CN1311823A

CN1311823A - Means and method for monitoring non-nucleoside reverse transcriptase inhibitor antiretroviral therapy

Info

Publication number: CN1311823A
Application number: CN 99808689
Authority: CN
Inventors: 珍尼特·惠特科姆
Original assignee: Virologic Inc
Current assignee: Monogram Biosciences Inc
Priority date: 1998-05-26
Filing date: 1999-05-26
Publication date: 2001-09-05
Anticipated expiration: 2019-05-26
Also published as: EP1082454A4; CN1204266C; WO1999061658A1; NZ508834A; AU772511B2; MXPA00011623A; AU4207599A; CA2329140A1; EP1082454A1; CN1532290A; BR9911600A; JP2002516119A

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

The approach of monitoring non-nucleoside reverse transcriptase inhibitor antiretroviral therapy and method

The application is the part continuation application of No. the 09/085th, 148, the U.S. Patent application submitted on May 26th, 1998, and requires the rights and interests of No. the 60/124th, 090, the U.S. Provisional Application submitted on March 12nd, 1999, and its content is incorporated herein by reference.

All kinds of in this application reference mark in bracket.The disclosed full content of these documents is incorporated herein by reference, more fully to describe the prior art situation in the technical field under the application.

Technical field

The present invention relates to antiretroviral drugs susceptibility and resistant proof, test is used for identifying that treatment human immunodeficiency virus (HIV) infects and the active drug preparation of acquired immune deficiency syndrome (AIDS) (AIDS).The invention further relates to phenotype and gene type assay method and monitor clinical HIV progression of infection and the reaction of antiretroviral therapy.The invention still further relates to the novel carriers, host cell and the composition that are used to finish the phenotype sensitivity test.The invention further relates to and use the range gene analytical procedure to differentiate the resistance that the HIV infected patient produces specific antiretroviral drugs preparation.The invention still further relates to according to the ability screening antiretroviral drug candidate that virus, the virus sequence of selecting and/or viral protein are suppressed.Present invention is specifically related to and use phenotype sensitivity test and/or genetic analysis test determination non-nucleotide reverse transcriptase inhibitors resistance.

Background of the present invention

The HIV infection character is to upgrade in the virus of pathogenic process high frequency, finally causes the extinction of cd4 cell and PD to worsen.Wei?X,Ghosh?SK,Taylor?ME,et?al.(1995)Nature?343,117-122,and?Ho?DD,Naumann?AU,Perelson?AS,et?al.(1995)Nature?373,123-126。The purpose of antiretroviral therapy is the inhibition that virus replication is obtained essence and continues, the control that virus is obtained to continue comprises a series of methods of treatment of employing, each methods of treatment comprises the use of uniting of 3 kinds or more antiretroviral drugs generally, therefore on the rational basis that selection beginning and follow-up methods of treatment should be based upon, fully understand resistance and crossed resistance for instructing the definite most important of methods of treatment.The basic rational basis of combination therapy is that medicine mating reaction and addition can obtain bigger inhibition to virus replication.Yet the tolerance of pharmaceutical preparation is still most important, because methods of treatment need keep many years.

Produce about 10 every day in patient's body of not receiving treatment ¹⁰Individual reovirion adds that hiv reverse transcriptase (RT) does not have the exonuclease correct functioning, can not proofread and correct transcription error, and this high-caliber virus is upgraded and caused having 10 every day in genomic each position of HIV ⁴To 10 ⁵Inferior sudden change, the result has produced genomic extensive sudden change rapidly.Although some template position or base pair replace (the Mansky LM that is more prone to make a mistake, Temin HM (1995) J Virol 69,5087-5094) (Schinazi RF, Lloyd RM, Ramanathan CS, et al. (1994) Antimicrob AgentsChemother 38,268-274), mathematical model still is presented in the infected individuals each may every day 10,000 sudden changes may take place in site.

The create antagonism resistance of reverse transcription disease cytotoxic drug must obtain modifying and keeping its function as the enzyme molecule of drug effect target position when inhibitor exists.The point mutation that causes single amino acids to replace may cause the change of enzyme shape, size, the perhaps change of reactive site, substrate binding site or neighboring area.In the sudden change that has detected on low-level before the initial therapy the antiretroviral drug resistant.(Mohri?H,Singh?MK,Ching?WTW?et?al.(1993)Proc?NatlAcad?Sci?USA?90,25-29)(Najera?I,Richman?DD,Olivares?I,et?al.(1994)AIDS?Res?Hum?Retroviruses?10,1479-1488)(Najera?I,Holguin?A,Quinones-Mateu?E,et?al.(1995)J?Virol?69,23-31)。Yet these mutant strains only account for the sub-fraction of whole viral total amount, and compare to be in wild-type virus and duplicate and compete inferior position.(Coffin?JM(1995)Science?267,483-489)。The selective pressure that antiretroviral therapy produces provides competitive edge for these anti-medicine mutant strains, they become advantage quasispecies (Frost SDW gradually like this, McLean AR (1994) AIDS 8,323-332) (Kellam P, Boucher CAB, Tijnagal JMGH (1994) J Gen Virol 75 341-351), finally causes anti-medicine and antiviral therapy failure in patient's body.

Non-nucleoside reverse transcriptase inhibitor

Non-nucleoside reverse transcriptase inhibitor (NNRTIs) is the compound that a class has the number of chemical group, and they have the strongly inhibited effect external to hiv reverse transcriptase.These compounds comprise pyridine derivatives, two aromatic heterocycle piperazines (BHAPs) for example draw furan pyridine (delavirdine) and Ai Te furan pyridine (atevirdine), dipyridyl diazepine Viramune (nevirapine), TSAO of adenosine derivative family and HEPT, a kind of α-aniline phenylacetamide (α-APA) compound network furan Ruide (loviride), with quino quinoline group inhibitor as (HBY-097), 1-and thioketones benzimidazole dihydrochloride (TIBO) compound and pyridine derivate (L-697,661), summary is seen (DeClercq E. (1996) Rev Med Virol6,97-117) (Emini EA (1996) Antiviral Drug Resistance antiviral resistance, ed.DD Richman, John Wiley ﹠amp; Sons, Led.).As if virus is carrying out just rapidly every kind of compound being produced high resistance in former weeks of single therapy usually, and this usually comprises single site mutation, and causes in many cases the considerable crossed resistance of other non-nucleoside reverse transcriptase inhibitor.The sudden change of great majority report occurs between codon 110-108 and the 181-190, they codings two beta sheet (Kohlstaedt LAs adjacent with the reversed transcriptive enzyme catalytic site, Wang J, Friedman JM, et al. (1992) Science 256,1783-90), the binding pocket of non-nucleoside reverse transcriptase inhibitor is that the non-substrate of reversed transcriptive enzyme is in conjunction with hydrophobic region as described in the document.Here, the direct and reversed transcriptive enzyme interaction of these preparations.They pass through to disturb the activity (D ' Aquilla RT. (1994) Clin Lab Med 14 of the movable or blocking-up of finger-type subdomain to the essential aspartic acid side chain direction inhibitory enzyme of catalytic activity, 393-432) (Arnold E., Ding J., Hughes SH, et al. (1995) Curr Opin Stmct Biol 5,27-38).

Existing reporting, cause that the sudden change that Viramune (nevirapine) susceptibility weakens is positioned at codon 98,100,103,106,108,181,188 and 190 (Richman DD, Havlir D, CorbeilJ. (1994) J Virol 68,1660-1666).Selection sudden change the most frequent in Viramune (nevirapine) single therapy process is a tyrosine ¹⁸¹-halfcystine (Y181C) sudden change, it can cause HIV that said preparation susceptibility is weakened 100 times, to pyridine derivate L-696,229 and L-697,661 susceptibility also can weaken (Arnold, the same).181 sudden changes also can restricted T SAO activity, but still keep active, and at external selection idiotype sudden change L-glutamic acid for 100 and 103 bit codon mutations ¹³⁸-halfcystine, closely interact at this site TSAO and reversed transcriptive enzyme (Richman, DD, the same) (Richman DD, Shih C-K, Lowy I, et al. (1991) Proc Natl Acad SciUSA 88,11241-11245).

Most patients can produce network furan Ruide (loviride) resistance after using 24 weeks of list-treatment, drawn codon 100-110; The collection of illustrative plates of 181-190, the most generally 103 codon mutations (Staszewski S, Miller V, Kober A, et al. (1996) Antiviral Ther 1,42-50).Use black pyridine (zidovudine) in network furan Ruide (loviride) and Ruide or Ruide crow pyridine (zidovudine) and come miaow crow pyridine (lamivudine) combination therapy, in 24 weeks, are deletion mutantion (Staszewski S of high frequency in the variation of codon 98 and 103 generations, Miller V, RehmetS, et al. (1996) AIDS 10, F1-7).

Although 101,103 and 181 sudden change also can cause the crossed resistance to two aromatic heterocycle piperazines (BHAPs), (Balzarini J, Karlsson A, Perez-Perez M-J, et al. (1992) Virology 192,246-253) replace L at 236 P of external because these medicament selection effects, make reversed transcriptive enzyme to other non-nucleoside reverse transcriptase inhibitor sensitivity, for example Viramune (nevirapine) resistance/half-inhibition concentration IC50 reduces 7 to 10 times, but do not influence susceptibility (Staszewski S., the same) to nucleoside analog.Although existing 103 of codons and 181 generation resistance gain mutations reported in single therapy, and with Ruide crow pyridine (zidovudine) combination therapy in 101,188,233 and 238 sudden changes of codon take place, but in the clinical treatment of Ai Te furan pyridine (atevirdine), also do not find 236 individualities of undergoing mutation of codon.

HBY-097 selects 190 sudden changes of codon at first external, and further constantly select reversed transcriptive enzyme codon 74 and 75 sudden changes, like this some mutated viruses reach over the ground gloomy (didanosine) and Si Tawu pyridine (stavudine) susceptibility reduce, but the susceptibility to Ruide crow pyridine (zidovudine) does not reduce (Kleim J-P, Rosner M, Winkler I, et al. (1995) J Acquir Immune Defic Syndr 10 Suppl 3,2)

Have typical case's sudden change of reporting 41 and 251 bit codons and can offset the resistance of 181 bit codon mutations Ruide crow pyridine (zidovudine), (Zhang D, Caliendo AM, Eron JJ, et al. (1994) Antimicrob Agents Chemother 38,282-287), it is feasible that the combination therapy possibility is carried out in these explanation some non-nucleoside reverse transcriptase inhibitors of application and Ruide crow pyridine (zidovudine).Although report the HIV mutant strain external to the pyridine (zidovudine) of Ruide crow, reach gloomy (didanosine), Viramune (nevirapine) triple resistances are arranged, (Larder BA, Kellam P, Kemp SD (1993) Nature 365,451-453), but unite and use these three kinds of pharmacological agent cd4 cell numbers less than 350/mm ³Patient after 48 weeks, immune and antiviral curative effect be better than really using separately Ruide crow pyridine (zidovudine) and reach gloomy (didanosine).

With Ruide crow pyridine (zidovudine) and pyridine derivate L-697,661 combination therapys can hinder is using this non-nucleoside reverse transcriptase to press down typical 181 sudden changes of initiation in the medicine single therapy process, delay the appearance to this chemical combination object height resistance, this research does not detect Ruide crow pyridine (zidovudine) susceptibility and changes.(Staszewski?S,Massari?FE,Kober?A,et?al.(1995)JInfect?Dis?171,1159-1165)。When using Viramune (nevirapine) treatment, can delaying drug resistance not occur with Ruide crow pyridine (zidovudine) treatment simultaneously or alternately; (Richman DD, the same) (Nunberg JH, Schleif WA, Boots EJ, et al. (1990) J Virol 65,4887-4892) (DeJong MD, Loewenthl M, Boucher CAB, et al. (1994) J Infect Dis 169,1346-1350) (Cheeseman SH, Harlir D, McLaughlin MM＜et al. (1995) JAcquir Immune Defic Syndr 8,141-151), yet observe 181 sudden changes in the combination therapy process, common mutations occurs in 190 bit codons (Richman DD, the same).This explanation is because 181 codon mutations can be offset Ruide crow pyridine (zidovudine) resistance external, and screening allows the susceptibility of this NNRTI medicine is reduced the sudden change that but is attended by Ruide crow pyridine (zidovudine) resistance simultaneously in the body so be unsuitable for using it for.

Because non-nucleoside reverse transcriptase inhibitor susceptibility is reduced rapidly, the use of these medicines is restricted, and single therapy is especially true.This carries out manually modified to these molecules with regard to initiation, in the hope of delaying the appearance of anti-medicine virus.A kind of " s-generation " non-nucleoside reverse transcriptase inhibitor, i.e. pyridine derivate L-702,019, to the half-inhibition concentration IC of wild-type and 181 mutant strain HIV-1 ₅₀3 times of changes are only arranged, and acquired multimutation cause high resistance (Goldman ME, O ' Brien JA, Ruffing TL, et al. (1993) Antimicrob Agents Chemother 37,947-949).

One of purpose of the present invention provides a kind of drug susceptibility and resistance detection method, and it can show whether the intravital virus population of patient has resistance to given formula of medicine.Another object of the present invention is to receive treatment the back when specific one or more medicines are produced resistances the patient, for the doctor provides a test that is used for replacing a kind of or multiple medications of treatment prescription.Another purpose of the present invention provides one can screen the test that treatment HIV infects and/or the AIDS active drug is filled a prescription.A further object of the present invention is to be that those produce resistances, has found out especially non-nucleoside reverse transcriptase inhibitor is produced the method that the patient of resistance provides suitable drug to select.Another purpose of the present invention provides test and the method for a kind of role of evaluation in the drug candidate biological effectiveness of specific virus, virogene and/or viral protein, particularly relevant with non-nucleoside reverse transcriptase inhibitor viral anti-medicine test and method.A further object of the present invention provides method and the composition of estimating HIV antiretroviral drugs resistance and susceptibility.Obviously this purpose of the present invention and other purpose technical requirements are an integral body.

Summary of the invention

The present invention relates to use phenotype and genetic analysis method monitor para-immunity defective virus progression of infection and it to resist the method for viral therapeutic response.The present invention also is based in part on and has found that reversed transcriptive enzyme (RT) genetic mutation that makes HIV antagonism viral therapy produce resistance can use phenotype or genetic analysis method directly to determine this discovery rapidly by patient's blood plasma HIV RNA, these methods are used polymerase chain reaction (PCR), perhaps do not use amplification step to use the method for the nucleic acid sequence of mensuration coding viral protein also can make the present invention monitor and/or improve antiretroviral therapy.The present invention also be based in part on found in patient's body of non-nucleoside reverse transcriptase inhibitor (efavirenz) treatment that hiv reverse transcriptase 225 bit codons suddenly change separately or with 103 bit codon simultaneous mutations, the increase of furan pyridine (delavirdine) susceptibility is relevant with drawing over the ground, and the susceptibility to Viramune (nevirapine) changes hardly, and these sudden changes were found in blood plasma HIV RNA after treatment beginning for some time.We find that hiv reverse transcriptase can be used as the index that forms resistance and final immunocompromised in the formation of 225 bit codon mutations and 103 bit codon mutations.The present invention also be based in part on found in accepting the patient that non-nucleoside reverse transcriptase presses down medicine (NNRTI) treatment, to take a turn for the worse record enzyme 236 bit codon mutations with draw the reduction of furan pyridine (delavirdine) susceptibility over the ground, do not reduce relevant the susceptibility of Viramune (nevirapine).We find that hiv reverse transcriptase can be used as the index that phenotype susceptibility/resistance changes in the formation of 190 and 103 and/or 101 bit codon mutations and 103 bit codon mutations, and this reduces relevant with antiviral therapy failure and immunological competence subsequently.The present invention also is based in part on and has found that hiv reverse transcriptase 190 bit codons suddenly change separately or 109 bit codons and 103 and/or 101 bit codon simultaneous mutations in accepting patient's body that non-nucleoside reverse transcriptase presses down agent class medicine (Chinese mugwort sends out the auspicious efavirenz now of furan) treatment, with draw furan pyridine (delavirdine) susceptibility to increase over the ground and the susceptibility of Viramune (nevirapine) reduced relevant, these sudden changes begin to find in blood plasma HIV RNA after for some time in the NNRTI treatment.We find that hiv reverse transcriptase can be used as the index that phenotype susceptibility/resistance changes in the formation of 236 and 103 bit codon mutations and/or 181 bit codon mutations, and this reduces relevant with viral therapy failure and immunological competence subsequently.

The present invention also be based in part on found in the patient of non-nucleoside reverse transcriptase inhibitor (Viramune nevirapine) treatment hiv reverse transcriptase 230 bit codons separately sudden change or with 181 bit codon simultaneous mutations, significantly reduction is relevant with the susceptibility of drawing furan pyridine (delavirdine) and Viramune (nevirapine) over the ground in these sudden changes, and these sudden changes were found in blood plasma HIV RNA after NNRTI treatment beginning for some time.We find that hiv reverse transcriptase can be used as the index that phenotype susceptibility/resistance changes in the formation of 230 and 181 bit codon mutations, and this reduces relevant with viral therapy failure and immunological competence subsequently.The present invention also is based in part on and has found hiv reverse transcriptase 181 bit codon mutations in the patient of non-nucleoside reverse transcriptase inhibitor (Viramune nevirapine) treatment, the susceptibility of significantly reduce with the susceptibility of drawing reduction of furan pyridine (delavirdine) susceptibility moderate and Viramune (nevirapine) over the ground, sending out furan auspicious now (efavirenz) to ending does not change not relevant, and this sudden change is treated at NNRTI and begun to find in blood plasma HIV RNA after for some time.We find that hiv reverse transcriptase is undergone mutation at 181 bit codons and can be used as the index that phenotype susceptibility/resistance changes that this is relevant with the reduction of immunological competence subsequently with the viral therapy failure.The present invention also is based in part on and has found hiv reverse transcriptase 188 bit codon mutations in the patient of non-nucleoside reverse transcriptase inhibitor (Chinese mugwort sends out the auspicious efavirenz now of furan) treatment, relevant with the substantive reduction of susceptibility of drawing slight reduction of furan pyridine (delavirdine) susceptibility and Viramune (nevirapine) over the ground, this sudden change was found in blood plasma HIV RNA after NNRTI treatment beginning for some time.We find that hiv reverse transcriptase is undergone mutation at 188 bit codons and can be used as the index that phenotype susceptibility/resistance changes that this reduces relevant with viral therapy failure and immunological competence subsequently.The present invention also is based in part on and has found hiv reverse transcriptase 188 bit codon mutations in the patient of non-nucleoside reverse transcriptase inhibitor treatment of no use, reduce with the susceptibility of drawing reduction of furan pyridine (delavirdine) susceptibility moderate and Viramune (nevirapine) over the ground is substantive, and it is relevant that Chinese mugwort is sent out the reduction of furan auspicious now (efavirenz) susceptibility moderate, and this sudden change was found in blood plasma HIV RNA after antiretroviral therapy treatment beginning for some time.We find that hiv reverse transcriptase is undergone mutation at 138 and 188 bit codons and can be used as the index that phenotype susceptibility/resistance changes that this is relevant with the reduction of immunological competence subsequently with the viral therapy failure.The present invention also is based in part on hiv reverse transcriptase 98 bit codon mutations among the patient who has found former non-nucleoside reverse transcriptase inhibitor treatment of no use, slightly reduction is relevant with drawing furan pyridine (delavirdine), Viramune (nevirapine) and Chinese mugwort to send out furan auspicious now (efavirenz) susceptibility over the ground, and this sudden change was found in blood plasma HIV RNA after antiretroviral therapy treatment beginning for some time.We find that hiv reverse transcriptase can be used as the index that phenotype susceptibility/resistance changes in the formation of 98 bit codon mutations, and this reduces relevant with antiviral therapy failure and immunological competence subsequently.

The present invention also be based in part on discovery when not knowing that the patient accepts the antiretroviral therapy situation hiv reverse transcriptase 98 bit codons separately sudden change or with 190 bit codon simultaneous mutations, furan auspicious now (efavirenz) the susceptibility essence reduction of raising of furan pyridine (delavirdine) susceptibility and Viramune (nevirapine) and Chinese mugwort is relevant with drawing over the ground, and these sudden changes are found in blood plasma HIV RNA.We find that hiv reverse transcriptase can be used as the index that forms resistance and final immunological competence reduction at 98 bit codon mutations and 190 bit codon mutations.The present invention also be based in part on found in the patient who accepts non-nucleoside reverse transcriptase inhibitor (draw furan pyridine delavirdine) treatment hiv reverse transcriptase 181 bit codons separately sudden change or with 98 bit codon simultaneous mutations, relevant with the susceptibility essence reduction of drawing furan pyridine (delavirdine) susceptibility significantly to reduce over the ground with a furan auspicious now (efavirenz) that ends, these sudden changes begin to find in blood plasma HIV RNA after for some time in treatment.We find that hiv reverse transcriptase can be used as the index that forms resistance and final immunological competence reduction at 98 and 181 bit codon mutations.The present invention also is based in part on and has found that 101 bit codons suddenly change separately, perhaps with 190 bit codon simultaneous mutations (for example hiv reverse transcriptase 190S), the susceptibility essence of drawing furan pyridine (delavirdine) susceptibility not have change and Viramune (nevirapine), Chinese mugwort to send out furan auspicious now (efavirenz) over the ground with the patient who accepts non-nucleoside reverse transcriptase inhibitor (Chinese mugwort sends out the auspicious efavirenz now of furan) treatment reduces relevant.These sudden changes were found in blood plasma HIV RNA after treatment beginning for some time.We find that hiv reverse transcriptase undergos mutation at 101 and 190 bit codons (for example hiv reverse transcriptase 190S), can be used as to form the index that resistance and final immunological competence reduce.The present invention also is based in part on hiv reverse transcriptase 108 bit codon mutations among the patient who has found former non-nucleoside reverse transcriptase inhibitor treatment of no use, send out furan auspicious now (efavirenz) susceptibility and do not change not relevantly with drawing over the ground that furan pyridine (delavirdine) susceptibility does not change, Viramune (nevirapine) susceptibility slightly reduces and end, this sudden change is treated in antiretroviral therapy and is begun to find in blood plasma HIV RNA after for some time.We find that hiv reverse transcriptase can be used as the index that phenotype susceptibility/resistance changes at 108 bit codon mutations, and this reduces relevant with antiviral therapy failure and immunological competence subsequently.

The present invention also be based in part among the patient who has found former non-nucleoside reverse transcriptase inhibitor of no use treatment hiv reverse transcriptase 101 bit codons separately sudden change or with 103 and/or 190 bit codon simultaneous mutations, furan auspicious now (efavirenz) the susceptibility change of furan pyridine (delavirdine), Viramune (nevirapine) and Chinese mugwort is relevant with drawing over the ground, and this sudden change was found in blood plasma HIV RNA after antiretroviral therapy treatment beginning for some time.101 and 190 bit codon mutations particularly, 190A for example, significantly reduction is relevant with drawing the not change of furan pyridine (delavirdine) susceptibility, the reduction of Viramune (nevirapine) susceptibility essence and Chinese mugwort to send out furan auspicious now (efavirenz) susceptibility over the ground, and these sudden changes are found in blood plasma HIVRNA after antiretroviral therapy is treated beginning for some time.We find that hiv reverse transcriptase can be used as the index that phenotype susceptibility/resistance changes at 101,103 and/or 190 bit codon mutations, and this reduces relevant with viral therapy failure and immunological competence subsequently.The present invention also be based in part on found in the patient of non-nucleoside reverse transcriptase inhibitor (Viramune nevirapine) treatment hiv reverse transcriptase 106 bit codons separately sudden change or with 189 and/or 181 and 227 bit codon mutations, with draw furan pyridine (delavirdine) over the ground, Viramune (nevirapine) is sent out furan auspicious now (efavirenz) susceptibility with Chinese mugwort and is changed relevant, particularly 106 and 181 bit codon mutations with draw furan pyridine (delavirdine) susceptibility significantly to reduce, it is relevant with the slight reduction of Chinese mugwort furan auspicious now (efavirenz) susceptibility that Viramune (nevirapine) susceptibility essence reduces, 106 and 189 bit codon mutations with draw furan pyridine (delavirdine) susceptibility slightly to reduce, Viramune (nevirapine) susceptibility moderate reduces not to be had to change not relevant with Chinese mugwort furan auspicious now (efavirenz) susceptibility, 106 and 227 bit codon mutations with draw furan pyridine (delavirdine) susceptibility slightly to reduce, it is relevant with the slight reduction of Chinese mugwort furan auspicious now (efavirenz) susceptibility that Viramune (nevirapine) susceptibility essence reduces, 181 and 227 bit codon mutations draw furan pyridine (delavirdine) susceptibility increase with ground, Viramune (nevirapine) susceptibility significantly reduces to be increased relevant with Chinese mugwort furan auspicious now (efavirenz) susceptibility, 106,181 and 227 bit codon mutations draw furan pyridine (delavirdine) susceptibility moderate reduction with ground, it is relevant with the slight reduction of Chinese mugwort furan auspicious now (efavirenz) susceptibility that Viramune (nevirapine) susceptibility essence reduces, and these sudden changes begin to find in blood plasma HIV RNA after for some time in NNRTI treatment treatment.We find that hiv reverse transcriptase can be used as the index that phenotype susceptibility/resistance changes at 106,189 and/or 181 and 227 bit codon mutations, and this reduces relevant with viral therapy failure and immunological competence subsequently.The present invention also be based in part on found in the patient of non-nucleoside reverse transcriptase inhibitor (Viramune nevirapine) treatment hiv reverse transcriptase 103 bit codons separately sudden change or with 100 and/or 188 bit codon mutations, these suddenly change and draw furan pyridine (delavirdine) over the ground, Viramune (nevirapine) is sent out furan auspicious now (efavirenz) susceptibility with Chinese mugwort and is changed relevant, particularly 103 and 188 bit codon mutations draw furan pyridine (delavirdine) susceptibility essence reduction with ground, it is relevant with Chinese mugwort furan auspicious now (efavirenz) susceptibility essence reduction that Viramune (nevirapine) susceptibility essence reduces, 100 and 103 bit codon mutations draw furan pyridine (delavirdine) susceptibility essence reduction with ground, it is relevant with Chinese mugwort furan auspicious now (efavirenz) susceptibility essence reduction that Viramune (nevirapine) susceptibility moderate reduces, 103,100 and 188 bit codon mutations draw furan pyridine (delavirdine) susceptibility essence reduction with ground, it is relevant with Chinese mugwort furan auspicious now (efavirenz) susceptibility essence reduction that Viramune (nevirapine) susceptibility essence reduces, and these sudden changes were found in blood plasma HIV RNA after NNRTI treatment treatment beginning for some time.We find that hiv reverse transcriptase can be used as the index that phenotype susceptibility/resistance changes at 103,100 and/or 188 bit codon mutations, and this reduces relevant with viral therapy failure and immunizing power subsequently.

In another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay to detect 225 bit codon mutations, also can detect comprise hiv reverse transcriptase 103 at other interior codon mutation, these sudden changes are relevant with the particular model that antiretroviral therapy is produced the reduction of resistance and immunizing power subsequently.In another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay to detect other site simultaneous mutation that 236 bit codons suddenly change separately or comprise 103 of hiv reverse transcriptases and/or 181, these sudden changes are with antiretroviral therapy to be produced resistance relevant with the reduction of immunizing power subsequently.In another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay to detect that 190 passwords (G190S) suddenly changes separately or with hiv reverse transcriptase on 101 bit codons (K101E) simultaneous mutation, these sudden changes are with relevant with the reduction of immunizing power subsequently to antiretroviral therapy generation resistance.

In yet another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay to detect that hiv reverse transcriptase 190 bit codons (G190A) suddenly changes separately or with 103 bit codons (K103N) simultaneous mutation, these sudden changes are with relevant with the reduction of immunizing power subsequently to antiretroviral therapy generation resistance.

In another embodiment of the invention, can use the pcr analysis method that comprises phenotype and gene type assay detect hiv reverse transcriptase 230 bit codons separately sudden change or with 181 bit codon simultaneous mutations, these sudden changes are with antiretroviral therapy to be produced resistance relevant with the reduction of immunizing power subsequently.

In another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay to detect hiv reverse transcriptase 181 bit codon mutations, this sudden change is with relevant with the reduction of immunizing power subsequently to antiretroviral therapy generation resistance.

In yet another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay to detect 188 bit codon mutations on the hiv reverse transcriptase, this sudden change is with relevant with the reduction of immunizing power subsequently to antiretroviral therapy generation resistance.

In yet another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay detect hiv reverse transcriptase 138 bit codons separately sudden change or with 188 bit codon simultaneous mutations, these sudden changes are with antiretroviral therapy to be produced resistance relevant with the reduction of immunizing power subsequently.

In another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay to detect hiv reverse transcriptase 98 bit codon mutations, this sudden change is with relevant with the reduction of immunizing power subsequently to antiretroviral therapy generation resistance.

In yet another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay detect hiv reverse transcriptase 98 bit codons separately sudden change or with 190 bit codon simultaneous mutations, these sudden changes are with antiretroviral therapy to be produced resistance relevant with the reduction of immunizing power subsequently.

In yet another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay detect on the hiv reverse transcriptase 181 passwords separately sudden change or with 98 bit codon simultaneous mutations, these sudden changes are with antiretroviral therapy to be produced resistance relevant with the reduction of immunizing power subsequently.

In another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay detect on the hiv reverse transcriptase 101 bit codons separately sudden change or with 190 bit codon simultaneous mutations (for example sudden change of 190s), these sudden changes are with antiretroviral therapy to be produced resistance relevant with the reduction of immunizing power subsequently.

In another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay to detect 108 bit codon mutations on the hiv reverse transcriptase, this sudden change is with relevant with the reduction of immunizing power subsequently to antiretroviral therapy generation resistance.

In yet another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay detect on the hiv reverse transcriptase 101 bit codons separately sudden change or with 103 and/or 190 bit codon simultaneous mutations, these sudden changes are with antiretroviral therapy to be produced resistance relevant with the reduction of immunizing power subsequently.

In yet another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay detect on the hiv reverse transcriptase 106 bit codons separately sudden change or with 189 and/or 181 and 227 bit codon simultaneous mutations, these sudden changes are with antiretroviral therapy to be produced resistance relevant with the reduction of immunizing power subsequently.

In yet another embodiment of the present invention, can use the pcr analysis method that comprises phenotype and gene type assay detect on the hiv reverse transcriptase 188 bit codons separately sudden change or with 100 and/or 103 bit codon simultaneous mutations, these sudden changes are with antiretroviral therapy to be produced resistance relevant with the reduction of immunizing power subsequently.In case in accepting patient's body of NNRTI antiretroviral therapy, detect 225 and 103 bit codon mutations, just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 236 and/or 103 and/or 181 bit codon mutations, just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 190 and/or 103 and/or 101 bit codon mutations, just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 230 and/or 181 bit codon mutations, just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 181 bit codon mutations, just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 188 bit codon mutations, just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 138 and/or 188 bit codon mutations, just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 98 bit codon mutations, just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 98 and/or 190 bit codon mutations, just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 181 and/or 98 bit codon mutations, just must consider to change the medicine prescription.Equally, in case detect 101 and/or 190 bit codon mutations in accepting patient's body of NNRTI antiretroviral therapy, for example 190S just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 108 bit codon mutations, just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 101 and/or 103 and/or 190 bit codon mutations (for example 190A), just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 106 and/or 189 and/or 181 and/or 227 bit codon mutations, just must consider to change the medicine prescription.Equally, in case in accepting patient's body of NNRTI antiretroviral therapy, detect 188 and/or 100 and/or 103 bit codon mutations, just must consider to change the medicine prescription.After using the pcr analysis method to estimate antiretroviral therapy, the time that should determine to revise the medicine prescription, this depends on patient's viral load amount that comprises, cd4 cell quantity and former this Several Factors of treatment history.

Another one of the present invention aspect has provided the method for estimating non-nucleoside reverse transcriptase antiretroviral drugs validity, comprising: (a) will have the originate resistant proof carrier of fragment and reporter gene of patient and import host cell; (b) cultivation is from the host cell of (a); (c) expression of examining report gene in the target host cell, wherein the expression of reporter gene depends on patient's fragment of originating; (d) will the two compare from the expression of detected reporter gene under the expression of step (c) reporter gene and the situation that does not add the NNRTI inverase when implementation step (a)-(c), wherein the experimental concentration of inverase NNRTI is at step (a)-(c); At step (b)-(c); Or in step (c), mark.

The present invention also provides evaluate patient to accept the method for non-nucleoside reverse transcriptase antiretroviral drugs treatment validity, comprising: the susceptibility typical curve of (a) drawing NNRTI class inverase; (b) use aforesaid sensitivity test to determine the susceptibility of patient NNRTI inverase; And (c) typical curve of determining in the susceptibility of NNRTI inverase in the step (b) and the step (a) is compared, wherein the anti-HIV susceptibility reduction of NNRTI shows the resistance that has formed inverase in patient's body.

The present invention also provides the method for estimating HIV antiretroviral drug candidate composite biological validity, comprising: (a) will have the originate resistant proof carrier of fragment and reporter gene of patient and import host cell; (b) cultivation is from the host cell of (a); (c) expression of examining report gene in the target host cell, wherein the expression of reporter gene depends on patient's fragment of originating; (d) will the two compare from the expression of detected reporter gene under the expression of step (c) reporter gene and the situation that does not add the NNRTI inverase when implementation step (a)-(c), wherein the experimental concentration of inverase NNRTI is at step (a)-(c); At step (b)-(c); Or in step (c), mark.

In the target cell resistant proof carrier finally depend on patient's fragment sequence of originating with the expression of reporter gene, the function of reporter gene is not essential.

Another aspect of the present invention is for instructing the antiretroviral drugs susceptibility/resistant proof at HIV/AIDS.The specific resistance test carrier carrier that is used for antiretroviral drugs susceptibility/resistant proof of HIV/AIDS among the present invention is identified.

Another aspect of the present invention has provided identifies and estimates method for the antiretroviral composite biological validity of HIV and/or AIDS treatment potentialization.On the other hand, the present invention is directed to a novel resistance test carrier.This carrier includes a patient originate fragment and a reporter gene.Patient's segment of originating further includes the sudden change of one or more pol genes.

Brief Description Of Drawings

Fig. 1

The resistant proof carrier.Include the originate resistant proof carrier of a fragment and a reporter gene of a patient with figure signal.

Fig. 2

Two cell analysis.Spectroscopic analysis method schematically.By making up the resistant proof carrier in fragment cloning to the reporter gene virus vector that the patient is originated, then with this resistant proof carrier and an expression vector cotransfection of expressing amphophilic murine leukemia virus (MLV) coat protein or other virus that can infect or cell protein, the pseudotyped viral particle of Chan Shenging includes proteolytic enzyme (PR) and reversed transcriptive enzyme (RT) gene product that comes from patient's sequence like this, and then collecting granules is used to infect new fresh cell.Use the proteolytic enzyme of defective and proteolytic enzyme and the reversed transcriptive enzyme that reversed transcriptive enzyme sequence proof uciferase activity depends on function.On the other hand, when virion infects or in cell, add reverse transcriptase inhibitors before this, this test is provided with a plurality of groups that do not contain medicine and add the different concns medicine, measures the content of luciferase, measures the inhibition per-cent (%) under the different tests drug level.

Fig. 3

Medicine phenotype susceptibility legend.By drawing uciferase activity to log ₁₀Concentration (um) suppresses the percentage analysis data.This figure is used to calculate the 50% (IC that suppresses virus replication ₅₀) and 95% (IC ₉₅) required drug level, suppress curve and can explain with resistance to higher drug level displacement.Demonstrate 3 kinds of typical curves among the figure and be a kind of ucleotides reverse transcriptase inhibitors (AZT), a kind of non-nucleoside reverse transcriptase inhibitor (draw furan pyridine delavirdine) and a kind of proteinase inhibitor (ritonavir).Compare with baseline (treatment before) sample or control drug susceptible virus, PNL4-3 or the HXB-2 that when baseline sample is difficult for obtaining, selects for use for example, the medicaments insensitive linearity curve has reflected that to higher drug level (moving to right) displacement drug susceptibility (resistance) reduces.

Fig. 4

Medicine phenotype susceptibility and resistance figure: 487 routine patients.Implement PCR phenotype sensitivity analysis drafting phenotypic drug sensitivity and resistance figure and draw furan pyridine (delavirdine) and Viramune (nevirapine) resistance to increase with showing, this is the example of first model of NNRTI susceptibility/resistance.Relevant by analysed for plasma source virus proof hiv reverse transcriptase 184 codon mutations (M184V) with the 3TC resistance, 103 suddenly change (K103N) with draw furan pyridine (delavirdine) relevant over the ground with Viramune (nevirapine) resistance.

Fig. 5

Reversed transcriptive enzyme rite-directed mutagenesis strain medicine phenotype susceptibility and resistance figure.Implement PCR phenotype sensitivity analysis drafting phenotype susceptibility and resistance figure and show 103 and 181 password rite-directed mutagenesis (K103N; Y181C) draw furan pyridine (delavirdine) and Viramune (nevirapine) resistance to increase, double-mutant strain shows that the additive effect of two sudden changes causes resistance further to increase with making.

Fig. 6

Medicine phenotype susceptibility and resistance figure; 487 routine patients.Implement the viral nucleic acid of PCR phenotype sensitivity analysis blood plasma source hiv reverse transcriptase, draw the phenotype sensitivity maps and show and draw furan pyridine (delavirdine) that resistance is arranged over the ground and Viramune (nevirapine) does not have resistance that this is the example of second model of NNRTI susceptibility/resistance.This patient originate virus to testing used proteinase inhibitor resistance is arranged, also AZT and 3TC are had obvious resistance, ddC, ddI and d4T susceptibility are slightly shifted.Utilization PCR and order-checking are carried out gene type assay blood plasma source virus and are estimated the demonstration hiv reverse transcriptase at 103 and 236 bit codon mutation (K103N; P236L).Previous report P236L mutant strain draw with causing furan pyridine (delavirdine) resistance and Viramune (nevirapine) super quick (Dueweke TJet al. (1993) Proc Natl Acad Sci 90,4713-4717).Yet in this patient's sample, ground draws furan pyridine (delavirdine) resistance the same with wild-type with Viramune (nevirapine) susceptibility.

Fig. 7

The medicine phenotype susceptibility of reversed transcriptive enzyme rite-directed mutagenesis strain (P236L) and resistance figure.The sensitivity analysis of enforcement PCR phenotype is drawn out and is shown that P236L site-directed mutagenesis mutant strain draws the phenotype susceptibility and the resistance figure of furan pyridine (delavirdine) and Viramune (nevirapine) susceptibility over the ground, the result is identical with the observations of the patient's Virus Sample shown in Fig. 6, next 2 grid index K103N site-directed mutagenesis mutant strains, 2 grid below index double-mutant strain K103N+P236L.On K103N sudden change basis P236L taking place again, causes and draw the high resistance of furan pyridine (delavirdine) over the ground, but do not exert an influence owing to K103N thereby to Viramune (nevirapine) resistance.Figure the right shows have similar results when P236L is additional to Y181 → sudden change.

Fig. 8 A

Medicine phenotype susceptibility resistance figure: 302 routine patients.This is the example of the 3rd model of NNRTI susceptibility/resistance.Patient's virus phenotype analytical proof draws furan pyridine (delavirdine) and Viramune (nevirapine) susceptibility to reduce over the ground.The feature of this model is to draw furan pyridine (delavirdine) susceptibility to reduce with ground to compare, Viramune (nevirapine) susceptibility is reduced by a larger margin, patient's virogene type analysis show reversed transcriptive enzyme K103N sudden change and Viramune (nevirapine) and draw furan pyridine (delavirdine) resistance and P225H relevant.

Fig. 8 B

Medicine phenotype susceptibility and resistance figure: 780 routine patients.This is second example of the 3rd model of NNRTI susceptibility/resistance.Patient's virus phenotype analytical draws furan pyridine (delavirdine) and Viramune (nevirapine) susceptibility all to reduce with showing.The feature of this model is to draw furan pyridine (delavirdine) susceptibility to reduce with ground to compare, Viramune (nevirapine) susceptibility is reduced by a larger margin, patient's virogene type analysis show reversed transcriptive enzyme K103N site undergo mutation with Viramune (nevirapine) and draw furan pyridine (delavirdine) resistance and P225H relevant.

Fig. 8 C

Medicine phenotype susceptibility and resistance figure: 302 routine patient's single virus clones.Have the K103N sudden change and do not have the P225H sudden change (virus R211K) has the K103N sudden change and has P225H sudden change (K103N, virus P225H) simultaneously for K103N, 1135M showing from patient 302 single virus clone gene type analysis.These virus clone phenotypic characteristics show that the P225H sudden change reduces the ground relevant with the K103N sudden change and draws furan pyridine (delavirdine) resistance (relatively bottom grid), but do not change Viramune (nevirapine) resistance relevant with the K103N sudden change (relatively top grid).

Fig. 8 D

Medicine phenotype susceptibility and resistance figure: the strain of reversed transcriptive enzyme rite-directed mutagenesis.The phenotypic characteristic that has the virus of reversed transcriptive enzyme rite-directed mutagenesis P225H shows that this sudden change increases and draws furan pyridine (delavirdine) resistance over the ground, but Viramune (nevirapine) resistance is not changed (relatively top grid).Have reversed transcriptive enzyme rite-directed mutagenesis P225H and add the phenotypic characteristic figure that K013N or P225H add the virus of Y181C and show that the P225H sudden change reduction ground relevant with K103N or Y181C sudden change draws furan pyridine (delavirdine) resistance, but do not reduce and K103N or Y181C relevant Viramune (nevirapine) resistance of suddenling change.Be to draw furan pyridine (delavirdine) rather than Viramune (nevirapine) (relatively more corresponding middle part and bottom grid) at ground.

Fig. 9 A

Medicine phenotype susceptibility and resistance figure: 644 routine patients.This is the example of the 4th model of NNRTI susceptibility/resistance.Patient's virus phenotype analytical is found that Viramune (nevirapine) susceptibility significantly reduces, draw furan pyridine (delavirdine) susceptibility not reduce, patient's virogene type analysis is found to exist reversed transcriptive enzyme sudden change G190S, and be attended by K101E sudden change that Ai Te furan pyridine (atevirdine) susceptibility reduces, MMP226, L-697,661 and UC-10,38,57 (Schinazi, Mellors, Larder resistance table).

Fig. 9 B

Reversed transcriptive enzyme rite-directed mutagenesis strain medicine phenotype susceptibility and resistance figure.The phenotypic characteristic that includes the virus of reverse transcription sudden change G190A or G190S proves that these sudden changes reduce Viramune (nevirapine) susceptibility first mate, draws furan pyridine (delavirdine) susceptibility slightly to raise (comparing top sheet) over the ground.

The present invention describes in detail

The present invention relates to monitor the patient who accepts the ARV clinical treatment, particularly accept non-The progression of infection of HIV in the patient body of efabirenz ART.

In one embodiment, the invention provides a kind of evaluate patient ART The method of validity comprises: (I) gathers biological sample from the patient that HIV infects; (ⅱ) true Decide the nucleic acid whether biological sample includes the hiv reverse transcriptase of encoding, have one at reverse transcriptase Or a plurality of mutational sites. Sudden change is proportionate with the change of phenotype sensitiveness/resistance.

In a specific embodiment, it is degeneration-resistant to the invention provides a kind of evaluate patient NNRTI Transcribe the method for viral therapy validity, comprising: (I) gathers biological from the patient that HIV infects Sample; (ⅱ) determining whether biological sample includes is coded in 225 and 103 bit codon mutations The nucleic acid of hiv reverse transcriptase, use the sensitivity analysis of medicine phenotype, the present invention has confirmed HIV 225 passwords of reverse transcriptase separately sudden change or with 103 password simultaneous mutations, these sudden changes and ground Draw furan pyridine (delavirdine) sensitiveness to increase, Viramune (nevirapine) Susceptible change is very little or Do not change and efavirenz (efavirenz) sensitiveness does not change relevant.

In another specific embodiment, it is anti-to the invention provides a kind of evaluate patient NNRTI The method of retrovirus treatment validity comprises: (I) gathered from the patient that HIV infects and given birth to Matter sample; (ⅱ) determining whether biological sample includes is coded in 236 and 103 and/or the 18l position The nucleic acid of the hiv reverse transcriptase of codon mutation uses phenotype sensitivity analysis the present invention to confirm The separately sudden changes or simultaneously prominent with 103 and/or 181 passwords of 236 passwords of hiv reverse transcriptase Become these sudden changes and DELAVIRDINE (delavirdine) Reduced susceptibility (resistance increase), the network of anti-the furan Flat (nevirapine) sensitiveness is constant relevant, 236 separately sudden changes or prominent under the background of Y181C Becoming does not affect the sensitiveness of efavirenz (efavirenz), but has largely recovered prominent by 103N Become the sensitiveness forfeiture that causes.

In another specific embodiment, it is anti-to the invention provides a kind of evaluate patient NNRTI The method of retrovirus treatment validity comprises: (I) gathered from the patient that HIV infects and given birth to Matter sample; (ⅱ) determining whether biological sample includes is coded in 230 and/or 181 bit codons The nucleic acid of the hiv reverse transcriptase of sudden change uses phenotype sensitivity analysis the present invention to confirm HIV 230 passwords of reverse transcriptase separately sudden change or with 181 password simultaneous mutations, these sudden changes and ground Draw that furan pyridine (delavirdine) sensitiveness significantly reduces (resistance increase), (nevirapine) is quick for Viramune Perception significantly reduces relevant.

In another specific embodiment, it is anti-to the invention provides a kind of evaluate patient NNRTI The method of retrovirus treatment validity comprises: (I) gathered from the patient that HIV infects and given birth to Matter sample; (ⅱ) determine whether biological sample includes the HIV that is coded in 181 bit codon mutations The nucleic acid of reverse transcriptase uses phenotype sensitivity analysis the present invention to confirm hiv reverse transcriptase 181 Bit codon mutation and DELAVIRDINE (delavirdine) sensitiveness moderate reduces (resistance increase), anti-furan The sensitiveness of the remarkable reduction of flat (nevirapine) sensitiveness of network and efavirenz (efavirenz) is not covert Close.

In another specific embodiment, it is anti-to the invention provides a kind of evaluate patient NNRTI The method of retrovirus treatment validity comprises: (I) gathered from the patient that HIV infects and given birth to Matter sample; (ⅱ) determine whether biological sample includes the HIV that is coded in 188 bit codon mutations The nucleic acid of reverse transcriptase uses phenotype sensitivity analysis the present invention to confirm hiv reverse transcriptase 188 Bit codon mutation and DELAVIRDINE (delavirdine) sensitiveness slightly reduces (resistance increase), anti-furan The sensitiveness of the substantive reduction of flat (nevirapine) sensitiveness of network and efavirenz (efavirenz) is remarkable Reduce relevant.

In another specific embodiment, it is anti-to the invention provides a kind of evaluate patient NNRTI The method of retrovirus treatment validity comprises: (I) gathered from the patient that HIV infects and given birth to Matter sample; (ⅱ) determining whether biological sample includes is coded in 138 and/or 188 bit codons The nucleic acid of the hiv reverse transcriptase of sudden change uses phenotype sensitivity analysis the present invention to confirm HIV 8 passwords of reverse transcriptase 13 separately sudden change or with 188 password simultaneous mutations, these sudden changes and ground Draw that furan pyridine (delavirdine) sensitiveness moderate reduces (resistance raising), (nevirapine) is quick for Viramune The substantive reduction of perception reduces relevant with the sensitiveness moderate of efavirenz (efavirenz).

In another specific embodiment, it is anti-to the invention provides a kind of evaluate patient NNRTI The method of retrovirus treatment validity comprises: (I) gathered from the patient that HIV infects and given birth to Matter sample; (ⅱ) determine whether biological sample includes the HIV that is coded in 98 bit codon mutations The nucleic acid of reverse transcriptase uses phenotype sensitivity analysis the present invention to confirm hiv reverse transcriptase 98 Bit codon mutation and DELAVIRDINE (delavirdine) sensitiveness slightly reduces (resistance increase), anti-furan The sensitiveness of the slight reduction of flat (nevirapine) sensitiveness of network and efavirenz (efavirenz) is slightly fallen Low relevant.

In another specific embodiment, it is anti-to the invention provides a kind of evaluate patient NNRTI The method of retrovirus treatment validity comprises: (I) gathered from the patient that HIV infects and given birth to Matter sample; (ⅱ) determining whether biological sample includes is coded in 98 and/or 190 bit codons The nucleic acid of the hiv reverse transcriptase of sudden change uses phenotype sensitivity analysis the present invention to confirm HIV 98 passwords of reverse transcriptase separately sudden change or with 190 password simultaneous mutations, these sudden changes and ground Draw furan pyridine (delavirdine) sensitiveness to increase (resistance reduction), Viramune (nevirapine) sensitiveness The substantive reduction reduces relevant with the sensitiveness substance of efavirenz (efavirenz). At another In the specific embodiment, the invention provides a kind of evaluate patient NNRTI ARV The method for the treatment of validity comprises: (I) gathers biological sample from the patient that HIV infects; (ⅱ) Determine whether biological sample includes the HIV that is coded in 181 and/or 98 bit codon mutations and reverse The nucleic acid of record enzyme uses phenotype sensitivity analysis the present invention to confirm that 181 of hiv reverse transcriptases are close Code separately sudden change or with 98 password simultaneous mutations, these sudden changes and DELAVIRDINEs (delavirdine) Sensitiveness significantly reduce (resistance increase), Viramune (nevirapine) sensitiveness substantive reduce and The slight reduction of the sensitiveness of efavirenz (efavirenz) is correlated with. In another specific embodiment In, the invention provides a kind of side of evaluate patient NNRTI ART validity Method comprises: (I) gathers biological sample from the patient that HIV infects; (ⅱ) determine biological sample Whether product include is coded in 101 and/or 190 bit codon mutations, and for example the HIV of 190S is contrary The nucleic acid of transcriptase uses phenotype sensitivity analysis the present invention to confirm 101 of hiv reverse transcriptases Password separately sudden change or with 190 password simultaneous mutations, these sudden changes and DELAVIRDINEs (delavirdine) Sensitiveness constant (wild type), the substantive reduction of Viramune (nevirapine) sensitiveness and Chinese mugwort send out a furan The substantive reduction of the sensitiveness of Rui Zi (efavirenz) is correlated with. In another specific embodiment, The invention provides a kind of method of evaluate patient NNRTI ART validity, Comprise: (I) gathers biological sample from the patient that HIV infects; (ⅱ) determine that biological sample is The no nucleic acid that includes the hiv reverse transcriptase that is coded in 108 bit codon mutations uses phenotype Sensitivity analysis the present invention has confirmed hiv reverse transcriptase 108 bit codon mutations and DELAVIRDINE (delavirdine) sensitiveness constant (wild type), Viramune (nevirapine) sensitiveness slightly reduce Substantive constant relevant with the sensitiveness of efavirenz (efavirenz). In another specific enforcement In the scheme, the invention provides a kind of evaluate patient NNRTI ART validity Method, comprising: (I) gathers biological sample from the patient that HIV infects; (ⅱ) determine to give birth to It is contrary whether matter sample includes the HIV that is coded in 101 and 103 and/or 190 bit codon mutations The nucleic acid of transcriptase uses phenotype sensitivity analysis the present invention to confirm 101 of hiv reverse transcriptases Password separately sudden change or with 130 and/or 190 password simultaneous mutations, these sudden changes and DELAVIRDINE (delavirdine) sensitiveness or constant (101 and 190) or moderate reduction (103 and 190, for example 190A) (resistance increase), the substantive reduction of Viramune (nevirapine) sensitiveness and a Chinese mugwort furan are auspicious Hereby substantive reduction of the sensitiveness of (efavirenz) is correlated with.

In another specific embodiment, it is anti-to the invention provides a kind of evaluate patient NNRTI The method of retrovirus treatment validity comprises: (I) gathered from the patient that HIV infects and given birth to Matter sample; (ⅱ) determining whether biological sample includes is coded in 106 and/or 189 and/or 181 And/or the nucleic acid of the hiv reverse transcriptase of 227 bit codon mutations, use the phenotype sensitivity analysis The present invention confirmed 106 passwords of hiv reverse transcriptase separately sudden change or with 106 and/or 189 and/ Or 181 and/or 227 password simultaneous mutations, these sudden changes and DELAVIRDINE (delavirdine), anti-The sensitiveness of furan network flat (nevirapine) and efavirenz (efavirenz) changes relevant. Especially, 106 and 181 passwords existence sudden change significantly reduces with DELAVIRDINE (delavirdine) sensitiveness, anti-furan The sensitiveness of the substantive reduction of flat (nevirapine) sensitiveness of network and efavirenz (efavirenz) is slight Reduce and be correlated with, 106 and 189 passwords exist sudden change and DELAVIRDINE (delavirdine) sensitiveness slight Reduce, Viramune (nevirapine) sensitiveness moderate reduces and efavirenz (efavirenz) quick Perception is constant relevant, and 106 and 227 passwords exist sudden change and DELAVIRDINE (delavirdine) sensitiveness Slight reduction, the substantive reduction of Viramune (nevirapine) sensitiveness and efavirenz (efavirenz) Sensitiveness slight reduce relevantly, 181 and 227 passwords exist sudden change and DELAVIRDINE (delavirdine) Sensitiveness increases, Viramune (nevirapine) sensitiveness significantly reduces and efavirenz (efavirenz) Sensitiveness increase relevantly, 106 and 181 passwords exist sudden change and DELAVIRDINE (delavirdine) quick The perception moderate reduces, Viramune (nevirapine) sensitiveness is substantive reduces and efavirenz (efavirenz) the slight reduction of sensitiveness is correlated with. In another specific embodiment, the present invention A kind of method of evaluate patient NNRTI ART validity is provided, has comprised: (I) From the patient that HIV infects, gather biological sample; (ⅱ) determine whether biological sample includes volume Code uses at the nucleic acid of the hiv reverse transcriptase of 188 and 100 and/or 103 bit codon mutations Phenotype sensitivity analysis the present invention has confirmed the separately sudden change or with 100 of 188 passwords of hiv reverse transcriptase And/or 103 password simultaneous mutations, these sudden changes and DELAVIRDINE (delavirdine), Viramune (nevirapine) change relevant with the sensitiveness of efavirenz (efavirenz). Especially, 103 Hes 188 passwords exist the substantive reduction of sudden change and DELAVIRDINE (delavirdine) sensitiveness, Viramune (nevirapine) the sensitiveness substance of the substantive reduction of sensitiveness and efavirenz (efavirenz) is fallen Low relevant, 100 and 103 passwords exist sudden change and DELAVIRDINE (delavirdine) sensitiveness substance Reduce, Viramune (nevirapine) sensitiveness moderate reduces and efavirenz (efavirenz) quick The substantive reduction of perception is correlated with, and 103 and 100 and 188 passwords exist sudden change and DELAVIRDINE (delavirdine) the substantive reduction of sensitiveness, the substantive reduction of Viramune (nevirapine) sensitiveness Relevant with the substantive reduction of the sensitiveness of efavirenz (efavirenz). In these cases, infect The phenotype sensitiveness of patient's HIV virus/resistance figure and genotype figure revised in case reflect right ARV preparation changes of reactivity. For the NNRTI ART, infect Patient's HIV virus may have resistance to one or more NNRTIs as described herein, but right Therefore other NNRTIs does not have resistance, needs after detecting sudden change or increases the anti-reverse transcription disease The dosage of toxin preparation perhaps changes other ARV preparation, perhaps at patient treatment Add one or more extra ARV preparations in the composition. For example, if the patient exists Accept efavirenz (efavirenz) (DMP-266) when treating 225 undergo mutation, the patient controls Treating composition may need to make following modification, (I) or change different NNRTI anti-reverse transcription diseases Toxin preparation, for example DELAVIRDINE (delavirdine) or Viramune (nevirapine), and stop Chinese mugwort and send out Furan auspicious hereby (efavirenz) treatment; Perhaps (ⅱ) increases the dosage of efavirenz (efavirenz); Perhaps (ⅲ) In the patient treatment compound, add other ARV preparation. Can be by detecting viral load Measure HIV DNA copy number for example and estimate the validity of modified treatment, HIV DNA copy number Reduce really relevant with the validity for the treatment of compound.

Refer to that with the term " positive correlation " here specific result causes specific most possible conclusion, Rather than other conclusion.

Preferred, the non-limiting particular of another one of the present invention is as follows: a kind of evaluation is suffered from The method of person NNRTI ART validity comprises: the trouble that (I) infects from HIV Gather biological sample among the person; (ⅱ) RNA of amplification HIV coding from biological sample is about to RNA Reverse transcription is cDNA, and with HIV primer amplification HIV sequence, the PCR that amplifies like this produces Thing includes pol gene; (ⅲ) carry out pcr amplification with primer, the PCR that amplifies like this Product includes wild type or 225 and 103 bit codon mutations; And (ⅳ) by the PCR product Determine whether to exist 225 or 103 bit codon mutations or the two simultaneous mutation. The present invention another Preferably, non-limiting particular is as follows: a kind of evaluate patient NNRTI anti-reverse transcription The method of viral therapy validity comprises: (I) gathers biological sample from the patient that HIV infects; (ⅱ) RNA of amplification HIV coding from biological sample, being about to the RNA reverse transcription is cDNA, And with HIV primer amplification HIV sequence, the PCR product that amplifies like this includes reverse transcriptase Gene; (ⅲ) carry out pcr amplification with primer, the PCR product that amplifies like this includes wild type Or 103 and/or 181 and 236 bit codon mutation; And (ⅳ) determine whether by the PCR product There are 236 and 103 and/or 181 bit codon mutations.

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluate patient The method of NNRTI ART validity comprises: the patient that (I) infects from HIV The middle biological sample of gathering; (ⅱ) RNA of amplification HIV coding from biological sample is about to RNA Reverse transcription is cDNA, and with HIV primer amplification HIV sequence, the PCR that amplifies like this produces Thing includes pol gene; (ⅲ) carry out pcr amplification with primer, the PCR that amplifies like this Product includes wild type or 101 and 190 (G190S) codon mutations; And (ⅳ) pass through The PCR product determines whether to exist 190 (G190S) and the sudden change of 101 password positions.

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluate patient The method of NNRTI ART validity comprises: the patient that (I) infects from HIV The middle biological sample of gathering; (ⅱ) RNA of amplification HIV coding from biological sample is about to RNA Reverse transcription is cDNA, and with HIV primer amplification HIV sequence, the PCR that amplifies like this produces Thing includes pol gene; (ⅲ) carry out pcr amplification with primer, the PCR that amplifies like this Product includes wild type or 103 and 190 (G190A) codon mutations; And (ⅳ) pass through The PCR product determines whether to exist 190 (G190A) and 103 bit codon mutations. The present invention again Preferred, a non-limiting particular is as follows: a kind of evaluate patient NNRTI is degeneration-resistant Transcribe the method for viral therapy validity, comprising: (I) gathers biological from the patient that HIV infects Sample; (ⅱ) RNA of amplification HIV coding from biological sample, soon the RNA reverse transcription is CDNA, and with HIV primer amplification HIV sequence, the PCR product that amplifies like this includes Pol gene; (ⅲ) carry out pcr amplification with primer, the PCR product that amplifies like this comprises Wild type or 230 and 181 bit codon mutations are arranged; And (ⅳ) determine whether by the PCR product There are 230 and 181 bit codon mutations.

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluate patient The method of NNRTI ART validity comprises: the patient that (I) infects from HIV The middle biological sample of gathering; (ⅱ) RNA of amplification HIV coding from biological sample is about to RNA Reverse transcription is cDNA, and with HIV primer amplification HIV sequence, the PCR that amplifies like this produces Thing includes pol gene; (ⅲ) carry out pcr amplification with primer, the PCR that amplifies like this Product includes wild type or 181 sudden changes; And (ⅳ) determine whether to exist by the PCR product 181 sudden changes. Another preferred, non-limiting particular of the present invention is as follows: a kind of The method of evaluate patient NNRTI ART validity comprises: (I) felt from HIV Gather biological sample among the patient who dyes; (ⅱ) RNA of amplification HIV coding from biological sample, namely Be cDNA with the RNA reverse transcription, and with HIV primer amplification HIV sequence, amplify like this The PCR product includes pol gene; (ⅲ) carry out pcr amplification with primer, amplify like this The PCR product include wild type or 188 sudden changes; And (ⅳ) determine by the PCR product be 188 sudden changes of no existence. Another preferred, non-limiting particular of the present invention is as follows: A kind of method of evaluate patient NNRTI ART validity comprises: (I) is from HIV Gather biological sample among the patient who infects; (ⅱ) RNA of amplification HIV coding from biological sample, Being about to the RNA reverse transcription is cDNA, and with HIV primer amplification HIV sequence, amplifies like this The PCR product include pol gene; (ⅲ) carry out pcr amplification with primer, like this amplification The PCR product that goes out includes wild type or 138 and 188 sudden changes; And (ⅳ) produce by PCR Thing determines whether to exist 138 and 188 sudden changes.

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluate patient The method of NNRTI ART validity comprises: the patient that (I) infects from HIV The middle biological sample of gathering; (ⅱ) RNA of amplification HIV coding from biological sample is about to RNA Reverse transcription is cDNA, and with HIV primer amplification HIV sequence, the PCR that amplifies like this produces Thing includes pol gene; (ⅲ) carry out pcr amplification with primer, the PCR that amplifies like this Product includes wild type or 98 bit codon mutations; And (ⅳ) determine whether by the PCR product There are 98 bit codon mutations. Another preferred, non-limiting particular of the present invention As follows: a kind of method of evaluate patient NNRTI ART validity comprises: (I) From the patient that HIV infects, gather biological sample; What (ⅱ) amplification HIV encoded from biological sample RNA, being about to the RNA reverse transcription is cDNA, and with HIV primer amplification HIV sequence, like this The PCR product that amplifies includes pol gene; (ⅲ) carry out pcr amplification with primer, this The PCR product that sample amplifies includes wild type or 98 and 190 bit codon mutations; And (ⅳ) Determine whether to exist 190 and 98 bit codon mutations by the PCR product. Another is excellent in the present invention Choosing, non-limiting particular is as follows: a kind of evaluate patient NNRTI anti-reverse transcription disease The method of poison treatment validity comprises: (I) gathers biological sample from the patient that HIV infects; (ⅱ) The RNA of amplification HIV coding from biological sample, being about to the RNA reverse transcription is cDNA, and uses HIV primer amplification HIV sequence, the PCR product that amplifies like this includes pol gene; (ⅲ) carry out pcr amplification with primer, the PCR product that amplifies like this includes wild type or 98 With 181 bit codon mutations; And (ⅳ) determine whether to exist 98 and 181 by the PCR product Codon mutation.

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluate patient The method of NNRTI ART validity comprises: the patient that (I) infects from HIV The middle biological sample of gathering; (ⅱ) RNA of amplification HIV coding from biological sample is about to RNA Reverse transcription is cDNA, and with HIV primer amplification HIV sequence, the PCR that amplifies like this produces Thing includes pol gene; (ⅲ) carry out pcr amplification with primer, the PCR that amplifies like this Product includes wild type or 101 and 190 bit codon mutations; And (ⅳ) by the PCR product Determine whether to exist 190, for example 190S and 101 bit codon mutations.

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluate patient The method of NNRTI ART validity comprises: the patient that (I) infects from HIV The middle biological sample of gathering; (ⅱ) RNA of amplification HIV coding from biological sample is about to RNA Reverse transcription is cDNA, and with HIV primer amplification HIV sequence, the PCR that amplifies like this produces Thing includes pol gene; (ⅲ) carry out pcr amplification with primer, the PCR that amplifies like this Product includes wild type or 108 bit codon mutations; And (ⅳ) determine by the PCR product be No existence 108 bit codon mutations. Another preferred, non-limiting particular implementation side of the present invention Case is as follows: a kind of method of evaluate patient NNRTI ART validity comprises: (I) gathers biological sample from the patient that HIV infects; (ⅱ) amplification HIV compiles from biological sample The RNA of code, being about to the RNA reverse transcription is cDNA, and with HIV primer amplification HIV sequence, The PCR product that amplifies like this includes pol gene; (ⅲ) carry out pcr amplification with primer, The PCR product that amplifies like this includes wild type or 101 and 103 and 190 bit codon mutations; And (ⅳ) determine whether to exist 101 and 103 and 190 bit codon mutations by the PCR product, 190A for example.

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluate patient The method of NNRTI ART validity comprises: the patient that (I) infects from HIV The middle biological sample of gathering; (ⅱ) RNA of amplification HIV coding from biological sample is about to RNA Reverse transcription is cDNA, and with HIV primer amplification HIV sequence, the PCR that amplifies like this produces Thing includes pol gene; (ⅲ) carry out pcr amplification with primer, the PCR that amplifies like this Product includes wild type or 106 and 189 and 181 and 227 bit codon mutations; And (ⅳ) logical Cross the PCR product and determine whether to exist 106 and 189 and 181 and 227 bit codon mutations.

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluate patient The method of NNRTI ART validity comprises: the patient that (I) infects from HIV The middle biological sample of gathering; (ⅱ) RNA of amplification HIV coding from biological sample is about to RNA Reverse transcription is cDNA, and with HIV primer amplification HIV sequence, the PCR that amplifies like this produces Thing includes pol gene; (ⅲ) carry out pcr amplification with primer, the PCR that amplifies like this Product includes wild type or 188 and 100 and 103 bit codon mutations; And (ⅳ) pass through PCR Product determines whether to exist 188 and 100 and 103 bit codon mutations. There is hiv reverse transcriptase 225 and 103 bit codon mutations show that the validity existing or treatment of NNRTI in the future need to change Advance, because 103 bit codon mutations reduce neurological susceptibility as shown in the present invention, this can lead to Cross 225 bit codon mutations and partly recover neurological susceptibility. Equally, use measure of the present invention Confirm to exist on the hiv reverse transcriptase 236 and 103 and/or 181 bit codon mutations to represent to have now Or the validity of the treatment of NNRTI in the future is lowered. Equally, use means of the present invention Confirm that with method hiv reverse transcriptase exists 190 (G190A) and 103 (K103N) position password Son sudden change expression the validity existing or treatment of NNRTI in the future is lowered. Equally, make Confirm that with measure of the present invention there are 190 (G190S) and 101 in hiv reverse transcriptase (K101E) bit codon mutation represent existing or the treatment of NNRTI in the future validity by Reduce. Equally, use measure of the present invention to confirm that hiv reverse transcriptase exists 230 and 181 Bit codon mutation represents that the validity existing or treatment of NNRTI in the future is lowered. With Sample uses measure of the present invention to confirm that there are 181 bit codon mutations in hiv reverse transcriptase Expression the validity existing or treatment of NNRTI in the future is lowered. Equally, use this Bright measure confirmation hiv reverse transcriptase exists 188 bit codon mutations to represent existing or will The validity of the NNRTI treatment that comes is lowered. Equally, use means of the present invention and side It is existing or future that method confirms that hiv reverse transcriptase exists 138 and 188 bit codon mutations to represent The validity of NNRTI treatment is lowered. Equally, use measure of the present invention to confirm Hiv reverse transcriptase exists 98 bit codon mutations to represent having of existing or the treatment of NNRTI in the future Effect property has been lowered. Equally, use measure confirmation hiv reverse transcriptase of the present invention to deposit The validity that represents existing or in the future NNRTI treatment at 98 and 190 bit codon mutations Be lowered. Equally, use measure of the present invention to confirm that there are 181 Hes in hiv reverse transcriptase 98 bit codon mutations represent that the validity existing or treatment of NNRTI in the future is lowered. Equally, use measure of the present invention to confirm that 101 and 190 of hiv reverse transcriptase existence are close Numeral sudden change, 190S for example, the validity of the existing or treatment of NNRTI in the future of expression by Reduce. Equally, use measure of the present invention to confirm that 108 of hiv reverse transcriptase existence are close Numeral sudden change expression the validity existing or treatment of NNRTI in the future is lowered. Equally, Use measure of the present invention to confirm that 101 and 103 and 190 of hiv reverse transcriptase existence are close Numeral sudden change, 190A for example, the validity of the existing or treatment of NNRTI in the future of expression by Reduce. Equally, use measure of the present invention to confirm that hiv reverse transcriptase exists 106 and 189 The validity that represents existing or in the future NNRTI treatment with 181 and 227 bit codon mutations Through being lowered. Equally, use measure of the present invention to confirm that hiv reverse transcriptase exists 188 The validity that represents existing or in the future NNRTI treatment with 100 and 103 bit codon mutations Through being lowered.

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluation is to HIV The patient who infects carries out the method for ART validity, the trouble of (a) infecting from HIV Gather biological sample among the person; (ⅱ) determining whether biological sample includes is coded in 236 and 103 And/or the nucleic acid of the hiv reverse transcriptase of 181 bit codon mutations, use the phenotype sensitivity analysis Find that these 3 sudden changes are really relevant with DELAVIRDINE (delavirdine) resistance, use phenotype sensitiveness Analyze and find that these 3 sudden changes are really relevant with Viramune (nevirapine) resistance. Real at another Execute in the scheme cipher coding leucine (L) of 236 sudden changes of hiv reverse transcriptase. Advance at one In the embodiment in one step, reverse transcriptase is at 103 codon mutations, 181 codon mutations or two The person suddenlys change in the lump and follows hiv reverse transcriptase 236 bit codon mutations. Further at another In the embodiment, 103 sudden change cipher coding asparagines (N), and 103 sudden change cipher codings Cysteine (C).

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluate patient The method of NNRTI ART validity (a) is gathered from the patient that HIV infects Biological sample; (ⅱ) determining whether biological sample includes is coded in 225 and 103 bit codons The nucleic acid of the hiv reverse transcriptase of sudden change uses the phenotype sensitivity analysis to find hiv reverse transcriptase 225 passwords suddenly change separately or cause that with 103 password simultaneous mutations DELAVIRDINE (delavirdine) is quick Perception increases, and Viramune (nevirapine) sensitiveness is not exerted an influence. In another enforcement In the scheme, 225 sudden change cipher coding histidines, 230 cipher coding leucines, 181 cipher codings Cysteine.

The invention provides patient that a kind of evaluation infects HIV carries out ART and has The method of effect property comprises: (a) gather biological sample from the patient that HIV infects; (ⅱ) true Decide the nucleic acid whether biological sample includes the hiv reverse transcriptase of 181 codon mutations of encoding, Use the phenotype sensitivity analysis to find that 181 codon mutations are really quick with DELAVIRDINE (delavirdine) The perception moderate reduces, Viramune (nevirapine) sensitiveness obviously reduces and efavirenz (efavirenz) sensitiveness is constant relevant. In one embodiment, 181 sudden change cipher codings are different bright Propylhomoserin.

The invention provides a kind of evaluation to the method that the patient of HIV infection carries out antiretroviral therapy validity, comprising: (a) from the patient that HIV infects, gather biological sample; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase of 188 codon mutations of encoding, utilization phenotype sensitivity analysis is found that 188 codon mutations draw that furan pyridine (delavirdine) susceptibility slightly reduces, Viramune (nevirapine) susceptibility essence reduces and send out furan auspicious now (efavirenz) susceptibility with Chinese mugwort with ground really and is significantly reduced relevant.In one embodiment, 188 sudden change cryptography halfcystines, Histidine or leucine.

The invention provides a kind of evaluation to the method that the patient of HIV infection carries out antiretroviral therapy validity, comprising: (a) from the patient that HIV infects, gather biological sample; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase of 190 codon mutations of encoding, utilization phenotype sensitivity analysis finds that 190 codon mutations draw with ground really that furan pyridine (delavirdine) susceptibility slightly reduces, Viramune (nevirapine) susceptibility reduces relevant significantly.In one embodiment, 190 sudden change cryptography L-Ala or Serines.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 230 and 181 bit codon mutations, the sensitivity analysis of utilization phenotype is found the independent sudden change of hiv reverse transcriptase 230 passwords or is drawn furan pyridine (delavirdine) susceptibility significantly to reduce with 181 password simultaneous mutations with causing, Viramune (nevirapine) susceptibility is significantly reduced.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 138 and 188 bit codon mutations, utilization phenotype sensitivity analysis discovery hiv reverse transcriptase 138 passwords suddenly change separately or draw furan pyridine (delavirdine) susceptibility moderate to reduce with 188 password simultaneous mutations with causing, Viramune (nevirapine) susceptibility essence are reduced and end send out the reduction of furan auspicious now (efavirenz) susceptibility moderate.In another embodiment, 138 sudden change cryptography L-Ala, 188 sudden change cryptography leucines.

The invention provides a kind of evaluation to the method that the patient of HIV infection carries out antiretroviral therapy validity, comprising: (a) from the patient that HIV infects, gather biological sample; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase of 98 codon mutations of encoding, utilization phenotype sensitivity analysis finds that 98 codon mutations draw with ground that furan pyridine (delavirdine) susceptibility slightly reduces, Viramune (nevirapine) susceptibility slightly reduces really and ends that to send out the slight reduction of furan auspicious now (efavirenz) susceptibility relevant.In one embodiment, 98 sudden change cryptography glycine.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 98 and 190 bit codon mutations, utilization phenotype sensitivity analysis discovery hiv reverse transcriptase 98 passwords suddenly change separately or draw furan pyridine (delavirdine) susceptibility to increase with 190 password simultaneous mutations with causing, Viramune (nevirapine) susceptibility essence are reduced and end send out the reduction of furan auspicious now (efavirenz) susceptibility essence.In another embodiment, 190 sudden change cryptography Serines, 98 sudden change cryptography glycine.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 181 and 98 bit codon mutations, utilization phenotype sensitivity analysis discovery hiv reverse transcriptase 181 passwords suddenly change separately or draw furan pyridine (delavirdine) susceptibility significantly to reduce with 98 password simultaneous mutations with causing, and a reduction of Viramune (nevirapine) susceptibility essence and Chinese mugwort furan auspicious now (efavirenz) susceptibility are slightly reduced.In another embodiment, 98 sudden change cryptography glycine, 181 sudden change cryptography halfcystines.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determining whether biological sample includes is coded in 101 and 190 bit codon mutations, the nucleic acid of the hiv reverse transcriptase of 190S for example, the sensitivity analysis of utilization phenotype is found the independent sudden change of hiv reverse transcriptase 101 passwords or is drawn furan pyridine (delavirdine) susceptibility constant with 190 password simultaneous mutations with causing, the reduction of furan auspicious now (efavirenz) susceptibility essence is sent out in reduction of Viramune (nevirapine) susceptibility essence and Chinese mugwort.In another embodiment, 190 sudden change cryptography Serines, 101 sudden change cryptography L-glutamic acid.

The invention provides a kind of evaluation to the method that the patient of HIV infection carries out antiretroviral therapy validity, comprising: (a) from the patient that HIV infects, gather biological sample; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase of 108 codon mutations of encoding, 108 codon mutations are found in utilization phenotype sensitivity analysis, and to draw that furan pyridine (delavirdine) susceptibility is constant, Viramune (inevirapine) susceptibility slightly reduces constant relevant with furan auspicious now (efavirenz) susceptibility that end with ground really.In one embodiment, 108 sudden change cryptography Isoleucines.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determining whether biological sample includes is coded in 101 and 190 bit codon mutations, the nucleic acid of the hiv reverse transcriptase of 190A for example, utilization phenotype sensitivity analysis discovery hiv reverse transcriptase 101 passwords suddenly change separately or draw furan pyridine (delavirdine) susceptibility constant with 190 password simultaneous mutations with causing, and a reduction of Viramune (nevirapine) susceptibility essence and Chinese mugwort furan auspicious now (efavirenz) susceptibility are significantly reduced.In another embodiment, 190 sudden change cryptography glycine, 101 sudden change cryptography L-glutamic acid.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 103 and 190 bit codon mutations, use the independent sudden change of phenotype sensitivity analysis discovery hiv reverse transcriptase 103 passwords or draw furan pyridine (delavirdine) susceptibility moderate to reduce with causing, the reduction of Viramune (nevirapine) susceptibility essence and furan auspicious now (efavirenz) susceptibility that ends are significantly reduced with 190 password simultaneous mutations.In another embodiment, 190 sudden change cryptography L-Ala, 103 sudden change cryptography l-asparagines.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 106 and 181 bit codon mutations, the sensitivity analysis of utilization phenotype is found the independent sudden change of hiv reverse transcriptase 106 passwords or is drawn furan pyridine (delavirdine) susceptibility significantly to reduce with 181 password simultaneous mutations with causing, the reduction of furan auspicious now (efavirenz) susceptibility essence is sent out in reduction of Viramune (nevirapine) susceptibility essence and Chinese mugwort.In another embodiment, 106 sudden change cryptography L-Ala, 181 sudden change cryptography halfcystines.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 106 and 189 bit codon mutations, utilization phenotype sensitivity analysis discovery hiv reverse transcriptase 106 passwords suddenly change separately or draw furan pyridine (delavirdine) susceptibility slightly to reduce with 189 password simultaneous mutations with causing, and are constant to a reduction of Viramune (nevirapine) susceptibility moderate and Chinese mugwort furan auspicious now (efavirenz) susceptibility.In another embodiment, 189 sudden change cryptography leucines, 106 sudden change cryptography L-Ala.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 106 and 227 bit codon mutations, utilization phenotype sensitivity analysis discovery hiv reverse transcriptase 106 passwords suddenly change separately or draw furan pyridine (delavirdine) susceptibility slightly to reduce with 227 password simultaneous mutations with causing, and a reduction of Viramune (nevirapine) susceptibility essence and Chinese mugwort furan auspicious now (efavirenz) susceptibility are slightly reduced.In another embodiment, 227 sudden change cryptography leucines, 106 sudden change cryptography L-Ala.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 181 and 227 bit codon mutations, utilization phenotype sensitivity analysis discovery hiv reverse transcriptase 181 passwords suddenly change separately or draw furan pyridine (delavirdine) susceptibility to increase with 227 password simultaneous mutations with causing, Viramune (nevirapine) susceptibility are significantly reduced and end send out the increase of furan auspicious now (efavirenz) susceptibility.

In another embodiment, 227 sudden change cryptography leucines, 181 sudden change cryptography halfcystines.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 106 and 181 and 227 bit codon mutations, the sensitivity analysis of utilization phenotype is found that hiv reverse transcriptase 106 passwords suddenly change separately or is drawn reduction of furan pyridine (delavirdine) susceptibility moderate and Chinese mugwort to send out furan auspicious now (efavirenz) susceptibility with 181 and 227 password simultaneous mutations with causing and slightly reduce.

In another embodiment, 106 sudden change cryptography L-Ala, 181 sudden change cryptography halfcystine and 227 cryptography leucines.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 103 and 188 bit codon mutations, utilization phenotype sensitivity analysis discovery hiv reverse transcriptase 103 passwords suddenly change separately or draw furan pyridine (delavirdine) susceptibility essence to reduce with 188 password simultaneous mutations with causing, Viramune (nevirapine) susceptibility essence are reduced and end send out the reduction of furan auspicious now (efavirenz) susceptibility essence.In another embodiment, 188 sudden change cryptography leucines, 103 sudden change cryptography l-asparagines.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 100 and 103 bit codon mutations, utilization phenotype sensitivity analysis discovery hiv reverse transcriptase 100 passwords suddenly change separately or draw furan pyridine (delavirdine) susceptibility essence to reduce with 103 password simultaneous mutations with causing, Viramune (nevirapine) susceptibility moderate are reduced and end send out the reduction of furan auspicious now (efavirenz) susceptibility essence.

In another embodiment, 100 sudden change cryptography Isoleucines, 103 sudden change cryptography l-asparagines.

Another is preferred in the present invention, the indefiniteness particular is as follows: a kind ofly estimate the method that the patient that HIV is infected carries out antiretroviral therapy validity, (a) gather biological sample from the patient that HIV infects; (ⅱ) determine whether biological sample includes the nucleic acid of the hiv reverse transcriptase that is coded in 100 and 103 and 188 bit codon mutations, utilization phenotype sensitivity analysis discovery hiv reverse transcriptase 100 passwords suddenly change separately or draw furan pyridine (delavirdine) susceptibility essence to reduce with 103 and 188 password simultaneous mutations with causing, Viramune (nevirapine) susceptibility moderate are reduced and end send out the reduction of furan auspicious now (efavirenz) susceptibility essence.

In another embodiment, 100 sudden change cryptography Isoleucines, 103 sudden change cryptography l-asparagines, and 188 cryptography leucines.

The present invention also provides means and the method for using the resistant proof carrier, and carrier contains the HIV gene, further contains the required NNRTI sudden change of drug screening.Especially, the invention describes the resistant proof carrier that includes hiv reverse transcriptase, reversed transcriptive enzyme has 225 and 103 required codon mutations of drug screening.The present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 236 and 103 and/or 181 codon mutations, the present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 190 (G190A) and 103 (K103N) codon mutation, and the present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 190 (G190S) and 101 (K103E) codon mutation.

The present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 230 and 181 codon mutations.

The present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 181 codon mutations.

The present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 188 codon mutations.

The present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 138 and 188 codon mutations.

The present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 98 codon mutations.

The present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 98 and 190 codon mutations.

The present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 181 and 98 codon mutations.

The present invention has also described and has included at 101 and 190 codon mutations, for example the hiv reverse transcriptase resistant proof carrier of 190S.

The present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 108 codon mutations.

The present invention has also described and has included at 101 and 103 and/or 190 codon mutations, for example the hiv reverse transcriptase resistant proof carrier of 190A.

The present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 106 and 189 and/or 181 and/or 227 codon mutations.

The present invention has also described the hiv reverse transcriptase resistant proof carrier that includes at 188 and 100 and/or 103 codon mutations.

The invention further relates to the novel carriers, host cell and the component that are used to separate and identify non-nucleoside HIV-1 reverse transcriptase inhibiting resistance mutant strain, and use these carriers, host cell and component to implement antiviral screening.The invention still further relates to the ability screening drug candidate that suppresses described mutant strain according to it.

Embodiment 1

Use the resistant proof carrier to measure medicine phenotype susceptibility and resistance

Use the above means of PCT international application (application number No.PCT/US/01609, January 29 1997 applying date) and method to carry out medicine phenotype susceptibility and resistant proof.

In these tests one section fragment from the patient (PDS) is arranged.This fragment, or by isolating viral RNA in the virus particle from the HIV patients serum, one section fragment utilizing reverse transcription-polymerase chain reaction from viral RNA, to increase again to come out; Or the paternal clone of antagonism test carrier DNA carries out rite-directed mutagenesis, the wild-type HIV-1 mutant that is obtained.This section from patient's fragment corresponding to hiv protease and reversed transcriptive enzyme zone.Use Standard operation procedure SOP isolated viral RNA, for example RNAgents Total RNA Isolation System (Pu Luomaige company, Wisconsin, USA Madison) or RNAzol (Tai Er-Tai Shite company, Texas, USA Fu Lande Wood).Reverse transcription-polymerase chain reaction schedule of operation was divided into for two steps.Utilize retroviral reversed transcriptive enzyme, the reversed transcriptive enzyme of Moloney MuLV (Luo Shi molecular system company for example, N.J. Blanche fort), the perhaps reversed transcriptive enzyme of avian myeloblastic leukosis virus AMV (Bao Ling Man, the Indiana, United States Indianapolis), duplicate cDNA from viral RNA.Use thermostability archaeal dna polymerase amplification cDNA then, Taq enzyme (Luo Shi molecular system for example, N.J. Blanche fort), Tth enzyme (Luo Shi molecular system, N.J. Blanche fort), the PrimeZyme enzyme (separates from Thermus brockianus, biometrics company, Germany's song Dettingen) amplification cDNA, or the description use of pressing " long segment PCR " is used in combination thermostability polysaccharase (Barnes, W.M., (1994) Proc.Natl.Acad.Sci, USA91, the 2216-2220) [high-fidelity that for example increases PCR (Taq+Pwo) (Bao Ling Man, the Indiana, United States Indianapolis) or GeneAmp XL PCR test kit (Tth+Vent), (Luo Shi molecular system company, N.J. Blanche fort)].Use the fragment of Apa I primer (PDSApa) and Age I primer (PDSAge) amplification from patient, by introducing Apa I and Age I restriction enzyme site respectively at PCR product 5 ' and 3 ' end, please respectively referring to seeing the PCT international application, application number PCT/US97/0169, January 29 1997 applying date.

Press PCT international application (application number PCT/US97/01609, January 29 1997 applying date) method described in, with viral RNA as template, with oligonucleotide PDSApa and PDSAge is primer, carry out this fragment of reverse transcription-polymerase chain reaction (PCR) amplification, with the DNA product of Apa I and Age I or isoschizomers PINAI digestion preparation 1.5KB, mix " test " fragment that comes from the patient by reverse transcription-pcr and make up the resistant proof carrier then.By guaranteeing in plasmid DNA, to contain the representative sample of HIV virus quasispecies in the particular patient serum corresponding to synthetic resistant proof carrier, when making up the specific resistance test carrier, gather the intestinal bacteria transformant, will a large amount of (＞100) independently the intestinal bacteria transformant merge as the preparation plasmid DNA.

The packaging expression vector of a coding amphotropic retrovirus MuLV 4070A env gene product can synthesize resistant proof vector virus particle in the resistant proof vector host cell.This particle is the infected person target cell effectively.Except the env gene, the resistant proof carrier of other all HIV genes of encoding all is used to the host cell of a kind of packing of transfection (in case host cell is transfected, just being called the resistant proof vector host cell).The packaging expression vector and the resistant proof carrier of coding amphotropic retrovirus MuLV4070A env gene product are used to synthetic (pseudotyped) resistant proof carrier infectivity particle with pseudo-type feature in the resistant proof vector host cell together.

When utilizing the resistance detection to carry out resistant proof, packing host and target host cell have been used.The target host cell is that the human embryonic kidney cell is 293 (cell cultivation equipment company, Univ California-San Francisco USAs) or Jurkat leukemia T clone (Arthur Weiss, Univ California-San Francisco USA).

Use the resistant proof carrier in two types host cell, to carry out resistant proof.The virion of resistant proof carrier is produced by first kind of host cell (resistant proof vector host cell), utilizes and uses resistant proof carrier and packaging host cell of the common transfection of packaging expression vector, prepares this resistant proof vector host cell.Infect second kind of host cell (target host cell), examining report expression of gene in the target host cell with resistant proof vector virus particle then.

Structure has the resistant proof carrier of the box gene of a functional luciferase, and with this resistant proof carrier DNA transfection host cell.The resistant proof carrier has the sequence from the patient, this sequence comprises reversed transcriptive enzyme and proteolytic enzyme sequence, and they have susceptibility or resistance to antiretroviral medicament (as nucleotide reverse transcriptase inhibitors, non-nucleotide reverse transcriptase inhibitors and proteinase inhibitor).With resistant proof carrier DNA transfection host cell, produce resistant proof vector virus particle.No matter whether proteinase inhibitor exists, and with this carrier DNA target cell infection, target cell does not add NRTI or NNRTI medicine during growth, or in the ever-increasing environment of drug level.The uciferase activity quantity that produces in the infected target cell when having medicine infected target cell when not adding medicine produces the amount of luciferase and compares.(half-inhibition concentration IC50) is not measured drug resistance to 50% needed dose of uciferase activity when not adding medicine with inhibition.With suppressing percentage ratio to Log ₁₀The IC50 value is measured in the drug level mapping.

Host cell is inoculated in the culture dish of diameter 10cm, adds resistant proof vector plasmid DNA and bag by the expression vector template, carries out transfection with calcium phosphate method after a couple of days.In 1 to 24 hour, contain the sedimentary substratum of DNA after the transfection with fresh cell culture medium replacement.After the transfection in 1 to 4 day, results contain resistant proof vector virus particulate cell culture medium, with behind the membrane filtration of 0.45mm-80 ℃ of preservations.HIV capsid protein (p24) expression level EIA method in the cell culture medium of results is pressed manufacturers instruction (SIAC company, Maryland, USA Fo Ledelike).Before the infection, earlier target cell (293 and 293/T) is inoculated in the cell culture medium.The infection experiment control group is (not containing DNA) pseudo-transfection or the resistant proof vector plasmid DNA transfection that does not contain the env expression plasmid.Infect the back and discarded substratum later in 1 to 3 day or more days, in every hole, add cell lysis buffer solution (Pu Luomaige company).Detect the uciferase activity (Fig. 3) of cell lysate.Suppress effect by following Equation for Calculating medicine:

% luciferase inhibiting rate=1-(RLUluc[ _Medicine]/RLUluc) * 100

RLUluc[wherein _Medicine] be the relative light unit of the plain enzymic activity of infected cell fluorescence when having medicine, RLUluc is the relative light unit of the uciferase activity of infected cell when not having medicine.Suppress the right log of per-cent with uciferase activity ₁₀From then on the drug level mapping finds the IC50 value on the sigmoid curve.Medicine suppresses curve and sees Fig. 3.

Embodiment 2: the analyzing gene type is set up the cognation between genotype and the phenotype susceptibility

The phenotype sensitivity analysis of patient HIV sample

Make up the resistant proof carrier as described in example 1 above.Antagonism test carrier or carry out the phenotype sensitivity test from the clone in resistant proof carrier storehouse accurately and is quantitatively measured the sensitive level of a series of antiretroviral drugs.This group antiretroviral drugs may comprise said nucleotide reverse transcriptase inhibitors (NRTIs) class medicine, non-nucleotide reverse transcriptase inhibitors (NNRTIs) class medicine and proteinase inhibitor (PRIs) class medicine.If have new drug or novel drugs target to utilize then can enlarge this and organize medicine.On each resistance carrier storehouse, measure the half-inhibition concentration IC50 of each trial drug.The susceptibility model and the known susceptibility model of all trial drugs are compared.Can do further inspection to patient's sample, find out the genotype relevant and change with observed susceptibility model.

The gene type assay of patient HIV sample

With gene survey type methods analyst resistant proof carrier storehouse or its clone described in the embodiment 2.In one embodiment of the invention, purified virus RNA carries out the reverse transcription-pcr amplification, with ABI chain terminator method automatic sequencing.The control sequence of measuring in sequence and the database compares.Perhaps, as possible, with receive treatment the patient before the sample gathered relatively.Check this genotype sequence, find out the sequence that is different from contrast or the preceding sample of treatment, and the dependency between foundation and the viewed phenotype.

The sensitivity analysis of directed mutants genotype

Under the clear and definite wild-type background of background, make up the resistant proof carrier that has specific sudden change, observe genotype and change and phenotype susceptibility, analysis cognation between the two.Sudden change can import the resistant proof carrier separately; Or import the resistant proof carrier with other medicines resistant mutation combination.These drug resistance sudden changes have been considered to regulate the susceptibility of HIV to an a certain certain drug or a class medicine.Available any rite-directed mutagenesis method that is widely known by the people will be suddenlyd change and be imported the resistant proof carrier.In one embodiment of the invention, use the large primer PCR method to carry out rite-directed mutagenesis.Then according to foregoing phenotype sensitivity analysis method, to having specific sudden change or one group of mensuration that the resistant proof carrier that suddenlys change carries out.The sensitivity maps that records and another wild-type resistant proof carrier are relatively.This carrier genetic background is clear and definite, lacks specific sudden change.The variation that observation takes place antiretroviral drugs sensitivity phenotype in the test, these change owing to the specific sudden change that imports the resistant proof carrier.

Embodiment 3

Set up the cognation of P225H gene type assay and phenotype susceptibility

Patient 97-302 phenotype analytical

As described in embodiment 1, make up the resistant proof carrier with patient 97-302 sample.This patient accepted about 10 months d4T, indinavir and DMP-226 treatment.Isolated viral RNA carries out reverse transcription-pcr and amplifies one section fragment from the patient.This fragment has comprised proteins encoded enzyme total length and the amino acid whose viral fragment of reversed transcriptive enzyme 1-313.This fragment insertion is had in the virus vector of reporter gene, obtain resistant proof carrier called after RTV-302.Use the phenotype sensitivity test accurately and quantitatively to measure the sensitivity levels of RTV-302 to a series of antiretroviral drugs.These medicines comprise said NRTI class medicine (AZT, 3TC, d4T, ddI, ddC), NNRTIs (draw furan pyridine, Viramune) and PRIs (indinavir, nelfinavir, ritonavir, saquinavir).Measure the half-inhibition concentration IC50 of each trial drug.Detecting patient's virus compares to the susceptibility of each trial drug and with known susceptibility model.Observe a kind of susceptibility model of patient RTV-302 to NNRTI class medicine.In this model, Viramune susceptibility significantly reduces (resistance increase), and ground draws furan pyridine susceptibility moderate to reduce (seeing Fig. 8 A).Further detecting patient 97-302 sample finds with observed to the relevant genotype variation of susceptibility model.

Patient 97-302 is genotypic to be determined

Use ABI chain terminator method to measure the dna sequence dna of RTV-302 automatically.(HIV sequence library Los Alamos NM) relatively, finds out the sequence different with control sequence to nucleotide sequence with wild-type branch B HIV-1.At reversed transcriptive enzyme K103N, sudden change is found in T200A and P225H site.K103N is relevant with NNRTI class drug resistance, uses the phenotype sensitivity test to prove that K103N sudden change and ground draw furan pyridine and the decline of Viramune resistance relevant, and the reduction degree is equal.Known between HIV wild-type (medicaments insensitive) variant, 1135M and T200A site have sequence polymorphism.Utilize rite-directed mutagenesis and phenotype sensitivity analysis, identify the P225H sudden change, set up the cognation between variation of 225 amino acids and the NNRTI class drug susceptibility.

Site-directed mutagenesis

Make up the resistant proof carrier, make it have the P225H sudden change, perhaps also have simultaneously contain some other drug resistance sudden change (K103N, Y181C).Known these drug resistance sudden changes can be regulated HIV to NNRTI class drug susceptibility.Use the large primer PCR method with rite-directed mutagenesis import the resistant proof carrier (Sakar G and Sommar SS (1994) Biotechniques8 (4), 404-407).Described phenotype sensitivity test before using detects the resistant proof carrier that contains the P225H sudden change, and another resistant proof carrier relatively.This carrier genetic background is clear and definite, is wild-type in 225 sites.Compare with wild-type RTV, draw the susceptibility model of furan pyridine to change NNRTI class medicine.P225H-RTV is in the wild-type background, when only containing the P225H sudden change.RTV compares with the wild-type contrast, and P225H-RTV draws the furan pyridine responsive more over the ground.P225H-RTV does not have noticeable change to the susceptibility of Viramune.The P225H sudden change also imports to a RTV who also contains other sudden change (K103N, Y181C or K103N+Y181C).Under all scenario, corresponding to not with the RTV carrier of P225H sudden change, the RTV carrier that has the P225H sudden change draws the furan pyridine to suppress responsive (Fig.8D) more over the ground.Under all scenario, the P225H sudden change can significantly not change Viramune susceptibility (Fig.8D).

Embodiment 4

Set up the cognation between HIV P236L loci gene type and the phenotype

The phenotype analytical of HIV patient 97-268 sample

As described in example 1 above, make up the resistant proof carrier with patient 97-268 sample.This patient accepted following pharmacological agent: AZT and 3TC (NRTI class medicine) in the past; Indinavirhe and saquinavir (PRJ class medicine); And draw furan pyridine (NNRTI class medicine).Treatment time is between 1 month to 2 months.Isolated viral RNA carries out reverse transcription-pcr, amplifies one section fragment from the patient (PDS).This fragment has comprised the virus sequence of proteins encoded enzyme total length and reversed transcriptive enzyme 1-313 amino acids.One of this section sequence insertion is had in the virus vector of reporter gene the resistant proof carrier of generation, called after RTV-268.Then RTV-268 is carried out phenotypic assay, accurately and quantitatively measure the sensitivity levels of RTV-268 one group of antiretroviral drugs.This group antiretroviral drugs comprised usually said NRTI class medicine (AZT, 3TC, d4T, ddI, ddC), NNRTI class medicine (draw furan pyridine, Viramune) and PRI class medicine (indinavir, nelfinavir, ritonavir, saquinavir).Measure the half-inhibition concentration IC50 of each trial drug.Check the susceptibility of patient's virus, and compare with the susceptibility that contrasts virus to each trial drug.Observe a kind of susceptibility model of patient's sample RTV-268 to NNTRI class medicine.In this model, Virus Sample draws the furan pyridine to have resistance and Viramune is not had resistance over the ground.Further this sample of inspection is found out the relevant genotype variation of susceptibility model.

The genotype of HIV patient 97-268

Use ABI chain terminator technology to RTV-268 DNA automatic sequencing.The gained nucleotide sequence is made comparisons with the consensus sequence of wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library).Analyzing this genotype finds out and contrasts different sequences.Compare with control sequence, find sudden change in the following site of reversed transcriptive enzyme: M41L, D67N, M184V, T200A, E203D, L210W, T215Y, K219Q and P236L.Known between HIV wild-type (drug resistance) variant, T200A and the sudden change of E203D place have sequence polymorphism.At M41L, D67N, L210W, T215Y is relevant with the AZT resistance with the sudden change of K219Q site.The M184V site mutation is relevant with the 3TC resistance.The P236L site mutation with draw furan pyridine resistance relevant, and with Viramune susceptibility increase relevant (Dueweke etc., Ibid.).Opposite with former report, the RTV-268 sample does not change Viramune susceptibility.Utilize rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay, identify the P236L sudden change, to set up the cognation between change of 236 amino acids and the change of phenotype susceptibility.

Rite-directed mutagenesis

Preparation resistant proof carrier makes these carriers only have the P236L sudden change, perhaps also have simultaneously known other sudden change relevant with resistance (K103N, Y181C).Known these relevant sudden changes with resistance can be regulated the susceptibility of HIV-1 to NNTRI class medicine.Utilize the large primer PCR method, (Saker and Sommar, Ibid.), will suddenly change imports the resistant proof carrier to carry out rite-directed mutagenesis.Resistant proof carrier P236L-RTV contains the P236L sudden change.P236L-RTV is carried out the phenotype sensitivity analysis, test-results and another resistant proof carrier are compared.This carrier genetic background is clear and definite, is wild-types in 236 sites.Test-results proof P236L-RTV has changed the susceptibility of NNRTI class medicine.P236L-RTV is in the wild-type background, only contains the P236L sudden change, and the susceptibility of drawing the furan pyridine over the ground is not as wild-type contrast RTV.Opposite with people's such as Dueweke report, observe the P236L sudden change and do not make Viramune susceptibility take place significantly to change.P236L sudden change also be imported into have K103N and (or) on the RTV carrier of Y181C sudden change.Compare with the RTV that does not have accordingly the P236L sudden change, in all cases, the RTV that has the P236L sudden change draws the furan pyridine to be not so good as the former sensitivity (resistance is higher) over the ground.In all cases, the P236L sudden change can significantly not change Viramune susceptibility.

Embodiment 5

Set up the cognation between HIV G190S loci gene type and the sensitivity phenotype

The phenotype analytical of HIV patient 97-644 sample

As described in example 1 above, make up the resistant proof carrier with patient 97-644 sample.This patient accepted following pharmacological agent: d4T (NRTI class medicine) in the past; Indinavir (PRI class medicine); And Chinese mugwort sends out furan auspicious now (NNRTI class medicine).Treatment time was not waited from 5 months to 17 months.Isolated viral RNA carries out the reverse transcription-pcr reaction, amplifies one section fragment from the patient (PDS).This fragment has comprised the virus sequence of proteins encoded enzyme total length and reversed transcriptive enzyme 1-313 amino acids.The insertion of this section sequence is had in the virus vector of reporter gene the resistant proof carrier of generation, called after RTV-644.Then RTV-644 is carried out phenotypic assay, accurately and quantitatively measure the sensitivity levels of RTV-644 one group of antiretroviral drugs.This group antiretroviral drugs comprised usually said NRTI class medicine (AZT, 3TC, d4T, ddI, ddC), NNRTI class medicine (draw furan pyridine, Viramune) and PRI class medicine (indinavir, nelfinavir, ritonavir, saquinavir).Measure the half-inhibition concentration IC50 of each trial drug.Check the susceptibility of patient's virus, and compare with a kind of contrast virus to each trial drug.Observe a kind of susceptibility model of patient's sample RTV-644 to NNRTI class medicine.In this model, Virus Sample has resistance and draws the furan pyridine to have resistance hardly over the ground Viramune.Further this sample of inspection is found out the genotype variation relevant with the susceptibility model.

The genotype of HIV patient 97-644

Use ABI chain terminator technology to RTV-644 DNA automatic sequencing.The gained nucleotide sequence is made comparisons with the consensus sequence of wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library).Analyzing this genotype finds out and contrasts different sequences.Compare with control sequence, find sudden change at reversed transcriptive enzyme K101E and G190S site.Known between HIV wild-type (medicaments insensitive) variant, the sudden change that is positioned at T200A and E203D two site has sequence polymorphism.Relevant in the sudden change of L101E site with some NNRTI class drug susceptibilities, but be not with all NNRTI class drug susceptibility is all relevant.The G190A sudden change is all relevant with the loviride resistance with Viramune with the G190S sudden change.Utilize rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay, identify G190S and G190A sudden change, to set up the cognation between variation of 190 amino acids and the variation of phenotype susceptibility.

Rite-directed mutagenesis

Structure has the resistant proof carrier of G190S and G190A sudden change.Utilize the large primer PCR reaction, (Saker and Sommar, Ibid.), will suddenly change imports the resistant proof carrier to carry out a rite-directed mutagenesis.Resistant proof carrier G190S-RTV has the G190S sudden change, and G190A-RTV has the G190A sudden change.Utilize the susceptibility of prominent G190S of analysis of phenotype sensitivity test or G190A, test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, is wild-type in the G190 site.Change has taken place to NNRTI class drug susceptibility in G190S-RTV and G190A-RTV.Under the wild-type background, these RTV significantly are lower than wild-type contrast RTV to Viramune susceptibility, draw furan pyridine susceptibility then to be better than wild-type contrast RTV slightly over the ground.

Embodiment 6

Be tested and appraised in the HIV-1 reversed transcriptive enzyme amino acid variation prediction to the reaction of non-nucleotide reverse transcriptase inhibitors

The phenotype of HIV-1 reversed transcriptive enzyme 236 amino acids sudden change and the dependency between the genotype

In one embodiment of the invention, make and estimate the effect that the variation of HIV-1 reverse transcriptase protein matter 236 amino acids causes with the following method, these methods comprise: (ⅰ) collect HIV-1 the infected's biological specimen; (ⅱ) whether the nucleotide sequence of judging coding HIV-1 reversed transcriptive enzyme in this biological specimen undergos mutation 236 of codons; 236 sudden changes of codon (P236L) with to draw furan pyridine susceptibility to reduce relevant, Viramune susceptibility is not change then.

These biological specimens are made up of whole blood and blood constitutent, comprise that peripheral blood lymphocytes (PBMC), serum, blood plasma (use the blood plasma of different antithrombotics preparations, for example EDTA, citric acid-glucose, heparin), biopsy, cerebrospinal fluid (CSF), perhaps other cell, tissue or body fluid.In another embodiment of the invention, can directly from biological specimen, separate HIV-1 nucleic acid or reverse transcriptase protein; Perhaps earlier from biological specimen, be purified into virion and then therefrom separate HIV-1 nucleic acid (geneome RNA) or reverse transcriptase protein.Can use diverse ways to analyze HIV-1 reversed transcriptive enzyme the 236th bit codon and whether undergo mutation, for example the direct viral nucleic acid of identification code HIV-1 reversed transcriptive enzyme or directly identify HIV-1 reverse transcriptase protein itself.Can utilize classics or novel protein sequence measurement directly to identify reverse transcription albumen, perhaps utilize antibody or other special conjugated protein or compound to carry out epi-position identification, can both measure HIV-1 reversed transcriptive enzyme the 236th amino acids.In addition, can also measure HIV-1 reversed transcriptive enzyme the 236th amino acids by the HIV-1 nucleotide sequence of amplification and identification code reverse transcriptase protein.Many methods HIV-1 nucleotide sequence that can be used to increase is arranged, and these methods comprise reverse transcription-pcr reaction, NASBA, SDR, RCR or 3SR, just can know as long as these methods have the technician of common skill.Directly measure the nucleotide sequence of coding HIV-1 reversed transcriptive enzyme and just can judge whether the 236th bit codon undergos mutation, measure nucleotide sequence and can use primer extension-chain termination method (SANGER, ABI/PE and Visible Genetics) or splitting of chain method (Maxam andGilbert); Perhaps for example flight time mass spectrum (matrix assistedlaser desorptoin-ionizationtime of flight, MALDI-TOF), these sequence measurements of developing recently of mass spectrum (Sequenom, Gene Trace Systems).In addition, also can utilize various probe hybridization methods to measure the nucleotide sequence of coding 236 amino acids, gene chip hybridization sequencing technologies (Affymetrix) for example, slit hybridization technique (LiPA; And differential hybridization technology (Chiron) Murex).

In first-selected embodiment of the present invention, use the resistant proof carrier DNA from biological specimen, prepare to carry out sensitivity phenotype's analysis, be that wild-type or mutant are measured to the amino acid of 236 of HIV-1 reversed transcriptive enzymes in the biological specimen.In one embodiment, collect plasma sample, purified virus RNA also amplifies one section HIV-1 proteolytic enzyme and reversed transcriptive enzyme zone nucleic acid fragment from the patient of encoding with the reverse transcription-pcr reaction technology.Connect and the bacterium conversion through DNA, this section mixed in the virus vector that has a reporter gene from patient's amplified fragments, thereby produce a resistant proof carrier.From bacterial cultures separation resistance test carrier, and by carrying out sensitivity phenotype's test described in the example 1.Test-results and a generation P236L sudden change patient sample compare.With fluoroscopic examination chain termination cycle sequencing (ABI/PE), nucleic acid (DNA) sequence in HIV-1 proteolytic enzyme and reversed transcriptive enzyme zone in mensuration patient 268 samples.This method is used to measure the consensus sequence (representative quasispecies) that mixes the various HIV-1 variants that exist in the representative censorship sample.This method also is used to measure the nucleotide sequence of every kind of variant.

The phenotype sensitivity maps of patient's sample and rite-directed mutagenesis draw 103 of furan pyridine and Viramune susceptibility and HIV-1 reversed transcriptive enzymes provably, and 181, or 236 do not undergone mutation relevant.The phenotype sensitivity maps of patient's sample and rite-directed mutagenesis draw the remarkable reduction (resistance increase) of furan pyridine susceptibility provably.103 of coding HIV-1 reversed transcriptive enzymes, 181 the nucleotide sequence of 236 amino acids L is undergone mutation when not undergoing mutation, and associated Viramune susceptibility does not but almost reduce.

The phenotype sensitivity maps and the rite-directed mutagenesis of patient's sample prove, 103 of reversed transcriptive enzymes, 181 or 103+181 amino acids appearance sudden change (K103N, Y181C, or K103N+Y181C) time, 236 proline(Pro) do not cause that delvirdine or Viramune susceptibility further increase.Yet, reversed transcriptive enzyme sudden change (the K103N that the phenotype sensitivity maps of patient's sample and rite-directed mutagenesis proof are being associated with the NNRTI resistance, Y181C, or K103N+Y181C) outside, 236 L draw furan pyridine susceptibility further to reduce (resistance increase) then almost not further reduction of susceptibility to Viramune with causing.

The phenotype of HIV-1 reversed transcriptive enzyme 225 amino acids sudden change and the dependency between the genotype

Utilize the phenotype sensitivity maps and the directed mutants of patient's sample to prove, reversed transcriptive enzyme sudden change (the K103N relevant with the NNRTI resistance do not appearring, Y181C) time, the change that proline(Pro) can not draw furan pyridine or Viramune susceptibility with causing appears in 225 of HIV-1 reversed transcriptive enzymes.Yet, utilize the phenotype sensitivity maps and the directed mutants of patient's sample also to prove, (K103N, in the time of Y181C), 225 Histidine occurs and draw furan pyridine susceptibility to increase and the almost not variation of Viramune susceptibility with causing the reversed transcriptive enzyme sudden change relevant with the NNRTI resistance not occurring.

Utilize the phenotype sensitivity maps of patient's sample to prove except producing the reversed transcriptive enzyme sudden change (K103N relevant with the NNRTI resistance with directed mutants, Y181C or K103N+Y181C) outside, 225 proline(Pro) occurs and do not draw furan pyridine and Viramune susceptibility further to reduce with not causing.In contrast, utilize the sensitivity phenotype of patient's sample the sudden change (K103N relevant to occur at reversed transcriptive enzyme with the NNRTI resistance with the directed mutants proof, Y181C or K103N+Y181C) time, 225 Histidine occurs and draw furan pyridine susceptibility to increase with causing, and Viramune susceptibility does not then almost change.

In the genotype of HIV-1 reversed transcriptive enzyme 190 amino acids sudden change and the cognation between the phenotype

Utilize the phenotype sensitivity maps and the positional mutation of patient's sample to prove, (K103N in the time of Y181C), if G occurs at 190, does not draw furan pyridine and Viramune susceptibility to change with not causing the sudden change relevant with NNRTI class drug resistance not occur at reversed transcriptive enzyme.Utilize the phenotype sensitivity maps of directed mutants to prove, when the sudden change relevant with NNRTI class drug resistance do not appear in reversed transcriptive enzyme, A occurs at 190, will draw furan pyridine susceptibility to increase with causing, Viramune susceptibility reduces.Utilize patient's sample phenotype sensitivity maps and directed mutants to prove, when the sudden change relevant with the NNRTI resistance do not appear in reversed transcriptive enzyme, 190 S occurred and draw furan pyridine susceptibility to increase and the reduction of Viramune susceptibility with causing.

Embodiment 8

Utilize resistant proof carrier and directed mutants, set up the cognation between the relevant phenotype of HIV Y181I loci gene type and NNRTI class drug susceptibility resistance

Preparation resistant proof carrier is also analyzed the phenotype of patient HIV sample 98-964

As described in example 1,98-964 makes up the resistant proof carrier with patient's sample.This patient accepted the treatment of following medicine: ddI in the past, d4T, AZT, 3TC and ddC (NRTI class medicine); Saquinavir and nelfinavir (PRI class medicine); Viramune (NNRTI class medicine) and HU.Isolated viral RNA carries out the reverse transcription-pcr reaction, amplifies one section fragment from the patient (PDS).This fragment has comprised the nucleic acid sequence of proteins encoded enzyme total length and reversed transcriptive enzyme 1-313 amino acids.This of fragment insertion is had in the virus vector of reporter gene, obtain a resistant proof carrier, called after RTV-964.Then RTV-964 is carried out phenotype analytical, accurately measure its susceptibility quantitatively one group of antiretroviral drugs.This group medicine comprised usually said NRTI class medicine (AZT, 3TC, d4T, ddI, ddC), NNRTI class medicine (draw furan pyridine, Viramune) and PRI class medicine (indinavir, nelfinavir, ritonavir, and saquinavir).On resistant proof carrier storehouse, measure the half-inhibition concentration IC50 of each trial drug.Procuratorial work resistant proof carrier compares with known drug susceptibility model the susceptibility model of each trial drug.Observe a kind of susceptibility model of patient RTV-964 to NNRTI class medicine.In this model, RTV-964 draws the susceptibility generation moderate of furan pyridine to reduce (10 times) over the ground, to the then significantly reduction (750 times) of susceptibility of Viramune.

Measure the genotype of patient HIV sample

Use ABI chain terminator technology to RTV-964 DNA automatic sequencing.Gained nucleic acid series is made comparisons with the consensus sequence of wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library).Check that this genotype finds out the sequence that is different from contrast.Compare with control sequence, in carrier RTV-964, find sudden change: M41L, K43E, D67N, K70R, L74I, V75S, Y181I, R211T, T215Y, D218E and K219Q as upper/lower positions.M41L, D67N, K70R, L74I, V75S, T215Y is relevant with NRTI class drug resistance with K219Q.Known between various wild-types (medicaments insensitive) variant of HIV, the sudden change of R211T position has sequence polymorphism.Proved in the past that Y181I was relevant with Viramune high level resistance.We set up the cognation between genotype variation and the phenotype with rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay inspection sudden change Y181I.

Utilize the effect of the rite-directed mutagenesis proof specific sudden change of HIV in antiretroviral susceptibility (phenotype)

Utilize the large primer PCR method to carry out rite-directed mutagenesis, and the Y181I that will suddenly change importing resistant proof carrier (Sakar and Sommar, Ibid.).Resistant proof carrier Y181I-RTV has the Y181I sudden change.By method noted earlier Y181I-RTV is carried out phenotypic assay, test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, is wild-type in 181 sites.We have drawn the furan pyridine with having measured NNRTI class medicine on the Y181I-RTV carrier, the auspicious susceptibility model now of Viramune and Ai Fa furan.Y181I-RTV only contains the Y181I sudden change, is in the wild-type background.RTV compares with the wild-type contrast, and ground draws furan pyridine susceptibility moderate to reduce (20 times), and Viramune susceptibility significantly reduces (740 times), and Chinese mugwort is sent out the auspicious wild-type susceptibility (1.4 times) that shows now of furan.

Embodiment 9

Utilize resistant proof carrier and directed mutants, set up HIV Y188 loci gene type and with the relevant phenotype of NNRTI class drug susceptibility resistance between cognation

Preparation resistant proof carrier is also analyzed the phenotype of patient HIV sample 97-300

As described in example 1,97-300 makes up the resistant proof carrier with patient's sample.This patient accepted the treatment of following medicine: d4T and 3TC (NRTI class medicine) in the past; Indinavir (PRI class medicine); Send out furan auspicious now (NNRTI class medicine) with Chinese mugwort.Isolated viral RNA carries out the reverse transcription-pcr reaction, and one section nucleic acid fragment from the patient (PDS) increases.This fragment has comprised that virus is used for the nucleotide sequence of proteins encoded enzyme total length and reversed transcriptive enzyme 1-313 amino acids.This of fragment insertion is had in the virus vector of reporter gene, obtain a resistant proof carrier, called after RTV-300.Then RTV-300 is carried out phenotype analytical, accurately measure its susceptibility quantitatively one group of antiretroviral drugs.This group medicine has comprised usually said NRTI class medicine (AZT, 3TC, d4T, ddI and ddC), NNRTI class medicine (draw furan pyridine, efarenz and Viramune) and PRI class medicine (indinavir, nelfinavir, ritonavir and saquinavir).On resistant proof carrier storehouse, measure the half-inhibition concentration IC50 of each trial drug.Check the susceptibility model of resistant proof carrier, compare with known drug susceptibility model to each trial drug.Observe a kind of susceptibility model of patient RTV-300 to NNRTI class medicine.In this model, RTV-300 draws the susceptibility generation moderate of furan pyridine to reduce (25 times) over the ground, Viramune susceptibility is then had substantially reduce (greater than 800 times).

Measure the genotype of patient HIV sample

Use the ABI chain terminator to RTV-300 DNA automatic sequencing.Gained nucleic acid series is made comparisons with the consensus sequence of wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library).Check that this genotype finds out and contrast different sequences.Compare with control sequence, in carrier RTV-300, find following sudden change: K32N, M184V and Y188L.M184V is relevant with the 3TC resistance.It is relevant to have proved in the past that sudden change Y188L and Chinese mugwort sent out the auspicious high-level now resistance of furan.Existing reporting with several NNRTI class medicines (E-ePseU, E-EPS, HEPT, Viramune, BHAP, U-8720, TIBO R82913, Loviride) treatment, other sudden change of screening Y188 position, i.e. Y188C and Y188H.We check the Y188L sudden change with rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay, set up the cognation between genotype change and the phenotype.

Utilize site-directed mutagenesis technique to confirm the effect of specific sudden change in antiretroviral drugs susceptibility (phenotype) among the HIV

(Saker and Sommar, Ibid.), the Y188L that will suddenly change imports the resistant proof carrier to use big primer-PCR method to carry out a rite-directed mutagenesis.Resistant proof carrier Y188L-RTV contains the Y188L sudden change.Use the previous phenotypic assay method of describing, antagonism test carrier Y188L-RTV carries out resistant proof, and test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, is wild-type in 188 sites.We draw furan pyridine, the auspicious phenotype susceptibility model now of Viramune and Ai Fa furan with measuring NNRTI class medicine on resistant proof carrier Y181L-RTV.Y188L-RTV is under the wild-type background, only contains the Y188L sudden change.With respect to wild-type contrast RTV, Y188L-RTV draws the susceptibility of furan pyridine that slight reduction (9 times) is arranged over the ground, and Viramune susceptibility is had substantive reduce (greater than 800 times), and Chinese mugwort is sent out the auspicious susceptibility now of furan remarkable reduction (109 times).Chinese mugwort is sent out the auspicious susceptibility now of furan reduce about 100 times, such height that this numerical value was not reported in the past.

Use site-directed mutagenesis technique and confirm the effect of specific sudden change in antiretroviral drugs susceptibility (phenotype) among the HIV

(Saker and Sommar, Ibid.), the Y188C that will suddenly change imports the resistant proof carrier to utilize big primer-round pcr to carry out a rite-directed mutagenesis.Resistant proof carrier Y188C-RTV contains the Y188C sudden change.Use the previous phenotypic assay method of describing, Y188C-RTV is carried out resistant proof, test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, is wild-type in 188 sites.We draw the auspicious phenotype susceptibility model now of furan pyridine, Viramune and Ai Fa furan with measuring NNRTI class medicine on resistant proof carrier Y188C-RTV.Y188C-RTV is under the wild-type background, only contains the Y188C sudden change.With respect to wild-type contrast RTV, Y188C-RTV draws the susceptibility of furan pyridine that slight reduction (3 times) is arranged over the ground, has moderate to reduce (30 times) to Viramune susceptibility, and Chinese mugwort is sent out the also moderate reduction (20 times) of the auspicious susceptibility now of furan.

(Saker and Sommar, Ibid.), the Y188H that will suddenly change imports the resistant proof carrier to use big primer-round pcr to carry out a rite-directed mutagenesis.Resistant proof carrier Y188H-RTV contains the Y188H sudden change.Use the previous phenotypic assay method of describing, Y188H-RTV is carried out resistant proof, test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, is wild-type in 188 sites.We draw the phenotype susceptibility model of furan pyridine and Viramune with measuring NNRTI class medicine on resistant proof carrier Y188H-RTV.Y188H-RTV is in the wild-type background, only contains the Y188H sudden change.With respect to wild-type contrast RTV, Y188H-RTV has moderate reduction (3.5 times) to the susceptibility of Viramune.Do not have to measure Chinese mugwort is sent out the auspicious susceptibility now of furan.

Embodiment 10

Use resistant proof carrier and directed mutants, set up HIV E138 and Y188 loci gene type and with the relevant phenotype of NNRTI class drug susceptibility resistance between cognation

Preparation resistant proof carrier is also analyzed the phenotype of patient HIV sample 97-209

As described in example 1,97-209 makes up the resistant proof carrier with patient's sample.This patient accepted the treatment of following medicine: AZT in the past, ddI, d4T and 3TC (NRTI class medicine); Indinavir (PRI class medicine); And adefovir (NNRTI class medicine).Isolated viral RNA carries out reverse transcription-pcr reaction, the one section fragment from the patient that increases (PDS).This fragment has comprised the nucleic acid sequence of proteins encoded enzyme total length and reversed transcriptive enzyme 1-313 amino acids.This of fragment insertion is had in the virus vector of reporter gene, produce a resistant proof carrier, called after RTV-209.Then RTV-209 is carried out phenotype analytical, accurately measure its susceptibility quantitatively one group of antiretroviral drugs.This group medicine has comprised said NRTI class medicine (AZT, 3TC, d4T, ddI and ddC), and NNRTI class medicine (draw furan pyridine, Viramune) and PRI class medicine (indinavir, nelfinavir, ritonavir, saquinavir).On resistant proof carrier storehouse, measure the half-inhibition concentration IC50 of each trial drug.Check the susceptibility model of resistant proof carrier, compare with known drug susceptibility model to each trial drug.Observe the susceptibility model of patient RTV-209 to NNRTI class medicine.In this model, RTV-209 draws the susceptibility moderate of furan pyridine to reduce (75 times) over the ground, and the susceptibility of Viramune is then had substantive reduce (greater than 800 times).

Measure the genotype of patient HIV sample

Use the ABI chain terminator to RTV-209 DNA automatic sequencing.The consensus sequence of gained nucleotide sequence and wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library) is made comparisons.Check that this genotype finds out and contrast different sequences.Compare with control sequence, in carrier RTV-209, find sudden change: A62V, S68G, V76I, F77L, F116Y, E138A, Q151M, M184V, Y188L and E291D as upper/lower positions.At A62V, V75I, F77L, F116, the sudden change on these sites of Q151M and M184V is relevant with NRTI class drug resistance.Proved in the past that the E138K site mutation was associated with several NNRTI class drug resistances, the sudden change of Y188L position is sent out the auspicious susceptibility now of furan with Chinese mugwort and is reduced relevant.Utilize rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay, we have checked sudden change Y188L and E138A, set up the cognation between genotype variation and the phenotype.

(Saker and Sommar, Ibid.), the E138A that will suddenly change imports separately or imports the resistant proof carrier with Y188L to use big primer-round pcr to carry out a rite-directed mutagenesis.Resistant proof carrier E138A-RTV contains sudden change E138A, and resistant proof carrier E138A-Y188L-RTV contains E138A and two sudden changes of Y188L.Use the previous phenotypic assay method of describing, these two resistant proof carriers are carried out resistant proof, test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, is wild-type in 138 sites.We are at resistant proof carrier E138A-RTV, and Y188L-RTV and E138A-Y188L-RTV go up and draw furan pyridine, the auspicious phenotype susceptibility model now of Viramune and Ai Fa furan with measuring NNRTI class medicine.E138A-RTV only contains the E138A sudden change.With respect to wild-type contrast RTV, E138A-RTV draws furan pyridine susceptibility to be in wild-type level (1.6 times) over the ground, and Viramune susceptibility is in wild-type level (1.3 times), Chinese mugwort is sent out the auspicious susceptibility now of furan be in wild-type level (1.4 times); Y188L-RTV draws furan pyridine susceptibility slightly to reduce over the ground, and substantive reduce (greater than 800 times) are taken place Viramune susceptibility, significantly reduces (110 times) and Chinese mugwort is sent out the auspicious susceptibility now of furan; E138A-Y188L-RTV draws the equal moderate of furan pyridine and the auspicious susceptibility now of Ai Fa furan to reduce over the ground, and ground draws the furan pyridine to reduce by 75 times, and Chinese mugwort sends out that furan is auspicious to reduce by 88 times now, and substantive reduce (greater than 800 times) are taken place for the susceptibility of Viramune.With independent sudden change when (referring to the single sudden change of E138A or Y188L) viewed effect compare, combinatorial mutagenesis (refer to E138A and Y188L two sudden change) draws the effect of furan pyridine susceptibility to increase over the ground.

Embodiment 11

Use resistant proof carrier and directed mutants, set up HIV A98 loci gene type and with the relevant phenotype of NNRTI class drug susceptibility resistance between cognation

Preparation resistant proof carrier is also analyzed the phenotype of patient HIV sample 98-675

As described in example 1,98-675 makes up the resistant proof carrier with patient's sample.This patient accepted the treatment of following medicine: ddI in the past, AZT and 3TC (NRTI class medicine); Saquinavir and nelfinavir (PRJ class medicine).Isolated viral RNA carries out reverse transcription-pcr reaction, the one section fragment from the patient that increases (PDS).This fragment has comprised the nucleic acid sequence of proteins encoded enzyme total length and reversed transcriptive enzyme 1-313 amino acids.This of fragment insertion is had in the virus vector of reporter gene, produce a resistant proof carrier, called after RTV-675.Then RTV-675 is carried out phenotype analytical, accurately and quantitatively measure its sensitivity levels one group of antiretroviral drugs.This group medicine comprises usually said NRTI class medicine (AZT, 3TC, d4T, ddI and ddC), and NNRTI class medicine (draw the furan pyridine, Chinese mugwort sends out the now auspicious and Viramune of furan) and PRI class medicine (indinavir, nelfinavir, ritonavir, saquinavir).On resistant proof carrier storehouse, measure the half-inhibition concentration IC50 of each trial drug.Check the susceptibility model of resistant proof carrier, compare with known drug susceptibility model to each trial drug.Observe a kind of susceptibility model of patient RTV-675 to NNRTI class medicine.In this model, observe RTV-675 and draw furan pyridine susceptibility to be in wild-type level (2.1 times) over the ground, the susceptibility of Viramune then there is slight reduction (6 times).

Measure the genotype of patient HIV sample

Utilize ABI chain terminator method to RTV-675 DNA automatic sequencing.The gained nucleotide sequence is made comparisons with the consensus sequence of wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library).Check that this genotype finds out and contrast different sequences; Find sudden change in following site: M41L, S48T, L74V, A98G, M184V, T215Y.These sudden changes are relevant with NRTI class drug resistance.The verified in the past sudden change on the A98G site is relevant with the Viramune resistance.Utilize rite-directed mutagenesis and external phenotype sensitivity test, we check that the A98G sudden change is to set up the cognation between genotype variation and the phenotype.

Utilize the effect of the site-directed mutagenesis technique proof specific sudden change of HIV in antiretroviral drugs susceptibility (phenotype)

(Saker and Sommar, Ibid.), the A98G that will suddenly change imports the resistant proof carrier, produces the resistant proof carrier A 98G-RTV that has this sudden change to utilize big primer-round pcr to carry out a rite-directed mutagenesis.Use the previous phenotypic assay method of describing, this resistant proof carrier is carried out resistant proof.Test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, is wild-type in 98 sites.We draw the furan pyridine with measuring NNTRI class medicine on A98G-RTV, Viramune, and Chinese mugwort sends out the auspicious phenotype susceptibility model now of furan.A98G-RTV is in the wild-type background, has only the A98G sudden change.With respect to wild-type contrast RTV, A98G-RTV draws the susceptibility of furan pyridine slightly to reduce by 3 times over the ground, and Viramune susceptibility has slightly been reduced by 8 times, Chinese mugwort is sent out furan is auspicious slightly to have reduced by 3 times now.

Embodiment 12

Utilize resistant proof carrier and directed mutants, set up the cognation between HIV A98 and G190 loci gene type and the relevant phenotype of NNTRI class drug resistance susceptibility

Preparation resistant proof carrier is also analyzed the phenotype of patient B HIV sample

As described in example 1 above, make up the resistant proof carrier with the patient B sample.Do not know the antiretroviral therapy of the suffered mistake of patient B.Isolated viral RNA carries out reverse transcription-pcr, amplifies one section fragment from the patient (PDS).This fragment comprises the virus sequence of proteins encoded enzyme total length and reversed transcriptive enzyme 1-133 amino acids.One of this section sequence insertion is had in the virus vector of reporter gene the resistant proof carrier of generation, called after RTV-B.From the RTV-B storehouse, filter out single clone.Then single clone is carried out phenotypic assay, accurately measure the sensitivity levels of the single clone of RTV-B quantitatively one group of antiretroviral drugs.This group antiretroviral drugs comprised usually said NRTI class medicine (AZT, 3TC, d4T, ddI, ddC), NNRTI class medicine (draw the furan pyridine, it is now auspicious that Chinese mugwort sends out furan) and PRI class medicine (indinavir, nelfinavir, ritonavir, saquinavir).On the resistant proof carrier cloning, measure the half-inhibition concentration IC50 of each trial drug.Check the susceptibility model of Total Test medicine, compare with known susceptibility model.Observe a kind of susceptibility pattern of 1 pair of NNTRI class medicine of patient RTV-B clone.In this model, clone 1 draws furan pyridine susceptibility to increase by 0.55 times over the ground, and substantive reduce (640 times) are taken place Viramune susceptibility, Chinese mugwort is sent out the auspicious susceptibility now of furan significantly reduce (250 times).

Measure the genotype of patient HIV sample

Use ABI chain terminator technology to RTV-B clone 1DNA automatic sequencing.The gained nucleotide sequence is made comparisons with the consensus sequence of wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library).Check that this genotype finds out the sequence that is different from contrast.Compare with control sequence, find sudden change in following site: M41L, A98G, M184V, L210W, R211, T215Y, E297A, G190S.M41L, M184V, L210W is relevant with the NRTI resistance with T215Y.Verified in the past relevant with the Viramune resistance in the A98G site mutation.Verified in the past relevant with the change of Viramune susceptibility in the G190A site mutation.Other sudden change to 190 promptly sports E by G, the existing report of Q and T.Utilize rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay, we check that sudden change A98G and G190S are to set up the cognation between genotype variation and the phenotype.

Use site-directed mutagenesis technique and confirm the effect of the specific sudden change of HIV in antiretroviral drugs susceptibility (phenotype)

(Saker and Sommar, Ibid.), will suddenly change A98G and G190S import the resistant proof carrier alone or in combination to utilize big primer-round pcr to carry out rite-directed mutagenesis.Resistant proof carrier A 98G-RTV contains the A98G sudden change; Resistant proof carrier G190S-RTV contains the G190S sudden change, and resistant proof carrier A 98G-G190S-RTV contains A98G and the two sudden changes of G190S.Use the previous phenotypic assay method of describing, these three the resistant proof carriers that have sudden change are carried out resistant proof, test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, and its 98 and 190 site all is a wild-type.On these three carriers, we have drawn furan pyridine, Viramune, Chinese mugwort to send out the auspicious susceptibility model now of furan with having measured NNTRI class medicine.A98G-RTV is under the wild-type background, only contains the A98G sudden change.With respect to wild-type contrast RTV, A98G-RTV draws the susceptibility of furan pyridine slightly to reduce by 3 times over the ground, reduces by 8 times to Viramune susceptibility is slight, Chinese mugwort is sent out the auspicious susceptibility now of furan moderate reduced by 3 times.G190S-RTV is under the wild-type background, only contains the G190S sudden change.With respect to wild-type contrast RTV, G190S-RTV draws furan pyridine susceptibility to increase by 0.5 times over the ground, and Viramune susceptibility moderate is reduced (75 times), Chinese mugwort is sent out the auspicious susceptibility now of furan slightly reduced by 8 times.With respect to wild-type contrast RTV, A98G-G190S-RTV draws furan pyridine susceptibility to increase by 0.8 times over the ground, but substantive the reduction all taken place for Viramune and the auspicious susceptibility now of Ai Fa furan, respectively greater than 800 times and 250 times.Though Chinese mugwort sends out the auspicious susceptibility now of furan and has only slight reduction when observing independent sudden change, the reduction that A98G and G190S simultaneous mutation cause the Viramune resistance greater than two separately the sudden change resistances reduce sums.

Embodiment 13

Utilize resistant proof carrier and directed mutants, set up the cognation between HIV Y181 and A98 loci gene type and the relevant phenotype of NNTRI class drug resistance susceptibility

Prepare the resistant proof carrier and patient 98-1057 sample is carried out phenotype analytical

As described in example 1 above, make up the resistant proof carrier with patient 98-1057 sample.This patient accepted following pharmacological agent: ddI in the past, d4T, AZT and 3TC (NRTI class medicine); Saquinavir and indinavir (PRI class medicine); And draw furan pyridine (NNRTI class medicine).Isolated viral RNA carries out reverse transcription-pcr, amplifies one section fragment from the patient (PDS).This fragment has comprised the virus sequence of proteins encoded enzyme total length and reversed transcriptive enzyme 1-313 amino acids.One of this section sequence insertion is had in the virus vector of reporter gene, produce the resistant proof carrier, called after RTV-1057.Then RTV-1057 is carried out phenotypic assay.Accurately and quantitatively measure the sensitivity levels of RTV-1057 to one group of antiretroviral drugs.This group antiretroviral drugs has comprised usually said NRTI class medicine (AZT, 3TC, d4T, ddI, ddC), NNRTI class medicine (draw the furan pyridine, it is now auspicious that Chinese mugwort sends out furan, Viramune) and PRI class medicine (indinavir, nelfinavir, ritonavir, saquinavir).On resistant proof carrier storehouse, measure the half-inhibition concentration IC50 of each trial drug.Check the susceptibility model of Total Test medicine, compare with known susceptibility model.Observe a kind of susceptibility model of patient RTV-1057 to NNTRI class medicine.In this model, RTV-1057 draws furan pyridine susceptibility moderate to reduce (35 times) over the ground, and Viramune susceptibility is significantly reduced (610 times).

Measure the genotype of patient HIV sample

Use ABI chain terminator technology to RTV-1057 DNA automatic sequencing.The consensus sequence of gained nucleotide sequence and wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library) is made comparisons.Check that this genotype finds out the sequence that is different from contrast.Compare with control sequence, find sudden change in following site: T39A, M41L, A62V, D67E, T69SST, A98G, I135T, Y181C, T200I and T215Y.These sudden changes are relevant with NNRTI class drug resistance.Known between HIV wild-type (to medicaments insensitive) variant, I135T and T200I sudden change have sequence polymorphism.Verified in the past relevant with the A98G site mutation with specific NNRTI class drug resistance at Y181C.Utilize rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay, we have checked that sudden change Y181C and A98G are to set up the cognation between genotype variation and the phenotype.

(Saker and Sommar, Ibid.), will suddenly change Y181C and A98G import the resistant proof carrier individually or simultaneously to utilize big primer-round pcr to carry out a rite-directed mutagenesis.Resistant proof carrier Y181C-RTV contains the Y181C sudden change; Resistant proof carrier A 98G-RTV contains the A98G sudden change, and resistant proof carrier Y181C-A98G-RTV contains A98G and the two sudden changes of G190S.Use the previous phenotypic assay method of describing, these three the resistant proof carriers that have sudden change are carried out resistant proof, test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, and its 181 and 98 site all is a wild-type.On these three carriers, we have drawn furan pyridine, Viramune, Chinese mugwort to send out the auspicious susceptibility model now of furan with having measured NNTRI class medicine.Y181C-RTV only contains the Y181C sudden change, is under the wild-type background.With respect to wild-type contrast RTV, Y181C-RTV draws the susceptibility moderate of furan pyridine to reduce (35 times) over the ground, and Viramune susceptibility is significantly reduced (161 times), Chinese mugwort is sent out the auspicious susceptibility now of furan slightly reduce (3 times).With respect to wild-type contrast RTV, A98G-RTV draws furan pyridine susceptibility slightly to reduce (3 times) over the ground, and Viramune susceptibility is slightly reduced (8 times), Chinese mugwort is sent out the auspicious susceptibility now of furan slightly reduce (3 times).With respect to contrast wild-type RTV, Y181C-A98G-RTV draws furan pyridine susceptibility significantly to reduce (240 times) over the ground, and substantive reduce (greater than 800 times) are taken place Viramune susceptibility, Chinese mugwort is sent out the auspicious susceptibility now of furan slightly reduce (7 times).These data declarations are compared with Y181C and the independent sudden change of A98G, and both simultaneous mutations cause the effect that Viramune susceptibility reduces, and suddenling change separately greater than two causes resistance reduction sum.

Embodiment 14

Utilize resistant proof carrier and directed mutants, set up the cognation between HIV K101 and G190 loci gene type and the relevant phenotype of NNTRI class drug resistance susceptibility

Prepare the resistant proof carrier and patient 98-644 and 98-1060 HIV sample are carried out phenotype analytical

Described in embodiment 1, make up resistant proof carrier, called after RTV-644 with patient 98-644 HIV sample.This patient accepted following pharmacological agent: d4T (NRTI class medicine) in the past, indinavir (PRI class medicine), and Chinese mugwort sends out furan auspicious now (NNRTI class medicine).Described in embodiment 1, make up another resistant proof carrier, called after RTV-1060 with patient 98-1060 HIV sample.This patient accepted following pharmacological agent: d4T (NRTI class medicine) in the past, indinavir (PRJ class medicine), and Chinese mugwort sends out furan auspicious now (NNRTI class medicine).Isolated viral RNA carries out reverse transcription-pcr, amplifies one section fragment from the patient (PDS).This fragment has comprised the virus sequence of proteins encoded enzyme total length and reversed transcriptive enzyme 1-313 amino acids.One of this section sequence insertion is had in the virus vector of reporter gene the resistant proof carrier of generation, called after RTV-644 and RTV-1060 respectively.Then RTV-644 and RTV-1060 are carried out phenotypic assay, accurately and quantitatively measure their sensitivity levels one group of antiretroviral drugs.This group antiretroviral drugs comprised usually said NRTI class medicine (AZT, 3TC, d4T, ddI, ddC), NNRTI class medicine (draw furan pyridine, Viramune) and PRI class medicine (indinavir, nelfinavir, ritonavir, saquinavir).On resistant proof carrier storehouse, measure the half-inhibition concentration IC50 of each trial drug.Check the susceptibility model of Total Test medicine, compare with known susceptibility model.Observe a kind of susceptibility model of patient RTV-644 to NNTRI class medicine.In this model, RTV-644 draws furan pyridine susceptibility to reduce very slight (2.5 times) over the ground, and Viramune susceptibility is significantly reduced (600 times).Observe the susceptibility model of patient RTV-1060 to NNTRJ class medicine.In this model, RTV-1060 draws furan pyridine susceptibility to show as wild-type level (1.5 times) over the ground, Chinese mugwort is sent out the auspicious susceptibility now of furan significantly reduce (900 times), and substantive reduce (greater than 800 times) are taken place Viramune susceptibility.

Measure the genotype of patient HIV sample

Use ABI chain terminator technology to RTV-1060 DNA automatic sequencing.The consensus sequence of gained nucleotide sequence and wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library) is made comparisons.Check that this genotype finds out and contrast different sequences.Compare with control sequence, sudden change is found in K101E and G190S site in RTV-644.Compare with control sequence, sudden change is found in following site in RTV-1060: K101E, G190S, T200A and T215Y.Sequence in the T215 site is that wild-type and mutant are mixed (existence).Verified in the past relevant with several NNTRI drug resistances in the K101E site mutation, comprise the high-level resistance of drawing the furan pyridine over the ground.Verified in the past relevant with the change of Viramune susceptibility in the G190A site mutation.Other sudden change in the G190 site promptly sports E, Q and T by G, and report is also arranged.Utilize rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay, we have checked sudden change K101E and G190S, set up the cognation between genotype variation and the phenotype.

(Saker and Sommar, Ibid.), will suddenly change K101E and G190S import the resistant proof carrier individually or simultaneously to utilize big primer-round pcr to carry out a rite-directed mutagenesis.Resistant proof carrier K101E-RTV contains the K101E sudden change; Resistant proof carrier G190S-RTV contains the G190S sudden change.Utilize previous described phenotypic assay method, these two the resistant proof carriers that have sudden change are carried out resistant proof, test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, and its 101 and 190 site all is a wild-type.On these two carriers, we have drawn furan pyridine, Viramune, Chinese mugwort to send out the auspicious susceptibility model now of furan with having measured NNTRI class medicine.K101E-RTV only contains the K101E sudden change, is under the wild-type background.With respect to wild-type contrast RTV, K101E-RTV draws the susceptibility of furan pyridine slightly to reduce (5 times) over the ground, Chinese mugwort is sent out the auspicious susceptibility now of furan slightly reduce (5 times), and Viramune susceptibility moderate is reduced (12 times).With respect to wild-type contrast RTV, G190S-RTV draws furan pyridine susceptibility to increase (0.5 times) over the ground, and Viramune susceptibility moderate is reduced (75 times), Chinese mugwort is sent out the auspicious susceptibility now of furan slightly reduce (7.6 times).With respect to contrast wild-type RTV, K101E-G190S-RTV draws furan pyridine susceptibility to be in wild-type level (1.4 times) over the ground, and substantive reduce (greater than 800 times) are taken place Viramune susceptibility, Chinese mugwort is sent out the auspicious susceptibility now of furan substantive reduce (250 times) take place.

In this embodiment, new susceptibility model has appearred in G190S and K101E simultaneous mutation.Observe G190S and K101E simultaneous mutation meeting and reverse the effect that furan pyridine susceptibility is drawn in the independent sudden change of G190S over the ground, the two simultaneous mutation to the influence of Viramune and the auspicious susceptibility of Ai Fa furan greater than its sudden change sum separately.

Embodiment 15

Utilize resistant proof carrier and directed mutants, set up the cognation between HIV V108I loci gene type and the relevant phenotype of NNTRI class drug susceptibility resistance

Prepare the resistant proof carrier and patient 98-652 HIV sample is carried out phenotype analytical

Described in embodiment 1, make up resistant proof carrier, called after RTV-652 with patient 98-652HIV sample.This patient did not accept antiretroviral therapy in the past.Isolated viral RNA carries out reverse transcription-pcr, amplifies one section fragment from the patient (PDS).This fragment has comprised the virus sequence of proteins encoded enzyme total length and reversed transcriptive enzyme 1-313 amino acids.One of this section sequence insertion is had in the virus vector of reporter gene, produce a resistant proof carrier, called after RTV-652.Then RTV-652 is carried out phenotypic assay, accurately and quantitatively measure their sensitivity levels one group of antiretroviral drugs.This group antiretroviral drugs comprised usually said NRTI class medicine (AZT, 3TC, d4T, ddI, ddC), NNRTI class medicine (draw furan pyridine, Viramune) and PRI class medicine (indinavir, nelfinavir, ritonavir, saquinavir).On resistant proof carrier storehouse, measure the half-inhibition concentration IC50 of each trial drug.Check the susceptibility model of Total Test medicine, compare with known susceptibility model.Observe a kind of susceptibility model of patient RTV-652 to NNTRI class medicine.In this model, RTV-652 draws furan pyridine susceptibility to increase (0.97 times) over the ground, and Viramune susceptibility is slightly reduced (5 times).

Measure the genotype of patient HIV sample

Use ABI chain terminator technology to RTV-652 DNA automatic sequencing.The consensus sequence of gained nucleotide sequence and wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library) is made comparisons.Check that this genotype finds out and contrast different sequences.Compare with control sequence, sudden change is found in K101E and G190S site in RTV-644.Compare with control sequence, find sudden change in following site: M41L, V108I, I135T, L210W and T215D.M41L, L210W is relevant with NRTI class drug susceptibility and resistance with the T215D site.Known between HIV wild-type (medicaments insensitive) variant, 1135T and R211K site mutation have sequence polymorphism.Known V108I is relevant with several NNRTI class drug resistances.Utilize rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay, we have checked that sudden change V108I is to set up the cognation between genotype variation and the phenotype.

Use site-directed mutagenesis technique confirms that the specific sudden change of HIV is had in antiretroviral drugs susceptibility (phenotype) effect

(Saker and Sommar, Ibid.), the V108I that will suddenly change imports the resistant proof carrier to utilize big primer-round pcr to carry out a rite-directed mutagenesis.Resistant proof carrier V108I-RTV contains the V108I sudden change.Utilize previous described phenotypic assay method, antagonism test carrier V108I-RTV carries out resistant proof, and test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, and its 108 site is a wild-type.On carrier V108I-RTV, we have drawn furan pyridine, Viramune, Chinese mugwort to send out the auspicious susceptibility model now of furan with having measured NNTRI class medicine.Resistant proof carrier V108I-RTV is under the wild-type background, only contains the V108I sudden change.With respect to wild-type contrast RTV, V108I-RTV draws the furan pyridine to show wild-type susceptibility (1.3 times) over the ground, and Chinese mugwort is sent out the auspicious wild-type susceptibility (1.7 times) that shows now of furan, and Viramune susceptibility is slightly reduced (3 times).

Embodiment 16

Utilize resistant proof carrier and directed mutants to set up cognation between HIV K103 K101 and G190 loci gene type and the relevant phenotype of NNRTI class drug susceptibility resistance

Prepare the resistant proof carrier and patient 98-955HIV sample is carried out phenotype analytical

Described in example 1,98-955 has made up the resistant proof carrier with patient's sample.This patient accepted nelfinavir (PRI class medicine) treatment in the past.Isolated viral RNA carries out reverse transcription-pcr, amplifies one section fragment from the patient (PDS).This fragment has comprised the viral nucleic acid of proteins encoded enzyme total length and reversed transcriptive enzyme 1-313 amino acids.One of this section PDS sequence insertion has in the virus vector of reporter gene, produces a resistant proof carrier, called after RTV-955.The sensitivity levels of RTV-955 to one group of antiretroviral drugs accurately and quantitatively measured in the property shown analysis then.This group antiretroviral drugs has comprised usually said NRTI class medicine (AZT, 3TC, d4T, ddI and ddC), NNRTI class medicine (draw furan pyridine, Chinese mugwort to send out auspicious now, the saquinavir of furan), PRI class medicine (indinavir, nelfinavir, ritonavir, saquinavir).On the resistant proof carrier, measure the half-inhibition concentration IC50 of each trial drug.Checking R TV-955 is to the susceptibility model of all trial drugs, and with known susceptibility model relatively.Observe a kind of susceptibility model of (patient) RTV-955, draw furan pyridine susceptibility slightly to reduce (4 times) and Viramune susceptibility significantly reduces (530 times) with finding NNRTI class medicine.

Measure the genotype of patient HIV sample

With ABI chain terminator technology to RTV-955 DNA automatic sequencing.The consensus sequence of gained nucleotide sequence and wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library) relatively.With control sequence relatively, check that this genotype finds out the sequence that is different from contrast.Suddenly change in following discovery: K20R, V35I, A62V, D67N, T69D, V75I, F77L, K101E, K103N, Y115F, F116Y, Q151M, I167V, Y181C, M184V, G190A, I202V, R211K, F214L, T215V and K219Q.At K101E, K103E, Y181C, G190A and F214L site mutation are that mutant and wild-type are mixed (existence).A26V, D67N, T69D, V75I, F77L, Y115F, F116Y, Q151M, M184V, T215V is relevant with the NRTI resistance with K219Q.Known between different HIV wild-types (medicaments insensitive) variant, at V35I, R211K and F214L site mutation have sequence polymorphism.Proved in the past that the K101E sudden change was relevant with NNRTI class drug resistance.Proved in the past that the Y181I site mutation was relevant with Viramune high level resistance.Verified in the past relevant with the resistance of drawing auspicious these three the NNRTI class medicines now of furan pyridine, Viramune and Ai Fa furan over the ground in the K103N site mutation.Utilize rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay, we have checked K101E, and the cognation between genotype variation and the phenotype is set up in J103N and G190A sudden change.

Use the effect of the site-directed mutagenesis technique proof specific sudden change of HIV in antiretroviral drugs susceptibility (phenotype)

Use big primer-round pcr to carry out a rite-directed mutagenesis, the K101E that will suddenly change, K103N and G190A import alone or in combination the resistant proof carrier (Saker and Sommar, Ibid.).Resistant proof carrier K101E-RTV contains the K101E sudden change; Resistant proof carrier K0103E-RTV contains the K103N sudden change; Resistant proof carrier G190A-RTV contains the G190A sudden change; Resistant proof carrier K101E-G190A-RTV and resistant proof carrier K103N-G190A-RTV each two sudden change respectively.Use the previous phenotype analytical method of describing, these the five kinds resistant proof carriers that contain sudden change are carried out resistant proof.Test-results and another resistant proof carrier are compared.This carrier genetic background is clear and definite, and 101,103,190 sites are wild-type.We on whole 5 carriers, measure its to NNTRI class medicine draw furan pyridine, Viramune and the auspicious phenotype now of Ai Fa furan susceptibility model.K101E-RTV is under the wild-type background, only contains the K101E sudden change.With respect to wild-type contrast RTV, K101E-RTV draws the susceptibility of furan pyridine slightly to reduce (5 times) over the ground, to the susceptibility moderate reduction (55 times) of Viramune, Chinese mugwort is sent out the auspicious susceptibility now of furan moderate reduce (30 times).G190A-RTV is in the wild-type background, only contains the G190A sudden change.With respect to wild-type contrast RTV, G190A-RTV sends out the auspicious susceptibility now of furan to Chinese mugwort increases by 8 times.With respect to wild-type contrast RTV, K101E-G190A-RTV draws the furan pyridine to show wild-type susceptibility (2 times) over the ground, Viramune susceptibility is had reduce (greater than 800) in fact, Chinese mugwort is sent out the auspicious susceptibility now of furan significantly reduce (120 times).In the resistant proof carrier that has the G190A single mutation, import second sudden change, draw furan pyridine susceptibility to take a turn for the worse with causing.The G190A mutant draws furan pyridine susceptibility to increase over the ground.But any one sudden change among G190A imports among K101E or the K103N both just causes the slight decline of drawing furan pyridine susceptibility over the ground.Secondly, G190A and K101E simultaneous mutation cause that Viramune and the auspicious susceptibility now of Ai Fa furan descend, and the degree of decline is greater than two kinds of (observed) effect sums of suddenling change separately.At last, G190A and K103N simultaneous mutation cause Viramune and the auspicious susceptibility now of Ai Fa furan are all reduced, and the reduction degree is greater than two kinds of (observed) effect sums of suddenling change separately.

Embodiment 17

Utilize resistant proof carrier and directed mutants, set up the cognation between HIV V106 V189 V181 and F227 loci gene type and the relevant phenotype of NNTRI class drug susceptibility resistance

Prepare the resistant proof carrier and patient 98-1033 and 98-757 HIV sample are carried out phenotype analytical

Described in example 1,98-1033 makes up the resistant proof carrier with patient's sample.This patient had before accepted the treatment of following medicine: AZT, d4T, 3TC and ddI (NRTI), saquinavir, indinavir and nelfinavir (PRIs), Viramune (NNTRI).Gather this 98-757 of another increment of this patient in another time.Described in example 1, made up another resistant proof carrier with sample 98-757.During 8 all additional treatment, this patient has accepted Viramune (NNTRI), d4T (NRTI), the treatment of these four kinds of medicines of saquinavir and nelfinavir (PRJ class medicine).Isolated viral RNA carries out reverse transcription-pcr, amplifies one section fragment from the patient (PDS).The virus sequence of this fragment coding proteolytic enzyme total length and reversed transcriptive enzyme 1-313 amino acids.One of this section sequence insertion is had in the virus vector of reporter gene, produce two resistant proof carriers, be designated as RTV-1033 respectively, RTV-757.Carry out phenotype analytical then, accurately and quantitatively measure RTV-1033 and RTV-757 sensitivity levels one group of antiretroviral drugs.This group antiretroviral drugs has comprised usually said NRTI class medicine (AZT, 3TC, d4T, ddI and ddC), NNRTI class medicine (draw furan pyridine and Viramune) and PRI class medicine (indinavir, nelfinavir, ritonavir and saquinavir).On resistant proof carrier storehouse, measure the half-inhibition concentration IC50 of each trial drug.Check (resistant proof carrier) susceptibility model, and compare with known susceptibility model to the Total Test medicine.Observe a kind of susceptibility model of patient's resistant proof carrier RTV-1033 to NNTRI class medicine.In this model, ground draws furan pyridine susceptibility moderate to reduce (30 times), and substantive reduce (greater than 800 times) take place Viramune susceptibility, and Chinese mugwort sends out the auspicious susceptibility now of furan and significantly reduces (200 times).Observe the susceptibility model of patient's resistant proof carrier RTV-757 to NNTRI class medicine.In this model, ground draws furan pyridine susceptibility slightly to reduce (10 times), and Viramune susceptibility has substantive reduce (greater than 800 times).

Measure the genotype of patient HIV sample

Use ABI chain terminator technology to RTV-1033 and RTV-757 DNA automatic sequencing.The consensus sequence of gained nucleotide sequence and wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library) is made comparisons.Check that this genotype finds out and contrast different sequences.In carrier RTV-1033, find sudden change: V35I, D67N, T69D, K70R, V106A, V189L, T200A, I202T, R211K, T215F, D218E, K219Q, H221Y, F227L, L228H and R284K as upper/lower positions.Compare with control sequence, in carrier RTV-757, find sudden change: V35I, D67N, T69D, K70R, V106A, V108I as upper/lower positions, L109V, Y180C, V189L, T200A, I202T, R211K, T215F, D218E, K219Q, H221Y, L228H, L283I and R284K.At V106A, the sequence in V108I and L109V site is that wild-type is mixed (existence) with mutant.D67N, T69D, K70R, T215F is relevant with the NTRI resistance with K219Q.Known between different HIV wild-types (medicaments insensitive) variant, V35I, T200A, these site mutation of R211K and R284K have sequence polymorphism.It is relevant to have proved in the past that V106A sudden change and Viramune resistance increased, and the sudden change of V189I position is relevant with the NNRTI resistance.V189I site amino acid mutation is L, and is relevant with the NNTRI resistance, and this point was not reported in the past.Proved in the past the V108I site mutation with to draw furan pyridine and Viramune resistance to increase relevant.Utilize rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay to check V106A, V189L, the cognation between genotype variation and the phenotype is set up in V181C and F227L sudden change.

Use big primer-round pcr to carry out a rite-directed mutagenesis, the V106A that will suddenly change, V189L, V181C, F227L import separately or make up import the resistant proof carrier (Saker and Sommar, Ibid.).Resistant proof carrier V106A-RTV contains the V106A sudden change; Resistant proof carrier V189L-RTV contains the V189L sudden change; Resistant proof carrier Y181C-RTV contains the Y181C sudden change; Resistant proof carrier F227L-RTV contains the F227L sudden change; Resistant proof carrier V106A-Y181C-RTV, V106A-V189L-RTV, each two kinds of sudden change of V106A-F227L-RTV and V181C-F227L-RTV.Resistant mutation carrier V106A-Y181C-F227L-RTV contains three kinds of sudden changes.Use the previous phenotype analytical method of describing, these nine the resistant proof carriers that contain sudden change are carried out resistant proof, test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, and 106,189,181,227 sites are wild-type.We have drawn the auspicious phenotype susceptibility model now of furan pyridine, Viramune and Ai Fa furan with having measured NNTRI class medicine on whole 9 carriers.The V106A-RTV background is a wild-type, only contains the V106A sudden change.With respect to wild-type contrast RTV, V106A-RTV draws the susceptibility of furan pyridine slightly to reduce (5 times) over the ground, to the susceptibility moderate reduction (60 times) of Viramune, Chinese mugwort is sent out the auspicious wild-type sensitivity levels (1.7 times) that shows now of furan.The V189L-RTV background is a wild-type, and it only contains the V189L sudden change.With respect to wild-type RTV (contrast), V189L-RTV draws the auspicious susceptibility now of furan pyridine, Viramune and Ai Fa furan all in the wild-type level over the ground, is respectively 1.8 times, and 1.3 times, 1.3 times.The Y181C-RTV background is a wild-type, only contains the Y181C sudden change.With respect to contrast wild-type RTV, Y181C-RTV draws furan pyridine susceptibility significantly to reduce (100 times) over the ground, and substantive reduce (greater than 800 times) are taken place Viramune susceptibility, and Chinese mugwort is sent out the auspicious susceptibility now of furan slightly descend (4 times).The F227L-RTV background is a wild-type, only contains the F227L sudden change.With respect to contrast wild-type RTV, F227L-RTV draws furan pyridine (0.03 times) and Chinese mugwort to send out furan auspicious now (0.48 times) susceptibility over the ground slightly to be increased, and Viramune susceptibility is slightly descended (3 times), to a Chinese mugwort auspicious susceptibility now of furan slightly descend (4 times).With respect to contrast wild-type RTV, V106A-Y181C-RTV draws furan pyridine susceptibility significantly to reduce (100 times) over the ground, and substantive reduce (800 times) are taken place Viramune susceptibility, and Chinese mugwort is sent out the auspicious susceptibility now of furan slightly descend (4 times).With respect to contrast wild-type RTV, V106A-V189L-RTV draws furan pyridine susceptibility slightly to descend (3 times) over the ground, and Viramune susceptibility moderate is reduced (50 times), and Chinese mugwort is sent out the auspicious wild-type susceptibility (1 times) that shows as now of furan.With respect to contrast wild-type RTV, V106A-F227L-RTV draws furan pyridine susceptibility slightly to descend (3 times) over the ground, and Viramune susceptibility is had substantive reduce (greater than 800 times), and Chinese mugwort is sent out the auspicious susceptibility now of furan slightly descend (8 times).With respect to contrast wild-type RTV, Y181C-F227L-RTV draws furan pyridine (0.89 times) and Chinese mugwort to send out furan auspicious now (0.99 times) susceptibility over the ground to be increased, and Viramune susceptibility is significantly reduced (285 times).With respect to contrast wild-type RTV, V106A-Y181C-F227L-RTV draws furan pyridine susceptibility moderate to descend (50 times) over the ground, to substantive reduce (greater than 800 times) of Viramune, Chinese mugwort is sent out the auspicious susceptibility now of furan slightly descend (12 times).

Embodiment 18

Utilize resistant proof carrier and directed mutants, set up the cognation between HIV Y188, L100 and K103 loci gene type and the relevant phenotype of NNTRI class drug susceptibility resistance

Preparation resistant proof carrier is also analyzed the phenotype of patient HIV sample 98-1058

Described in example 1,98-1058 makes up the resistant proof carrier with patient's sample.This patient had before accepted ddI, d4T, AZT, 3TC, ddC and abacavir (NRTI class medicine), indinavir and amprenavir (PRI class medicine).Isolated viral RNA carries out reverse transcription-pcr, amplification one section fragment from the patient in source (PDS).The virus sequence of this fragment coding proteolytic enzyme total length and reversed transcriptive enzyme 1-313 amino acids.One of this section sequence insertion is had in the virus vector of reporter gene, produce the resistant proof carrier, called after RTV-1058.Select each mono-clonal of RTV-1058, carry out phenotype analytical then, accurately and quantitatively measure the sensitivity levels of RTV-1058 one group of antiretroviral drugs.This group antiretroviral drugs has comprised usually said NRTI class medicine (AZT, 3TC, d4T, ddI and ddC), NNRTI class medicine (draw furan pyridine and Viramune) and PRI class medicine (indinavir, nelfinavir, ritonavir and saquinavir).On resistant proof carrier storehouse, measure the half-inhibition concentration IC50 of each trial drug.Check the susceptibility model of resistant proof carrier, and compare with known susceptibility model to the Total Test medicine.Observation is from three clones' (clone 4,5,10) of patient RTV-1033 susceptibility model.Clone 4 draws furan pyridine susceptibility that remarkable reduction (85 times) is arranged over the ground, to substantive reduce (greater than 800 times) of the susceptibility generation of Viramune.Clone 5 draws the susceptibility of furan pyridine that substantive reduce (250 times) take place over the ground, and Viramune susceptibility is significantly reduced (120 times).Clone 10 draws the susceptibility of furan pyridine that substantive reduce (greater than 250 times) take place over the ground, and substantive reduce (greater than 800 times) are also taken place Viramune susceptibility.

Measure the genotype of patient HIV sample

Use the ABI chain terminator to RTV-1058 DNA automatic sequencing.The consensus sequence of gained nucleotide sequence and wild-type clade B HIV-1 (New Mexico, Los Alamos, HIV sequence library) is made comparisons.Check that this genotype finds out and contrast different sequences.In RTV-1033, find sudden change: M41L, A62V, D67N, T69SST, L74V, L100I, K103N, V181I, I135T, T200S, L210W, R211K and T215Y as upper/lower positions.L74V and L100I site are that wild-type and mutant sequence are mixed existence.Clone 4 contains K103N and Y188L sudden change.Clone 5 contains L100I and K103N site mutation.Clone 10 contains L100I, K103N and Y188L sudden change.M41L, A62V, D67N, T69SST, L74V, L210W is relevant with the NTRI resistance with T215Y.Known between different HIV wild-types (medicaments insensitive) variant, I135T, T200S and R211T site mutation have sequence polymorphism.Proved in the past the L100I site mutation with draw the furan pyridine relevant with the Viramune resistance, the K103N site mutation with draw the auspicious resistance now of furan pyridine, Viramune and Ai Fa furan relevant.Utilize rite-directed mutagenesis and extracorporeal sensitivity phenotypic assay, we have checked sudden change Y188L, and L100I and K103N set up the cognation between genotype variation and the phenotype.

(Saker and Sommar, Ibid.), the Y188L that will suddenly change, L100I and K103N import separately or combination imports the resistant proof carrier to use big primer-round pcr to carry out a rite-directed mutagenesis.Resistant proof carrier Y188L-RTV contains the Y188L sudden change; Resistant proof carrier L100I-RTV contains the L100I sudden change; Resistant proof carrier K103N-RTV contains the K103N sudden change; Resistant proof carrier K103N-Y188LRTV, L100I-K103N-RTV respectively contain two kinds of sudden changes.Resistant mutation carrier L100I-K103N-Y188L-RTV contains three kinds of sudden changes.Use the previous phenotype analytical method of describing, these 6 the resistant proof carriers that contain sudden change are carried out resistant proof, test-results and another resistant proof carrier are relatively.This carrier genetic background is clear and definite, and 188,100 and 103 all is wild-type.We have drawn the auspicious phenotype susceptibility model now of furan pyridine, Viramune and Ai Fa furan with having measured NNTRI class medicine on whole 6 carriers.Y188L-RTV only contains the Y188L sudden change.With respect to wild-type contrast RTV, Y188L-RTV draws the susceptibility of furan pyridine slightly to reduce (9 times) over the ground, and substantive reduce (greater than 800 times) are taken place Viramune susceptibility, Chinese mugwort is sent out the auspicious susceptibility now of furan moderate reduce (110 times).L100I-RTV only contains the L100I sudden change.With respect to wild-type RTV (contrast), L100I-RTV draws furan pyridine susceptibility moderate to reduce (30 times) over the ground, Chinese mugwort is sent out the auspicious susceptibility now of furan slightly reduce (10 times), and Viramune susceptibility is slightly reduced (3 times).K103N only contains the K103N sudden change.With respect to contrast wild-type RTV, K103N-RTV draws furan pyridine, Viramune and the auspicious susceptibility now of Ai Fa furan moderate to reduce over the ground, and ground draws the furan pyridine to reduce by 50 times, and Viramune reduces by 55 times, and Chinese mugwort sends out that furan is auspicious to reduce by 30 times now.With respect to contrast wild-type RTV, K103N-Y188L-RTV draws the auspicious susceptibility now of furan pyridine, Viramune and Ai Fa furan that substantive the reduction taken place over the ground, and ground draws the furan pyridine to reduce greater than 250 times, and Viramune reduces greater than 800 times, and furan is auspicious reduces now greater than 250 times for Chinese mugwort.With respect to contrast wild-type RTV, draw furan pyridine and Viramune that substantive the reduction taken place L100I-K103N-RTV, ground draws the furan pyridine to reduce greater than 250 times, and Chinese mugwort sends out that furan is auspicious to be reduced now greater than 250 times, to the susceptibility moderate reduction (70 times) of Viramune.With respect to contrast wild-type RTV, L100I-K103N-Y188L-RTV draws the furan pyridine over the ground, and the auspicious susceptibility now of Viramune and Ai Fa furan takes place by substantive the reduction, and ground draws furan pyridine susceptibility to reduce greater than 250 times, Viramune reduces greater than 800 times, and Chinese mugwort sends out that furan is auspicious to be reduced now greater than 250 times.Sudden change is made up by new mode, has produced unexpected susceptibility model.These new models are different with the model that single sudden change is had.

Claims

1. method of assessing HIV infected patient non-nucleoside reverse transcriptase antiretroviral therapy validity comprises:

A collects the infected patient plasma sample;

B detects the nucleic acid that whether contains the coding hiv reverse transcriptase of undergoing mutation at 236 bit codons in patient's plasma sample;

Wherein, the existence of this sudden change with draw that furan pyridine susceptibility reduces, Viramune susceptibility changes very little or do not change relevant.

2. the process of claim 1 wherein 236 sudden change codon coding leucines.

3. the process of claim 1 wherein that there is extra single mutation in reversed transcriptive enzyme at 103 bit codons or 181 bit codons, or the two simultaneous mutation.

4. the method for claim 3, wherein, 103 sudden change codons coding l-asparagines (N), 181 sudden change codon encoding aminothiopropionic acids (C).

5. the process of claim 1 wherein that the HIV infected patient is just being accepted the treatment of antiretroviral drugs.

6. method of assessing HIV infected patient antiretroviral therapy validity comprises:

A collects the biological specimen of HIV infected patient;

The nucleotide sequence that whether contains the coding hiv reverse transcriptase of undergoing mutation in b detection patient's the biological specimen at 225 bit codons;

7. the method for claim 6, wherein, 225 sudden change codon encoding histidines.

8. the method for claim 6, wherein, the HIV infected patient is just being accepted the treatment of antiretroviral drugs.

9. the method for claim 6, wherein, there is extra single mutation in this reversed transcriptive enzyme at 103 bit codons or 181 bit codons, or the two simultaneous mutation.

10. method of assessing HIV infected patient antiretroviral therapy validity comprises:

A collects the biological specimen of HIV infected patient;

The nucleic acid that whether contains the coding hiv reverse transcriptase of undergoing mutation in b detection patient's the biological specimen at 190 bit codons;

Wherein, the existence of this sudden change with draw the reduction of the raising of furan pyridine susceptibility and Viramune susceptibility relevant.

11. the method for claim 10, wherein, 190 sudden change codons coding L-Ala or Serine.

12. a method of assessing candidate's antiretroviral drugs compound biological effectiveness comprises:

A imports one to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this patient fragment of originating further comprises 236 and 103 and/or 181 bit codon mutations;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

D comparison step (c) the two reporter gene expression of surveying when not adding antiretroviral candidate medicine implementation step (a)-(c);

Wherein, at step (a)-(c); Step (b)-(c); Or the antiretroviral candidate medicine that has experimental concentration in the step (c).

13. the method for the anti-candidate's antiretroviral drugs compound biological effectiveness of assessment comprises:

A imports one to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment further comprises 225 and 103 bit codon mutations;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

14. a method of assessing the biological effectiveness of candidate HIV antiretroviral drugs compound comprises:

A imports one to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment coding has the reversed transcriptive enzyme of sudden change at 236 bit codons:

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

15. a method of assessing the anti-HIV retrovirus of candidate medical compounds biological effectiveness comprises:

A imports one to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment coding has the reversed transcriptive enzyme of sudden change at 225 bit codons;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

16. a method of assessing candidate HIV antiretroviral drugs compound biological effectiveness comprises:

A imports one to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment coding has the reversed transcriptive enzyme of sudden change at 190 bit codons;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

17. one kind has the originate resistant proof carrier of a fragment and a reporter gene of a HIV patient, this fragment coding has the reversed transcriptive enzyme of sudden change at 190 bit codons, and wherein, this report expression of gene depends on patient's fragment of originating.

18. one kind has the originate resistant proof carrier of a fragment and a reporter gene of a HIV patient, this fragment coding has the reversed transcriptive enzyme of sudden change at 225 bit codons, and wherein, this report expression of gene depends on patient's fragment of originating.

19. one kind has the originate resistant proof carrier of a fragment and a reporter gene of a HIV patient, this fragment coding has the reversed transcriptive enzyme of sudden change at 236 bit codons, and wherein, this report expression of gene depends on patient's fragment of originating.

20. the resistant proof carrier of claim 17, wherein, the fragment that includes the patient source of 190 bit codon mutations further comprises 103 bit codon mutations.

21. the resistant proof carrier of claim 18, wherein, the fragment that includes the patient source of 225 bit codon mutations further comprises 103 bit codon mutations.

22. the resistant proof carrier of claim 19, wherein, the fragment that includes the patient source of 236 bit codon mutations further comprises 103 and/or 181 bit codon mutations.

23. a method of assessing HIV infected patient non-nucleoside reverse transcriptase antiretroviral therapy validity comprises:

A collects HIV infected patient plasma sample.

B detects whether contain the hiv reverse transcriptase that coding 230 bit codons are undergone mutation in patient's plasma sample;

Wherein, the existence of sudden change with draw the drug susceptibility of furan pyridine and Viramune to reduce relevant.

24. the method for claim 23, wherein, 230 sudden change codon coding leucines (L).

25. the method for claim 23, wherein, there is additional mutations in reversed transcriptive enzyme at 181 bit codons, or simultaneous mutation.

26. the method for claim 25, wherein, 281 sudden change codon encoding aminothiopropionic acids (C).

27. the method for claim 23, wherein, the HIV infected patient is just being accepted the treatment of antiretroviral drugs.

28. an assessment is carried out the method for the validity of non-nucleoside reverse transcriptase antiretroviral therapy to the HIV infected patient, comprising:

A collects HIV infected patient plasma sample;

B detects whether to contain the nucleic acid that is coded in the hiv reverse transcriptase that 181 bit codons undergo mutation in patient's plasma sample;

Wherein, the existence of this sudden change with draw the drug susceptibility of furan pyridine and Viramune to reduce and Chinese mugwort sends out the auspicious susceptibility now of furan and changes very little or do not change relevant.

29. the method for claim 28, wherein, 181 sudden change codon encoding aminothiopropionic acids (C).

30. the method for claim 28, wherein, there is extra single mutation in reversed transcriptive enzyme at 98,106 or 227 bit codons, or the combination of these single mutation.

31. the method for claim 30, wherein, 98 sudden change codon coding glycine (G), 106 sudden change codon coding L-Ala (A), 227 sudden change codon coding leucines (L).

32. the method for claim 28, wherein, the HIV infected patient is just being accepted the treatment of antiretroviral drugs.

33. a method of assessing the validity of HIV infected patient non-nucleoside reverse transcriptase antiretroviral therapy comprises:

A collects the plasma sample of HIV infected patient.

B detects whether to contain the nucleic acid that is coded in the hiv reverse transcriptase that 188 bit codons undergo mutation in patient's the plasma sample;

Wherein, the existence of sudden change reduces relevant with drawing furan pyridine, Viramune and Ai Faluoruizi drug susceptibility over the ground.

34. the method for claim 33, wherein, 188 sudden change codon codings leucine (L), halfcystine (C) or Histidines (H).

35. the method for claim 33, wherein, there is single mutation in reversed transcriptive enzyme at 138,103 or 100 bit codons, or the combination of these sudden changes.

36. the method for claim 35, wherein, 138 sudden change codon coding L-Ala (A), 103 sudden change codon coding l-asparagines (N), 100 sudden change codon coding Isoleucines (I).

37. the method for claim 33, wherein, the HIV infected patient is just being accepted the treatment of antiretroviral drugs.

38. a method of assessing the validity of the sweet class reversed transcriptive enzyme of the non-nuclear of HIV infected patient antiretroviral therapy comprises:

A collects the plasma sample of HIV infected patient;

B detects whether to contain the nucleic acid that is coded in the hiv reverse transcriptase that 190 bit codons undergo mutation in patient's the plasma sample;

Wherein, the existence of sudden change draws furan pyridine susceptibility to improve with ground, and Viramune is relevant with the reduction of Ai Faluoruizi drug susceptibility.

39. the method for claim 38, wherein, 190 sudden change codons coding L-Ala (A) or Serine (S).

40. the method for claim 38, wherein, there is the combination of extra single mutation or these single mutation in reversed transcriptive enzyme at 98,101 or 103 bit codons.

41. the method for claim 40, wherein, 98 sudden change codon coding glycine (G), 101 sudden change codon coding L-glutamic acid (E), 103 sudden change codon coding l-asparagines (N).

42. the method for claim 38, wherein, the HIV infected patient is just being accepted the treatment of antiretroviral drugs.

43. a method of assessing the validity of the sweet class reversed transcriptive enzyme of the non-nuclear of HIV infected patient antiretroviral therapy comprises:

A collects the plasma sample of HIV infected patient;

B detects whether to contain the nucleic acid that is coded in the hiv reverse transcriptase that 106 bit codons undergo mutation in patient's the plasma sample;

Wherein, the existence of sudden change with draw that furan pyridine and Viramune susceptibility reduce, Ai Faluoruizi susceptibility changes very little or do not change relevant.

44. the method for claim 43, wherein, 106 sudden change codon coding L-Ala (A).

45. the method for claim 43, wherein, there is the combination of extra single mutation or these single mutation in reversed transcriptive enzyme at 227 and 189 bit codons.

46. the method for claim 45, wherein, 227 sudden change codon coding leucines (L), 189 sudden change codon coding leucines (L).

47. the method for claim 43, wherein, the HIV infected patient is just being accepted the treatment of antiretroviral drugs.

48. a method of assessing the validity of the sweet class reversed transcriptive enzyme of the non-nuclear of HIV infected patient antiretroviral therapy comprises:

A collects the plasma sample of HIV infected patient;

B detects whether to contain the nucleic acid that is coded in the hiv reverse transcriptase that 103 bit codons undergo mutation in patient's the plasma sample;

Wherein, the existence of sudden change with draw furan pyridine, Viramune and Ai Faluoruizi susceptibility to reduce relevant.

49. the method for claim 48, wherein, 103 sudden change codon coding l-asparagines (N).

50. the method for claim 48, wherein, reversed transcriptive enzyme exists additional mutations or simultaneous mutation at 100 bit codons.

51. the method for claim 50, wherein, 100 sudden change codon coding Isoleucines (I).

52. the method for claim 48, wherein, the HIV infected patient is just being accepted the treatment of antiretroviral drugs.

53. a method of assessing HIV antiretroviral therapy candidate medicine biological effectiveness comprises:

A imports to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment further comprises 230 and 181 bit codon mutations;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

54. a method of assessing HIV antiretroviral therapy candidate medicine biological effectiveness comprises:

A imports to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment further comprises 181 and 98 and/or 106 and/or 227 bit codon mutations;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

55. a method of assessing HIV antiretroviral therapy candidate medicine biological effectiveness comprises:

A imports to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment further comprises 188 and 138 and/or 103 and/or 100 bit codon mutations;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

56. a method of assessing HIV antiretroviral therapy candidate medicine biological effectiveness comprises:

A imports to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment further comprises 190 and 98 and/or 101 and/or 103 bit codon mutations;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

57. a method of assessing HIV antiretroviral therapy candidate medicine biological effectiveness comprises:

A imports to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment further comprises 106 and 227 and/or 189 bit codon mutations;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

58. a method of assessing HIV antiretroviral therapy candidate medicine biological effectiveness comprises:

A imports to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment further comprises 103 and 100 bit codon mutations;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

59. a method of assessing HIV antiretroviral therapy candidate medicine biological effectiveness comprises:

A imports to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment coding has the reversed transcriptive enzyme of sudden change at 230 bit codons;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

60. a method of assessing HIV antiretroviral therapy candidate medicine biological effectiveness comprises:

A imports to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment coding has the reversed transcriptive enzyme of sudden change at 227 bit codons;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

61. a method of assessing HIV antiretroviral therapy candidate medicine biological effectiveness comprises:

A imports to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment coding has the reversed transcriptive enzyme of sudden change at 188 bit codons;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

62. a method of assessing HIV antiretroviral therapy candidate medicine biological effectiveness comprises:

A imports to host cell and has the originate resistant proof carrier of a fragment and a reporter gene of a patient, and this fragment coding has the reversed transcriptive enzyme of sudden change at 189 bit codons;

Host cell among the b culturing step a;

The expression of c examining report gene in the target host cell;

63. one kind has the originate resistant proof carrier of a fragment and a reporter gene of a HIV patient, this fragment further comprises reversed transcriptive enzyme 230 bit codon mutations, and wherein, the expression of reporter gene depends on patient's fragment of originating.

64. one kind has the originate resistant proof carrier of a fragment and a reporter gene of a HIV patient, this fragment further comprises reversed transcriptive enzyme 227 bit codon mutations, and wherein, the expression of reporter gene depends on patient's fragment of originating.

65. one kind has the originate resistant proof carrier of a fragment and a reporter gene of a HIV patient, this fragment further comprises reversed transcriptive enzyme 188 bit codon mutations, and wherein, the expression of reporter gene depends on patient's fragment of originating.

66. one kind has the originate resistant proof carrier of a fragment and a reporter gene of a HIV patient, this fragment further comprises reversed transcriptive enzyme 189 bit codon mutations, and wherein, the expression of reporter gene depends on patient's fragment of originating.

67. one kind has the originate resistant proof carrier of a fragment and a reporter gene of a HIV patient, this fragment further comprises the combination of 181 of reversed transcriptive enzymes, 98,106 and 227 bit codon mutations or these sudden changes, wherein, the expression of reporter gene depends on patient's fragment of originating.

68. one kind has the originate resistant proof carrier of a fragment and a reporter gene of a HIV patient, this fragment further comprises the combination of 106 of reversed transcriptive enzymes, 227 and 189 bit codon mutations or these sudden changes, wherein, the expression of reporter gene depends on patient's fragment of originating.

69. one kind has the originate resistant proof carrier of a fragment and a reporter gene of a HIV patient, this fragment further comprises 103 of reversed transcriptive enzymes and 101 bit codon mutations, and wherein, the expression of reporter gene depends on patient's fragment of originating.

70. the resistant proof carrier of claim 17, wherein, the fragment that has a patient source of sudden change at 190 bit codons also comprises the combination of 98 and 101 bit codon mutations or these sudden changes.

71. the resistant proof carrier of claim 63, wherein, the patient who has a sudden change at the 230 bit codons fragment of originating also comprises 181 bit codon mutations.

72. the resistant proof carrier of claim 65, wherein, the patient who has a sudden change at the 188 bit codons fragment of originating further comprises the combination of 138,103 and 190 bit codon mutations or these sudden changes.