CA2244735A1

CA2244735A1 - Method of managing the chemotherapy of patients who are hiv positive based on the phenotypic drug sensitivity of human hiv strains

Info

Publication number: CA2244735A1
Application number: CA 2244735
Authority: CA
Inventors: Marie-Pierre De Bethune; Kurt Hertogs; Rudi Pauwels
Original assignee: Individual
Current assignee: Virco BVBA
Priority date: 1996-01-26
Filing date: 1997-01-24
Publication date: 1997-07-31
Anticipated expiration: 2017-01-24
Also published as: CA2244735C

Abstract

A method of managing HIV chemotherapy of patients who are HIV positive comprises transfecting a cell line susceptible to infection by HIV with a sequence, preferably that coding for RT and protease, from the pol gene of HIV obtained from a patient and a HIV-DNA construct from which the sequence has been deleted, culturing the transfected cells so as to create a stock of chimeric viruses, assessing the phenotypic sensitivity of the chimeric viruses to an inhibitor of the enzyme encoded by the pol gene of HIV and assigning a value thereto, constructing a data set comprising the value for chimeric virus sensitivity and the corresponding value for a chimeric wild-type strain of HIV, repeating the sensitivity assessment for at least two further inhibitors and thereby constructing at least three such data sets in total, representing the data sets in two-dimensional or three-dimensional graphical form such that the difference between the chimeric and wild-type sensitivities in the case of each data set provides a visual measure of the resistance of the chimeric stock to treatment by the inhibitor in question, and selecting the optimum inhibitor(s) on the basis of the graphical representation of the resistances so measured. The method yields phenotypic information on individual HIV infected patients on a large scale, economically and rapidly. The method is applicable to all currently available chemotherapeutic regimens and it is expected to be equally applicable to future chemotherapeutic regimens.

Claims

1. A method of managing HIV chemotherapy of patients who are HIV positive, which comprises transfecting a cell line susceptible to infection by HIV with a sequence from the pol gene of HIV, obtained by isolating viral RNA from a sample of a biological material from a patient and reverse transcribing the desired region of said pol gene, and a HIV-DNA construct from which said sequence has been deleted, culturing said transfected cells so as to create a stock of chimeric viruses providing an indication of the resistance profile of the circulating virus, assessing the phenotypic sensitivity of said chimeric viruses to an inhibitor of said enzyme encoded by the pol gene of HIV and assigning a value thereto, constructing a data set comprising said value for chimeric virus sensitivity and the corresponding value for a chimeric wild-type strain of HIV, repeating the sensitivity assessment for at least two further inhibitors and thereby constructing at least three such data sets in total, representing said data sets in two dimensional or three dimensional graphical form such that the difference between the chimeric and wild-type sensitivities in the case of each data set provides a visual measure of the resistance of the chimeric stock to treatment by the inhibitor in question, and selecting the optimum inhibitor(s) on the basis of the graphical representation of the resistances so measured.

2. A method of managing HIV chemotherapy according to Claim 1, wherein the data sets are represented on a polygonal or quasi-circular graph comprising:

(a) a plurality of normalised axes extending radially from an origin, each axis corresponding to one data set or inhibitor or combination thereof;

(b) the axes being normalised such that the sensitivity values for wild-type HIV for the various inhibitors are equal on each axis, the data points for wild-type HIV being optionally represented and connected to form a regular polygon whose vertices lie on the axes and whose center is defined by the origin;

(c) on each axis a data point representing the sensitivity value of the chimeric HIV stock against the inhibitor corresponding to said axis is plotted, the chimeric data points being optionally connected to form a regular or irregular polygon the shape of which represents the resistance of the chimeric stock to a range of inhibitors.

3. A method according to Claim 2, wherein each axis has a logarithmic scale.

4. A method according to Claim 2 or 3, wherein eccentric data points in the chimeric polygon, if represented, identify inhibitors whose usefulness can be assumed to be of little or no benefit to the patient, while data points lying within, on or close outside the wild-type polygon identify inhibitors whose usefulness can be assumed to be of substantial benefit to the patient.

5. A method according to any preceding claim, wherein each of said three or more data sets further comprises a value for worst-case measurable resistance for the inhibitor in question, said worst case values being represented on said graphical representations such that the data point for the chimeric stock can be compared both to wild-type and to worst-case HIV, thereby providing an assessment of the relative value of the inhibitor in a particular case.

6. A method of managing HIV chemotherapy of patients who are HIV positive, which comprises the steps of:

(a) periodically assessing the phenotypic sensitivity of a patient's HIV strains according to any one of Claims 1-5;

(b) maintaining the chemotherapy with the selected inhibitor while the patient's HIV strains remain susceptible to the selected chemotherapy;

(c) selecting a different inhibitor if and when the susceptibility of the original inhibitor decreases.

7. A method according to any one of Claims 1-6, wherein the phenotypic sensitivity of said chimeric viruses to inhibitors of at least two enzymes encoded by the pol gene of HIV is simultaneously assessed.

8. A method of determining the phenotypic drug sensitivity of individual HIV strains in a patient to inhibitors of at least two enzymes encoded by the pol gene of HIV, which comprises transfecting a cell line susceptible to infection by HIV with a sequence from the pol gene of HIV, obtained by isolating viral RNA from a sample of a biological material from a patient and reverse transcribing the desired region of said pol gene, and a HIV-DNA construct from which said sequence has been deleted, culturing said transfected cells so as to create a stock of chimeric viruses providing an indication of the resistance profile of the circulating virus and assessing the phenotypic sensitivity of said chimeric viruses to inhibitors of said enzymes encoded by the pol gene of HIV.

9. A method according to any preceding claim, wherein said biological material is selected from plasma, serum or a cell-free body fluid selected from semen and vaginal fluid.

10. A method according to any one of Claims 1-8, wherein the biological material is whole blood to which an RNA stabiliser has been added.

11. A method according to any preceding claim, wherein the biological material is tissue material selected from brain tissue or lymph nodal tissue.

12. A method according to any one of Claims 8-11, wherein the at least two enzymes are selected from HIV RT, protease and integrase.

13. A method according to any one of Claims 1-6 and 7-12, wherein the cell line susceptible to infection by HIV is a CD4+ T-cell line.

14. A method according to Claim 13, wherein the CD4+ T-cell line is the MT4 cell line or the HeLa CD4+ cell line.

15. A method according to any one of Claims 1-6 and 7-14, wherein the desired region of the patient's pol gene is reverse transcribed using a specific downstream primer.

16. A method according to Claim 15, wherein the sequence to be reverse transcribed is that coding for reverse transcriptase and protease.

17. A method according to Claim 16, wherein the downstream primer is OUT3: 5'-CAT TGC TCT CCA ATT ACT GTG ATA TTT
CTC ATG-3' (SEQ ID NO: 1).

18. A method according to any one of Claims 15-17, wherein the product of reverse transcription is amplified using a nested PCR
technique.

19. A method according to any one of Claims 1-6 and 7-18, wherein the HIV-DNA construct is one from which the RT and protease genes are deleted and is the plasmid pGEMT3-.DELTA.PRT as deposited at the Belgian Coordinated Collections of Microorganisms-BCCM
LMBP-Collection on November 8, 1996 under the number LMBP3590.

20. A method according to any one of Claims 1-6 and 7-19, wherein the transfection is achieved by electroporation.

21. A method according to any one of Claims 1-6 and 7-19, wherein the transfection is achieved by the use of cationic lipids.

22. A method according to any one of Claims 1-6 and 7-21, wherein the phenotypic drug sensitivity of the chimeric viruses to different RT, protease and integrase inhibitors is assessed in an automated cell-based assay.

23. A method according to any one of Claims 1-6 and 7-22, wherein the phenotypic drug sensitivity of the chimeric viruses and of the wild HIV strain to one or more RT, protease or integrase inhibitor(s) is expressed as an inhibitory concentration (IC value).

24. A method according to any one of Claims 1-6 and 7-23, wherein RT inhibitors are selected from nucleoside RT inhibitors such as AZT, ddI, ddC, 3TC, d4T, non-nucleoside RT inhibitors such as loviride, nevirapine and tivirapine, protease inhibitors such as saquinavir, indinavir and ritonavir and integrase inhibitors such as caffeic acid phenylethyl ester (CAPE).

25. A method according to Claim 1 of managing HIV
chemotherapy of patients who are HIV positive, substantially as hereinbefore described and exemplified.

26. A method according to Claim 8 of determining the phenotypic drug sensitivity of individual HIV strains in a patient to inhibitors of at least two enzymes encoded by the pol gene of HIV, substantially as hereinbefore described and exemplified.