WO2009114083A2 - Determinants of antiviral response - Google Patents

Description

DETERMINANTS OF ANTIVIRAL RESPONSE

Field of the Invention

The present invention relates to antiretroviral drug susceptibility tests that can be used to identify HIV-I -infected patients whose HIV-I virus will respond to treatment with maturation inhibitors. The invention further relates to novel vectors, host cells and compositions for carrying out these novel antiviral drug susceptibility tests. This invention also relates to the screening of candidate drugs for their capacity to inhibit cleavage of the HIV-I Gag polyprotein at the CA- SPl cleavage site.

Related References U.S. Patent Applications 10/766,528; 10/851,637; 11/597,431 ; and published

International Appl. No. WO 2005/113059 refer to compositions and methods useful in the inhibition of HIV-I replication by disruption of the processing of the viral capsid-spacer peptide 1 protein. Each of these applications disclose mutations selected for in vitro and engineered mutations within the capsid ("CA") to spacer peptide 1 ("SPl") region of Gag.

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus- 1 (HIV-I) is a retrovirus that infects and invades cells of the immune system; it breaks down the body's immune system and renders the patient susceptible to opportunistic infections and neoplasms. The immune defect appears to be progressive and irreversible, with a high mortality rate that approaches 100% over several years. Though combination antiretroviral therapy has improved the prognosis of HIV-I infected patients, the increasing number of primary infections that are resistant to marketed antiretroviral therapies has become a significant clinical challenge in many regions, including the USA and Western Europe.

Drugs acting by novel mechanisms of action are desperately needed to treat patients harboring viruses that are not sensitive to currently approved drugs. One such class of novel mechanism drugs is maturation inhibitors. Three maturation inhibitors have been used in the clinical setting, including: bevirimat (also known as 3-<3-(3',3'-Dimethylsuccinyl)-betulinic acid, or DSB, or BVM, bevirimat dimeglumine, or MPC-4326), PA-040, and MPC-9055. Many other maturation inhibitors with the same mechanism of action have been profiled in preclinical settings including 3-O-(3',3'-Dimethylsuccinyl)betulinic acid-N-(2-carboxybenzyl) amide, 3-0- (3',3'-Dimethylsuccinyl)betulinic acid-N-(4-methoxyphenethyl) amide) and 3-O-(3',3'-- Dimethylsuccinyl)betulinic acid-N-(3-pyridinyl) amide) and other compounds mentioned in USPN 5,679,828; USPN 6,172,110; USPA 10/870,555; 10/949,875; USPA 11/272,019; and USPA 1 1/134,904.

HIV-I maturation inhibitors exhibit a distinct mode and site of action relative to other FDA approved antiretrovirals and have exhibited reduced potential for cross-resistance to other HIV-I pharmaceuticals. Maturation inhibitors prevent the cleavage of capsid protein ("CA" or "p24") from its precursor ("CA-SPl" or "p25") thereby preventing the capsid from condensing into the conical shape critical for infectivity; the resultant virions are immature and noninfectious.

The nonconserved nature of the HIV-I genome and the associated high mutation rate in HIV-I replication pose a challenge to the successful treatment of HIV-I -infected patients for two primary reasons: (1) HIV-I mutates to become drug resistant; and (2) the viruses that exist both within a particular patient and across the patient population contain genetic polymorphisms. U.S. patent applications 10/766,528, 10/851,637, and 11/597,431 address the effect of in vitro resistance-selected mutations and deliberately engineered mutations in HIV-I on the in vitro sensitivity of the virus to maturation inhibitors.

Amino acid residues in the N-terminal half of SPl appear to be involved in bevirimat activity, whereas residues in the C-terminal half of SPl may be less significant to antiviral response profiles.

Due in part to the conserved nature of the CA-SPl junction region, the development of resistance to bevirimat in vitro requires multiple passages and often yields viral mutants exhibiting reduced replication capacity or viral fitness. Mutations conferring resistance to bevirimat map to amino acid residues at, or near, the CA-SPl cleavage site. Numerous independent rounds of serial drug passages have consistently identified six mutations that are capable of conferring resistance to bevirimat in vitro. All six of these mutations involve substitutions within 5 amino acids of the CA-SPl cleavage site, with three found in the C- terminus of CA (H358Y, L363F, L363M) and three found in the N-terminus of SPl (A364V, A366T, and A366V). None of these mutations have been reported as resistance-conferring or compensatory mutations for any other class of antiviral pharmaceuticals, including protease inhibitors. Single point deletion scanning of CA-SPl indicated that residues 364, 365 and 366 are critical for virus replication or production; residues 367, 368, 369, 370 and 371 as capable of allowing some virus replication or production, but have an effect on bevirimat susceptibility; and residues 372, 373, 374, 375 and 376 as less relevant to virus replication or production. See, for example WO 2005/113059. Considering the value that a novel class of antiretrovirals could provide to HIV- I/AIDS patients, a method of differentiating HIV-I strains that are likely to respond in vivo to treatment with a maturation inhibitor, including bevirimat, from HIV-I strains that are not likely to respond in vivo to treatment with a maturation inhibitor would satisfy a critical need and represent a significant advance in the art. Such a method of differentiating would ideally include a rapid, sensitive genotypic or phenotypic assay to measure the susceptibility of HIV-I to a maturation inhibitor.

Similarly, a method of identifying maturation inhibitor drug candidates having activity against a broad population of viral genotypes or against HIV-I that has reduced susceptibility to existing maturation inhibitors would represent a significant advance in the design and discovery of next generation maturation inhibitors.

BRIEF SUMMARY OF THE INVENTION

In order to maximize the efficacy of maturation inhibitor therapy, and to assist physicians in choosing the appropriate treatment regimen for a given HIV-infected patient, accurate determination of the susceptibility of a patient's viral strains toward a maturation inhibitor such as bevirimat. Such information is needed to inform decisions about appropriate treatment options.

Of the clinical phase maturation inhibitors, bevirimat is presently being studied in Phase lib clinical trials. Analysis of the viral load reductions observed in clinical trial participants receiving bevirimat revealed that patient sensitivity to bevirimat was generally bi-modal, with patients being classified as either "responders" or "non-responders". "Responders" are defined as patients who show at least a 0.5 log 10 viral load reduction following short-term (e.g. 7-21 days) monotherapy or functional monotherapy with a maturation inhibitor; and "non-responders" are defined as patients who show less than a 0.5 log 10 viral load reduction after this period of monotherapy or functional monotherapy. Monotherapy is the treatment of a patient with a single anti-retroviral drug, whereas functional monotherapy is the treatment of a patient with an antiretroviral drug in combination with a pre-existing regimen of antiretroviral drugs that have lost their anti-HIV activity due to development of drug resistance. A-

Until the aforementioned dichotomy in clinical response was observed, the effect of naturally occurring polymorphisms on patient response to bevirimat was not appreciated.

As used herein, "MOI" means multiplicity of invention or the ratio of number of infectious particles to a known number of cells. It has been discovered that the susceptibility of HIV-I to a maturation inhibitor, including

3-(9-(3',3'-dimethylsuccinyl)-betulinic acid, is affected by polymorphic determinants of antiviral activity. These polymorphic determinants of antiviral activity differ from drug-induced resistance mutations, since viruses comprising these polymorphic determinants of antiviral activity remain susceptible to maturation inhibitor treatment in vitro under certain circumstances as demonstrated herein, whereas viruses comprising drug-induced resistance mutations are not susceptible to maturation inhibitor treatment under these circumstances. Moreover, none of these polymorphic determinants of antiviral activity were detected in repeated independent in vitro resistance selection experiments.

Viruses comprising one or more of these polymorphic determinants of antiviral activity, which were taken from patients who did not respond to maturation inhibitor treatment, were grown in vitro and were treated with a maturation inhibitor. It was found that at low MOI, the replication of these viruses was inhibited by a maturation inhibitor in vitro, despite the fact that they were obtained from a patient who did not respond to maturation inhibitor treatment. However at high MOI, the EC50 of the maturation inhibitor exhibited a 10, 100 or 1,000 fold increase compared to the EC50 for that virus under low MOI conditions, reflecting the lack of response in the patient from whom the virus was obtained. Viruses without the polymorphisms were generally susceptible under both low and high MOI conditions, while viruses with resistance mutations were generally not susceptible under both conditions.

Using the sequences, methods, and kits of the invention will allow for better tailoring of HIV-I therapy to patients. In this way, patients will be treated in accordance with whether they are infected with HIV-I that is more susceptible, or less susceptible, to maturation inhibitors. For patients infected with HIV-I carrying these polymorphism(s), combination therapy of maturation inhibitors with other classes of antiretrovirals is expected to result in effective treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the effect of virus titration on BVM sensitivity in NL4.3 HIV-I. In this in vitro assay designed to measure spreading HIV-I replication, wild-type NL4.3 virus is readily inhibited by bevirimat even at high levels of virus input (e.g. 1 :2 dilution of virus), or high multiplicity of infection. This is apparent from the low level of virus infection in the presence of bevirimat (+ BVM, dark bars, Fig. 1), relative to the high levels of virus infection in the absence of bevirimat (- BVM, light grey bars, Fig. 1). Figure 2 depicts the effect of virus titration on BVM sensitivity with A364V resistance mutation engineered into the NL4.3 HIV-I backbone. The previously reported bevirimat- resistance mutation, A364V, which had been identified in previous in vitro resistance selection experiments, shows high levels of virus infection in the presence of bevirimat (+ BVM, dark bars, Fig. 2) as compared to the absence of bevirimat (- BVM, light brey bars, Fig. 2). At every virus dilution there was less than a two-fold reduction in virus infection in the presence of bevirimat as compared to the level of replication in the absence of bevirimat. In fact, at a 1 :2048 dilution, it appeared as though the virus actually replicated better in the presence of compound than in the absence of compound. Therefore, with this mutation, virus is resistant to bevirimat inhibition regardless of the dilution of virus supernatant (or MOI). Figure 3 depicts the effect of virus titration on DSB sensitivity with V370A resistance mutation engineered into the NL4.3 HIV-I backbone. A chimeric viral genome was constructed in which an approximately 500 bp fragment of the gag gene encoding the CA-SPl region from a virus isolated from an HIV-I -positive volunteer that responded poorly to bevirimat treatment was substituted into an otherwise wild-type NL4.3 HIV-I backbone. The only change from wild-type within six residues upstream of the CA-SPl cleavage site and eight residues downstream of the CA-SPl cleavage site was the V370A polymorphism. Unlike the wild-type and A364V viruses, this chimeric virus showed an MOI-dependent sensitivity to bevirimat. At high levels of virus (high MOI, e.g. dilutions of 1 :2 or 1 :8, Fig. 3), the virus appeared to be resistant to inhibition by bevirimat (i.e. no significant difference in the level of virus infection in the presence or absence of compound). However, at low levels of virus input (low MOI, e.g. dilutions from 1 :512 to 1:32768), the virus was inhibited quite effectively by bevirimat (note reduction in virus infection in the presence of bevirimat [dark bars, Fig. 3] as compared to the absence of bevirimat [light grey bars, Fig. 3]. Similar results have also been seen when the V370A mutation was introduced into NL4.3 by site-directed mutagenesis (data not shown). Therefore, the polymorphism V370A appears to render virus susceptible to bevirimat in an MOI-dependent manner in in vitro infection assays.

Figure 4 depicts polymorphisms identified in the Los Alamos Database. Figure 5 depicts a summary of PA-457 activity on point deletion mutants.

Figure 6 depicts the amino acid residues surrounding the CA-SPl cleavage site in the GAG polyprotein that have been found to be determinants of bevirimat response in clade B HIV- 1. Figure 7 depicts the relative in vitro sensitivities of clade B HIV-I patient isolates to bevirimat.

DETAILED DESCRIPTION OF THE INVENTION

Some embodiments of the present invention relate to sequences comprising polymorphic determinants of antiviral activity, and methods for evaluating the susceptibility of HIV-I to maturation inhibitor therapy. In particular, polymorphic determinants of antiviral activity in the HIV-I CA-SPl region and their effect on clinical efficacy of maturation inhibitors are determined. The methods rely on providing a patient HIV-I sample and evaluating the predicted efficacy of maturation inhibitor therapy through genotyping the virus, phenotyping the virus, or a combination thereof. Such methods are useful in multiple fields including diagnostics, drug screening, pharmacogenetics and drug development.

Numerical references to specific amino acid residues herein refer to the Gag sequence of HIV-I HXB2CG sequence, though, in the context of polymorphisms, it is understood that the corresponding polymorphisms in any HIV-I strain, including viral strains isolated from patients, are envisioned by the present invention, as amino acid differences outside the region of the CA- SP 1 cleavage site between various HIV-I strains should not influence the polymorphisms and methods described herein. This can be seen by the fact that each polymorphism had the same effect in vivo in the original patient strain in which each was found as it had in vitro when transferred to a different genetic backbone. Thus, the polymorphism(s), when located in any HIV-I strain, such as clinically-isolated strains and strains commonly used in the laboratory or otherwise known or engineered, will have a similar effect on clinical antiviral activity of maturation inhibitors. For example, such polymorphisms may be introduced into other HIV-I strains including the IIIB consensus or NL4.3 viral sequence. In some circumstances the numbering of the amino acid residues may differ from that of HXB2CG but will be readily identified by comparing a sequence of multiple amino acids, preferably at least 10, to identify regions of homology with the HXB2CG sequence. In some embodiments, Clade B HIV-I strains are useful in accordance with the present invention. As used herein, a "polymorphic determinant of antiviral activity" is one or more change, deletion or insertion with respect to an HIV-I HXB2CG sequence at a residue selected from the group consisting of 369, 370, 371 and 380. In some embodiments, the polymorphic determinant of antiviral activity is selected from the group consisting of Q369H, V370A, V370M, T371A, T371S, Q369H + V370A, Q369H + V370M, Δ370 + T371A, and R380K. In some embodiments, the polymorphic determinant of antiviral activity is Q369H. In some embodiments, the polymorphic determinant of antiviral activity is V370A. In some embodiments, the polymorphic determinant of antiviral activity is V370M. In some embodiments, the polymorphic determinant of antiviral activity is T371A. In some embodiments, the polymorphic determinant of antiviral activity is T371S. In some embodiments, the polymorphic determinants of antiviral activity are Q369H in conjunction with V370A. In some embodiments, the polymorphic determinants of antiviral activity are Q369H in conjunction with V370M. In some embodiments, the polymorphic determinants of antiviral activity are Δ370 in conjunction with T371 A. In some embodiments, the polymorphic determinant of antiviral activity is located at residue R380K. Polymorphic determinants of antiviral activity located at residues 369, 370, and 371 are associated with reduced sensitivity to maturation inhibitors including bevirimat. The polymorphic determinant of antiviral activity located at residue 380 is associated with increased sensitivity to maturation inhibitors including bevirimat.

Blood plasma concentrations of maturation inhibitors are important in patient response if the levels are below a minimum effective concentration. For bevirimat, this concentration appears to be about 20 μg/ml. Plasma concentrations used herein refer to the trough concentration of a maturation inhibitor at steady state, i.e. the minimum plasma concentration on any day after sufficient days of dosing have taken place that the pharmacokinetic profile is similar on consecutive days. In some embodiments, steady state concentrations are reached after about 5 days of maturation inhibitor therapy. In some embodiments, steady state concentrations are reached after about 7 days of maturation inhibitor therapy. In some embodiments, steady state concentrations are reached after about 10 days of maturation inhibitor therapy. Patients with bevirimat plasma concentrations below the minimum effective concentration generally do not respond to bevirimat, or respond with a level of response that is less than about a 1.0 loglO reduction in viral load after short term monotherapy or functional monotherapy. Patients with bevirimat plasma concentrations above about 20 μg/ml and who do not have a polymorphic determinant of activity at 369, 370, or 371 respond to the drug usually have a response close to or greater than about a 1.0 log 10 reduction in viral load after short term monotherapy or functional monotherapy. Further increases in plasma concentration above about 20 μg/ml do not usually appear to affect the level of response.

For patients exhibiting bevirimat trough plasma concentrations greater than about 20 μg/ml, generally it is not bevirimat plasma concentrations that limit response, rather it is the presence or absence of polymorphisms in Gag that determine whether a patient responds to maturation inhibitor treatment. For example a patient with one or more of polymorphisms present at residues 369, 370, and 371 are generally associated with a lack of response to the maturation inhibitor. Polymorphisms at residue 380, when present, are generally associated with patient response to the maturation inhibitor; as such the MOI-effect described herein does not relate to polymorphisms at residue 380.

Without being bound by theory, it is believed that the ratio of free (not protein-bound) bevirimat to Gag in vivo is low relative to the same ratio in a low-MOI infection assay in vitro. The ratio in vivo may be similar to that at a high-MOI infection assay in vitro. This is supported by the fact that the high-MOI conditions in the in vitro infection assay described above appear to be more predictive of patient response than the low-MOI conditions.

Without being bound by theory, it is believed that polymorphic determinants of antiviral activity, including but not limited to V370A, induce an MOI-dependent effect on clinical response to a maturation inhibitor through one or both of the following mechanisms. First, rather than directly affecting the binding of bevirimat to Gag, these polymorphic determinants of antiviral activity might rather increase the rate of cleavage of CA from CA-SPl by HIV-I protease. Such an effect on the cleavage rate could, for example, result from a higher intrinsic rate of enzymatic cleavage, higher affinity binding to the site by protease, greater accessibility of the site to protease, or a combination of any of the foregoing. In a situation where the concentration of free drug is much higher than the concentration of Gag, as in a low-MOI in vitro assay, the maturation inhibitor might be able to overcome this increased rate of cleavage by more effectively competing with the protease for binding to the cleavage site. Second, these polymorphic determinants of antiviral activity could improve the replication fitness of the virus such that it can tolerate a lower level or less efficient or slower rate of cleavage of CA from CA- SPl caused by the presence of a maturation inhibitor. Again, this effect might be overcome by the maturation inhibitor under low MOI conditions. However, under the higher MOI conditions used in the high-MOI in vitro assay, or in the in vivo clinical studies, these mechanisms could reduce the antiviral activity of a maturation inhibitor.

Some embodiments of the present invention provide a method for measuring the susceptibility of HIV-I to a maturation inhibitor. In some embodiments, the method is an assay for determining the susceptibility of an HIV-I viral strain present in a patient to a maturation inhibitor comprising analyzing HIV-I of a patient to determine whether the HIV-I is expected to be susceptible to the maturation inhibitor. In some embodiments, the method is an assay for determining the susceptibility of an HIV-I infected patient to a maturation inhibitor comprising analyzing HIV-I of a patient to determine whether the HIV-I is expected to be susceptible to the maturation inhibitor. In some embodiments the assay is a genotypic assay. In some embodiments the assay is a phenotypic assay. In some embodiments, the assay is a combination of a genotypic assay and a phenotypic assay.

Genotyping

Generally, genotyping is the process of elucidating the genotype of a subject with a biological assay. Also known as a genotypic assay, techniques include PCR, RT-PCR, DNA fragment analysis, sequencing, and nucleic acid hybridization to microarrays or beads. Some common genotyping techniques useful in accordance with the present invention include Restriction Fragment Length Polymorphism (RFLP), Terminal Restriction Fragment Length Polymorphism (t-RFLP), Amplified Fragment Length Polymorphisms (AFLP), Branched-DNA Signal Amplification, and Multiplex Ligation-dependent Probe Amplification (MLPA).

Some embodiments of the present invention provide a method of determining if an individual is infected with HIV-I that is susceptible to treatment by a maturation inhibitor. In one embodiment, the method comprises taking a sample from the patient, reverse transcribing the viral RNA to DNA, genotyping the viral DNA and determining whether the viral DNA contains polymorphic determinants of antiviral activity in the sequence encoding the region within 18 residues of the CA-SPl cleavage site. In some embodiments, the method further comprises amplifying the viral DNA.

In some embodiments, the genotyping comprises: a) obtaining nucleic acid from HIV-I derived from a patient sample; b) analyzing the patient HIV-I nucleic acid sequence; and, c) predicting the efficacy of a maturation inhibitor against the patient's HIV-I based on the HIV-I nucleic acid sequence. In some embodiments, the genotyping comprises: a) analyzing a nucleic acid sequence from the patient-derived HIV-I ; and, b) determining if one or more polymorphic determinants of antiviral activity in Gag is present in the nucleic acid sequence; wherein the presence polymorphic determinants of antiviral activity in Gag is indicative of the susceptibility of the HIV-I to maturation inhibitor treatment. [0031] In some embodiments, the determining comprises: c) comparing polymorphic determinants of antiviral activity in the patient-derived

HIV-I nucleic acid sequence to a HXB2CG HIV-I nucleic acid sequence; and d) predicting whether the HIV-I is susceptible to the maturation inhibitor if the number of polymorphic determinants of antiviral activity in the patient-derived HIV-I nucleic acid sequence relative to the HXB2CG HIV-I nucleic acid sequence is 0, 1 , or 2.

In some embodiments, the determining comprises: contacting the patient sample with a probe specific to a polynucleotide encoding an amino sequence at least about 60%, 70%, 80%, 90% or homologous to, or identical to a polynucleotide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, with the proviso that underlined polymorphisms must be present as described herein. In some embodiments, the probe is specific to one or more residues selected from the group consisting of 369, 370 and 371. In some embodiments, the probe is specific to residue 380. In some embodiments, the determining comprises c) consulting a database of genotypes, mutants, or polymorphisms. Such a database may list the sensitivity or not of said polymorphism, and may even list the level of maturation inhibitor (EC50) that has been found to be effective in vivo (e.g., clinically) or in vitro for that genotype, mutant or polymorphism.

In some embodiments, the genetic sequence is obtained from a patient sample of at least one of: a plasma sample, a blood sample, a saliva sample, a mucous sample, a serum sample, a semen sample, a urine sample, and a tissue sample.

In some embodiments, the one or more polymorphic determinants of antiviral activity is found at one or more residues selected from the group consisting of 369, 370, 371, and 380.

In some embodiments, the polymorphic determinants of antiviral activity are found at residues 369 and 371.

In some embodiments, the polymorphic determinants of antiviral activity are found at residues 369 and 380. In some embodiments, the polymorphic determinants of antiviral activity are found at residues 370 and 371.

In some embodiments, the polymorphic determinants of antiviral activity are found at residues 370 and 380. In some embodiments, the polymorphic determinants of antiviral activity are found at residues 371 and 380.

In some embodiments, the one or more polymorphic determinants of antiviral activity is selected from the group consisting of Q369H, V370A, V370M, T371A, and T371S.

In some embodiments, the one or more polymorphic determinants of antiviral activity is found at residue 369.

In some embodiments, the one or more polymorphic determinants of antiviral activity are found at residues 369 and 370.

In some embodiments, the one or more polymorphic determinants of antiviral activity are found at residues 369, 370 and 371. These polymorphisms are distinguished from the HIV-I mutants identified in WO

2005/1 13059 by their ability to be inhibited by maturation inhibitors at low but not high MOI. In contrast, the mutants disclosed in WO 2005/113059 are generally not inhibited by maturation inhibitors at either low or high MOI. The mutants disclosed in WO 2005/1 13059 also arise as a result of in vitro resistance selection, where HIV is serially passaged in the presence of a maturation inhibitor.

Phenotyping

Some embodiments of the present invention provide a method for identifying whether a maturation inhibitor inhibits HIV-I maturation comprising the steps of: (a) obtaining virus or nucleic acid from a patient infected by HIV-I that (i) encodes a Gag protein, or (ii) encodes part of a Gag protein that comprises the CA-SPl cleavage site, or (iii) encodes a HIV-I polyprotein comprising 5-15 (e.g., 15, 12, 10, 7, or 5) amino acids upstream and 5-20 (e.g., 5, 7, 10, 12, 18, or 20) amino acids downstream of the CA-SPl cleavage site of Gag; and optionally encoding part or all of Pol, and optionally encoding a full-length Gag-Pol sequence that comprises said nucleic acid of (i), (ii) or (iii); (b) co-transferring or infecting into a first cell (i) the virus or the nucleic acid from (a) such that the first cell produces HIV-I virions comprising the Gag-Pol protein encoded by the nucleic acid obtained from the patient (c) measuring the amount of virus infection or replication as a signal (s) comparing the HIV-I replication or the amount of signal in step (c) with the amount of replication or signal produced in the absence of the compound, wherein a reduced amount of signal measured in the presence of the compound indicates that the compound inhibits HIV-I maturation. In some embodiments, the phenotyping is performed in human cells, for example embryonic kidney cells, T cells, peripheral blood mononuclear cells ("PBMCs"), astroglioma cells, or osteosarcoma cells. In some embodiments, the human embryonic kidney cells are 293 cells. In some embodiments, the cells are human T cells. In some embodiments, the cells are human PBMCs. In some embodiments, the cells are astroglioma cells, for example cells are U87 cells. In some embodiments, the cells are osteosarcoma cells, for example HT4 cells.

In some embodiments, the phenotyping comprises: a) obtaining a nucleic acid segment from HIV-I derived from the patient; b) contacting a patient-derived nucleic acid segment with a maturation inhibitor, wherein the patient-derived nucleic acid segment further comprises an indicator gene; c) detecting the expression of the indicator gene; and d) comparing the expression indicator gene to a control expression of the indicator gene, thereby determining the susceptibility of the HIV-I to the maturation inhibitor.

In some embodiments, the patient-derived nucleic acid segment is obtained from a patient sample of at least one of: a plasma sample, a blood sample, a saliva sample, a mucous sample, a serum sample, a semen sample, a urine sample, and a tissue sample. In some embodiments, the phenotyping further comprises the step of amplifying the genetic sequence. In some embodiments, the step of amplifying the genetic sequence comprises a step selected from the group consisting of PCR, RT-PCR, DNA fragment analysis, sequencing, nucleic acid hybridization to microarrays or beads restriction fragment length polymorphism (RFLP), terminal restriction fragment length polymorphism (t-RFLP), amplified fragment length polymorphisms (AFLP), Branched-DNA Signal Amplification, and multiplex ligation-dependent probe amplification (MLPA).

In some embodiments, the indicator gene is a luciferase gene, a secreted alkaline phosphatase (SEAP) gene, or a green fluorescent protein (GFP) gene.

In some embodiments, the genetic sequence of the HIV-I comprises an HIV gag-pol gene.

In some embodiments, the method of determining susceptibility of a population of HIV-I in a patient to a viral maturation inhibitor comprises: a) contacting the population of virus with cells in the presence of the viral maturation inhibitor wherein the population of virus comprises: (i) a viral expression vector that lacks a nucleic acid encoding a Gag-Pol protein and which comprises an indicator nucleic acid that produces a signal, and (ii) a population of Gag-Pol proteins derived from the population of virus in the patient; b) measuring the signal produced by the cells in the presence of the viral maturation inhibitor; and, c) comparing the signal measured in step (b) to the signal produced by the cells in the absence of the viral maturation inhibitor, wherein a change in signal indicates altered susceptibility of virus to the viral maturation inhibitor.

In some embodiments, the population of virus is produced by co-transferring (e.g., transfecting or electroporating) into a cell (i) a population of gag-pol genes, or fragments thereof, obtained from an HIV-infected patient; and (ii) a viral expression vector lacking a nucleic acid encoding a Gag-Pol protein, wherein the vector comprises an indicator nucleic acid that produces a detectable signal.

In some embodiments, the viral expression vectors comprise an HIV-I nucleic acid. In some embodiments, the viral expression vectors comprise an HIV gag-pol gene, or fragments thereof.

In some embodiments, the method comprises repeating the phenotypic analysis with varying concentrations of compound, and, optionally comprising the step of comparing the amounts of signal generated for each of the concentrations. In some embodiments, the method further comprises generating a plot of viral infectivity based on compound concentrations. In some embodiments, the method further comprises determining an EC50 of the maturation inhibitor.

Kits

In some embodiments, the present invention comprises a diagnostic kit for predicting a patient's response to a maturation inhibitor comprising a maturation inhibitor and components for use in a method for determining the susceptibility of a patient's HIV-I to the maturation inhibitor.

In some embodiments, the method for determining the susceptibility of a patient's HIV-I to the maturation inhibitor is phenotyping the virus. In some embodiments, the method for determining the susceptibility of a patient's HIV-I to the maturation inhibitor is genotyping the virus. In some embodiments, the method for determining susceptibility of a patient's HIV-I to the maturation inhibitor is a combination of phenotyping and genotyping. Vectors

Some embodiments of the present invention provide a vector system (e.g., a retroviral vector system) encoding the polymorphic determinants described herein. Such a vector system produces virus particles containing Gag-Pol proteins derived from a variety of sources. Some embodiments provide the identification of cell lines that express viral receptors and are permissive for viral replication.

Some embodiments of the present invention provide a vector that comprises most of the HIV-I viral genome, and further comprises a reporter gene (e.g., luciferase), also known herein as an indicator gene or indicator nucleic acid. Some embodiments of the present invention comprise a vector that comprises a polynucleotide disclosed herein. In some embodiments, a cell is provided that comprises any of the poly-nucleotides disclosed herein. In some embodiments, a tissue is provided that comprises a cell disclosed herein. In some embodiments, an organism is provided that comprises a cell disclosed herein. In some embodiments, an organism is provided that comprises a cell capable of expressing a polypeptide which is expressed from the polynucleotide disclosed herein. In some embodiments, the organism is a non-human transgenic animal that comprises a cell capable of expressing a polypeptide which is expressed from the polynucleotide disclosed herein. In some embodiments, the non-human transgenic animal is selected from the group consisting of a mouse, rat, dog, cat, cow, pig, horse, rabbit, frog, chicken, monkey, macaque, chimpanzee, guinea pig, marmoset, and sheep. In one aspect, the non-human transgenic animal is a mouse.

Amino Acid Sequences

Some embodiments of the present invention provide an amino acid sequence comprising a sequence useful for identifying maturation inhibitors to which HIV-I will be susceptible in vivo. This aspect of the invention is also directed to a vector, virus and host cell comprising the amino acid sequence, and a method of making the vectors, viruses and host cells comprising the amino acid sequence.

In the present application with regard to amino acid sequences (Gag p24 capsid— SPl- NC): polymorphisms are underlined; the CA-SPl cleavage site and the SP 1-NC cleavage site are each represented by a hyphen " — "; and spaces between each 10 residues are provided for the reader's convenience and are understood to not reflect any structure inherent in the amino acids. SEQ ID NO:72: HXB2CG consensus: PIVQNIQGQM VHQAISPRTL NA WVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRVHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTN NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE VKNWMTETLL VQNANPDCKT ILKALGPAAT LEEMMTACQG VGGPGHKARV L— AEAMSQVTNS ATIM— MQRGNFRNQR KIVKCFNCGK EGHTARNCRA PRKKGCWKCG KEGHQMKDCT ERQAN.

Amino acid sequences useful in accordance with the present invention include amino acid sequences identical to, or at least about 60%, 70%, 80%, or 90% homologous to, one of the following, with the proviso that underlined polymorphisms must be present as described herein:

SEQ ED NO: 1 : PrVQNIQGQM VHQAISPRTL NA WVKVVEEK AFSPEVEPMF SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRVHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTN NPPrPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE VKNWMTETLL VQNANPDCKT ILKALGPAAT LEEMMTACQG VGGPGHKARV L— AEAMSHVTNS ATIM— MQRGNFRNQR KIVKCFNCGK EGHTARNCRA PRKKGCWKCG KEGHQMKDCT ERQAN.

SEQ DD NO:2: PIVQNIQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRVHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTN NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE VKNWMTETLL VQNANPDCKT ILKALGPAAT LEEMMTACQG VGGPGHKARV L— AEAMSQATNS ATIM— MQRGNFRNQR KIVKCFNCGK EGHTARNCRA PRKKGCWKCG KEGHQMKDCT ERQAN. SEQ DD NO:3: PIVQNIQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF

SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRVHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTN NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE VKNWMTETLL VQNANPDCKT ILKALGPAAT LEEMMTACQG VGGPGHKARV L— AEAMSQMJNS ATIM— MQRGNFRNQR KIVKCFNCGK EGHTARNCRA PRKKGCWKCG KEGHQMKDCT ERQAN. SEQ ID NO:4: PIVQNIQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRVHPVHAGP

IAPGQMREPR GSDIAGTTST LQEQIGWMTN NPPIPVGEIY KRWIILGLNK IVRMYSPTSI

LDIRQGPKEP FRDYVDRFYK TLRAEQASQE VKNWMTETLL VQNANPDCKT ILKALGPAAT LEEMMTACQG VGGPGHKARV L— AEAMSQVANS ATM— MQRGNFRNQR KIVKCFNCGK EGHTARNCRA PRKKGCWKCG KEGHQMKDCT ERQAN.

SEQ ID NO:5: PIVQNIQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRVHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTN NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LD1RQGPKEP FRDYVDRFYK TLRAEQASQE VKNWMTETLL VQNANPDCKT ILKALGPAAT LEEMMTACQG VGGPGHKARV L— AEAMSQVSNS ATM— MQRGNFRNQR KIVKCFNCGK EGHTARNCRA PRKKGCWKCG KEGHQMKDCT ERQAN. SEQ ID NO:6: PIVQNIQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF

SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRVHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTN NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE VKNWMTETLL VQNANPDCKT ILKALGPAAT LEEMMTACQG VGGPGHKARV L— AEAMSHMTNS ATM— MQRGNFRNQR KIVKCFNCGK EGHTARNCRA PRKKGCWKCG KEGHQMKDCT ERQAN.

SEQ ID NO:7: PIVQNIQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRVHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTN NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE VKNWMTETLL VQNANPDCKT ILKALGPAAT LEEMMTACQG VGGPGHKARV L— AEAMSHATNS ATM— MQRGNFRNQR KIVKCFNCGK EGHTARNCRA PRKKGCWKCG KEGHQMKDCT ERQAN.

SEQ ID NO:8: PIVQNIQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRVHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTN NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE VKNWMTETLL VQNANPDCKT 1LKALGPAAT LEEMMTACQG VGGPGHKARV L— AEAMSQVTNS ATIM — MQKGNFRNQR KIVKCFNCGK EGHTARNCRA PRKKGCWKCG KEGHQMKDCT ERQAN.

Amino acid sequences useful in accordance with the present invention include amino acid sequences identical to, or at least about 60%, 70%, 80%, or 90% homologous to one of the following, with the proviso that underlined polymorphisms must be present as described herein:

SEQ ID NO:9: — AEAMSHVTNS ATIM — .

SEQ ID NO: 10:— AEAMSQATNS ATM — .

SEQ ID NO: 11 :— AEAMSQMTNS ATM — . SEQ ID NO: 12:— AEAMSQVANS ATM — .

SEQ JD NO: 13:— AEAMSQVSNS ATM — .

SEQ ID NO: 14:— AEAMSHMTNS ATIM — .

SEQ ED NO: 15:— AEAMSHATNS ATM — .

SEQ ID NO: 16:— AEAMSQVTNS ATM — .

Polynucleotides & Polypeptides

Some embodiments of the present invention provide isolated polypeptides and polynucleotides. In one embodiment, the invention includes a polypeptide comprising an amino acid sequence at least about 60%, 70%, 80%, or 90% homologous to, or identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO: 15, with the proviso that underlined polymorphisms must be present as described herein.

Some embodiments of the present invention provide a method of treating HIV-I infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide comprising an amino acid sequence being at least about 60%, 70%, 80%, 90% or homologous to, or identical to a sequence selected from the group consisting of SEQ DD NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ DD NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO:15, with the proviso that underlined polymorphisms must be present as described herein. Some embodiments of the present invention provide a method of treating HIV-I infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide encoded by a polynucleotide sequence encoding a sequence at least about 60%, 70%, 80%, 90% or homologous to, or identical to a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO.12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, with the proviso that underlined polymorphisms must be present as described herein. Some embodiments of the present invention provide a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) by administration of a compound. In related embodiments, such a compound binds to a polypeptide with an amino acid sequence encoding a sequence at least about 60%, 70%, 80%, 90% or homologous to, or identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, with the proviso that underlined polymorphisms must be present as described herein.

Some embodiments of the present invention comprise a vector that comprises a polynucleotide disclosed herein. Some embodiments of the present invention comprise a host cell comprising a disclosed herein. Some embodiments of the present invention comprise a method of producing a polypeptide comprising incubating a host cell containing a vector disclosed herein in a medium and recovering the polypeptide from the medium.

Antibodies

In one embodiment, the invention is directed to an antibody. Such an antibody may bind to a polypeptide with an amino acid sequence being at least about 60%, 70%, 80%, 90% or homologous to, or identical to a sequence selected from the group consisting of SEQ ID NO:1,

SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ

ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ

ID NO: 14, and SEQ ID NO: 15, with the proviso that underlined polymorphisms must be present as described herein. Such an antibody may specifically bind a polymorphic determinant of antiviral activity, or combination thereof, as described herein.

The invention is also directed to polynucleotides encoding such antibodies.

Mutant Viruses The invention is also drawn to HIV-I viruses comprising polymorphic determinants described herein. Such viruses may comprise the polypeptides and/or the polynucleotides described herein. In one such embodiment, the invention is an isolated mutant recombinant HIV- 1 virus, wherein the processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in said virus is not significantly inhibited by 3-(9-(3',3'-dimethylsuccinyl) betulinic acid. In related embodiments, this virus is not inhibited by 3-O-(3',3'-dimethylsuccinyl) betulinic acid. In another embodiment, 3-O-(3',3'-dimethylsuccinyl) betulinic acid does not inhibit the interaction of protease with the Gag polypeptide in this virus. In another, the virus comprises an amino acid sequence being at least about 60%, 70%, 80%, 90% or homologous to, or identical to a sequence selected from the group consisting of SEQ JD NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ED NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ DD NO: 11, SEQ TD NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO:15, with the proviso that underlined polymorphisms must be present as described herein. Mutant viruses may be used in the methods of the invention described elsewhere herein. For example, such viruses are useful in a method of identifying a compound which inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), the method comprising comparing the ability of said compound to inhibit HIV-I replication compared with the replication of a the mutant virus outlined above. Such inhibition may be examined in a cell, or in an animal, or in vitro.

Such viruses may be chimeric. Chimeric viruses useful in accordance with the present invention include viruses selected from the group consisting of HIV-2, HTLV-I, HTLV-II, SIV, avian leukosis virus (ALV), endogenous avian retrovirus (EAV), mouse mammary tumor virus (MMTV), feline immunodeficiency virus (Hy), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), Visna-maedi virus, and feline leukemia virus (FeLV) wherein a portion of the virus's genome is deleted and a polynucleotide comprising a nucleic acid encoding an amino acid sequence being at least about 60%, 70%, 80%, 90% or homologous to, or identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ED NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO:14, and SEQ ID NO: 15 is inserted into the virus genome. Such viruses may be used in the methods of the invention described elsewhere herein; such methods include assays performed in a cell, or in an animal, or in vitro, with the proviso that underlined polymorphisms must be present as described herein.

The invention is also drawn to a non-human animal infected with a virus comprising an amino acid sequence being at least about 60%, 70%, 80%, 90% or homologous to, or identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, and SEQ ID NO: 15, with the proviso that underlined polymorphisms must be present as described herein.

Drug Screening Methods

Some embodiments of the present invention provide a polynucleotide comprising a sequence useful for identifying maturation inhibitors to which HIV-I will be susceptible in vivo. This aspect of the invention is also directed to a vector, virus and host cell comprising the polynucleotide, and a method of making the vectors, viruses and host cells comprising the polynucleotide. In some embodiments the polynucleotide has at least about 60%, 70%, 80%, 90% or homology to, or is identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, with the proviso that underlined polymorphisms must be present as described herein.

Some embodiments of the present invention provide a method for identifying a maturation inhibitor candidate compound comprising (a) contacting HIV-I infected cells with a maturation inhibitor candidate compound; and (b) assaying the cell culture for the presence of mature virions, wherein the presence of mature virions indicates that the maturation inhibitor candidate compound is a maturation inhibitor compound; wherein the HIV-I comprises a polynucleotide having at least about 60%, 70%, 80%, 90% or homology to, or is identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, with the proviso that underlined polymorphisms must be present as described herein.

Some embodiments of the present invention provide a method for identifying a maturation inhibitor compound comprising (a) contacting HIV-I infected cells with a maturation inhibitor candidate compound; and (b) analyzing virus particles that are released to detect the presence of p25; wherein the HIV-I comprises a polynucleotide having at least about 60%, 70%, 80%, 90% or homology to, or being identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, and SEQ ID NO:15, with the proviso that underlined polymorphisms must be present as described herein. In some embodiments, the analyzing comprises western blotting of viral proteins and detecting using an antibody to p25. In some embodiments, the analyzing comprises gel electrophoresis. In some embodiments, the analyzing comprises imaging metabolically labeled proteins. In some embodiments, the analyzing comprises immunoassays that use an antibody to p25 or SPl to distinguish p25 from p24; for example, a microwell assay can be performed where p25 in detergent-solubilized virus is captured using an antibody specific for SPl that is bound to the plastic microwell plate. Following a washing step, bound p25 is detected using an antibody to p25 that is conjugated to an appropriate detection reagent (e.g. alkaline phosphatase for an enzyme-linked immunosorbent assay). Virus released by cells treated with a maturation inhibitor compound has increased levels of p25 compared with untreated virions.

Some embodiments of the present invention provide a method for identifying a maturation inhibitor compound involving contacting HIV-I infected cells with a maturation inhibitor candidate compound, and thereafter analyzing virus particles released by the contacted cells, by thin-sectioning and transmission electron microscopy, and identifying if virion particles are detected with non-condensed cores and a distinctive thin electron-dense layer near the viral membrane; wherein the presence of virion particles having non-condensed cores indicates that the maturation inhibitor candidate compound is a maturation inhibitor; and wherein the HIV-I comprises a polynucleotide having at least about 60%, 70%, 80%, 90% or homology to, or is identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 1 1, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ED NO: 15, with the proviso that underlined polymorphisms must be present as described herein.

The assay of this invention can be used with other viral infections arising from infections due to other viruses within these families as well as viral infections arising from viruses in other viral families. In addition, the drug susceptibility and resistance test of this invention is useful for screening for compounds to treat viral diseases for which there is no currently available therapy.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The mere mentioning of the publications and patent applications does not necessarily constitute an admission that they are prior art to the instant application.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Examples

Example 1

Demonstration of MOI-dependent sensitivity to bevirimat in in vitro virus infection assays

HeLa cells were transfected with proviral plasmid DNA constructs encoding: wild-type

NL4.3 HIV-I (Fig. 1); NL4.3 containing an A364V site-directed resistance mutation (Fig. 2); or chimeric NL4.3 containing an approximately 500 bp fragment from a patient virus isolate harboring the V370A polymorphism in SPl (Fig. 3). Culture supernatants containing virus were collected 48 hrs post-transfection. Four-fold serial dilutions of virus supernatants were titered on U87-CD4-CXCR4 cells in the presence (dark bars) or absence (light grey bars) of bevirimat at a final concentration of 1 μg/ml. Cultures were maintained with media changes (with or without fresh drug, respectively) on days one, three, and six post-infection. On day six, the U87-CD4- CXCR4 culture supernatants were collected and used to infect TZM-bl indicator cells (Tranzyme) for 48 hrs. Viral infection was quantitated by incubating the TZM-bl cells with GalScreen reagent as recommended by the manufacturer (Applied Biosystems). Increased levels of beta-galactosidase reporter gene expression (higher relative light units [RLU] resulting from the GalScreen assay) correlate with increased levels of virus infection.

Example 2 V370 and T371 as polymorphic determinants of antiviral activity

Published studies demonstrated that mutations within the amino terminal half of SPl

(through T371) affected susceptibility to BVM (see Figure 5); whereas, mutations in the C- terminal half of SPl tend to have little or no effect on BVM activity. Most of the residues in the amino -terminal region of SPl (364-369) are relatively conserved in the Los Alamos database in terms of frequency of polymorphisms, suggesting their importance in virus replication. Residues 370 and 371, however, are more variable. Residues in the C-terminal region of SPl are highly variable. The CA-SPl cleavage site has been predicted from computer modeling to exist as an alpha-helix at some point during Gag maturation. It is noted that the alpha-helical structure predicted by cryotomography terminates in the vicinity of positions 370-371.

Example 3 Analysis of clinical trial data

Analysis of clinical trial data from 44 HIV-infected patients to whom bevirimat was administered at a dose of either 250 mg of BVM in a 10% hyroxypropyl-β-cyclodextrin ("HPβCD") solution, 300 mg of BVM in a HPβCD solution, 350 mg of BVM in a HPβCD, 400 mg of BVM in a HPβCD solution or a tablet consisting of 400 mg BVM (bioequivalent to approximately 138 mg of BVM in a HPβCD solution) confirms the importance of these polymorphic determinants of antiviral activity. The patients in the study were multi-drug resistant and had failed their most recent antiretroviral drug regimens. They were dosed with bevirimat once daily for 14 days in combination with their failed regimen, so-called "functional monotherapy". The primary virological endpoint, provided below, was viral load reduction on day 15.

Within the entire group of 44 patients, 20 (45%) had a 0.5 log 10 viral load reduction or greater with 15 (34%) having a 1.0 log 10 or greater reduction. The mean viral load reduction for this group was 0.60. Of the 36 patients for whom a Gag sequence was obtained and who had a 20 μg/mL or greater bevirimat trough plasma concentration on day 15, 19 (53%) had a 0.5 log 10 viral load reduction or greater with 14 (39%) having a 1.0 log 10 or greater reduction. The mean viral load reduction for this group was 0.70. Of the 23 patients having a 20 μg/mL or greater bevirimat trough plasma concentration on day 15 and having one or more polymorphisms at a residue selected from the group consisting of 369, 370 and 371, 7 (30%) had a 0.5 log 10 viral load reduction or greater with 4 (17%) having a 1.0 log 10 or greater reduction. The mean viral load reduction for this group was 0.38.

Of the 13 patients having a 20 μg/mL or greater bevirimat trough plasma concentration on day 15 and having no polymorphisms at residues 369, 370 and 371, 12 (92%) had a 0.5 log 10 viral load reduction or greater, with 10 (77% having a 1.0 log 10 or greater reduction. The mean viral load reduction for this group was 1.26 log 10.

Of the 13 patients having a 20 μg/mL or greater bevirimat trough plasma concentration on day 15 and having a polymorphism at residue 380, 10 (77%) had a 0.5 log 10 viral load reduction or greater, with 9 (69%) having a 1.0 log 10 or greater reduction. The mean viral load reduction for this group was 1.14 log 10.

Of the 6 patients having a 20 μg/mL or greater bevirimat trough plasma concentration on day 15 and having a polymorphism at residue 380 but having no polymorphisms at residues 369, 370 and 371, all 6 (100%) had a 1.0 log 10 or greater reduction. The mean viral load reduction for this group was 1.49 log 10.

Example 4 Susceptibility of non-clade B HIV-I isolates to bevirimat in vitro

Background: The HIV-I maturation inhibitor bevirimat (BVM, MPC-4326) binds to Gag and specifically inhibits CA-SPl processing. Recent clinical studies identified key baseline polymorphisms at Gag positions 369/370/371 in SPl that correlated with variable patient responses. Polymorphisms at these 3 positions are found in -30% of patients with clade B virus. Since the clade B consensus sequence at these positions (QVT) differs from that of other clades, we examined the susceptibility of non-clade B isolates to BVM in vitro to determine which polymorphisms affect BVM activity in these other clades. Methods: A panel of 25 non-clade B viruses was compiled with multiple representatives from each clade with global prevalence >1% worldwide (clades A, C, CRF01_AE, CRF02_AG, D, G). The panel consisted of 10 isolates with known, distinct CA-SPl genotypes and 15 randomly selected patient plasma samples from Switzerland. The complete Gag-PR region from each isolate was amplified and cloned into a pNL4-3 background. The InPheno replicative in- vitro phenotyping assay, deCIPhR (dual-enhancement of Cell Infection to Phenotype Resistance), was used to quantitate susceptibility to BVM. Fold-change (FC) in IC50 was compared to FC values for BVM-treated patient isolates and site-directed mutant controls.

Results: None of the 25 viruses in the test panel contained the wild-type QVT clade B consensus sequence at positions 369-371 ; nonetheless, 7/25 isolates (28%) were highly susceptible to BVM (FC <2). These included 5 viruses containing the clade A and CRF02_AG consensus sequence, QVQ. 9/25 isolates (36%) had intermediate susceptibility (FC 2-10), and 9/25 isolates (36%) fell into the least susceptible category (FC >10). Of the intermediate/least susceptible viruses, 13/18 contained V370A, V370M, or ΔV370 polymorphisms, all of which are key polymorphisms in clade B.

Polymorphisms at Gag 369, 370, and 371 correlate with reduced in vitro susceptibility of clade B patient isolates to bevirimat:

Table 1 : Phenotypes of clade B patient isolates correlate with polymorphisms at Gag 369, 370, and 371. i InPheno deCIPhR™ ; GAG+PR Phenotype . I Assay

SEQ

Patient

CA-SPl Sequence Fold Change in IC50 ID ID

No.

WiId- r, ' ...GHKARVL-AEAMSQVTNSATIM ^' 1 17

129 ... GHKARVL-AEAMSQVTSSATMM 0.57 18 180 ... GHKARVL-AEAMSQVTNPPTIM 0.84 19 6 ... GHKARVL-AEAMSQVTNSATIM 0.87 17

126 ... GHKARVL-AEAMSQVTGSAAVM 1.02 20 ^{" '}

1 J ...GHKARVL- AEAMSQVTNS ATVM 1.14 21

182 ... GHKARVL- AEAMSQVTNP ATM 1.16 22 14 ..^".GHKARVL-AEAMSQVTPS ATVM 1 51 ^{" "}23 16 ... GHKARVL-AEAMSQVTNPSNIM 1.69 24

3 ^" ... SHK -AR - I -L- A - EA -MS - Q - V .TG - -P - AN - -IM- . 1.79 25

127a ...GHKARVL-AEAMSQMTNSATAM 3.6 26 127b ^{" "} 7. GHKARVL-^' AEAMSQMTNS AT AM 4.25 ^{" "}26^{" " ' "} 4 ... GHKARVL-AEAMSQMTNP ATΓM 4.6 27 24 ...SHKARVL-AEAMSQV-NPTNIM

_ - ₁₀- - - 4.61 28

^~ ... NHKARIL- AE AMCH VTNS AT v¥ 4.85 - ^~2§

^"Ϊ2 ...GHKARVL-AEAMSQMTNS ATTM ; ^{' " " "}l2^~8 ^' 30 ^{" "}8 ...SHKARVL-AEAMCQA-NSTTVM : 14.5 31

125a ^{" " " "} . GHKARVL-AEAMSQATASNVIM ^" : ._ _ 15__.3^ ^' 32

^" Ϊ5 ^"" ...GHK^RVL-AEAMSQA-NSSSΓM ^{r " "} 33

^" 125b ... GHKARVL-AE AMSQ AT ASNVIM ^{" "} 22.2 32

30 ... GHKARVL- AEAMSQ ATNS AAlM [ 32.3 34

Site-directed mutagenesis demonstrates that some, but not all, changes at 369-371 are sufficient to reduce susceptibility to bevirimat: Table 2: Site-directed mutagenesis at 369, 370, & 371.

* Resistance mutations identified by in vitro selection

The Gag 36-371 consensus sequence differes in non-subtype B virus clades: Table 3: Consensus sequence at 369, 370 & 371 for different virus clades.

Many polymorphisms common in non-clade B isolates are susceptible to bevirimat:

Table 4. Phenotype of selected non-clade B patient isolates.

Observations from Table 4 dataset:

All of the 25 non-clade B viruses selected contained polymorphisms that differ from the clade B consensus sequence, QVT, at positions 369-371 in Gag

Despite this, 7/25 isolates (28%) were highly susceptible to BVM (FC < 2), and an additional 9/25 isolates (36%) had intermediate susceptibility (FC = 2-10) ^■ The clade A and CRF02_AG consensus sequence, QVQ, was particularly sensitive to BVM, with FC < 3.5 for all 6 isolates

^■ The current clade B patient genotype screening algorithm appears to be too stringent for use in evaluating patients with non-clade B virus and we are continuing to refine the clade B algorithm as we collect new data

^■ Additional in vitro phenotyping studies will help to further refine the genotyping algorithm to permit the identification of patients with non-clade B viruses that are suitable for treatment with BVM

Conclusions: Our analysis demonstrates that some, but not all, polymorphisms at Gag 369/370/371 in SPl reduce the susceptibility of viruses to BVM in vitro. Specifically, the T371Q polymorphism that gives rise to the clade A and CRF02 AG consensus sequence, QVQ, appears to have no effect on BVM susceptibility. This suggests that, following more extensive testing, it may be possible to exclude the T371Q polymorphism from a future genotyping algorithm used to identify patients suitable for BVM treatment. Additional phenotyping and genotyping should help to further refine the genotyping algorithm for non-clade B patients.

Claims

WHAT IS CLAIMED IS:

1. A method of determining susceptibility of an HIV-I infected patient to a maturation inhibitor comprising: analyzing genetic material of a patient to determine whether the HIV- 1 virus is expected to be susceptible to the maturation inhibitor.

2. A method of determining susceptibility of an HIV-I virus present in a patient to a maturation inhibitor comprising: analyzing genetic material of a patient to determine whether the HIV-I virus is expected to be susceptible to the maturation inhibitor.

3. The method of Claim 1 wherein the analyzing is genotyping.

4. Method of Claim 3 wherein the genotyping comprises:

(a) obtaining an HIV-I genetic sequence from a patient sample;

(b) determining the genotype of the genetic sequence; and

(c) predicting the efficacy of a maturation inhibitor against HIV-I based on the genotype of the HIV-I.

5. The method of Claim 3 wherein the genotyping comprises:

(a) analyzing a genetic sequence of the HIV-I from the patient; and,

(b) determining if one or more polymorphic determinants of response in GAG is present in the genetic sequence; wherein the presence a polymorphic determinants of response in GAG is indicative of a change in the susceptibility of the HIV-I.

6. The method of either Claim 4 or 5 wherein the determining comprises:

(a) comparing polymorphic determinants of response in the HIV-I genetic sequence to a wild type or consensus HIV-I sequence; and

(b) predicting whether the HIV-I will be susceptible to the maturation inhibitor if the number of polymorphic determinants of response in the patient sample's HIV-I genetic sequence relative to the wild type or consensus HIV-I sequence is 0, 1, or 2.

7. Method of either Claim 4 or 5 wherein the HIV-I genetic sequence is obtained from a patient sample of at least one of: a plasma sample, a blood sample, a saliva sample, a mucous sample, a serum sample, a semen sample, a urine sample, and a tissue sample.

8. The method of Claim 5 wherein the one or more polymorphic determinant of response is found at one or more residues selected from the group consisting of 363, 364, 369, 370, 371, and 380.

9. The method of Claim 5 wherein the one or more polymorphic determinant of response is selected from the group consisting of Q369A, Q369H, V370A, V370M, T371A, T371 S, T371N, R380K, L363M, A364V, ΔQ369, ΔV370, and ΔT371.

10. The method of Claim 5 wherein the one or more polymorphic determinant of response is found at one of residues 369, 370 and 371.

11. The method of Claim 5 wherein the one or more polymorphic determinants of response are found at residues 369 and 370, 370 and 371, or 369 and 371.

12. A method of determining susceptibility of a HIV-I virus in a patient to bevirimat comprising the steps of:

(a) obtaining virus or nucleic acid from a patient infected by HIV-I that (i) encodes a Gag protein, or (ii) encodes part of a Gag protein that comprises the CA-SPl cleavage site, or (iii) encodes a HIV-I polyprotein comprising about 5 to about 15 amino acids upstream and about 5 to about 20 amino acids downstream of the CA- SPl cleavage site of Gag, and optionally encoding part or all of Pol, and optionally producing a full-length sequence encoding Gag-Pol that comprises said nucleic acid of (ii) or (iii);

(b) co-transferring or infecting into a first cell (i) the virus or the nucleic acid from (a) such that the first cell produces HIV-I virions comprising the Gag-Pol protein encoded by the nucleic acid obtained from the patient;

(c) measuring the amount of virus infection or replication as a signal;

(d) comparing the HIV-I replication or the amount of signal with the amount of replication or signal produced in the presence and absence of bevirimat, wherein a reduced amount of signal measured in the presence of bevirimat indicates that the compound inhibits HIV-I maturation.

13. The method of claim 12, wherein the population of virus is produced by co-transferring into a cell (i) a gag-pol nucleic acids derived from an HIV-I infected patient; and (ii) a viral expression vector lacking a nucleic acid encoding a Gag-Pol protein, wherein the vector comprises an indicator nucleic acid that produces a detectable signal.

14. The method of claim 13, wherein the viral expression vector comprises an HIV-I nucleic acid.

15. The method of claim 13, wherein the viral expression vector comprises an HIV-I gag-pol gene.

16. The method of either Claim 12 or 13, wherein the steps are repeated with varying concentrations of bevirimat, and further comprising the step of comparing the amounts of signal generated for each of the concentrations.

17. The method of claim 16, further comprising generating a plot of viral infectivity based on bevirimat concentrations.

18. The method of claim 16, further comprising determining an IC50 for bevirimat.

19. The method of Claim 1 wherein the analyzing is phenotyping.

20. The method of Claim 19 wherein the phenotyping comprises conducting the assay described in Example 1 using proviral plasmid DNA constructs encoding an HIV-I isolate obtained from said patient.

21. The method of Claim 20 wherein the HIV-I isolate obtained from said patient is obtained from a patient sample of at least one of: a plasma sample, a blood sample, a saliva sample, mucous sample, and a tissue sample.

22. The method of Claim 4 or 5 wherein the determining comprises contacting the HIV-I genetic sequence obtained from a patient sample patient sample with a nucleic acid probe.

23. The method of Claim 22, wherein the nucleic acid probe is designed to detect the presence of absence of a specific nucleotide sequence encoding the amino acid residues at positions 369, 370 and 371 of the HIV-I Gag polyprotein.

24. A method of treating HIV-I -infection in a patient by administering a maturation inhibitor that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein the maturation inhibitor binds to a polypeptide identical to, or at least about 60%, 70%, 80%, or 90% homologous to a polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

25. The method of Claim 24 wherein the HIV-I does not respond to other HIV-I therapies.

26. The method of Claim 25 wherein the HIV-I is resistant to a second drug used to treat HIV-I infection.

27. The method of Claim 26 wherein the second drug is a protease inhibitor, a reverse transcriptase inhibitor, a nucleoside analog, a vaccine, an attachment inhibitor, an immunomodulator, a fusion inhibitor, a CCR5 antagonist, a CXCR4 antagonist, an integrase inhibitor or any other HIV-I inhibitor.

28. The method of Claim 27 wherein the drug is a polymerase inhibitor.

29. The method of Claim 27 wherein the drug is a nucleoside analog.

30. The method of Claim 27 wherein the drug is a fusion inhibitor.

31. The method of Claim 27 wherein the drug is an immunomodulator.

32. The method of Claim 27 wherein the drug is a reverse transcriptase inhibitor.

33. The method of Claim 27 wherein the drug is a CCR5 antagonist.

34. The method of Claim 27 wherein the drug is a CXCR4 antagonist.

35. The method of Claim 27 wherein the drug is an integrase inhibitor.