EP1407042A2 - Dispositif et methodes de surveillance d'une therapie antiretrovirale inhibitrice de la protease et determination de decisions therapeutiques dans le traitement du vih/sida - Google Patents
Dispositif et methodes de surveillance d'une therapie antiretrovirale inhibitrice de la protease et determination de decisions therapeutiques dans le traitement du vih/sidaInfo
- Publication number
- EP1407042A2 EP1407042A2 EP02744311A EP02744311A EP1407042A2 EP 1407042 A2 EP1407042 A2 EP 1407042A2 EP 02744311 A EP02744311 A EP 02744311A EP 02744311 A EP02744311 A EP 02744311A EP 1407042 A2 EP1407042 A2 EP 1407042A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- mutation
- codon
- hiv
- patient
- protease
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/702—Specific hybridization probes for retroviruses
- C12Q1/703—Viruses associated with AIDS
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16041—Use of virus, viral particle or viral elements as a vector
- C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
- C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Definitions
- This invention relates to antiretroviral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AI DS ) .
- HIV human immunodeficiency virus
- AI DS acquired immunodeficiency syndrome
- the invention further relates to the means and methods of monitoring the clinical progression of HIV infection and its response to antiretroviral therapy using phenotypic or genotypic susceptibility assays .
- the invention also relates to novel vectors , host cells and compositions for carrying out phenotypic susceptibility tests .
- the invention further relates to the use of various genotypic methodologies to identify patients who do not respond to a particular antiretroviral drug regimen .
- This invention also relates to the screening of candidate antiretroviral drugs for their capacity to inhibit viral replication , -Z- selected viral sequences and/or viral proteins. More particularly, this invention relates to the determination of protease inhibitor (PRI) susceptibility using phenotypic or genotypic susceptibility tests.
- PRI protease inhibitor
- This invention also relates to a means and method for accurately and reproducibly measuring viral replication fitness .
- HIV infection is characterized by high rates of viral turnover throughout the disease process, eventually leading to CD4 depletion and disease progression.
- the aim of antiretroviral therapy is to achieve substantial and prolonged suppression of viral replication. Achieving sustained viral control is likely to involve the use of sequential therapies, generally each therapy comprising combinations ' of three or more antiretroviral drugs. Choice of initial and subsequent therapy should, therefore, be made on a rational basis, with knowledge of resistance and cross-resistance patterns being vital to guiding those decisions.
- the primary rationale of combination therapy relates to synergistic or additive activity to achieve greater inhibition of viral replication.
- the tolerability of drug regimens will remain critical, however, as therapy will need to be maintained over many years.
- the target enzyme For antiretroviral drug resistance to occur, the target enzyme must be modified while preserving its function in the presence of the inhibitor. Point mutations leading to an amino acid substitution may result in changes in shape, size or charge of the active site, substrate binding site or in positions surrounding the active site of the enzyme. Mutants resistant to antiretroviral agents have been detected at low levels before the initiation of therapy. (Mohri H, Singh K, Ching WTW, et al . (1993) Proc Natl Acad Sci USA 90, 25-29) (Najera I, Rich an DD, Olivares I, et al.
- the selective pressure of antiretroviral therapy provides these drug-resistant mutants with a competitive advantage and thus they come to represent the dominant quasi species (Frost SDW, McLean AR (1994) AIDS 8, 323-332) (Kellam P, Boucher CAB, Tijnagal J GH (1994) J Gen Virol 75, 341-351) ultimately leading to a rebound in viral load in the patient.
- protease enzyme of HIV was crystallized and its three-dimensional structure was determined, (Navia MA, Fitzgerald PMD, McKeever BM, Leu CT, Heimbach JC, Herber WK, Sigal IS, Darke PL, Springer JP (1989) Nature 337:615-620 and Winters MA, Schapi-ro JM, Lawrence J, Merigan TC (1997) In Abstracts of the International Workshop on HIV Drug Resistance, Treatment Strategies and Eradication, St. Russia, Fla.) allowing for the rapid development of protease inhibitors. Initially, it was hypothesized that HIV protease, unlike reverse transcriptase, would be unable to accommodate "mutations leading to drug resistance.
- HIV protease was classified as an aspartic proteinase on the basis of putative active-site homology (Toh H, Ono M, Saigo K, Miyata T (1985) Nature 315:691), its inhibition by peptastin (Richards AD, Roberts R, Dunn BM, Graves MC, Kay J (1989) FEBS Lett 247:113), and its crystal structure (Navia MA, Fitzgerald PMD, McKeever BM, Lau CT, Heimbach JC, Herber WK, Sigal IS, Darke PL, Springer JP (1989) Nature 337:615-620).
- the enzyme functions as a homodimer composed of two identical 99-amino acid chains (Debouck C, Navia MA, Fitzgerald PMD, McKeever BM, Leu CT, Heimbach JC, Herber WK, Sigal IS, Darke PL, Springer JP (1988) Proc. Natl. Acad. Sci. USA 84:8903-8906), with each chain containing the characteristic Asp-Thr-Gly active-site sequence at positions 25 to 27 (Toh H, Ono M, Saigo K, Miyata T (1985) Nature 315:691).
- HIV protease processes gag (p55) and gag-pol (pl ⁇ O) polyprotein products into functional core proteins and viral enzymes (Kohl NE, Diehl RE, Rands E, Davis LJ, Hanobik MG, Wolanski B, Dixon RA (1991) J. Virol. 65:3007-3014 and Kramer RA, Schaber MD, Skalka AM, Ganguly K, Wong-Staal F, Reddy EP (1986) Science 231:1580-1584).
- the polyproteins are cleaved by the enzyme at nine different cleavage """ sites to yield the structural proteins ( ⁇ l7, p24, p7, and p6) as well as the viral enzymes reverse transcriptase, integrase, and protease (Pettit SC, Michael SF, Swanstrom R (1993) Drug Discov. Des. 1:69-83).
- wild-type virus particles produced by infected cells treated with protease inhibitors contain unprocessed precursors and are noninfectious (Crawford S, Goff SP
- Saquinavir developed by Hoffmann-La Roche, was the first protease inhibitor to undergo clinical evaluation, demonstrating that HIV-1 protease was a valid target for the treatment of HIV infection (Jacobsen H, Brun-Vezinet F, Duncan I, Hanggi M, Ott M, Vella S, Weber J, Mous J ( 1994 ) J. Virol. 68:2016-2020).
- Saquinavir is a highly active peptidomimetic protease inhibitor with a 90% inhibitory concentration (IC90) of 6 nM (id) .
- saquinavir can select for variants with one or both of two amino acid substitutions in the HIV-1 protease gene, a valine-for-glycine substitution at position 48 (G48V ) , a methionine-for-leucine substitution at residue 90 (L90M ) , and the double substitution G48V-L90M (Eberle J, Bechowsky B, Rose D, Hauser U, vonder Helm K, Guertler L, Nitschko H ( 1995 ) AIDS Res. Hum.
- G48V is the first mutation to appear, and continued selection results in highly resistant double-mutant variants. A substitution at either residue results in a 3- to 10-fold decreased susceptibility to the inhibitor, whereas the simultaneous occurrence of both substitutions causes a more severe loss of susceptibility of >100-fold ( id) .
- the frequency of resistance is lower (22% ) in patients receiving combination therapy with zidovudine, zalcitabine, and saquinavir (Collier AC, Coombs R, Schoenfeld DA, Bassett RL, Joseph Timpone MS, Baruch A, Jones M, Facey K, Whitacre C, McAuliffe VJ, Friedman HM, Merigan TC, Reichmann RC, Hooper C, Corey L (1996) N. Engl. J. Med. 334:1011-1017).
- the L90M exchange is the predominant mutation selected in vivo while the G48V (2%) or the double mutant ( ⁇ 2%) is rarely found (id) .
- Integrase is an attractive target for antivirals because it is essential for HIV replication and, unlike protease and reverse transcriptase, there are no known counterparts in the host cell. Furthermore, integrase uses a single active site to accommodate two different configurations of DNA substrates, which may constrain the ability of HIV to develop drug resistance to integrase inhibitors.
- Hazuda et al . (Science 287: 646-650, 2000) have described compounds (termed L-731, 988 and L-708,906) which specifically inhibit the strand-transfer activity of HIV-1 integrase and HIV-1 replication in vitro.
- Viruses grown in the presence of these inhibitors display reduced inhibitor susceptibility and bear mutations in the integrase coding region at amino acid positions 66 (T66I) , 153 (S153Y) , and 154 (M154I) .
- Site-directed mutants of a laboratory strain of HIV-1 (HXB2) with these amino acid changes confirmed their direct role in conferring reduced integrase inhibitor susceptibility. In addition some of these mutants displayed delayed growth kinetics, suggesting that viral fitness was impaired.
- the present invention relates to methods of monitoring, via phenotypic and genotypic methods the clinical progression of human immunodeficiency virus infection and its response to antiviral therapy.
- the invention is also based, in part , on the discovery that genetic changes in HIV protease (PR) which confer changes in susceptibility to antiretroviral therapy may be rapidly determined directly from patient plasma HIV RNA using phenotypic or genotypic methods.
- PR HIV protease
- the methods utilize nucleic acid amplification based assays, such as polymerase chain reaction (PCR) .
- PCR polymerase chain reaction
- PCR based a ssays may be u s e d to detect a substitution at codon 88 from asparagine to a serine residue either alone or in combination with o n e or more mutations at other codons selected from the g roup consisting of 10 , 20 , 36, 46, 63 and/or 77 or a co m b ination thereof of HIV PR .
- a mutation at codon 88 f r o m an asparagine residue to a serine residue (N88S ) alo ne correlates with an increase in susceptibili ty to a m p r e navir and a mutation at codon 88 from an asparagine r e s i d ue to a serine residue in combination with mutations a t c o d ons 63 and/or 77 or a combination thereof correlates w it h a n increase in susceptibility to amprenavir and a dec re a se in nelfinavir and indinavir susceptibility .
- a mutation at codon 90 from a leucine residue to a methionine in combination with secondary mutations at codons 73 and/or 71 or 73, 71 and/or 77 correlates with a reduction in susceptibility to indinavir and saquinavir, respectively.
- a mutation at codon 90 from a leucine residue to a methionine in combination with at least 3 secondary mutations correlates with a reduction in susceptibility to indinavir and saquinavir .
- a method for assessing the effectiveness of a protease inhibitor antiretroviral drug comprising: (a) introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; (b) culturing the host cell from step (a); (c) measuring expression of the indicator gene in a target host cell wherein expression of the indicator gene is dependent upon the patient derived segment; and (d) comparing the expression of the indicator gene from step (c) with the expression of the indicator gene measured when steps (a) - (c) are carried out in the absence of the PRI anti-HIV drug, wherein a test concentration of the PRI, anti-HIV drug is presented at steps (a) - (c) ; at steps (b) - (c) ; or at step (c) .
- the expression of the indicator gene in the resistance test vector in the target ceil is ultimately dependent upon the action of the HIV enzymes (PR and RT) encoded by the patient-derived segment DNA sequences.
- the indicator gene may be functional or non-functional.
- this invention is directed to antiretroviral drug susceptibility and resistance tests for HIV/AIDS.
- Particular resistance test vectors of the invention for use in the HIV/AIDS antiretroviral drug susceptibility and resistance test are identified.
- Yet another aspect of this invention provides for the identification and assessment of the biological effectiveness of potential therapeutic antiretroviral compounds for the treatment of HIV and/or AIDS.
- the invention is directed to a novel resistance test vector comprising a patient-derived segment further comprising one or more mutations on the PR gene and an indicator gene.
- Still another aspect of this invention provides for the identification and assessment of the fitness of a virus infecting a patient.
- the invention is directed to a novel resistance test vector comprising a patient-derived segment further comprising one or more mutations on the PR gene and an indicator gene, enabling the measurement of viral fitness.
- a resistance test vector is generated by cloning the patient-derived segment into an indicator gene viral vector. The resistance test vector is then co-transfected with an expression vector that produces amphotropic murine leukemia virus (MLV) envelope protein or other viral or cellular proteins which enable infection.
- MMV amphotropic murine leukemia virus
- Pseudotyped viral particles are produced containing the protease (PR) and the reverse transcriptase (RT) gene products encoded by the patient-derived DNA sequences . The particles are then harvested and used to infect fresh cells . Using defective PR and RT sequences it was shown that luciferase activity is dependent on functional PR and RT . PR inhibitors are added to the cells following transfection and are thus present during particle maturation.
- RT inhibitors are added to the cells at the time of or prior to viral particle infection.
- the assay is performed in the absence of drug and in the presence of drug over a wide range of concentrations . Luciferase activity is determined and the percentage (%) inhibition is calculated at the different drug concentrations tested. Fig. 3
- phenotypic drug susceptibility profiles Data are analyzed by plotting the percent inhibition of luciferase activity vs. loglO concentration. This plot is used to calculate the drug concentration that is required to inhibit virus replication by 50% (IC50) or by 95% (IC95) . Shifts in the inhibition curves towards higher drug concentrations are interpreted as evidence of drug resistance. Three typical curves for a nucleoside reverse transcriptase inhibitor (AZT) , a non-nucleoside reverse transcriptase inhibitor (efavirenz) , and a protease inhibitor (indinavir) are shown.
- AZA nucleoside reverse transcriptase inhibitor
- efavirenz non-nucleoside reverse transcriptase inhibitor
- indinavir protease inhibitor
- Phenotypic PRI susceptibility profile of a protease mutant generated by site-specific oligonucleotide-directed mutagenesis was carried out giving the phenotypic drug susceptibility profile of a virus having substitutions at codons 63, 77 -JJ-
- the profile demonstrates resistance to both nelfinavir and indinavir, and increased susceptibility to amprenavir.
- the assay can also be performed at defined drug concentrations .
- Luciferase activity produced by patient derived viruses is compared to the luciferase activity produced by well- characterized reference viruses. Replication fitness is expressed as a percent of the reference. Figure B . '
- Virus stocks produced from fitness test vectors derived from patient samples were used to infect cells. Luciferase activity was measured at various times after infection. Patient derived viruses may produce more, approximately the same, or less luciferase activity than the reference virus (Ref) and are said to have greater, equivalent, or reduced replication fitness, respectively.
- the drug susceptibility profiles of three representative patient derived viruses are _ shown (PI, P2, P3) .
- Figure C Identifying alterations in protease or reverse transcriptase function associated with differences in replication fitness of patient viruses.
- Replication fitness is expressed as a percent of the reference virus (top) .
- Fitness measurements are compared to protease processing of the p55 gag polyprotein (middle) and reverse transcriptase activity (bottom) .
- Protease processing is measured by Western blot analysis using an antibody that reacts with the mature capsid protein (p24). The detection of unprocessed p55 or incompletely processed p41 polyproteins are indicators of reduced cleavage.
- Reverse transcriptase activity is measured using a quantitative RT-PCR assay and is expressed as a percent of the reference virus.
- Reduced replication fitness is associated with ' high numbers of protease mutations, and the L90M mutation.
- Patient viruses were sorted based on the number of protease mutations. Viruses with large numbers of protease mutations or the L90M protease mutation generally exhibit reduced replication fitness.
- Low replication capacity is associated with specific protease mutations.
- Patient viruses were sorted based on replication capacity. Specific protease mutations either alone (D30N) or in combination (L90M plus others) were observed with high frequency in viruses with reduced replication fitness.
- Virus samples were collected weekly during a period of treatment interruption and evaluated for phenotypic drug susceptibility. Fitness values shown represent percent of the reference virus. The increase in fitness between week 9 and week 10 corresponds to improved protease processing (bottom) and reversion of the drug resistant phenotype to a drug sensitive phenotype ( Figure M) .
- Figure 0. Increased replication fitness during treatment interruption. Replication fitness was measured at the time of treatment interruption and various times during the period of treatment interruption. Generally, replication fitness was significantly higher in samples that corresponded to timepoints after the virus had reverted from a drug resistant phenotype to a drug sensitive phenotype.
- the present invention provides for a method of evaluating the effectiveness of antiretroviral therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV PR having a mutation at one or more positions in the PR.
- the mutation (s) correlate positively with alterations in phenotypic susceptibility.
- the invention provides for a method of evaluating the effectiveness of PRI antiretroviral therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV PR having a mutation at codon 88 from an asparagine residue to a serine residue (N88S) .
- This invention established, using a phenotypic susceptibility assay, that a mutation at codon 88 to a serine residue of HIV protease is correlated with an increase in amprenavir susceptibility.
- the invention provides for a method of evaluating the effectiveness of PRI antiretroviral therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV PR having a mutation at codon 88 from an asparagine residue to a serine residue (N88S) either alone or in combination with mutations at codons 63 and/or 77 or a combination thereof.
- This invention established, using a phenotypic susceptibility assay, that a mutation at codon 88 to a serine residue of HIV protease is correlated with an increase in amprenavir susceptibility and a mutation at codon 88 to a serine residue in combination with mutations at codons 63 and/or 77 or a combination thereof of HIV protease are correlated with an increase in amprenavir susceptibility and a decrease in nelfinavir and indinavir susceptibility.
- the invention provides for a method of evaluating the effectiveness of PRI antiretroviral therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV PR having a mutation at codon 88 from an asparagine residue to a serine residue (N88S) either alone or in combination with mutations at codons 46, 63 and/or 77 or a combination thereof.
- This invention established, using a phenotypic in ⁇
- a mutation at codon 88 to a serine residue of HIV protease is correlated with an increase in amprenavir susceptibility and a mutation at codon 88 to a serine residue in combination with mutations at codons 46, 63 and/or 77 or a combination thereof of HIV protease are correlated with an increase in amprenavir susceptibility and a decrease in nelfinavir and indinavir susceptibility.
- the invention provides for a method of evaluating the effectiveness of PRI antiretroviral therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV PR having a mutation at codon 88 from an asparagine residue to a serine residue (N88S) either alone or in combination with mutations at codons 10, 20, 36, 46, 63 and/or 77 or a combination thereof.
- This invention established, using a phenotypic susceptibility assay, that a mutation at codon 88 to a serine residue of HIV protease is correlated with an increase in amprenavir susceptibility and a mutation at codon 88 to a serine residue in combination with mutations at codons 10, 20, 36, 46, 63 and/or 77 or a combination thereof of HIV protease are correlated with an increase in amprenavir susceptibility and a decrease in nelfinavir and indinavir susceptibility.
- the phenotypic susceptibility profile and genotypic profile of the HIV virus infecting the patient has been altered reflecting a change in response to the antiretroviral agent.
- the HIV virus infecting the patient may be resistant to one or more PRIs but hypersensitive to another of the PRIs as described herein. It therefore may be desirable after detecting the mutation (s), to either increase the dosage of the antiretroviral agent, change to another antiretroviral agent, or add one or more additional antiretroviral agents to the patient's therapeutic regimen.
- the patient's therapeutic regimen may desirably be altered by either (i) changing to a different PRI antiretroviral agent, such as saquinavir, ritonavir or amprenavir and stopping nelfinavir treatment; or (ii) increasing the dosage of nelfinavir; or (iii) adding another antiretroviral agent to the patient's therapeutic regimen.
- a different PRI antiretroviral agent such as saquinavir, ritonavir or amprenavir and stopping nelfinavir treatment
- increasing the dosage of nelfinavir or adding another antiretroviral agent to the patient's therapeutic regimen.
- the effectiveness of the modification in therapy may be further evaluated by monitoring viral burden such as by HIV RNA copy number. A decrease in HIV RNA copy number correlates positively with the effectiveness of a treatment regimen.
- codon number refers to the position of the amino acid that the codon codes for. Therefore a codon referencing a particular number is equivalent to a "postion" referencing a particular number, such as for example, "codon 88" or "position 88".
- a method of evaluating the effectiveness of PRI therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; (ii) purifying and converting the viral RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the PR gene; (iii) performing PCR using primers that result in PCR products comprising wild type or serine at codon 88; and (iv) determining, via the products of PCR, the presence or absence of a serine residue at codon 88.
- a method of evaluating the effectiveness of PRI therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; (ii) purifying and converting the viral RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the PR gene; (iii) performing PCR using primers that result in PCR products comprising wild type or serine at codon 88 and mutations at codons 63 and/or 77; and (iv) determining, via the products of PCR, the presence or absence of a serine residue at codon 88 and the presence or absence of mutations at codons 63 and/or 77.
- a method of evaluating the effectiveness of PRI therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; (ii) purifying and converting the viral RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the PR gene; (iii) performing PCR using primers that result in PCR products comprising wild type or serine at codon 88 and mutations at codons 63, 77 and/or 46 or a combination thereof; and (iv) determining, via the products of PCR, the presence or absence of a serine residue at codon 88 and the presence or absence of mutations at codons 63, 77 and/or 46 or a combination thereof.
- a method of evaluating the effectiveness of PRI therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; (ii) purifying and converting the viral RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the PR gene; (iii) performing PCR using primers that result in PCR products comprising wild type or serine at codon 88 and mutations at codons 63, 77, 46, 10, 20, and/or 36 or a combination thereof; and (iv) determining, via the products of PCR, the presence or absence of a serine residue at codon 88 and the presence or absence of mutations at codons 63, 77, 46, 10, 20, and/or 36 or a combination thereof.
- Another preferred, non-limiting, specific embodiment of the invention is as follows: a method of evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises nucleic acid encoding HIV protease having a mutation at codon 88 to serine. Using the phenotypic susceptibility assay, it was observed that the presence of the mutation at codon 88 to serine of HIV PR causes a an increase in amprenavir susceptibility.
- Another preferred, non-limiting, specific embodiment of the invention is as follows: a method of evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises nucleic acid encoding HIV protease having a mutation at codon 88 to serine and additional mutatio (s) at codons 63 and/or 77 or a combination thereof.
- Another preferred, non-limiting, specific embodiment of the invention is as follows: a method of evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises nucleic acid encoding HIV protease having a mutation at codon 88 to serine and additional mutation (s) at codons 63, 77, 46, 10, 20 and/or 36 or a combination thereof.
- This invention provides a method for identifying a compound which is capable of affecting the function of the protease of HIV-1 comprising contacting the compound with the polypeptide (s) comprising all or part of the HIV-1 protease, wherein codon 88 is changed to a serine residue, wherein a positive binding indicates that the compound is capable of affecting the function of said protease.
- This invention also provides a method for assessing the viral fitness of patient's virus comprising: (a) determining the luciferase activity in the absence of drug for the reference control using the susceptibility test described above; (b) determining the luciferase activity in the absence of drug for the patient virus sample using the susceptibility test described above; and (c) comparing the luciferase activity determined in step (b) with the luciferase activity determined in step (a), wherein a decrease in luciferase activity indicates a reduction in viral fitness of the patient's virus.
- a resistance test vector is constructed using a patient derived segment from a patient virus which is unfit, and the fitness defect is due to genetic alterations in the patient derived segment, then the virus produced from cells transfected with the resistance test vector will produce luciferase more slowly. This defect will be manifested as reduced luciferase activity (in the absence of drug) compared to the drug sensitive reference control, and may be expressed as a percentage of the control.
- It is a further embodiment of this invention to provide a means and method for measuring replication fitness for viruses including, but not limited to human immunodeficiency virus (HIV) , hepadnaviruses (human hepatitis B virus), flaviviruses (human hepatitis C virus) and herpesviruses (human cytomegalovirus) .
- viruses including, but not limited to human immunodeficiency virus (HIV) , hepadnaviruses (human hepatitis B virus), flaviviruses (human hepatitis C virus) and herpesviruses (human cytomegalovirus) .
- This invention further relates to a means and method for measuring the replication fitness of HIV-1 that exhibits reduced drug susceptibility to reverse transcriptase inhibitors and protease inhibitors.
- This invention relates to a means and method for identifying mutations in protease or reverse transcriptase that alter replication fitness.
- a means and methods are provided for identifying mutations that alter replication fitness for other components of HIV-1 replication, including, but not limited to integration, virus assembly, and virus attachment and entry.
- This invention also relates to a means and method for quantifying the affect that specific mutations in protease or reverse transcriptase have on replication fitness.
- a means and method are provided for quantifying the affect that specific protease and reverse transcriptase mutations have on replication fitness in other viral genes involved in HIV-1 replication, including, but not limited to the gag, pol, and envelope genes.
- This invention also relates to the high incidence of patient samples with reduced replication fitness.
- This invention relates to the correlation between reduced drug susceptibility and reduced replication fitness.
- This invention further relates to the occurrence of viruses with reduced fitness in patients receiving protease inhibitor and/or reverse transcriptase inhibitor treatment .
- This invention further relates to the incidence of patient samples with reduced replication fitness in which the reduction in fitness is due to altered protease processing of the gag polyprotein (p55) .
- This invention further relates to the incidence of protease mutations in patient samples that exhibit low, moderate or normal (wildtype) replication fitness. This invention further relates to protease mutations that are frequently observed, either alone or in combination, in viruses that exhibit reduced replication capacity.
- This invention also relates to the incidence of patient samples with reduced replication fitness in which the reduction in fitness is due to altered reverse transcriptase activity.
- This invention relates to the occurrence of viruses with reduced replication fitness in patients failing antiretroviral drug treatment.
- This invention further relates to a means and method for using replication fitness measurements to guide the treatment of HIV-1.
- This invention further relates to a means and method for using replication fitness measurements to guide the treatment of patients failing antiretroviral drug treatment.
- This invention further relates to the means and methods for using replication fitness measurements to guide the treatment of patients newly infected with HIV-1.
- This invention provides the means and methods for using replication fitness measurements to guide the treatment of viral diseases, including, but not limited to HIV-1, hepadnaviruses (human hepatitis B virus), flaviviruses (human hepatitis C virus) and herpesviruses (human cytomegalovirus) .
- viral diseases including, but not limited to HIV-1, hepadnaviruses (human hepatitis B virus), flaviviruses (human hepatitis C virus) and herpesviruses (human cytomegalovirus) .
- the invention provides a method for determining replication capacity for a patient's virus comprising :
- step (c) harvesting viral particles from step (b) and infecting target host cells
- step (f) normalizing the expression of the indicator gene by measuring an amount of virus in step (c) .
- patient-derived segment encompasses segments derived from human and various animal species. Such species include, but are not limited to chimpanzees, horses, catties, cats and dogs.
- Patient-derived segments can also be incorporated into resistance test vectors using any of several alternative cloning techniques as set forth in detail in US Patent Number 5,837,464 (International Publication Number WO 97/27319) which is hereby incorporated by reference. For example, cloning via the introduction of class II restriction sites into both the plasmid backbone and the patient-derived segments or by uracil DNA glycosylase primer cloning.
- the patient-derived segment may be obtained by any method of molecular cloning or gene amplification, or modifications thereof, by introducing patient sequence acceptor sites, as described below, at the ends of the patient-derived segment to be introduced into the resistance test vector.
- restriction sites corresponding to the patient-sequence acceptor sites can be incorporated at the ends of the primers used in the PCR reaction.
- restriction sites can be incorporated at the ends of the primers used for first or second ' strand cDNA synthesis, or in a method such as primer-repair of DNA, whether cloned or uncloned DNA, said restriction sites can be incorporated into the primers used for the repair reaction.
- the patient sequence acceptor sites and primers are designed to improve the representation of patient-derived segments . Sets of resistance test vectors having designed patient sequence acceptor sites provide representation of patient-derived segments that may be underrepresented in one resistance test vector alone .
- Resistance test vector means one or more vectors which taken together contain DNA comprising a patient-derived segment and an indicator gene. Resistance test vectors are prepared as described in US Patent Number 5,837,464
- a resistance test vector (also referred to as a resistance test vector system) is prepared by introducing patient sequence acceptor sites into a packaging vector, amplifying or cloning patient-derived segments and inserting the amplified or cloned sequences precisely into the packaging vector at the patient sequence acceptor sites and co-transfecting this packaging vector with an indicator gene viral vector.
- “Indicator or indicator gene,” as described in US Patent Number 5,837,464 (International Publication Number WO 97/27319) refers to a nucleic acid encoding a protein, DNA or RNA structure that either directly or through a reaction gives rise to a measurable or noticeable aspect, e.g. a color or light of a measurable wavelength or in the case of DNA or RNA used as an indicator a change or generation of a specific DNA or RNA structure.
- Preferred examples of an indicator gene is the E.
- the indicator or indicator gene may be functional or non-functional as described in US Patent Number 5,837,464 (International Publication Number WO 97/27319) .
- the phenotypic drug susceptibility and resistance tests of this invention may be carried out in one or more host cells as described in US Patent Number 5,837,464 ( International Publication Number WO 97/27319) which is incorporated herein by reference.
- Viral drug susceptibility is determined as the concentration of the anti-viral agent at which a given percentage of indicator gene expression is inhibited (e.g. the IC50 for an anti-viral agent is the concentration at which 50% of indicator gene expression is inhibited) .
- a standard curve for drug susceptibility of a given anti-viral drug can be developed for a viral segment that is either a standard laboratory viral segment or from a drug-naive patient
- the method is provided , wherein the mutation at codon 82 is a substitution of alanine (A), phenylalanine (F), serine (S), or threonine (T) for valine (V) .
- patient-derived segment encompasses segments derived from human and various animal species. Such species include, but are not limited to chimpanzees, horses, catties, cats and dogs. Patient-derived segments can also be incorporated into resistance test vectors using any of several alternative cloning techniques. For example, cloning via the introduction of class II restriction sites into both the plasmid backbone and the patient-derived segments or by uracil DNA glycosylase primer cloning (refs).
- refs uracil DNA glycosylase primer cloning
- a replication-competent viral genome is enfeebled in a manner such that it cannot replicate on its own.
- the packaging expression vector can produce the trans-acting or missing genes required to rescue a defective viral genome present in a cell containing the enfeebled genome, the enfeebled genome cannot rescue itself.
- col i phoA gene which encodes alkaline phosphatase, green fluorescent protein and the bacterial CAT gene which encodes chloramphenicol acetyltransferase .
- Additional preferred examples of an indicator gene are secreted proteins or cell surface proteins that are readily measured by assay, such as radioimmunoassay (RIA) , or fluorescent activated cell sorting (FACS), including, for example, growth factors, cytokines and cell surface antigens (e.g. growth hormone, 11-2 or CD4, respectively) .
- RIA radioimmunoassay
- FACS fluorescent activated cell sorting
- “Indicator gene” is understood to also include a selection gene, also referred to as a selectable marker.
- Suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR) , thymidine kinase, hygromycin, neomycin, zeocin or E. coli gpt .
- DHFR dihydrofolate reductase
- the indicator gene and the patient-derived segment are discrete, i.e. distinct and separate genes.
- a patient-derived segment may also be used as an indicator gene.
- one of said viral • genes may also serve as the indicator gene.
- One example of a functional indicator gene in the case of HIV or HBV places the indicator gene and its promoter (a CMV IE enhancer/promoter) in the same or opposite transcriptional orientation as the HIV-LTR or HBV enhancer-promoter, respectively, or 'the CMV IE enhancer/promoter associated with the viral vector.
- a CMV IE enhancer/promoter a promoter associated with the viral vector.
- the indicator gene may be "non-functional" in that the indicator gene is not efficiently expressed in a packaging host cell transfected with the resistance test vector, which is then referred to a resistance test vector host cell, until it is converted into a functional indicator gene through the action of one or more of the patient-derived segment products.
- An indicator gene is rendered non-functional through genetic manipulation according to this invention.
- an indicator gene is rendered non-functional due to the location of the promoter, in that, although the promoter is in the same transcriptional orientation as the indicator gene, it follows rather than precedes the indicator gene coding sequence.
- This misplaced promoter is referred to as a "permuted promoter.”
- the orientation of the non-functional indicator gene is opposite to that of the native or foreign promoter/enhancer of the viral vector.
- the coding sequence of the non-functional indicator gene can neither be transcribed by the permuted promoter nor by the viral promoters.
- the non-functional indicator gene and its permuted promoter is rendered functional by the action of one or more of the viral proteins .
- T7 promoter T7 phage RNA polymerase promoter
- the indicator gene cannot be transcribed by the T7 promoter as the indicator gene cassette is positioned upstream of the T7 promoter.
- the non-functional indicator gene in the resistance test vector is converted into a functional indicator gene by reverse transcriptase upon infection of the target cells, resulting from the repositioning of the T7 promoter, by copying from the 5' LTR to the 3' LTR, relative to the indicator gene coding region.
- a permuted promoter may be any eukaryotic or prokaryotic promoter which can be transcribed in the target host cell.
- the promoter will be small in size to enable insertion in the viral genome without disturbing viral replication.
- a promoter that is small in size and is capable of transcription by a single subunit RNA polymerase introduced into the target host cell such as a bacteriophage promoter, will be used. Examples of such bacteriophage promoters and their cognate RNA polymerases include thos ⁇ of phages T7, T3 and Sp6.
- the indicator gene is rendered non-functional through use of an "inverted intron," i.e. an intron inserted into the coding sequence of the indicator gene with a transcriptional orientation opposite to that of the indicator gene.
- the overall transcriptional orientation of the indicator gene cassette including its own, linked promoter is opposite to that of the viral control elements, while the orientation of the artificial intron is the same as the viral control elements. Transcription of the indicator gene by its own linked promoter does not lead to the production of functional transcripts as the inverted intron cannot be spliced in this orientation.
- the indicator gene Transcription of the indicator gene by the viral control elements does, however, lead to the removal of the inverted intron by RNA splicing, although the indicator gene is still not functionally expressed as the resulting transcript has an antisense orientation.
- the indicator gene can be functionally transcribed using its own linked promoter as the inverted intron has been previously removed.
- the indicator gene itself may contain its own functional promoter with the entire transcriptional unit oriented opposite to the viral control elements.
- the non-functional indicator gene is in the wrong orientation to be transcribed by the viral control elements and it cannot be functionally transcribed by its own promoter, as the inverted intron cannot be properly excised by splicing.
- transcription by the viral promoters results m the removal of the inverted intron by splicing.
- the indicator gene can now be functionally transcribed by its own promoter.
- the inverted intron consisting of a splice donor and acceptor site to remove the intron, is preferably located in the coding region of the indicator gene in order to disrupt translation of the indicator gene.
- the splice donor and acceptor may be any splice donor and acceptor.
- a preferred splice donor-receptor is the CMV IE splice donor and the splice acceptor of the second exon of the human alpha globin gene ("intron A”) .
- indicator gene viral vector refers to a vector (s) comprising an indicator gene and its control elements and one or more viral genes.
- the indicator gene viral vector is assembled from an indicator gene, cassette and a "viral vector," defined below.
- the indicator gene viral vector may additionally include an enhancer, splicing signals, polyadenylat ion sequences, transcriptional terminators, or other regulatory sequences. Additionally the indicator gene viral vector may be functional or nonfunctional. In the event that the viral segments which are the target of the anti-viral drug are not included in the indicator gene viral vector they are provided in a second vector.
- An "indicator gene cassette" comprises an indicator gene and control elements.
- “Viral vector” refers to a vector comprising some or all of the following: viral genes encoding a gene product, control sequences, viral packaging sequences, and in the case of a retrovirus, integration sequences.
- the viral vector may additionally include one or more viral segments one or more of which may be the target of an anti-viral drug.
- Two examples of a viral vector which contain viral genes are referred to herein as an "genomic viral vector” and a “subgenomic viral vector.”
- a “genomic viral vector” is a vector which may comprise a deletion of a one or more viral genes to render the virus replication incompetent, but which otherwise preserves the mRNA expression and processing characteristics of the complete virus.
- the genomic viral vector comprises the HIV gag-pol , vif, vpr, ta t , rev, vpu , and nef genes (some, most or all of env may be deleted) .
- a "subgenomic viral vector” refers to a vector comprising the coding region of one or more viral genes which may encode the proteins that are the target (s) of the anti-viral drug.
- a preferred embodiment is a subgenomic viral vector comprising the HIV ga g-pol gene.
- a preferred embodiment is a subgenomic viral vector comprising the HBV P gene.
- HXB2 Fisher et al . , (1986) Na ture, 320, 367-371) and NL4-3, (Adachi et al . , (1986) J. Virol . , 59, 284-291) .
- HBV a large number of full length genomic sequences have been characterized and could be used for construction of HBV viral vectors: GenBank Nos. M54923, M38636, J02203 and X59795.
- the viral coding genes may be under the control of a native enhancer/promoter or a foreign viral or cellular enhancer/promoter.
- a preferred embodiment for an HIV drug susceptibility and resistance test is to place the genomic or subgenomic viral coding regions under the control of the native enhancer/promoter of the HIV-LTR U3 region or the CMV immediate-early (IE) enhancer/promoter.
- a preferred embodiment for an HBV drug susceptibility and resistance test is to place the genomic or subgenomic viral coding regions under the control of the CMV immediate-early (IE) enhancer/promoter.
- an indicator gene viral vector that contains one or more viral genes which are the targets or encode proteins which are the targets of an anti-viral drug(s) then said vector contains the patient sequence acceptor sites.
- the patient-derived segments are inserted m the patient sequence acceptor site m the indicator gene viral vector which is then referred to as the resistance test vector, as described above.
- Patient sequence acceptor sites are sites m a vector for insertion of patient-derived segments and said sites may be: 1) unique restriction sites introduced by site-directed mutagenesis into a vector; 2) naturally occurring unique restriction sites m the vector; or 3) selected sites into which a patient-derived segment may be inserted using alternative cloning methods (e.g. UDG cloning) .
- the patient sequence acceptor site is introduced into the indicator gene viral vector.
- the patient sequence acceptor sites are preferably located within or near the coding region of the viral protein which is the target of the anti-viral drug.
- the viral sequences used for the introduction of patient sequence acceptor sites are preferably chosen so that no change, or a conservative change, is made in the amino acid coding sequence found at that position.
- the patient sequence acceptor sites are located within a relatively conserved region of the viral genome to facilitate introduction of the patient-derived segments.
- the patient sequence acceptor sites are located between functionally important genes or regulatory sequences.
- Patient-sequence acceptor sites may be located at or near regions in the viral genome that are relatively conserved to permit priming by the primer used to introduce the corresponding restriction site into the patient-derived segment.
- primers may be designed as degenerate pools to accommodate viral sequence heterogeneity, or may incorporate residues such as deoxyinosine (I) which have multiple base-pairing capabilities.
- Sets of resistance test vectors having patient sequence acceptor sites that define the same or overlapping restriction site intervals may be used together in the drug resistance and susceptibility tests to provide representation of patient-derived segments that contain internal restriction sites identical to a given patient sequence acceptor site, and would thus be underrepresented in either resistance test vector alone.
- the resistance test vector is introduced into a host cell.
- Suitable host cells are mammalian cells.
- Preferred host cells are derived from human tissues and cells which are the principle targets of viral infection.
- HIV include human cells such as human T cells, monocytes, macrophage, dendritic cells, Langerhans cells, hematopoeitic stem cells or precursor cells, and other cells.
- suitable host cells include hepatoma cell lines (HepG2, Huh 7), primary human hepatocytes, mammalian cells which can be- infected by pseudotyped HBV, and other cells.
- a packaging host cell is a host cell which is transfected with one or more packaging expression vectors and when transfected with a resistance test vector is tnen referred to herein as a "resistance test vector host cell” and is sometimes referred to as a packaging host cell/resistance test vector host cell.
- Preferred host cells for use as packaging host cells for HIV include 293 human embryonic kidney cells (293, Graham, F.L. et al., J. Gen Virol. 36: 59, 1977), BOSC23 (Pear et al., Proc. Na tl . Acad . Sci . 90, 8392, 1993), tsa54 and tsa201 cell lines (Heinzel et al., J. Virol .
- Preferred host cells for use as target host cells include human T cell leukemia cell lines including Jurkat (ATCC T1B-152), H9 (ATCC HTB-176), CEM (ATCC CCL-119), HUT78 (ATCC T1B-161), and derivatives tnereof.
- replication capacity is defined herein is a measure of how well the virus replicates. This may also be referred to as viral fitness. In one embodiment, replication capacity can be measured by evaluating the ability of the virus to replicate in a single round of replication .
- control resistance test vector is defined as a resistance test vector comprising a standard viral sequence (for example, HXB2, PNL4-3) and an indicator gene.
- normalizing is defined as standardizing the amount of the expression of indicator gene measured relative to the number of viral particles- giving rise to the expression of the indicator gene. For example, normalization is measured by dividing the amount of luciferase activity measured by the number of viral particles measured at the time of infection.
- Plasmids and vectors are designated by a lower case p followed by letters and/or numbers.
- the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
- equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
- Plasmids of the invention employs standard ligation and restriction techniques which are well understood in the art (see Ausubel et al . , (1987) Current Protocols in Molecular Biology, Wiley Interscience or Maniatis et al . , (1992) in Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory, N.Y.). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired. The sequences of all DNA constructs incorporating synthetic DNA were confirmed by DNA sequence analysis (Sanger et al . (1977) Proc. Natl. Acad. Sci. 74, 5463-5467).
- “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences, restriction sites, in the DNA.
- the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements are known to the ordinarily skilled artisan.
- For analytical purposes typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme m about 20 ⁇ l of buffer solution. Alternatively, an excess of restriction enzyme is used to insure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37°C are workable, although variations can be tolerated.
- cleaved fragments After each incubation, protein is removed by extraction with phenol/chloroform and the nucleic acid recovered from aqueous fractions by precipitation with ethanol .
- size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods of Enzymology 65:499-560 (1980) .
- Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 minutes at 20°C in 50 M Tris (pH 7.6) 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 5-10 mM dNTPs .
- the Klenow fragment fills in at 5' sticky ends but chews back protruding 3' single strands, even though the four dNTPs are present.
- selective repair can be performed by supplying only one of the dNTPs, or with selected dNTPs, within the limitations dictated by the nature of the sticky ends.
- the mixture is extracted with phenol/chloroform and ethanol precipitated.
- Treatment under appropriate conditions with SI nuclease or Bal-31 results in hydrolysis of any single-stranded portion.
- Ligations are performed in 15-50 ⁇ l volumes under the following standard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 mg/ml BSA, 10 mM- 50 mM NaCl, and either 40 ⁇ M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticky end” ligation) or ImM ATP, 0.3 - 0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt end” ligation) .
- Intermolecular "sticky end” ligations are usually performed at 33-100 ⁇ g/ml total DNA concentrations (5-100 mM total end concentration) .
- Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at l ⁇ M total ends concentration.
- Transient expression refers to unamplified expression within about one day to two weeks of transfection.
- the optimal time for transient expression of a particular desired heterologous gene may vary depending on several factors including, for example, any transacting factors which may be employed, translational control mechanisms and the host cell.
- Transient expression occurs when the particular plasmid that has been transfected functions, i.e., is transcribed and translated. During this time the plasmid DNA which has entered the cell is transferred to the nucleus. The DNA is in a nonintegrated state, free within the nucleus. Transcription of the plasmid taken up by the cell occurs during this period. Following transfection the plasmid DNA may become degraded or diluted by cell division. Random integration within the cell chromatin occurs .
- promoters and control sequences which are derived from species compatible with the host cell are used with the particular host cell.
- Promoters suitable for use with prokaryotic hosts illustratively include the beta-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan
- trp tac promoter system and hybrid promoters such as tac promoter.
- other functional bacterial promoters are suitable.
- eukaryotic microbes such as yeast cultures may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available.
- Promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, simian virus 40
- SV40 adenovirus
- retroviruses hepatitis B virus and preferably cytomegalovirus
- heterologous mammalian promoters e.g. 3 _ actin promoter.
- the early and late promoters of the SV 40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
- the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment.
- promoters from the host cell or related species also are useful herein.
- the vectors used herein may contain a selection gene, also termed a selectable marker.
- a selection gene encodes a protein, necessary for the survival or growth of a host cell transformed with the vector.
- suitable selectable markers for mammalian cells include the dihydrofolate reductase gene (DHFR), the ornithme decarboxylase gene, the multi-drug resistance gene (mdr), the adenosme dea mase gene, and the glutamine synthase gene.
- the first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media.
- the second category is referred to as dominant selection w ⁇ ch refers to a selection scheme used in any cell type and ⁇ oes not require the use of a mutant cell line.
- Tnese schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin (Southern and Berg (1982)
- Transfection means introducing DNA into a host cell so that the DNA is expressed, whether functionally expressed or otherwise; tne DNA may also replicate either as an extrachromosomal element or by chromosomal integration.
- the method used herein for transfection of the host cells is the calcium phosphate co-precipitation method of Graham and van der Eb (1973) Virology 52, 456-457.
- Alternative methods for transfection are electroporation, the DEAE-dextran method, lipofection and biolistics (K ⁇ egler (1990) Gene Transfer and Expression: A Laboratory Manual, Stockton Press) .
- Host cells may be transfected with the expression vectors of the present invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes.
- Host cells are cultured in F12:DMEM (Gibco) 50:50 with added glutamine.
- the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
- patient-derived segment (s) corresponding to the HIV protease and reverse transcriptase coding regions were either patient-derived segments amplified by the reverse transcription-polymerase chain reaction method (RT-PCR) using viral RNA isolated from viral particles present in the serum of HIV-infected individuals or were mutants of wild type HIV-1 made by site directed mutagenesis of a parental clone of resistance test vector DNA.
- Isolation of viral RNA was performed using standard procedures (e.g. RNAgents Total RNA Isolation System, Promega, Madison WI or RNAzol, Tel-Test, Friendswood, TX) .
- the RT-PCR protocol was divided into two steps .
- a retroviral reverse transcriptase e.g. Moloney MuLV reverse transcriptase (Roche Molecular Systems, Inc., Branchburg, NJ) , or avian myeloblastosis virus (AMV) reverse transcriptase,
- Resistance tests were carried out with resistance test vectors using two host cell types. Resistance test vector viral particles were produced by a first host cell (the resistance test vector host cell) that was prepared by transfecting a packaging host cell with the resistance test vector and the packaging expression vector. The resistance test vector viral particles were then used to infect a second host cell (the target host cell) in which the expression of the indicator gene is measured (see Fig. 2) .
- % luciferase inhibition [1 - (RLUluc [drug] RLUluc) ] x 100
- RLUluc [ ⁇ rug] is the relative light unit of luciferase activity in infected cells m the presence of drug and RLUluc s the Relative Light Unit of luciferase activity in infected cells in the absence of ⁇ rug.
- IC50 values were obtained from the sigmcidal curves that were generated from tne data by plotting the percent inhibition of luciferase activity vs. the log. drug concentration. Examples of drug inhibition curves are shown in (Fig. 3) .
- Resistance test vectors are constructed as described m example 1. Resistance test vectors, or clones derived from the resistance test vector pools, are tested in a phenotypic assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
- This panel of anti-retroviral drugs may comprise members of the classes known as nucleoside-analog reverse transcriptase inhibitors
- NRTIs non-nucleoside reverse transcriptase inhibitors
- NRTIs neuropeptide kinase inhibitors
- PRIs protease inhibitors
- the panel of drugs can be expanded as new drugs or new drug targets become available.
- An IC50 is determined for each resistance test vector pool for each drug tested. The pattern of susceptibility to all of the drugs tested is examined and compared to known patterns of susceptibility.
- a patient sample can be further examined for genotypic changes correlated with the pattern of susceptibility observed . Genotypic analysis of patient HIV samples
- Resistance test vector DNAs are analyzed by any of the genotyping methods described in
- Example 1 In one embodiment of the invention, patient
- HIV sample sequences are determined using viral RNA purification, RT/PCR and ABI chain terminator _automated sequencing.
- the sequence that is determined is compared to control sequences present in the database or is compared to a sample from the patient prior to initiation of therapy, if available.
- the genotype is examined for sequences that are different from the control or pre-treatment sequence and correlated to the observed phenotype. Phenotypic susceptibility analysis of site directed mutants
- Genotypic changes that are observed to correlate with changes in phenotypic patterns of drug susceptibility are evaluated by construction of resistance test vectors containing the specific mutation on a defined, wild-type
- Mutations may be incorporated alone and/or in combination with other mutations that are thought to modulate the susceptibility of HIV to a certain drug or class of drugs. Mutations are introduced into the resistance test vector- through any of the widely known methods for site-directed mutagenesis. In one embodiment of this invention the mega-primer PCR method for site-directed mutagenesis is used. A resistance test vector containing the specific mutation or group of mutations are then tested using the phenotypic susceptibility assay described above and the susceptibility profile is compared to that of a genetically defined wild-type (drug susceptible) resistance test vector which lacks the specific mutations. Observed changes in the pattern of phenotypic susceptibility to the antiretroviral drugs tested are attributed to the specific mutations introduced into the resistance test vector.
- RTV-0732 DNA was analyzed by ABI chain terminator automated sequencing.
- the nucleotide sequence was compared to the consensus sequence of a wild type clade B HIV-1 (HIV Sequence Database Los Alamos, NM) .
- the nucleotide sequence was examined for sequences that are different from the control sequence.
- PR mutations were noted at positions K14R, I15V, K20T, E35D, M36I, R41K, I62V, L63Q and N88S.
- K14R, I15V, E35D, R41K and I62V are naturally occurring polymorphisms in HIV-1 PR and are not associated with reduced susceptibility to any drug.
- M36I has previously oeen described to be associated with resistance to ritonavir and nelfinavir (Shihazi, 1998).
- N88S has previously been described to be associated with resistance to nelfinavir (Patick AAC, 42: 2637 (1998) and an mvestigational PRI, SC55389A (Smidt, 1997).
- a resistance test vector was constructed as described in example 1 from a patient sample designated as 627. This patient had been treated with indinavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-627. RTV-627 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility tc a panel of anti-retroviral drugs.
- a pattern of susceptibility to the PRIs was observed for patient sample RTV-627 m which there was a decrease in indinavir and nelfinavir susceptibility (increased resistance) and an increase in amprenavir and saquinavir susceptibility.
- Patient sample 627 was examined further for genotypic changes associated with the pattern of susceptibility .
- RTV-627 DNA was analyzed by ABI chain terminator automated sequencing.
- the nucleotide sequence was compared to the consensus sequence of a wild type clade B HIV-1 (HIV Sequence Database Los Alamos, NM) .
- the nucleotide sequence was examined for sequences that are different from the control sequence.
- PR mutations were noted at positions 13I/V, E35D, M46L, L63P, I64V, I73V and N88S.
- I13V, E35D and I64V are naturally occurring polymorphisms in HIV-1 PR and are not associated with reduced susceptibility to any drug.
- M46L has previously been described to be associated with resistance to indinavir and amprenavir.
- L63P has previously been described to be associated with resistance to indinavir and nelfinavir.
- N88S has previously been described to be associated with resistance to nelfinavir (Patick, 1998) and an investigational PRI, SC55389A (Smidt, 1997) .
- a resistance test vector was constructed as described in example 1 from a patient sample designated as 1208. This patient had been previously treated with nelfinavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-1208. RTV-1208 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
- This panel of anti-retroviral drugs comprised members of the classes known as NRTIs (AZT, 3TC, d4T, ddl, ddC, and abacavir) , NNRTIs (delavirdine, nevirapine and efavirenz), and PRIs (indinavir, nelfinavir, ritonavir, saquinavir and amprenavir) .
- An IC50 was determined for each drug tested. Susceptibility of the patient virus to each drug was examined and compared to known patterns of susceptibility.
- RTV-1208 DNA was analyzed by ABI chain terminator automated sequencing. The nucleotide sequence was compared to the consensus sequence of a wild type clade B
- HIV-1 HIV Sequence Database Los Alamos, NM
- the nucleotide sequence was examined for sequences _ that are different from the control sequence.
- PR mutations were noted at positions I62V, L63P, V77I, and N88S.
- I62V is a naturally occurring polymorphism in HIV-1 PR and is not associated with reduced susceptibility to any drug.
- L63P has previously been described to be associated with resistance to indinavir and nelfinavir.
- V77I has previously been described to be associated with resistance to nelfinavir.
- N88S has previously been described to be associated with resistance to nelfinavir (Patick, 1998) and an investigational PRI, SC55389A (Smidt, 1997).
- a resistance test vector was constructed as described in example 1 from a patient sample designated as 360. This patient had been previously treated with indinavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-360. RTV-360 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
- This panel of anti-retroviral drugs comprised members of the classes known as NRTIs (AZT, 3TC, d4T, ddl, ddC, and abacavir) , NNRTIs (delavirdine, nevirapine and efavirenz), and PRIs (indinavir, nelfinavir, ritonavir, saquinavir and amprenavir) .
- An IC50 was determined for each drug tested. Susceptibility of the patient virus to each drug was examined and compared to known patterns of susceptibility.
- a pattern of susceptibility to the PRIs was observed for patient sample RTV-360 in which there was a decrease in indinavir and nelfinavir susceptibility (increased resistance) and an increase in amprenavir susceptibility.
- Patient sample 360 was examined further for genotypic changes associated with the pattern of susceptibility. Determination of genotype of patient 360
- RTV-360 DNA was analyzed by ABI chain terminator automated sequencing.
- the nucleotide sequence was compared to the consensus sequence of a wild type clade B HIV-1 (HIV Sequence Database Los Alamos, NM) .
- the nucleotide sequence was examined for sequences that are different from the control sequence.
- PR mutations were noted at positions I13V, K20M, M36V, N37A, M46I, I62V, L63P, N88S, and I93L.
- I13V, N37A and I62V are naturally occurring polymorphisms in HIV-1 PR and are not associated with reduced susceptibility to any drug.
- K20M ⁇ has previously been described to be associated with resistance to indinavir.
- M46I has previously been described to be associated with resistance to indinavir, ritonavir, nelfinavir and amprenavir.
- L63P has previously been described to be associated with resistance to indinavir and nelfinavir.
- N88S has previously been described to be ' associated with resistance to nelfinavir (Patick, 1998) and an investigational PRI, SC55389A (Smidt, 1997).
- a resistance test vector was constructed as described in example 1 from a patient sample designated as 0910. This patient had been previously treated with nelfinavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-0910. RTV-0910 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
- This panel of anti-retroviral drugs comprised members of the classes known as NRTIs (AZT, 3TC, d4T, ddl, ddC, and abacavir) , NNRTIs (delavirdine, nevirapine and efavirenz) , and PRIs (indinavir, nelfinavir, ritonavir, saquinavir and amprenavir) .
- An IC50 was determined for each drug tested. Susceptibility of the patient virus to each drug was examined and compared to known patterns of susceptibility.
- a resistance test vector was constructed as described in example 1 from a patient sample designated as 3542. This patient had been treated with indinavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences cooing for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-3542. RTV-3542 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
- the biological sample comprises whole blood, blood components including peripheral mononuclear cells (PBMC), serum, plasma (prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin) , tissue biopsies, cerebral spinal fluid (CSF) , or other cell, tissue or body fluids.
- PBMC peripheral mononuclear cells
- plasma prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin
- tissue biopsies tissue biopsies
- cerebral spinal fluid (CSF) cerebral spinal fluid
- the HIV-1 nucleic acid (genomic RNA) or reverse transcriptase protein can be isolated directly from the biological sample or after purification of virus particles from the biological sample. Evaluating whether the amino acid at position 88 of the HIV-1 protease is mutated to serine, can be performed using various methods, such as direct characterization of the viral nucleic acid encoding protease or direct characterization of the protease protein itself.
- Defining the amino acid at position 88 of protease can be performed by direct characterization of the protease protein by conventional or novel amino acid sequencing methodologies, epitope recognition by antibodies . or other specific binding proteins or compounds.
- the amino acid at position 88 of the HIV-1 protease protein can be defined by characterizing amplified copies of HIV-1 nucleic acid encoding the protease protein.
- Amplification of the HIV-1 nucleic acid can be performed using a variety of methodologies including reverse transcription-polymerase chain reaction (RT-PCR), NASBA, SDA, RCR, or 3SR.
- the nucleic acid sequence encoding HIV protease at codon 88 can be determined by direct nucleic acid sequencing using various primer extension-chain termination (Sanger, ABI/PE and Visible Genetics) or chain cleavage (Maxam and Gilbert) methodologies or more recently developed sequencing methods such as matrix assisted laser desorption-ionization time of flight (MALDI-TOF) or mass spectrometry (Sequenom, Gene Trace Systems) .
- the nucleic acid sequence encoding amino acid position 88 can be evaluated using a variety of probe hybridization methodologies, such as genechip hybridization sequencing (Affymetrix) , line probe assay (LiPA; Murex) , and differential hybridization (Chiron) .
- evaluation of protease inhibitor susceptibility and of whether amino acid position 88 of HIV-1 protease was wild type or serine was carried out using a phenotypic susceptibility assay or genotypic assay, respectively, using resistance test vector DNA prepared from the biological sample.
- the plasma sample was collected, viral RNA was purified and an RT-PCR methodology was used to amplify a patient derived segment encoding the HIV-1 protease and reverse transcriptase regions. The amplified patient derived segments were then incorporated, via DNA ligation and bacterial transformation, into an indicator gene viral vector thereby generating a resistance test vector.
- Resistance test vector DNA was isolated from the bacterial culture and the phenotypic susceptibility assay was carried out as described in Example 1.
- the results of the phenotypic susceptibility assay with a patient sample having an N88S mutation in PR is shown in Figure 4.
- the nucleic acid (DNA) sequence of the patient derived HIV-1 protease and reverse transcriptase regions from patient sample 0732 was determined using a fluorescence detection chain termination cycle sequencing methodology __ (ABI/PE) .
- the method was used to determine a consensus nucleic acid sequence representing the combination of sequences of the mixture of HIV-1 variants existing in the subject sample (representing the quasispecies) , and to determine the nucleic acid sequences of individual variants .
- Phenotypic and genotypic correlation of mutations at amino acid 88 of HIV-1 Protease Phenotypic susceptibility profiles of patient samples and site directed mutants showed that amprenavir susceptibility correlated with the presence of the N88S mutation in HIV-1 protease. Phenotypic susceptibility profiles of patient samples and site directed mutants showed that a significant increase in amprenavir susceptibility (decreased resistance) correlated with a mutation in the nucleic acid sequence encoding the amino acid serine (S) at position 88 of HIV-1 protease.
- Phenotypic susceptibility profiles of patient samples and site directed mutants showed reduction in amprenavir susceptibility (decreased resistance) and a decrease in susceptibility to nelfinavir and indinavir with the amino acid serine at position 88 when the PR mutations at positions 63, 77 or 46 were also present (L63P, V77I, or M46L) .
- EXAMPLE 6 Using Resistance test vectors and site directed mutants to correlate genotypes associated with alterations in PRI susceptibility with viral fitness .
- Luciferase activity measured in the absence of drug for the seven resistance test vectors constructed from the patient viruses containing the N88S PR mutation ranged from 0.7 to 16% of control (Table 3). Although these viruses also contain multiple mutations in reverse transcriptase, which could also contribute to a reduction in viral fitness, the data suggest that viruses containing the N88S mutation are less fit than wild type. To confirm this observation, the luciferase expression level for the site-directed mutant resistance test vectors was also examined .
- the biological sample comprises whole blood, blood components including peripheral mononuclear cells (PBMC) , serum, plasma (prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin) , tissue biopsies, cerebral spinal fluid (CSF) , or other cell,' tissue or body fluids.
- PBMC peripheral mononuclear cells
- plasma prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin
- tissue biopsies tissue biopsies
- cerebral spinal fluid (CSF) cerebral spinal fluid
- other cell ' tissue or body fluids.
- the amino acid at position 82 of the HIV-1 protease protein can be defined by characterizing amplified copies of HIV-1 nucleic acid encoding the protease protein.
- Amplification of the HIV-1 nucleic acid can be performed using a variety of methodologies including reverse transcription-polymerase chain reaction (RT-PCR) , NASBA, SDA, RCR, or 3SR.
- Genotypes are analyzed as lists of amino acid differences between virus in the patient sample and a reference laboratory strain of HIV-1, NL4-3. Genotypes and corresponding phenotypes (fold-change in IC50 values) are entered in a relational database linking these two results with patient information. Large datasets can then be assembled from patient virus samples sharing particular characteristics, ' such as the presence of any given mutation, or combination of mutations or reduced susceptibility to any drug or combination of drugs .
- Phenotypic susceptibility profiles of 75 patient virus samples which contained a mutation at position 82 (V82A, F, S, or T) , but no other primary mutations, were analyzed. According to most published guidelines, such viruses are expected to be resistant to ritonavir, nelfinavir, indinavir, and saquinavir. However, 8%, 20%, 23%, and 73% of these samples were phenotypically susceptible to these four protease inhibitors, respectively (see Table 6) . Thus, particularly for indinavir and saquinavir, there was poor correlation between the presence of mutations at position 82 and drug susceptibility.
- Indinavir resistance in viruses containing mutations at position 82 was evaluated with respect to the presence of other specific mutations. Decreased indinavir susceptibility (fold-change in IC 50 greater than 2.5) in viruses containing V82A, F, S, or T but no other primary mutations was correlated with the presence of mutations at secondary positions. Reduced indinavir susceptibility was observed in 20 samples containing mutations at both positions 24 and 82 (100%) and in 27 samples with both 71 and 82 (100%) (See Table 7). The combination of mutations at position 82 with mutations at other positions (e.g. 54, 46, 10, and 63) also significantly increased the proportion of samples that had reduced indinavir susceptibility (Table 7).
- Saquinavir resistance in viruses containing mutations at position 82 was evaluated with respect to the presence of other specific mutations. Decreased saquinavir susceptibility (fold-change in IC 50 greater than 2.5) in viruses containing V82A, F, S, or T but no other primary mutations was correlated with the presence of mutations at secondary positions. Reduced saquinavir susceptibility was observed in 4 of 5 samples containing mutations at both positions 20 and 82 (80%) and in 8 of 11 samples with both 36 and 82 (73%) (See Table 8) . The combination of mutations at position 82 with mutations at other positions (e.g. 24, 71, 54, and 10) also significantly increased the proportion of samples that had reduced saquinavir susceptibility (Table 8) .
- Indinavir resistance in viruses containing mutations at position 82 was evaluated with respect to the presence of a defined number of other mutations. Decreased indinavir susceptibility (fold-change in IC 5r greater than 2.5) in viruses containing V82A, F, S, or T but no other primary mutations was correlated with the number of mutations at secondary positions. Reduced indinavir susceptibility was observed in 100% of samples with V82A, F, S, or T and at least 6 other secondary mutations (See Table 9) . The proportion of samples that had reduced indinavir susceptibility increased significantly in samples with V82A, F, S, or T combined with 3 to 5 other secondary mutations (Table 9) .
- changes in the amino acid at position 90 of the protease protein of HIV-1 are evaluated using the following method comprising: (i) collecting a biological sample from an HIV-1 infected subject; (ii) evaluating whether the biological sample contains nucleic acid encoding HIV-1 protease having a leucine to methionine (L90M) substitution at codon 90; and (iii) determining susceptibility to protease inhibitors (PRI) •
- the biological sample comprises whole blood, blood components including peripheral mononuclear cells (PBMC), serum, plasma (prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin) , tissue biopsies, cerebral spinal fluid (CSF) , or other cell, tissue or body fluids.
- PBMC peripheral mononuclear cells
- plasma prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin
- tissue biopsies tissue biopsies
- cerebral spinal fluid (CSF) cerebral spinal fluid
- the HIV-1 nucleic acid (genomic RNA) or reverse transcriptase protein can be isolated directly from the biological sample or after purification of virus particles from the biological sample. Evaluating whether the amino acid at position 90 of the HIV-1 protease is mutated to methionine, can be performed using various methods, such as direct characterization of the viral nucleic acid encoding protease or direct characterization of the protease protein itself.
- Defining the amino acid at position 90 of protease can be performed by direct characterization of the protease protein by conventional or novel amino acid sequencing methodologies, epitope recognition by antibodies or other specific binding proteins or compounds.
- the amino acid at position 90 of the HIV-1 protease protein can be defined by characterizing amplified copies of HIV-1 nucleic acid encoding the protease protein.
- Amplification of the HIV-1 nucleic acid can be performed using a variety of methodologies including reverse transcription-polymerase chain reaction (RT-PCR), NASBA, SDA, RCR, or 3SR.
- the nucleic acid sequence encoding HIV protease at codon 90 can be determined by direct nucleic acid sequencing using various primer extension-chain termination (Sanger, ABI/PE and Visible Genetics) or chain cleavage (Maxam and Gilbert) methodologies or more recently developed sequencing methods such as matrix assisted laser desorption-ionization time of flight (MALDI-TOF) or mass spectrometry (Sequenom, Gene Trace Systems) .
- the nucleic acid sequence encoding amino acid position 90 can be evaluated using a variety of probe hybridization methodologies, such as genechip hybridization sequencing (Affymetrix) , line probe assay (LiPA; Murex) , and differential hybridization (Chiron) .
- evaluation of protease inhibitor susceptibility and of whether amino acid position 90 of HIV-1 protease was wild type or methionine was carried out using a phenotypic susceptibility assay or genotypic assay, respectively, using resistance test vector DNA prepared from the biological sample.
- the plasma sample was collected, viral RNA was purified and an RT-PCR methodology was used to amplify a patient derived segment encoding the HIV-1 protease and reverse transcriptase regions. The amplified patient derived segments were then incorporated, via DNA ligation and bacterial transformation, into an indicator gene viral vector thereby generating a resistance test vector.
- Resistance test vector DNA was isolated from the bacterial culture and the phenotypic susceptibility assay was carried out and analyzed as described in Example 1.
- the nucleic acid (DNA) sequence of the patient derived HIV-1 protease and reverse transcriptase regions was determined using a fluorescence detection chain termination cycle sequencing methodology (ABI/PE) .
- the method was used to determine a consensus nucleic acid sequence representing the combination of sequences of the mixture of HIV-1 variants existing in the subject sample
- Genotypes are analyzed as lists of amino acid differences between virus in the patient sample and a reference laboratory strain of HIV-1, NL4-3. Genotypes and corresponding phenotypes (fold-change in IC50 values) are entered in a relational database linking these two results with patient information. Large datasets can then be assembled from patient virus samples sharing particular characteristics, such as the presence of any given mutation, or combination of mutants, or reduced susceptibility to any drug or combination of drugs.
- Indinavir resistance in viruses containing mutations at position 90 was evaluated with respect to the presence of other specific mutations. Decreased indinavir susceptibility (fold-change in IC 50 greater than 2.5) in viruses containing L90M but no other primary mutations was correlated with the presence of mutations at secondary positions. Reduced indinavir susceptibility was observed in 17 of 19 samples containing mutations at both positions 73 and 90 (89%) and n 16 of 18 samples with both 71 and 90 (89%) (See Table 10). The combination of mutations at position 90 with mutation at position 46 also significantly increased the proportion of samples that had reduced indinavir susceptibility (Table 10) .
- Saquinavir resistance in viruses containing mutations at position 90 was evaluated with respect to the presence of other specific mutations. Decreased saquinavir susceptibility (fold-change IC 50 greater than 2.5) viruses containing L90M but no other primary mutations was correlated with the presence of mutations at secondary positions. Reduced saquinavir susceptibility was observed in 15 of IS samples containing mutations at both positions 73 and 90 (79%) and m 14 of 18 samples with both 71 and 90 (78%) (See Table 11) . The combination of mutations at position 90 with mutations at other positions (e.g. 77 and 10) also significantly increased the proportion of samples that had reduced saquinavir susceptibility (Table 1) .
- Saquinavir susceptibility of viruses containing combinations of mutations at amino acid 90 and many secondary mutations in HIV-1 Protease was evaluated with respect to the presence of a defined number of other mutations. Decreased saquinavir susceptibility (fold-change in IC 5C greater than 2.5) in viruses containing L90M but no other primary mutations was correlated with the number of mutations at secondary positions. Reduced saquinavir susceptibility was observed in 100% of samples with L90M and at least 5 other secondary mutations (See Table 12). The proportion of samples that had reduced saquinivir susceptibility increased significantly in samples with L90M combined with 3 or 4 other secondary mutations (Table 12) .
- changes in the amino acid at position 82 and 90 of the protease protein of HIV-1 are evaluated using the following method comprising: (i) collecting a biological sample from an HIV-1 infected subject; (ii) evaluating whether the biological sample contains nucleic acid encoding HIV-1 protease having a valine to alanine (V82A) , phenylalanine (V82F) , serine (V82S), or threonine (V82T) substitution at codon 82 or a leucine to methionine at position 90 (L90M) ; and (iii) determining susceptibility to protease inhibitors (PRI).
- V82A valine to alanine
- V82F phenylalanine
- V82S serine
- V82T threonine
- the biological sample comprises whole blood, blood components including peripheral mononuclear cells (PBMC), serum, plasma (prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin) , tissue biopsies, cerebral spinal fluid (CSF) , or other cell, tissue or body fluids.
- PBMC peripheral mononuclear cells
- plasma prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin
- tissue biopsies tissue biopsies
- cerebral spinal fluid (CSF) cerebral spinal fluid
- the HIV-1 nucleic acid (genomic RNA) or reverse transcriptase protein can be isolated directly from the biological sample or after purification of virus particles from the biological sample.
- Evaluating whether the amino acid at position 82 of the HIV-I protease is mutated to alanine, phenylalanine, serine, or threonine or at position 90 to methionine can be performed using various methods, such as direct characterization of the viral nucleic acid encoding protease or direct characterization of the protease protein itself. Defining the amino acid at positions 82 and 90 of protease can be performed by direct characterization of the protease protein by conventional or novel amino acid sequencing methodologies, epitope recognition by antibodies or other specific binding proteins or compounds.
- the amino acid at positions 82 and 90 of the HIV-1 protease protein can be defined by characterizing amplified copies of HIV-1 nucleic acid encoding the protease protein.
- Amplification of the HIV-1 nucleic acid can be performed using a variety of methodologies including reverse transcription-polymerase chain reaction (RT-PCR) , NASBA, SDA, RCR, or 3SR.
- the nucleic acid sequence encoding HIV protease at codons 82 and 90 can be determined by direct nucleic acid sequencing using various primer extension-chain termination (Sanger, ABI/PE and Visible Genetics) or chain cleavage (Maxam and Gilbert) methodologies or more recently developed sequencing methods such as matrix assisted laser desorption-ionization time of flight (MALDI-TOF) or mass spectrometry (Sequenom, Gene Trace Systems) .
- the nucleic acid sequence encoding amino acid positions 82 and 90 can be evaluated using a variety of probe hybridization methodologies, such as genechip hybridization sequencing (Affymetrix) , line probe assay (LiPA; Murex) , and differential hybridization (Chiron).
- evaluation of protease inhibitor susceptibility and of whether amino acid positions 82 and 90 of HIV-1 protease was wild type or alanine, phenylalanine, serine, or threonine in the case of position 82 and methionine at position 90 was carried out using a phenotypic susceptibility assay or genotypic assay, respectively, using resistance test vector DNA prepared from the biological sample.
- plasma sample was collected, viral RNA was purified and an RT-PCR methodology was used to amplify a patient derived segment encoding the HIV-1 protease and reverse transcriptase regions.
- a means and method is provided for accurately measuring and reproducing the replication fitness of HIV-1.
- This method for measuring replication fitness is applicable to other viruses, including, but not limited to hepadnaviruses (human hepatitis B virus), flaviviruses (human hepatitis C virus) and herpesviruses (human cytomegalovirus)-.
- This example further provides a means and method for measuring the replication fitness of HIV-1 that exhibits reduced drug susceptibility to reverse transcriptase inhibitors and protease inhibitors.
- This method can be used for measuring replication fitness for other classes of inhibitors of HIV-1 replication, including, but not limited to integration, virus assembly, and virus attachment and entry.
- Replication fitness tests are carried out using the means and methods for phenotypic drug susceptibility and resistance tests described in US Patent Number 5,837,464 (International Publication Number WO 97/27319) which is hereby incorporated by reference.
- patient-derived segment (s) corresponding to the HIV protease and reverse transcriptase coding regions were either patient-derived segments amplified by the reverse transcription-polymerase chain reaction method (RT-PCR) using viral RNA isolated from viral particles present in the serum of HIV-infected individuals or were mutants of wild type HIV-1 made by site directed mutagenesis of a parental clone of resistance test vector DNA.
- Resistance test vectors are also referred to as "fitness test vectors" when used to evaluate replication fitness. Isolation of viral RNA was performed using standard procedures (e.g. RNAgents Total RNA Isolation System, Promega, Madison WI or RNAzol, Tel-Test, Friendswood, TX) .
- the RT-PCR protocol was divided into two steps.
- a retroviral reverse transcriptase e.g. Moloney MuLV reverse transcriptase (Roche Molecular Systems, Inc., Branchburg, NJ) , or avian myeloblastosis virus (AMV) reverse transcriptase, (Boehringer Mannheim, Indianapolis, IN)
- AMV avian myeloblastosis virus
- the cDNA was then amplified using a thermostable DNA polymerase [e.g.
- thermostable polymerases as described for the performance of "long PCR” (Barnes, W.M., (1994) Proc. Natl. Acad. Sci, USA 91, 2216-2220) [e.g. Expand High Fidelity PCR System (Taq + Pwo) , (Boehringer Mannheim. Indianapolis, IN) OR GeneAmp XL PCR kit (Tth + Vent), (Roche Molecular Systems, Inc., Branchburg, NJ) ] .
- WO 97/27319 Number WO 97/27319 (see Fig. 1) using an amplified DNA product of 1.5 kB prepared by RT-PCR using viral RNA as a template and oligonucleotides PCR6 (#1) , PDSApa (#2) and PDSAge (#3) as primers, followed by digestion with Apal and Agel or the isoschizomer PinAl .
- PCR6 #1
- PDSApa #2
- PDSAge #3
- the plasmid DNA corresponding to the resultant fitness test vector comprises a representative sample of the HIV viral quasi-species present in the serum of a given patient, many (>100) independent E. coli transformants obtained in the construction of a given fitness test vector were pooled and used for the preparation of plasmid DNA.
- the fitness test vectors containing a functional luciferase gene cassette were constructed and host cells were transfected with the fitness test vector DNA.
- the fitness test vectors contained patient-derived reverse transcriptase and protease DNA sequences that encode proteins which were either susceptible or resistant to the antiretroviral agents, such as nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors. _
- Reverse transcriptase activity can be measured by any number of widely used assay procedures, including but not limited to homopolymeric extension using (e.g. oligo dT:poly rC) or real time PCR based on molecular beacons (reference Kramer) or 5' exonuclease activity (Lie and Petropoulos, 1996) .
- virion associated reverse transcriptase activity was measured using a quantitative PCR assay that detects the 5' exonuclease activity associated with thermo-stable DNA polymerases ( Figure C) .
- the fitness of the patient virus was compared to a reference virus to determine the relative fitness compared to "wildtype" viruses that have not been exposed to reverse transcriptase inhibitor drugs.
- the fitness of the patient virus was compared to viruses collected from the same patient at different timepoints, for example prior to initiating therapy, before or after changes in drug treatment, or before or after changes in virologic (RNA copy number) , im unologic (CD4 T-cells) , or clinical (opportunistic infection) markers of disease progression. Genotypic analysis of patient HIV samples
- Fitness test vector DNAs are analyzed by any of the genotyping methods described in Example 1.
- patient HIV sample sequences were determined using viral RNA purification, RT/PCR and ABI chain terminator automated sequencing. The sequence was determined and compared to reference sequences present in the database or compared to a sample from the patient prior to initiation of therapy. The genotype was examined for sequences that are different from the reference or pre-treatment sequence and correlated to the observed fitness.
- Genotypic changes that are observed to correlate with changes in fitness were evaluated by construction of fitness vectors containing the specific mutation on a defined, wild-type (drug susceptible) genetic background. Mutations may be incorporated alone and/or in combination with other mutations that are thought to modulate the fitness of a virus. Mutations were introduced into the fitness test vector through any of the widely known methods for site-directed mutagenesis. In one embodiment of this invention the mega-primer PCR method for site-directed mutagenesis is used. A fitness test vector containing the specific mutation or group of mutations were then tested using the fitness assay described in Example 10 and the fitness was compared to that of a genetically defined wild-type (drug susceptible) fitness test vector which lacks the specific mutations.
- a means and method for identifying mutations in protease that alter replication fitness is provided.
- Protease activity can be measured by any number of widely used assay procedures, including but not limited to in vitro reactions that measure protease cleavage activity
- protease cleavage of the gag polyprotein (p55) was measured by
- the fitness of the patient virus was compared to a reference virus to determine the relative fitness compared to "wildtype" viruses that have not been exposed to protease inhibitor drugs.- In another embodiment, the fitness of the patient virus was compared to viruses collected from the same patient at different timepoints, for example prior to initiating therapy, before or after changes in drug treatment, or before or after changes in virologic
- Genotypic changes that are observed to correlate with changes in fitness are evaluated by construction of fitness vectors containing the specific mutation on a defined, wild-type (drug susceptible) genetic background. Mutations may be incorporated alone and/or in combination with other mutations that are thought to modulate the fitness of a virus. Mutations are introduced into the fitness test vector through any of the widely known methods for site-directed mutagenesis. In one embodiment of this invention the mega-primer PCR method for site-directed mutagenesis is used. A fitness test vector containing the specific mutation or group of mutations are then tested using the fitness assay described in Example 10 and the fitness is compared to that of a genetically defined wild-type (drug susceptible) fitness test vector which lacks the specific mutations.
- fitness test vectors containing site directed mutations in reverse protease that result in amino acid substitutions at positions 30, 63, 77, 90 are constructed and tested for fitness ( Figure E) .
- the fitness results enable the correlation between specific protease amino acid substitutions and changes in viral fitness.
- This example describes the high incidence of patient samples with reduced replication fitness.
- This example also describes the general correlation between reduced drug susceptibility and reduced replication fitness.
- This example further describes the occurrence of viruses with reduced fitness in patients receiving protease inhibitor and/or reverse transcriptase inhibitor treatment.
- This example further describes the incidence of patient samples with reduced replication fitness in which the reduction in fitness is due to altered protease processing of the gag polyprotein (p55) .
- This example further describes the incidence of protease mutations in patient samples that exhibit low, moderate or normal (wildtype) replication fitness.
- protease mutations that are frequently observed, either alone or in combination, in viruses that exhibit reduced replication capacity.
- This example also describes the incidence of patient samples with reduced replication fitness in which the reduction in fitness is due to altered reverse transcriptase activity.
- This example describes the occurrence of viruses with reduced replication fitness in patients failing antiretroviral drug treatment.
- Fitness/resistance test vectors were constructed as described in example 10. Fitness and drug susceptibility was measured in 134 random patient samples that were received for routing phenotypic testing by the ViroLogic Clinical Reference Laboratory. Fitness assays were performed as described in Example 10. Drug susceptibility testing and genotyping of the protease region was performed as described in Example 1. Reverse transcriptase activity was measured as described in Example 11. Protease processing was measured as described in Example 12.
- Drug susceptibility of patient viruses Reduced drug susceptibility was observed for a majority of the patient virus samples (Table A) . 66 percent of the viruses exhibited large (define as >10X of the reference) reductions in susceptibility to one or more NRTI drugs. 52 percent of the viruses exhibited large reductions in susceptibility to one or more NNRTI drugs. 45 percent of the viruses exhibited large reductions in susceptibility to one or more PRI drugs .
- Viruses with reduced drug susceptibility were much more likely to display reduced replication fitness ( Figures F, G, H, and I) .
- protease mutations D30N, M46I/L, G48V, I54L/A/S/T/V, and I84V were observed at high incidences in viruses with reduced protease processing of the p55 gag polyprotein (Figure L) .
- a means and method for using replication fitness measurements to guide the treatment of HIV-1 is provided.
- This example further provides a means and method for using replication fitness measurements to guide the treatment of patients failing antiretroviral drug treatment.
- This example further provides the means and methods for using replication fitness measurements to guide the treatment of patients newly infected with HIV-1.
- physicians may choose to perform routine replication fitness assays for patients that have multi- drug resistant virus.
- This assay could be used to monitor the replication fitness of patient viruses when complete suppression of virus replication is not possible due to multi-drug resistance.
- the assay would be used to guide treatment decisions that prevent the drug resistant virus with low replication fitness from increasing its replication fitness. In this way, physicians may prolong the usefulness of antiretroviral drugs despite the presence of drug resistant virus in the patient.
- This example provides a means and method for identifying mutations in protease that affect susceptibility (increased or decreased) to saquinavir.
- the effects of combination of mutations at position 82 are evaluated using the following method comprising: (i ) collecting a biological sample from an HIV-1 infected subject; (ii) evaluating whether the HIV-1 in the sample contains nucleic acid encoding protease having a valine to alanine (V82A) , phenylalanine (V82F) , serine (V82S) , or threonine (V82T) substitution at position 82 or a leucine to methionine substitution at position 90 (L90M) ; and (iii) determining susceptibility to protease inhibitors (PRIs) .
- PRIs susceptibility to protease inhibitors
- the biological sample comprises whole blood, ' blood components including peripheral mononuclear cells (PBMC) , serum, plasma (prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin) , tissue biopsies, cerebral spinal fluid (CSF) , or other cell, tissue or body fluids.
- PBMC peripheral mononuclear cells
- plasma prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin
- tissue biopsies tissue biopsies
- cerebral spinal fluid (CSF) cerebral spinal fluid
- the HIV-1 nucleic acid (genomic RNA) or reverse transcriptase protein can be isolated directly from the biological sample or after purification of virus particles from the biological sample.
- Evaluating whether the amino acid at position 82 of the HIV-1 protease is mutated to alanine, phenylalanine, or threonine can be performed using various methods, such as direct characterization of the viral nucleic acid encoding protease or direct characterization of the protease protein itself. Defining the amino acid at position 82 of protease can be performed by direct characterization of the protease protein by conventional or novel amino acid sequencing methodologies, epitope recognition by antibodies or other specific binding proteins or compounds. Alternatively, the amino acid at position 82 of the HIV-1 protease protein can be defined by characterizing amplified copies of HIV-1 nucleic acid encoding the protease protein.
- Amplification of the HIV-1 nucleic acid can be performed using a variety of methodologies including reverse transcription-polymerase chain reaction (RT-PCR) , NASBA, SDA, RCR, or 3SR.
- RT-PCR reverse transcription-polymerase chain reaction
- the nucleic acid sequence encoding HIV protease at codon 82 can be determined by direct nucleic acid sequencing using various primer .extension-chain termination (Sanger, ABI/PE and Visible Genetics) or chain cleavage (Maxam and Gilbert) methodologies or more recently developed sequencing methods such as matrix assisted laser desorption-ionization time of flight (MALDI-TOF) or mass spectrometry (Sequenom, Gene Trace Systems).
- MALDI-TOF matrix assisted laser desorption-ionization time of flight
- MSequenom Gene Trace Systems
- nucleic acid sequence encoding amino acid position 82 can be evaluated using a variety of probe hybridization methodologies, such as genechip hybridization sequencing (Affymetrix) , line probe assay (LiPA; Murex) , and differential hybridization (Chiron) .
- probe hybridization methodologies such as genechip hybridization sequencing (Affymetrix) , line probe assay (LiPA; Murex) , and differential hybridization (Chiron) .
- evaluation of the effects of mutations at amino acid position 82 of HIV-1 protease on protease inhibitor susceptibility was carried out using a phenotypic susceptibility assay using resistance test vector DNA prepared from the biological sample.
- plasma samples were collected, viral RNA was purified and an RT-PCR methodology was used to amplify a patient derived segment encoding the HIV-1 protease and reverse transcriptase regions.
- the amplified patient derived segments were then incorporated, via DNA ligation and bacterial transformation, into an indicator gene viral vector thereby generating a resistance test vector.
- Resistance test vector DNA was isolated from the bacterial culture and the phenotypic susceptibility assay was carried out as described in Example 1.
- the genotype of the protease region was determined by dideoxy chain- termination sequencing of the resistance test vector DNA. The results are summarized for saquinavir (SQV) in Figure 6. Samples were categorized as having mutations in protease encoding alanine (A), phenylalanine (F), or threonine (T) at position 82, instead of the wild-type valine (V) , and the percentage of samples in each category displaying hyper-sensitivity to saquinavir (i.e., fold- change vs. reference of 0 . 4 or less) was determined.
- SQV saquinavir
- This example provides the means and methods for identifying mutations that alter replication fitness for various components of HIV-1 replication, including, but not limited to integration, virus assembly, and virus attachment and entry.
- This example also provides a means and method for quantifying the affect that specific mutations in protease, reverse transcriptase, or integrase have on replication fitness. This method can be used for quantifying the effect that specific integrase mutations have on replication fitness and can be used to quantify the effect of other mutations in other viral genes involved in HIV-1 replication, including, but not limited to the gag, pol, and envelope genes.
- Table 3 Relative luciferase activity levels for patient sample virus-derived resistance test vector pools.
- the luciferase activity (relative light units, RLU) measured in the absence of drug for the patient sample was compared to that of the drug sensitive reference control from the same assay run, and expressed as a percentage of control. These values are from one assay each. All the samples that contain the N88S mutations in PR were found to have reduced luciferase activity compared to control.
- Table 4 Relative luciferase activity levels for resistance test vectors containing site-directed mutations.
- the luciferase activity (relative light units, RLU) measured in the absence of drug for the mutant was compared to that of the drug sensitive reference control from the same assay run, and expressed as a percentage of control. These values are from one to five assays each, and each value was obtained using an independent clone for mutants which were tested multiple times. All the constructs that contain the N88S mutations in PR were found to have reduced luciferase activity compared to control. All the constructs with the K20T mutation were essentially inactive in the assay.
- a method is provided of assessing the effectiveness of protease antiretroviral therapy of an HIV-infected patient comprising:
- the above method is provided of assessing the effectiveness of protease antiretroviral therapy, having a mutation at codon 82 and a secondary mutation at codons 32 or 39, or a mutation at codon 90 and a secondary mutation at codons 64 or 93, wherein the change in susceptibility in step (c) is an increase in susceptibility to saquinavir.
- the above method is provided of assessing the effectiveness of protease antiretroviral therapy, having a mutation at codon 82 and a secondary mutation at - codons selected from the group consisting of 73, 55, 48, 20, 43, 53, 90, 13, 48, 23, 84, 53, 74, 60, 33, 36, 35, 32, and 46 or a mutation at codon 90 and a secondary mutation at codons selected from the group consisting of 95, 55, 54, 82, 85, 84, 20, 72, 62, 74, 53, 48, 23, 58, 36, 64, 77, and 93.
- the above method is provided of assessing the effectiveness of protease antiretroviral therapy, wherein the protease inhibitor is selected from the group consisting of indinavir, amprenavir, and saquinavir.
- the above method is provided of assessing the effectiveness of protease antiretroviral therapy, having a mutation at codon 82 and a secondary mutation at codons 32 or 46, or a mutation at codon 90 and a secondary mutation at codons 64, 77, or 93, wherein the change in susceptibility in step (c) is an increase in susceptibility to saquinavir.
- a resistance test vector comprising an HIV patient-derived segment comprising nucleic acid encoding protease having a mutation at codon 82 and secondary mutations at codons selected from the group consisting of 73, 55, 48, 20, 43, 53, 90, 13, 84, 23, 33, 74, 32, 39, 60, 36, and 35, or a mutation at codon 90 and secondary mutations at codons selected from the group consisting of 53, 95, 54, 84, 82, 46, 13, 74, 55, 85, 20, 72, 62, 66, 84, 48, 33, 73, 71, 64, 93, 23, 58, and 36 and an indicator gene, wherein the expression of the indicator gene is dependent upon the patient-derived segment.
- the second column represents a positive (+) or negative (-) correlation between the change in resistance from the number of wild-type reference samples to those samples having the secondary mutation.
- the fifth column designates "wt %", as the percentage of wild-type reference samples showing the indicated fold resistance, (i.e, > 10 fold or or 2.5 fold) to the specified protease inhibitor.
- the present invention provides a method for determining cross resistance of an HIV virus to RTV and SQV which comprises determining (i) whether position 30 of the HIV protease is D, and (ii) whether the virus is resistant to NFV, wherein a mutation from D to N at position 30 of HIV protease and resistance of the virus to NFV are indicative of cross resistance to IDV and SQV.
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Abstract
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US09/874,472 US20030108857A1 (en) | 2001-06-04 | 2001-06-04 | Means and methods for monitoring protease inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS |
WOPCT/US02/01682 | 2002-01-18 | ||
PCT/US2002/001682 WO2002068618A1 (fr) | 2001-01-19 | 2002-01-18 | Procedes pour controler une therapie antiretrovirale par inhibiteur de protease |
PCT/US2002/018684 WO2002099387A2 (fr) | 2001-06-04 | 2002-06-04 | Dispositif et methodes de surveillance d'une therapie antiretrovirale inhibitrice de la protease et determination de decisions therapeutiques dans le traitement du vih/sida |
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US7186506B1 (en) | 2000-06-12 | 2007-03-06 | Monogram Biosciences, Inc. | Means and methods for monitoring protease inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS |
US7138231B2 (en) | 2000-09-15 | 2006-11-21 | Monogram Biosciences, Inc. | Means and methods for monitoring protease inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS |
US6869759B1 (en) | 1999-06-22 | 2005-03-22 | Virologic, Inc. | Means and methods for monitoring protease inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS |
US7384734B2 (en) | 2002-02-15 | 2008-06-10 | Monogram Biosciences, Inc. | Compositions and methods for determining the susceptibility of a pathogenic virus to protease inhibitors |
AU2003256460A1 (en) | 2002-07-01 | 2004-01-19 | Virologic, Inc. | Compositions and methods for determining the susceptibility of a pathogenic virus to protease inhibitors |
WO2004003512A2 (fr) | 2002-07-01 | 2004-01-08 | Virologic, Inc. | Compositions et methodes permettant de mesurer la sensibilite d'un virus pathogene a des inhibiteurs de la protease |
EP1726643A1 (fr) * | 2005-05-27 | 2006-11-29 | Direvo Biotech AG | Méthode pour la provision, l'identification, la sélection des protéases avec une sensititivité modifiée contre des substances modulatrices |
US20100136516A1 (en) * | 2008-12-01 | 2010-06-03 | 454 Life Sciences Corporation | System and method for detection of HIV integrase variants |
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PATICK A K ET AL: "Genotypic and phenotypic characterization of human immunodeficiency virus type 1 variants isolated from patients treated with the protease inhibitor nelfinavir" ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON, DC, US, vol. 42, no. 10, October 1998 (1998-10), pages 2637-2644, XP002978189 ISSN: 0066-4804 * |
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