MXPA00011623A - Means and methods for monitoring non-nucleoside reverse transcriptase inhibitor antiretroviral therapy. - Google Patents

Abstract

This invention relates to antiviral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of human immunodificiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) and further relates to the means and methods of monitoring the clinical progression of HIV infection and its response to antiretroviral therapy, particularly non-nucleoside reverse transcriptase inhibitor therapy using phenotypic susceptibility assays or genotypic assays.

Description

MEANS AND METHODS TO VERIFY ANTIRETROVIRAL THERAPY WITH NON-NUCLEOSIDIC INHIBITOR OF THE INVERSE TRANSCRIPTASE This application is a continuation in part of U.S. Patent Serial No. 09 / 085,148, filed on May 26, 1998 and claims the benefits of U.S. Provisional Patent Application No. 60 / 124,090, filed on March 12, 1999, the content of which is therefore incorporated as a reference in this application. Through this Application, several references are mentioned in parentheses. The descriptions of those publications in their entirety are therefore incorporated by reference in this application to more fully describe the state of the art to which this invention pertains.

TECHNICAL FIELD This invention relates to tests of susceptibility and resistance to antiretroviral drugs to be used in the identification of effective drug regimens for the treatment of infection by the human immunodeficiency virus (HIV) and the syndrome of in-one-disease acquired (AIDS). The invention also relates to means and methods for verifying the clinical progress of HIV infection and its response to antiretroviral therapy using phenotypic or genotypic susceptibility assays. The invention also relates to vectors, host cells and novel compositions for carrying out phenotypic susceptibility tests. The invention also relates to the use of various genotypic methodologies to identify patients whose infection has become • 10 resistant to a particular antiretroviral drug regimen. This invention also relates to the separation of candidate antiretroviral drugs for their ability to inhibit viruses, selected from viral sequences and / or viral proteins. More In particular, this invention relates to the determination of phenotypic susceptibility tests • and / or genotypic tests that use resistance to the non-nucleoside inhibitor of reverse transcriptase.

Background of the Invention HIV infection is characterized by the high rates of viral turnover through the disease process, which eventually lead to CD4 depletion and the progression of the disease. ei X, 25 Ghosh SK, Taylor Shorter length, et al (1995) Na ture 343, 117-122 and Ho DD, Naumann AU, Perelson AS, et al. (1995) Na ture 373, 123-126. The main purpose of therapy • Antiretroviral therapy is to achieve a substantial and prolonged suppression of viral reproduction. Achieving sustained control probably involves the use of sequential therapies, generally each therapy comprising combinations of three or more antiretroviral drugs. The choice of initial and subsequent therapy, therefore, should be made on a rational basis, with the • 10 knowledge of the patterns of resistance and cross resistance that are vital to guide those decisions. The main reason for the combination therapy is related to the synergistic or additive activity to achieve a greater inhibition of viral reproduction. The tolerance 15 of the drug regimens will remain critical, however, since the therapy needs to be-maintained • for many years. In an untreated patient, something like 1010 viral particles per day is produced. Coupled with the 20 HIV transcriptase (RT) failure to correct transcription errors by correction of exonucleolytic tests, this high level of viral turnover results in 104 to 105 mutations per day at each position in the HIV genome. The result is the fast 25 establishment of an extensive genotypic variation.

Although some standard positions or substitutions of base pairs may be more prone to errors (Mansky LM, Temin HM (1995) J Virol 69, 5087-5094) (Schinazi RF, Loyod RM, Ramanathan CS, et al (1994) Antimicrob Agents Chemother 38, 268-274), mathematical models suggest that, at each possible single point, a mutation can occur up to 10,000 times per day in infected individuals. For resistance to the antiretroviral drug to occur, the target enzyme must be modified and this function preserved in the presence of the inhibitor. Mutations that lead to the substitution of an amino acid can result in changes in the shape, size or charge of the active site, the binding site of the substrate or surrounding regions of the enzyme. Mutants resistant to retroviral agents have been detected at levels before the start of therapy.

(Mohri H, Singh MK, Ching T, et al. (1993) Proc Na ti Acad Sci USA 90, 25-29) (Nájera I, Rich an DD, Olivares I, et al. (1994) AIDS Res Hum Retroviruses 10, 1479-1488) (Nájera I, Holguin A, Quiñones-Mateu E, et al. . (1995) J Virol 69, 23-31). However, those mutant strains represent only a small proportion of the total viral load and may have a competitive reproduction or disadvantage compared to the wild type virus (Coffin JM (1995) Science 267, 483-489). The selective pressure of antiretroviral therapy gives these drug-resistant mutants a competitive advantage and thus they become quasi-dominant species (Frost SDW, McLean Ar (1994) AIDS 8, 323-332) (Kellam P, Boucher CAB, Tijnagal JMGH (1994) J Gen Virol 75, 341-351), ultimately leading to drug resistance and virological failure in the patient.

Non-Nucleoside Inhibitors of Reverse Transcriptase Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a chemically diverse group of compounds which are potent inhibitors of HIV-1 RT in vitro. These compounds include the pyridinone derivatives, bis (heteroaryl) piperazines (BHAP) such as delavirdine and atevirdine, dipyridodiazepinone, nevirapine, the thymine derivative groups TSAO and HEPT, and the a-anilino phenylacetate compound. (a-APA) loviride, and inhibitors of the quinoxaline class such as (HBY-097), the benzodiazepinone-and--thione compounds (TIBO) and the pyridinone derivatives (L-697, 661). For other reviews see (DeClerq E. (1996) Rev Med Virol 6, 97-117) (Emini EA (1996) Aniviral Drug Resistance, ed DD Richman, John Wiley &Sons, Ltd. The high level of resistance to individual compounds seems • develop rapidly, often within a few weeks of initiating monotherapy, often involving only single point mutations and in many cases leading to considerable cross-resistance to other NNRTIs. Most of the reported mutations occur in groups 100-108 and 181-190 of the codon coding for the two β-sheets adjacent to the site 10 catalytic RT enzyme (Kohlstaedt LA, Wang J, Fried an JM et al. (1992) Science 256, 1783-90). The NNRTI binding receptacle, as has been described, is a binding region without hydrophobic substrate of the RT where these agents interact directly with the RT. 15 They inhibit activity by interfering with the mobility of the subdomain "thumb" or the disturbance of the • orientation of the conserved aspartic acid side chains essential for catalytic activity (D'Arquilla RT. (1994) Clin Lab Med 14, 393-423) (Arnold 20 E., Ding J., Hughes SH, et al. (1995) Curr Opin Struct Biol 5, 27-38) Curr Opin Struct Biol 5, 27-38). Mutations conferring reduced susceptibility to nevirapine have been described in codons 98, 100, 103, 106, 108, 181, 188 and 190 (Richman 25 DD, Havlir D, Corbeil J. (1994) J Virol 68, 1660-1666).

The most frequent variant selected during monotherapy with nevirapine is a Tyr181_Cys (Y181C) mutation which results in a 100-fold reduction in sensitivity to this agent, with reduced susceptibility to the pyridinone derivatives L-696, 229 and L- 697, 661 (Arnold, Ibid). TSAO also has limited activity in the presence of the 181 mutation, but maintains activity in the presence of mutations with codons 100 and 103 and selects a mutation in vitro • 10 unique, GLU138_Lys (E138K), in the region where it interacts most closely with RT (Richman, DD, Ibid) (Richamn DD, Shih CK, Lo and I, et al. (1991) Proc Na ti Acad Sci USA88 11241-11245). Resistance to loviride used as 15 monotherapy develops in the majority of patients by week 24. This has been treated in • a range of codons 100-110; 181-190), most commonly codon 103 (Staszewski S, Miller V, Kober A, et al. (1996) Theriviral Therl, 42-50. 20 combined using loviride with zidovudine or zidovudine plus lamivudine, variants at codons 98 and 103 were defects due to more frequent mutations at 24 weeks (Staszwesk, S, Miller V, Rehmet S, et al., 1996) AIDS 10, Fl- 7).

Although mutations 101, 103 and 181 also confer cross-resistance to BHAP (Balzarini J, Karlsson A, Pérez-Pérez MJ, et al. (1992) Virology 192, 246-253) the substitution of characteristic P236L selected by these agents in vi tro seems to sensitize the RT to some other NNRTI, reducing the IC50 for nevirapine, for example, from 7 to 10 times, without having influence on the sensitivity to nucleoside analogues (Stasweski, S., Ibid). This mutation in the • 10 codon 236 has not been observed in clinical isolates during therapy with atevirdine, although other mutations have been reported in codons 103, and 181 that confer resistance during monotherapy as well as codons 101, 188, 233 and 238 during the therapy 15 combined with zidovudine. Although the HBY-097 can initially • select a mutation in codons 190 in vi tro, an additional pass consistently selects mutations in codons 74 and 75 of RT, with some mutant viruses 20 showing decreased sensitivity to didanosine and stavudine, but not to zidovudine (Kleim J-P, Roser M, Winkler I, et al. (1995) J Acquir Immune Defic Syndr 10 Suppl 3.2). It has been reported that the mutation at codon 181 25 antagonizes resistance to zidovudine due to mutations at typical codons 41 and 215, (Zhang D, Caliendo AM, Eron JJ, et al. (1994) Antimicrob Agents Chemother 38, 282-287) suggesting that the combination therapy with Some NNRTI and zidovudine may be feasible. Although the HIV mutant with triple resistance to zidovudine, didanosine and nevirapine has been described in vi tro (Larder BA, Kellam P, Kemp SD (1993) Na ture 365,451-453) treatment with this triple combination provides immunological and virological responses higher than the treatment with zidovudine plus didanosine only during a period of 48 weeks in patients with CD4 counts of < 350 / mm3. The combination therapy with zidovudine and the pyridinone derivative L-697, 661 prevents the appearance of the mutation of codon 181 typically selected during monotherapy with this NNRTI, and retarding the onset of high level resistance to this compound. Changes in susceptibility to zidovudine were not examined in this study. (Staszewski S, Massari FE, Kober A, et al. (1995) J Infect Dis 171, 1159-1165). Concomitantly or alternatively, zidovudine therapy does not delay the onset of resistance during therapy with nevirapine; (Richman DD, Ibid) (Nunberg JH, Schleif WA, Boots EK, et al. (1990) J Virol 65, 4887-4892) (DeJong MD, Loewenthl M, Boucher CAB, et al. (1994) J Infect Dis 169 , 1346-1350) (Cheese an SH, Havlir D, McLaughlin MM, et al. (1995) J Acquir Immune Defic Syndr 8, 141-151), however, mutant 181 is not observed during the combination, making the change most common in codon 190 (Richman DHP, Ibid). This suggests that the mutation of codon 181 is antagonistic to resistance to zidovudine in vitro, is a selection compatible, or not preferred in vivo, that favors other mutations that allow the reduction in susceptibility to this NNRTI concomitant with resistance to zidovudine . The rapid development of lower susceptibility to NNRTI suggests limited utility of these agents, particularly with monotherapies, and has led to the modification of these molecules in an attempt to delay the emergence of the drug-resistant virus. A second generation of NNRTI, the pyridinone derivative L-702.019, demonstrated only a three-fold change in IC50 between natural HIV-1 and the mutant at codon 181, and required multiple mutations to generate high-level resistance (Goldman ME, 0 'Brian JA, Ruffing TL, et al. (1993) Animicrob Agents Chenother 37, 947-949). An object of this invention is to provide a drug susceptibility and resistance test capable of showing whether a viral population in a patient is resistant to a given prescribed drug. Another object of this invention is to provide a test that will allow • the physician substitute one or more drugs in a therapeutic regimen for a patient who has become resistant to a drug or drugs given after the course of therapy. Yet another object of this invention is to provide a test that will allow the selection of an effective drug regimen for the treatment of HIV infections. 10 and / or AIDS. Still another object of this invention is to provide the means to identify the drugs to which the patient has become resistant, in particular to identify resistance to non-nucleoside inhibitors of reverse transcriptase. Another object even more of this The invention is to provide a test and methods for evaluating the biological effectiveness of pharmaceutical compounds • candidates acting on viruses, viral genes and / or specific viral proteins particularly with respect to resistance to viral drugs associated with 20 non-nucleoside inhibitors of reverse transcriptase. It is also an object of this invention to provide the means and compositions for evaluating the resistance and susceptibility of HIV to antiretroviral drugs. These and other objects of this invention will be apparent from 25 of the specification as a whole.

Brief Description of the Invention The present invention relates to methods • to verify, using phenotypic and genotypic methods, the clinical progress of infection by human immunodeficiency virus and its response to antiviral therapy. The invention is also based, in part, on the discovery that genetic changes in HIV reverse transcriptase (RT) that confers resistance to antiretroviral therapy may be • determined directly from the HIV RNA of the patient's plasma using phenotypic or genotypic methods. The methods use the polymerase chain reaction (PCR). Alternatively, methods to evaluate viral protein viral nucleic acid in The absence of an amplification step could use the teachings of this invention to verify and / or modify • antiretroviral therapy. This invention is based in part on the discovery of a mutation at codon 225, either alone or in combination with a mutation in the 20 codon 103 of HIV reverse transcriptase in patients treated with non-nucleoside inhibitor (efavirenz) of reverse transcriptase, in which the presence of mutations correlate with an increase in susceptibility to delavirdine and little 25 or no change in susceptibility to nevirapine.

Mutations were found in HIV RNA in plasma after a period of time after initiation * of therapy. It was found that the development of the codon 225 mutant in addition to the mutation in codon 103 in the 5 RT of HIV was an indicator of the development of resistance and finally of immunological decline. This invention is based in part on the discovery of a mutation at codon 236 of the RT that was discovered to occur in patients treated with non-nucleoside inhibitor of the • 10 reverse transcriptase (NNRTI) in which the presence of the mutation correlates with a decreased susceptibility to delavirdine and an absence of reduced susceptibility to nevirapine. It was found that the development of mutations in the codon 15 190 103 and / or 101 of the HIV RT was an indicator of the development of alterations in susceptibility. • • phenotypic resistance that has been associated with virological failure and subsequent immunological decline. This invention is based in part on the discovery of A mutation in codon 190 alone or in combination with a mutation in codon 190 alone or in combination with a mutation in codon 103 and / or 101 of HIV reverse transcriptase in patients treated with non-nucleoside inhibitor (efavirenz) of the reverse transcriptase in 25 the presence of mutations it correlates with an increased susceptibility to delavirdine and a decrease in susceptibility to nevirapine. Mutations were found in HIV RNA in plasma after a period of time after the start of NNRTI therapy. It was found that the development of codon 236 and 103 and / or 181 mutations in HIV RT was an initiator of the development of alterations in phenotypic susceptibility / resistance that has been associated with virological failure and subsequent immunological decline. This invention is based in part on the discovery of a mutation in codon 230 alone or in combination with a mutation in codon 181 of HIV reverse transcriptase in patients treated with non-nucleoside inhibitor (nevirapine) of reverse transcriptase in which the presence of mutations correlates with a significant decrease in susceptibility to both delavirdine and nevirapine. Mutations were found in the HIV RNA after a period of time after the start of NNRTI therapy. It was found that the development of mutations in codon 230 and 181 of HIV RT was an indicator of the development of alterations in phenotypic susceptibility / resistance that has been associated with virological failure and subsequent immunological decline. This invention is based in part on the discovery of a mutation at codon 181 of • HIV reverse transcriptase in patients treated with non-nucleoside inhibitor (nevirapine) reverse transcriptase in whom the presence of the mutation correlates with a moderate decrease in susceptibility to delavirdine and a significant decrease in susceptibility to nevirape and without • 10 changes to susceptibility to efavirenz. The mutation was found in HIV RNA in plasma after a period of time after the start of NNRTI therapy. It was found that the development of codon 181 mutation in HIV RT was an indicator of 15 development of alterations in susceptibility / phenotypic resistance that has been • associated with virological failure and subsequent immunological decline. This invention is based in part on the discovery of a mutation at codon 188 of 20 HIV reverse transcriptase in patients treated with non-nucleoside inhibitor (efavirenz) reverse transcriptase in whom the presence of the mutation correlates with a slight decrease in susceptibility to delavirdine and a decrease 25 substantial in susceptibility to nevirapine. The mutation was found in HIV RNA in plasma after a period of time after the start of NNRTI therapy. It was found that the development of codon 188 mutation in HIV RT was an indicator of the development of alterations in phenotypic susceptibility / resistance that has been associated with virological failure and subsequent immunological decline. This invention is based in part on the discovery of a mutation in codon 188 of HIV reverse transcriptase in patients without previously reported exposure to non-nucleoside reverse transcriptase inhibitors in which the presence of mutations correlates with a moderate decrease in susceptibility to delavirdine and a substantial decrease in susceptibility to nevirapine and a moderate decrease in susceptibility to efavirenz. The mutation was found in HIV RNA in plasma after a period of time after the start of antiretroviral therapy. We found that the development of codon 138 and 188 mutations in HIV RT was an indicator of the development of alterations in phenotypic susceptibility / resistance that has been associated with virological failure and subsequent immunological decline. This invention is based in part on the discovery of a mutation in codon 98 of HIV reverse transcriptase in patients without previously reported exposure to non-nucleoside reverse transcriptase inhibitors in which the presence of the mutation correlates with a slight decrease in susceptibility to delavirdine, nevirapine and efavirenz. The mutation was found in HIV RNA in plasma after a period of time after the start of antiretroviral therapy. It was found that the development of codon 198 mutation in HIV RT was an indicator of the development of alterations in phenotypic susceptibility / resistance that has been associated with virological failure and subsequent immunological decline. This invention is based in part on the discovery of a new mutation in codon 98 alone or in combination with a mutation in codon 190 of HIV reverse transcriptase in patients whose antiretroviral treatment was unknown in whom the presence of mutations was correlates with an increased susceptibility to delavirdine and a substantial decrease in susceptibility to both nevirapine and efavirenz. Mutations were found in HIV RNA in plasma. It was found that the development of the mutant of codon 98 in addition to the mutation in codon 190 of the RT of "HIV is an indicator of the development of resistance and finally of immunological decline." This invention is based in part on the discovery of a mutation in codon 181 alone or in combination with a mutation in codon 98 of HIV reverse transcriptase in patients treated with non-nucleoside inhibitor (delavirdine) of reverse transcriptase in which the presence of mutations correlates with a decrease • Significant in susceptibility to delavirdine and a substantial decrease in susceptibility to efavirenz. Mutations were found in HIV RNA in plasma after a period of time of initiation of therapy. It was found that the development of the mutant 15 in codon 98 in addition to the mutation in codon 181 in HIV RT was an indicator of the development of • resistance and finally of immunological decline. This invention is based in part on the discovery of a mutation in codon 101 alone or in combination with a 20 mutation in codon 190, for example 190s of HIV reverse transcriptase in patients treated with non-nucleoside inhibitor (efavirenz) of reverse transcriptase in which the presence of the mutations correlates with changes in the susceptibility to 25 delavirdine and a substantial decrease in susceptibility to both nevirapine and efavirenz. Mutations were found in HIV RNA in plasma after a period of time after the start of therapy. It was found that the development of the mutant in codon 101 in addition to the mutation in codon 190, for example 190s in HIV RT, was an indicator of the development of resistance and finally of immunological decline. This invention is based in part on the discovery of a mutation at codon 108 of the • 10 HIV reverse transcriptase in patients without previously reported exposure to non-nucleoside reverse transcriptase inhibitor in whom the presence of the mutation does not correlate with changes in susceptibility to delavirdine and a slight decrease 15 in susceptibility to nevirapine and no change in susceptibility to efavirenz. The mutation was found • in HIV RNA in plasma after a period of time after the start of antiretroviral therapy. It was found that the mutation development of codon 108 20 of HIV RT was an indicator of the development of alterations in phenotypic susceptibility / resistance that has been associated with virological failure and the subsequent immunological decline. This invention is based in part on the 25 discovery of a mutation in codon 101 alone or in combination with a mutation in codon 103 and / or 190 of HIV reverse transcriptase in patients with • previously unreported exposure to non-nucleoside reverse transcriptase inhibitors in which 5 the presence of mutations correlates with changes in susceptibility to delavirdine, nevirapine, and efavirenz. Specifically, the presence of mutations in 101 and 190, for example 190A, is not related to changes in susceptibility to delavirdine and a • 10 substantial decrease in susceptibility to nevirapine and a significant decrease in susceptibility to efavirenz. The presence of mutations in 103 and 190 correlates with a moderate decrease in susceptibility to delavirdine, a decrease 15 substantial in susceptibility to nevirapine and a significant decrease in susceptibility to • efavirenz. Mutations were found in HIV RNA in plasma after a period of time after the start of antiretroviral therapy. It was found that the The development of mutations in codon 101 and 103 and / or 190 in HIV RT is an indicator of the development of alterations in phenotypic susceptibility / resistance that has been associated with virological failure and subsequent immunological decline. This invention is 25 is based in part on the discovery of a mutation in codon 106 alone or in combination with a mutation in codon 189 and / or 181 and 227 of HIV reverse transcriptase in patients treated with non-nucleoside inhibitor (nevirapine) of transcriptase Inverse, in which the presence of mutations correlates with changes in susceptibility to delavirdine, nevirapine and efavirenz. Specifically, the presence of mutations in 106 and 181 correlates with a significant decrease in susceptibility to delavirdine, a substantial decrease in susceptibility to nevirapine and a slight decrease in susceptibility to efavirenz. The presence of mutations in 106 and 189 correlates with a slight decrease in susceptibility to delavirdine, a moderate decrease in susceptibility to nevirapine, and no change in susceptibility to efavirenz. The presence of mutations in 106 and 227 correlated with a slight decrease in susceptibility to delavirdine, a substantial decrease in susceptibility to nevirapine, and a slight decrease in susceptibility to efavirenz. The presence of mutations in 181 and 227 is correlated with an increase in susceptibility to delavirdine, a significant decrease in susceptibility to nevirapine and an increase in susceptibility to efavirenz. The presence of mutations in 106 and 181 and 227 correlates with a moderate decrease in susceptibility to delavirdine, a decrease • Substantial susceptibility to nevirapine and a slight decrease in susceptibility to efavirenz. Mutations were found in HIV RNA in plasma after a period of time after the start of NNRTI therapy. It was found that the development of mutations in codon 106 and 189 and / or 181 and 227 is a # 10 indicator of the development of alterations in susceptibility / phenotypic resistance that has been associated with virological failure and subsequent immunological decline. This invention is based in part on the discovery of a mutation in codon 103 alone 15 or in combination with a mutation in codon 100 and / or 188 of HIV reverse transcriptase in treated patients • with non-nucleoside inhibitor (nevirapine) of reverse transcriptase, in which the presence of mutations correlates with changes in the 20 susceptibility to delavirdine, to nevirapine and efavirenz. Specifically, the presence of mutations in 103 and 188 correlates with a substantial decrease in susceptibility to delavirdine, a substantial decrease in susceptibility to nevirapine, and a 25 Substantial decrease in susceptibility to efavirenz.

The presence of mutations in 100 and 103 correlates with a substantial decrease in susceptibility to delavirdine, a moderate decrease in susceptibility to nevirapine, and a substantial decrease in susceptibility to efavirenz. The presence of mutations in 103 and 100 and 188 correlates with a substantial decrease in susceptibility to delavirdine, a substantial decrease in susceptibility to nevirapine and a substantial decrease in susceptibility to efavirenz. Mutations were found in HIV RNA in plasma after a period of time after the start of NNRTI therapy. It was found that the development of mutations in codon 103 and / or 188 in HIV RT is an indicator of the development of alterations in phenotypic susceptibility / resistance that has been associated with virological failure in the subsequent immunological decline. In a further embodiment of the present invention, PCR-based assays can be used, including phenotypic and genotypic assays to detect codon 225 mutations in combination with mutations in other codons including 103 of HIV RT that correlate with a specific pattern of resistance to antiretroviral therapies and the subsequent immunological decline. In another embodiment of the invention, PCR-based assays can be used, • including phenotypic and genotypic assays to detect mutations in codon 236 alone or in combination with mutations in other codons including 103 and / or 181 of HIV RT and immunological decline. In yet another embodiment of the invention, PCR-based assays, including phenotypic and genotypic assays, can be used to detect F 10 mutations in codon 190 (G190S) alone or in combination with a mutation in codon 101 (K101E) of HIV RT that correlate with resistance to antiretroviral therapy and immunological decline. In still another embodiment of the invention, 15 can be used based on PCR assays, including phenotypic and genotypic assays for F detect mutations in codon 190 (G190A) alone or in combination with a mutation in codon 103 (K103N) of HIV RT that correlates with resistance 20 antiretroviral therapy and immunological decline. In yet another embodiment of the invention, PCR-based assays may be used, including phenotypic and genotypic assays to detect mutations at codon 230 alone or in combination 25 with mutations in codon 181 of HIV RT that correlate with resistance to antiretroviral therapy and immunological decline. In yet another embodiment of the invention, they can • PCR-based assays should be used, including 5 phenotypic and genotypic assays to detect a mutation in codon 181 of HIV RT that correlates with resistance to antiretroviral therapy and immunological decline. In yet another embodiment of the invention, they can 10 PCR-based assays, including phenotypic and genotypic assays, should be used to detect a mutation at codon 188 of HIV RT that correlates with resistance to antiretroviral therapy and immunological decline. In yet another embodiment of the invention, PCR-based assays, including • phenotypic and genotypic assays to detect mutations in codon 138 alone or in combination with mutations in codon 188 of HIV RT that is 20 correlate with resistance to antiretroviral therapy and immunological decline. In yet another embodiment of the invention, PCR-based assays may be used, including phenotypic and genotypic assays to detect a 25 mutation in codon 98 of HIV RT that correlates with resistance to antiretroviral therapy and immunological decline. In yet another embodiment of the invention, PCR-based assays can be used, including phenotypic and genotypic assays to detect mutations in codon 98 alone or in combination with mutations in codon 190 of HIV RT that correlate with resistance to antiretroviral therapy and immunological decline. In yet another embodiment of the invention, PCR-based assays can be used, including phenotypic and genotypic assays to detect mutations in codon 181 alone or in combination with mutations in codon 98 of HIV RT that correlate with resistance to antiretroviral therapy and immunological decline. In yet another embodiment of the invention, PCR-based assays, including phenotypic and genotypic assays for ~ detecting mutations in codon 101 alone or in combination with a mutation in codon 190, eg, 190s of HIV RT, can be used. which correlates with resistance to antiretroviral therapy and immunological decline. In yet another embodiment of the invention, PCR-based assays, including phenotypic and genotypic assays, can be used to detect a mutation at codon 108 of HIV RT that correlates with resistance to therapy • antiretroviral and immunological decline. In yet another embodiment of the invention, PCR-based assays can be used, including phenotypic and genotypic assays to detect mutations in codon 101 alone or in combination with a mutation in codon 103 and / or 190 of HIV RT • 10 that correlates with resistance to antiretroviral therapy and immunological decline. In yet another embodiment of the invention, PCR-based assays may be used, including phenotypic and genotypic assays to detect 15 mutations in codon 106 alone or in combination with a mutation at codon 189 and / or 181 and 227 of the RT of the • HIV that correlates with resistance to antiretroviral therapy and immunological decline. In yet another embodiment of the invention, they can 20 PCR-based assays, including phenotypic and genotypic assays to detect mutations in codon 188 alone or in combination with a mutation in codon 100 and / or 103 of HIV RT that correlates with resistance therapy, should be used. 25 antiretroviral and immunological decline. Once the mutations in codon 225 and 103 have been detected in a patient undergoing antiretroviral therapy with NNRTI, it should be considered an alteration in the therapeutic regimen. Similarly, once mutations have been detected at codon 236 and / or 103 and / or 181 in a patient undergoing certain retroviral therapy with NNRTI, it must be considered an alteration in the therapeutic regimen. Similarly, once mutations have been detected in codon 190 and / or 103 and / or 101 in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once mutations have been detected in codon 230 and / or 181 in a patient undergoing certain retroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once a mutation in codon 181 has been detected in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once a mutation in codon 188 has been detected in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once mutations have been detected in codon 138 and / or 188 in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once a mutation in codon 98 has been detected in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once mutations have been detected in codon 98 and / or 190 in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once mutations have been detected in codon 181 and / or 98 in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once mutations have been detected in codon 101 and / or 190, for example 190S, in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once a mutation in codon 108 has been detected in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once mutations have been detected in codon 101 and / or 103 and / or 190, for example 190A, in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once mutations have been detected in codon 106 and / or 189 and / or 181 and / or 227 in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. Similarly, once mutations have been detected in codon 188 and / or 100 and / or 133 in a patient undergoing certain antiretroviral therapy with NNRTI, an alteration in the therapeutic regimen should be considered. The time at which a modification of the therapeutic regimen should be made after the evaluation of antiretroviral therapy using PCR-based assays may depend on several factors, including the patient's viral load, CD4 count and previous treatment history. . In another aspect of the invention, there is provided a method for evaluating the effectiveness of a non-nucleoside antiretroviral drug for reverse transcriptase comprising: (a) introducing a resistance test vector comprising a segment derived from the patient and a reporter gene in a a host cell; (b) culturing the host cell of step (a); (c) measuring the expression of the reporter gene in a target host cell wherein the expression of the reporter gene depends on the segment derived from the patient; and (d) comparing the expression of the reporter gene from step (c) with the expression of the measured reporter gene when steps (a) - (c) are carried out in the presence of the anti-HIV NNRTI drug, where the concentration of the Drug test NNRTI, anti-HIV is presented in steps (a) - (c); in steps (b) - (C); or in step (c). This invention also provides a method for evaluating the effectiveness of non-nucleoside antiretroviral therapy for reverse transcriptase in a patient, comprising: (a) developing a standard curve of drug susceptibility for an anti-HIV NNRTI drug; (b) determining the susceptibility to the anti-HIV NNRTI drug in the patient using the susceptibility test described above; and (c) comparing the susceptibility to the anti-HIV NNRTI drug in step (b) with the standard curve determined in step (a), where a decrease in NNRTI susceptibility indicates the development of resistance to the anti-HIV drug in the patient. This invention also provides a method for evaluating the biological effectiveness of an antiretroviral drug compound against HIV candidate comprising: (a) introducing a resistance test vector comprising a segment derived from a patient and a reporter gene in a host cell; (b) F cultivate the host cell of step (a); (c) measuring the expression of the reporter gene in a target host cell, wherein the expression of the reporter gene depends on the segment derived from the patient; and (d) comparing the expression of the reporter gene from step (c) with the expression of the reporter gene measured when steps (a) - (c) are carried out in the absence of the pharmaceutical compound 10 candidate antiviral, where a test concentration is present in the candidate antiviral drug compound in steps (a) - (c); in steps (b) - (c); or in step (c). The expression of the reporter gene in the vector of 15 endurance test in the target cell ultimately depends on the action of the segment sequence F derived from the patient. The reporter gene may be functional or non-functional. In another aspect, this invention is directed to 20 tests of susceptibility and resistance to antiretroviral drugs for HIV / AIDS. The particular resistance test vectors of the invention are identified for use in the antiretroviral drug susceptibility and resistance test for the 25 HIV / AIDS.

In still another aspect, this invention provides the identification and evaluation of the biological effectiveness of potential therapeutic antiretroviral compounds for the treatment of HIV and / or AIDS. In another aspect, the invention is directed to a novel resistance test vector comprising a segment derived from the patient further comprising one or more mutations on the RT gene and an indicator gene.

Brief Description of the Drawings Figure 1 Resistance Test Vector. A schematic representation of the resistance test vector comprising a segment derived from a patient and an initiator gene.

Figure 2 Two Cellular Assays. Schematic representation of the trial. A resistance test vector was generated by cloning the patient-derived segment into a viral vector of the reporter gene. The resistance test vector was then cotransfected with an expression vector that produces the envelope protein of murine leukemia virus (MLV), amphotropic, or other viral or cellular proteins that allow infection. Pseudotyped viral particles containing the protease gene (PR) and reverse transcriptase (RT) products encoded by the patient-derived sequences were produced. The particles were harvested and then used to infect fresh cells. Using defective PR and RT sequences, it was shown that luciferase activity depends on functional PR and RT. PR inhibitors were added to the cells after transfection and thus are present during the maturation of the particle. The RT inhibitors, on the other hand, were added to the cells at the time or before the infection with the viral particle. The assay was carried out in the absence of drug and in the presence of drug over a wide range of concentrations. The amount of luciferase was determined and the percentage (%) of inhibition was calculated at the different drug concentrations tested.

Figure 3 Examples of profiles of phenotypic susceptibility to the drug. The data were analyzed by plotting the percent inhibition of luciferase activity against the concentration (uM). This graph was used to calculate the drug concentration that is required to inhibit the reproduction of the virus by 50% (IC50) or by 95% (IC50). Deviations in the inhibition curves towards higher drug concentrations were interpreted as evidence of resistance to • drug. Three typical curves are shown for a nucleoside reverse transcriptase inhibitor (AZT), a non-nucleoside inhibitor of reverse transcriptase (delavirdine), and a protease inhibitor (ritonavir). A reduction in the susceptibility (resistance) to the drug is reflected in a deviation in the curve of F 10 susceptibility to the drug towards higher drug concentrations (to the right) compared to a baseline (pretreatment) sample or a virus control susceptible to the drug, such as PNL4-3 or HXB-2, when a drug is not available. basal sample. 15 Figure 4 F Profile of phenotypic susceptibility and resistance to the drug: patient 487. A phenotypic susceptibility test based on PCR was carried out 20 giving the profile of phenotypic susceptibility and resistance to the drug showing greater resistance to both delavirdine and nevirapine. This is an example of the first pattern of susceptibility / resistance to NNRTI. The evaluation of this plasma virus showed that the HIV reverse transcriptase has mutations in codons 184 (M184V) associated with resistance to 3TC and in 103 (K103N) associated with resistance to both delavirdine and nevirapine.

Figure 5 Profile of phenotypic susceptibility and resistance to the drug of the reverse transcriptase mutants directed to the site. A phenotypic susceptibility assay based on PCR was conducted giving the profile of phenotypic susceptibility and resistance to the drug for site-directed mutants having mutations at codons 103 and 181 (K103N; Y181C) demonstrating resistance to both delavirdine and nevirapine. The double mutants demonstrate the additive effect of both mutations that result in a greater increase in resistance.

Figure 6 Profile of phenotypic susceptibility and resistance to the drug: Patient 268. A phenotypic susceptibility test was carried out based on PCR, giving the profile of phenotypic susceptibility and resistance to the drug showing the evaluation of the plasma virus with HIV reverse transcriptase which has resistance to delavirdine but not to nevirapine. This is an example of the second NNRTI susceptibility / resistance pattern. This patient virus resistant to all protease inhibitors tested and also has significant resistance to AZT and 3TC and shows slight deviations in susceptibility to ddC, ddl, and d4T. Evaluation of this plasma virus using PCR and sequencing based on the genotypic assay showed that HIV reverse transcriptase has mutations at codons 103 and 236 (K103N; P236L). It was previously reported that the P236L mutation causes resistance to delavirdine and hypersensitivity to nevirapine (Due eke TJ et al. (1993) Proc Na ti ACad Sci 90, 4713-4717). However, in this patient sample, although there was resistance to delavirdine, the susceptibility to nevirapine was the same as the natural one.

Figure 7 Genotypic susceptibility and resistance profile to the drug of the site-directed reverse transcriptase mutant (P236L). A phenotypic susceptibility assay based on PCR was carried out, giving the profile of susceptibility and phenotypic resistance to the drug showing the susceptibility to delavirdine and nevirapine of the mutagenesis mutant directed to the P236L site. This result is identical to that observed in the virus sample of the patient shown in Figure 6. The next two panels show the mutagenesis mutant directed to the K103N site and the two lower panels show the double mutant K103N + P236L. The P236L mutation additive to K103N causing severe resistance to delavirdine without having an effect on nevirapine resistance due to K103N at the same time. The right side of Figure 3 shows a similar result when the P236L mutation is added to the mutation Y181- > C.

Figure 8A Profile of Susceptibility and Phenotypic Drug Resistance: Patients 302. This is an example of the third pattern of susceptibility to resistance to NNRTI. The phenotypic analysis of the patient's virus showed less susceptibility to both delavirdine and nevirapine. This pattern is characterized by a greater reduction in susceptibility to nevirapine compared to reduced susceptibility to delavirdine. The genotypic analysis of the patient's virus showed the presence of the RT mutations K103N associated with resistance to nevirapine and delavirdine and P225H.

Figure 8B Susceptibility and Resistance Profile • Phenotypic to the Drug: Patients 780. This is a second example of the third pattern of susceptibility / resistance to NNRTI. The phenotypic analysis of the patient's virus showed less susceptibility to both delavirdine and nevirapine. This pattern is characterized by a greater reduction in susceptibility to nevirapine compared to reduced susceptibility to • 10 delavirdine. Genotypic analysis of the patient's virus demonstrated the presence of K103N RT mutations associated with resistance to nevirapine and delavirdine and P225H. 15 Figure 8C Susceptibility and Resistance Profile • Phenotypic to the Drug: Individual Virus Clones of the Patient 302. Genotypic analysis of individual virus clones of patient 302 revealed viruses that contain 20 the K103N mutation without the P225H mutation (K103N, I135M, R211K) and viruses containing the K103N mutation with the P225H mutation (K103N, P225H). The phenotypic characterization of these virus clones indicates that the P225H mutation reduces the amount of resistance to delavirdine 25 associated with the K103N mutation (compare both panels), but does not alter the amount of resistance to nevirapine associated with the K103N mutation (compare the upper panels).

Figure 8D Profile of Phenotypic Susceptibility and Resistance to Drugs: Reverse Transcriptase Mutants Directed to the Site. The phenotypic characterization of the site of a virus containing the P225H mutation of the RT directed to the site indicates that this mutation increases susceptibility to delavirdine, but not to nevirapine (compare the upper panels). The phenotypic characterization of a virus containing the P225H plus K103N or P225H plus Y181C mutations of the RT directed to the site indicates that the P225H mutation decreases the amount of delavirdine resistance associated with either the K103N or Y181C, but does not decrease the amount of resistance to nevirapine associated with K103N or Y181C to delavirdine, but not to nevirapine (compare the corresponding mid and lower panels).

Figure 9A Profile of Susceptibility and Phenotypic Resistance to Drugs: Patients 644. This is an example of the short pattern of susceptibility and resistance to NNRTI. The phenotypic analysis of the patient's virus showed a great reduction in susceptibility to nevirapine, but not to delavirdine. Genotypic analysis of the patient's virus demonstrated the presence of G190S mutations of the RT, as well as the K101E mutation associated with reductions in susceptibility to atevirdine, DMP266, L-697, 661 and UC-10,38,57 (Schinazi, Mellors, Larder's Resistance Table).

Figure 9B Profile of Susceptibility and Phenotypic Resistance to Drugs: Reverse Transcriptase Mutants Directed to the Site. Phenotypic characterizations of viruses containing any of the RT mutations directed to the G190A, or G190S site indicate that these mutations greatly reduce susceptibility to nevirapine, and slightly increase susceptibility to delavirdine (compare upper panels) .

Detailed Description of the Invention The present invention relates to physicians to verify the clinical progress of HIV infection in patients receiving antiretroviral therapy, particularly antiretroviral therapy with non-nucleoside reverse transcriptase inhibitor. In a modality, the present invention provides a method for evaluating the effectiveness of antiretroviral therapy of the patient comprising (i) collecting a biological sample from a patient infected with HIV, and (ii) determining whether the biological sample comprises the nucleic acid encoding the HIV RT that has a mutation in one or more positions in the RT. Mutations correlate positively with mutations in phenotypic susceptibility / resistance. In a specific embodiment, the invention provides a method for evaluating the effectiveness of antiretroviral therapy with NNRTI of a patient comprising (i) collecting a biological sample from an HIV-infected patient.; and (ii) determining whether the biological sample comprises nucleic acid encoding the HIV RT having a mutation at codon 225 and 103. This invention established, using phenotypic susceptibility assays, that codon 225 mutations alone or in combination with a mutation in codon 103 of HIV reverse transcriptase correlates with an increase in susceptibility to delavirdine, little or no change in susceptibility to nevirapine and little or no change in susceptibility to efavirenz. In another specific embodiment, the invention • provides a method for evaluating the effectiveness of antiretroviral therapy with NNRTI of the patient which comprises (i) collecting a biological sample from a patient infected with HIV; and (ii) determining whether the biological sample comprises the nucleic acid encoding the HIV RT having a mutation in the 10 codons 236 and 103 and / or 181. This invention established, using a phenotypic susceptibility assay, that mutations in codon 236 alone or in combination with a mutation in codon 103 and / or 181 of HIV reverse transcriptase are correlate with a decrease in 15 susceptibility to delavirdine (increased resistance) and no change in susceptibility in the • Nevirapine. Mutation 236 alone or with a history of Y181C has an effect on susceptibility to efavirenz but establishes the significant portion of the loss of 20 susceptibility caused by the 103N mutation. In another specific embodiment, the invention provides a method for evaluating the effectiveness of antiretroviral therapy with the patient's NNRTI, comprising (i) collecting a biological sample from a 25 patient infected with HIV; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV RT having a mutation at codons 230 and / or 181. This invention established, using a phenotypic susceptibility assay, that mutations in codon 230 alone or in combination with a mutation in codon 181 of HIV reverse transcriptase are correlated with a significant decrease in susceptibility to delavirdine (increased resistance), the significant decrease in susceptibility in nevirapine. In another specific embodiment, the invention provides a method for evaluating the effectiveness of antiretroviral therapy with a patient's NNRTI, comprising (i) collecting a biological sample from a patient infected with HIV; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV RT having a mutation at codon 181. This invention established, using a phenotypic susceptibility assay, that the mutation at codon 181 of the reverse transcriptase of the HIV is correlated with a significant decrease in delavirdine susceptibility (increased resistance), a significant decrease in susceptibility to nevirapine, and no change in susceptibility to efavirenz.

In another specific embodiment, the invention provides a method for evaluating the effectiveness of antiretroviral therapy with a patient's NNRTI, comprising (i) collecting a biological sample from a patient infected with HIV; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV RT having a mutation at codon 188. This invention established, using a phenotypic susceptibility assay, that the mutation in codon 188 of the reverse transcriptase of the HIV is correlated with a decrease in susceptibility to delavirdine (increased resistance), a substantial decrease in susceptibility to nevirapine, and a significant decrease in susceptibility to efavirenz. In another specific embodiment, the invention provides a method for evaluating the effectiveness of antiretroviral therapy with a patient's NNRTI, comprising (i) collecting a biological sample from a patient infected with HIV; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV RT having a mutation at codons 138 and / or 188. This invention established, using a phenotypic susceptibility assay, that mutations in codon 138 alone or in combination with a mutation in codon 188 of HIV reverse transcriptase are correlated with a moderate decrease in the • susceptibility to delavirdine (increased resistance), or a substantial decrease in susceptibility to nevirapine and a moderate decrease in susceptibility to efavirenz. In another specific embodiment, the invention provides a method for evaluating the effectiveness of antiretroviral therapy with NNRTI of a patient, which 10 comprises (i) collecting a biological sample from a patient infected with HIV; and (ii) determining whether the biological sample comprises the nucleic acid encoding the HIV RT having a mutation at codon 98. This invention established, using an assay 15 of phenotypic susceptibility, that mutations in codon 98 of HIV reverse transcriptase are • correlated with a slight decrease in susceptibility to delavirdine (increased resistance), a substantial decrease in 20 susceptibility to nevirapine and a slight significant decrease in susceptibility to efavirenz. In another specific embodiment, the invention provides a method for evaluating the effectiveness of antiretroviral therapy with NNRTI of a patient, which 25 comprises (i) collecting a biological sample from a patient infected with HIV; and (ii) determining whether the biological sample comprises the nucleic acid encoding the HIV RT having a mutation at codons 98 and / or 190. This invention established, using a phenotypic susceptibility assay, that mutations in the codon 98 alone or in combination with a mutation in codon 190 of HIV reverse transcriptase are correlated with an increase in susceptibility to delavirdine (resistance • 10 decreased), a substantial decrease in susceptibility to nevirapine and a substantial decrease in susceptibility to efavirenz. In another specific embodiment, the invention provides a method for evaluating the effectiveness of antiretroviral therapy 15 with a patient's NNRTI, which comprises (i) collecting a biological sample from a patient infected with HIV; Y • (ii) determining whether the biological sample comprises nucleic acid encoding the HIV RT having a mutation at codons 181 and / or 98. This invention 20 established, using a phenotypic susceptibility assay, that the mutation in codon 181 alone or in combination with a mutation in codon 98 of HIV reverse transcriptase is correlated with a decrease in susceptibility to delavirdine. 25 (increased resistance), a substantial decrease in susceptibility to nevirapine, and a significant decrease in susceptibility to efavirenz. In another specific embodiment, the invention provides a method • to evaluate the effectiveness of antiretroviral therapy 5 with a patient's NNRTI, which comprises (i) collecting a biological sample from a patient infected with HIV; and (ii) determining whether the biological sample comprises the nucleic acid encoding the HIV RT having a mutation at codons 101 and / or 190, for example • 10 190S. This invention established, using a phenotypic susceptibility assay, that mutations in codon 101 alone or in combination with a mutation in codon 190 of HIV reverse transcriptase are correlated with the absence of changes in the 15 susceptibility to delavirdine (natural), a substantial decrease in susceptibility to • nevirapine and a substantial decrease in susceptibility to efavirenz. In another specific embodiment, the invention provides a method for 20 evaluate the effectiveness of antiretroviral therapy with NNRTI of a patient, comprising (i) collecting a biological sample from a patient infected with HIV; Y (ii) determine if the biological sample comprises the nucleic acid encoding the HIV RT that has A mutation in codon 108. This invention established, using a phenotypic susceptibility assay, that a mutation in codon 108 of HIV reverse transcriptase is correlated with the absence of changes in susceptibility to delavirdine (natural), a slight 5 decrease in susceptibility to nevirapine and in the absence of changes in susceptibility to efavirenz. In another specific embodiment, the invention provides a method for evaluating the effectiveness of antiretroviral therapy with a patient's NNRTI, F 10 comprising (i) collecting a biological sample from a patient infected with HIV; and (ii) determining whether the biological sample comprises the nucleic acid encoding the HIV RT having a mutation at codons 101 and 103 and / or 190. This invention 15 established, using a phenotypic susceptibility assay, that mutations in codon 101 alone or F in combination with a mutation in codon 103 and / or 190 of HIV reverse transcriptase are correlated with any of the absence of 20 change (101 and 190) or a moderate decrease (103 and 190, for example 190A) in delavirdine susceptibility (increased resistance), a substantial decrease in susceptibility to nevirapine and a significant decrease in 25 susceptibility to efavirenz.

In another specific embodiment, the invention provides a method for evaluating the effectiveness of the • antiretroviral therapy with a patient's NNRTI, comprising (i) collecting a biological sample from a patient infected with HIV; and (ii) determining whether the biological sample comprises the nucleic acid encoding the HIV RT having a mutation at codons 106 and / or 189 and / or 181 and / or 227. This invention established, using a susceptibility assay • 10 phenotypic, that mutations in codon 106 alone or in combination with a mutation in codon 189 and / or 181 and / or 227 of HIV reverse transcriptase are correlated with changes in susceptibility to delavirdine, nevirapine and efavirenz . Especially, the 15 presence of mutations in 106 and 181 correlates with a significant decrease in susceptibility to delavirdine, a substantial decrease in susceptibility to nevirapine and a slight decrease in susceptibility to efavirenz. The presence of 20 mutations in 106 and 189 correlate with a slight decrease in susceptibility to delavirdine, a moderate decrease in susceptibility to nevirapine and the absence of changes in susceptibility to efavirenz. The presence of mutations in 106 and 227 is 25 correlated with a slight decrease in susceptibility to delavirdine, a substantial decrease in susceptibility to nevirapine, and a slight decrease in susceptibility to efavirenz. The presence of mutations in 181 and 227 correlated with an increase in susceptibility to delavirdine, a significant decrease in susceptibility to nevirapine and an increase in susceptibility to efavirenz. The presence of mutations in 106 and 181 and 227 is correlated with a moderate decrease in susceptibility to delavirdine, a substantial decrease in susceptibility to nevirapine, and a slight decrease in susceptibility to efavirenz. In another specific embodiment the invention provides a method for evaluating the effectiveness of antiretroviral therapy with NNRTI of a patient, comprising (i) collect a biological sample from a patient infected with HIV; and (ii) determining whether the biological sample comprises the nucleic acid encoding the HIV RT having a mutation at codons 188 and 100 and / or 103. This invention established, using a phenotypic susceptibility assay, that mutations in codon 188 alone or in combination with a mutation at codon 100 and / or 103 in HIV reverse transcriptase are correlated with changes in susceptibility to delavirdine, nevirapine and efavirenz.

Specifically, the presence of mutations in 103 and 188 correlates with a substantial decrease in susceptibility to delavirdine, a substantial decrease in susceptibility to nevirapine, and a substantial decrease in susceptibility to efavirenz. The presence of mutations in 100 and 103 correlates with a substantial decrease in susceptibility to delavirdine, a moderate decrease in susceptibility to nevirapine, and a substantial decrease in susceptibility to efavirenz. The presence of mutations in 103 and 100 and 188 correlates with a substantial decrease in susceptibility to delavirdine, a substantial decrease in susceptibility to nevirapine and a substantial decrease in susceptibility to efavirenz. Under the above circumstances, the profile of phenotypic susceptibility / resistance and the genotypic profile of the HIV virus infecting the patient has been altered reflecting some change in the response to the antiretroviral agent. In the case of antiretroviral therapy with NNRTI, the HIV virus that infects the patient may be resistant to one or more but not to another of the NNRTIs as described here. Therefore, it may be desirable that after detecting the mutation, the dose of the antiretroviral agent is increased, changed to another antiretroviral agent, or one or more additional antiretroviral agents are added to the patient's therapeutic regimen. For example, if the patient had been being treated with efavirenz (DMP-266) when the 225 mutation arose, the patient's therapeutic regimen could be desirably altered (i) by switching to a different NNRTI antiretroviral agent, such as delavirdine or nevirapine and stopping treatment with efavirenz; or (ii) increasing the dose of efavirenz; (iii) adding another antiretroviral agent to the patient's therapeutic regimen. The effectiveness of the modification of the therapy can be evaluated by checking the viral load such as the. number of copies of HIV RNA. A decrease in the number of HIV RNA copies correlates positively with the effectiveness of a treatment regimen. The phrase "correlates positively", as used here, indicates that a particular result produces a particular conclusion more likely than other conclusions. Another specific, non-limiting, preferred embodiment of the invention is the following: A method for evaluating the effectiveness of NNRTI therapy of a patient, comprising (i) collecting a biological sample from an HIV-infected patient; (ii) amplifying the RNA encoding HIV in the biological sample by converting RNA to cDNA and amplifying the HIV sequences using HIV primers that result in a PCR product comprising that RT gene; (iii) performing the PCR using primers that result in PCR products comprising natural codons or mutants 225 and 103; (iv) determine, via PCR products, the presence or absence of a mutation at codon 225 or 103 or both. Another specific, non-limiting, preferred, further embodiment of the invention is the following: A method for evaluating the effectiveness of NNRTI therapy of a patient comprising (I) collecting a plasma sample from an HIV-infected patient; (ii) amplifying the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplifying the HIV sequences using HIV primers that result in a PCR product comprising the RT gene; (iii) PCR using PCR primers resulting in PCR products comprising natural codons or mutations in 103 and / or 181 and 236; and (iv) determining, via the PCR products, the presence or absence of a mutation in codons 236 and 106 and / or 181. Another specific, non-limiting, preferred, further embodiment of the invention is as follows: method for evaluating the effectiveness of NNRTI therapy of a patient, comprising (I) collecting a plasma sample from a patient infected with HIV; (ii) amplifying an RNA encoding HIV in a plasma sample by converting the RNA into cDNA and amplifying the HIV sequences using HIV primers that result in a PCR product comprising that RT gene: (iii) ) performing the PCR using primers that result in PCR products comprising the natural codon or mutations in 101 and 190 (G190S); and (iv) determining, via the PCR products, the presence or absence of a mutation at codon 190 (G190S) and 101. Another specific, non-limiting, preferred, further embodiment of the invention is as follows: to evaluate the effectiveness of a patient's NNRTI therapy, comprising (i) collecting a plasma sample from an HIV-infected patient; (ii) amplifying the RNA encoding HIV in the plasma sample by converting the RNA into cDNA and amplifying the HIV sequences using HIV primers that result in a PCR product comprising the RT gene; (iii) perform the PCR using primers that result in PCR products that comprise the natural codon or mutations in codon 103 and 190 (G190A) and (iv) determine, via the PCR products, the presence or absence of a mutation in codon 190 (190A) and 103. Another specific, non-limiting, preferred, further embodiment of the invention is the following: A method for evaluating the effectiveness of NNRTI therapy of a patient, comprising (i) ) collect a plasma sample from a patient infected with HIV; (ii) amplifying the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplifying the HIV sequences using HIV primers that result in a PCR product comprising the RT gene; (iii) performing the PCR using primers that result in a PCR product comprising the natural codon or mutations in codon 230 and 181, and (iv) determining, via the PCR products, the presence or absence of a mutation in codon 230 and 181. Another specific, non-limiting, preferred, further embodiment of the invention is the following: A method for evaluating the effectiveness of NNRTI therapy of a patient, comprising (i) collecting a plasma sample of a patient infected with HIV; (ii) amplifying the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplifying the HIV sequences using HIV primers resulting in a PCR product comprising the RT gene; (iii) performing the PCR using primers that result in PCR products that comprise the wild-type or mutation in 181; and (iv) determining, via the PCR product, the presence or absence of a mutation in codon 181. Another specific, non-limiting, preferred, further mode of the invention is as follows: A method for evaluating the effectiveness of NNRTI therapy of a patient, comprising (i) collecting a plasma sample from a patient infected with HIV; (ii) amplifying the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplifying the HIV sequences using HIV primers resulting in a PCR product comprising the RT gene; (iii) PCR using primers that result in PCR products that include the wild type or mutation at codon 188; and (iv) determining, via the PCR products, the presence or absence of a mutation in codon 188. Another specific, non-limiting, preferred, further embodiment of the invention is as follows: A method for evaluating the effectiveness of NNRTI therapy of a patient, comprising (i) collecting a plasma sample from a patient infected with HIV; (ii) amplifying the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplifying the HIV sequences using HIV primers that result in a PCR product comprising the RT gene; (iii) carry out the PCR using primers that result in PCR products comprising the wild type 5 or mutations in codon 138 and 188; and (iv) determining, via the PCR products, the presence or absence of a mutation at codons 138 and 188. Another specific, non-limiting, preferred, further embodiment of the invention is as follows: • 10 method for evaluating the effectiveness of a patient's NNRTI therapy, comprising (i) collecting a "plasma sample from an HIV-infected patient; (ii) amplifying the RNA encoding HIV in the plasma sample converting RNA to cDNA and amplifying 15 HIV sequences using HIV primers that result in a PCR product comprising the gene • of the RT; (iii) performing the PCR using primers that result in PCR products comprising the wild-type or mutation at codon 98; and (iv) Determine, via the PCR product, the presence or absence of a mutation in codon 98. Another specific, non-limiting, preferred, further mode of the invention is as follows: A method for evaluating the effectiveness of the therapy with NNRTI of a patient, comprising (i) 25 collecting a plasma sample from a patient infected with HIV; (ii) amplifying the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplifying the HIV sequences using HIV primers resulting in a PCR product comprising the RT gene; (iii) PCR using primers that result in PCR products that include the wild type or mutations in codon 98 and 190; and (iv) determining, via the PCR products, the presence or absence of a mutation in codon 190 and 98. Another specific, non-limiting, preferred, further embodiment of the invention is as follows: A method for evaluating the effectiveness of a patient's NNRTI therapy, comprising (i) collecting a plasma sample from a patient infected with HIV; (ii) amplifying the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplifying the HIV sequences using HIV primers that result in a PCR product comprising the RT gene; (iii) PCR using primers that result in PCR products that include the wild type or mutations in codon 98 and 181; and (iv) determining, via the PCR products, the presence or absence of a mutation in codons 98 and 181. Another specific, non-limiting, preferred, further mode of the invention is as follows: method for evaluating the effectiveness of a patient's NNRTI therapy, comprising (i) collecting a plasma sample from an HIV-infected patient; (ii) amplifying the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplifying the HIV sequences using HIV primers resulting in a PCR product comprising the RT gene; (iii) performing the PCR using primers that result in PCR products comprising the wild type or mutations in codon 101 and 190; and (iv) determining, via the PCR product, the presence or absence of a mutation in codon 190, for example 190S and 101. Another specific, non-limiting, preferred embodiment of the invention is as follows: method for evaluating the effectiveness of a patient's NNRTI therapy, comprising (i) collecting a plasma sample from an HIV-infected patient; (ii) amplifying the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplifying the HIV sequences using HIV primers resulting in a PCR product comprising the RT gene; (iii) performing the PCR using primers that result in PCR products comprising the wild-type or mutation at codon 108; and (iv) determining, via the PCR product, the presence or absence of a mutation in codon 108. Another specific, non-limiting, preferred, further embodiment of the invention • is as follows: A method for evaluating the effectiveness of a patient's NNRTI therapy, comprising (i) collecting a plasma sample from an HIV-infected patient; (ii) amplify the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplify the HIV sequences using primers • HIV 10 which results in a PCR product comprising the RT gene; (iii) PCR using primers that result in PCR products that comprise the wild type or mutation in codons 101 and 103 and 190; and (iv) determine, via the 15 PCR products, the presence or absence of a mutation at codon 101 and 103 and 190, for example 190A. Another specific, non-limiting, preferred, further modality of the invention is the following: A method to evaluate the effectiveness of the therapy with 20 NNRTI of a patient, comprising (i) collecting a plasma sample from a patient infected with HIV; (ii) amplify the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplifying the HIV sequences using HIV primers that give As a result a PCR product comprising the RT gene; (iii) performing PCR using primers that result in PCR products comprising the wild-type or mutations at codon 106 and 189 and 181 and 227; and (iv) determining, via the PCR product, the presence or absence of a mutation at codons 106 and 189 and 181 and 227. Another specific, non-limiting, preferred, further embodiment of the invention is as follows: method for evaluating the effectiveness of a patient's NNRTI therapy, comprising (i) collecting a plasma sample from an HIV-infected patient; (ii) amplifying the RNA encoding HIV in the plasma sample by converting the RNA to cDNA and amplifying the HIV sequences using HIV primers resulting in a PCR product comprising the RT gene; (iii) performing the PCR using primers that result in PCR products comprising the wild-type or mutations at codon 188 and 100 and 103; and (iv) determining, via the PCR product, the presence or absence of a mutation at codon 188 and 100 and 103. The presence of the mutation in codon 225 and 103 of the HIV RT indicates that the effectiveness of therapy with current or prospective NNRTI may require alteration, since as shown by this invention, the mutation in codon 103 reduces susceptibility, which susceptibility can be partly restored by the mutation in codon 225. Using the methods of this invention would indicate the change in therapy with NNRTI. Similarly, using the means and methods of this invention, the presence of the mutation in codon 236 and / or 103 and / or 181 of the HIV RT indicates that the effectiveness of the current prospective NNRTI therapy has decreased . Similarly, using the means and methods of this invention, the mutation in codon 190 • 10 (G190A) and 103 (K103N) of the HIV RT indicate that the effectiveness of current or prospective NNRTI therapy has decreased. Similarly, using the means and methods of this invention, the presence of the mutation at codon 190 (G190A) and 101 (K101E) of HIV RT 15 indicates that the effectiveness of current or prospective NNRTI therapy has decreased. Similarly, using • the means and methods of this invention, the presence of the mutation in codon 230 and 181 of the HIV RT indicates that the effectiveness of the current NNRTI therapy or 20 prospective to diminished. Similarly, using the means and methods of this invention, the presence of the mutation in codon 181 of HIV RT indicates that the effectiveness of current or prospective NNRTI therapy has been diminished. Similarly, using In the means and methods of this invention, the presence of the mutation in codon 188 of the HIV RT indicates that the effectiveness of current or prospective NNRTI therapy has decreased. Similarly, using the means and methods of this invention, the presence of the mutation in codon 138 and 188 of the HIV RT indicates that the effectiveness of current or prospective NNRTI therapy has been diminished. In a similar way, using the means and methods of this invention, the presence of the mutation in codon 98 of the HIV RT indicates that the effectiveness of current or prospective NNRTI therapy has decreased. Similarly, using the means and methods of this invention, the presence of the mutation in codon 98 and 190 of HIV RT indicates that the effectiveness of current or prospective NNRTI therapy has been diminished. Similarly, using the means and methods of this invention, the presence of the mutation in codon 181 and 98 of the HIV RT indicates that the effectiveness of current or prospective NNRTI therapy has been diminished. Similarly, using the means and methods of this invention, the presence of the mutation in codon 101 and 190, for example 190S, of HIV RT indicates that the effectiveness of current or prospective NNRTI therapy has been diminished. Similarly, using the means and methods of this invention, the presence of the mutation in codon 108 of the HIV RT indicates that the effectiveness of current or prospective NNRTI therapy has been decreased. Similarly, using the means and methods of this invention, the presence of the mutation in codon 101 and 103 and 190, for example 5 190A, of HIV RT indicates that the effectiveness of current or prospective NNRTI therapy diminished. Similarly, using the means and methods of this invention, the presence of the mutation in codon 106 and 189 and 181 and 227 of the HIV RT indicates that the • 10 effectiveness of current or prospective NNRTI therapy has decreased. Similarly, using the means and methods of this invention, the presence of the mutation in codon 188 and 100 and 103 of the HIV RT indicates that the effectiveness of the current or prospective NNRTI therapy. 15 a diminished. Another specific modality, not limiting, • preferred, of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient comprising: (a) 20 to collect a biological sample from a patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding the HIV reverse transcriptase having a mutation in antibodies 236 and 103 and / or 181. 25 phenotypic susceptibility, it was observed that the presence of the three mutations correlates positively with resistance to delavirdine. Using the phenotypic susceptibility test, it was observed that the presence of the three mutations correlates positively with resistance to nevirapine. In another embodiment, the mutant codon 236 of HIV RT codes for leucine (L). In a further embodiment, the reverse transcriptase has a mutation in codon 103, a mutation in codon 181 or a combination thereof, in addition to a mutation in codon 236 of HIV RT. In a still further embodiment, the mutant codon 103 codes for an asparagine (N) and the mutant codon 181 codes for a cysteine (C). Another specific, non-limiting, preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of a patient infected with HIV comprising: (a) collecting a biological sample from a patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codons 225 and 103. Using a phenotypic susceptibility assay, the presence of codon mutations was observed 225 alone or in combination with a mutation in codon 103 of HIV RT causes an increase in susceptibility to delavirdine and has no effect on susceptibility to nevirapine. In yet another embodiment, the codon mutant 225 codes for a histidine, codon 230 codes for a leucine and codon 181 codes for a cysteine. This invention provides a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient comprising: (a) collecting a biological sample from the patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding the HIV reverse transcriptase having a mutation at codon 181. Using the phenotypic susceptibility assay, it was observed that the presence of the mutations in codon 181 was positively correlated with a moderate decrease in susceptibility to delavirdine and a significant decrease in susceptibility to nevirapine and the absence of changes in susceptibility to efavirenz. In one embodiment, mutant codon 181 codes for an isoleucine. This invention provides a method for evaluating the effectiveness of antiretroviral therapy of a patient infected with HIV comprising: (a) collecting a biological sample from the patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 188. Using the phenotypic susceptibility assay, the presence of the codon 188 mutations was observed. positively correlates with a slight decrease in susceptibility to delavirdine and a substantial decrease in susceptibility to • 10 nevirapine and a significant decrease in susceptibility to efavirenz. In one embodiment, the mutant codon 108 codes for a cysteine, histidine or leucine. This invention provides a method for 15 evaluate the effectiveness of antiretroviral therapy of a patient infected with HIV comprising: (a) • collect a biological sample from the patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding the 20 HIV reverse transcriptase having a mutation at codon 190. Using the phenotypic susceptibility assay, it was observed that the presence of mutations in codon 190 correlates positively with a slight increase in susceptibility to delavirdine. 25 and a large decrease in susceptibility to nevirapine. In one embodiment, the mutant codon 190 codes for an alanine or a serine. Another specific, non-limiting, preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of a patient infected with HIV comprising: (a) collecting a biological sample from a patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding the • HIV reverse transcriptase having a mutation at codon 230 and 181. Using a phenotypic susceptibility assay, it was observed that the presence of mutations in codons 230 alone or in combination with a mutation in codon 181 of the RT of the 15 HIV causes a significant decrease in susceptibility to delavirdine and a decrease • significant in susceptibility to nevirapine. Another specific, non-limiting, preferred embodiment of the invention is the following: a method 20 to evaluate the effectiveness of antiretroviral therapy of an HIV-infected patient comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises the nucleic acid encoding the HIV reverse transcriptase having a mutation at codon 138 and 188. Using the phenotypic communication assay, it was observed that the presence of mutations in codons 138 alone or in combination with a mutation in codon 188 of the RT of the HIV causes a moderate decrease in susceptibility to delavirdine and a substantial decrease in susceptibility to nevirapine and a moderate decrease in susceptibility to efavirenz. In another embodiment, mutant codon 138 codes for an alanine and codon 188 codes for a leucine. This invention provides a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) collecting a biological sample from a patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 98. Using the phenotypic susceptibility assay, it was observed that the presence of mutations in codon 98 correlates positively with a slight decrease in susceptibility to delavirdine and a slight decrease in susceptibility to delavirdine and a slight decrease in susceptibility to nevirapine and a slight decrease in susceptibility to efavirenz. In one embodiment, mutant codon 98 codes for glycine. Another specific, non-limiting, preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of a patient afflicted with HIV, comprising: (a) collecting a biological sample from a patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 98 and 190. Using the phenotypic susceptibility assay, the presence of codon mutations was observed 98 alone or in combination with a mutation in codon 190 of HIV RT causes an increase in susceptibility to delavirdine and a substantial decrease in susceptibility to nevirapine and a substantial decrease in susceptibility to efavirenz. In yet another embodiment, mutant codon 190 codes for a serine and codon 98 for a glycine. Another specific, non-limiting, preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) collecting a biological sample from the HIV-infected patient; and (b) determining whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codons 181 and 98. Using the phenotypic susceptibility assay, the presence of codon mutations was observed 181 alone or in combination with a mutation in codon 98 of HIV RT causes a significant decrease in susceptibility to delavirdine and a substantial decrease in susceptibility to nevirapine and a slight decrease in susceptibility to efavirenz. In yet another embodiment, mutant codon 98 codes for a glycine and codon 181 codes for a cysteine. Another specific, non-limiting, preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation in the -codon 101 and 190 e.g. 190S. Using the phenotypic susceptibility assay, it was observed that the presence of the mutations in codons 101 alone or in combination with a mutation in codon 190 of the HIV RT does not cause changes in the susceptibility to delavirdine and a substantial decrease in the susceptibility to nevirapine and a substantial decrease in susceptibility to efavirenz. In yet another embodiment, mutant codon 190 codes for a serine and codon 101 codes for a glutamic acid. This invention provides a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) collecting a biological sample from a patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 108. Using the phenotypic susceptibility assay, it was observed that the presence of mutations at codon 108 correlates positively with the absence of changes in susceptibility to delavirdine and a slight decrease in susceptibility to nevirapine and in the absence of changes in susceptibility to efavirenz. In one embodiment, the mutant codon 108 codes for isoleucine. Another specific, non-limiting, preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) collecting a biological sample from a patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 101 and 190, for example 190A. Using the phenotypic susceptibility assay, it was observed that the presence of the mutations in codons 101 alone or in combination with a mutation in codon 190 of the HIV RT does not cause changes in the susceptibility to delavirdine and a substantial decrease in the susceptibility to nevirapine and a significant decrease in susceptibility to efavirenz. In yet another embodiment, mutant codon 190 codes for a glycine and codon 101 for a glutamic acid. Another specific, non-limiting, preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 103 and 980. Using the phenotypic susceptibility assay, the presence of codon mutations was observed 1-03 alone or in combination with a mutation in codon 190 of HIV RT causes a moderate decrease in susceptibility • to delavirdine and a substantial decrease in nevirapine susceptibility "a significant decrease in susceptibility to efavirenz." In yet another embodiment, mutant codon 190 codes for an alanine and codon 103 codes for an asparagine. , not limiting, The preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determine if the biological sample 15 comprises the nucleic acid encoding the HIV reverse transcriptase which has a mutation in • codon 106 and 181. Using the phenotypic susceptibility assay, it was observed that the presence of mutations in codons 106 only or in 20 combination with a mutation at codon 181 of HIV RT causes a significant decrease in susceptibility to delavirdine and a substantial decrease in susceptibility to nevirapine and a substantial decrease in susceptibility to 25 efavirenz. In yet another embodiment, mutant codon 106 codes for an alanine and codon 181 codes for a cysteine. Another specific, non-limiting, preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) collecting a biological sample from a patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding the • HIV reverse transcriptase having a mutation at codon 106 and 189. Using the phenotypic susceptibility assay, it was observed that the presence of mutations in codons 106 alone or in combination with a mutation in codon 189 of the RT of the 15 HIV causes a slight decrease in susceptibility to delavirdine and a - moderate decrease in • susceptibility to nevirapine and the absence of changes in susceptibility to efavirenz. In yet another embodiment, the mutant codon 189 codes for a leucine and the 20 codon 106 codes for an alanine. Another specific, non-limiting, preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) 25 to collect a biological sample from a patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding the HIV reverse transcriptase having a mutation at codon 106 and 227. Using the phenotypic susceptibility assay, it was observed that the presence of the mutations in the codons 106 alone or in combination with a mutation at codon 227 of HIV RT causes a slight decrease in susceptibility to delavirdine and a substantial decrease in • 10 susceptibility to nevirapine and a slight decrease in susceptibility to efavirenz. In yet another embodiment, the mutant codon 227 codes for a leucine and the codon 106 codes for an alanine. Another specific modality, not limiting, The preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of • an HIV-infected patient, which includes: (a) collecting a biological sample from a patient infected with HIV; and (b) determine if the biological sample 20 comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codons 181 and 227. Using the phenotypic susceptibility assay, it was observed that the presence of mutations in codons 181 alone or in 25 combination with a mutation in codon 227 of HIV RT causes an increase in susceptibility to delavirdine and a significant decrease in susceptibility to nevirapine and an increase in • susceptibility to efavirenz. In another embodiment --- more, mutant codon 227 codes for a leucine and codon 181 codes for a cysteine. Another specific, non-limiting, preferred embodiment of the invention is the following: a method for • 10 evaluate the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises the nucleic acid encoding the 15 HIV reverse transcriptase that has a mutation at codon 106 and 181 and 227. Using the • phenotypic susceptibility, it was observed that the presence of mutations in codons 106 alone or in combination with a mutation in codons 181 and 227 of the 20 RT of HIV causes a moderate decrease in susceptibility to .delavirdine and a slight decrease in susceptibility to efavirenz. In yet another embodiment, mutant codon 106 codes for an alanine, codon 181 codes for a 25 cysteine and codon 227 code for a leucine.

Another specific, non-limiting, preferred embodiment of the invention is the following: a method for -fl ^ evaluate the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) 5 collect a biological sample from a patient infected with HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding the HIV reverse transcriptase having a F ^ codon 103 and 188. Using the assay of # 10 phenotypic susceptibility, it was observed that the presence of mutations in codons 103 alone or in combination with a mutation in codon 188 of HIV RT cause a substantial decrease in susceptibility to delavirdine and a decrease 15 substantial in susceptibility to nevirapine and a substantial decrease in susceptibility to ^ efavirenz. In yet another embodiment, mutant codon 188 codes for a leucine and codon 103 codes for an asparagine. Another specific, non-limiting, preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) collecting a biological sample from an infected patient 25 for HIV; and (b) determining whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codons 100 and 103. Using the phenotypic susceptibility assay, the presence of codon mutations was observed 100 alone or in combination with a mutation in codon 103 of HIV RT causes a substantial decrease in susceptibility to delavirdine and a moderate decrease in susceptibility to nevirapine and a substantial decrease in susceptibility to efavirenz. In yet another embodiment, mutant codon 100 codes for an isoleucine and codon 103 codes for an asparagine. Another specific, non-limiting, preferred embodiment of the invention is the following: a method for evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient, comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codons 100 and 103 and 188. Using the phenotypic susceptibility assay, the presence of the mutations was observed in codons 1QD alone or in combination with a mutation in codon 103 and 188 of HIV RT cause a substantial decrease in susceptibility to delavirdine and a moderate decrease in susceptibility to nevirapine and a substantial decrease in susceptibility to efavirenz. In yet another embodiment, mutant codon 100 codes for an isoleucine, codon 103 codes for an asparagine and codon 188 codes for a leucine. This invention also provides the means and methods for using the resistance test vector comprising an HIV gene further comprising a mutation of NNRTI to select "drugs." More particularly, the invention describes the resistance test vector comprising the HIV reverse transcriptase which has mutations at codons 225 and 103 for selecting drug. The invention also discloses the resistance test vector comprising HIV reverse transcriptase having mutations at codons 236 and 103 and / or 181. The invention also discloses the resistance test vector comprising the HIV reverse transcriptase which has mutations in codons 190 (G190A) and 103 (K103N). The invention also discloses the resistance test vector comprising HIV reverse transcriptase having mutations at codons 190 (G190S) ~ and 101 (K101E). The invention also describes the vector of • resistance test comprising HIV reverse transcriptase 5 having mutations at codons 230 and 181. The invention also discloses the resistance test vector comprising HIV reverse transcriptase having a mutation at codon 181. 10 invention also discloses the resistance test vector comprising HIV reverse transcriptase having a mutation at codon 188. The invention also discloses the resistance test vector comprising reverse transcriptase of HIV having mutations in codons 138. and 188.

• The invention also discloses the resistance test vector comprising HIV reverse transcpptase having a mutation at codon 98. The invention also discloses the resistance test vector comprising the HIV reverse transcriptase which has mutations in the codons 98 and 190. The invention also discloses the resistance test vector comprising HIV reverse transcriptase having mutations at codons 181 and 98. The invention also discloses the resistance test vector comprising HIV reverse transcriptase. which has mutations in codons 101 and 190, for example 190S. The invention also describes the resistance test vector comprising HIV reverse transcriptase having a mutation at codon 108. The invention also discloses the resistance test vector comprising HIV reverse transcriptase which has mutations at codons 101 and 103 and / or 190, for example 190A. The invention also discloses the resistance test vector comprising HIV reverse transcriptase having mutations at codons 106 and 189 and / or 181 and / or 227. The invention also discloses the resistance test vector comprising reverse transcriptase of HIV having mutations at codons 188 and 103 and / or 103. The invention also relates to novel vectors, host cells and compositions for the isolation and identification of mutant resistance to the nucleoside inhibitor of HIV-1 reverse transcriptase and the use of such vectors, host cells and compositions to carry out the drug selection F antiviral. This invention also relates to the selection of candidate drugs for their ability to inhibit such a mutant.

EXAMPLE 1: Drug Susceptibility and Phenotype Testing Using Resistance Test Vectors • 10 Phenotypic drug susceptibility and resistance resistance tests were carried out using the means and methods described in the PCT International Application No. PCT / US97 / 01609, "filed on January 29, 1997, which is incorporated herein by reference.15 In those experiments the patient-derived segments corresponding to the regions they encode • for protease and HIV reverse transcriptase were patient derived segments amplified by the reverse transcription-chain reaction method of the 20 polymerase (RT-PCR) using viral RNA isolated from viral particles present in the serum of HIV-infected individuals or were natural HIV-1 mutants made by site-directed mutagenesis of a DNA clone of the test vector of resistance.

The isolation of the viral RNA is carried out using standard procedures (for example RNAgents Total RNA Isolation System, Promega, Madison Wl or RNAzol, Tel-Test, Friends ood, TX). The RT protocol of the PCR was • divided into two steps. A retroviral reverse transcriptase 5 [for example, MuLV reverse transcriptase from Moloney (Roche Molecular Systems, Inc., Branchburg, NJ), or reverse transcriptase from bird myeloblastosis virus (AMV), (Boehriger Mannheim, Indianapolis, IN) ] was used to copy the viral RNA into cDNA. The cDNA • 10 was then amplified using a thermostable DNA polymerase [e.g., Taq (Roche Molecular Systems, Inc., Branchburg, NJ), Tth (Roche Molecular. Systems, Inc., Branchburg, NJ), Primezyme (isolated from Thermus brocJianus, Biometra, Gottingen, Germany)] or a 15 combination of thermostable polymerases as described for the performance of "long PCR" [Barnes, W.M., (1994) Proc. Nati Acad. Sci, USA 91, 2216-22Z0) [for example, Expanded High Fidelity PCR System (Taq + Pwo), (Boehringer Mannheim, Indianapolis, In) or the 20 PCR GeneAmp XL (Tth + Vent), (Roche Molecular Systems, Inc., Branchburg, NJ)]. The primers, Apal primer (PDSAapa) and Agel primer (PDSAge) used to amplify the patient-derived segments of "test" contained sequences 25 which result in the introduction of Apal and Agel recognition sites in the 5 'and 3' terms of the PCR product, respectively as described in PCT International Application No. PCT / US97 / 01609, filed on January 29 , 1997. Resistance test vectors incorporating the "test" patient derived segments were constructed as described in PCT International Application No. PCT / US97 / 01609, filed on January 29, 1997 using an amplified DNA product. 1.5 kB prepared by RT-PCR using viral RNA as standard and oligonucleotides PDSApa (1) and PDSAge (2) as primers, followed by digestion with Apal and Agel or the PINAI isosquizimer. To ensure that the plasmid DNA corresponding to the resulting resistance test vector comprises a representative sample of viral HIV species present in the serum of a given patient, many (> 100) independent E. coli transformants were pooled in the construction of a given resistance test vector and were used for the preparation of the plasmid DNA. A packaging expression vector encoding an amphotrophic MulV 4070a env gene product allows the production in a vector host cell of all the resistance of viral particles of the resistance test vector which can efficiently infect human target cells . Resistance test vectors encoding all HIV genes with the exception of env were used to transfect a host cell or packaging (once the host cell is transfected it is referred to as a resistance test vector host cell). The packaging expression vector codes for the 4070A env gene product of the amphotrophic MuLV is used with the vector of resistance tests to allow the production of the host cell of the vector in viral particle resistance tests of the test vectors. resistance pseudotyped as infectious. Resistance tests carried out with resistance test vectors were carried out using packaging host cells and target host which consisted of the human embryonic kidney cell line 293 (Cell Culture Facility, UC San Francisco, SF, CA) or the line of Jurkat leukemia cells (Arthur Weiss, UC San Francisco, SF, CA). Resistance tests were carried out with resistance test vectors using two types of host cell. Viral particles of the resistance test vector were produced by means of a first host cell (the host cell of the resistance test vector) which was prepared by transfecting a host cell packed with the resistance test vector and the expression vector of packaging. The viral particles of the resistance test vector were then used to infect a second host cell (the target host cell) in which the expression of the reporter gene was measured. Resistance test vectors containing a functional luciferase gene were constructed and host cells were transfected with DNA from the resistance test vector. The resistance test vectors contained the reverse transcriptase and protease sequences derived from the patient that were susceptible or -resistant to antiretroviral agents, such as nucleoside reverse transcriptase inhibitors., non-nucleoside inhibitors of reverse transcriptase and protease inhibitors. Viral particles of the resistance test vector produced by the transfection of the DNA of the host cell resistance test vector, in the presence or absence of protease inhibitors, were used to infect target host cells that were grown in the absence of NRTI or NNRTI or in the presence of increasing concentrations of the drug. The amount of luciferae activity produced in the target host cells infected in the presence of drugs compared to the amount of luciferase produced in the target host cells infected in the absence of the drug. Drug resistance was measured as the amount of drug required to inhibit 50% of the luciferase activity in the absence of the drug (50% inhibitory concentration, IC 50). The IC 50 values were determined by plotting the percent inhibition of the drug against the concentration of the drug. The host cells were seeded on disks of 10 cm in diameter and were transfected several days later by culturing with the plasmid DNA of the resistance test vector and the envelope expression vector. The transfections were carried out using a calcium phosphate precipitation method. The cell culture media containing the DNA precipitate were replaced with fresh medium, from one to 24 hours, after transfection. The cell culture media containing viral particles of the resistance test vector were harvested - at four days after transfection and passed through a 0.45 mm filter before being stored at -80 ° C. The protein levels of the HIV capsule (p24) in the harvested cell culture medium were determined by an EIA method as described by the manufacturer (SIAC; Frederick, MD). Prior to infection, the target cells (293 and 293 / T) were cultured in cell culture medium. Infections were carried out using cell culture media of simulated transfections (without DNA) or transfections containing the plasmid DNA of the resistance test vector without the expression plasmid of the envelope. One to three or more days after infection the media were removed and cell lysis buffer was added (Promega) to each well. The used - cell phones were tested to determine the activity of luciferase (Figure 3). The inhibitory effect of the drug was determined using the following equation: % inhibition of luciferase = 1 - (RLUlu-c- Ifármaco] -r RLUluc) x 100 where RLUluc [drug] is the unit of relative light of luciferase activity in infected cells in the presence of drug and RLUluc is the Relative Light Unit of luciferase activity in infected cells in the absence of the drug. The IC50 values were obtained from the sig nal curves that were generated with the data plotting the percent inhibition of luciferase activity against the logio of the drug concentration. The inhibition curves of the drug are shown in Figure-3. 5 EXAMPLE 2: Correlation of Phenotypic Susceptibility and Genotypic Analysis Phenotypic susceptibility analysis of patient HIV samples The vectors of the resistance test are # 10 constructed as described in Example 1. Resistance test vectors or clones derived from sets of resistance test vectors were tested in a phenotypic assay to determine the level of susceptibility in an accurate and quantitative manner. 15 of a panel of antiretroviral drugs. This panel of antiretroviral drugs may comprise members of • classes known as nucleoside analog reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors 20 (NNRTI), and protease inhibitors (PRI). The drug panel can be used in new drugs or new target drugs can become available. One IC50 is determined for each resistance test vector pooled by each drug. The boss 25 of susceptibility for all tested drugs was examined and compared with known susceptibility patterns. A patient sample can be further examined for genotypic changes related to • the pattern of susceptibility observed.

Genotypic analysis of HIV samples from the patient DNA of the resistance test vector was analyzed, either as a whole or as clones, by any of the genotypic methods described in • Example 2. In one embodiment of the invention, the patient's HIV sample sequences were determined using viral RNA purification, RT / PCR and automated sequencing with ABI chain terminator. The sequence that was determined was compared with 15 control sequences present in the database and compared with a sample of the patient before the start of • therapy, if available. The genotype was examined for sequences that are different from the control or sequence before treatment and correlated with 20 the observed phenotype.

Analysis of the phenotypic susceptibility of mutants directed to the site The genotypic changes that were observed were 25 correlated with changes in phenotypic patterns of drug susceptibility were evaluated by constructing vectors of the resistance test containing the specific mutation on a background • genetic (susceptible to the drug) natural, defined. The 5 mutations can be incorporated alone - and / or in combination with other known drug resistance mutations that are thought to modulate the susceptibility of HIV to certain drugs or classes of drugs. Mutations are introduced into the vector of • 10 resistance test through any of the widely known methods for site-directed mutagenesis. In one embodiment of this invention, the PCR method with mega-primer was used for site-directed mutagenesis. A test vector of The resistance containing the specific mutation or group of mutations was then tested using the F phenotypic susceptibility described above and the susceptibility profile was compared with that of a resistance test vector (susceptible to the drug) 20 natural, genetically defined, lacking specific mutations. The changes observed in the pattern of phenotypic susceptibility to the antiretroviral drugs tested were attributed to the specific mutations introduced into the test vector of 25 resistance.

EXAMPLE 3 Correlation of Phenotypic Susceptibility and Analysis Genotypic: P225H Phenotypic analysis of patient 97-302 A resistance test vector was constructed as described in Example 1 of a patient sample designated 97-302. This patient had been treated with DGT, indinavir and DMP-266 for a period of approximately 10 months. Isolation of the viral RNA and RT / PCR was used to generate a segment derived from the patient that comprised the viral sequences coding for the whole PR and aa 1-313 of the RT. The patient-derived segment was inserted into the viral vector of the reporter gene to generate a resistance test vector designated RTV-302. RTV-302 was tested using a phenotypic susceptibility assay to accurately and quantitatively determine the level of susceptibility to a panel of antiretroviral drugs. This panel of antiretroviral drugs comprised members of the classes known as NRTI (AZT, 3TC, d4T, ddl and ddC), NNRTI (delavirdine and nevirapine), and PRI (indinavir, nelfinapir, ritonavir, and esquinavir). An IC50 was determined for each drug tested. The susceptibility of the patient virus to each drug was examined and compared with known susceptibility patterns. A pattern of susceptibility to NNRTI was observed for the RTV-302 sample • of the patient in whom there was a significant decrease in susceptibility to nevirapine (increased resistance and a modest decrease in susceptibility to delavirdine (See Figure 8A). Patient sample 97-302 was also examined to determine genotypic changes associated with the observed susceptibility pattern • 10 Determination of the patient's genotype 97-302 The DNA of the RTV-302 was analyzed by automated sequencing with ABI chain terminator The nucleotide sequence was compared with the 15 Consensus sequence of a natural class B HIV-1 (HIV Sequence Datábase Los Alamos, NM). Sequence • nucleotide was examined for sequences that are different from the control sequence. The RT mutations were noticed in the mutations K103N, I135M, T200A, 20 and P225H. K103N is associated with resistance to NNRTI and has been demonstrated, using the phenotypic susceptibility assay, which is associated with reduced susceptibility to both delavirdine and nevirapine to an equal degree. Mutations in I135M and 25 T200A are known polymorphisms of the variants (sensitive to drugs) natural to HIV. The mutation, P225H, was characterized using directed mutagenesis • the site and the phenotypic susceptibility test to correlate changes in amino acid 225 with changes in phenotypic susceptibility to NNRTIs.

Site-directed mutagenesis Resistance test vectors containing the P225H mutation were constructed only and • 10 in combination with other known drug resistance mutations (K103N, Y181C), which are known to modulate the susceptibility of HIV to NNRTT. Mutations were introduced into the resistance test vector using the PCR method with mega-primer for the 15 site-directed mutagenesis. (Sakar G and Sommar SS (1994) Biotechniques 8 (4), 404-407). A resistance test vector containing the P225H (P225H-RTV) mutation was tested using the phenotypic susceptibility assay described above and the results were 20 compared with a genetically defined resistance test vector that was natural at position 225. The pattern of phenotypic susceptibility to NNRTI, delavirdine in P225H-RTV was altered compared to the natural one. In the context of a natural background 25 (ie P225H mutation only) in P225H-RTV was more susceptible to delavirdine than natural control RTV. There was no significant change in the F susceptibility to nevirapine in P225H-RTV. The P225H mutation was also introduced an RTV that contained additional mutations in K103N, Y181C or both (K103N + Y181C). In all cases, the RTV were more susceptible to inhibition by delavirdine if the P225H mutation was present compared to the corresponding RTV that lacks the P225H mutation (Figure F 10 8D). In all cases, the P225H mutation did not significantly change the susceptibility to nevirapine (Figure 8D).

EXAMPLE 4 15 Correlation of Phenotypic Susceptibility and Genotypic Analysis: P236L • Phenotypic Analysis of HIV Patient 97-268 A resistance test vector was constructed as described in Example 1 of a sample of 20 patient designated as 97-268. This patient had been treated with AZT and 3TC (NRTI), indinavir and saquinavir (PRI) and delavirdine (an NNRTI) for variable periods of 1 month to 2 years. Isolation of viral RNA and RT / PCR were used to generate a segment derived from the 25 patient who understood the viral sequences coding for the entire PR and amino acids 1-313 of the RT. The patient-derived segment was inserted into a viral vector of the reporter gene to generate a resistance test vector designated RTV-268. RTV-268 5 was then tested using the phenotypic susceptibility test to determine the exact and quantitative level of susceptibility to a panel of antiretroviral drugs. This panel of antiretroviral drugs comprised members of the classes • 10 known as NRTI (AZT, 3TC, d4T, ddl and ddC), NNRTI (delavirdine and nevirapine), and PRI (indinavir, nelfinapir, ritonavir, and saquinavir). An IC50 was determined for each drug tested. The susceptibility of the patient's virus to each drug was examined and compared with the 15 susceptibility of a reference virus. A pattern of susceptibility to NNRTI was observed for the sample of • RTV-268 from the patient, a virus sample in which it was observed to be more resistant to delavirdine without resistance to delavirdine. The sample was examined additionally 20 for genotypic changes associated with the susceptibility pattern.

Genotype of the patient with HIV 97-268 The DNA of the RTV 268 was analyzed by automated sequencing with ABI chain terminator. The nucleotide sequence was compared with the consensus sequence for the HIV-1 class B natural. The nucleotide sequence was examined for sequences that # are different from the control sequence. Mutations of RT were noted at positions M41L, D67N, M184V, T200A, E203D, L210W, T215Y, K219Q, and P236L compared to the control sequence. Mutations in T200A and E203D are known polymorphisms in the variants (sensitive to drugs) natural to HIV. Mutations in • 10 positions M41L, D67N, L210W, T215Y, and K219Q were associated with resistance to AZT. The mutation in N184V was associated with resistance to 3TC. The mutation in P263L was associated with resistance to delavirdine and increased susceptibility to nevirapine (Dueweke et al. 15 al., Ibid. ). In contrast to previous reports, the RTV-268 sample showed no changes in susceptibility F to nevirapine. The mutation, P236L, was characterized using site-directed mutagenesis and the phenotypic susceptibility test in vi tro to correlate 20 changes in amino acid 236 with changes in phenotypic susceptibility.

Site-directed mutagenesis 25-resistance test vectors containing the P236L mutation were constructed alone and in combination with other known drug resistance mutations (K103N, Y181C), which is known to modulate the susceptibility of HIV-1 to NNRTI. Mutations were introduced into the resistance test vector using the mega-primer PCR method for site-directed mutagenesis. (Sakar G and Sommar, Ibid). A resistance test vector containing the P236L (P236H-RTV) mutation was tested using the phenotypic susceptibility assay and the results were compared to those of a genetically defined resistance test vector that was natural at position 236. The P236L -RTV exhibited changes in phenotypic susceptibility to NNRTIs. In the context of a natural background in other circumstances (ie P236L mutation only) P236L-RTV was less susceptible to delavirdine than a natural reference RTV. In contrast to Dueweke et al. no significant changes were observed in the susceptibility to nevirapine for P236L-RTV. The P236L mutation was also introduced in an RTV that contained mutations in K103N, Y181C or both (K103N + Y181C). In all cases, the RTV were less susceptible (more resistant) to delavirdine if the P36KL mutation was present compared to the corresponding RTV that lacks the P236L mutation. In all cases, the P316L mutation did not significantly alter the susceptibility to nevirapine.

Example 5 Correlation of Phenotypic Susceptibility and Genotypic Analysis: G190S Phenotypic Analysis of HIV Patient 97-644 A resistance test vector was constructed as described in Example 1 of a patient sample designated 97-644. This patient had been treated with d4T (NRTI), indinavir (PRI) and efavirenz (NNRTI) for varying periods of 5 to 17 months. Viral RNA isolation and RT / PCR were used to generate a segment derived from the patient that comprised the viral sequences coding for the entire PR and amino acids 1-313 of the RT. The patient-derived segment was inserted into a viral vector of the reporter gene to generate a resistance test vector designated RTV-644. The RTV-644 was then tested using the phenotypic susceptibility assay to accurately and quantitatively determine the level of susceptibility to a panel of antiretroviral drugs. This panel of antiretroviral drugs comprised members of the classes known as NRTI (AZT, 3TC,, d4T, ddl and ddC), NNRTI (delavirdine and nevirapine), and PRI (indinavir, nelfinapir, ritonavir, and saquinavir). An IC50 was determined for each drug tested. The susceptibility of the patient's virus to each drug was examined and compared to the susceptibility of a reference virus. A pattern of NNRTI susceptibility was observed for the patient's RTV-644 sample, a virus sample in which it was found to be more resistant to nevirapine with little or no resistance to delavirdine. The sample was examined additionally for • 10 genotypic changes associated with the susceptibility pattern.

Genotype of the patient with HIV 97-644 The DNA of the RTV-644 was analyzed by the 15 automated sequencing with chain terminator ABI. The nucleotide sequence was compared with the F consensus sequence for HIV-1 class B natural. The nucleotide sequence was examined for sequences that are different from the control sequence. The mutations 20 of the RT were noted at positions K101E and G190S compared to the control sequence. Mutations in T200A and E203D are known polymorphisms in the variants (sensitive to drugs) natural to HIV. The mutation in K101E was associated with resistance to 25 some but not all of the NNRTI. Mutation in G190A but not specifically G190S was associated with resistance to nevirapine and loviride. Mutations in G190S and G190A characterized using site-directed mutagenesis and the phenotypic susceptibility test in vi tro to correlate changes to amino acid 190 with changes in phenotypic susceptibility.

Site-directed mutagenesis F Test vectors were constructed 10 resistance contained in the G190S and G190A mutations. Mutations were introduced into the resistance test vector using the mega-primer PCR method for site-directed mutagenesis. (Sakar G and So mar, Ibid). A test vector of 15 resistance containing the G190S or G190A (G190S-HTV, or G190A-RTV) mutations using the F phenotypic susceptibility and the results were compared with those of a genetically defined resistance test vector that was natural at position G190. He 20 G190A-RTV and G190A-RTV exhibited changes in phenotypic susceptibility to NNRTI. In the context of a natural history in other circumstances, RTV was markedly less susceptible to nevirapine and slightly less susceptible to delavirdine than an RTV 25 of natural reference.

EXAMPLE 6 Prediction of the Response to Non-Nucleoside Inhibitors of Reverse Transcriptase by Characterization of Amino Acid Changes in HIV Reverse Transcriptase Phenotypic and genotypic correlation of mutations at amino acid 236 of HIV-1 Reverse Transcriptase In a modality of this invention, changes in the amino acid at position 236 of the HIV-1 reverse transcriptase protein were evaluated using the following method comprising: antiretroviral of an HIV-infected patient comprising: (i) collecting a biological sample from a patient infected with HIV. HIV-1; (ii) assess whether the biological sample - comprises the nucleic acid encoding HIV-1 reverse transcriptase having a mutation at codon 236. The presence of a mutation at codon 236 (P236L) correlates with a reduction in Susceptibility to delavirdine and little or no change in susceptibility to nevirapine. The biological sample comprises "whole blood, blood components including peripheral mononuclear cells (PBMC), serum, plasma (prepared using various anticoagulants such as EDTA, citric acid-dextrose, heparin), tissue biopsies, cerebrospinal fluid (CSF), or other cells, tissue or body tissues In another embodiment, the HIV-1 nucleic acid (genomic RNA) or reverse transcriptase protein can be isolated directly from the biological sample or after purification of virus particles from the biological sample. of whether the amino acid at position 236 of the HIV-1 reverse transcriptase mutated can be made using several methods, such as the direct characterization of the viral nucleic acid encoding the reverse transcriptase or the direct characterization of the reverse transcriptase protein itself The definition of the amino acid at position 236 of the reverse transcriptase can be made by direct characterization of the reverse transcriptase protein by conventional or novel amino acid sequencing methodologies, recognition of epitopes by antibodies or other proteins or specific binding compounds. Alternatively, the amino acid at position 236 of the HIV-1 reverse transcriptase protein can be defined by characterizing amplified copies of the HIV-1 nucleic acid encoding the reverse transcriptase protein. Amplification of HIV-1 nucleic acid can be done using a variety of methodologies including reverse transcription-reaction in. polymerase chain (RT-PCR), NASBA, SDA, RCR, and 3SR, as is known to those • experts in the art. The evaluation of whether the nucleic acid coding for HIV reverse transcriptase has a mutation at codon 236 can be effected by direct nucleic acid sequencing using various chain-termination extension methodologies (Sanger, ABI / PE and Visible). Genetics) or chain splitting (Maxa and Gilbert) or methods of • 10 more recently developed sequence sequences such as the laser desorption ionization flight time aided by a matrix (MALDI-TOF) or mass spectrometry (Sequenom, Gene Trace Systems). Alternatively, the nucleic acid sequence encoding 15 for the amino acid at position 236 can be evaluated using a variety of probe hybridization methodologies, such as genechip hybridization sequencing (Affymetrix), linear probe assay (LiPA, Murex), and differential hybridization (Chiron). In a preferred embodiment of this invention, the evaluation of whether the amino acid at position 236 of the HIV-1 reverse transcriptase was natural or mutant was carried out using a phenotypic susceptibility assay using the DNA of the HIV test vector. 25 prepared resistance of the biological sample. In one embodiment, a plasma sample was collected, the viral RNA was purified and the RT-PCR methodology was used • amplify a segment derived from the patient that codes for the protease and reverse transcriptase regions of HIV-1. The amplified patient-derived segments were then incorporated, via DNA ligation and bacterial transformation, into a viral vector of the reporter gene thereby generating a resistance test vector. The DNA of the test vector • 10 resistance was isolated from the bacterial culture and the phenotypic susceptibility test was carried out as described in Example 1. The results of the phenotypic susceptibility test with a patient sample having a P236L mutation. The sequence of The nucleic acid (DNA) of the patient derived from the protease and HIV-1 reverse transcriptase regions of the patient sample 268 was determined using a sequencing methodology of the fluorescence detection chain termination cycle. 20 (ABI / PE). The method was used to determine a consensus nucleic acid sequence representing the combination of sequences from the mixture of HIV-1 variants that exist in the subject's sample (representing the quasispecies), and to determine the 25 nucleic acid sequences of individual variants.

Phenotypic susceptibility profiles of patient samples and site-directed mutants showed that susceptibility to delavirdine and nevirapine correlates with the absence of RT mutations at positions 103, 181 or 236 of HIV-reverse transcriptase 1. The phenotypic susceptibility profiles of patient and site-directed mutants showed a significant reduction in delavirdine susceptibility (increased resistance and little or no reduction in nevirapine susceptibility correlated with a mutation in the nucleic acid sequence that encodes the amino acid leucine (L) at position 236 of the HIV-1 reverse transcriptase and the absence of mutations at positions 103 and 181. The phenotypic susceptibility profiles of the patient samples and the site-directed mutants did not show further reduction in susceptibility to delavirdine or nevirapine (increased resistance) with the amino acid proline at position 236 when the RT mutations at positions 103, 181 or 103 and 181 were present (K103N, Y181C, or K103N + Y181C) However, the profiles of phenotypic susceptibility of patient samples and mutants directed to the thio showed an additional reduction in delavirdine susceptibility (increased resistance) and little to no further reduction in susceptibility to nevirapine with the amino acid leucine (L) at position 236 and in addition the RT mutations associated with resistance to the NNRTI (K103N, Y181C, or K103N + Y 181C).

Phenotypic correlation of amino acid 225 mutations of HIV-1 reverse transcriptase Phenotypic susceptibility profiles from patient samples and site-directed mutants did not show changes in susceptibility to delavirdine or nevirapine when the amino acid proline (P) was present at position 225 of HIV-1 reverse transcriptase in the absence of RT mutations associated with resistance to NNRTI (K103N, Y181C). However, phenotypic susceptibility profiles of patient samples and site-directed mutants showed an increased susceptibility to delavirdine and little to no change in susceptibility to nevirapine when the amino acid histidine (H) was present at position 225 in the absence of RT mutations (K103N, Y181C) associated with NNRTI resistance. Phenotypic susceptibility profiles of patient samples and site-directed mutants showed no additional reduction in delavirdine susceptibility or susceptibility to nevirapine when the amino acid proline (P) at position 225 was present in addition to the RT mutations associated with resistance to NNRTI (K103N, Y181C or K103N, + Y181C). In contrast, the phenotypic susceptibility profiles of patient samples and site-directed mutants showed an increased susceptibility to delavirdine and little to no change in susceptibility to nevirapine when the amino acid histidine (H) was present at position 225 in the presence of RT mutations associated with resistance to NNRTI (K103N, Y181C K103N, + Y181C).

Phenotypic and genotypic correlation of mutations of amino acid 190 of HIV-1 reverse transcriptase The phenotypic susceptibility profiles of patient samples and site-directed mutants showed no changes in susceptibility to delavirdine and nevirapine when the amino acid glycine (G ) at position 190 was present in the absence of RT mutations associated with NNRTI resistance (K103N, Y181C). Phenotypic susceptibility profiles of the site-directed mutants showed an increased susceptibility to delavirdine and a decrease in susceptibility to nevirapine when the amino acid alanine (A) was present at position 190 in the absence of associated RT mutations with the resistance to NNRTI. Phenotypic susceptibility profiles of mutant patient samples directed to the site showed an increase in susceptibility to delavirdine and a decrease in susceptibility to nevirapine when the amino acid serine (S) was present at position 190 in the absence of mutations of the RT associated with resistance to NNRTI.

EXAMPLE 8 Use of the Vectors of the Resistance Test and Mutants Directed to the Site to Correlate Genotypes and Phenotypes Associated with Susceptibility and Resistance to NNRTI Drugs in HIV: Y181I Preparation of resistance vectors and phenotypic analysis of patient HIV samples 98-964 A test vector was constructed resistance as described in Example 1 of the sample of a patient designated 98-964. This patient had previously been treated with ddl, d4T, AZT, 3TC, ddC, (NRTI), saquinavir and nalfinavir (PRI) and nevirapine (an NNRTI) and HU. Viral RNA isolation and RT / PCR were used to generate a segment derived from the patient that comprised the viral sequence coding for all the PR and aa 1-313 of the RT. The PDS was inserted in the vector F viral of the indicator gene to generate a test vector * and of resistance designated as RTV-9-64. The RTV-964 was then tested in a phenotypic assay to accurately and quantitatively determine the level of susceptibility to a panel of antiretroviral drugs. This panel of antiretroviral drugs comprised members of the classes known as NRTI (AZT, 3TC, d4T, ddl and ddC), 10 NNRTI (delavirdine and nevirapine), and PRI (indinavir, nelfinapir, ritonavir, and saquinavir) _. An IC50 was determined for the pooled resistance test vector of each drug tested. The susceptibility pattern of all the tested drugs was examined and compared with 15 known susceptibility patterns. A pattern of susceptibility to NNRTI was observed for RTV-964 of the patient in which there was a moderate (10-fold) decrease in susceptibility to delavirdine and a significant decrease (750-fold) in the 20 susceptibility to nevirapine.

Determination of the genotype of HIV samples from the patient The DNA of the RTV-964 was analyzed by 25 automated sequencing with ABI chain terminator. The nucleotide sequence was compared with the consensus sequence of a natural HIV-1 class B (HIV • Sequence Datábase Los Alamos, NM). The genotype was examined for sequences that are different from the control sequence. RT mutations were noted at positions M41L, K43E, D67N, K70R, L74I, V75S, Y181I, R211T, T215Y, D218E, and K219Q compared to the control sequence. The M41L, D67N, K70R, L74I, V75S, T215Y and K219Q were associated with resistance to 10 NNRTI. A mutation in R211T is a known polymorphism in the sequence between different variants (sensitive to drugs) natural to HIV. Previously it had been shown that Y181I is associated with a high level of resistance to nevirapine. We examined the mutation, 15 Y181I, using site-directed mutagenesis and phenotypic susceptibility testing in vi tro, to correlate • the changes observed in the genotype with the phenotype.

Site-directed mutagenesis was used to confirm the role of specific mutations in phenotypic susceptibility to antiretroviral drugs in HIV. The Y181I mutation was introduced into the resistance test vector using the mega-primer method for site-directed mutagenesis ( Sakar 25 G and Sommar, Ibid.). A resistance test vector containing the mutation Y181I (Y181I-RTV) was then tested using the phenotypic assay described at the beginning and the results were compared with those determined using a genetically defined resistance test vector that was natural at the position 181. We determined the pattern of phenotypic susceptibility to NNRTI, delavirdine, nevirapine and efavirenz, in Y181I-RTV. In the natural history (ie mutation Y181I only) the Y181I-RTV presented a moderate parity of susceptibility (20 times) to delavirdine and a significant loss of susceptibility (740 times) to nevirapine compared to natural control RTV. The Y181I-RTV showed natural susceptibility (1.4 times) to efavirenz.

EXAMPLE 9 Use of the Vectors of the Resistance Test and Mutants Directed to the Site to Correlate Genotypes and Phenotypes Associated with the Susceptibility and Resistance to NNRTI Drugs in HIV: Y188 Preparation of the vectors of the resistance test and phenotypic analysis of samples of Patient HIV 97-300 A resistance test vector was constructed as described in Example 1 from a patient sample designated 97-300. This patient had previously been treated with d4T and 3TC (NRTI), indinavir (a PRI) and efavirenz (an NNRTI). Isolation of the viral RNA and RT / PCR were used to generate a segment derived from the patient that comprised the viral sequences coding for the whole PR and the aa 1-313 of the RT. The PDS was inserted into the viral vector of the reporter gene to generate a resistance test vector designated as RTV-300. The RTV-300 was then tested in a phenotypic assay to accurately and quantitatively determine the level of susceptibility to a panel of antiretroviral drugs. This panel of antiretroviral drugs comprised members of the classes known as NRTI (AZT, 3TC, d4T, ddl and ddC), NNRTI (delavirdine, efavirenz and nevirapine), and PRI (indinavir, nelfinapir, ritonavir, and saquínavír). An IC50 was determined for the pooled resistance test vector of each drug tested. The susceptibility pattern of all tested drugs was examined and compared with known susceptibility patterns. A pattern of susceptibility to NNRTI was observed for the patient's RTV-300 in which there was a moderate (25-fold) decrease in susceptibility to delavirdine and a substantial (greater than 800-fold) decrease in susceptibility to nevirapine.

Determination of the genotype of HIV samples from the patient The DNA of the RTV-300 was analyzed by automated sequencing with ABI chain terminator. The nucleotide sequence was compared with the consensus sequence of a natural class B HIV-1 (HIV Sequence Datábase Los Alamos, NM). The genotype was examined by sequences that are different from the control sequence. Mutations were noted at positions K32N, M184V and Y188L compared to the control sequence. The mutation in M184V was associated with resistance to 3TC. Previously it had been shown that Y188L was associated with a high level of resistance to efavirenz. It has been reported that other mutations at position Y188L (ie Y188C and Y188H) have been selected for treatment with several NNRTIs (E-ePseU, E-EPS, HEPT, Nevirapine, BHAP, U-8720E, TIBO R82913, Loviride) . We examined the mutation, Y188L, using site-directed mutagenesis and phenotypic susceptibility testing in vi tro to correlate the changes observed in the genotype with the phenotype.

Site-directed mutagenesis was used to confirm the role of specific mutations in phenotypic susceptibility to antiretroviral drugs in HIV The Y1881- mutation was -introduced into the resistance test vector using the mega-primer method for site-directed mutagenesis ( Sakar and Sommar, Ibid.). A resistance test vector containing the Y188L (Y188L-RTV) mutation was then tested using the phenotypic assay described at the beginning and the results compared with those determined using a genetically defined resistance test vector that was natural at position 188 We determined the pattern of phenotypic susceptibility to NNRTI, delavirdine, nevirapine and efavirenz, in Y188L-RTV. In the natural history (ie Y188L mutation only) the Y188L-RTV had a slightly lower susceptibility (9 times) to delavirdine and a substantial loss of susceptibility (greater than 800 times) to nevirapine and a significant loss of susceptibility (109 times) to efavirenz compared to natural control RTV. The approximately 100-fold susceptibility test to efavirenz was not as high as previously reported.

Site-directed mutagenesis was used to confirm the role of specific mutations in phenotypic susceptibility to antiretroviral drugs in HIV The Y188C mutation was introduced into the resistance test vector using the mega-primer method for site-directed mutagenesis (Sakar G and Sommar, Ibid.). A resistance test vector containing the mutation Y188C (Y188C-RTV) was then tested using the phenotypic assay described at the beginning and the results compared with those determined using a genetically defined resistance test vector that was natural at position 188 We determined the pattern of phenotypic susceptibility to NNRTI, delavirdine, nevirapine and efavirenz, in Y188C-RTV. In the natural history (ie, the Y188C mutation only) Y188C-RTV had a slight loss in susceptibility (3 times) to delavirdine and a moderate loss of susceptibility (30 times) to nevirapine and efavirenz (20 times) compared to the natural control RTV.

Site-directed mutagenesis was used to confirm the role of specific mutations in phenotypic susceptibility to antiretroviral drugs in HIV The Y188H mutation was introduced into the resistance test vector using the mega-primer method for site-directed mutagenesis (Sakar et al. So ar, Ibid.). A resistance test vector containing the Y188H (Y188H-RTV) mutation was then tested using the phenotypic assay described at the beginning and the results were compared with those determined using a genetically defined resistance test vector that was natural at the position 188. We determined the pattern of phenotypic susceptibility to NNRTI, delavirdine and nevirapine, in Y188H-RTV. At • 10 natural antecedent (ie mutation Y188H only) Y18SH-RTV had a moderate loss of susceptibility (3.5 times) to nevirapine compared to a natural control RTV. The phenotypic susceptibility of Y188H to efavirenz was not determined. EXAMPLE 10 • Use of Resistance Testing Vectors and Mutants Directed to the Site to Correlate Genotypes and Phenotypes Associated with Drug Susceptibility and 20 Resistance to NRTI Drugs in HIV: E138 and Y188. Preparation of vectors of resistance tests and phenotypic analysis of samples of patients with HIV 97-209 A resistance test vector was constructed 25 as described in Example 1 of a patient sample designated as 97-209. This patient was previously treated with AZT, ddl, d4T and 3TC (NRTI), indinavir (a PRI) and adefovir. Isolation of viral RNA was used and • RT / PCR to generate a patient-derived segment that comprised the viral sequences coding for the entire PR and aa 1 - 313 of the RT. The PDS was inserted into a viral vector of the reporter gene to generate the resistance test vector designated RT-209. RT-209 was then tested in a genotypic assay to determine • 10 accurately and quantitatively levels of susceptibility to a panel of antiretroviral drugs. This panel of antiretroviral drugs comprised members of the classes known as NRTI (AZT, 3TC, d4T, ddl and ddC), NNRTI (delavirdine, efavirenz and nevirapine), and PRI (indinavir, 15 nelfinavir, ritonavir, and saquinavir). An IC50 was determined for the resistance test vector pooled by each drug-tested. The susceptibility pattern for all drugs tested. The susceptibility pattern for all the drugs tested was examined and compared with 20 of known susceptibility. A pattern of susceptibility to NNRTI of the patient's RTV-209 was observed in which there was a moderate (75-fold) decrease in susceptibility to delavirdine and a substantial (greater than 800-fold) decrease in susceptibility to 25 nevirapine.

Determination of the genotype of patient HIV samples The DNA of the RTV-209 was, analyzed by the automated string terminator sequence ABI. The nucleotide sequence was compared with the consensus sequence of a natural class B HIV-1 (HIV Sequence Datábase Los Alamos, NM). The genotype was examined by sequences that are different from the control sequence. Mutations were noted at positions A62V, S68G, V76I, F77L, F116Y, E138A, Q151M, M184V, Y188L and E291D compared to the control sequence- Mutations in A62V, V75I, F77L, F116Y, Q151M and M184V are associated with resistance to the NRTI. A mutation in E138K had previously been shown to be associated with resistance to several NNRTIs and a mutation in Y188L had previously been shown to be associated with a decreased susceptibility to efavirenz. We examined the Y188L and E138A mutations using site-directed mutagenesis and in vitro phenotypic susceptibility tests to correlate the changes observed in the genotype with the phenotype.

Site-directed mutagenesis was used to confirm the role of specific mutations in phenotypic susceptibility to antiretroviral drugs in HIV E138A mutation alone and in combination with Y188L was introduced into resistance vectors using the mega-primer method for mutagenesis directed to the site (Sakar and Sommar, Ibid.). The resistance test vectors containing the E138A (E138A-RTV) mutations or the E138 mutations together with the Y1881 mutation (E138A-Y188L-RTV) were then tested using the phenotypic assay described at the beginning and the results were compared with those determined using a genetically defined resistance test vector that was natural at position 188 and 138. We determined the pattern of phenotypic susceptibility to NNRTI, delavirdine, nevirapine and efavirenz, in E138A-RTV, Y188L-RTV and E138-Y188L-RTV . In a natural history (ie, the E138A mutation only) E138A-RTV presented natural susceptibility to delavirdine (1.6 times), nevirapine (1.3 times) and efavirenz (1.4 times). The Y188L-RTV presented a slight loss of susceptibility (greater than 800 times) to nevirapine and a significant loss of susceptibility (110-fold) to efavirenz. The E138A-Y18-8L-RTV presented a moderate loss of susceptibility (75 times) to delavirdine and efavirenz (88 times) and a substantial loss of susceptibility to nevirapine (greater than 800 times) compared to a natural control RTV . The combination of mutations resulted in a greater effect on delavirdine susceptibility compared to the effect observed for each mutation alone. - '9 EXAMPLE 11 10 Use of Resistance Testing Vectors and Mutants Directed to the Site to Correlate Genotypes and Phenotypes Associated with the Susceptibility and Resistance to NNRTI Drugs in HIV: A98 Preparation of resistance test vectors in phenotypic analysis of HIV samples from patient 98- F 675. A resistance test vector was constructed as described in Example 1 of a sample of a 20 designated patient 98-675. This patient had previously been treated with ddl, AZT, and 3TC (NRTI), and saquinavir and nelfinavir (PRI). Isolation of the viral RNA and RT / PCR were used to generate the patient-derived segment that comprised the viral sequences that 25 code for all PR and aa 1 - 313 of the RT. The PDS was inserted into a viral vector of an indicator to generate a resistance test vector designated RTV-675. The RTV-675 was then tested in a phenotypic assay to accurately and quantitatively determine the 5 level of susceptibility to a panel of antiretroviral drugs. This panel of antiretroviral drugs comprised members of the classes known as NRTI (AZT, 3TC, d4T, ddl and ddC), NNRTI F (delavirdine, efavirenz and nevirapine) and PRI (indinavir, 10 nelfinavir, ritonavir, and saquinavir). An IC50 was determined for the resistance test vector pooled for each drug tested. The pattern of susceptibility to all tested drugs was examined and compared to known susceptibility patterns. A 15 pattern of susceptibility to NNRTI for RTV-675 in a patient in whom susceptibility was observed Natural F (2.1 times) for delavirdine and a slight decrease (ß fold) was observed for susceptibility to nevirapine. 20 Determination of the genotype of patient HIV samples The DNA of the RTV-675 was analyzed by automated sequencing and ABI chain termination. The nucleotide sequence was compared with the sequence 25 consensus of a natural HIV-1 class B (HIV Sequence Datábase Los Alamos, NM). The genotype was examined by sequences that are different from the control sequence. Mutations were noted at positions M41L, S48t, L74V, A98G, M184V and T215Y associated with NRTI resistance. A mutation in A98G had previously been shown to be associated with resistance to nevirapine. We examined the A98G mutation using site-directed mutagenesis and phenotypic susceptibility testing in vitro to correlate the changes observed in the genotype with • 10 the phenotype.

Site-directed mutagenesis was used to confirm the role of specific mutations in phenotypic susceptibility to antiretroviral drugs in HIV 15 - The A98G mutation in the resistance test vector using the mega-primer method for site-directed mutagenesis (Sakar and Sommar , Ibid.). A resistance test vector containing the mutation A98g (A98G-RTV) was then tested using the assay 20 phenotypic described at the beginning and the results were compared with those determined using a genetically defined resistance test vector that was natural - position 98. We determined the pattern of genotypic susceptibility to NNRTI, delavirdine, 25 nevirapine and efavirenz, in the A98G-RTV. In a natural history (ie, the A98G mutation only) the A98G-RTV had a slight loss of susceptibility to delavirdine (3 times), nevirapine (8 times) and efavirenz (3 times) compared to "natural RTV control.

Example 12 Use of Resistance Test Vectors and Mutants Directed to the Site to Correlate Genotypes and Phenotypes Associated with the Susceptibility and Resistance to NNRTI Drugs in HIV: A98 and G190 Preparation of resistance test vectors and genotypic analysis of HIV samples from patient B. A vector of resistance tests was constructed as described in Example 1 of a patient sample designated as B. The antiretroviral treatment that this patient received was do not know Viral RNA isolation and RT-PCR were used to generate a segment derived from the patient that comprised the viral sequences coding for the whole PR and aa 1 - 313 of the RT. The PDS was inserted into a viral vector of the reporter gene to generate a resistance test vector designated RTV-B. Individual clones of the assembled RTV-B were selected and then tested in a phenotypic assay to accurately and quantitatively determine the level of susceptibility to a panel of antiretroviral drugs. This panel of antiretroviral drugs comprised members of the classes known as NRTI (AZT, 3TC, d4T, ddl and ddC), NNRTI (delavirdine, and nevirapine), and PRI (indinavir, nelfinavir, ritonavir and saquinavir). An IC50 was determined for the clone of the resistance test vector for each drug tested. The susceptibility pattern of all tested drugs was examined and compared with known susceptibility patterns. A pattern of susceptibility to NNRTI was observed for clone 1 of RTV-B of the patient in which there was an increase in susceptibility (0.55 times) to delavirdine, a substantial loss of susceptibility (640 times) to nevirapine and a significant loss of susceptibility (250 times) to efavirenz.

Determination of the genotype of HIV samples from the patient The DNA of clone 1 of the RTV-B was analyzed by automated sequencing with ABI chain terminator. The nucleotide sequence was compared to the consensus sequence of a natural HIV-1 class (HIV Sequence Datábase Los Alamos, NM). The genotype was examined by sequences that are different from the control sequence. Mutations were noted at positions M41L, A98G, M184V, L210W, R211, T215Y, E297A and G190S compared to the control sequence. The M41L, M184V, L210W and T215Y are associated with the NRTI resistor. A mutation in A98G that had previously been shown to be associated with resistance to nevirapine. A mutation in position G190A had previously been shown to be associated with changes in susceptibility to nevirapine. Other changes in position 190 have also been reported (ie, E, Q, and T). We examined the A98G and G190S mutations, using site-directed mutagenesis and in vitro genotypic susceptibility testing to correlate the changes observed in the genotype with the phenotype.

Site-directed mutagenesis to confirm the role of specific mutations in phenotypic susceptibility to antiviral drugs in HIV A98 and G190S mutations were introduced alone or in combination into the resistance test vector using the mega-primer method for site-directed mutagenesis (Sakar and Sommar, Ibid. ). The resistance test vectors containing the A98G mutation (A98G-RTV), the G190S mutation (G190S-RTV) and both mutations (A98G-G190S-RTV) were then tested using the phenotypic assay described at the beginning and the results compared. with those determined using a test vector of • genetically defined resistance that was natural at positions 98 and 190. We determined the pattern of 5 phenotypic susceptibility to the NNRTIs, delavirdine, nevirapine and efavirenz, in all three vectors. In a natural history (ie the A98G mutation only) the A98G-RTV had a slight loss to susceptibility to delavirdine (3 times), nevirapine (8 • 10 times) and efavirenz (3 times) compared to the natural RTV control. In a natural history (ie the G190S mutation only) the G190S-RTV presented an increase in susceptibility (0.5 times) to delavirdine, a moderate loss of susceptibility (75 15 times) to nevirapine and a slight loss of susceptibility (8 times) to efavirenz compared to • a natural control RTV. The A98G-G190-RTV presented an increase in susceptibility of (0.8 times) to delavirdine, but a substantial loss of 20 susceptibility to both nevirapine (more than 800 times) and efavirenz (more than 250 times) compared to a natural control RTV. Although only a slight loss of susceptibility to efavirenz was observed for individual mutations, the combination of A98G and 25 G190S resulted in a substantial loss in susceptibility to efavirenz. Likewise, this combination of mutations resulted in a greater loss of susceptibility to nevirapine than the sum of the two mutations alone.

EXAMPLE 13 Use of Resistance Test Vectors in Mutants Directed to the Site to Correlate Genotypes and Phenotypes Associated with the Susceptibility and Resistance to NNRTI Drugs in HIV: Y181 and A98 Preparation of resistant test vectors and phenotypic analysis of patient samples 98-1057 A resistance test vector was constructed as described in Example 1 of a sample from a patient designated 98-1057. This patient had previously been treated with ddl, d4T, AZT, and 3TC (NRTI), saquinavir and indinavir (PRI) and delavirdine (an NNRTI). Viral RNA isolation and RT / PCR were used to generate a segment derived from the patient that comprised the viral sequences coding for the entire RP and aa 1-313 of the RT. The PDS was inserted into the viral vector of the reporter gene to generate the resistance test vector designated RTV-1057. The RTV-1057 was then tested in a phenotypic assay to accurately and quantitatively determine the level of susceptibility to a panel of drugs • antiretrovirals. This panel of antiretroviral drugs comprised the member of classes 5 known as NRTI (AZT, 3TC, d4T, ddl, and ddC), NNRTI (delavirdine, efavirenz and nevirapine) and PRI (indinavir, nelfinavir, ritonavir, "and saquinavir). An IC50 was determined for the resistance test vector collected for each drug tested.

All the drugs tested were examined and compared with known susceptibility patterns. A pattern of susceptibility to NNRTI was observed for the patient's RTD-1057 in which there was a moderate decrease in susceptibility to delavirdine (35 times) and a 15 significant decrease (610 times) and susceptibility to nevirapine. • Determination of the genotype of HIV samples from patient 20 The DNA of RTV-1057 was analyzed by automated sequencing with ABI chain terminator. The nucleotide sequence was compared with the consensus sequence of HIV-1 class B natural (HIV Sequence Datábase, Los Alamos, NM). The genotype was 25 examined by sequences that are different from the control sequence. Mutations were observed in positions T39A, M41L, A62V, D67E, T69SST, A98G, I135T, • Y181C, T200I and T215Y compared to the control sequence. The M41L, A62V, D67E, T69SST and T215Y are 5 associated with NRTI resistance. Mutations at positions I135T and T200I are known polymorphisms in the sequence between different (drug-sensitive) natural variants of HIV. The Y181C and the A98G have previously shown that they are associated with • 10 resistance to certain NNRTI. We examined the Y181C and A98G mutations using site-directed mutagenesis and phenotypic evidence of their in vitro susceptibility to correlate the changes observed in the genotype with the genotype. 15 Site-directed mutagenesis was used to confirm the role of specific mutations in phenotypic susceptibility to antiretroviral drugs in HIV The Y181C and A98G mutations were introduced 20 alone or in combination in resistance test vectors using the mega-primer method for site-directed mutagenesis (Sakar and Sommar, Ibid.). The resistance test vectors containing the mutation Y181C (Y181C-RTV) and the mutation A98G (A98G-RTV) 25 and both mutations (A98G-G190S-RTV) were then tested using the phenotypic assay described at the beginning and the results were compared with those determined using a genetically defined resistance test vector that was natural at position 181 and 98. We determined the pattern of phenotypic susceptibility to NNRTI, delavirdine, nevirapine and efavirenz, in the three vectors. In a natural history (ie the Y181C mutation only) Y181C-RTV had a moderate loss of susceptibility (35 times), to delavirdine, a significant loss of susceptibility (161 times) to nevirapine and a slight loss of susceptibility (3 times) to efavirenz compared to the natural RTV control. Y181C-A98C-RTV presented a significant loss of susceptibility (240-fold) to delavirdine, a substantial loss of susceptibility (greater than 800 times) to nevirapine, and a slight loss of susceptibility (7-fold) to efavirenz compared to a RTV natural control. These data indicate that the combination of the two mutations, Y181C and A98G, resulted in a greater loss of susceptibility to both delavirdine to both nevirapine than the sum of the effects observed for those two mutations individually.

EXAMPLE 14 Use of Resistance Testing and Site-directed Mutants to Correlate Genotypes and Phenotypes Associated with Susceptibility and Resistance to NNRTI Drugs in HIV: K101 and G190 Preparation of resistance test vectors and phenotypic analysis of HIV samples from Patients 98-644 and 98-1060 A resistance test vector was constructed as described in Example 1 of a sample from a patient designated 98-1644. This patient had previously been treated with d4T (an NRTI), indinavir (a PRI) and efavirenz (an NNRTI). A second resistance test vector was constructed as described in Example 1 of a sample from a patient designated 98-1060. This patient had previously been treated with d4T (an NRTI), indinavir (a PRI) and efavirenz (an NNRTI). Viral RNA isolation and RT / PCR were used to generate a segment derived from the patient that comprised the viral sequences coding for the entire RP and aa 1-313 of the RT. The PDS was inserted into the viral vector of the reporter gene to generate resistance test vectors designated as RTV-1644 and RTV-1060. The RTV-644 and the RTV-1060 were then tested in a phenotypic assay to accurately and quantitatively determine the level of susceptibility to a panel of drugs • antiretrovirals. This panel of antiretroviral drugs comprised the member of classes 5 known as NRTI (AZT, 3TC, d4T, ddl, and ddC), NNRTI (delavirdine and nevirapine) and PRI (indinavir, nelfinavir, ritonavir, and saquinavir). An IC50 was determined for the resistance test vector collected for each drug tested. The susceptibility pattern 10 for all tested drugs was examined and compared with known susceptibility patterns. A pattern of susceptibility to NNRTI was observed for the RTD-644 of the patient in which there was a slight decrease (2.5 times) in the susceptibility to delavirdine and 15 a significant decrease (600 fold) in susceptibility to nevirapine. A pattern was observed • the susceptibility to NNRTI for the RTD-644 of the patient in which there was a very slight decrease (2.5 fold) in susceptibility to delavirdine and 20 a significant decrease (600-fold) in susceptibility to nevirapine. A pattern of susceptibility to NNRTI was observed for RTV-1060 of the patient in which a natural susceptibility (1.5 fold) to delavirdine was observed. One was observed 25 significant decrease in susceptibility to efavirenz (900-fold) and a substantial decrease in delavirdine (greater than 800-fold) for RTV-1060.

Determination of the genotype of the patient's HIV samples The DNA of the RTV-644 and RTV-1060 was analyzed by automated sequencing with ABI chain terminator. The nucleotide sequence was compared with the consensus sequence of HIV-1 class B natural (HIV Sequence Datábase, Los Alamos, NM). The genotype was examined by sequences that are different from the control sequence. Mutations were noted at positions K101E and G190S for RTV-644 compared to the control sequence and mutations were noted at positions K101E, G109S, T200A and T215Y for RTV-1060 compared to the control sequence. The sequence at position T215 was a natural mixture and mutation. A mutation in position K101E had previously been shown to be associated with resistance to several NNRTIs including a high level of resistance to delavirdine. A mutation in position G190A had previously been shown to be associated with changes in susceptibility to nevirapine. Other changes in position 190 have also been reported (ie E, Q and T). We examined the K101E and G190S mutations, using site-directed mutagenesis and in vitro phenotypic susceptibility testing to correlate the changes observed in the genotype with the phenotype. 5 Site-directed mutagenesis was used to confirm the role of specific mutations in phenotypic susceptibility to antiretroviral drugs in HIV K191E and G190S mutations were introduced alone or in combination in resistance test vectors • 10 using the mega-primer method for site-directed mutagenesis (Sakar and Sommar, Ibid.). The test vectors containing the K101E mutation (KIOIE-RTV, the G190S mutation (G190S-RTV) were then tested using the phenotypic assay described at the beginning and the 15 results were compared with those determined using a genetically defined resistance test vector that • was natural at positions 101 and 190. We determined the pattern of phenotypic susceptibility to the NNRTIs, delavirdine, nevirapine and efavirenz, in the three vectors. In 20 the natural background (ie K101E mutation only) the KIOIE-RTV presented a slight loss of susceptibility (5 times) to delavirdine and efavirenz (5 times) and a moderate loss of susceptibility (12 times) to nevirapine compared to natural control RTV. He 25 K101E-G190S-RTV presented an increase in susceptibility to delavirdine (0.5 times), a moderate loss of susceptibility to nevirapine (75 times) and a slight loss of susceptibility (7.6 times) to efavirenz compared to a control RTV natural. The K101E-G190S-RTV presented natural susceptibility (1.4 times) to delavirdine and a substantial loss of susceptibility to both of nevirapine (greater than 800 times) and efavirenz (greater than 250 times) compared to a natural RTV control. In this example, the combination of • 10 mutations, G190S and K101E, presented a novel phenotypic pattern. The combination resulted in the regression of the effect on delavirdine susceptibility observed for the G190S mutation and greater than the additive effect on the susceptibility for both of the 15 nevirapine and efavirenz.

• EXAMPLE 15 Use of the Vectors of the Resistance Test and Mutants Directed to the Site to Correlate Genotypes 20 and Phenotypes Associated with the Susceptibility and Resistance to NNRTI Drugs in HIV: V108I Preparation of resistance vectors and phenotypic analysis of HIV samples from patient 98-652 A resistance test vector was constructed 25 as described in Example 1 of a patient sample designated 98-652. This patient had not been previously treated with antiretrovirals. Isolation of the viral RNA and RT / PCR were used to generate a segment derived from the patient that comprised the viral sequences coding for the whole PR and the aa 1-313 of the RT. The PDS was inserted into a viral vector of the reporter gene to generate a resistance test vector designated as RTV-652. The RTV-652 was then tested in a phenotypic assay to accurately and quantitatively determine the level of susceptibility to a panel of antiretroviral drugs. This panel of antiretroviral drugs comprised members of the classes known as NRTI (AZT, 3TC, d4T, ddl and ddC), NNRTI (delavirdine and nevirapine), and PRI findinavir, nelfinapir, ritonavir, and saquinavir). An IC50 was determined for the pooled resistance test vector of each drug tested. The susceptibility pattern of all tested drugs was examined and compared with known susceptibility patterns. A pattern of susceptibility to NNRTI was observed for patient RTV-652 in which there was an increase in susceptibility (0.97 times) to delavirdine and a slight (5-fold) decrease in susceptibility to nevirapine was observed.

Determination of the genotype of patient's HIV samples • The DNA of RTV-652 was analyzed by automated sequencing with ABI chain terminator. The 5 nucleotide sequence was compared with the consensus sequence of a natural class B HIV-1 (HIV Sequence Datábase Los Alamos, NM). The genotype was examined for sequences that are different from the control sequence. RT mutations were noted at positions M41L, V108I, I13-5T, L210W, 10 R211K and T215D compared to the control sequence. M41L, L210W and T215D were associated with resistance to NNRTI. It is known that mutations at positions I135T and R211K are sequence polymorphisms between different variants (sensitive to drugs) natural to HIV. It's known 15 that V108I is associated with resistance to several NNRTIs. We examined the mutation, V108I, using mutagenesis • directed to the site and in phenotypic susceptibility tests in vitro to correlate the changes observed in the genotype with the phenotype. 20 Site-directed mutagenesis was used to confirm the role of specific mutations in phenotypic susceptibility to antiretroviral drugs in HIV The V108I mutation was introduced into the vector of 25 resistance test using the mega-primer method for site-directed mutagenesis (Sakar and Sommar, Ibid.). A resistance test vector containing the V108I (V108I-RTV) mutation was then tested using the phenotypic assay described at the beginning and the results compared with those determined using a genetically defined resistance test vector that was natural at the position 108. We determined the pattern of phenotypic susceptibility to NNRTI, delavirdin, nevirapine and efavirenz, in V108I-RTV. In an antecedent • 10 natural (ie V108I mutation only) V108I-RTV had a natural susceptibility (1.3 times) to delavirdine and efavirenz (1.7 times) and a slight loss of susceptibility (3 times) to nevirapine compared to a control RTV natural. 15 EXAMPLE 16 • Use of the Vectors of the Resistance Test and Mutants Directed to the Site to Correlate Genotypes and Phenotypes Associated with Susceptibility and 20 Resistance to NNRTI Drugs in HIV: K103 and K101 and G190 Preparation of resistance test vectors and phenotypic analysis of patient HIV samples 98-955 A resistance test vector was constructed 25 as described in Example 1 of a sample from a patient designated 98-955. This patient had been treated with nelfinavir (a PRI). The • Isolation of the viral RNA and RT / PCR to generate a segment derived from the patient that comprised the 5 viral sequences coding for the whole PR and the aa 1-313 of the RT. The PDS was inserted into a viral vector of the reporter gene to generate a resistance test vector designated as RTV-955. The RTV-955 was then tested in a phenotypic assay to determine • 10 accurately and quantitatively the level of susceptibility to a panel of antiretroviral drugs. This panel of antiretroviral drugs comprised members of the classes known as NRTI (AZT, 3TC, d4T, ddl and ddC), NNRTI (delavirdine, efavirenz and nevirapine), and PRI 15 (indinavir, nelfinapir, ritonavir, and saquinavir). An IC50 was determined for the pooled resistance test vector of each drug tested. The susceptibility pattern of all tested drugs was examined and compared with susceptibility patterns 20 known. A pattern of susceptibility to NNRTI was observed for patient RTV-955 in which there was a slight (4-fold) decrease in susceptibility to delavirdine and a significant (530-fold) decrease in susceptibility to nevirapine. 25 Determination of genotype of patient HIV samples The DNA of RTV-955 was analyzed by automated sequencing with ABI chain terminator. The nucleotide sequence was compared with the consensus sequence of a natural class B HIV-1 (HIV Sequence Datábase Los Alamos, NM). The genotype was examined for sequences that are different from the control sequence. RT mutations were noted at positions K20R, V35I, A62V, D67N, T69D, V75I, F77L, K101E, K103N, Y115F, F116Y, Q151M, I167V, Y181C, M184V, G190A, I202V, R211K, F214L, T215V, and K219Q compared to the control sequence. Mutations at positions K101E, K103N, Y181C, G190A and F214L were mixtures of natural and mutation. The A62V, D67N, T69D, V75I, F77L, Y115F, F116Y, Q151M, M184V, T215V, and K219Q were associated with NNRTI resistance. Mutations in V35I, R211K and F214L are sequence polymorphisms between different variants (sensitive to drugs) natural to HIV. A mutation in position K101E has previously been shown to be associated with resistance to NNRTI. A mutation in Y181I has previously been shown to be associated with a high level of resistance to nevirapine. A mutation in K103N has previously been shown to be associated with resistance to all three NNRTIs, delavirdine and nevirapine and efavirenz. We examined mutations K101E, J103N and G190A using site-directed mutagenesis and phenotypic susceptibility tests in vi tro to correlate the changes observed in the genotype with the phenotype.

Site-directed mutagenesis was used to confirm the role of specific mutations in phenotypic susceptibility to antiretroviral drugs in HIV • 10 K101E, K103N, and G190A mutations were introduced alone or in combination with resistance vectors using the mega-primer method for site-directed mutagenesis (Sakar and Sommar, Ibid.). The resistance test vectors 15 containing the K101E mutation (KIOIE-RTV), the K103N mutation (K103N-RTV), the G190A mutation (G190A-RTV) and two • mutations (K101E-G190-RTV) and (K103N-G190A-RTV) were then tested using the phenotypic assay described at the beginning and the results were compared with We determined the phenotypic susceptibility pattern to the NNRTIs, delavirdine, nevirapine and efavirenz, in the five vectors. In a 25 natural history (ie the K101E mutation only) K101E showed a slight (5-fold) loss of susceptibility to delavirdine and efavirenz (5 times) and a moderate loss of susceptibility (12 times) to nevirapine (55 times) and efavirenz (30 times) compared to a natural control RTV. In a natural history (ie, the G190A mutation only), G190A showed greater susceptibility (8 times) to efavirenz compared to a natural control RTV. K101E-G190A-RTV presented natural susceptibility (2 times) to delavirdine, a substantial loss of susceptibility (greater than 800 times) to nevirapine and a significant loss of susceptibility (120 times) to efavirenz compared to a natural control RTV. K103N-G190A-RTV had a moderate (40-fold) loss of susceptibility to delavirdine, a substantial loss of susceptibility (greater than 800-fold) to nevirapine, and a significant loss of susceptibility (215-fold) to efavirenz compared with a RTV natural control. The introduction of a second mutation to a vector containing G190A resulted in a regression of the effect on delavirdine susceptibility observed for the G190A mutation alone. The G190A mutation exhibited an increased susceptibility to delavirdine. However, the addition of either K101E or K103N mutations to G190A resulted in a slight loss of susceptibility to delavirdine. Besides, the • combination of G190A and K101E resulted in a greater additive effect on the loss of susceptibility to nevirapine and efavirenz. Finally, these data indicated that the combination of the two G190A and K103N mutations resulted in a greater loss of susceptibility to both nevirapine and efavirenz than the sum of the effects observed for those two 10 mutations individually.

EXAMPLE 17 Use of the Vectors of the Resistance Test and Mutants Directed to the Site to Correlate Genotypes 15 and Phenotypes Associated with the Susceptibility and Resistance to NNRTI Drugs in HIV: V106 and V189 and V181 and • F227 Preparation of the resistance test vectors and phenotypic analysis of patient HIV samples 98-20 2033 and 98-757 A resistance test vector was constructed as described in Example 1 of a sample from a designated patient like 98-1033. This patient had previously been treated with AZT, d4T, 3TC and ddl (NRTI), 25 saquinavir, indinavir and nelfinavir (PRI) and nevirapine (an NNRTI). A second resistance test vector was constructed as defined in Example 1 of a sample obtained from the same patient at a different time and designated as 98-757. This patient had received an additional 5 weeks of treatment with nevirapine (one NNRTI), d4T (one NNRTI) and saquinavir and nelfinavir (PRI). Isolation of the viral RNA and RT / PCR were used to generate a segment derived from the patient that comprised the viral sequences that codify for all • 10 the PR and aa 1-313 of the RT. The PDS was inserted into a viral vector of the reporter gene to generate resistance test vectors designated as RTV-1033 and RTV-757. The RTV-1033 and the RTV-757 were then tested in a phenotypic assay to determine exact and 15 quantitatively the levels of susceptibility to a panel of antiretroviral drugs. This panel of • antiretroviral drugs comprised members of the classes known as NRTI (AZT, 3TC, d4T, ddl and ddC), NNRTI (delavirdine, and nevirapine), and PRI (indinavir, 20 nelfinapir, ritonavir, and saquinavir). An IC50 was determined for the pooled resistance test vector of each drug tested. The susceptibility pattern of all tested drugs was examined and compared with known susceptibility patterns. A 25 NNRTI susceptibility pattern for patient RTV-1033 of the patient in which there was a moderate (30-fold) decrease in susceptibility to delavirdine and a substantial (greater than 800-fold) decrease in susceptibility to nevirapine and a significant decrease (200-fold) in susceptibility to efavirenz. A pattern of susceptibility to NNRTI was observed for RTV-757 of the patient in which there was a slight (10-fold) decrease in susceptibility to delavirdine and a substantial (greater than 800-fold) decrease in susceptibility to nevirapine.

Determination of the Genotype of the Patient's HIV Samples The DNA of the RTV-1033 and RTV-757 was analyzed by automated sequencing with ABI chain terminator. The nucleotide sequence was compared with the consensus sequence of an HIV-1 class B (HIV Sequence Datábase Los Alamos, NM). The genotype was examined by sequences that are different from the control sequence. Mutations were observed at positions V35I, D67N, T69D, K70R, V106A, V189L, T200A, I202T, R211K, T215F, D218E, K219Q, H221Y, F227L, L228H and R284 for RTV-1033 compared to the control sequence. Mutations were observed at positions V35I, D67N, T69D, K70R, V106A, V108I, L109V, Y108C, V189L, T200A, I202T, R211K, T215F, D218E, K219Q, H221Y, L228H, L283I and R284K for the RTV-757 compared with the control sequence. The sequences at positions V106A, V108I and L109V were a mixture of natural and mutation. The D67N, T69D, K70R, T215F and K219Q are associated with NRTI resistance. Mutations in V35I, T200A, R211K and R284K are known polymorphisms in the sequence between different variants (sensitive to drugs) natural to HIV. A mutation in V106A has previously been shown to be associated with an increase in resistance to nevirapine. A mutation in V189I has previously been shown to be associated with NNRTI resistance but a mutation to L in this position has not previously been reported as associated with resistance to NNRTI. A mutation in V108I has previously been shown to be associated with increased resistance to both delavirdine and nevirapine. A mutation in Y181C has previously been shown to be associated with increased resistance to both delavirdine and nevirapine. We examined the mutations V106A, V189L, V181C and F227L using site-directed mutagenesis _- and in vitro phenotypic susceptibility tests to correlate the changes observed in the genotype with the phenotype.

Site-directed mutagenesis was used to confirm the role of specific mutations in susceptibility • Phenotypic to antiretroviral drugs in HIV Mutations V106A, V189L, V181C and F227L 5 were introduced alone or in combination in resistance test vectors using the mega-primer method for site-directed mutagenesis (Skar and Sommar, Ibid.). The test vectors containing the mutation V106A (V106A-RTV), the mutation V189L (V189L-RTV), the 10 mutation V181C (V181C-RTV), and the mutation F227L (F2271-RTV) and two mutations (V106A-Y181C-RTV) and (V106A-V189L-RTV) and (V106A-F227-RTV) and (V181C-F227- RTV) and three mutations, (V106A-Y181C-F227L-RTV) were then tested using the phenotypic assay described above.

At the beginning, the results were compared with those determined using a test vector of • genetically defined resistance that was natural at positions 106, 189, 181 and 227. We determined the pattern of phenotypic susceptibility to NNRTIs, 20 delavirdine, nevirapine and efavirenz, in the nine vectors. In a natural history (ie, the V106A mutation only) V106A-RTV had a slight (5-fold) loss of susceptibility to delavirdine and a moderate loss of susceptibility 25 (60 times) to nevirapine and a natural susceptibility (1.7 times) to efavirenz compared to a natural control RTV. In a natural history (ie, the # V189L mutation only) V189-RTV presented a natural susceptibility to delavirdine (1.8 times), 5 nevirapine (1.3 times) and efavirenz (1.3 times) compared to a natural control RTV. In a natural history (ie, mutation of V181C only) Y181C-RTV had a significant (100-fold) loss of susceptibility to delavirdine and a loss • substantial susceptibility (greater than 800 times) to nevirapine and a slight loss of susceptibility (4 times) to efavirenz compared to a natural control RTV. In a natural history (ie, the F227L mutation only) the F227L-RTV presented greater susceptibility 15 (0.03) times to delavirdine and efavirenz (0.48 times) and a slight loss of susceptibility (3 times) to nevirapine compared to a natural RTV control. V106A-Y181C-RTV had a significant (100-fold) loss of susceptibility to delavirdine and a substantial loss of 20 susceptibility (greater than 800 times) to nevirapine and a slight loss of susceptibility (4 times) to efavirenz compared to a natural control RTV. V106A-V189L-RTV showed a slight loss of susceptibility (3 times) to delavirdine, a moderate loss of susceptibility 25 (50 times) to nevirapine and a natural susceptibility (1 fold) to efavirenz compared to a natural control RTV. The V106A-F227-RTV presented a slight loss of • susceptibility (3 times) to delavirdine and a substantial loss of susceptibility (greater than 800 times) to nevirapine and a slight loss of susceptibility (8 times) to efavirenz compared to a natural control RTV. Y181C-F227L-RTV presented greater susceptibility (0.89 times) to delavirdine and efavirenz (0.99 times) and a significant loss of susceptibility (285 times) to nevirapine. 10 compared to a natural control RTV. V106A-Y181C-F227L-RTV presented a moderate (50-fold) loss of susceptibility to delavirdine and a substantial loss of susceptibility (greater than 800 times) to nevirapine and a slight loss of susceptibility (12-fold) to efavirenz in 15 comparison with a natural control RTV.

EXAMPLE 18 Use of Mutant Resistance Test Vectors Directed To The Site To Correlate Genotypes And 20 Phenotypes Associated with the Susceptibility and Resistance to NNRTI Drugs in HIV: Y188 and L100 and K103 Preparation of Resistance Test Vectors and Phenotypic Analysis of the Patient's HIV 98-1058 A resistance test vector was constructed 25 as described in Example 1 from a sample of a patient designated 98-1058. This patient had been previously treated with ddl, d4T, AZT, 3TC, • ddC and abacavir (NRTI), indinavir and amprenavir (PRI) and nevirapine (an NNRTI). Isolation of the viral RNA and RT / PCR was used to generate a segment derived from the patient that comprised the viral sequence coding for all the RP and aa 1 - 313 of the RT. The PDS was inserted into a viral vector of the indicator gene to generate a resistance test vector designated as • 10 RTV-1058. Individual clones were selected from RTV-1058 and then tested in a phenotypic assay to accurately and quantitatively determine the level of susceptibility to a panel of antiretroviral drugs. The panel of antiretroviral drugs 15 comprised members of classes known as NRTI (AZT, 3TC, d4T, ddl and ddC), NNRTI (delavirdine and nevirapine), a PRI (indinavir, nelfinavir, ritonavir, and saquinavir). An IC50 was determined for the resistance test vector collected for each drug tested. 20 The pattern of susceptibility to all drugs tested and examined compared to known susceptibility patterns. A pattern of susceptibility to NNRTI was observed for clones 4, 5 and 10 of the patient's RTV-1058. Clone 4 presented a loss Significant susceptibility (85 times) for delavirdine a substantial loss of susceptibility (greater than 800 times) for nevirapine. The clone 5 • presented a substantial loss of susceptibility (250 times) to delavirdine and a significant loss of 5 susceptibility (120 times) to nevirapine: The clone 10 presented a substantial loss of susceptibility (greater than 250 times) to delavirdine and (more than 800 times) to nevirapine. 10 Determination of the genotype of HIV samples from the patient The DNA of the RTV-1058 was analyzed by the automated ABI chain terminator sequencing. The nucleotide sequence was compared with the sequence of 15 consensus of an HIV-1 class B (HIV Sequence Datábase Los Alamos, NM). The genotype was examined by sequences that • they are different from the control sequence. Mutations were observed at positions M41L, A62V, D67N, T69SST, L74V, L100I, K103N, V118I, I135T, T200S, L210W, R211K and 20 T215Y- compared to the control sequence. The L74V and the L100I were natural mixtures and mutations. Clone 4 had mutations at positions K103N and Y188L. Clone 5 had mutations at positions L100I and K103N. Clone 10 had mutations at positions L10I, K103N 25 and Y188L. The M41L, A62V, D67N, T69SST, L74V, L210W and T215Y are associated with NRTI resistance. Mutations at positions I135T, T200S and R211T are • Known polymorphisms in the sequences between different variants (sensitive to drugs) natural to HIV. A mutation in L100I had previously been shown to be associated with resistance to delavirdine and nevirapine. A mutation in K103L had previously been shown to be associated with resistance to F delavirdine, nevirapine and efavirenz. Examining the 10 mutations, Y188L, L100I and K103N, using site-directed mutagenesis and in vitro phenotypic susceptibility testing to correlate the changes observed in the genotype with the phenotype. 15 Site-directed mutagenesis was used to confirm the role of specific mutations in phenotypic susceptibility to antiretroviral drugs in HIV The Y188L, L100I and K103N mutations were introduced alone and in combination in vectors of 20 resistance tests using the mega-primer method for site-directed mutagenesis (Sakar and Sommar, Ibid.). The resistance test vectors containing the mutation Y188L (Y188L-RTV), the mutation L100I (L100I-RTV), the mutation K103N (K103N-RTV), the two 25 mutations (K103N-Y188L-RTV) and (L100I-K103N-RTV), and all three mutations (L100I-K103N-Y188L-RTV) were then tested using the phenotypic assay described at the beginning the results were compared with those determined using a genetically defined resistance test vector that was natural at positions 188, 100, and 103. We determined the pattern of phenotypic susceptibility to NNRTIs, delavirdine, nevirapine and efavirenz, in vectors. In a natural history (ie, mutation Y188L only) Y188L-RTV had a slight loss of susceptibility (9 times) to delavirdine, a substantial loss of susceptibility (greater-800 times) to nevirapine and a moderate loss of susceptibility (110 times) to efavirenz compared to a natural control RTV. In a natural history (that is, the mutation (L100I only) the L1Q0I-RTV presented a moderate loss of susceptibility (30 times) to delavirdine, and efavirenz (10 times) and presented a slight moderate loss of susceptibility (10 times) and a slight loss of susceptibility (3 times) ) to nevirapine compared to a natural control RTV. In a natural history (ie, the K103N mutation only) K103N-RTV had a moderate loss of susceptibility to delavirdine, (50 times), nevirapine (55 times) and efavirenz (30 times) compared to a RTV control natural. K103N-Y188L-RTV presented substantial losses of susceptibility to delavirdine (greater than 250 times), nevirapine (greater than 800 times) and efavirenz (greater than 250 times) compared to a natural RTV control. The L100I-K103N-RTV presented substantial losses of susceptibility (greater than 250 times) to delavirdine and efavirenz (greater than 250 times) and a moderate loss of susceptibility (70 times) to nevirapine compared to a natural RTV control. The LL100I-K103N-Y188L-RTV presented a substantial loss of susceptibility to delavirdine (greater than 250 times) nevirapine (greater than 800 times) and efavirenz (greater than 250 times) compared to a natural RTV control. The novel combinations resulted in unpredictable resistance patterns that were different from those patterns observed for each of the mutations alone. It is noted that with regard to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (1)

1. REVIVALS Having described the invention as above, the content of the following claims is claimed as property. A method for evaluating the effectiveness of the non-nucleoside antiretroviral therapy of the reverse transcriptase of an HIV-infected patient, characterized in that it comprises: (a) collecting a plasma sample from the HIV-infected patient; and (b) evaluating whether the plasma sample contains the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 236; in which the presence of the mutation correlates with a lower susceptibility to delavirdine and little or no change in susceptibility to nevirapine. 2. The method according to claim 1, characterized in that the mutation in codon 236 codes for a leucine. 3. The method according to claim 1, characterized in that the reverse transcriptase has an additional mutation at codon 103, codon 181 or a combination thereof. 4. The method according to claim 3, characterized in that the mutation in codon 103 codes for an asparagine (N) and the mutation in codon 181 codes for a cysteine (C). 5. The method according to claim 1, characterized in that the HIV-infected patient is being treated by an anti-retroviral agent. 6. A method for assessing the effectiveness of antiretroviral therapy of a patient infected with HIV, characterized in that it comprises: (a) collecting a biological sample from the patient infected with HIV; and (b) evaluating whether the biological sample comprises the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 225; in which the presence of the mutation correlates with an increased susceptibility to delavirdine and little or no change in susceptibility to nevirapine. 7. The method according to claim 6, characterized in that the codon mutant 225 codes for a histidine (H). 8. The method according to claim 6, characterized in that an HIV-infected patient is being treated with an antiretroviral agent. The method according to claim 6, characterized in that the reverse transcriptase has an additional mutation at codon 103, 181 or a combination thereof. 10. The method for evaluating the effectiveness of antiretroviral therapy of a patient infected with HIV, characterized in that it comprises: (a) collecting a biological sample from the patient infected with HIV; and (b) evaluating whether the biological sample comprises the nucleic acid encoding the HIV reverse transcriptase having a mutation at codon 190; in which the presence of the mutation correlates with an increase in susceptibility to delavirdine and an increase in susceptibility to nevirapine 11. The method according to claim 10, characterized in that the mutation in codon 190 encodes for an alanine or a serine 12. A method for evaluating the biological effectiveness of an antiretroviral pharmaceutical compound for HIV candidate, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient comprising, in addition, a mutation in codon 236 and a mutation in codon 103 and / or 181 and a gene 5 indicator in a host cell; (b) culturing the host cell of step (a); (c) measuring the indicator in a target host cell; and (d) compare the measurement of the step indicator "^ 10 (c) with the measurement of the indicator measured when steps (a) - (c) are carried out in the absence of the candidate antiretroviral drug compound; wherein a test concentration of the candidate antiretroviral drug compound is present in steps (a) - (c); in steps (b) - (c); or in step (c). 13. A method for evaluating the biological effectiveness of an antiretroviral drug compound for candidate HIV, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient further comprising a mutation in the codon 225 and a mutation in codon 103 and a reporter gene in a host cell; 25 (b) cultivating the host cell of step (a); (c) measuring the indicator in a target host cell; and • (d) compare the measurement of the indicator in step (c) with the measurement of the indicator measured when the steps 5 (a) - (c) are carried out in the absence of the candidate antiretroviral drug compound; wherein a test concentration of the candidate antiretroviral drug compound is present in steps (a) - (c); in steps (b) - (c); or in step 10 (c). 14. A method for evaluating the biological effectiveness of an antiretroviral drug compound for HIV candidate, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient encoding the reverse transcriptase having a mutation in codon 236 and a reporter gene in a host cell; (b) culturing the host cell of step (a); 20 (c) measuring the expression of the reporter gene in a target host cell; and (d) comparing the expression of the reporter gene of step (c) with the expression of the reporter gene measured when steps (a) - (c) are carried out in the absence of the 25 antiretroviral pharmaceutical compound - candidate; where a test concentration of the candidate antiretroviral drug compound is present in • steps (a) - (c); in steps (b) - (c); or in step (c). 15. A method for evaluating the biological effectiveness of an antiretroviral pharmaceutical compound for candidate HIV, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the - & & 10 patient encoding the reverse transcriptase having a mutation at codon 225 and a reporter gene in a host cell; (b) culturing the host cell of step (a); (c) measuring the expression of the reporter gene in a target host cell; and (d) compare the expression of the indicator gene of F step (c) with the expression of the reporter gene measured when steps (a) - (c) are carried out in the absence of the candidate antiretroviral drug compound; Wherein a test concentration of the candidate antiretroviral drug compound is present in steps (a) - (c); in steps (b) - (c); or in step (c). 16. A method to evaluate the biological effectiveness of an antiretroviral pharmaceutical compound 25 for the HIV candidate, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient encoding the reverse transcriptase having a mutation at codon 190 and a reporter gene in a host cell; (b) culturing the host cell of step (a); (c) measuring the expression of the reporter gene in a target host cell; and (d) comparing the expression of the reporter gene from step (c) with the expression of the reporter gene measured when steps (a) - (c) are carried out in the absence of the candidate antiretroviral drug compound; wherein a test concentration of the candidate antiretroviral drug compound is present in steps (a) - (c); in steps (b) - (c); or in step (c). 17. A resistance test vector, characterized in that it comprises a segment derived from a patient with HIV which also comprises the reverse transcriptase having a mutation in codon 190 and a reporter gene, where the expression of the reporter gene depends on the segment derived from the patient. 18. A resistance test vector, characterized in that it comprises a segment derived from a patient with HIV which also comprises the reverse transcriptase having a mutation in the codon 225- and a reporter gene, where the expression of the reporter gene depends on the segment derived from the patient. • 19. A resistance test vector, characterized in that it comprises a segment derived from a 5 patient with HIV also comprising the reverse transcriptase having a mutation in codon 236 and a reporter gene, where the expression of the reporter gene depends on the segment derived from the patient. 20. The resistance test vector according to claim 17, characterized in that the segment derived from the patient having a mutation in codon 190 also comprises a mutation in codon 103. 21. The resistance test vector 15 according to claim IB, characterized in that the segment derived from the patient having a • mutation in codon 225 further comprises a mutation in codon 103. 22. The resistance test vector according to claim 17, characterized in that the segment derived from the patient having a mutation in codon 236 comprises, in addition, a mutation in codon 103 and / or 181. 23. A method for evaluating the effectiveness of non-nucleoside antiretroviral therapy for reverse transcriptase of an HIV-infected patient, characterized in that it comprises: (a) collecting a sample of plasma of a patient infected with HIV; and 5 (b) evaluating whether the plasma sample contains the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 230; in which the presence of the mutation correlates with a decrease in susceptibility to • 10 delavirdine and nevirapine. 24. The method according to claim 23, characterized in that the mutation in codon 230 codes for a leucine (L). 25. The method according to claim 23, characterized in that the reverse transcriptase has additional mutations at codon 181 or • a combination of them. 26. The method according to claim 25, characterized in that the mutation in the 20 codon 281 codes for a cysteine (C). 27. The method according to claim 23, characterized in that the patient infected with HIV is being treated with antiretroviral agent. 28. A method for evaluating the effectiveness of the non-nucleoside antiretroviral therapy of the reverse transcriptase of an HIV-infected patient, characterized in that it comprises: (a) collecting a plasma sample from an HIV-infected patient; and (b) evaluating whether the plasma sample contains the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 181; in which the presence of the mutation correlates with a decrease in susceptibility to delavirdine and nevirapine and little or no change in susceptibility to efavirenz. 29. The method according to claim 28, characterized in that the mutation in codon 181 codes for a cysteine (C). 30. The method according to claim 28, characterized in that the reverse transcriptase has additional mutations at codon 98, codon 106, codon 227 or a combination thereof. 31. The method according to claim 30, characterized in that the mutation in codon 98 codes for a glycine (G), the mutation in codon 106 codes for an alanine (A) and the mutation in codon 227 codes for a leucine (L). 32. The method according to claim 28, characterized in that the patient infected with HIV is being treated with antiretroviral agent. 33. A method for evaluating the effectiveness of non-nucleoside antiretroviral therapy for reverse transcriptase of an HIV-infected patient, characterized in that it comprises: (a) collecting a plasma sample from an HIV-infected patient; and (b) evaluate if the plasma sample contains the • nucleic acid encoding HIV reverse transcriptase having a mutation at codon 188; in which the presence of the mutation correlates with a decrease in susceptibility to delavirdine and nevirapine and efavirenz. 34. The method according to claim 33, characterized in that the mutation in the • codon 188 codes for a leucine (L), cysteine (C) or histidine (H). 35. The method according to claim 33, characterized in that the reverse transcriptase has additional mutations at codon 138, codon 103, codon 100 or a combination thereof. 36. The method according to claim 35, characterized in that the mutation in the 25 codon 138 codes for an alanine (A) and the mutation in codon 103 codes for an asparagine (N) and the mutation in codon 100 codes for an isoleucine (I) • 37. The method according to claim 33, characterized in that the patient infected with HIV is being treated with antiretroviral agent. 38. A method for evaluating the effectiveness of non-nucleoside antiretroviral therapy for reverse transcriptase of an HIV-infected patient, characterized in that it comprises: (a) collecting a plasma sample from a patient infected with HIV; and (b) evaluating whether the plasma sample contains the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 190; in which the presence of the mutation correlates with an increase in susceptibility to delavirdine and a decrease in susceptibility to nevirapine and efavirenz. 39. The method according to claim 38, characterized in that the mutation in codon 190 codes for an alanine (A) or a serine (S). 40. The method according to claim 38, characterized in that the reverse transcriptase has additional mutations at codon 98, codon 101, codon 103 or a combination thereof. • 41. The method according to claim 40, characterized in that the mutation in the 5 codon 98 codes for a glycine (G), 101 codes for a glutamic acid (E) and 103 codes for an asparagine (N). 42. The method according to claim 38, characterized in that the patient infected with HIV is being treated with antiretroviral agent. • 10 43. A method for evaluating the effectiveness of the non-nucleoside antiretroviral therapy of the reverse transcriptase of an HIV-infected patient, characterized in that it comprises: (a) collecting a plasma sample from a patient infected with HIV; and (b) evaluate if the plasma sample contains the • nucleic acid encoding HIV reverse transcriptase that has a mutation at codon 106; in which the presence of the mutation correlates with a decrease in susceptibility to delavirdine and nevirapina and little or no change in susceptibility to efavirenz. 44. The method according to claim 43, characterized in that the mutation in codon 106 codes for an alanine (A). 45. The method according to claim 43, characterized in that the transcriptase - & ^^ $ ^^^ inverse has additional mutations in codon 227, and _ codon 189, or combinations thereof. 46. The method according to claim 45, characterized in that the mutation in codon 227 codes for a leucine (L), and 198 codes for a leucine (L). 47. The method of compliance with • 10 claim 43, characterized in that the patient infected with HIV is being treated with antiretroviral agent. 48. A method to assess the effectiveness of non-nucleoside antiretroviral therapy for the 15 reverse transcriptase of an HIV-infected patient, characterized in that it comprises: • (a) collecting a plasma sample from a patient infected with HIV; and (b) evaluating whether the plasma sample contains the nucleic acid encoding HIV reverse transcriptase having a mutation at codon 103; in which the presence of the mutation correlates with a decrease in susceptibility to delavirdine and nevirapine and efavirenz. 49. The method according to claim 48, characterized in that the mutation in codon 103 codes for an asparagine (N). 50. The method? according to claim 48, characterized in that the reverse transcriptase has additional mutations at codon 100 or a combination thereof. 51. The method according to claim 50, characterized in that the mutation in codon 100 codes for an isosoleucin (I). 52. The method according to claim 48, characterized in that the patient infected with HIV is being treated with antiretroviral agent. 53. A method for evaluating the biological effectiveness of an antiretroviral drug compound for HIV candidate, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient which also comprises a mutation in the codon 230 and a mutation in codon 181 and a reporter gene in a host cell; (b) culturing the host cell of step (a); (c) measuring the indicator in a target host cell; and (d) compare the measurement of the indicator in step (c) with the measurement of the indicator measured when- the steps • (a) - (c) are carried out in the absence of the candidate antiretroviral drug compound; Wherein a test concentration of the candidate antiretroviral drug compound is present in steps (a) - (c); in steps (b) - (c); or in step (c). 54. A method to assess the effectiveness • Biological of an antiretroviral pharmaceutical compound for HIV candidate, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient that also comprises a mutation in the codon 15 181 and a mutation in codon 98 and / or 106 and / or 227 and a reporter gene in a host cell; (b) culturing the host cell of step (a); (c) measuring the indicator in a target host cell; and 20 (d) compare the measurement of the step indicator (c) with the measurement of the indicator measured when steps (a) - (c) are carried out in the absence of the candidate antiretroviral drug compound; where a compound test concentration The candidate antiretroviral pharmacist is present in steps (a) in steps (b) - (c); or in step 55. A method for evaluating the biological effectiveness of an antiretroviral drug compound for candidate HIV, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient further comprising a mutation at codon 188 and a mutation at codon 138 and / or 103 and / or 100 and a reporter gene in a host cell; (b) culturing the host cell of step (a); (c) measuring the indicator in a target host cell; and (d) compare the measurement of the indicator in step (c) with the measurement of the indicator measured when the steps (a) - (c) are carried out in the absence of the candidate antiretroviral drug compound; wherein a test concentration of the candidate antiretroviral drug compound is present in steps (a) - (c); in steps (b) - (c); or in step (c). 56. A method for evaluating the biological effectiveness of an antiretroviral drug compound for HIV candidate, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the > patient further comprising a mutation in codon 190 and a mutation in codon 98 and / or 101 and / or 103 and a 5 reporter gene in a host cell; (b) culturing the host cell of step (a); (c) measuring the indicator in a target host cell; and (d) compare the measurement of the step indicator • 10 (c) with measurement of the indicator measured when steps (a) - (c) are carried out in the absence of the candidate antiretroviral drug compound; where a test concentration of the candidate antiretroviral drug compound is present in 15 steps (a) - (c); in steps (b) - (c); or in step (c). • 57. A method for evaluating the biological effectiveness of an antiretroviral drug compound for the candidate HIV, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient which also comprises a mutation in codon 106 and a mutation in codon 127 and / or 189 and a reporter gene in a host cell; 25 (b) cultivating the host cell of step (a); (c) measuring the indicator in a target host cell; and (d) compare the measurement of the indicator in step (c) with the measurement of the indicator measured when the steps -5 (a) - (c) are carried out in the absence of the candidate antiretroviral drug compound; wherein a test concentration of the candidate antiretroviral drug compound is present in steps (a) - (c); in steps (b) - (c); or in step 10 (c). 58. A method for evaluating the biological activity of an antiretroviral drug compound for HIV candidate, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient further comprising a mutation in the codon 103 and a mutation in codon 100 and a reporter gene in a host cell; (b) culturing the host cell of step (a); 20 (c) measuring the indicator in a target host cell; and (d) comparing the measurement of the indicator of step (c) with the measurement of the indicator measured when steps (a) - (c) are carried out in the absence of the candidate antiretroviral drug compound 5; wherein a test concentration of the candidate antiretroviral drug compound is present in steps (a) - (c); in steps (b) - (c); or in step (c). 59. A method for evaluating the biological effectiveness of an antiretroviral drug compound for HIV candidate, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient, which codes for the reverse transcriptase that has a mutation in codon 230 and a reporter gene in a host cell; (b) culturing the host cell of step (a); (c) measuring the expression of the reporter gene in a target host cell; and (d) comparing the expression of the reporter gene from step (c) with the expression of the reporter gene measured when steps (a) - (c) are carried out in the absence of the candidate antiviral drug compound, where a test concentration of Pharmaceutical antiviral candidate compound is present in steps (a) - (b) in steps (b) - (c); or in step (c). 60. A method for evaluating the biological effectiveness of an antiretroviral drug compound for HIV candidate, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient, which codes for the reverse transcriptase that has a mutation in codon 227 and a reporter gene in a host cell; (b) culturing the host cell of step (a); (c) measuring the expression of the reporter gene in a target host cell; and (d) compare expression of the indicator gene • step 10 (c) with the expression of the reporter gene measured when steps (a) - (c) are carried out in the absence of the candidate antiviral drug compound, where a test concentration of the candidate antiviral drug compound is present in the 15 step (a) - (b) in steps (b) - (c); or in step "_x 61. A method for evaluating the biological effectiveness of an antiretroviral drug compound for HIV candidate, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient, which encodes the transcriptase reverse that has a mutation in codon 188 and a reporter gene in a host cell; 25 (b) cultivating the host cell of step (a); (c) measuring the expression of the reporter gene in a target host cell; and (d) comparing the expression of the reporter gene from step (c) with the expression of the measured reporter gene 5 when steps (a) - (c) are carried out in the absence of the candidate antiviral drug compound, where a test concentration of the candidate antiviral drug compound is present in steps (a) - (b) in steps (b) ) - (c); or in step • 10 (c). 62. A method for evaluating the biological effectiveness of an antiretroviral drug compound for candidate HIV, characterized in that it comprises: (a) introducing a resistance test vector comprising a segment derived from the patient, which codes for reverse transcriptase - * • having a mutation in codon 189 and a reporter gene in a host cell; (b) culturing the host cell of step (a); 20 (c) measuring the expression of the reporter gene in a target host cell; and (d) comparing the expression of the indicator gene of step (c) with the expression of the reporter gene measured when steps (a) - (c) are carried out in the absence 25 of the candidate antiviral drug compound, wherein a test concentration of the candidate antiviral drug compound is present in steps (a) - (b) in steps (b) - (c); or in step (c). 63. A resistance test vector, characterized in that it comprises a segment derived from a patient derived with HIV that also comprises the reverse transcriptase that has the mutation in the codon 230 and a reporter gene, where the expression of the reporter gene depends on the segment derived from the patient. 64. A resistance test vector, characterized in that it comprises a segment derived from a patient derived with HIV that also comprises the reverse transcriptase that has the mutation in the codon 227 and a reporter gene, where the expression of the reporter gene depends on the segment derived from the patient. 65. A resistance test vector, characterized in that it comprises a segment derived from a patient derived with HIV that also comprises the reverse transcriptase that has the mutation in codon 188 and - a reporter gene, where the expression of the reporter gene depends of the segment derived from the patient. 66. A resistance test vector, characterized in that it comprises a segment derived from a patient derived with HIV which also comprises the reverse transcriptase having the mutation in codon 189 and a reporter gene, where the expression of the reporter gene depends on the segment derived from the patient. # 67. A resistance test vector, characterized in that it comprises a segment derived from a patient with HIV which also comprises a reverse transcriptase having mutations in codon 181, codon 98, codon 106 and codon 227 or a combination of them and a reporter gene, where the expression of the gene • indicator depends on the segment derived from the patient. 68. A resistance test vector, characterized in that it comprises a segment derived from a patient with HIV also comprising a reverse transcriptase having mutations at codon 106, codon 227, and codon 189 or a combination thereof. and a gene 15 indicator, where the expression of the reporter gene depends on the segment derived from the patient. 69. A resistance test vector, characterized in that it comprises a segment derived from a patient with HIV that also comprises a transcriptase Inverse has mutations in codon 103 and codon 101 or a combination thereof and a reporter gene, where the expression of the reporter gene depends on the segment derived from the patient. 70. The resistance test vector according to claim 17, characterized in that the segment derived from the patient having a mutation in codon 190 also comprises mutations in codon 98 and codon 101 or a combination thereof. . 71. The resistance test vector according to claim 63, characterized in that the segment derived from the patient having a mutation in codon 230 further comprises a mutation in codon 181. 72. The resistance test vector of according to claim 65, characterized in that the segment derived from the patient having a mutation in codon 188 further comprises mutations in codon 138, codon 103 and codon 190 or a combination thereof.