT production using a commercially available kit (CAT-ELISA, Boehringer Mannheim).
  • AF2P12-1 CIRPNNNTRTSIRIGPGQAFYATG 11GDIRQAYC CCR5 (SEQ ID NO.1)
  • AF2P12-2 CIRPNNNTRTSIRIGPGQAFYATGN11GGIRQAYC CCR5 (SEQ ID NO.35)
  • AF2P12-4 CIRPNNNTRTSIRIGPGQAFYATGN11GDIRQAYC CCR5 (SEQ ID NO.1)
  • AF2P12-6 CIRPNNNTRTSIRIGPGQAFYATGN11GDIRQAYC CCR5 (SEQ ID NO.1)
  • AF2P12-8 CIRPNNNTRTSIRIGPGQAFYATGN11GDIRQAYC CCR5 (SEQ ID NO.1)
  • AF2P12-9 CIRPNNNTRTSIRIGPGQAFYATGN11GDIRQAYC CCR5 (SEQ ID NO.1)
  • AF2P12-10 CIRPNNNTRTSIRIGPRQAFYATGN11GDIRQAYC CXCR4 (SEQ ID NO.2)
  • AF3P-2 RKSVHIGPGQAFYATGDI IGNIRKAHC negative (SEQ ID NO. .4)
  • AF5P CTRPNNNTRKSVHIGPGQAFYATGDI IGDIRQAYC CCR5 SEQ ID NO.38
  • AF5P CTRPNNNTRKSVHIGPGQAFYATGDI IGDIRQAYC CCR5 SEQ ID NO.39
  • AF5P CTRPNNNTRKSVHIGPGQAFYATGDI IGDIRQAYC CCR5 SEQ ID NO.38
  • AF6P-1 CTRPINNRRKSIHMGPGQAFYGT DDIIGDIRKARC CCR5 (SEQ ID NO.8)
  • Peripheral blood was collected and separate into plasma and cell components.
  • Other fluids and tissues derived from an HIV-infected individual can also be used, with minor modifications to the RNA extraction protocol outlined below.
  • HIV-1 RNA was quantitated in plasma by using NucliSens (Organon Teknika Corp., Durham, NC), with a lower limit of quantitation set at approximately 80 copies/ml.
  • HIV-1 RNA was extracted from plasma using Qiagen's Viral RNA Kit and
  • Samples were standardized by extracting a volume of plasma equal to 10000 copies of HIV-1 RNA. For example, if the patient's plasma viral load is 25000 copies/ml, 0.4 ml of plasma in the extraction should be used.
  • the virus was resuspended in 100 ul of Rnase-free water (to give a final concentration of ⁇ 100 copies of HIV-1 RNA per ul) and optionally treated with Rnase-free Dnase to remove any contaminating DNA.
  • This dilution series is sufficient to ensure minority species amplification.
  • Conditions are adaptable to achieve limiting dilutions.
  • RNA template 1 ul aliquots of RNA template were distributed into the wells of a PE2400 or PE9700 PCR tray -retainer and 8-24 tubes containing of each RNA dilution were prepared.
  • An example of the template set-up for a PE2400 is shown in Figure 3.
  • An RT reaction mix was prepared:
  • HIVGaolF 5'-GGCTTAGGCATCTCCTATGGCAGGAAGAA-3'
  • HIVGaolR 5'-GGCTTAGGCATCTCCTATGGCAGGAAGAA-
  • primer HIVGao2R (20 uM) 1 ul Taq polymerase (50U/ul) 0.5 ul
  • Primer sequences for HIVGao2F and HIVGao2R are:
  • HIVGao2F 5 ' -AGAAAGAGC AGAAGAC AG
  • GGC AATGA-3 ' HIVGao2R: 5'-AGCCCTTCCAGTCCCCCCTTTTCTTTTA-3'
  • Each well of a new PE2400 base received a 98 ul aliquot, followed by 2 ul of each primary PCR reaction serving as a as template for the nested PCR reaction.
  • the same cycle parameters as indicated for the primary PCR were applied.
  • PCR reaction products were purified using Qiagen's Gel Extraction Kit according to the manufacturer's standard protocol.
  • Plasmid DNA was prepared according to standard procedures for ABI sequencing. ABI sequencing of RT-PCR products or clones:
  • the purpose of this procedure is to describe the actions followed when receiving and preparing plasma specimens for HIV-1 coreceptor utilization analysis (QXR).
  • Samples were removed from tubes in a sterile decontaminated hood. If lavender- top tubes of whole blood were sent, it was centrifuged at room temperature for 10 minutes at 1,100 x g (2300 rpm). Tubes were removed from the centrifuge and checked for complete separation. The plasma layer was transferred to freezer vials.
  • the purpose of this procedure is to extract HIV- 1 viral RNA from plasma.
  • the extracted RNA is subsequently used for analysis of HIV- 1 coreceptor utilization.
  • ADL Lysis Buffer
  • AW1 Wash Buffer 1
  • AW2 Wash Buffer 2
  • Samples that may contain cells should first be centrifuged for 10 minutes at 2,000 rpm, and only the clarified supernatant used.
  • RNA samples with HIV-1 RNA loads ⁇ 1.0 x 10 5 copies/ml were pelleted by centrifuging the tubes for 90 minutes at 10,000 x g at 4°C. All tubes to be used were labeled with the correct identifiers. 560 ⁇ of Lysis Buffer (AVL) was pipetted into an appropriately labeled 1.5-ml screw-cap tube, then 140 ⁇ plasma was added and mixed by pulse-vortexing for 15 seconds. Samples were lysed for at least 10 minutes at room temperature (although samples may be lysed for up to 24 hours at room temperature or 7 days at 4°C without significant effect on the yield or quality of the purified RNA).
  • ADL Lysis Buffer
  • the 1.5-ml screw-cap tubes were briefly centrifuged (2-3 seconds at 8,000 rpm) to remove drops from the inside of the lid. 560 ⁇ of absolute ethanol was added and mixed by pulse-vortexing for 15 seconds. The 1.5-ml screw-cap tubes were briefly centrifuged (2-3 seconds at 8,000 rpm) to remove drops from the inside of the lid. 630 ⁇ of the solution was carefully applied to an appropriately labeled QIAampTM spin column. The sample or solution from the lysis tube was then carefully applied to the column or tube by pipetting the sample into the tube without wetting the rim or outside of the column. Tubes were centrifuged for 60 seconds at 6,000 x g. The QIAampTM spin columns were transferred into clean 2-ml collection tubes.
  • the supernatant-containing collection tubes were discarded into a waste bucket.
  • the remaining 630 ⁇ of the solution was carefully applied, without wetting the rim or outside of the column, to an appropriately labeled QIAampTM spin column.
  • the tubes were centrifuged for 60 seconds at 6,000 x g, and the QIAampTM spin columns were transferred into clean 2-ml collection tubes.
  • the supernatant-containing collection tubes were discarded into a waste bucket.
  • the QIAampTM spin columns were opened carefully and 500 ⁇ of Wash Buffer 1 (AW1) was added. Tubes were centrifuged for 60 seconds at 6,000 x g.
  • the QIAampTM spin columns were transferred into clean 2-ml collection tubes.
  • the supernatant-containing collection tubes were discarded into a waste bucket.
  • the QIAampTM spin columns were carefully opened and 500 ⁇ of Wash Buffer 2 (AW2) was added. Tubes were centrifuged for 3 minutes at 20,000x g. The supernatant was aspirated from the collection tubes using either a transfer pipettes or vacuum with trap. The pipette or tip was changed after each aspiration. Tubes were centrifuged for 60 seconds at 20,000x g to eliminate any chance of wash buffer carryover. The QIAampTM spin columns were transferred into clean 1.5 ml microcentrifuge tubes. The supernatant-containing collection tubes were discarded into a waste bucket. 60 ⁇ of Elution Buffer (AVE) was added to each column. The pipette tip was changed for each tube.
  • AVE Elution Buffer
  • the columns were incubated for 60 seconds at room temperature, followed by centrifugation for 60 seconds at 6,000 x g. 10 ⁇ of eluted ribonucleic acid was transferred into a new 1.5-ml screw-cap tube for coreceptor utilization analysis. The remaining viral RNA (-48-50 ⁇ ) was transferred into another 1.5-ml screw-cap tube for long-term storage at ⁇ -70°C.
  • RT Reverse Transcription
  • PCR Polymerase Chain Reaction
  • HIV-1 Human Immunodeficiency Virus type 1
  • RNA PCR core Kit reagents were thawed to room temperature, except for enzymes, which were removed from freezer only when needed. Reagents were mixed by vortexing and then microcentrifuged briefly before placing tubes in an ice bucket. A sterile 1.5 ml microcentrifuge tube was placed in the ice bucket.
  • Enough RT master mix was prepared to accommodate the number of planned reactions plus one (to accommodate pipetting eror), based on the following amounts of reagents per reaction: 2 ⁇ 10X RT-PCR Buffer II, 4 ⁇ 25 mM MgCl, 2 ⁇ 10 mM dCTP, 2 ⁇ 10 mM dGTP, 2 ⁇ 10 mM dTTP, 2 ⁇ 10 mM dATP, 1 ⁇ 50 ⁇ Random Hexamers, 1 ⁇ , RNase Inhibitor (20 U/ ⁇ ), 1 ⁇ , MuLV RT (50 U/ ⁇ ). Master mix and retainer assembly was transferred to a sterile laminar flow hood.
  • RNA Template Addition 2 ⁇ 10X RT-PCR Buffer II, 4 ⁇ 25 mM MgCl, 2 ⁇ 10 mM dCTP, 2 ⁇ 10 mM dGTP, 2 ⁇ 10 mM dTTP, 2 ⁇ 10 mM dATP, 1
  • MicroAmp reaction tubes were labeled and placed in retainer/tray assembly.
  • RT master mix was mixed by gently pipetting up and down a few times. 17 ⁇ of master mix was pipetted into each of the reaction tubes. 3 ⁇ 1 of viral RNA extracted from patient samples was added.
  • One extraction positive control (HIV-1 LAV) and one extraction negative control (Sera Care Plasma) were included with each RT-PCR run. Tubes were capped with cap strips and retainer/tray assembly was removed from the laminar flow hood and transferred to thermocycler.
  • the RT reaction mixtures were incubated at 25°C for 10 minutes, then at 42°C for 60 minutes followed by heat inactivation at 95°C for 5 minutes.
  • the completed RT reaction can be stored at 4°C (short-term) or -20°C (long-term) until ready for cDNA amplification.
  • RNA PCR Core Kit reagenets were thawed at room temperature, except for enzymes, which were removed from freezer only when needed. Reagents were mixed by vortexing and then briefly microcentrifuged and placed in an ice bucket. A sterile 1.5 ml microcentrifuge tube was placed in the ice bucket.
  • Enough cDNA amplification/primary PCR master mix was prepared to accommodate the number of planned reactions plus one (to accommodate pipetting error), based on the following amounts of reagents per reaction: 8 ⁇ ⁇ 10X PCR buffer II, 2 25 mM MgCI 2 , 1 ⁇ , of each primer (25 ⁇ ) (Table 3), 67.5 ⁇ , sterile water, and 0.5 ⁇ ⁇ Taq polymerise (5U ⁇ L). Primary PCR master mix and retainer assembly containing completed RT reactions were transferred to sterile laminar flow hood in template addition area.
  • PCR master mix was mixed by gently gently pipetting up and down a few times. 80 ⁇ ⁇ of master mix was overlayed into each of the RT reaction tubes, giving a total reaction volume of 100 ⁇ ⁇ . Tubes were capped with cap strips and retainer/tray assembly was removed from laminar flow hood and transferred to a thermocycler, which was programmed for cDNA amplification as follows: PCR mixtures were pre-incubated at 94°C for 5 minutes, followed by 35 cycles of three-step incubations at 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 1 minute, followed by a 5 minute incubation at 72°C. The completed primary PCR reaction was stored at 4°C (short-term) or -20°C (long-term) until ready for nested amplification. Secondary/Nested PCR Master Mix Preparation:
  • RNA PCR Core Kit reagents were thawed at room temperature, except for enzymes, which were removed from freezer only when needed. Reagents were vortexed to mix and then briefly microcentrifuged and placed in an ice bucket. A sterile 1.5 ml microcentrifuge tube was placed in the ice bucket. Enough cDNA amplification/primary PCR master mix was prepared to accommodate the number of planned reactions plus one (to accommodate pipetting error), based on the following amounts of reagents per reaction: 10 ⁇ . 10X PCR Buffer II, 6 ⁇ .
  • MicroAmp reaction tubes were labeled and placed in retainer/tray assembly.
  • PCR master mix was mixed by gently pipetting up and down a few times. 98 ⁇ ⁇ of master mix was added into each of the reaction tubes. 2 ⁇ . of the primary PCR reaction was added to corresponding secondary PCR reaction tube for a total volume of ⁇ ⁇ . Tubes were capped with cap strips and the retainer/tray assembly was removed from the laminar flow hood and transferred to a thermocycler which was programmed for cDNA amplification as follows: re-incubated at 94°C for 5 minutes, followed by 35 cycles of three- step incubations at 94°C for 15 seconds, 55°C for 30 seconds, 72°C for 1 minute, followed by a 5 minute incubation at 72°C. The completed primary PCR reaction was stored at 4°C (short-term) or -20°C (long-term).
  • 6X gel-loading buffer was prepared as follows: 0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol, and water up to desired final volume. A stock solution can be prepared and stored at room temperature. 20 ⁇ , of 6x gel-loading buffer was added to each secondary/nested PCR reaction tube. Samples were mixed by pipetting up and down.
  • 5X-TBE buffer was diluted to 0.5X with distilled water. Ethidium bromide was added to a final concentration of 0.5 ⁇ g/ml.
  • a 4% (w/v) GTG NuSieve agarose solution was prepared by adding 6g agarose to 150ml 0.5x TBE/EtBr in a 250 ml glass Erlenmeyer flask. The agarose/TBE solution was gently mixed for 10 minutes at room temperature (to allow the agarose to hydrate), followed by heating in the microwave at 40% power for 10 minutes, mixing occasionally, until all agarose is completely dissolved. The dissolved agarose solution was gently cooled under cold running water and then poured into a previously set-up gel tray (with appropriate size gel comb), while making sure to minimize bubbles. The agarose was allowed to completely solidify for approximately 30-60 minutes at room temperature.
  • the comb was gently removed and the gel apparatus was prepared to receive running buffer.
  • 0.5x TBE buffer containing 0.5 ⁇ g/ ⁇ ethidium bromide, was slowly poured into the electrophoresis rig until the gel was completely submerged. ⁇ of the 100-bp DNA ladder was loaded into the first well of the agarose gel.
  • the gel was visualized using the preparative setting on the UV transilluminator.
  • Each sample band was cut out of the gel with a clean razor blade or scalpel and place in a pre- weighed 1.5 ml microcentrifuge tube.
  • the band was cut as close to its edges as possible, in preparation for the QIAquick separation kit which allows for a maximum of 400 ⁇ g of agarose. Blades were changed between bands to avoid sample cross-contamination.
  • Amplified DNA was extracted from each agarose slice using Qiagen's QIAquick separation protocol (e.g. Qiagen's QIAquick Gel Extraction Kit Protocol (03/2001 Handbook)).
  • Qiagen's QIAquick separation protocol e.g. Qiagen's QIAquick Gel Extraction Kit Protocol (03/2001 Handbook)
  • Purified DNA was analyzed spectrophotometrically and was adjusted to -25-50 ng/ ⁇ . Approximately 90 ⁇ ⁇ DNA was used for the subsequent coreceptor analysis procedures. The purified DNA was transferred to sterile 1.5 ml screw-cap tubes and was either stored at 4°C (short-term) or -20°C (long-term) until ready for HTA analysis or TOPO TA cloning.
  • the purpose of this procedure was to amplify a portion of the envelope gene of Human Immunodeficiency Virus type 1 (HIV-1), using cloned plasmid DNA.
  • Fluorescent- labeled PCR primers were used to generate fluorescein-conjugated DNA probes. The resulting probes were subsequently used for qualitative and quantitative analysis of HIV- 1 coreceptor utilization.
  • Two sets of fluorescently-labeled primers were used to generate fluorescein-conjugated DNA probes, with the forward primer of each pair covalently linked at the 5 ' end to fluorescein. For primers see Table 4.
  • V3-7232R 5'- TGC TCT ACT AAT GTT ACA ATG TGC TTG TCT TAT-3'
  • V3HTA-EcoRI-R 5'- AAT TCG CCC TTT GCT CTA CTA ATG -3'
  • RNA PCR Core Kit reagents were thawed at room temperature, except for enzymes, which were removed from freezer only when needed. Reagents were vortexed to mix and then briefly microcentrifuged and placed in an ice bucket. A sterile 1.5 ml microcentrifuge tube was placed in the ice bucket.
  • Enough cDNA amplification/primary PCR master mix was prepared to accommodate the number of planned reactions plus one (to accommodate pipeting error), based on the amounts of reagents per reaction as follows: 10 ⁇ L ⁇ 10X PCR buffer II, 6 ⁇ L ⁇ 25 mM MgCI 2 , 4 ⁇ L ⁇ 10 mM dNTP blend, 1 ⁇ L ⁇ of each primer to make probe (at 25 ⁇ ) ⁇ 4), 76.5 ⁇ ⁇ sterile water, 0.5 ⁇ ⁇ Taq polymerase (5 ⁇ / ⁇ ). At least four reactions were planned (one for each probe). A negative control containing sterile water instead of plasmid DNA was also prepared. This "qualitative" PCR master mix and retainer tray assembly were transferred to a sterile laminar flow hood in the template addition area.
  • RNA PCR Core Kit reagents were thawed at room temperature, except for enzymes, which were removed from freezer only when needed. Reagents were vortexed to mix and then briefly microcentrifuged and placed in an ice bucket. A sterile 1.5 ml microcentrifuge tube was placed in the ice bucket.
  • Enough cDNA amplification/primary PCR master mix was prepared to accommodate the number of planned reactions plus one (to accommodate pipeting error), based on the amounts of reagents per reaction as follows: 10 ⁇ L ⁇ 10X PCR buffer II, 6 ⁇ L ⁇ 25 mM MgCI 2 , 4 ⁇ L ⁇ 10 mM dNTP blend, 1 ⁇ L ⁇ of each primer to make probe (at 25 ⁇ ) ⁇ 4), 76.5 ⁇ ⁇ sterile water, 0.5 ⁇ ⁇ Taq polymerase (51 ⁇ / ⁇ ). At least four reactions were planned (one for each probe). A negative control containing sterile water instead of plasmid DNA was also prepared. This "quantitative" PCR master mix and retainer tray assembly were transferred to a sterile laminar flow hood in the template addition area.
  • MicroAmp reaction tubes were labeled and placed in retainer/tray assembly. Each of the "qualitative" and “quantitative” PCR master mixes were mixed by gently pipetting up and down a few times. 99 ⁇ ⁇ of each master mix were added into reaction tubes. 1 ⁇ ⁇ of each plasmid DNA template (SFi 6 2, JR-CSF, Sw54, and Sw87; derived from primary HIV-1 strains of the same name) was added to corresponding PCR reaction tube for a total volume of ⁇ .
  • SFi 6 2, JR-CSF, Sw54, and Sw87 derived from primary HIV-1 strains of the same name
  • Tubes were capped with cap strips and retainer/tray assembly was removed from laminar flow hood and transferred to thermocycler, which was programmed for cDNA amplification as follows: pre-incubation at 94°C for 5 minutes, followed by 35 cycles of three-step incubations at 94°C for 15 seconds, 55°C for 30 seconds, 72°C for 1 minute, followed by a 5 minute incubation at 72°C.
  • the completed PCR reaction can be stored at 4°C (short-term) or -20°C (long-term) until ready for cDNA amplification.
  • PCR products were analyzed and gel-purified on a 4% agarose gel as described above.
  • HTA Heteroduplex Tracking Assay
  • This assay uses a heteroduplex tracking (HTA) technique to analyze a portion of the Human Immunodeficiency Virus type 1 (HIV-1) envelope gene encompassing the key determinates of coreceptor utilization. Sequence difference between CCR5- and CXCR4- using variants result in distinct heteroduplex electrophoretic mobilities that allow the overall number and relative proportion of distinct variants to be estimated, even in samples consisting of heterogeneous CCR5 and CXCR4 pools. Plasma specimens showing heteroduplex patterns indicative of CXCR4 strains are then subjected to further analysis to quantitate the portion of CCR5 and CXCR4 viruses in the patient quasispecies. Interpretation of the gels is based on the banding pattern seen in each gel lane.
  • HTA heteroduplex tracking
  • 12% acrylamide solution was prepared to accommodate the number of planned gels, based on the following amounts of reagents per 75 mL gel: 22.5 mL 40% (29: 1) acrylamide/bis-acrylamide stock solution, 36.9 mL deionized water, 15 mL 5x Tris-Borate- EDTA (TBE) stock buffer, 52.5 TEMED, 525 ⁇ 10% AMPS, freshly prepared in deionized water.
  • TBE Tris-Borate- EDTA
  • the reservoirs of the electrophoresis tank were filled with lx TBE (made with 1 part 5x TBE and 4 parts deionized water).
  • probe and target DNA were thawed at room temperature. Probe and target DNA were vortexed to mix and then microcentrifuged briefly and placed in an ice bucket. Between two and four sterile 1.5 ml microcentrifuge tubes were placed in the ice bucket (one tube per probe). Enough HTA annealing mix was prepared to accommodate the number of planned reactions plus one (to accommodate pipeting error), based on the following amounts of reagents per reaction: 3 ⁇ ⁇ ⁇ HTA annealing buffer, 5 ⁇ L FITC- labeled probe, 2 ⁇ sterile water. MicroAmp reaction tubes were labeled and placed in retainer/tray assembly.
  • HTA annealing mix was mixed by gently pipeting up and down a few times. 10 ⁇ ⁇ of master mix were aliquotted into each of the reaction tubes. 20 ⁇ ⁇ of viral DNA derived from viral RNA from patient samples was then added. There were two to four reactions for each patient sample - one for each probe used. One positive control (the purified HIV- 1 LAV extraction control) and one negative control (water only) were included in each run. These controls were also used to determine the amount of homoduplex and heteroduplex DNA present in each experiment. Tubes were capped with cap strips and the retainer/tray assembly was removed from the laminar flow hood and transferred to a thermocycler. The HTA annealing reaction was run for 2 minutes at 94° C, followed by quenching to 4°C (short-term). The resulting reactions were placed on ice and immediately loaded on a 12% non-denaturing polyacrylamide gel.
  • 6X gel-loading buffer was prepared by combining: 0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol, water up to desired final volume. A stock solution may be prepared and stored at room temperature. 6 ⁇ ⁇ of 6x gel-loading buffer was added to each HTA annealing reaction tube and was mixed by pipeting up and down. Using a sequencing gel loading tip, the entire HTA annealing reaction was gently loaded into the polyacrylamide gel wells.
  • the electrodes were connected to the power supply and the gel was run at a constant voltage until the last of the upper xylene cyanol dye front runs off the bottom of the gel (approximately 6 hours at 250V or overnight at 90V), or until the polyacrylamide gel marker dyes had migrated the desired distance.
  • the Fluorlmager 595 controls were adjusted to the following settings: 1) single label dye; 2) 488 nm excitation; 3) no emission filter; 4) no calibration; 5) 1000V PMT; 6) high sensitivity; 7) 200 ⁇ pixels; and 8) 16-bit resolution.
  • ImageQuaNTTM software package was used to display the gel image, once the scan was complete.
  • HIV- 1 R A isolated and amplified from patient plasma was cloned into a plasmid vector (pCR ® 2.1-TOPO ® , Invitrogen), used to transform chemically competent Escherichia coli, and plated onto selective bacterial media.
  • pCR ® 2.1-TOPO ® Invitrogen
  • the following reagents per reaction were gently mixed and incubated for 5 minutes at room temperature and then placed on ice: 4 ⁇ extracted DNA amplicon/sterile water, 1 ⁇ salt solution, and 1 ⁇ ⁇ TOPOTM vector.
  • Enough OneShot ® E.coli cells were thawed on ice to accommodate the number of planned cloning reactions.
  • 2 ⁇ of the TOPO ® cloning reaction was added to a vial of OneShot ® E.coli and mixed gently using a pipette tip. E. coli was incubated on ice for 30 minutes and heat-shocked for 30 seconds at 42°C. 250 xL of room temperature SOC medium was added to the cells.
  • HIV-1 RNA isolated and amplified from patient plasma was cloned into a plasmid vector, grown overnight in 1-3 mL of Escherichia coli bacterial culture, and purified using a commercially available plasmid miniprep kit (Perfectprep®, Eppendorf, Westbury, NY). Analysis for the viral specific sequences was carried out by digestion of the recombinant plasmid with restriction enzyme EcoRI (20 U/ ⁇ ).
  • a sterile 1.5 ml microcentrifuge tube was also placed in the ice bucket. Digests were performed in duplicate (one digest for agarose gel analysis and one digest for quantitative HTA analysis). Tubes were capped with strips and transferred to the thermocycler, which was programmed for EcoRI digestions as follows: 37°C for 37 minutes followed by 95°C for 1 minute. The completed restriction enzyme digests can be stored at 4°C (short-term) or - 20°C (long-term) until ready for gel and HTA analysis.
  • This assay uses the heteroduplex tracking (HTA) technique described in Example 1 to analyze a portion of the Human Immunodeficiency Virus type 1 (HIV-1) envelope gene encompassing the key determinates of coreceptor utilization.
  • HTA Heduplex tracking
  • Individual clones from patient plasma specimens which showed heteroduplex patterns indicative of CXCR4 strains were subjected to analysis to accurately quantitate the portion of CCR5 and CXCR4 viruses in the patient quasispecies.
  • DNA heteroduplex tracking analysis was performed with the coreceptor utilization profile of a minimum of twenty positive transformants from each patient sample determined by using two probes to screen each clone. Probes were prepared from one laboratory CCR5 isolate (SF162 or JR-CSF) and one primary CCR5 isolate (Sw54 or Sw87).
  • FIG. 6 is a schematic representation of HTA analysis of four different targets: probe only, a CCR5 virus V3 region, a CXCR4 virus V3 region, and mixed quasispecies containing both CCR5 and CXCR4 virus V3 regions.
  • a common problem is low or no target DNA yield following PCR, reflecting either PCR efficiency or sample preparation problems.
  • This problem was alleviated in part by use of a commercially available RNA extraction kit (Qiagen Viral RNA Kit), and in part by use of a small amount of pooled HIV- 1 LAV, which is always simultaneously extracted as a positive RNA control. This practice is part of the standard operating procedure.
  • Primers used herein were designed to match the clade B consensus sequence as posted on the Los Alamos National Laboratories HIV database. Using this primer set, inventors currently have a success rate of 98.4% in amplifying envelope sequences from patient samples with a viral load of at least 1000 copies per milliliter of plasma.
  • Inventors ran a series of HTA' s using different amounts of input template and multiple parallel amplifications to prove that inventors can consistently amplify all of the majority and minority variants in a patient sample.
  • Three levels of sequence difference between target DNA mixtures were selected to span the diversity found in the HIV-1 envelope gene.
  • Duplicate 10-fold serial dilutions of viral RNA were then amplified by PCR and analyzed by HTA using Applicants' various probes.
  • HTA The main advantage of HTA is its ability to simultaneously analyze multiple genetic variants coamplified by PCR. Using optimized reaction conditions, HTA's can be used to detect variants that represent less than 1% of the total quasispecies population (5). The ability of the coreceptor-specific HTA to detect rare variants has been examined by reconstituting mixtures of virus using laboratory isolates with known coreceptor usage. The sensitivity of the HTA method to detect R5 and X4 isolates was independently ascertained by using reconstituted samples with QXR values at or near 0 and 1 , respectively. These experiments have demonstrated that inventors can routinely and reproducibly detect CCR5 and CXCR4 variants that represent as little as 0.2% of the total viral population.
  • Example 8 HIV-1 coreceptor usage and CXCR4-specific viral load predict clinical disease progression
  • the purpose of this example is to show the relationship of HIV- 1 coreceptor usage to clinical endpoints, and in particular the identification of patients at high risk for AIDS or death before or during cART.
  • the SHCS is a prospective, clinic-based, observational study of HIV-1 -infected adults initiated in 1988, with documentation of follow-up visits every six months (Ledergerber et al. (1994)). A subset of patients were selected from 2674 who initiated cART between 1995 and 1998 and who were described in Applicants' previous report on clinical progression and persistent viremia (Ledergerber et al. (1999)). The study was approved by Institutional Review Boards at each site and each patient signed informed consent. Selection of study subjects and samples
  • inventors identified the 170 patients who subsequently progressed to a new clinical AIDS-defining event or death while receiving cART. To qualify for the present study, patients needed sufficient plasma available from the SHCS visit preceding the initiation of cART, called baseline, and an HIV-1 load >1000 copies/mL at that visit. The median interval between the initiation of cART and the baseline visit was 18 days
  • inventors identified pre- and post-cART aliquots from 91 patients who did not progress within the period of the original study (up to December 31, 1998) and who were matched to progressors according to the clinic site and year cART was initiated. With the requirement for one aliquot remaining in stock, 4 specimens lost to handling, and Applicants' inability to amplify from 7, inventors quantified coreceptor usage in 84 baseline and 31 follow-up samples from non-progressors. In total, inventors analysed 180 baseline and 70 follow-up samples.
  • CD4 lymphocyte counts were measured by using flow cytometry and HIV-1 R A levels, by using the Cobas Amplicor test, with a level of detection of 500 copies/mL (Roche Diagnostics, Rotnch, Switzerland) (Ledergerber et al. (1999)).
  • Inventors quantified the proportion of HIV- 1 variants using R5 or X4 in each plasma sample by employing a non-radioactive, DNA heteroduplex tracking assay (HTA) developed based upon previous methods (Delwart et al. (1997) Methods 12:348-54); and Nelson et al. (1997) J. Virol. 71:8750-8).
  • HTA DNA heteroduplex tracking assay
  • X4 variants ordinarily coexist in a viral swarm along with R5, (Berger et al. (1998); Shankarappa et al. (1999); Scarlatti et al. (1997); Koot et al. (1993); and Connor et al. (1997)), it was necessary to quantify the proportion of viruses in plasma using each coreceptor.
  • QXR Quantity of X4 and R5
  • V3 variable domain of the envelope gene
  • inventors developed a nucleic acid-based assay focusing on this region of the HIV-1 genome.
  • Viral RNA was extracted from patient samples by using a QIAamp viral RNA extraction kit (Qiagen, Valencia, CA), with samples from different patients processed separately to minimise possible cross-contamination or mislabeling.
  • RT-PCR Reverse transcription and PCR amplification
  • DNA heteroduplex formation was carried out by annealing fluorescein-labeled probes derived from four CCR5-using HIV-1 strains with a 10-fold excess of unlabeled target DNA. Sequence differences between envelope variants resulted in distinct heteroduplex
  • HTA HTA to characterize -400 biologic and molecular HIV-1 clones of known coreceptor specificity.
  • the predictive value of the HTA method for detecting R5 and X4 strains was 100% and 98.7 %, respectively.
  • the sensitivity of the HTA method also allows rare variants to be detected and quantified; HIV-1 subpopulations that represent as little as 1% of the total quasispecies be can readily identified (Delwart et al. (1997).
  • Those samples harbouring X4 strains (QXR ⁇ 1) were subjected to more detailed analysis, during which V3 loops were cloned and individually analysed by using HTA.
  • inventors After determining the coreceptor usage of each clone, inventors then calculated QXR for each plasma specimen by applying a mathematical model derived previously (Philpott et al. (2001). The X4-specific HIV-1 load was calculated by multiplying the total viral load by the proportion of the viral population using
  • X4-specific viral load (total HIV-1 load) (1-QXR) Analyses of these and other plasma samples demonstrated that inventorswere capable of determining HIV-1 coreceptor usage in 97% of samples with HIV-1 RNA loads >1000 copies/mL and 85% of those with viral loads ⁇ 1000 copies/mL.
  • Virologic responses were measured in terms of the percentage of patients with HIV-1 RNA ⁇ 500 copies/mL six months after initiating cART.
  • inventors determined the change in CD4 counts between values obtained at baseline and those obtained at the visit closest to six months.
  • QXR the proportion of plasma HIV-1 using CCR5
  • the association between virologic responses and baseline QXR was assessed by comparing the percentages of patients with undetectable HIV-1 RNA load across the different strata by using Fisher's exact test. Immunologic responses across two strata were compared by Wilcoxon rank-sum tests.
  • Kaplan-Meier curves and Cox proportional hazard regression models were applied to quantify the association of baseline or follow-up QXR (equal 1 vs. less than 1) with subsequent clinical progression, defined as a new clinical AIDS-defming event or death.
  • inventors included an additional model analysing the relationship of X4 viral load to HIV- 1 disease progression by stratifying X4-specific viral load into three strata:
  • Table 5 Characteristics of 180 patients at initiation of cART (baseline).
  • IQR Interquartile range. ⁇ Stratification according to median of 61 values with non-zero values of X4-specific viral load.
  • QXR ⁇ 1 signifying a mixture of R5 and X4 variants
  • HIV-1 RNA viral loads at 6 months were available for 162/180 patients with baseline QXR values.
  • CD4 cell counts at 6 months available for 157/180 patients with baseline QXR values and for 58/70 with follow-up QXR values.
  • Table 7 Univariable and multivariable Cox proportional hazard regression models of time from starting cART to new clinical AIDS defining illness or death by using baseline QXR or baseline X4-specific load together with CD4 cell counts and viral load as predictors..
  • HR Hazard ratio
  • CI Confidence interval Multivariable model includes baseline QXR and is adjusted for X4-specific and total viral load as well as CD4 ⁇ cell count.
  • ⁇ Multivariable model includes baseline X4-specific viral load, stratified according to the median of 61 non-zero QXR values, and is adjusted for QXR, total viral load, and CD4 ⁇ cell count.
  • the adjusted multivariable hazard ratio (HR) for clinical progression was 4.8 (95% CI: 2.3-10.0) for QXR ⁇ 1 at baseline.
  • X4-specific HIV-1 load was a similarly independent predictor, with HRs of 3.7 (1.2- 1 1.3) for baseline X4-specific viral loads of 2.2-4.3 logio copies/mL and 5.9 (2.2-15.0) for X4 loads >4.3 logio copies/mL.
  • This example identifies HIV- 1 coreceptor usage as a powerful predictor of response to cART.
  • Patients harbouring X4 variants not only exhibited a diminished immunologic response compared to those without X4 strains, but also displayed a markedly increased risk of progressing to AIDS or death despite treatment.
  • the increased probability of clinical progression was observed in patients who displayed QXR ⁇ 1 before initiating cART and in those with persistent viraemia and QXR ⁇ 1 after 6 months of therapy.
  • QXR and X4-specific viral load identifies a subset of individuals at increased risk of clinical progression, they promise to be useful in clinical management.
  • the quantification of QXR and X4-specific load may inform the decision to begin cART in untreated patients. It would be of interest to consider a clinical trial evaluating the initiation of cART in asymptomatic individuals with QXR ⁇ 1, even those with CD4 counts >350 cells/uL.
  • the aim of initiating cART in such patients would be to shift the predominant viral population from X4 to R5 (Philpott et al. (2001); Equils et al. (2000); and Skrabal et al. (2003)) as well as to reduce HIV-1 levels and thereby slow disease progression.
  • NIH www.aidsreagent.org
  • Table 8 HIV-1 coreceptor usage determined by phenotypic assay, heteroduplex tracking assay and V3 sequence analysis
  • HTA heteroduplex tracking assay
  • R5 HIV-1 strain using the CCR5 coreceptor
  • X4 HIV-1 strain using the CXCR4 coreceptor
  • NSI nonsyncytium inducing
  • SI syncytium inducing
  • NA not available.
  • °MT-2 cells were employed as an indicator cell line for the production of syncytia in the presence of certain strains of HIV-1 .
  • Syncytia induction during HIV-1 infection has a high correlation with X4 coreceptor usage; non-syncytia induction correlates strongly with R5 usage [www.aidsreagent.org].
  • dMagi cells expressing a modified CCR5 or CXCR4 gene served as indicator cell lines for HIV-1 coreceptor usage [43].
  • eHOS-CD4+ cells transfected with genes encoding either CCR5 or CXCR4 in addition to CD4 were employed as indicator lines for coreceptor usage [28].
  • V3 nucleotide sequences (GenBank accession nos. EF688428-EF688458 for Wadsworth Center sequences), including a BLAST search and construction of a phylogenetic tree, indicated that the sequences are unique, and there was no evidence of contamination. V3 amino acid sequences were deduced and the HIV-1 coreceptor usage for each strain was predicted on the basis of the V3 loop's overall charge and the presence of basic residues at positions 1 1, 24, and 25.
  • V3 sequences in this panel were aligned and compared with the R5 reference strain SF 162 (Table 1), revealing that X4 sequences often displayed amino acid insertions and disparities as compared to most of the R5 sequences. This observation supports the idea that identification of tropism by HTA rests largely on the disparity between genomes coding for R5 vs. X4 usage rather than upon differences among individual R5 or X4 sequences themselves. Because all four probes in this assay are R5-specific, annealing of an X4-specific V3 genome to these probes may result in DNA mismatches, unpaired loops of DNA, and slower electrophoretic migration.
  • Statistical analyses of the sequence prediction method based upon the 46 strains demonstrating sole usage of either R5 or X4 coreceptor, indicated that for X4 strains, the sequence prediction method's sensitivity was 100%, specificity 85%, PPV 67%, and NPV 100%. For R5 strains, the sensitivity of the sequence prediction was 85%, specificity 100%, PPV 100%, and NPV 67%.
  • the coreceptor usage was predicted from population-based sequences and indicated the tropism of the predominant strains; it therefore did not predict mixed coreceptor usage.
  • the HTA is capable of detecting both predominant and rare variants, reflecting the actual molecular quasispecies. Because population-based sequences identify the predominant base at each position, they may fail to reflect sequences of nucleotides that are linked on the same molecule of HIV- 1 RNA; this phenomenon has been demonstrated by studies of drug resistance mutations in individual viral variants.
  • HTA was used to analyse two additional sets of patient-derived variants. First, Applicants characterized -400 biologic and molecular HIV-1 clones of known coreceptor specificity from 15 patients. The PPV of the HTA for detecting R5 and X4 strains was 100% and 98.1%, respectively. The HTA also confirmed the phenotypic results obtained by
  • a new approach to HTA, described in this Example, is focused on methods to slow the electrophoretic mobility of HIV-1 strains using the X4 coreceptor, relative to those that use R5, in order to amplify the separation of coreceptor-specific strains and facilitate their identification.
  • the modifications apply to all X4 strains, but particularly those that do not have significant insertions or deletions in their V3 sequence compared to R5 strains.
  • CDGE Constant denaturing gel electrophoresis
  • the separation principle of CDGE is based on the melting behavior of DNA molecules.
  • double-stranded DNA is subjected to conditions that will melt the DNA in discrete segments called melting domains.
  • the melting temperature (Tm) of each domain is sequence-specific.
  • the Tm usually depends upon a specific nucleotide sequence composition. Mismatches, insertions, and deletions, however, will result in large decrements in Tm.
  • Tm of the lowest melting domain is reached, the DNA will become partially melted, creating branched molecules. Partial melting of the DNA reduces its mobility in a polyacrylamide gel. Since the Tm of a particular melting domain is sequence-specific, the presence of a mutation will alter the melting profile of that DNA when compared to the wild type. Thus, different DNA sequences containing different mutations will exhibit different mobilities compared to the wild-type DNA.
  • heteroduplex DNA was formed after hybridization of the fluorescent probe with the target V3 DNA obtained from a patient sample.
  • the electrophoresis was performed on a native polyacrylamide gel.
  • CDGE actually amplifies the sequence difference under the conditions of the denatured gel matrix.
  • the heteroduplex V3 DNAs of R5 and X4 viruses will display either a distinct number of melting domains, or the Tm of their melting domains will differ.
  • electrophoresis of heteroduplex DNA under CDGE increases the separation of DNA molecules with different mutations.
  • the CDGE method provides a more sensitive way to detect X4 virus sequences.
  • a 30 to 50bp GC rich sequence is added to the 5 'end of one of the PCR primers, co-amplified, and thus introduced into the amplified DNA fragments.
  • the GC-rich sequence acts as a high-melting domain which not only prevents the two DNA strands from complete dissociation into single strands but also increases the number of melting domains to be analyzed (Glavac and Dean, 1995).
  • the application of the GC clamp greatly increases the detection sensitivity and resolution; the disadvantage of GC clamp, however, is that it decreases the efficiency and specificity of PCR. It also is impractical to add a 30 to 50bp GC rich sequence to Applicants' V3 2 nd PCR, which only has about 120bp. Applicants therefore applied a novel technique to avoid these pitfalls. Locked nucleic acids
  • LNA Locked nucleic acids
  • LNA is a bicyclic ribose derivative with a bridging methylene group between 2'- O and 4'- C.
  • LNAs are a promising tool for increasing oligonucleotide hybridization strength and specificity (Michael and Jesper, 2003).
  • the LNA modification confers the largest increase in base pairing stability of any commercially available DNA or RNA analogue: a positive AT m of up to 10 °C has been obtained upon substitution of one LNA nucleotide in DNA, and multiple incorporations give larger effects.
  • Mutation Detection Enhancement (MDE®) Gel Solution is a commercially available polyacrylamide-like matrix (Lonza, Allendale,NJ)that has a high sensitivity to DNA conformational differences.This gel's unique structure causes DNA separation to occur on the basis of both size and conformation. MDE® Gel Solution's formulation enables the detection of more mutations by traditional heteroduplex and SSCP analysis than detection using standard polyacrylamide. In order to increase the sensitivity of detecting X4 sequences, Applicants used MDE® Gel in Applicants' modified CDGE assay for HIV-1 coreceptor usage. Improvement in probe design and preparation
  • the probe is still the key for an assay's specificity and sensitivity.
  • Applicants designed a new probe and improved the method of probe preparation. The improvements are described here:
  • the sequence of the new probe is a consensus sequence derived from the sequences of the V3 portion of the HIV-1 env gene of all the R5-specific sequences downloaded from the Los Alamos database and the R5 -specific sequences studied in Applicants' lab.
  • the variants included in this consensus have been identified as using the R5 receptor only.
  • Cloning vector StrataClone PCR Cloning Kit (Stratagene) was used for cloning, with pSC-A-amp/kan as the cloning vector and StrataClone SoloPack Competent Cells as the bacteria used for transformation.
  • the size of the recombinant plasmid is ⁇ 4.4kb.
  • a 138 bp DNA fragment was custom-synthesized to encompass the following: 1) an HIV-1 env V3 consensus sequence encoding CCR5 tropism and 2) flanking sequences for primer binding. The fragment was cloned into the pSC-A-amp/kan vector, and the clone is ampicillin and kanamycin resistant ⁇ see Figure 13).
  • HTAs of HIV- 1 strains previously characterized for tropism by cell-based phenotypic assays were performed by using both the improved HTA-CDGE and the original HTA method, denoted here as HTA-QXR ( Figures 8-12). Lanes depicting virus strains identified as using R5 are labeled in red; virus strains that use X4 are seen in lanes numbered in blue.
  • the improved HTA-CDGE used a new consensus R5 probe described above although the original HTA-QXR method used the R5 strain HIV- 1 JR-CSF as probe. The viruses used in each gel are indicated on the Figures.
  • Figures 11 and 12 illustrate that for the pairs of R5 and X4 variants derived from the same patient that Applicants examined, the improved HTA- CDGE was much more effective than the original HTA-QXR method in distinguishing the X4 from the R5 strains.
  • RMP Relative Mobility Percentage
  • MD-a is the migration distance of heteroduplex band A
  • MD-b is the migration distance of heteroduplex band B
  • MDP is the migration distance of single-stranded probe
  • Table 9 displays results of analyses of data from both HTA-CDGE and the original HTA-QXR by using RMP (Relative Mobility Percentage) measurements.
  • RMP Relative Mobility Percentage
  • the HTA-CDGE results in a broader span of mobilities than the HTA-QXR [the RMP's of HTA-CDGE range from 51.33 (ELI) to 158.18(Ba-L) and those of HTA-QXR from 87.64 (92HT599) to 138.60(JRFL)]. It was also noticed that in the HTA-QXR, there are overlaps in RMP between some R5 and X4 viruses, which could be the main reason for Applicants' previous inability to detect some X4 viruses such as HIV-1 MN. There were no overlaps between X4 and R5 strains when the HTA-CDGE procedure was used.
  • the novel CDGE-HTA method has a high specificity in HIV- 1 tropism detection. It has the potential to be a rapid, automated, economic, highly sensitive and specific genotypic test for HIV- 1 tropism, and promises to be useful in clinical management.
  • a diagnostic method comprising determining the viral load of a population of acquired immunodeficiency (AIDS) virus using the CXCR4 coreceptor (X4-specific viral load) in a patient-derived biological sample comprising the steps of:
  • initiation or change of antiretroviral therapy may be considered anytime that the X4- specific viral load is greater than zero.
  • viruses in a population are a mixture of those that use the R5 and X4 coreceptors.
  • the biological sample is a bodily fluid selected from the group consisting of blood, plasma, and spinal fluid.
  • the at least one set of oligonucleotide primers includes a second set of oligonucleotide primers, the second set consisting of the second set of primers in Table 3.
  • heteroduplex tracking assay comprises the steps of:
  • the labeled probe comprises a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a chemiluminescent moiety, or an enzymatic moiety.
  • a method of determining when to initiate antiretroviral therapy in a patient comprising determining the viral load of a population of AIDS virus using the CXCR4 coreceptor (X4-specific viral load) in a patient-derived biological sample comprising the steps of:
  • viruses in a population are a mixture of those that use the R5 and X4 coreceptors.
  • the biological sample is a bodily fluid selected from the group consisting of blood, plasma, and spinal fluid.
  • the at least one set of oligonucleotide primers includes a second set of oligonucleotide primers, the second set consisting of the second set of primers in Table 3.
  • heteroduplex tracking assay comprises the steps of:
  • the labeled probe comprises a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a
  • chemiluminescent moiety or an enzymatic moiety.
  • antiretroviral therapy is selected from the group consisting of combination antiretroviral therapy (cART), protease inhibitors, fusion inhibitors, integrase inhibitors, coreceptor specific agents, nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • cART combination antiretroviral therapy
  • protease inhibitors fusion inhibitors
  • integrase inhibitors integrase inhibitors
  • coreceptor specific agents nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • nucleoside analogue reverse transcriptase inhibitor is 3TC.
  • nucleoside analogue reverse transcriptase inhibitor is AZT.
  • a method of monitoring the efficacy of antiretroviral therapy in a patient comprising determining the viral load of a population of AIDS virus using the CXCR4 coreceptor (X4- specific viral load) in a patient-derived biological sample comprising the steps of:
  • viruses in a population are a mixture of those that use the R5 and X4 coreceptors.
  • the biological sample is a bodily fluid, such as blood, plasma, and spinal fluid.
  • the at least one set of oligonucleotide primers includes a second set of oligonucleotide primers, the second set consisting of the second set of primers in Table 3.
  • heteroduplex tracking assay comprises the steps of: (a) amplifying the individual molecular clone or a portion thereof by PCR to provide amplified DNA comprising the genetic determinates of coreceptor usage or a portion thereof;
  • the labeled probe comprises a detectable moiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, a
  • chemiluminescent moiety or an enzymatic moiety.
  • antiretroviral therapy is selected from the group consisting of combination antiretroviral therapy (cART), protease inhibitors, fusion inhibitors, integrase inhibitors, coreceptor specific agents, nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • cART combination antiretroviral therapy
  • protease inhibitors fusion inhibitors
  • integrase inhibitors integrase inhibitors
  • coreceptor specific agents nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
  • nucleoside analogue reverse transcriptase inhibitor is 3TC.
  • nucleoside analogue reverse transcriptase inhibitor is AZT.
  • nonnucleoside analogue reverse transcriptase inhibitor is nevirapine.
  • a diagnostic method for determining the viral load of a population of acquired immunodeficiency virus using the CXCR4 coreceptor (X4-specific viral load) in a patient- derived biological sample is provided.
  • a diagnostic method comprising determining the viral load of a population of acquired immunodeficiency (AIDS) virus using the CXCR4 coreceptor (X4-specific viral load) in a patient-derived biological sample comprising the steps of: (a) screening individual molecular clones of patient-derived acquired immunodeficiency primary isolate with a V3 loop sequencing assay to determine CCR5 coreceptor usage and CXCR4 coreceptor usage of each individual molecular clone;
  • a diagnostic method comprising determining the viral load of a population of acquired immunodeficiency (AIDS) virus using the CXCR4 coreceptor (X4-specific viral load) in a patient-derived biological sample comprising the steps of:
  • CDGE constant denaturing gel electrophoresis
  • the biological sample is a bodily fluid selected from the group consisting of blood, plasma, and spinal fluid.
  • the individual molecular clones each comprise a DNA sequence corresponding to a portion of the HIV genome, the DNA sequence comprising at least a portion of the genetic determinates of coreceptor usage.
  • the at least one set of oligonucleotide primers includes a second set of oligonucleotide primers, the second set consisting of the second set of primers in Table 3.
  • heteroduplex tracking assay comprises the steps of:

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Abstract

La présente invention concerne une méthode de diagnostic permettant d'effectuer le suivi de l'utilisation des corécepteurs pendant le traitement et la prise en charge clinique d'une infection par le virus de l'immunodéficience humaine (VIH). La présente invention concerne en outre une méthode de diagnostic appliquée à des individus positifs au VIH recevant un traitement antirétroviral hautement actif (TAHA) pour effectuer le suivi de la suppression des souches spécifiques de CCR5 ou de CXCR4. Les méthodes de diagnostic peuvent être utilisées pour permettre de décider de l'opportunité du début du TAHA, du type d'agents à sélectionner, et permettre l'amélioration des prévisions de pronostic de la maladie au cours du temps. Les méthodes de l'invention sont des méthodes à base de cellules, notamment des tests de fusion cellulaire, et des méthodes à base de molécules, notamment un test des échantillons de VIH prélevés chez le patient à des fins d'utilisation des corécepteurs. Cette invention concerne en outre une nouvelle méthode améliorée de diagnostic par CDGE-HTA pour détecter des souches de VIH-I qui présentent une sensibilité et une spécificité élevées de détection du tropisme du VIH.
PCT/US2010/048604 2009-09-11 2010-09-13 Détection de souches x4 du vih-1 par test de suivi des hétéroduplexes Ceased WO2011032078A1 (fr)

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