Compositions and methods for determining the susceptibility of a pathogenic virus to protease inhibitors Number:7,384,734 from the United States Patent and Trademark Office (PTO) owispatent The present invention provides an approach for developing an algorithm for determining the effectiveness of anti-viral drugs based on a comprehensive analysis of paired phenotypic and genotypic data guided by phenotypic clinical cut-offs. In one aspect, the algorithm allows one to provide a patient with effective treatment. It helps predict whether an infected individual will respond to treatment with an anti-viral compound, thereby allowing an effective treatment regimen to be designed without subjecting the patient to unnecessary side effects. Also, by avoiding the administration of ineffective drugs, considerable time and money is saved.<H susceptibility, Compositions, Compositions, an, 7384734.html, Patent, pto, 7384734

Title: Compositions and methods for determining the susceptibility of a pathogenic virus to protease inhibitors

Abstract: The present invention provides an approach for developing an algorithm for determining the effectiveness of anti-viral drugs based on a comprehensive analysis of paired phenotypic and genotypic data guided by phenotypic clinical cut-offs. In one aspect, the algorithm allows one to provide a patient with effective treatment. It helps predict whether an infected individual will respond to treatment with an anti-viral compound, thereby allowing an effective treatment regimen to be designed without subjecting the patient to unnecessary side effects. Also, by avoiding the administration of ineffective drugs, considerable time and money is saved.

Patent Number: 7,384,734 Issued on 06/10/2008 to Parkin, et al.

Inventors: Parkin; Neil T. (Belmont, CA), Chappey; Colombe (San Francisco, CA), Petropoulos; Christos J. (South San Francisco, CA)
Assignee: Monogram Biosciences, Inc. (South San Francisco, CA)

Appl. No.: 10/367,223

Filed: February 13, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
60357171	Feb., 2002
60359342	Feb., 2002
60392377	Jun., 2002

Current U.S. Class: 435/5 ; 424/188.1

Field of Search: 435/5 424/188.1,208.1

References Cited [Referenced By]

U.S. Patent Documents


5436131	July 1995	Condra et al.
5766842	June 1998	Melnick et al.
5837464	November 1998	Capon et al.
6033902	March 2000	Haseltine et al.
6103462	August 2000	Paulous et al.
6242187	June 2001	Capon et al.
2002/0064838	May 2002	Parkin et al.
2003/0108857	June 2003	Parkin et al.
2004/0106106	June 2004	Parkin et al.

Foreign Patent Documents


WO99/67427	Jun., 1999	WO
WO00/78996	Dec., 2000	WO
WO02/22076	Mar., 2002	WO
WO02/068618	Sep., 2002	WO
WO02/099387	Dec., 2002	WO
WO03/070700	Aug., 2003	WO
WO2004/003512	Jan., 2004	WO
WO2004/003514	Jan., 2004	WO

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Primary Examiner: Parkin; Jeffrey S.
Attorney, Agent or Firm: Jones Day

Parent Case Text

This application is entitled to and claims priority to U.S. Provisional Application Nos. 60/357,171, filed Feb. 15, 2002; 60/359,342, filed Feb. 22, 2002; and 60/392,377, filed Jun. 26, 2002, the contents of which are hereby incorporated by reference in their entireties.

Claims

What is claimed is:

1. A method for determining whether a human immunodeficiency virus (HIV) has an increased likelihood of having reduced susceptibility to treatment with lopinavir, comprising: (a) detecting, in said HIV, the presence or absence of one or more of the HIV protease mutations listed in Table 7; (b) assigning a weighting factor to each mutation as provided in Table 7; and (c) adding said weighting factors to get a total score for said HIV, wherein said HIV has an increased likelihood of being resistant to treatment with lopinavir if said total score is equal to or greater than 6.

2. The method of claim 1, wherein said HIV has an increased likelihood of being resistant to treatment with lopinavir if said total score is equal to or greater than 7.

3. The method of claim 1, wherein said HIV has an increased likelihood of being resistant to treatment with lopinavir if said total score is equal to or greater than 8.

4. The method of claim 1, wherein the mutation is detected in a nucleic acid of said HIV that encodes the protease.

5. The method of claim 4, wherein said presence or absence of said mutation in said protease is detected by hybridization with a sequence-specific oligonucleotide probe to a nucleic acid sequence of said human immunodeficiency virus encoding said mutation, wherein the occurrence of hybridization indicates said presence or absence of said mutation.

6. The method of claim 5 wherein said sequence-specific oligonucleotide probe hybridizes to a nucleic acid encoding said mutation and the presence of hybridization indicates the presence of said mutation.

7. The method of claim 4, wherein said presence or absence of said mutation in said protease is detected by nucleic acid sequencing.

8. The method of claim 1, wherein said human immunodeficiency virus is human immunodeficiency virus type 1 (HIV-1).

9. The method of claim 1 wherein the method comprises detecting the presence or absence of a mutation associated with reduced susceptibility to treatment with lopinavir at 2 or more of the amino acid positions.

10. A method for determining whether an individual infected with a human immunodeficiency virus (HIV) has an increased likelihood of having reduced susceptibility to treatment with lopinavir, comprising: (a) detecting, in a sample from said individual, the presence or absence of one or more of the HIV protease mutations listed in Table 7; (b) assigning a weighting factor to each mutation as provided in Table 7; and (c) adding said weighting factors to get a total score for said individual wherein said individual has an increased likelihood of being resistant to treatment with lopinavir if said total score is equal to or greater than 6.

11. The method of claim 10, wherein said individual has an increased likelihood of being resistant to treatment with lopinavir if said total score is equal to or greater than 7.

12. The method of claim 10, wherein said individual has an increased likelihood of being resistant to treatment with lopinavir if said total score is equal to or greater than 8.

13. The method of claim 10, wherein the mutation is detected in a nucleic acid of said HIV that encodes the protease.

14. The method of claim 13, wherein said presence or absence of said mutation in said protease is detected by hybridization with a sequence-specific oligonucleotide probe to a nucleic acid sequence of said human immunodeficiency virus encoding said mutation, wherein the occurrence of hybridization indicates said presence or absence of said mutation.

15. The method of claim 14 wherein said sequence-specific oligonucleotide probe hybridizes to a nucleic acid encoding said mutation and the presence of hybridization indicates the presence of said mutation.

16. The method of claim 13, wherein said presence or absence of said mutation in said protease is detected by nucleic acid sequencing.

17. The method of claim 10, wherein said human immunodeficiency virus is human immunodeficiency virus type 1 (HIV-1).

18. The method of claim 10 wherein the method comprises detecting the presence or absence of a mutation associated with reduced susceptibility to treatment with lopinavir at 2 or more of the amino acid positions.

19. The method of claim 10, wherein the individual is undergoing or has undergone prior treatment with lopinavir or a different protease inhibitor.

Description

1. FIELD OF INVENTION

This invention relates to compositions and methods for determining the susceptibility of a pathogenic virus to an anti-viral compound. The compositions and methods are useful for identifying effective drug regimens for the treatment of viral infections, and identifying and determining the biological effectiveness of potential therapeutic compounds.

2. BACKGROUND OF THE INVENTION

More than 60 million people have been infected with the human immunodeficiency virus ("HIV"), the causative agent of acquired immune deficiency syndrome ("AIDS"), since the early 1980s. See Lucas, 2002, Lepr Rev. 73(1):64-71. HIV/AIDS is now the leading cause of death in sub-Saharan Africa, and is the fourth biggest killer worldwide. At the end of 2001, an estimated 40 million people were living with HIV globally. See Norris, 2002, Radiol Technol. 73(4):339-363.

Modern anti-HIV drugs target different stages of the HIV life cycle and a variety of enzymes essential for HIV's replication and/or survival. Amongst the drugs that have so far been approved for AIDS therapy are nucleoside reverse transcriptase inhibitors such as AZT, ddI, ddC, d4T, 3TC, abacavir, nucleotide reverse transcriptase inhibitors such as tenofovir, non-nucleoside reverse transcriptase inhibitors such as nevirapine, efavirenz, delavirdine and protease inhibitors such as saquinavir, ritonavir, indinavir, nelfinavir, amprenavir and lopinavir.

One consequence of the action of an anti-viral drug is that it can exert sufficient selective pressure on virus replication to select for drug-resistant mutants (Herrmann et al., 1977, Ann NY Acad Sci 284:632-637). With increasing drug exposure, the selective pressure on the replicating virus population increases to promote the more rapid emergence of drug resistant mutants.

With the inevitable emergence of drug resistance, strategies must be designed to optimize treatment in the face of resistant virus populations. Ascertaining the contribution of drug resistance to drug failure is difficult because patients that are likely to develop drug resistance are also likely to have other factors that predispose them to a poor prognosis (Richman, 1994, AIDS Res Hum Retroviruses 10:901-905). In addition, each patient typically harbors a diverse mixture of mutant strains of the virus with different mutant strains having different susceptibilities to anti-viral drugs.

The traditional tools available to assess anti-viral drug resistance are inadequate; the classical tests for determining the resistance of HIV to an anti-viral agent are complex, time-consuming, expensive, potentially hazardous and not custom tailored to the treatment of a given patient. See Barre-Sinoussi et al., 1983, Science 220:868-871; Popovic et al., 1984, Science 224:497-500), and variations of it (see, e.g., Goedert et al., 1987, JAMA 257:331-334; Allain et al., 1987, N. Engl. J. Med. 317:1114-1121; Piatak et al., 1993, Science 259:1749-1754; Urdea, 1993, Clin. Chem. 39:725-726; Kellam and Larder, 1994, Antimicrobial Agents and Chemo. 38:23-30.

Two general approaches are now used for measuring resistance to anti-viral drugs. The first, called phenotypic testing, directly measures the susceptibility of virus taken from an infected person's virus to particular anti-viral drugs. Petropoulos et al., 2000, Antimicrob. Agents Chemother. 44:920-928 and Hertogs et al., 1998, Antimicrob Agents Chemother 42(2):269-76 provide a description of phenotypic assays in widespread use today. Gunthard et al., 1998, AIDS Res Hum Retroviruses 14:869-76 and Schuurman et al., 1999, J Clin Microbiol. 37:2291-96 discuss currently prevalent genotypic assays. Hirsch et al., 2000, JAMA 283:2417-26 provide a general analysis of the currently available assays for testing drug susceptibility.

The second method, called genotypic testing, detects mutations in the virus that affect drug susceptibility and can associate specific genetic mutations with drug resistance and drug failure. Genotypic testing examines virus taken from a patient, looking for the presence of specific genetic mutations that are associated with resistance to certain drugs. Genotypic testing has a few advantages over phenotypic testing, most notably the relative simplicity and speed with which the test can be performed. The testing can take as little as a few days to complete, and because it is less complex, it is somewhat cheaper to perform. However, interpretation of genotypic data is dependent on previous knowledge of the relationships between specific mutations and changes in drug susceptibility.

Carrillo et al., 1998, J. Virol. 72:7532-41 describe the in vitro selection and characterization of HIV-1 variants having reduced susceptibility to lopinavir. Nine different mutations at 8 amino acid positions were associated with reduced susceptibility to lopinavir. A subsequent study found 23 different mutations at 11 positions in the HIV protease that correlated with reduced in vitro susceptibility to lopinavir in plasma samples of HIV-infected patients who had been treated previously with at least one protease inhibitor (Kempf et al., 2001, J. Virol. 75:7462-69). A crude algorithm that attempted to correlate the phenotypic resistance to lopinavir with the number of mutations observed at the 11 identified positions, and therefore to predict the effectiveness of lopinavir treatment, was postulated (Kempf et al., 2000, Antiviral Therapy 5 (suppl. 3):70, abstract 89). According to the algorithm, a virus was susceptible to treatment with lopinavir if it had five or fewer mutations at the 11 identified positions in its protease. If the number of mutations at these 11 positions was six or more, then the virus was predicted to be resistant to lopinavir treatment. Id.

Efforts to date to use genotypic correlates of reduced susceptibility to predict the effectiveness of anti-viral drugs, especially drugs targeted against the ever-evolving HIV are, at best, imperfect. An algorithm that can more accurately predict whether a given anti-viral drug or combination of drugs would be effective in treating a given patient would save time and money by identifying drugs that are not likely to succeed before they are administered to the patient. More importantly, it would improve the quality of life of the patient by sparing him or her the trauma of treatment with potent toxins that result in no improvement with respect to his or her HIV infection. Therefore, an urgent need exists for a more accurate algorithm for predicting whether a particular drug would be effective for treating a particular patient. Moreover, a genotype based assay can be faster and more cost effective than phenotypic assays.

3. SUMMARY OF THE INVENTION

The present invention provides methods and compositions for developing and using algorithms for determining the effectiveness of an anti-viral therapy or combination of therapies. The algorithms are based on an analysis of paired phenotypic and genotypic data guided by phenotypic clinical cut-offs (the point at which resistance to a therapy begins and sensitivity ends). The algorithms significantly improve the quality of life of a patient by accurately predicting whether a given anti-viral drug would be effective in treating the patient, thereby sparing him or her the trauma of treatment with potent toxins that result in no improvement in his or her HIV infection.

In one aspect, the present invention provides algorithms that allow one to provide a patient with an effective treatment regimen by predicting whether an infected individual will respond to treatment with an anti-viral agent or combination of agents, thereby allowing an effective treatment regimen to be designed without subjecting the patient to unnecessary side effects. Also, by avoiding the administration of ineffective drugs, considerable time and money is saved.

In another aspect, the present invention provides methods for determining the susceptibility of a virus to an anti-viral treatment, comprising detecting, in the viral genome or viral enzymes, the presence or absence of mutations associated with reduced susceptibility to the anti-viral treatment.

In another aspect, the present invention provides methods for determining the effectiveness of an anti-viral treatment of an individual infected with a virus, comprising detecting, in a sample from said individual, the presence or absence of mutations associated with reduced susceptibility to the anti-viral treatment.

The present invention also provides methods of monitoring the clinical progression of viral infection in individuals receiving an anti-viral treatment by determining, as described above, the effectiveness of the same or a different anti-viral treatment.

In one embodiment, the present invention provides nucleic acids and polypeptides comprising a mutation in the protease of a human immunodeficiency virus ("HIV") associated with reduced susceptibility to a protease inhibitor. Examples of protease inhibitors include, but are not limited to, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir and lopinavir. In one embodiment, the protease inhibitor is lopinavir.

In one aspect, the invention provides a method for determining whether a human immunodeficiency virus is likely to be resistant or susceptible to treatment with a protease inhibitor, comprising: detecting, in said HIV, the presence or absence of one or more of the HIV protease mutations listed in Table 7, assigning a weighting factor to each mutation as provided in Table 7, and adding said weighting factors to get a total score for said individual wherein said individual is likely to be resistant to treatment with said protease inhibitor if said total score is equal to or greater than a cut-off score and said individual is likely to be susceptible to treatment with said protease inhibitor if said total score is less than said cut-off score. In one embodiment, the cut-off score is 6. In another embodiment, the cut-off score is 7. In another embodiment, the cut-off score is 8.

In another aspect, the invention provides a method for determining whether a human immunodeficiency virus has an increased likelihood of having reduced susceptibility to treatment with a protease inhibitor, comprising: detecting, in said HIV, the presence or absence of one or more of the HIV protease mutations listed in Table 7; assigning a weighting factor to each mutation as provided in Table 7; and adding said weighting factors to get a total score for said HIV, wherein said HIV has an increased likelihood of being resistant to treatment with said protease inhibitor if said total score is equal to or greater than a cut-off score. In one embodiment, the cut-off score is 6. In another embodiment, the cut-off score is 7. In another embodiment, the cut-off score is 8.

In another aspect, the invention provides a method for determining whether an individual infected with a human immunodeficiency virus is likely to be resistant or susceptible to treatment with a protease inhibitor, comprising: detecting, in a sample from said individual, the presence or absence of one or more of the HIV protease mutations listed in Table 7, assigning a weighting factor to each mutation as provided in Table 7, and adding said weighting factors to get a total score for said individual wherein said individual is likely to be resistant to treatment with said protease inhibitor if said total score is equal to or greater than a cut-off score and said individual is likely to be susceptible to treatment with said protease inhibitor if said total score is less than said cut-off score. In one embodiment, the cut-off score is 6. In another embodiment, the cut-off score is 7. In another embodiment, the cut-off score is 8.

In another aspect, the invention provides a method for determining whether a HIV has an increased likelihood of having a reduced susceptibility to treatment with a protease inhibitor, comprising detecting in the protease of said HIV or in a nucleic acid of said HIV that encodes the protease, the presence or absence of a mutation associated with reduced susceptibility to treatment with said protease inhibitor at amino acid position 20, 33, 34, 43, 46, 48, 50, 54, 55, 58, 63, 66, 73, 74, 76, 79, 82, 84 or 89 of the amino acid sequence of said protease, wherein the presence of said mutation indicates that the HIV has an increased likelihood of having reduced susceptibility to treatment with the protease inhibitor, with the proviso that said mutation is not K20M, K20R, M46I, M46L, I54L, I54T, I54V, L63P, V82A, V82F, V82T or I84V. In one embodiment, the mutation is detected in the protease of said HIV. In another embodiment, the mutation is detected in a nucleic acid of said HIV that encodes the protease.

In another embodiment, the mutation associated with reduced susceptibility to treatment with said protease inhibitor is at amino acid position 20, 33, 34, 46, 50, 54, 63, 66, 73, 74, 76, 79, 82, 84 or 89 of the amino acid sequence of said protease, with the proviso that said mutation is not K20M, K20R, L33F, L33M, K43T, M46I, M46L, I50V, I54A, I54L, I54M, I54S, I54T, I54V, L63P, G73A, G73S, G73T, T74S, V82A, V82F, V82I, V82S, V82T or I84V.

In another embodiment, the mutation associated with reduced susceptibility to treatment with said protease inhibitor is at amino acid position 10, 11, 32, 47, 53, 71 or 95 of the amino acid sequence of said protease, with the proviso that said mutation is not V32I or I47V. In one embodiment, the mutation associated with reduced susceptibility to treatment with said protease inhibitor is selected from the group consisting of: L10F, F53L and A71L.

In another embodiment, the mutation associated with reduced susceptibility to treatment with said protease inhibitor is selected from the group consisting of: K20I, M46V, I50L, I54A, I54M, I54S, L63T, V82S, I84A, I84L, L33F, L33I, L33V, E34D, E34K, E34Q, K43T, G48V, I50L, I50V, K55R, Q58E, G73C, G73T, T74A, T74P, T74S, L76V, P79A, P79D, P79E, L89I and L89M. In another embodiment, the mutation is selected from the group consisting of: K20I, M46V, I50L, L63T, I84A, I84L, L33I, L33V, E34D, E34K, E34Q, I50V, I54M, G73C, T74A, T74P, L76V, P79A, P79D, P79E, L89I and L89M. In another embodiment, the mutation is selected from the group consisting of: K20I, M46V, I50L, L63T, I84A, I84L, L33I, L33V, E34D, E34K, E34Q, G73C, T74A, T74P, L76V, P79A, P79D, P79E, L89I and L89M.

In another aspect, the invention provides a method for determining whether an individual infected with HIV has an increased likelihood of having a reduced susceptibility to treatment with a protease inhibitor, comprising detecting, in a sample from said individual, the presence or absence of a mutation associated with reduced susceptibility to treatment with said protease inhibitor at amino acid position 20, 33, 34, 43, 46, 48, 50, 54, 55, 58, 63, 66, 73, 74, 76, 79, 82, 84 or 89 of the amino acid sequence of the protease of the HIV, wherein the presence of said mutation indicates that the individual has an increased likelihood of having reduced susceptibility to treatment with the protease inhibitor, with the proviso that said mutation is not K20M, K20R, M46I, M46L, I54L, I54T, I54V, L63P, V82A, V82F, V82T or I84V. In one embodiment, the mutation is detected in the protease of said HIV. In another embodiment, the mutation is detected in a nucleic acid of said HIV that encodes the protease.

In another embodiment, the mutation associated with reduced susceptibility to treatment with said protease inhibitor is at amino acid position 20, 33, 34, 46, 50, 54, 63, 66, 73, 74, 76, 79, 82, 84 or 89 of the amino acid sequence of the protease of the HIV, with the proviso that said mutation is not K20M, K20R, L33F, L33M, K43T, M46I, M46L, I50V, I54A, I54L, I54M, I54S, I54T, I54V, L63P, G73A, G73S, G73T, T74S, V82A, V82F, V82I, V82S, V82T or I84V.

In another embodiment, the mutation associated with reduced susceptibility to treatment with said protease inhibitor is at amino acid position 10, 11, 32, 47, 53, 71 or 95 of the amino acid sequence of the protease of the HIV, with the proviso that said mutation is not V32I or I47V. In one embodiment, the mutation associated with reduced susceptibility to treatment with said protease inhibitor is selected from the group consisting of: L10F, F53L and A71L.

In another embodiment, the mutation associated with reduced susceptibility to treatment with said protease inhibitor is selected from the group consisting of: K20I, M46V, I50L, I54A, I54M, I54S, L63T, V82S, I84A, I84L, L33F, L33I, L33V, E34D, E34K, E34Q, K43T, G48V, I50L, I50V, K55R, Q58E, G73C, G73T, T74A, T74P, T74S, L76V, P79A, P79D, P79E, L89I and L89M. In another embodiment, the mutation is selected from the group consisting of: K20I, M46V, I50L, L63T, I84A, I84L, L33I, L33V, E34D, E34K, E34Q, I50V, I54M, G73C, T74A, T74P, L76V, P79A, P79D, P79E, L89I and L89M. In another embodiment, the mutation is selected from the group consisting of: K20I, M46V, I50L, L63T, I84A, I84L, L33I, L33V, E34D, E34K, E34Q, G73C, T74A, T74P, L76V, P79A, P79D, P79E, L89I and L89M.

In another embodiment, the human immunodeficiency virus is human immunodeficiency virus type 1 ("HIV-1").

In another aspect, the invention provides an isolated oligonucleotide encoding a protease in a HIV that comprises a mutation at amino acid position 20, 33, 34, 43, 46, 48, 50, 54, 55, 58, 63, 66, 73, 74, 76, 79, 82, 84 or 89 of an amino acid sequence of said protease in said human immunodeficiency virus, wherein the mutation is associated with reduced susceptibility to a protease inhibitor, with the proviso that said mutation is not K20M, K20R, M46I, M46L, I54L, I54T, I54V, L63P, V82A, V82F, V82T or I84. In one embodiment, the oligonucleotide is between about 10 and about 100 nucleotides long. In another embodiment, the oligonucleotide is between about 10 and about 90 nucleotides long. In another embodiment, the oligonucleotide is between about 10 and about 80 nucleotides long. In another embodiment, the oligonucleotide is between about 10 and about 70 nucleotides long. In another embodiment, the oligonucleotide is between about 10 and about 60 nucleotides long. In another embodiment, the oligonucleotide is between about 10 and about 50 nucleotides long. In another embodiment, the oligonucleotide is between about 10 and about 40 nucleotides long. In another embodiment, the oligonucleotide is between about 10 and about 30 nucleotides long. In another embodiment, the oligonucleotide is between about 10 and about 20 nucleotides long.

In another embodiment, the isolated oligonucleotide comprises mutation associated with reduced susceptibility to a protease inhibitor at amino acid position 20, 33, 34, 43, 46, 48, 50, 54, 55, 58, 63, 66, 73, 74, 76, 79, 82, 84 or 89 of an amino acid sequence of said protease in said HIV with the proviso that said mutation is not K20M, K20R, L33F, L33M, K43T, M46I, M46L, I50V, I54A, I54L, I54M, I54S, I54T, I54V, L63P, G73A, G73S, G73T, T74S, V82A, V82F, V82I, V82S, V82T or I84V. The oligonucleotide can be between about 10 and about 100, between about 10 and about 90, between about 10 and about 80, between about 10 and about 70, between about 10 and about 60, between about 10 and about 50, between about 10 and about 40, between about 10 and about 30 or between about 10 and about 20 nucleotides long.

In another embodiment, the isolated oligonucleotide encodes a protease in a HIV that comprises a mutation at codon 20, 33, 34, 43, 46, 48, 50, 54, 55, 58, 63, 66, 73, 74, 76, 79, 82, 84 or 89, wherein the mutation is associated with reduced susceptibility to protease inhibitor, with the proviso that the codons do not encode M or R at position 20, I or L at position 46, L, T or V at position 54, P at position 63, A, F or T at position 82 or V at position 84. The oligonucleotide can be between about 10 and about 100, between about 10 and about 90, between about 10 and about 80, between about 10 and about 70, between about 10 and about 60, between about 10 and about 50, between about 10 and about 40, between about 10 and about 30 or between about 10 and about 20 nucleotides long.

In another embodiment, the invention provides an isolated oligonucleotide encoding a protease in a HIV that comprises mutations at codon 20, 33, 34, 50, 54, 63, 66, 73, 74, 76, 79, 82 or 89, wherein the mutation is associated with reduced susceptibility to a protease inhibitor, with the proviso that the codons do not encode M or R at position 20, F or M at position 33, I or L at position 46, A, L, S, T or V at position 54, P at position 63, S or T at position 73, S at position 74, A, F or T at position 82 or V at position 84. The oligonucleotide can be between about 10 and about 100, between about 10 and about 90, between about 10 and about 80, between about 10 and about 70, between about 10 and about 60, between about 10 and about 50, between about 10 and about 40, between about 10 and about 30 or between about 10 and about 20 nucleotides long.

In another embodiment, the invention provides a polypeptide that comprises residues 1-10 of the amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises residues 11-20 of the amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises residues 21-30 of the amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises residues 31-40 of the amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises residues 41-50 of the amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises residues 51-60 of the amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises residues 61-70 of the amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises residues 71-80 of the amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises residues 81-90 of the amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises residues 91-99 of the amino acid sequence of SEQ ID NO:1.

In another embodiment, the polypeptide is at least 70%, but less than 100%, identical to a polypeptide having the amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1.

In one embodiment, the invention provides a method wherein the presence or absence of a mutation in a protease is detected by hybridization with a sequence-specific oligonucleotide probe to a nucleic acid sequence of human immunodeficiency virus encoding said mutation, wherein the occurrence of hybridization indicates said presence or absence of said mutation.

In another embodiment, the invention provides a method wherein the presence or absence of a mutation in a protease is detected by nucleic acid sequencing.

In one embodiment, the individual is undergoing or has undergone prior treatment with said or different protease inhibitor.

In one embodiment, the amino acid at position 20 of said protease is an amino acid having a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 20 of said protease is I. In another embodiment, the amino acid at position 33 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 33 of said protease is I, F or V. In another embodiment, the amino acid at position 33 of said protease is I or V. In another embodiment, the amino acid at position 34 of said protease is an amino acid having a basic, polar or hydrophilic side chain. In another embodiment, the amino acid at position 34 of said protease is K. In another embodiment, the amino acid at position 46 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 46 of said protease is V. In another embodiment, the amino acid at position 50 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 50 of said protease is L or V. In another embodiment, the amino acid at position 54 of said protease is an amino acid with a neutral, hydrophobic, non-polar, hydrophilic or polar side chain. In another embodiment, the amino acid at position 54 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 54 of said protease is A or M. In another embodiment, the amino acid at position 54 of said protease is M. In another embodiment, the amino acid at position 54 of said protease is an amino acid with a neutral, hydrophilic or polar side chain. In another embodiment, the amino acid at position 54 of said protease is S. In another embodiment, the amino acid at position 63 of said protease is an amino acid with a neutral, hydrophilic or polar side chain. In another embodiment, the amino acid at position 63 of said protease is T. In another embodiment, the amino acid at position 66 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 66 of said protease is F or V. In another embodiment, the amino acid at position 73 of said protease is an amino acid with a neutral, hydrophilic or polar side chain. In another embodiment, the amino acid at position 73 of said protease is C or T. In another embodiment, the amino acid at position 73 of said protease is C. In another embodiment, the amino acid at position 74 of said protease is an amino acid with a neutral, hydrophobic, non-polar, hydrophilic or polar side chain. In another embodiment, the amino acid at position 74 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 74 of said protease is A or P. In another embodiment, the amino acid at position 74 of said protease is an amino acid with a neutral, hydrophilic or polar side chain. In another embodiment, the amino acid at position 74 of said protease is S. In another embodiment, the amino acid at position 76 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 76 of said protease is V. In another embodiment, the amino acid at position 79 of said protease is an amino acid with a neutral, hydrophobic, non-polar, acidic, hydrophilic or polar side chain. In another embodiment, the amino acid at position 79 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 79 of said protease is A. In another embodiment, the amino acid at position 79 of said protease is an amino acid with an acidic, hydrophilic or polar side chain. In another embodiment, the amino acid at position 79 of said protease is D or E. In another embodiment, the amino acid at position 82 of said protease is an amino acid with a neutral, hydrophilic or polar side chain. In another embodiment, the amino acid at position 82 of said protease is S. In another embodiment, the amino acid at position 84 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 84 of said protease is L. In another embodiment, the amino acid at position 89 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 89 of said protease is I or M. In another embodiment, the amino acid at position 43 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 43 of said protease is T. In another embodiment, the amino acid at position 48 of said protease is an amino acid with a neutral, hydrophobic or non-polar side chain. In another embodiment, the amino acid at position 48 of said protease is V. In another embodiment, the amino acid at position 55 of said protease is an amino acid with a basic, hydrophilic or polar side chain. In another embodiment, the amino acid at position 55 of said protease is R. In another embodiment, the amino acid at position 58 of said protease is an amino acid with an acidic, hydrophilic or polar side chain. In another embodiment, the amino acid at position 58 of said protease is E.

In another aspect, the invention provides a method for detecting the presence or absence of a mutation associated with reduced susceptibility to treatment with said protease inhibitor at at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 of the amino acid positions. In one embodiment, the invention provides a method for detecting the presence or absence of a mutation associated with reduced susceptibility to treatment with said protease inhibitor at 2 or more amino acid positions. In another embodiment, the invention provides a method for detecting the presence or absence of a mutation associated with reduced susceptibility to treatment with said protease inhibitor at 3 or more amino acid positions. In another embodiment, the invention provides a method for detecting the presence or absence of a mutation associated with reduced susceptibility to treatment with said protease inhibitor at 4 or more amino acid positions. In another embodiment, the invention provides a method for detecting the presence or absence of a mutation associated with reduced susceptibility to treatment with said protease inhibitor at 5 or more amino acid positions. In another embodiment, the invention provides a method for detecting the presence or absence of a mutation associated with reduced susceptibility to treatment with said protease inhibitor at 6 or more amino acid positions. In another embodiment, the invention provides a method for detecting the presence or absence of a mutation associated with reduced susceptibility to treatment with said protease inhibitor at 7 or more amino acid positions. In another embodiment, the invention provides a method for detecting the presence or absence of a mutation associated with reduced susceptibility to treatment with said protease inhibitor at 8 or more amino acid positions. In another embodiment, the invention provides a method for detecting the presence or absence of a mutation associated with reduced susceptibility to treatment with said protease inhibitor at 9 or more amino acid positions.

In another aspect, the invention provides a method for determining whether a HIV has an increased likelihood of having a reduced susceptibility to treatment with a first protease inhibitor, comprising detecting in the protease of said HIV the presence or absence of a mutation associated with reduced susceptibility to treatment with a second protease inhibitor at amino acid position 10, 11, 32, 33, 34, 43, 46, 47, 48, 50, 54, 58, 71, 76, 79, 82, 84 or 95 of the amino acid sequence of said protease, wherein the presence of said mutation indicates that the HIV has an increased likelihood of having reduced susceptibility to treatment with said first protease inhibitor, with the proviso that said mutation is not V32I, M46I, M46L, I47V, I50V, I54L, I54M, V82A, or I84V. In one embodiment, the first protease inhibitor is lopinavir or amprenavir. In another embodiment, the second protease inhibitor is lopinavir or amprenavir.

In another aspect, the invention provides a method of determining whether an individual infected with HIV has an increased likelihood of having a reduced susceptibility to treatment with a first protease inhibitor, comprising detecting, in a sample from said individual, the presence or absence of a mutation associated with reduced susceptibility to treatment with a second protease inhibitor at amino acid position 10, 11, 32, 33, 34, 43, 46, 47, 48, 50, 54, 58, 71, 76, 79, 82, 84 or 95 of the amino acid sequence of the protease of the HIV, wherein the presence of said mutation indicates that the individual has an increased likelihood of having reduced susceptibility to treatment with said first protease inhibitor, with the proviso that said mutation is not V32I, M46I, M46L, I47V, I50V, I54L, I54M, V82A, or I84V. In one embodiment, the first protease inhibitor is lopinavir or amprenavir. In another embodiment, the second protease inhibitor is lopinavir or amprenavir.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation of the genomic structure of HIV-1.

FIG. 2 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples obtained from a training data set with 2038 patients as a function of the number of the resistance-associated mutations in the protease. The genotypic "cutoff value" is 6, i.e., a HIV is defined as genotypically resistant to lopinavir if its total score is 6 or greater and genotypically sensitive if its total score is less than 6.

FIG. 3 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples as a function of the number of the resistance-associated mutations in the protease after the removal of samples containing mixtures of amino acids at any of the positions associated with reduced susceptibility to lopinavir. Those samples that contained both a wild-type and a mutant were excluded from the analysis.

FIG. 4 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples containing the mutation I50V in the protease as a function of the total number of the resistance-associated mutations in those samples.

FIG. 5 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples containing the mutations V82A, F, S, T or I in the protease as a function of the total number of the resistance-associated mutations in those samples.

FIG. 6 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples containing the mutations I54A, L, M, S, T or V in the protease as a function of the total number of the resistance-associated mutations in those samples.

FIG. 7 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples as a function of the number of the resistance-associated mutations in the protease after the removal of samples without any primary mutations associated with protease inhibitors and without an IC.sub.50 fold change ("FC") greater than two for any protease inhibitor.

FIG. 8 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples as a function of the number of the resistance-associated mutations in the protease after the removal of samples without any primary mutations associated with protease inhibitors and without an IC.sub.50 fold change ("FC") greater than two for any protease inhibitor and the removal of samples containing mixtures of amino acids at any of the positions associated with reduced susceptibility to lopinavir. Only those samples that contained both, a wild-type or reference strain and a mutant were removed.

FIG. 9 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples containing the mutation I50V in the protease as a function of the total number of the resistance-associated mutations in those samples after the removal of samples without any primary mutations associated with protease inhibitors and without an IC.sub.50 fold change ("FC") greater than two for any protease inhibitor.

FIG. 10 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples containing the mutations V82A, F, S, T or I in the protease as a function of the total number of the resistance-associated mutations in those samples after the removal of samples without any primary mutations associated with protease inhibitors and without an IC.sub.50 fold change ("FC") greater than two for any protease inhibitor.

FIG. 11 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples containing the mutations I54A, L, M, S, T or V in the protease as a function of the total number of the resistance-associated mutations in those samples after the removal of samples without any primary mutations associated with protease inhibitors and without an IC.sub.50 fold change ("FC") greater than two for any protease inhibitor.

FIG. 12A shows the amino acid sequence of the NL4-3 HIV (GenBank Accession No. AF324493) protease (SEQ. ID. NO: 1).

FIG. 12B shows the nucleic acid sequence for the NL4-3 HIV (GenBank Accession No. AF324493) protease gene (SEQ. ID. NO: 2).

FIG. 13 shows the effect of the amino acid at position 82 on lopinavir fold change. The median (horizontal line), 25th and 75th percentile (box), 10th and 90th percentile (whiskers) and outliers (dots) are shown.

FIG. 14 shows the effect of amino acid at position 54 on lopinavir fold change. The median (horizontal line), 25th and 75th percentile (box), 10th and 90th percentile (whiskers) and outliers (dots) are shown.

FIG. 15 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples obtained from a training data set with 2195 patients as a function of the number of the resistance-associated mutations in the protease. The genotypic "cutoff value" is 8, i.e., a HIV is genotypically resistant to lopinavir if its total score is 8 or greater and genotypically sensitive if its total score is less than 8.

FIG. 16 is a scatter plot that shows the susceptibility to lopinavir (Log lopinavir fold change) of HIV samples obtained from data set of 1099 samples as a function of the number of the resistance-associated mutations in the protease. The genotypic "cutoff value" is 7, i.e., a HIV is genotypically resistant to lopinavir if its total score is 7 or greater and genotypically sensitive if its total score is less than 7.

FIG. 17 shows the effect of mutations associated in HIV with resistance to Amprenavir ("APV") on resistance to lopinavir. The median (horizontal line), 25th and 75th percentile (box), 10th and 90th percentile (whiskers) and outliers (dots) are shown.

FIG. 18 shows a bivariate scatter plot of lopinavir fold change ("log LPV") versus amprenavir fold change ("log APV").

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for developing an algorithm for determining the effectiveness of anti-viral drugs based on a comprehensive analysis of paired phenotypic and genotypic data guided by phenotypic clinical cut-offs. The present invention also provides methods for determining the susceptibility of a virus to an anti-viral treatment, methods for determining the effectiveness of an anti-viral treatment of an individual infected with a virus, and methods of monitoring the clinical progression of viral infection in individuals receiving anti-viral treatment. In another aspect, the present invention also provides nucleic acids and polypeptides comprising a mutation in the protease of a human immunodeficiency virus ("HIV") associated with reduced susceptibility to protease inhibitors, e.g., lopinavir.

5.1 Abbreviations

"LPV" is an abbreviation for the protease inhibitor lopinavir.

"APV" is an abbreviation for the protease inhibitor amprenavir.

"PI" is an abbreviation for protease inhibitor.

"PT-R" and "PT-S" are abbreviations for "phenotypically resistant" and "phenotypically sensitive," respectively.

"GT-R" and "GT-S" are abbreviations for "genotypically resistant" and "genotypically sensitive," respectively.

"PCR" is an abbreviation for "polymerase chain reaction."

"FC" is an abbreviation for "fold change."

The amino acid notatio