MEANS AND METHODS FOR MONITORING NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR ANTIRETROVIRAL THERAPY

PROCEDES ET MOYENS DE SURVEILLANCE DE THERAPIE ANTIRETROVIRALE DES INHIBITEURS DE TRANSCRIPTASE INVERSE DES NON-NUCLEOSIDES

English Abstract

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

French Abstract

La présente invention concerne des tests de susceptibilité et de résistance aux médicaments antiviraux pour la recherche de posologies médicamenteuses convenant au traitement de l'infection par le VIH et au traitement du SIDA. L'invention concerne également des moyens et des procédés permettant de faire un suivi clinique de l'évolution de l'infection par le VIH et sa réponse à la thérapie antirétrovirale, et plus particulièrement la thérapie antirétrovirale des inhibiteurs de transcriptase inverse des non-nucléosides par utilisation d'essais de susceptibilité phénotypiques ou d'essais génotypiques.

Claims

98
What is claimed is:
1. A method of assessing the effectiveness of
non-nucleoside reverse transcriptase antiretroviral therapy
of an HIV-infected patient comprising:
(a) collecting a plasma sample from the HIV-infected
patient; and
(b) evaluating whether the plasma sample contains
nucleic acid encoding HIV reverse transcriptase
having a mutation at colon 236;
in which the presence of the mutation correlates with
decreased susceptibility to delavirdine and little or
no change in nevirapine susceptibility.
2. The method of claim 1, wherein the mutation at colon
236 codes for a leucine.
3. The method of claim 1, wherein reverse transcriptase
has an additional mutations) at colon 103, colon 181
or a combination thereof.
4. The method of claim 3, wherein the mutation at colon
103 encodes an asparagine (N) and the mutation at colon
181 encodes a cysteine C).
5. The method of claim 1, wherein the HIV-infected patient
is being treated with an antiretroviral agent.
6. A method of assessing the effectiveness of
antiretroviral therapy of an HIV-infected patient
comprising:
(a) collecting a biological sample from an
HIV-infected patient; and

99
(b) evaluating whether the biological sample comprises
nucleic acid encoding HIV reverse transcriptase
having a mutation at codon 225;
in which the presence of the mutation correlates with
an increase in delavirdine susceptibility and little or
no change in nevirapine susceptibility.
7. The method of claim 6, wherein the mutated codon 225
encodes a histidine (H).
8. The method of claim 6, wherein the HIV-infected patient
is being treated with an antiretroviral agent.
9. The method of claim 6, wherein the reverse
transcriptase has an additional mutation(s) at codon
103, 181 or a combination thereof.
10. A method of assessing the effectiveness of
antiretroviral therapy of an HIV-infected patient
comprising:
(a) collecting a biological sample from an
HIV-infected patient; and
(b) evaluating whether the biological sample
comprises nucleic acid encoding HIV reverse
transcriptase having a mutation at codon 190;
in which the presence of the mutation correlates with
an increase in delavirdine susceptibility and a
decrease in nevirapine susceptibility.
11. The method of claim 10, wherein the mutation at codon
190 encodes an alanine or a serine.
12. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:

100
(a) introducing a resistance test vector comprising a
patient-derived segment further comprising a
mutation at codon 236 and a mutation at codon 103
and/or 181 and an indicator gene into a host cell;
(b) culturing the host cell from step (a);
(c) measuring the indicator in a target host cell; and
(d) comparing the measurement of the indicator. from
step (c) with the measurement of the indicator
measured when steps (a) - (c) are carried out in
the absence of the candidate antiretroviral drug
compound;
wherein a test concentration of the candidate
antiretroviral drug compound is present at steps (a)
- (c); at steps (b) - (c); or at step (c).
13. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment further comprising a
mutation at codon 225 and a mutation at codon 103
and an indicator into a host cell;
(b) culturing the host cell from step (a);
(c) measuring the indicator in a target host cell; and
(d) comparing the measurement of the indicates from
step (c) with the measurement of the indicator
measured when steps (a) - (c) are carried out in
the absence of the candidate antiretroviral drug
compound;
wherein a test concentration of the candidate
antiretroviral drug compound is present at steps (a)
- (c); at steps (b) - (c) ; or at step (c)

101
14. A method for assessing the biological effectiveness of
candidate HIV antiretroviral drug compound
comprising:

102
(a) introducing a resistance test vector comprising a
patient-derived segment which encodes the reverse
transcriptase having a mutation at colon 236 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; 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 candidate
anti-viral drug compound,
wherein a test concentration of the candidate
anti-viral drug compound is present at steps (a) - (c); at
steps (b) - (c); or at step (c).
15. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment which encodes the reverse
transcriptase having a mutation at colon 225,
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; and

103
(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 candidate anti-viral drug
compound,
wherein a test concentration of the candidate
anti-viral drug compound is present at steps (a) - (c); at
steps (b) - (c); or at step (c).
16. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment which encodes the reverse
transcriptase having a mutation at codon 190, 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; 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 candidate anti-viral drug
compound,
wherein a test concentration of the candidate
anti-viral drug compound is present at steps (a) - (c); at
steps (b) - (c); or at step (c).

104
17. A resistance test vector comprising an HIV
patient-derived segment further comprising reverse
transcriptase having a mutation at colon 190 and an
indicator gene, wherein the expression of the indicator
gene is dependent upon the patient derived segment.
18. A resistance test vector comprising an HIV
patient-derived segment further comprising reverse
transcriptase having a mutation at colon 225 and an
indicator gene, wherein the expression of the indicator
gene is dependent upon the patient derived segment.
19. A resistance test vector comprising an HIV
patient-derived segment further comprising reverse
transcriptase having a mutation at colon 236 and an
indicator gene, wherein the expression of the indicator
gene is dependent upon the patient derived segment.
20. A resistance test vector of claim 17, wherein the
patient-derived segment having a mutation at cotton
190 further comprises a mutation at colon 103.
21. A resistance test vector of claim 18, wherein the
patient-derived segment having a mutation at colon
225 further comprises a mutation at colon 103.
22. The resistance test vector of claim 17, wherein the
patient-derived segment having a mutation at colon
236 farther comprises a mutation at colon 103 and/or
181.
23. A method of assessing the effectiveness of
non-nucleoside reverse transcriptase antiretroviral therapy
of an HIV-infected comprising:

105
(a) collecting a plasma sample from an HIV-infected
patient; and
(b) evaluating whether the plasma sample contains
nucleic acid encoding HIV reverse transcriptase
having a mutation at codon 230;
in which the presence of the mutation correlates with
decreased susceptibility to delavirdine and nevirapine.
24. The method of claim 23, wherein the mutation at colon
230 codes for a leucine (L).
25. The method of claim 23, wherein reverse transcriptase
has an additional mutation(s) at colon 181 or a
combination thereof.
26. The method of claim 25, wherein the mutation at colon
281 encodes a cysteine (C).
27. The method of claim 23, wherein the HIV-infected
patient is being treated with an antiretroviral agent.
28. A method of assessing the effectiveness of
non-nucleoside reverse transcriptase antiretroviral therapy
of an HIV-infected comprising:
(a) collecting a plasma sample from an HIV-infected
patient; and
(b) evaluating whether the plasma sample contains
nucleic acid encoding HIV reverse transcriptase
having a mutation at colon 181;
in which the presence of the mutation correlates with
decreased susceptibility to delavirdine and nevirapine
and little or no change in efavirenz susceptibility.
29. The method of claim 28, wherein the mutation at colon
181 codes for a cysteine (c).

106
30. The method of claim 28, wherein reverse transcriptase
has an additional mutation(s) at codon 98, codon 106,
codon 227 or a combination thereof.
31. The method of claim 30, wherein the mutation at codon
98 encodes a glycine (G), the mutation at codon 106
encodes an alanine (A) and the mutation at codon 227
encodes a leucine (L)
32. The method of claim 28, wherein the HIV-infected
patient is being treated with an antiretroviral agent.
33. A method of assessing the effectiveness of
non-nucleoside reverse transcriptase antiretroviral therapy
of an HIV-infected comprising:
(a) collecting a plasma sample from an HIV-infected
patient; and
(b) evaluating whether the plasma sample contains
nucleic acid encoding HIV reverse transcriptase
having a mutation at codon 188;
in which the presence of the mutation correlates with
decreased susceptibility to delavirdine and nevirapine
and efavirenz.
34. The method of claim 33, wherein the mutation at codon
188 codes for a leucine (L), cysteine (C) or histidine
(H).
35. The method of claim 33, wherein reverse transcriptase
has an additional mutation(s) at codon 138, codon 103,
codon 100 or a combination thereof.
36. The method of claim 35, wherein the mutation at codon
138 encodes an alanine (A), the mutation at codon 103

107
encodes an asparagine (N) and the mutation at codon 100
encodes an isoleucine (I).
37. The method of claim 33, wherein the HIV-infected
patient is being treated with an antiretroviral agent.
38. A method of assessing the effectiveness of
non-nucleoside reverse transcriptase antiretroviral therapy
of an HIV-infected comprising:
(a) collecting a plasma sample from an HIV-infected
patient; and
(b) evaluating whether the plasma sample contains
nucleic acid encoding HIV reverse transcriptase
having a mutation at codon 190;
in which the presence of the mutation correlates with
increased susceptibility to delavirdine and decreased
susceptibility to nevirapine and efavirenz.
39. The method of claim 38, wherein the mutation at codon
190 codes for an alanine (A) or a serine (S).
40. The method of claim 38, wherein reverse transcriptase
has an additional mutation(s) at codon 98, codon 101 or
codon 103 or a combination thereof.
41. The method of claim 40, wherein the mutation at codon
98 encodes a glycine (G), 101 encodes a glutamic acid
(E) and 103 encodes an asparagine (N).
42. The method of claim 38, wherein the HIV-infected
patient is being treated with an antiretroviral agent.

108
43. A method of assessing the effectiveness of
non-nucleoside reverse transcriptase antiretroviral therapy
of an HIV-infected comprising:
(a) collecting a plasma sample from an HIV-infected
patient; and
(b) evaluating whether the plasma sample contains
nucleic acid encoding HIV reverse transcriptase
having a mutation at codon 106;
in which the presence of the mutation correlates with
decreased susceptibility to delavirdine and nevirapine
and little or no change in efavirenz susceptibility.
44. The method of claim 43, wherein the mutation at codon
106 encodes an alanine (A).
45, The method of claim 43, wherein reverse transcriptase
has an additional mutation(s) at codon 227 and codon
189 or a combination thereof.
46. The method of claim 45, wherein the mutation at codon
227 encodes a leucine (L) and 189 encodes a leucine
(L).
47. The method of claim 43, wherein the HIV-infected
patient is being treated with an antiretroviral agent.
48. A method of assessing the effectiveness of
non-nucleoside reverse transcriptase antiretroviral therapy
of an HIV-infected comprising:
(a) collecting a plasma sample from an HIV-infected
patient; and
(b) evaluating whether the plasma sample contains
nucleic acid encoding HIV reverse transcriptase
having a mutation at codon 103;

109
in which the presence of the mutation correlates with
decreased susceptibility to delavirdine and nevirapine
and efavirenz.
49. The method of claim 48, wherein the mutation at codon
103 codes for asparagine (N).
50. The method of claim 48, wherein reverse transcriptase
has an additional mutation(s) at codon 100 or a
combination thereof.
51. The method of claim 50, wherein the mutation at codon
100 encodes an isoleucine (I).
52. The method of claim 48, wherein the HIV-infected
patient is being treated with an antiretroviral agent.
53. A method for assessing the biological effectiveness of
candidate HIV antiretroviral. drug compound comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment further comprising a
mutation at codon 230 and a mutation at codon 181
and an indicator gene into a host cell;
(b) culturing the host cell from step (a);
(c) measuring the indicator in a target host cell; and
(d) comparing the measurement of the indicator from
step (c) with the measurement of the indicator
measured when steps (a) - (c) are carried out in
the absence of the candidate antiretroviral drug
compound;
wherein a test concentration of the candidate
antiretroviral drug compound is present at steps (a)
- (c); at steps (b) - (c); or at step (c).

110
54. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment further comprising a
mutation at codon 181 and a mutation at codon 98
and/or 106 and/or 227 and an indicator gene into
a host cell;
(b) culturing the host cell form step (a);
(c) measuring the indicator in a target host cell; and
(d) comparing the measurement of the indicator from
step (c) with the measurement of the indicator
measured when steps (a) - (c) are carried out in
the absence of the candidate antiretroviral drug
compound;
wherein a test concentration of the candidate
antiretroviral drug compound is present at steps (a)
- (c); at steps (b) - (c); or at step (c).
55. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment further comprising a
mutation at codon 188 and a mutation at codon 138
and/or 103 and/or 100 arid an indicator gene
into a host cell;
(b) culturing the host cell from step (a);
(c) measuring the indicator: in a target host cell; and
(d) comparing the measurement of the indicator from
step (c) with the measurement of the indicator
measured when steps (a) - (c) are carried out in
the absence of the candidate antiretroviral drug
compound;

111
wherein a test concentration of the candidate
antiretroviral drug compound is present at. steps (a)
- (c); at steps (b) - (c) or at step (c).
56. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment further comprising a
mutation

112
at colon 190 and a mutation at colon 98 and/or 101
and/or 103 and an indicator gene into a host cell;
(b) culturing the host cell from step (a);
(c) measuring the indicator in a target host cell; and
(d) comparing the measurement of the indicator form
step (c) with the measurement of the indicator
measured when steps (a) - (c) are carried out in
the absence of the candidate antiretroviral drug
compound;
wherein a test concentration of the candidate
antiretroviral drug compound is present at steps (a)
- (c); at steps (b) - (c); or at step (c).
57. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment further comprising a
mutation at colon 106 and a mutation at colon 227
and/or 189 and an indicator gene into a host
cell;
(b) culturing the host cell from step (a);
(c) measuring the indicator in a target host cell; and
(d) comparing the measurement of the indicator from
step (c) with the measurement of the indicator
measured when steps (a) - (c) are carried out in
the absence of the candidate antiretroviral drug
compound;
wherein a test concentration of the candidate
antiretroviral drug compound is present at steps (a)
- (c); at steps (b) - (c); or at step (c).
58. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:

113
(a) introducing a resistance test vector comprising a
patient-derived segment further comprising a
mutation at codon 103 and a mutation at codon 100
and an indicator gene into a host cell;
(b) culturing the host cell from step (a);
(c) measuring the indicator in a target host cell; and
(d) comparing the measurement of the indicator from
step (c) with the measurement of the indicator
measured when steps (a) - (c) are carried out in
the absence of the candidate antiretroviral drug
compound;
wherein a test concentration of the candidate
antiretroviral drug compound is present at steps (a)
- (c); at steps (b) - (c); or at step (c).
59. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment which encodes the reverie
transcriptase having a mutation at codon 230 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; 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 candidate
anti-viral drug compound,
wherein a test concentration of the candidate
anti-viral drug compound is present at steps (a) - (c); at
steps (b) - (c); or at step (c).

114
60. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment which encodes the reverse
transcriptase having a mutation at codon 227 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; 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 candidate anti-viral
drug compound,
wherein a test concentration of the candidate
anti-viral drug compound is present at steps (a) - (c); at
steps (b) -(c); or at step (c).
61. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment which encodes the reverse
transcriptase having a mutation at codon 188 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; 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 candidate anti-viral drug
compound,

115
wherein a test concentration of the candidate
anti-viral drug compound is present at steps (a) - (c); at
steps (b) - (c); or at step (c).
62. A method for assessing the biological effectiveness of
a candidate HIV antiretroviral drug compound
comprising:
(a) introducing a resistance test vector comprising a
patient-derived segment which encodes the
reversetranscriptase having a mutation at codon
189 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; 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 candidate anti-viral drug
compound,
wherein a test concentration of the candidate
anti-viral drug compound is present at steps (a) - (c); at
steps (b) - (c); or at step (c).
63. A resistant test vector comprising an HIV
patient-derived segment further comprising reverse
transcriptase having a mutation at codon 230 and an
indicator gene, wherein the expression of the indicator
gene is dependent upon the patient derived segment.
64. A resistance test vector comprising an HIV
patient-derived segment further comprising reverse
transcriptase having a mutation at codon 227 and an
indicator gene, wherein the expression of the indicator
gene is dependent upon the patient derived segment.

116
65. A resistance test vector comprising an HIV
patient-derived segment further comprising reverse
transcriptase having a mutation at codon 188 and an
indicator gene, wherein the expression of the indicator
gene is dependent upon the patient derived segment.
66. A resistance test vector comprising an HIV
patient-derived segment further comprising reverse
transcriptase having a mutation at codon 189 and an
indicator gene, wherein the expression of the indicator
gene is dependent upon the patient derived segment.
67. A resistance test vector comprising an HIV
patient-derived segment further comprising reverse
transcriptase having a mutations at codon 181, codon
98, codon 106 and codon 227 or a combination thereof
and an indicator gene, wherein the expression of the
indicator gene is dependent upon the patient derived
segment.
68. A resistance test vector comprising an HIV
patient-derived segment further comprising reverse
transcriptase having a mutations at codon 106, codon
227, and codon 189 or a combination thereof and an
indicator gene, wherein the expression of the indicator
gene is dependent upon the patient derived segment.
69. A resistance test vector comprising an HIV
patient-derived segment further comprising reverse
transcriptase having a mutations at codon 103 and codon
101 and an indicator gene, wherein the expression of
the indicator gene is dependent upon the patient
derived segment.

117
70. The resistance test vector of claim 17, wherein the
patient-derived segment having a mutation at colon 190
further comprises mutations at codon 98 and codon 101
or a combination thereof.
71. The resistance test vector of claim 63, wherein the
patient-derived segment having a mutation at codon 230
further comprises a mutation at codon 181.
72. The resistance test vector of claim 65, wherein the
patient-derived segment having a mutation at codon 188
further comprises mutations at codon 138, codon 103 and
codon 190 or a combination thereof.

Description

CA 02329140 2000-11-24
WO 99161658 PCT/US99/11629
MEANS AND METHODS FOR MONITORING NON-NUCLEOSIDE REVERSE
TRANSCRIPTASE INHIBITOR ANTIRETROVIRAL THERAPY
This application is a continuation-in-.part of U.S. Serial
No. 09/085,148, filed May 26, 1998 and claims the benefit
of U.S. Provisional Application No. 60/124,090, filed
March 12, 1999, the contents of which are hereby
incorporated by reference into this application.
Throughout this application, various references are
referred to within parenthesis. Disclosures of these
publications in their entireties are hereby incorporated by
reference into this application to more fully describe the
state of the art to which this .invention pertains.
Technical Field
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
immunodef iciency syndrome (AIDS). 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 whose infection has become resistant to
a particular antiretroviral drug regimen. This invention
also relates to the screening of candidate antiretroviral
drugs for their capacity to inhibit viruses, selected viral
sequences and/or viral proteins. More particularly, this
invention relates to the determination of non-nucleoside
reverse transcriptase inhibitor resistance using phenotypic
susceptibility tests and/or genotypic tests.
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Background of the Invention
HIV infection is characterized by high rates of viral
turnover throughout the disease process, eventually leading
to CD4 depletion and disease progression. Wei X, Ghosh SP;,
Taylor ME, et al. (1995) Nature 343, 117-122 and Ho DD,
Naumann AU, Perelson AS, et al. (1995) Nature 373, 123-12E~.
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 add~.tive 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.
In an untreated patient, some 101°new viral particles are
produced per day. Coupled with the failure of HIV reverse
transcriptase (RT) to correct transcription errors by
exonucleolytic proofreading, this high level of viral
turnover results in 10' to 105 mutations per day at each
position in the HIV genome. The result is the rapid
establishment of extensive genotypic variation. While soc~e
template positions or base pair substitutions may be more ,
error prone (Mansky LM, Temin HM (1995) J Virol 69, 5087-
5094) (Schinazi RF, Lloyd RM, Ramanathan CS, et al. (1994)
Antimicrob Agents Chemother 38, 268-274), mathematical
modeling suggests that, at every possible single point,
mutation may occur up to 10,000 times per day in infected
individuals.
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
SUBSTITUTE SHEET (RULE 26)

CA 02329140 2000-11-24
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an amino acid substitution may result in change in shape,
size or charge of the active site, substrate binding site
or surrounding regions of the enzyme. Mutants resistant to
antiretroviral agents have been detected at. low levels
before the initiation of therapy. (Mohri H, Singh MK,
Ching WTW, et al. (1993) Proc Natl Acad Sci USA 90, 25-29)
(Najera I, Richman DD, Olivares I, et al. (1994) AIDS R~.~s
Hum Retroviruses 10, 1479-1488) (Najera I, Holguin A,
Quinones-Mateu E, et al. (1995) J Virol 69, 23-31).
However, these mutant strains represent only a small
proportion of the total viral load and may have a
replication or competitive disadvantage compared with wild-
type virus. (Coffin JM (1995) Science 267, 483-489). The
selective pressure of antiretroviral therapy provides these
drug-resistant mutants with a competitive advantage and
thus they come to represent t:he dominant quasispecies
(Frost SDW, McLean AR (1994) ATDS 8, 323-332) (Kellam P,
Boucher CAB, Tijnagal JMGH (1994) J Gen Virol 75, 341-351)
ultimately leading to drug resistance and virologic failure
in the patient.
Non-nucleoside Reverse Transcriptase Inhibitors
Non-nucleoside reverse transcriptase inhibitors (NNRTIs)
are a chemically diverse group of compounds which are
potent inhibitors of HIV-1 RT ~:n vitro. These compounds
include pyridinone derivatives, bis(heteroaryl)piperazines
(BHAPs) such as delavirdine and atevirdine, the
dipyridodiazepinone nevirapine, the thymine derivative
groups TSAO and HEPT, an a-anilino phenylacetamides (a-APA)
compound loviride, and the qu_inoxaline-class inhibitors
such as (HBY-097), the benzodiazE:pin-one and -thione (TIBO)
compounds and the pyridinone derivatives (L-697,661). For
overviews see (DeClercq E. (1996) Rev Med Virol 6, 97-117)
(Emini EA (1996) Antiviral Drug Resistance, ed. DD Richman,
John Wiley & Sons, Ltd. High-level resistance to
individual compounds appears too develop rapidly, often
within a few weeks of initiating monotherapy, frequently
involving only single-point mutations and in many cases
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leading to considerable cross-resistance to other NNRTIs.
Most mutations reported occur in the codon groups 100-lOF3
and 181-190 which encode for the two (~-sheets adjacent to
the catalytic site of the RT enzyme (Kohlstaedt LA, Wang J,
Friedman JM, et al. (1992) Science 256, 1783-90) The NNRT:L
binding pocket, as it has been described, is a hydrophobic
non-substrate binding region of: RT where these agents
directly interact with RT. They inhibit activity by
interfering with mobility of the 'thumb' subdomain, o:r
disrupting the orientation of conserved aspartic acid side
chains essential for catalytic activity (D'Aquilla RT.
(1994) Clin Lab Med 14, 393-423) (Arnold E., Ding J.,
Hughes SH, et al. (1995) Curr Op~in Struct Biol 5, 27-38).
Mutations conferring reduced susceptibility to nevirapine
have been described at codons 98, 100, 103, 106, 108, 181,
188 and 190 (Richman DD, Havlir D, Corbeil J. (1994) J
Virol 68, 1660-1666). The most frequently selected variant
during nevirapine monotherapy is a Tyrlsl Cys (Y181C)
mutation which results in a 100-fold reduction in
sensitivity to this agent, with reduced susceptibility to
the pyridinone derivatives L-696,:229 and L-697,661 (Arnold,
Ibid). TSAO also has limited activity in the presence of
the 181 mutation, but maintains activity in the presence of
mutations at codons 100 and 103 and in vitro selects for a
unique mutation, GLU'38 Lys (E138K), in the region where it
most closely interacts with RT (Richman, DD, Ibid) (Richman
DD, Shih C-K, Lowy I, et al.(1991) Proc Nail Acad Sci USA
88, 11241-11245).
Resistance to loviride when used as monotherapy develops .in
most patients by week 24. It has been mapped to a range of
codons 100-110; 181-190), most commonly codon 103
(Staszewski S, Miller V, Kober A, et al. (1996) Antiviral
Ther 1, 42-50. During combination therapy using loviride
with zidovudine or zidovudine plus lamivudine, variants at
codons 98 and 103 were the most frequent mutations defected
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at 24 weeks (Staszewski S, Miller V, Rehmet S, et al.
(1996) AIDS 10, Fl-7) .
Although the 101, 103 and 181 mutations also confer cross-
5 resistance to BHAPs, (Balzarini J, Karlsson A, Perez-Perez
M-J, et al. (1992) Virology I92, 246-253) the
characteristic P236L substitution selected for by these
agents in vitro appears to sensitize RT to some other
NNRTIs, reducing the IC50 for nevirapine, for example, '7-
to 10-fold, without influencing sensitivity to nucleoside
analogues (Staszewski S., Ibid). This mutation at codon
236 has not been observed in clinical isolates during
atevirdine therapy, although other resistance-conferring
mutations at codons 103 and 181 have been reported during
monotherapy as well as at codons 101, 188, 233 and 2:38
during combination therapy with zidovudine.
While HBY-097 may initially select for a mutation at codons
190 in vitro, further passage consistently selects for
mutations at RT codon 74 and 75, with some mutant viruses
showing decreased sensitivity to didanosine and stavudine,
but not zidovudine (Kleim J-P, Rosner M, Winkler I, et al.
(1995) J Acquir Immune Defic Syndr 10 Suppl 3, 2).
Mutation at codon 181 has bean reported to antagonize
zidovudine resistance due to the typical 41 and 215 codon
mutations, (Zhang D, Caliendo AM, Eron JJ, et al. (1994)
Antimicrob Agents Chemother 38, 282-287) suggesting that
combination therapy with some NNRTIs and zidovudine may be
feasible. Although an HIV mutant with triple resistance to
zidovudine, didanosine and nevirapine has been described in
vitro, (Larder BA, Kellam P, Kemp SD (1993) Nature 365,
451-453) treatment with this triple combination does
provide superior immunological and virological responses to
treatment with zidovudine plus didanosine alone over a 48
week period in patients with CD4 cell counts <350/mm3.
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Combination therapy with zidovudine and the pyridinone
derivative L-697,661 prevents the appearance of the codon
181 mutation typically selected during monotherapy with
this NNRTI, delaying the appearance of high-level
resistance to this compound. Changes in susceptibility to
zidovudine were not examined in this study. (Staszewski
S, Massari FE, Kober A, et al. (1995) J Infect Dis 171,
1159-1165). Concomitant or alternating zidovudine therapy
does not delay the appearance of resistance during
nevirapine therapy; (Richman DD, Ibid) (Nunberg JH, Schleif
WA, Boots EJ, et al. (1990) J Viro1 65, 4887-4892) (DeJong
MD, Loewenthl M, Boucher CAB, et al. (1994) J Infect Dis
169, 1346-1350) (Cheeseman SH, Havlir D, McLaughlin MM, e:t
al. (1995) J Acquir Tmmune Defic Syndr 8, 141-151) however,
the 181 mutant is not being observed during combination,
the most common change being at codon 190 (Richman DL),
Ibid). This suggests that the codon 181 mutation which is
antagonistic to zidovudine resistance in vitro is not
compatible, or not preferred in vivo, selection favoring
other mutations which allow for reduced susceptibility to
this NNRTI concomitant with zidovudine resistance.
The rapid development of reduced susceptibility to the
NNRTIs suggests limited utility of these agents,
particularly as monotherapies, and has led to the
modification of these molecules in an attempt to delay tine
appearance of drug-resistant virus. A 'second generation'
NNRTI, the pyridinone derivative L-702,019, demonstrated
only a 3-fold change in ICso between wild-type and codon 181
mutant HIV-1, and required multiple mutations to engender
high-level resistance (Goldman ME, O'Brien JA, Buffing TL,
et al. (1993) Antimicrob Agents Chemother 37, 947-949).
It is an object of this invention to provide a drug
susceptibility and resistance test capable of showing
whether a viral population in a patient is resistant to a
given prescribed drug. Another object of this invention is
to provide a test that will enable the physician to
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substitute one or more drugs in a therapeutic regimen for
a patient that has become resistant to a given drug or
drugs after a course of therapy. Yet another object of
this invention is to provide a test that will enable
selection of an effective drug regimen for the treatment of
HIV infections and/or AIDS. Yet another object of this
invention is to provide the means for identifying the drugs
to which a patient has become resistant, in particular
identifying resistance to non-nucleoside reverse
transcriptase inhibitors. Still another object of this
invention is to provide a test and methods for evaluating
the biological effectiveness of candidate drug compounds
which act on specific viruses, viral genes and/or viral
proteins particularly with respect to viral drug resistance
associated with non-nucleoside reverse transcriptase
inhibitors. It is also an object of this invention to
provide the means and compositions for evaluating HIV
antiretroviral drug resistance and susceptibility. This
and other objects of this invention will be apparent from
the specification as a whole.
_Summary of the Invention
The present invention relates to methods of monitoring,
using 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 reverse transcriptase (RT) which confer resistance to
antiretroviral therapy may be rapidly determined directly
from patient plasma HIV RNA using phenotypic or genotypic
methods. The methods utilize polymerase chain reaction
(PCR) based assays. Alternatively, methods evaluating
viral nucleic acid of viral protein in the absence of an
amplification step could utilize the teaching of this
invention to monitor and/or modify antiretroviral therapy.
This invention is based in part on the discovery o:f a
mutation at codon 225 either alone or in combination with
a mutation at codon 103 of HIV reverse transcriptase in
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non-nucleoside reverse transcriptase inhibitor (efavirenz)
treated patients) in which the presence of the mutations
correlate with an increase in delavi.rdine susceptibility
and little or no change in nevirapine susceptibility. The
mutations were found in plasma ~iIV RNA after a period of
time following initiation of therapy. The development of
the mutant at colon 225 in addition to the mutation at
colon 103 in HIV RT was found t:o be an indicator of the
development of resistance and ultimately of immunological
decline. This invention is based in part on the discovery
of a mutation at colon 236 of RT was discovered to occur in
non-nucleoside reverse transcriptase inhibitor (NNRTI)
treated patients in which the presence of the mutation
correlates with decreased susceptibility to delavirdine and
no reduction in nevirapine susceptibility. The development
of the colon 190 and 103 and/or 101 mutations in HIV RT was
found to be an indicator of the development of alterations
in phenotypic susceptibility/resistance which has been
associated with virologic failure and subsequent
immunological decline. This invention is based in part on
the discovery of a mutation at colon 190 either alone or i.n
combination with a mutation at colon 190 either alone or i.n
combination with a mutation at colon 103 and/or 101 of HI:V
reverse transcriptase in non-nucleoside reverse
transcriptase inhibitor (efavirenz) treated patients) i.n
which the presence of the mutations correlate with an
increase in delavirdine susceptibility and a decrease in
nevirapine susceptibility. The: mutations were found in
plasma HIV RNA after a period of time following initiation
of NNRTI therapy. The development of the colon 236 and lU3
and/or 181 mutations in HIV RT was found to be an indicator
of the development of alterations in phenotypic
susr_eptibility/resistance which has been associated with
virologic failure and subsequent. immunological decline.
This invention is based in part on the discovery of a
mutation at colon 230 either alone or in combination with
a mutation at ccdon 181 of HIV' reverse transcriptase in
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non-nucleoside reverse transcriptase inhibitor (nevirapine)
treated patients) in which the presence of the mutations
correlate with a significant dec:rease in both delavirdine
and nevirapine susceptibility. The mutations were found i.n
plasma HIV RNA after a period of time following initiation
of NNRTI theraphy. The development of the codon 230 and
181 mutations in HIV RT were found to be an indicator of
the development of alterations in phenotypic
susceptibility/resistance which has been associated with
virologic failure and subsequent immunological decline.
This invention is based in part on the discovery of a
mutation at codon 181 of HIV reverse transcriptase in non-
nucleoside reverse transcriptase inhibitor (nevirapinE:)
treated patients) in which the presence of the mutation
correlates with a moderate decrease in delavirdine
susceptibility and a significant decrease in nevirapine
susceptibility and no change in efavirenz susceptibility.
The mutation was found in plasma HIV RNA after a period of
time following initiation o:f NNRTI therapy. T:he
development of the codon 181 mutation .in HIV RT was found
to be an indicator of the development of alterations in
phenotypic susceptibiltiy/resi.stance which has been
associated with virologic failure and subsequent
immunological decline. This invention is based in part on
the discovery of a mutation at codon 188 of HIV reverse
transcriptase in non-nucleoside reverse transcripta.se
inhibitor (efavirenz) treated patients) in which the
presence of the mutation correlates with a slight decrease
in delavirdine susceptibility and a substantial decrease in
nevirapine susceptibility. The mutation was found in
plasma HIV RNA after a period o:f time following initiation
of NNRTI therapy. The development of the codon 7_88
mutation in HIV RT was found to be an indicator of t:he
development of alterations in phenotypic
susceptibility/resistance which has been associated with
viologic failure and subsequent immunological decline.
This invention is based in part on the discovery lof a
mutation at codon 188 of HIV reverse transcriptase in
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patients) with no previously reported exposure to non-
nucleoside reverse transcriptase inhibitors in which the
presence of the mutations correlate with a moderate
decrease in delavirdine susceptibility and a substatial
5 decrease in nevirapine susceptibility and a maderate
decrese in efavirenz susceptibility. The mutation was
found in plasma HIV RNA after a period of time following
initiation of anti-retroviral th~°_rapy. The development of
the codon 138 and 188 mutations in HIV RT was found to be
10 an indicator of the development of alterations in
phenotypic susceptibility/resistance which has been
associated with virologic failure and subsequent
immunological decline. This invention is based in part on
the discovery of a mutation at codon 98 of HIV reverse
transcriptase in patients) wir_h no previously reported
exposure to non-nucleoside reverse transcriptase inhibtors
in which the presence of the mutation correlates with
slight decrease in delavirdine, nevirapine and efavirenz
susceptibility. The mutation was found in plasma HIV RNA
after a period of time following initiation of ant:i-
retroviral therapy. The devE:lopment of the codon 98
mutation in HIV RT was found t:o be an indicator of the
development of alterations in phenotypic
susceptibility/resistance which has been associated with
virologic failure and subsequenr_ immunological decline.
This invention is based in part on the discovery of a
mutation at codon 98 either alone or in combination with a
mutation at codon 190 of HIV reverse transcriptase, in
patients) whose anti-retroviral treatment was unknown in
which the presence of the mutations correlate with an
increase in delavirdine susceptibility and substantial
decrease in both nevirapine and efavirenz susceptibilti.y.
The mutations were found in plasma HIV RNA. The
development of the mutant at codon 98 in addition to t: he
mutation at codon 190 in HI'J RT was found to be an
indicator of the development of resistance and ultimately
of immunological decline. This invention is based in part
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on the discovery cf a mutation at codon 181 either alone or
in combination with a mutation at codon 98 of HIV reverse
transcriptase in non-nucleoside reverse transcr:iptase
inhibitor (delavirdine) treated patients) in which the
presence of the mutations correlate with an significant
decresase in delavirdine susceptibility and a substantial
decrease in efavirenz susceptibwlity. The mutations were
found in plasma HIV RNA sfter a period of time following
initiation of therapy. The development of the mutant at
codon 98 in addition to the mutation at codon 181 in HIV RT
was found to be an indicator of the development of
resistance and ultimately of immunological decline. This
invention is based in part on the discovery of a mutation
at codon 101 either alone or in combination with a mutation
at codon 190, for example 190s of HIV reverse transcriptase
in non-nucleoside reverse transcriptase inhibitor
(efavirenz) treated patients) in which the presence of the
mutations correlate with no change in delavirdine
susceptibiltiy and a substantial decrease in both
nevirapine and efavirenz susceptibiltiy. The mutations
were found in plasma HIV RNA after a period of time
following initiation of therapy. The development of the
mutant at codon 101 in addition to the mutation at codon
190, for example 190s in HIV RT was found to be an
indicator of the development of resistance and ultimately
of immuological decline. This invention is based in part
on the discovery of a mutation at codon 108 of HIV reverse
transcriptase in patients) with no previously reported
exposure to non-nucleoside reverse transcriptase inhibitor
in which the presence of the mutation correlates with no
change in delavirdine susceptibility and a slight decrease
in nevirapine susceptibility and no change in efavirenz
susceptibility. The mutation was found in plasma HIV RNA
after a period of time following initiation of anti-
retroviral therapy. The development of the codon 1.08
mutation in HIV RT was found to be an indicator of the
development of alterations in phenotypic
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susceptibility/resistance which has been associated with.
virologic failure and subsequent immunological decline.
This invention is based in part. on the discovery of a
miutation at codon 101 either alone or in combination with
a mutation at codon 103 and/or 190 of HIV reverse
transcriptase in patients with no previously reported
exposure to non-nucleoside reverse transcriptase inhibitors
in which the presence of the mutatins correlate with
changes in delavirdine, nevirapine and efavirenz
susceptibility. Specifically, the: presence of mutations a't
101 and 190, for example 190A, correlates with no change in
delavirdine susceptibility and a substantial decrease in
nevirapine susceptibility and a significant decrease i.:n
efavirenz susceptibility. The presence of mutations at 103
and 190 correlates with a moderate decrease in delavirdine
susceptibility, a substantial decrease in nevirapine
susceptibiltiy and a significant decresase in efavirenz
susceptibility. The mutations were found in plasma HIV RNA
after a period of time following initiation of anti-
retroviral therapy. The development of the codon 101 and
103 and/or 190 mutations in HIV RT was found to be an
indicator of the development of alterations in phenotypic
susceptibiltiy/resistance which has been associated with
virologic failure and subsequent immunological decline.
This invention is based in part on the discovery of a
mutation at codon 106 either alone or in combination with
a mutation at codon 189 and/or :181 and 227 of HIV reverse
transcriptase in non-nucleoside reverse transcripta:~e
inhibitor (nevirapine) treated patients) in which the
presence of the mutations correlate with changes :in
delavirdine, nevirapine and efavirenz susceptibility.
Specifically, the presence of mutations at 106 and 181
correlates with a significant decrease in delavirdi:ne
susceptibility, a substantial decresase in neviradine
susceptibility and a slight decrease in efavirenz
susceptibility. The presence of mutations at 106 and 189
correlates with a slight decrease in delavirdine
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susceptibility, a moderate ciecresase in nevirapine
susceptibitlity and no change in efavirenz susceptibility.
The presence of mutations at 106 and 227 correlates with a
slight decrease in delavirdine susceptibility, a
substantial decresase in nevirapine susceptibility and a
slight decrease in efavirenz susceptibility. The presence
of mutations at 181 and 227 correlates with an increase in
delavirdine susceptibility, a significant decrease in
nevirapine susceptibility and an incease in efavirenz
susceptibility. The presence of mutations at 106 and 1E31
and 227 correlates with a moderate decrease in delavirdine
susceptibility , a substantial decrease in nevirapine
susceptibility and a slight decrease in efavirenz
susceptibility. The mutations were found in plasma HIV R~1A
after a period of time following initiation of NNR'TI
therapy. The development of the codon 106 and 189 and/or
181 and 227 mutations in HI~~ RT was found to be an
indicator of the development of alterations in phenotypic
susceptibility/resistance which. has been associated with
virologic failure and subsequent immuological decline.
This invention is based in part on the discovery of a
mutation at codon 103 either alone or in combination with
a mutation at codon 100 and/or 188 of HIV reverse
transcriptase in non-nucleo=;ide reverse transcriptase
inhibitor ~nevirapine) treated patients) in which t:he
presence of the mutations correlate with changes in
delavirdine, nevirapine and efavirenz susceptibility.
Specifically, the presence of mutations at 103 and 188
correlates with a substantial decrease in delavird:ine
susceptibility, a substantial decrease in nevirap:ine
susceptibility and a substantial decrease in efaviranz
susceptibility. The presence of mutations at 100 and 103
correlates with a substantial decrease in delavirdine
susceptibility, a moderate decrease in nevirapine
susceptibility and a substantial decrease in efavirenz
susceptibility. The presence of mutations at 103 and 100
and 188 correlates with a substantial decrease in
delavirdine susceptibility, a substantial decrease in
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nevirapine susceptibility and a substantial decrease in
efavirenz susceptibility. The mutations were found in
plasma HIV RNA after a period of time following initiation
of NNRTI therapy. The developemnt of the codon 103 and 100
and/or 188 mutations in HIV RT was found to be an indicator
of the development of alterations in phenotypic
susceptibility/resistance which has been associated with
virologic failure and subsequent immunological decline.
In a further embodiment of the invention, PCR based assay:,
including phenotypic and genoty~>ic assays, may be used t:o
detect mutations at codon 225 in combination with mutations
at other codons including 103 of HIV RT which correlate
with a specific pattern of resistance to antiretroviral
therapies and subsequent immunologic decline. In yet
another embodiment of the invention, PCR based assays,
including phenotypic and genotypic assays, may be used i=o
detect mutations at codon 236 either alone or in
combination with mutations at other codons including 103
and/or 181 of HIV RT which correlate with resistance to
antiretroviral therapy and immunologic decline. In yet
another embodiment of the invention, PCR based assays,
including phenotypic and genotypic assays, may be used to
detect mutations at codon 190 (G190S) either alone or in
combination with mutation at codon 101 (K101E) of HIV RT
which correlates with resistancE: to antiretroviral therapy
and immunologic decline.
In still another embodiment of the invention, PCR based
assays, including phenotypic and genotypic assays, may be
used to detect mutations at codon 190 (G190A) either alone
or in combination with mutation at codon 103 (K103N) of FiIV
RT which correlates with resistance to antiretroviral
therapy and immunologic decline.
In yet another embodiment of the invention, PCR based
assays, including phenotypic and genotypic assays, may be
used to detect mutations at codon 230 either alone or in
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combination with mutation at codon 181 of HIV RT which
correlates with resistance to antiretroviral therapy and
immunologic decline.
5 In yet another embodiment of t:he invention, PCR based
assays, including phenotypic and genotypic assays, may be
used to detect a mutation at codon 181 of HIV RT which
correlates with resistance to antiretroviral therapy and
immunologic decline.
In yet another embodiment of the invention, PCR based
assays, including phenotypic and. genotypic assays, may be
used to detect a mutation at codon 188 of HIV RT which
correlates with resistance to antiretYoviral therapy anal
immunologic decline.
In yet another embodiment of the invention, PCR based
assays, including phenotypic and genotypic assays, may be
used to detect mutations at codon 138 either alone or in
combination with mutation at codon 188 of HIV RT which
correlates with resistance to antiretroviral therapy and
immunologic decline.
In yet another embodiment of the invention, PCR based
assays, including phenotypic and genotypic assays, may be
used to detect a mutation at codon 98 of HIV RT which
correlates with resistance to antiretroviral therapy and
immunologic decline.
In yet another embodiment of the invention, PCR based
assays, including phenotypic and genotypic assays, may be
used to detect mutations at codon 98 either alone or in
combination with mutation at codon 190 of HIV RT which
correlates with resistance to antiretroviral therapy and
immuolgoic decline.
In yet another embodiment of the invention, PCR based
assays, including phenotypic and genotypic assays, may be
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used to detect mutations at colon 181 either alone or i.n
combination with mutation at c~odon 98 of HIV RT whic:h
correlates with resistance to antiretroviral therapy and
immunologic decline.
In yet another embodiment of the invention, PCR based
assays, including phenotypic and genotypic assays, may be
used to detect mutations at colon 1.01 either alone or in
combination with mutation at colon 190, for example 190s of
HIV RT which correlates with resistance to antiretrovir<~1
therapy and immunologic decline.
In yet another embodiment of the invention, PCR based
assays, including phenotypic and genotypic assays, may be
used to detect a mutation at colon 108 of HIV RT which
correlates with resistance to antiretroviral therapy and
immunologic decline.
In yet another embodiment of the invention, PCR based
assays, including phenotypic and genotypic assays, may be
used to detect mutations at colon 101 either alone or in
combination with mutations at colon 103 and/or 190 of HIV
RT which correlates with re:~istance to antiretoviral
therapy and immunologic decline.
In yet another embodiment of the invention, PCR based
assays, including phenotypic and genotypic assays, may be
used to detect mutations at colon 106 either alone or in
combination with mutations at colon 189 and/or 181 and 227
of HIV RT which correlates with resistance to
antiretroviral therapy and immunologic decline.
In yet another embodiment of the invention, PCR based
assays, including phenotypic and genotypic assays, may be
used to detect mutations at colon 188 either alone or in
combination with mutation at colon 100 and /ar 103 of HIV
RT which correlates with resistance to antiretroviral
therapy and immunologic declaine. Once mutations at colon
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225 and 103 have been detected in a patient undergoing
NNRTI antiretroviral therapy, an alteration in the°_
therapeutic regimen must be con:~idered. Similarly, once
mutations at colon 236 and/or 103 and/or 181 have been
detected i.n a patient undergoing certain NNRTI
antiretroviral therapy, an alteration in the therapeutic
regimen must be considered. Similarly, once mutations at
colon 190 and/or 103 and/or 101 have been detected in a
patient undergoing certain NNRTI antiretroviral therapy, an
alteration in the therapeutic regimen must be considered.
Similarly, once mutations at colon 230 and/or 181 have been
detected in a patient undergoing certain NNRTI
antiretroviral therapy, an alteration in the therapeutic
regimen must be considered. Similarly, once a mutation a.t
colon _181 has been detected in a patient undergoing certain
NNRTI antiretroviral therapy, an alteration in the
therapeutic regimen must be considered. Similarly, once a
mutation at colon 188 has been detected in a patient
undergoing certain NNRTI antiretroviral therapy, an
alteration in the therapeutic regimen must be considered.
Similarly, once mutations at colon 138 and/or 188 have been
detected in a patient undergoing certain NNRTI
antiretroviral therapy, an alteration in the therapeutic
regimen must be considered. Similarly, once a mutation at
colon 98 has been detected in a .patient undergoing certain
NNRTI antiretroviral therapy, an alteration in the
therapeutic regimen must be considered. Similarly, once
mutations at colon 98 and/or 190 have been detected in a
patient undergoing certain NNRTI antiretroviral therapy, an
alteration in the therapeutic regimen must be considered.
Similarly, once mutations at colon 181 and/or 98 have been
detected in a patient undergoing certain NNRTI
antiretroviral therapy, an alteration in the therpeutic
regimen must be considered. Similarly, once mutations at
colon 101 and/or 190, for example 1905, have been detected
in a patient undergoing certain NNRTI antiretroviral
therapy, an alteration in the therapeutic regimen must be
considered. Similarly, once a mutation at colon 108 has
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been detected in a patient underfoing certain NNRT:I
antiretroviral therapy, an alteration in the therapeuti~~
regimen must be considered. Similarly, once mutations at
codon 101 and/or 103 and/or 190, for example 190A, have
been detected in a patient undergoing certain NNRTI
antiretroviral therapy, an alteration in the therapeutic
regimen must be considered. Similarly, once mutations at
codon 106 and/or 189 and/or 181 and/or 227 have been
detected in a patient undergoing certain NNRTI
antiretroviral therapy, an alteration in the therapeutic
regimen must be considered. Similarly, once mutations at
codon 188 and/or 100 and/or 103 have been detected in a
patient undergoing certain NNRTI antiretroviral therapy, a.n
alteration in the therapeutic regimen must be considered.
The timing at which a modification of the therapeutic
regimen should be made, following the assessment of the
antiretroviral therapy using PCR based assays, may depend
on several factors including the patient's viral load, CD4
count, and prior treatment history.
In another aspect of the invention there is provided a
method for assessing the effectiveness of a non-nucleoside
reverse transcriptase 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 NNRTI anti-HIV
drug, wherein a test concentration of the NNRTI, anti-HIV
drug is presented at steps (a) - (c) ; at steps (b) - (c) ;
or at step (c) .
This invention also provides a method for assessing the
effectiveness of non-nucleoside reverse transcriptase
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antiretroviral therapy in a patient comprising: (a)
developing a standard curve of drug susceptibility for an
NNRTI anti-HIV drug; (b) determining NNRTI anti-HIV drug
susceptibility in the patient using the susceptibility test
described above; and (c) comparing the NNRTI anti-HIV drug
susceptibility in step (b) with the standard curve
determined in step (a), wherein a decrease in NNRTI anti-
HIV susceptibility indicates development of anti-HIV drug
resistance in the patient.
This invention also provides a method for evaluating the
biological effectiveness of a candidate HIV antiretroviral
drug compound 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 indicate>r
gene from step (c) with the expression of the indicator
gene measured when steps (a) - (c) are carried out in the
absence of the candidate anti-viral drug compound, wherein
a test concentration of the <:andidate anti-viral drug
compound is present 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 cell is ultimately dependent upon the
action of the patient-derived segment sequences. The
indicator gene may be functional. or non-functional.
In another aspect 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.
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In yet another aspect 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. In another
5 aspect, the invention is directed to a novel resistance
test vector comprising a patient-derived segment further
comprising one or more mutations on the RT gene and an
indicator gene.
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Brief Description of the Drawincts
Fig. 1
Resistance Test Vector. A diagrammatic representation of
the resistance test vector comprising a patient derived
segment and an indicator gene.
Fig. 2
Two Cell Assay. Schematic Representation of the Assay. 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. Pseudotyped viral
particles are produced containing the protease (PR) and the
reverse transcriptase (RT) gene products encoded by the
patient-derived 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, on the
other hand, are added to the cells at the time of or prior
to viral particle infection. ThE: assay is performed in t:he
absence of drug and in the presence of drug over a wide
range of concentrations. The amount of luciferase is
determined and the percentage (4) inhibition is calculated
at the different drug concentrations tested.
Fig. 3
Examples of phenotypic drug susceptibility profiles. Data
are analyzed by plotting th.e percent inhibition of
luciferase activity vs. logy concentration (uM). This plot
is used to calculate the drug concentration that is
reauired to inhibit virus replication by 50% (ICSO) or by
950 (IC95). Shifts in the ~~nhibition curves towards higher
drug concentrations are interpreted as evidence of drug
resistance. Three typical curves for a nucleoside reverse
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transcriptase inhibitor (AZT), a non-nucleoside reverse
transcriptase inhibitor (delavirdine), and a protease
inhibitor (ritonavir) are shown. A reduction in drug
susceptibility (resistance) is reflected in a shift in the
drug susceptibility curve toward higher drug concentrations
(to the right) as compared to a baseline (pre-treatment)
sample or a drug susceptible virus control, such as PNL4-3
or HXB-2, when a baseline sample is not available.
Fig. 4
Phenotypic drug susceptibility and resistance profile:
patient 487. A PCR-based phenotypic susceptibility assay
was carried out giving the phenotypic drug susceptibility
and resistance profile showing increased resistance to both
delavirdine and nevirapine. This is an example of the
first pattern of NNRTI susceptibility/resistance.
Evaluation of this virus from plasma showed HIV reverse
transcriptase having mutations at codons 184 (M184V)
associated with 3TC resistance and at 103 (K103N)
associated with both delavirdine and nevirapine resistance.
Fig. 5
Phenotypic drug susceptibility and resistance profile of
site directed reverse transcriptase mutants. A PCR-based
phenotypic susceptibility assay was carried out giving t:he
phenotypic drug susceptibility and resistance profile for
site directed mutants having mutations at codons 103 a:nd
181 (K103N; Y181C) demonstrating resistance to both
delavirdine and nevirapine. The double mutant demonstrates
the additive effect of both mutations resulting in a
further increase in resistance.
Fig. 6
Phenotypic drug susceptibility and resistance profile:
Patient 268. A PCR-based phenotypic susceptibility assay
was carried out giving the phenotypic drug susceptibility
and resistance profile showing the evaluation of virus from
plasma with HIV reverse transcriptase having phenotypic
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resistance to delavirdine but not nevirapine. This is an
example of the second pattern of NNRT:L
susceptibility/resistance. This f>atient virus is resistant
to all of the protease inhibitors tested and also has
significant resistance to AZT and 3TC and shows slight
shifts in susceptibility to ddC, ddI, and d4T. Evaluation
of this virus from plasma using a PCR and sequencing based
genotypic assay showed HIV reverse transcriptase having
mutations at codons 103 and 236 (K103N; P236L). The P236L
mutation was previously reported to cause delavirdine
resistance and nevirapine hypersensitivity (Dueweke TJ et
al. (1993) Proc Natl Acad Sci 90,. 4713-4717). However, in
this patient sample, while there was delavirdine resistance
nevirapine susceptibility was the same as wild type.
Fig. 7
Phenotypic drug susceptibility and resistance profile of
site-directed reverse transcriptase mutant (P236L). A PCR-
based phenotypic susceptibility assay was carried out
giving the phenotypic drug susceptibility and resistance
profile showing the susceptibility to delavirdine and
nevirapine of the P236L site-directed mutagenesis mutant.
This result is identical to that observed in the patient
virus sample shown in Figure 6. The next two panels show
the K103N site-directed mutagenesis mutant and the two
panels below show the double mutant K103N + P236L. The
P236L mutation is additive to the K103N causing severe
resistance to delavirdine while having no effect on
nevirapine resistance due to K103N. The right side of the
figure shows a similar result when the P236L mutation is
added to the Y181->C mutation.
Fig. SA
Phenotypic Drug Susceptibility and Resistance Profile:
Patients 302. This is one example of the third pattern of
NNRTI susceptibility/resistance. Phenotypic analysis of
the patient virus demonstrated reduced susceptibility to
both delavirdine and nevirapine. This pattern is
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characterized by a larger reduction of nevirapin.e
susceptibility compared to the reduction of delavirdine
susceptibility. Genotypic analysis of the patient virus
demonstrated the presence of the RT mutations K103N
associated with nevirapine and <ielavirdine resistance arLd
P225H.
Fig. 8B
Phenotypic Drug Susceptibility and Resistance Profile:
Patients 780. This is a second example of the third
pattern of NNRTI susceptibility/resistance. Phenotypic
analysis of the patient virus demonstrated reduced
susceptibility to both delavirdine and nevirapine. This
pattern is characterized by a larger reduction of
nevirapine susceptibility compared to the reduction of
delavirdine susceptibility. C~enotypic analysis of t;he
patient virus demonstrated the presence of the RT mutations
K103N associated with nevirapine and delavirdine resistance
and P225H.
Fig. 8C
Phenotypic Drug Susceptibility and Resistance Profile:
Individual Virus Clones of Patient 302. Genotypic analysis
of individual virus clones from patient 302 revealed
viruses containing the K103N mutation without the P225H
mutation (K103N, I135M, R211K) and viruses containing the
K103N mutation with the P225H mutation (K103N, P225H:).
Phenotypic characterization of these virus clones indicates
that the P225H mutation reduces the amount delavirdine
resistance associated with the K103N mutation (compare
bottom panels), but does not alter the amount of nevirapine
resistance associated with the K103N mutation (compare t:op
panels) .
Fig. 8D
Phenotypic Drug Susceptibility and Resistance Profile: Site
Directed Reverse Transcriptase Mutants. Phenotypic
characterization of a virus cont:ainirig the site directed RT
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mutation P225H indicates that this mutation increases
susceptibility to delavirdine, but not nevirapine (compare
top panels). Phenotypic characterization of a virus
containing the site directed RT mutations P225H plus KO1~N
5 or P225HV plus Y181C indicate that the P225H mutation
decreases the amount of delavirdine resistance associated
with either K103N or Y181C, but does not decrease the
amount of nevirapine resistance associated with K103N or
Y181C. to delavirdine, but not nevirapine (compare
10 corresponding middle and bottom panels).
Fig_ 9A
Phenotypic Drug Susceptibility and Resistance Profile:
Patients 644. This is one example of the fourth pattern of
15 NNRTI susceptibility and resistance. Phenotypic analysis
of the patient virus demonstrated by a large reduction in
susceptibility to nevirapine,, but not delavirdine.
Genotypic analysis of the patient virus demonstrated the
presence of the RT mutations G190S, as well as the K101E
20 mutation associated with reductions in susceptibility to
atevirdine, DMP266, L-697,661 and UC-10,38,57 (Schinazi,
Mellors, Larder resistance table).
Fig. 9B
25 Phenotypic Drug Susceptibility and Resistance Profile: Site
Directed Reverse Transcriptase Mutants. Phenotypic
characterizations of viruses containing either site
directed RT mutations G190A, oz' G190S indicate that these
mutations greatly reduce susceptibility to nevirapine, and
slightly increase susceptibility to delavirdine (compare
top panels).
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Detailed Description of the Invention
The present invention relates to methods of monitoring the
clinical progression of HIV infection in patients receiving
antiretroviral therapy, particularly non-nucleoside reverse'
transcriptase inhibitor antiretroviral therapy.
In one embodiment, the present invention provides for <~
method of assessing the effectiveness of antiretrovira:l
therapy of a patient comprising (s_) collecting a biological
sample from an HIV-infected patient; and (ii) determining
whether the biological sample comprises nucleic acid
encoding HIV RT having a mutation at one or more positions
in the RT. The mutations) correlate positively with
alterations in phenotypic susceptibility/resistance.
In a specific embodiment, the invention provides for a
method of assessing the effectiveness of NNRTI
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 RT having a mutation at
codon 225 and 103. This invention established, using a
phenotypic susceptibility assays, that mutations at codon
225 either alone or in combination with a mutation at codon
103 of HIV reverse transcriptase are correlated with an
increase in delavirdine -susceptibility, little or no chance
in nevirapine susceptibility and little or no change in
efavirenz susceptibility.
In another specific embodiment, the invention provides for
a method of evaluating the effectiveness of NNR'rI
antiretroviral therapy of a patient comprising (:i)
collecting a biological sample from an HIV-infected
patient; and (ii) determining whEaher the biological sample
comprises nucleic acid encoding HIV RT having a mutation at
codon(s) 236 and 103 and/or 181. This invention
established, using a phenotypic susceptibility assay, that
mutations at codon 236 either alone or in combination with
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a mutation at codon 103 and/or 181 of HIV reverse
transcriptase are correlated with a decrease in delavirdine
susceptibility (increased resistance) and no change in
nevirapine susceptibility. The 236 mutation alone or on a
Y181C background has no effect on efavirenz susceptibility
but restores a significant portion of the loss of
susceptibility caused by a 103N mutation.
In another specific embodiment, t:he invention provides for
a method of evaluating the effectiveness of NNRTI
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 RT having a mutation at
codon(s) 230 and/or 181. This invention established, using
a phenotypic susceptibility assay, that mutations at codon
230 either alone or in combination with a mutation at codon
181 of HIV reverse transcriptase are correlated with a
significant decrease in delavirdine susceptibility
(increased resistance), significant decrease in nevirapine
susceptibility.
In another specific embodiment, the invention provides for
a method of evaluating the effectiveness of NNR'rI
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 RT having a mutation at
codon 181. This invention established, using a phenotypic
susceptibility assay, that a mutation at codon 181 of HIV
reverse transcriptase is correlated with a moderate
decrease in delavirdine susceptibility (increased
resistance), significant decrease in nevirapine
susceptibility and no change in efavirenz susceptibility.
In another specific embodiment, the invention provides for
a method of evaluating the effectiveness of NNRTI
antiretroviral therapy of a patient comprising (i)
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collecting a biological sample from an HIV-infected
patient, and (ii) determining whether the biological sample
comprises nucleic acid encoding HIV RT having a mutation at
colon 188. This invention established, using a phenotypic
susceptibility assay, that a mutation at colon 188 of HIV
reverse transcriptase are correla~ed with a slight decrease
in delavirdine susceptibility ;increased resistance), a
substantial decrease in nevira~>ine susceptibility and a
significant decrease in efavirenz susceptibility.
In other specific embodiment, the invention provides for a
method of evaluating the effectiveness of NNRTI
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 H:IV RT having a mutation a t
codon(s) 138 and/or 188. This invention established, using
a phenotypic susceptibility assay, that mutations at colon
138 either alone or in combination with a mutation at
colon 188 of HIV reverse transcriptase are correlated with
a moderate decrease in delavirdine susceptibility
(increased resistance), a ;substantial decrease :in
nevirapine susceptibility and a moderate decrease in
efavirenz susceptibility.
In another specific embodiment, the invention provides for
a method of evaluating the effectiveness of NNRTI
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 RT having a
mutation at codon(s) 98. This invention established, using
a phenotypic susceptibility assays, that mutations at colon
98 of HIV reverse transcriptase are correlated with a
slight decrease in delavirdine susceptibility (increase
resistance), a slight decrease in nevirapine susceptibility
and a slight decrease in efavirenz susceptibility.
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In another specific embodiment, the invention provides for
a method of evaluating the effectiveness of NNRTI:
antiretroviral therapy of a patient comprising (i)
collecting a biological samplf=_ from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding HIV RT having a mutation at.
codon(s) 98 and/or 190. This invention established, using
a phenotypic susceptibility assay, that mutations at codon
98 either alone or in combination. with a mutation at codon
190 of HIV reverse transcriptase are correlated with an
increase in delavirdine susceptibility (decreased
resistance), a substantial decrease in nevirapin~=
susceptibility and a substantial decrease in efavirenz
susceptibility. In other specific embodiment, the
invention provides for a method of evaluating the
effectiveness of NNRTI antiretroviral therapy of a patient
comprising (i) collecting a biological sample from an HIV-
infected patient; and (ii) determining whether the
biological sample comprises nuc:Leic acid encoding HIV RT
having a mutation at codon(s) 181 and/or 98. This
invention established, using a phenotypic susceptibility
assay, that mutations at codon 181 either alone or in
combination with a mutation at codon 98 of HIV reverse
transcriptase are correlated with. a significant decrease in
delavirdine susceptibility (increased resistance), a
substantial decrease in nevirapine susceptibility and a
slight decrease in efavirenz susceptibility. In another
specific embodiment, the invention provides for a method of
evaluating the effectiveness of NNRTI 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 RT having a mutation at codon(s) 101 and/or
190, for example 1905. This invention established, using
a phenotypic susceptibility assay, that mutations at codon
101 either alone or in combination with a mutation at codon
190 of HIV reverse transcriptase are correlated with no
change in delavirdine susceptibility (wild-type), a
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substantial decrease in nevirapine susceptibility and a
substantial decrease in efavirenz susceptibility. In
another specific embodiment, the invention provides for a
method of evaluating the Effectiveness of NNRT7:
5 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 HTV RT having a mutation at
codon(s) 108. This invention established, using a
10 phenotypic susceptibility assay, that a mutation at codon
108 of HIV reverse transcriptase are correlated with a no
change in delavirdine susceptibility (wild-type), a slight
decrease in nevirapine susceptibility and no change i:n
efavirenz susceptibility. In anc>ther specific embodiment,
15 the invention provides fox a method of evaluating the
effectiveness of NNRTI antiretroviral therapy of a patient
comprising (i) collecting a biological sample from an HIV-
infected patient; and (ii) determining whether the
biological sample comprises nuc:Leic acid encoding HIV RT
20 having a mutation at codon(s) 101 and 103 and/or 190. This
invention established, using a phenotypic susceptibility
assay, that mutations at codon 101, either alone or in
combination with a mutation at codon 103 and/or 190 of HIV
reverse transcriptase are correlated with a either no
25 change (101 and 190) or a moderate decrease (103 and 190,
for example 190A) in delavirdine susceptibility (increasE:d
resistance), a substantial decrease in nevirapine
susceptibility and a significant decrease in efavirenz
susceptibility.
In another specific embodiment, the invention provides for
a method of evaluating the effectiveness of NNR'rI
antiretroviral therapy of a patient comprising (:i)
collecting a biological sample from an HIV- infected
patient; and (ii) determining wheaher the biological sample
comprises nucleic acid encoding HIV RT having a mutation at
codon(s) 106 and/or 189 and/or 181 and/or 227. This
invention established, using a phenotypic susceptibility
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assay, that mutations at codon 106 either alone or in
combination with a mutation at codon 189 and/or 181 and/or
227 of HIV reverse transcriptase are correlated with
changes in delavirdine, nevirapine and efavirenz
susceptibility. Specifically, the: presence of mutations at.
106 and 181 correlates with a significant decrease in
delavirdine susceptibility, a substantial decrease in
nevirapine susceptibility and a slight decrease in
efavirenz susceptibility. The presence of mutations at 106
and 189 correlates with a slight. decrease in delavirdine
susceptibility, a moderate decrease in nevirapine
susceptibility and no change in efavirenz susceptibility.
The presence of mutations at 106 and 227 correlates with a
slight decrease in delavirdine susceptibility, a
substantial decrease in nevirapine susceptibility and a
slight decrease in efavirenz susceptibility. The presence
of mutations at 181 and 227 correlates with an increase in
delavirdine susceptibility, a significant decrease in
nevirapine susceptibility and <~n increase in efavirenz
susceptibility. The presence of mutations at 106 and 181
and 227 correlates with a moderate decrease in delavirdin.e
susceptibility, a substantial decrease in nevirapin.e
susceptibility and a slight decrease in efavirer.~z
susceptibility. In another ;specific embodiment, the
invention provides for a method of evaluating the
effectiveness of NNRTI 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 RT
having a mutation at codon(s) 188 and 100 and/or 103. Th_Ls
invention established, using a phenotypic susceptibilit=y
assay, that mutations at codon 188 either alone or :in
combination with a mutation at codon 100 and/or 103 of HIV
reverse transcriptase are correlated changes in
delavirdine, nevirapine and efavirenz susceptibility.
Specifically, the presence of mutations at 103 and 188
correlates with a substantial decrease in delavirdine
susceptibility, a substantial decrease in nevirapine
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susceptibility and a substatntial decrease in efaviren:~
susceptibility. The presence of mutations at 100 and 103
correlates with a substantial decrease in delavirdine
susceptibility, a moderate decrease in nevirapine
susceptibility and a substantial decrease in efavirenz
susceptibility. The presence of mutations at 103 and 100
and 188 correlates with a substantial decrease in
delavirdine susceptibility, a substantial decrease in
nevirapine susceptibility and a. substantial decrease in
efavirenz susceptibility. Under the foregoing
circumstances, the phenotypic susceptibility/resi~~tance
profile and genotypic profile cf the HIV virus infecting
the patient has been altered reflecting some change in the
response to the antiretroviral agent. In the case on NNRTI
antiretroviral therapy, the HIV virus infecting the patient
may be resistant to one or more but not another of th.e
NNRTIs as described herein. It therefore may be desirable
after detecting the mutation, to either increase the dosage
of the antiretroviral agent, change to another
antiretroviral agent, or add one or more additional
antiretroviral agents to the pati.ent's therapeutic regimen.
For example, if the patient was being treated with
efavirenz (DMP-266) when the 225 mutation arose, the
patient's therapeutic regimen may desirably be altered by
either (i) changing to a different NNRTI antiretroviral
agent, such as delavirdin a or nevirapine and stopping
efavirenz treat; or (ii) increasing the dosage of
efavirenz; or (iii) adding another antiretroviral agent to
the patient's therapeutic regimen. The effectiveness of
the modification in therapy may be 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 regiment.
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The phrase "correlates positively," as used herein,
indicates that a particular result renders a particular
conclusion more likely than other conclusions.
Another preferred, non-limiting, specific embodiment of the
invention is as follows: A method of assessing the
effectiveness of NNRTI therapy of a patient comprising (:i)
collecting a biological sample from an HIV-infected patient;
(ii) amplifying the HIV-encoding RNA in the biological
sample by converting the RNA to cDNA and amplifying HIV
sequences using HIV primers that result in a PCR product
that comprises that RT gene; (iii) performing PCR using
primers that result in PCR producers comprising wild type or
mutant 225 and lU3 codons; and (iv) determining, via the
I5 products of PCR, the presence or absence of a mutation at
codon 225 or 103 or both. Yet another preferred, non-
limiting specific embodiment, of the invention is as
follows: A method of assessing the effectiveness of NNR.TI
therapy of a patient comprising (I) collecting a plasma
sample from an HIV-infected pat=lent; (ii) amplifying the
HIV-encoding RNA in the plasma sample by converting the F;NA
to cDNA and amplifying HIV sequences using HIV primers that
result in a PCR product that comprises the RT gene; (ii_i)
performing PCR using primers that result in PCR producers
comprising the wild type or mutations at codons I03 and/or
181 and 236; and (iv) determining, via the products of PCR,
the presence or absence of a mutation at codon 236 and :'_03
and/or 181.
Yet another preferred, non-limiting specific embodiment, of
the invention is as follows: A method of assessing 'the
effectiveness of NNRTI therapy of a patient comprising (I)
collecting a plasma sample from an HIV-infected patient;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA tc cDNA and amplifying HIV sequences
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using HIV primers that result in a PCR product that
comprises that RT gene: (iii) performing PCR using primers
that result in PCR products comprising the wild type or
mutations at codon 101 and 190 (G190S); and (iv)
determining, via the products of PCR, the presence or
absence of a mutation at codon 190 (G190S) and 101.
Yet another preferred, non-limiting specific embodiment, of
the invention is as follows: P, method of assessing the
effectiveness of NNRTI therapy of a patient comprising (i)
collecting a plasma sample from an HIV-infected patient;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA to cDNA and amplifying HIV sequences
using HIV primers that result in a PCR product that
comprises the RT gene; (iii) performing PCR using primers
that result in PCR products comprising the wild type or
mutations at codon 103 and 190 (G190A) and (iv) determining,
via the products of PCR, the presence or absence of a
mutation at codon 190 (G190A) and 103. Yet another
preferred, non-limiting specific embodiment, of the
invention is as follows: A method of assessing the
effectiveness of NNRTI therapy of a patient comprising li)
collecting a plasma sample from an HIV-infected patient;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA to cDNA and amplifying HTV sequences
using HIV primers that result in a PCR product that
comprises the RT gene; (iii) performing PCR using primers
that result in PCR products comprising the wild ~ype or
mutations at codon 230 and 181, and (iv) determining, via
the products of PCR, the presence or absence of a mutation
at codon 230 and 181.
Yet another preferred, non-limiting specific embodiment, of
the invention is as follows: A method of assessing the
effectiveness of NNRTI therapy of a_patient comprising (i)
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collecting a plasma sample from. an HIV-infected patient;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA to cDNA and amplifying HIV sequences
using HIV primers that result in a PCR product that
5 comprises the RT gene; (iii) performing PCR using primers
that result in PCR products comprising the wild type or
mutation at 181; and (iv) determining, via the products of
PCR, the presence or absence of a mutation at colon 181.
Yet another preferred, non-limit_Lng specific embodiment, of
10 the invention is as follows: A method of assessing the
effectiveness of NNRTI therapy of a patient comprising (i)
collecting a plasma sample frorn an HIV-infected patier.;t;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA to cDNA anc~ amplifying HIV sequences
15 using HIV primers that result in a PCR product that
comprises the RT gene; (iii) performing PCR using primE:rs
that result in PCR products comprising the wild type or
mutation at colon 188; and (iv) determining, via t=he
products of PCR, the presence or absence of a mutation at
20 colon 188. Yet another preferred, non-limiting specific
embodiment, of the invention is as follows: A method of
assessing the effectiveness of NNRTI therapy of a patient
comprising (i) collecting a plasma sample from an H:IV-
infected patient; (ii) amplifying the HIV-encoding RNA in
25 the plasma sample by converting the RNA to cDNA and
amplifying HIV sequences using H",IV primers that result in a
PCR product that comprises the R'.' gene; (iii) performing PCR
using primers that result in PCR products comprising the
wild type or mutations at colon 138 and 188; and (iv)
30 determining, via the products of PCR, the presence or
absence of a mutation at colon 138 and 188.
Yet another preferred, non-limiting specific embodiment, of
the invention is as follows: A method of assessing the
35 effectiveness of NNRTI therapy of a.patient comprising (i)
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collecting a plasma sample frorn an HIV-infected patient;
(ii) amplifying the HIV-enccding RNA in the plasma sample by
converting the RNA to cDNA and amplifying HIV sequences
using HIV primers that result= in a PCR product that
comprises the RT gene; (iii) p~°rforming PR using primers
that result in PCR products comprising the wild t:.ype or
mutation at codon 98 and (iv) determining, via the products
of PCR, the presence or absence of a mutation at codon 98.
Yet another preferred, non-limiting specific embodiment, of
the invention is as follows: A method of assessing t=he
effectiveness of NNRTI therapy of a patient comprising (i)
collecting a plasma sample from an HIV-infected patient;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA to cDNA anal amplifying HIV sequences
using HIV primers that result in a PCR product that
comprises the RT gene; (iii) performing PCR using primers
that result in PCR products comprising the wild type or
mutations at codon 98 and 190; and (iv) determining, via the
products of PCR, the presence or absence of a mutation at
codon 190 and 98. Yet another preferred, non-limiting
specific embodiment, of the invention is as follows: A
method of assessing the effectiveness of NNRTI therapy of a
patient comprising (i) col.lecti.ng a plasma sample from an
HIV-infected patient; (ii) amplifying the HIV-encoding RNA
in the plasma sample by converting the RNA to cDNA and
amplifying HIV sequences using HIV primers that result in a
PCR product that comprises the R'r gene; (iii) performing PCR
using primers that result in fCR products comprising the
wild type or mutations at codon 98 and 181; and (iv)
determining, via the products of PCR, the presence or
absence of a mutation at codon 98 and 181.
Yet another preferred, non-limiting specific embodiment, of
the invention is as follows: A method of assessing the
effectiveness of NNRTI therapy of a_patient comprising (i)
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collecting a plasma sample from an HIV-infected patient;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA to cDNA _and amplifying HIV sequences
using HIV primers that result in a PCR product that.
comprises the RT gene; (iii) performing PCR using primers
that result in PCR products comprising the wild type or
mutations at codon 101 and 190; and (iv) determining, via
the products of PCR, the presence or absence of a mutation
at codon 190, for example 1905 and 101.
Yet another preferred, non-limiting specific embodiment, of
the invention is as follows: A method of assessing the
effectiveness of NNRTI therapy e~f a patient comprising (i)
collecting a plasma sample from an HIV-infected patient;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA to cDNA anti amplifying HIV sequences
using HIV primers that result= in a PCR product that
comprises the RT gene; (iii) performing PCR using primers
that result in PCR products comprising the wild type or a
mutation at codon 108; and (iv) determining, via t:he
products of PCR, the presence or absence of a mutation at
codon 108. Yet another preferred, non-limiting specific
embodiment, of the invention is as follows: A method of
assessing the effectiveness of NNRTI therapy of a patient
comprising (i) collecting a plasma sample from an H:LV-
infected patient; (ii) amplifying the HIV-encoding RNA in
the plasma sample by converting the RNA to cDNA and
amplifying HIV sequences using HIV primers that result in a
PCR product that comprises the R7.' gene; (iii) performing PCR
using primers that result in PCR products comprising the
wild type or a mutation at codon 101 and 103 and 190 and
(iv) determining, via the products of PCR, the presence or
absence of a mutation at codorv 101 and 103 and 190, for
example 190A.
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Yet another preferred, non-limiting specific embodiment, of
the invention is as follows: A method of assessing the
effectiveness of NNRTI therapy of a patient comprising (i)
collecting a plasma sample from an HIV-infected patient;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA to cDNA and amplifying HIV sequencf:s
using HIV primers that result in a PCR product. tha t
comprises that RT gene; (iii) performing PCR using primers
that result in PCR products comprising the wild type or
mutations at codon 106 and and 189 arid 181 and 227 and (iw)
determining, via the products of PCR, the presence or
absence of a mutation at codon 106 and 189 and 181 and 227.
Yet another preferred, non-limiting specific embodiment, of
the invention is as follows: A method of assessing the
effectiveness of NNRTI therapy of a patient comprising (i)
collecting a plasma sample from an HIV-infected patient;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA to cDNA and amplifying HIV sequences
using HIV primers that result in a PCR product that
comprises the RT gene; (iii) performing PCR using primers
that result in PCR products comprising the wild type or
mutations at codon 188 and 100 and 103 and (iv) determining,
via the products of PCR, the presence or absence of a
mutation at codon 188 and 100 and 103. The presence of t:he
mutation at codon 225 and 103 of HIV RT indicates that t:he
effectiveness of the current or prospective NNRTI therapy
may require alteration, since as shown by this invention
mutation at codon 103 reduces susceptibility which
susceptibility can in part be restored by mutation at codon
225. Using the methods of this invention change in the
NNRTI therapy would be indicated. Similarly, using the
means and methods of this invention the presence of the
mutation at codon 236 and 103 and/or 181 of the HIV RT
indicates that the effectivE:ness of the current or
prospective NNRTI therapy has been diminished. Similarly,
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using the means and methods of this invention the presence
of the mutation at colon 190 (G190A) and 103 (K103N) of the
HIV RT indicates that the effectiveness of the current or
prospective NNRTI therapy has been diminished. Similarly,
using the means and methods of this invention the presence
of the mutation at colon 190 (G190S) and 101 (K101E) of t:he
HIV RT indicate that the effecriveness of the current or
prospective NNRTI therapy has been diminished. Similarly,
using the means and methods of this invention the presence
of the mutation at colon 230 and 181 of the HIV RT indicat:es
that the effectiveness of the current or prospective NNRTI
therapy has been diminished. Similarly, using the means and
methods of this invention the presence of the a mutation at
colon 181 of the HIV RT indicates that the effectiveness of
the current or prospective NNRTI therapy has been
diminished. Similarly, using the means and methods of this
invention the presence of the mutation at colon 188 of the
HIV RT indicates that the effectiveness of the current of
prospective NNRTI therapy has been diminished. Similar:Ly,
using the means and methods of this invention the presence
of the mutation at colon 138 and 188 of the HIV RT indicates
that the effectiveness of the current or prospective NN:RTI
therapy has been diminished. Similarly, using the means and
methods of this invention the presence of the mutation at
colon 98 of the HIV RT indicates that the effectiveness of
the current or prospective NNRTI therapy has been
diminished. Similarly, using the means and methods of this
invention the presence of the mutation at colon 98 and 190
of the HIV RT indicates that the effectiveness of the
current or prospective NNRTI therapy has been diminished.
Similarly, using the means and methods of this invention the
presence of the mutation at colon 181 and 98 of the HIV' RT
indicates that the effectivE=_ness of the current or
prospective NNRTI therapy has been diminished. Similarly,
using the means and methods of this invention the presence
of the mutation at colon 101 and 190, for example 190S, of
the HIV RT indicates that the effectiveness of the current
or prospective NNRTI therapy has been diminished.
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Similarly, using the means and methods of this invention the
presence of a mutation at codon 1.08 of the HIV RT indicates
that the effectiveness of the current or prospective NNRTI
therapy has been diminished. Sim:Llarly, using the means and
5 methods of this invention the presence of the mutation at
101 and 103 and 190, for example 190A, of the HIV RT
indicates that the effectiveness of the current or
prospective NNRTI therapy has been diminished. Similarly,
using the means and methods cf t:his invention the presence
10 of the mutation at codon 106 and 189 and 181 and 22'1 of the
HIV RT indicates that the effectiveness of the current or
prospective NNRTI therapy has been diminished. Similarly,
using the means and methods of the invention the presence of
the mutation at codon 188 and 100 and 103 of the HIV RT
15 indicates that the effectiveness of the current or
prospective NNRTI therapy has been diminished.
Another preferred, non-limiting, specific embodiment of the
invention is as follows. a method of evaluating the
20 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
reverse transcriptase having a mutation at codon 236 and 103
25 and/or 181. Using the phenotypic susceptibility assay, it
was observed that the presence of the three mutations
correlates positively with delavirdine resistance. Using
the phenotypic susceptibility assay, it was observed that
the presence of the three mutations correlates positively
30 with nevirapine resistance. In another embodiment, the
mutated codon 236 of HIV RT a_ncodes leucine (L). In a
further embodiment, the reverse transcriptase has a mutation
at codon 103, a mutation at codon 181 or a combination
thereof in addition to the mutation at codon 236 o.f HIV RT.
35 In a still further embodiment, the mutated codon 103 encodes
an asparagine (N) and the mutated codon at 181 encodes a
cysteine (C) .
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Another preferred, non-limiting, specific embodiment of the
invention is a follows: a method of assessing the
effectiveness of antiretroviral therapy of an HIV-infectE=_d
patient comprising: (a) collecting a biological sample from
an HIV-infected patient; and (b) determining whether the
biological sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at codon 225 a:nd
103. Using the phenotypic susceptibility assay, it was
observed that the presence of the mutations at codons 225
alone or in combination with a mutation at codon 103 of HIV
RT cause an increase in delavirdine susceptibility while
having no effect ors nevirapine susceptibility. In yet
another embodiment, the mutated codon 225 codes for a
histidine, codon 230 codes for a :Luecine and codon 181 codes
for a cysteine.
This invention provides a method of assessing 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 t:he
biological sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at codon 181. Using
the phenotypic susceptibility assay it was observed that t=he
presence of mutations at codor:. 18 i correlates positively
with a moderate decrease in delavirdine susceptibility and
a significant decrease in nevirapine susceptibility and no
change in efavirenz susceptibility. In an embodiment, the
mutated codon 181 for a isol.euci.ne.
This invention provides a method of assessing 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
reverse transcriptase having a mutation at codon 188. Using
the phenotypic susceptibility assay it was observed that
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the presence of mutations at codor-i 188 correlates positively
with a slight decrease in delavirdine susceptibility and a
substantial decrease in nevirapine susceptibility and
significant decrease in efavirenz susceptibility. In an
embodiment, the mutated codon x_88 codes for a cysteine,
histidine, or leucine.
This invention provides a method of assessing tire
effectiveness of antiretroviral therapy of an HIV-infected
patient comprising: (a) collecting a biological sample from
an HIV-infected patient; and (b) determining whether t:he
biological sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at codon 190. Using
the phenotypic susceptibility assay it was observed that the
presence of mutations at codon 190 correlates positively
with a slight increase in delavirdine susceptibility and a
large decrease in nevirapine susceptibility. In an
embodiment, the mutated codon 190 codes for an alanine or a
serine.
Another preferred, non-limiting, specific embodiment of the
invention is as follows: a method of assessing the
effectiveness of antiretroviral therapy of an HIV-infected
patient comprising: (a) collecting a biological samp~'~e from
an HIV-infected patient; and (b) determining whether the
biological sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at codon 230 and
181. Using the phenotypic susceptibility assay, it was
observed that the presence of the mutations at codons 230
alone or in combination with a mutation at codon 181 of HIV
RT causes a significant decrease in delavirdine
susceptibility and a significant decrease in nevirapine
susceptibility.
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Another preferred, non-limiting, specific embodiment of the
invention is as follows: a method of assessing the
effectiveness of antiretroviral therapy of an HIV-infected
patient comprising: (a) collecting a biological sample from
an HIV-infected patient; and (}y determining whether the
biological sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at codon 138 and
188. Using the phenotypic susceptibility assay, it was
observed that the presence of the mutations at codons 138
alone or in combination with a mutation at codon 188 of H:IV
RT causes a moderate decrease in delavirdine susceptibility
and a substantial decrease in newirapine susceptibility a.nd
a moderate decrease in efavirenz susceptibility. In yet
another embodiment, the mutated codon 138 codes for a
alanine and codon 188 codes for a leucine.
This invention provides a method of assessing t:he
effectiveness of antiretroviral therapy of an HIV-infected
patient comprising: (a) collecting a biological sample from
an HIV-infected patient; and (b) determining whether t:he
biological sample comprises z:ucleic acid encoding HIV
reverse transcriptase having a mutation at codon 98. Using
the phenotypic susceptibility asp>ay it was observed that ~~he
presence of mutations at codon 98 correlates positively with
a slight decrease in delavirdine susceptibility and a slight
decrease in delavirdine susceptibility and a slight
decrease in nevirapine susceptibility and a slight decrease
in efavirenz susceptibility. In an embodiment, the mutated
codon 98 codes for glycine.
Another preferred, non-limiting, specific embodiment of the
invention is as follows: a method of assessing 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
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biological sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at codon 98 and 190.
Using tote phenotypic susceptibility assay, it was observed
that the presence of the mutations at codons 98 alone or in
combination with a mutation at codon 190 of HIV RT causes an
increase in delavirdine susceptibility and a substantial
decrease in nevirapine susceptibility and a substantial
decrease in efavirenz susceptibility. In yet another
embodiment, the mutated codon 190 codes for a serine and
codon 98 for a glycine.
Another preferred, non-limiting, specific embodiment of the
invention is as follows: a method of assessing the
effectiveness of antiretroviral_ therapy of 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
reverse transcriptase having a mutation at codon 181 and 98.
Using the phenotypic susceptibility assay, it was observed
that the presence of the mutations at codons 181 alone or in
combination with a mutation at codon 98 of HIV RT cause~~ a
significant decrease in delavirdine susceptibility and a
substantial decrease in nevirapine susceptibility and a
slight decrease in efavirenz susceptibility. In yet another
embodiment, the mutated codon 98 codes for a glycine and
codon 181 codes for a cysteine.
Another preferred, non-limiting, specific embodiment of v he
invention is as follows: a method of assessing '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 EiIV
reverse transcriptase having a mutation at codon 101 and
190, for example 190S. Using tl-.e phenotypic susceptibility
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assay, it was observed that the presence of the mutations at
codons 101 alone or in combination with a mutation at codon
190 of HIV RT causes no change in delavirdine susceptibility
and a substantial decease in nevirapine susceptibility and
5 a substantial decrease in efavire.nz susceptibility. In y~st
another embodiment, the mutated codon 190 codes for a serine
and codon 101 codes for a glutamine acid.
This invention provides a method of assessing t:he
10 effectiveness of antiretroviral therapy of an HIV-infected
patient comprising: (a) collecting a biological sample from
an HIV-infected patient; and (r~) determining whether the
biological sample comprises nucleic. acid encoding HIV
reverse transcriptase having a mutation at codon 108. Using
15 the phenotypic susceptibility assay it was observed that the
presence of mutations at codon 108 correlates positively
with no change in delavirdine susceptibility and a slight
decrease in nevirapine susceptibility and no change in
efavirenz susceptibility . In an embodiment, the mutated
20 codon 108 codes for a isoleucine.
Another preferred, non-limiting, specific embodiment of the
invention is as follows: a method of assessing the
effectiveness of antiretroviral therapy of an HIV-infected
25 patient comprising: (a) collecting a biological sample from
an HIV-infected patient; and (b} determining whether the
biological sample comprises nucleic acid encoding EiIV
reverse transcriptase having a mutation at codon 101 and
190, for example °y90A. Using the phenotypic susceptibility
30 assay, it was observed that the presence of the mutations at
codons 101 alone or in combination with a mutation at codon
190 of HIV RT causes no change in delavirdine susceptibility
and a substantial decease in nevirapine susceptibility and
a significant decrease in efavirenz susceptibility. In yet
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another embodiment, the mutated codon 190 codes for a
glycine and codon 101 codes for a glutamine acid.
Another preferred, non-limiting, specific embodiment of the
invention is as follows: a ;nethod of assessing 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 H:IV
reverse transcriptase having a mutation at codon 103 and
190. Using the phenotypic susceptibility assay, it w;ss
observed that the presence of the mutations at codons 103
alone or in combination with a mutation at codon 190 of HIV
RT causes a moderate decrease in delavirdine susceptibility
and a substantial decrease in nevirapine susceptibility and
a significant decrease in efavirenz susceptibility . In yet
another embodiment, the mutated codon 190 codes for a
alanine and codon 103 codes for a asparagine.
Another preferred, non-limiting, specific embodiment of the
invention is as follows: a method of assessing the
effectiveness of antiretroviral therapy of an HIV-infected
patient comprising: (a) collecting a biological samp~~e from
an HIV-infected patient; and (b) determining whether the
biological sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at codon 106 and
181. Using the phenotypic susceptibility assay, it was
observed that the presence of the mutations at colons 106
alone or in combination with a mutation at colon 181 of H:IV
RT causes a significant decease in delvaridine
susceptibility and a substantial decrease in nevirapi.ne
susceptibility and a substantial decrease in efavire~nz
susceptibility . In yet another embodiment, the mutated
colon 106 codes for a alanine and colon 181 codes for a
cysteine.
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Another preferred, non-limiting, specific embodiment of the
invention is as follows: a method of assessing 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 t:he
biological sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at colon 106 and
189. Using the phenotypic susceptibility assay, it was
observed that the presence of the mutations at colons 7_06
alone or in combination with a mutation at colon 189 of HIV
RT causes a slight decrease in delavirdine susceptibility
and a moderate decrease in nevirapine susceptibility and no
change in efavirenz susceptibility. In yet another
embodiment, the mutated colon 189 codes for a leucine and a
colon 106 codes for a alanine.
Another preferred, non-limiting, specific embodiment of 'the
invention is as follows: a method of assessing the
effectiveness of antiretroviral therapy of an HIV-infected
patient comprising: (a) collecting a biological sample from
an HIV-infected patient; and ib) determining whether the
biological sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at colon 106 and
227. Using the phenotypic susceptibility assay, it was
observed that the presence of the mutations at colons 106
alone or in combination with a mutation at colon 227 of HIV
RT causes a slight decrease in delavirdine susceptibility
and a substantial decrease in nE:virapine susceptibi.iity and
a slight decrease in efavirenz susceptibility. In yet
another embodiment, the mutated colon 227 codes for a
leucine and colon 106 codes for a alanine.
Another preferred, non-limiting, specific embodiment of the
invention is as follows: a method of assessing the
effectiveness of antiretroviral therapy of an HIV-infected
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patient comprising: (a) collecting a biological sample from
an HIV-infected patient; and (b) determining whether the
biological cample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at codon 181 and
227. Using the phenotypic susceptibility assay, it was
observed that the presence of the mutations at codons 181
alone or in combination with a mutation at codon 227 of HIV-
RT causes an increase in delavirdine susceptibility and an
significant decrease in nevirapine susceptibility and and an
increase in efavirenz susceptibility.
In yet another embodiment, the mutated codon 227 codes f:or
a leucine and codon 181 nodes fcr cysteine.
Another preferred, non-limiting, specific embodiment of t:he
invention is as follows: a method of assessing t:he
effectiveness of antiretroviral therapy of an HIV-.infect:ed
patient comprising: (a) collecting a biological sample from
an HIV-infected patient; and (b) determining whether t:he
biological sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at codon 106 and :L81
and 227. Using the phenotypic :susceptibility assay, it was
observed that the presence of t:he mutations at codons 106
alone or in combination with a mutation.at codon 181 and 227
of HIV RT causes a moderate decrease in delavird.ine
susceptibility and a slight: decrease in efavir~~nz
susceptibility.
In yet another embodiment, the mutated codon 106 codes for
a alanine, codon 181 codes for a cysteine and codon 227
codes for a leucine.
Another preferred, non-limiting, specific embodiment of the
invention is as follows: a method of assessing the
effectiveness of antiretroviral therapy of an HIV-infected
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patient comprising: (a) collecting a biological sample from
an HIV-infected patient; and (b) determining whether the
biological sample comprises nucleic acid encoding H=LV
reverse transcriptase having a mutation at colon 103 and
S 188. Using the phenotypic susceptibility assay, it was
observed that the presence of the mutations at colons 103
alone or in combination with a mutation at colon 188 of H:IV
RT causes a substantial decrease in delavirdine
susceptibility and a substantial decrease in nevirapine
susceptibility and a substantial decrease in efavirenz
susceptibility. In yet another embodiment, the mutated
colon 188 codes for a leucine and colon 103 codes for a
asparagine.
Another preferred, non-limiting, specific embodiment of the
invention is as follows: a method of assessing t:he
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
reverse transcriptase having a mutation at colon 100 and
103. Using the phenotypic susceptibility assay, it was
observed that the presence of the mutations at colons 100
alone or in combination with a mutation at colon 103 of HIV
RT causes a substantial decrease in delavirdine
susceptibility and a moderate decrease in nevirapine
susceptibility and a substantial decrease in efavirenz
susceptibility.
In yet another embodiment, the mutated colon 100 codes for
a isoleucine, colon 103 codes for a asparagine.
Another preferred, non-limiting, specific embodiment of the
invention is as follows: a method of assessing the
effectiveness of antiretroviral therapy of an HIV-infected
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patient comprising: (a) collectirng a biological sample from
an HIV-infected patient; and (b;~ determining whether the
biological sample comprises nucleic acid encoding HIV
reverse transcriptase having a mu~ation-at colon 100 and 103
5 and 188. Using the phenotypic susceptibility assay, it was
observed that the presence of the mutations at colons 100
alone or in combination with a mutation at colon 103 and 188
of HIV RT causes a substantial decrease in delavirdine
susceptibility and a moderatE: decrease in nevirapine
10 susceptibility and a substant~.al decrease in efavirenz
susceptibility.
In yet another embodiment, the mutated colon 100 codes for
a isoleucine, colon 103 codes for a asparagine and colon 1.88
15 codes for a leucine.
This invention also provides the means and methods to use
the resistance test vector comprising an HIV gene further
comprising an NNRTI mutation for drug screening. More
20 particularly, the invention describes the resistance test
vector comprising the HIV reverse transcriptase having
mutations at colons 225 and 10:3 for drug screening. ~~he
invention also describes the resistance test vector
comprising the HIV reverse transc:riptase having mutations at
25 colons 236 and 103 and/or 181. The invention also describes
the resistance test vector comprising the HIV reverse
transcriptase having mutations at: colons 190 (G190A) and 103
(K103N). The invention also describes the resistance test
vector comprising the HIV reverse transcriptase having
30 mutations at colons 190 (G190S) and 101 (K101E).
The invention also describes the resistance test vector
comprising the HIV reverse transcriptase having mutations at
colons 230 and 181.
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The invention also describes the resistance test vector
comprising the HIV reverse transcriptase having a mutation
at codon 181.
The invention also describes the resistance test vecvor
comprising the HIV reverse transcriptase having a mutation
at codon 188.
The invention also describes the resistance test vector
comprising the HIV reverse transcriptase having mutations at
codons 138 and 188.
The invention also describes the resistance test vector
comprising the HIV reverse transcriptase having a mutation
at 98.
The invention also describes the resistance test vector
comprising the HIV reverse transcriptase having mutations at
codons 98 and 190.
The invention also describes the resistance test vector
comprising the HIV reverse transcriptase having muta~ions at
codons 181 and 98.
The invention also describes the resistance test vector
comprising the HIV reverse transcriptase having mutatio n; at
codons 101 and 190, for example 1905.
The invention also describes the resistance test vector
comprising the HIV reverse transcriptase having a mutation
at codon 108.
The invention also describes the resistance test veca or
comprising the HIV reverse transcriptase having mutations at
codons 101 and 103 and/or 190, for example 190A.
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The invention also describes the resistance test vector
comprising the HIV reverse transcriptase having mutations at
codons 106 and 189 and/or 181 and/or 227.
The invention also describes the resistance test vector
comprising the HIV reverse transc:riptase having mutations at
codons 188 and 100 and/or 103.
The invention further relates to novel vectors, host cells
and compositions for isolation and identification of the
non-nucleoside HIV-1 reverse transcriptase inhibitor
resistance mutant and using such vectors, host cells and
compositions to carry out anti-viral drug screening. This
invention also relates to the screening of candidate drugs
for their capacity to inhibit said mutant.
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EXAMPLE 1: Phenotypic Drug Susceptibility and Resistance
Test Using Resistance Test Vectors
Phenotypic drug susceptibility and resistance tests a.re
carried out using the means and methods described in PCT
International Application No. PCT/US9'7/01609, filed January
29, 1997 which is hereby incorporated by reference.
In these experiments patient-derived segments)
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 vir al
particles present in the serum of HIV-infected individuals
or were mutants of wild type HIV-1 made by site direct=ed
mutagenesis of a parental clones of resistance test veci~or
DNA. Isolation of viral RNA was performed using standard
procedures (e. g. RNAgents Total RNA Isolation SystE=_m,
Promega, Madison WI or RNAzol, Tel-Test, Friendswood, T;~).
The RT-PCR protocol was divided into two steps. A
retroviral reverse transcriptase [e. g. Moloney MuLV reverse
transcriptase (Roche Molecular Systems, Inc., Branchbu.rg,
NJ), or avian myeloblastosis virus (AMV) reverse
transcriptase, (Boehringer Mannheim, Indianapolis, IN)] was
used to copy viral RNA into cDNA. The cDNA was then
amplified using a thermostablE: DNA polymerase [e.g. Taq
(Roche Molecular Systems, Inc., Branchburg, NJ), Tth (Roche
Molecular Systems, Inc., Branchburg, NJ), frimeZyme
(isolated from Thermus brockianus, Biometra, Gottingen,
Germany)] or a combination of 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)]..
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The primers, Apal primer (PDSApa) and AgeI primer i;PDSAge)
used to amplify the "test" patient-derived segments
contained sequences resulting in Apal and Agel recognition
sites being introduced into the ~' and 3' termini of the PCR
product, respectively as described in PCT International
Application No. PCT/US97/01609, filed January 29, 1997.
Resistance test vectors incorporating the "test" patient-
derived segments were constru~~ted as described in I?CT
International Application No. PCT/US97/01609, filed January
29, 1997 using an amplified DNA product of 1.5 kB prepa:red
by RT-PCR using viral RNA as a tE~mplate and oligonucleotides
PDSApa (1) and PDSAge (2) as primers, followed by digestion
with ApaI and AgeI or the isoschizimer PINAI. To ensure
that the plasmid DNA corresponding to the resultant
resistance test vector comprise~~ 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 resistance test
vector were pooled and used for the preparation of plasmid
DNA.
A packaging expression vector encoding an amphotrophic NIuLV
4070A env gene product enables production in a resistance
test vector host cell of resistance test vector viral
particles which can efficientl~r infect human target ce7_ls.
Resistance test vectors encoding all HIV genes with the
exception of env were used to transfect a packaging host
cell (once transfected the host cell is referred to as a
resistance test vector host cell.). The packaging expression
vector which encodes the amphotrophic MuLV 4070A env gene
product is used with the resi:>tance test vector to enable
production in the resistance test vector host cell of
infectious pseudotyped resistance test vector viral
particles.
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Resistance tests performed with resistance test vectors were
carried out using packaging hcst and target host cells
consisting of the human embryonic kidney cell line 293 (Cell
Culture Facility, UC San Francisco, SF, CA) or the Jurk.at
5 leukemic T-cell line (Arthur Weiss, UC San Francisco, ~~F,
CA) .
Resistance tests were carried out with resistance test
vectors using two host cell types. Resistance test vector
10 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 expres~;ion vector. The resistance
test vector viral particles were then used to .infect: a
15 second host cell (the target host cell) in which the
expression of the indicator gene is measured.
The resistance test vectors containing a functional
luciferase gene cassette were constructed and host cells
20 were transfected with the resistance test vector DNA. The
resistant test vectors contained patient-derived reverse
transcriptase and protease sequences that were either
susceptible or resistant to the antiretroviral agents, such
as nucleoside reverse transcriptase inhibitors, non-
25 nucleoside reverse transcriptase inhibitors and protease
inhibitors. The resistance test vector viral particles
produced by transfecting the resistance test vector DNA into
host cells, either in the presence or absence of protease
inhibitors, were used to infect target host cells grown.
30 either in the absence of NRTI or NNRTI or in the presence of:
increasing concentrations of the drug. The amount: of:
luciferase activity produced :~n infected target host cells
in the presence of drug was compared to the amount of
luciferase produced in infected target host cells in the
35 absence of drug. Drug resistance was_measured as the amount
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of drug required to inhibit by 50o the luciferase activity
detected in the absence of drug (inhibitory concentration
500, IC50). The IC50 values were determined by plotting
percent drug inhibition vs. log,;, drug concentration.
Host cells were seeded in 10-cm-diameter dishes and were
transfected several days after plating with resistance tE:st
vector plasmid DNA and the envelope expression vector.
Transfections were performed using a calcium-phosphate
precipitation procedure. The cell culture media containing
the DNA precipitate was replaced with fresh medium, from one
to 24 hours, after transfect:ion. Cell culture media
containing resistance test vector viral particles was
harvested one to four days after transfection and was pa w ed
through a 0.45-mm filter befcre being stored at -80°C. I~IV
capsid protein (p24) levels in the harvested cell cult ure
media were determined by an EIA method as described by the
manufacturer (SIAC; Frederick, MD). Before infection,
target cells (293 and 293/T) were plated in cell culture
media. Control infections were performed using cell culture
media from mock transfections (no DNA) or transfections
containing the resistance test vector plasmid DNA without
the envelope expression plasmid. pne to three or more days
after infection the media was removed and cell lysis buffer
(Promega) was added to each well. Cell lysates were assayed
for luciferase activity (Fig.3). The 'inhibitory effect of
the drug was determined using the following equation:
o luciferase inhibition = 1 - (RLUluc [drug) - RLUluc) x 100
where RLUluc (drug] is the relative light unit of lucifei:ase
activity in infected cells in the presence of drug and
RLUluc is the Relative Light Unit of luciferase activit~,r in
infected cells in the absence of drug. IC50 values were
obtained from the sigmoidal curves that were generated :Eram
the data by plotting the percent inhibition of lucife:rase
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activity vs. the 1og10 drug concentration. The drug
inhibition curves are shown in (Fig.3).
EXAMPLE 2: Correlating Phenotypic Susceptibility And
Genotypic Analysis
Phenotypic susceptibility analysis of patient HIV samples
Resistance test vectors are constructed as described in
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 (NNRTIs), and protease inhibitors (PRIs). ~'he
panel of drugs can be expanded as new drugs or new drug
targets become available. An C50 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, either pools or clones, are
analyzed by any of the genotyping methods described in
Example 2. 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
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different from the control or pre-treatment sequence and
correlated to the observed phenc.-~type.
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
(drug susceptible) genetic background. Mutations may be
incorporated alone and/or in combination with other known
drug resistance 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 is then t=ested using the phenotypic
susceptibility assay described above and the susceptibi7_ity
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
is attributed to the specific mutations introduced into the
resistance test vector.
EXAMPLE 3
Correlating Phenotypic Susceptibility And Genotypic
Analysis: P225H
Phenotypic analysis of Patient 97-302
A resistance test vector was constructed as described in
example 1 from a patient sample: designated as 97-302. 'This
patient had been treated with d4T, indinavir and DMP-266 for
a period of approximately 10 months. Isolation of viral RNA
and RT/PCR was used to generate a patient derived segment
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that comprised viral sequences coding for all of PR and as
1 - 313 of RT. The patient derived segment was inserted
into a indicator gene viral vector to generate a resistance
test vector designated RTV-302. RTV-302 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, 3fC,
d4T, ddI and ddC), NNRTIs (delavirdine -and nevirapine), and
PRIs (indinavir, nelfinavir, ritonavir, and saquinavir). 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 NNRTIs was observed for patient sample
RTV-302 in which there was significant decrease in
nevirapine susceptibility (incre:ased resistance) and modest
decrease in delavirdine susceptibility (See Figure 8A).
Patient sample 97-302 was examined further for genotypic
changes associated with the observed pattern of
susceptibility.
Determination of genotype of patient 97-302
RTV-302 DNA was analyzed by AB:L chain terminator automated
sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type Glade B HIV-1 (HIV
Sequence Database Los Alamos, NM). The nucleotide sequence
was examined for sequences that are different prom the
control sequence. RT mutations were noted at positions
K103N, I135M, T200A, and P225H. K103N is associated with
resistance to the NNRTIs and has been shown using the
phenotypic susceptibility assay to be associated with
reduced susceptibility to both delavirdine and nevirapine
to an equal extent. The mutations at I135M and T200A are
known polymorphisms of the wild-type (drug-sensitive)
variants of HIV. The mutation, P225H, was characterized
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using site directed mutagenesis and phenotypic
susceptibility testing to correlate the changes at amino
acid 225 with changes in NNRTI phenotypic susceptibility.
5 Site directed mutagenesis
Resistance test vectors were constructed containing the
P225H mutation alone arid in combination with other known
drug resistance mutations (K103N, Y181C) known to modulate
the HIV susceptibility to NNRTIs. Mutations were int:roduc:ed
10 into the resistance test vector using the mega-primer fCR
method for site-directed mutagenesis. (Sakar G and Somrnar
SS (1994) Biotechniques 8(4), 404-407). A resistance test
vector containing the P225H mutation (P225H-RTV) was tested
using the phenotypic susceptibility assay described above
15 and the results were compared to that of a genetically
defined resistance test vector that was wild type at
position 225. The pattern of phenotypic susceptibility to
the NNRTI, delavirdine in the P225H-RTV was altered as
compared to wild type. In the context of an otherwise wild
20 type background (i.e. P225H mutation alone) the P225H-,ATV
was more susceptible to delavirdine than the wild type
control RTV. No significant change in nevirapine
susceptibility was observed in the P225H-RTV. The P225H
mutation was also introduced into a RTV containing
25 additional mutations at K103N, 5.'181C or both (K103NtY181C).
In all cases, RTVs were more susceptible to inhibition by
delavirdine if the P225H mutation was present as compared. to
the corresponding RTV lacking the P225H mutation (Fig. 8D).
In all cases the P225H mutation did riot significantly change
30 nevirapine susceptibility (Fig. 8D).
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EXAMPhE 4
Correlating Phenotypic Susceptibility And Genotyp:ic
Analysis: P236Z
Phenotypic analysis of HIV patient 97-268
A resistance test vector was constructed as described in
Example 1 from a patient sample designated 97-268. This
patient had been treated with AZT and 3TC (NRTIs), indinavir
and saquinavir (PRIs) and delavirdine (an NNRTI) for
periods varying from 1 month to 2 years. 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 amino acids 1 - 313 of RT. 'rhe patient derived segment
was inserted into a indicator gene viral vector to generate
a resistance test vector designated RTV-268. RTV-268 was
then tested using the 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 t:he
classes known as NRTIs (AZT, 3TC, d4T, ddI and ddC) , NNR'.CIs
(delavirdine and nevirapine), and PRIs (indinav:Lr,
nelfinavir, ritonavir, and saquinavir). An IC50 w as
determined for each drug tested. Susceptibility of 'the
patient virus to each drug was examined and compared to the
susceptibility of a reference virus. A pattern of
susceptibility to the NNRTIs was observed for the patient
sample RTV-268 in which the virus sample was observed to be
resistant to delavirdine with no resistance to delavirdine.
The sample was examined further for genotypic changes
associated with the pattern of susceptibility.
Genotype of HIV patient 97-268
RTV-268 DNA was analyzed by ABC chain terminator automated
sequencing. The nucleotide sequence was compared to the
consensus sequence of wild type Glade B HIV-1. The
nucleotide sequence was evaluated f-or sequences different
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from the control sequence. R':' mutations were noted at
positions M41L, D67N, M184V, T200A, E203D, L210W, T215'~,
K219Q, and P236L compared to the control sequence. The
mutations at T200A and E203D are known polymorphisms in
wild-type (drug-sensitive) variants of HIV. Mutations at
positions M41L, D67N, L210W, T215Y, and~K219Q are associated
with AZT resistance. The mutation at M184V is associated
with 3TC resistance. The mutation at P236L is associated
with resistance to delavirdine and increased susceptibility
to nevirapine (Dueweke et al., Ibid.). In contrast to
previous reports, the RTV-268 ~cample showed no change in
nevirapine susceptibility. The mutation, P236L, was
characterized using site directed mutagenesis and in vitro
phenotypic susceptibility testing to correlate changes at
amino acid 236 with changes in phenotypic susceptibility.
Site directed mutagenesis
Resistance test vectors were constructed containing the
P236L mutation alone and in ccmbination with other known
drug resistance mutations (K103Cd, Y181C) that are known to
modulate the susceptibility of fiIV-1 to NNRTIs. Mutations
were introduced into the resistance test vector using the
mega-primer PCR method for site-directed mutagenesis (Sa1<;ar
and Sommar, Ibid.). A resistance test vector containing t:he
P236L mutation (P236L-RTV) was tested using the phenotypic
susceptibility assay and the results were compared to that
of a genetically defined resistance test vector that was
wild type at position 236. P236L-RTV exhibited changes in
NNRTI phenotypic susceptibility. In the context of an
otherwise wild type background (i.e. P236L mutation alone)
the P236L-RTV is less susceptible to delavirdine than a wild
type reference RTV. In contrast to Dueweke et al. no
significant change in nevirapine susceptibility was observed
for P236L-RTV. The P236L mutation was also introduced into
a RTV containing mutations at K103N, Y181C or both
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(K103N+Y181C). In all cases, the RTV's were less
susceptible (more resistant) to deiavirdine if the P236L
mutation was present as compared to the corresponding RTV
lacking the P236L mutation. In all cases the P236L mutation
did not significantly alter nev~.rapine susceptibility.
Example 5
Correlating Phenotypic Susceptibility And Genotypic
Analysis: G190S
Phenotypic analysis of HIV patient 97-644
A resistance test vector was constructed as described in
Example 1 from a patient sample designated 97-644. T his
patient had been treated with d4T (NRTI), indinavir (PRI)
and efavirenz (NNRTI) for a period varying from 5 to 17
months. 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 amino acids 1 - 313 of
RT. The patient derived segment was inserted into a
indicator gene viral vector to generate a resistance test
vector designated RTV-644. RTV-644 was then tested using
the 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 NRTTs (A2T,
3TC, d4T, ddI and ddC), NNRTIs (delavirdine a.nd
nevirapine), and PRIs (indinavir, nelfinavir, ritonavi.r,
and saquinavir). An IC50 was determined for each drug
tested. Susceptibility of the patient virus to each drug
was examined and compared to the susceptibility of a
reference virus. A pattern of susceptibility to the NNRTIs
was observed for the patient sample RTV-644 in which t:he
virus sample was observed to be resistant to nevirap=.ne
with little or no resistance t.o delavirdine. The sample
was examined further for genotypic changes associated with
the pattern of susceptibility.
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Genotype of HIV patient 97-644
RTV-644 DNA was analyzed by ABI chain terminator automated
sequencing. The nucleotide sequence was compared to the
consensus sequence of wild type Glade B HIV-1. The
S nucleotide sequence was evaluated for sequences different
from the control sequence. R'w' mutations were noted at
positions K101E and G190S compared to the control
sequence. The mutations at T200A and E203D are known
polymorphisms in wild-type (drug-sensitive) variants of
HIV. The mutation at K101E is associated with resistance
to some but not all NNRTIs. The mutation, G190A but not
specifically G190S is associ<.~ted with nevirapine a:nd
loviride resistance. The mutations G190S and G190A were
characterized using site directed mutagenesis and in vitro
phenotypic susceptibility testing to correlate changes at
amino acid 190 with changes in phenotypic susceptibility.
Site directed mutagenesis
Resistance test vectors were constructed containing the
G190S and G190A mutations. Mutations were introduced into
the resistance test vector using the mega-primer PCR method
for site-directed mutagenesis (Sakar and Sommar, Ibid.).
Resistance test vectors containing the G190S or G190A
mutations (G190S-RTV, or G190A-~RTV) were tested using the
phenotypic susceptibility assay and the results were
compared to that of a genetically defined resistance test
vector that was wild type at position 6190. G190S-RTV and
G190A-RTV exhibited change~> in NNRTI phenotypic
susceptibility. In the context: of an otherwise wild type
background these RTVs were markedly less susceptible to
nevirapine and slightly more susceptible to delavirdine
than a wild type reference RTV.
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Example 6
Predicting Response to Non-nucleoside Reverse Transcriptase
Inhibitors by Characterization of Amino Acid Changes in HI'il-
1 Reverse Transcriptase
5 Phenotypic and genotypic correlation of mutations at amino
acid 236 of HIV-1 Reverse Transcriptase
In one embodiment of this invention, changes in the amino
acid at position 236 of the reverse transcriptase protein of
HIV-1 is evaluated using the following method comprising:
10 (i) collecting a biological sample from an HIV-1 infected
subject; (ii) evaluating whether the biolagical sample
contains nucleic acid encoding t-fIV-1 reverse transcriptase
having a mutation at codon 236. The presence of a mutation
at codon 236 (P236L) is correlated with a reduction in
15 delavirdine susceptibility and little or no change in
nevirapine susceptibility.
The biological sample comprises whole blood, blood
components including peripheral mononuclear cells (PBMC:),
20 serum, plasma (prepared using various anticoagulants such as
EDTA, acid citrate-dextrose, ~neparin), tissue biopsies,
cerebral spinal fluid (CSF), or other cell, tissue or body
fluids, In another embodiment., the HIV-1 nucleic acid
(genomic RNA) or reverse transcriptase protein can be
25 isolated directly from the b_Lological sample or after
purification of virus particles from the biological sampJ_e.
Evaluating whether the amino acid at position 236 of t=he
HIV-1 reverse transcriptase is mutated, can be perforrned
using various methods, such as direct characterization of
30 the viral nucleic acid encoding reverse transcriptase or
direct characterization of the reverse transcriptase protein
itself. Defining the amino acid at position 236 of reverse
transcriptase can be performed by direct characterization of
the reverse transcriptase protein by conventional cr novel
35 amino acid sequencing methodoloqies,-epitope recognition by
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antibodies or other specific binding proteins or compounds.
Alternatively, the amino acid at position 236 of the HIV--1
reverse transcriptase protein can be defined by
characterizing amplified copies of HIV-1 nucleic acid
encoding the reverse transcriptase protein. Amplification
of the HIV-1 nucleic acid can be performed using a varie!:.y
of methodologies including reverse transcription-polymeraae
chain reaction (RT-PCR), NASBA, SDA, RCR, or 3SR as would be
known to the ordinarily skilled artisan. Evaluating whethE=_r
the nucleic acid encoding HIV r~=verse transcriptasE= has a
mutation at codon 236 can be pE:rformed 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). Alternatively,
the nucleic acid sequence encoding amino acid position 236
can be evaluated using a variety of probe hybridization
methodologies, such as genechip hybridization sequencing
(Affymetrix), line probe assay (LiPA; Murexj, and
differential hybridization (Chiron).
In a preferred embodiment of th:~.s invention, evaluation of
whether amino acid position 236 of HIV-1 reverse
transcriptase was wild type or mutant was carried out using
a phenotypic susceptibility assay using resistance test
vector DNA prepared from the biological sample. Tn one
embodiment, 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 a.nd
reverse transcriptase regions. The amplified patient
derived segments were then incorporated, via DNA l igation
and bacterial transformation, into an indicator gene viral
vector thereby generating a resistance test vector.
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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
a P236L mutation. The nucleic acid (DNA) sequence of the
patient derived HIV-1 protease and reverse transcriptaae
regions from patient samp~'~e 268 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 susceptibility profiles of patient samples and
site directed mutants showed that delavirdine and nevirapine
susceptibility correlated with t:he absence of RT mutations
at positions 103, 181 or 236 of HIV-1 reverse transcriptase.
Phenotypic susceptibility profiles of patient samples and
site directed mutants showed a significant reduction in
delavirdine susceptibility (increased resistance) and little
or no reduction in nevirapine su~~ceptibility correlated with
a mutation in the nucleic acid sequence encoding the amino
acid leucine (L) at position 236 of HIV-1 reverse
transcriptase and the absence of mutations at positions 103
and 181.
Phenotypic susceptibility profiles of patient samples and
site directed mutants showed no additional reduction in
delavirdine or nevirapine susceptibility (increa:~ed
resistance) with the amino acid p roline at position 236 when
the RT mutations at positions 103, 181 or 103 and 181 wE:re
present (K103N, Y181C, or K103N ~- Y181C). However,
phenotypic susceptibility profiles of patient samples and
site directed mutants showed an additional reduction in
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delavirdine susceptibility (.increased resistance) and litt=Le
or no additional reduction in nevirapine susceptibility wit=h
the amino acid leucine (L) at position 236 in addition 1~0
the RT mutations associated wits NNRTI resistance (K103N,
Y181C, or K103N + Y181C).
Phenotypic and genotypic correlation of mutations at amino
acid 225 of HIV-1 Reverse Transcriptase
Phenotypic susceptibility profi~.es of patient samples and
site directed mutants showed no change in susceptibility t o
delavirdine or nevirapine when t=he amino acid proline (:P)
was present at position 225 of HIV-1 reverse transcriptase
in the absence of RT mutations associated with NNR'TI
resistance (K103N, Y181C). However, phenotypic
susceptibility profiles of patient samples and site directed
mutants showed an increase in del~avirdine susceptibility a:nd
little or no change nevirapine susceptibility when the amino
acid histidine (H) was present: at position 225 in the
absence of RT mutations (K103N, Y181C) associated with NNRTI
resistance.
Phenotypic susceptibility profiles of patient samples and
site directed mutants showed no additional reduction in
delavirdine susceptibility or nevirapine susceptibility when
the amino acid proline (P) at position 225 was present in
addition to the RT mutations associated with NNRTI
resistance (K103N, Y181C, or K103N + Y181C). In contrast
phenotypic susceptibility profiles of patient samples and
site directed mutants showed an increase in delavirdine
susceptibility and little or no change in nevirapine
susceptibility when the amino acid histidine (H) was present
at position 225 in the presence of RT mutations associated
with NNRTI resistance (K103N, Y1.81C, ~r K103N + Y18~_C).
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Phenotypic and genotypic correlation of mutations at amino
acid 190 of HIV-1 Reverse Transcriptase
Phenotypic susceptibility profiles of patient samples and
site directed mutants showed no change in susceptibility to
delavirdine or nevirapine when the amino acid glycine (G) at
position 190 was present in the absence of RT mutatio;~s
associated with NNRTI resistance (K103N, Y181C). Phenotypic
susceptibility profiles of site directed mutants showed an
increase in delavirdine susceptibility and a decrease in
nevirapine susceptibility when the amino acid alanlne ~.y~
was present at position 190 in the absence of RT mutatio:ns
associated with NNRTI resistance. Phenotypic susceptibility
profiles of patient samples and site directed mutants showed
an increase in delavirdine susceptibility and a decrease in
nevirapine susceptibility when the amino acid serine (S) was
present at position 190 in the absence of RT mutations
associated with NNRTI resistance.
EXAMPLE 8
Using Resistance Test Vectors and Site Directed Mutants To
Correlate Genotypes And Phenotypes Associated With NNR.TI
Drug Susceptibility And Resistance in HIV: Y181I
Preparation os resistant test. vectors and phenotypic
analysis of patient 98-964 HIV samples
A resistance test vector was constructed as described in
Example 1 from a patient sample designated 98-964. This
patient had been previously treated with ddI, d4T, AZT, 3T'C,
ddC, (NRTIs), saquinavir and nelfinavir (PRIs) and
nevirapine (an NNRTI) and HU. Isolation of viral RNA a,nd
RT/PCR was used to generate a patient derived segment that
comprised viral sequence coding 'for all of PR and as 1- .'.13
of RT. The PDS was inserted into an indicator gene viral
vector to generate a resistance test vector designated RTV-
964. RTV-964 was then 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 comprised members of t:he
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classes known as NRTIs (AZT, 3TC, d4T, ddI and ddC), NNRT:Ls
(delavirdine and nevirapine), and PRIs (indinavi:r,
nelfinavir, ritonavir, and saquinavir) . An IC50 was
determined for the resistance test vector pool for each drag
5 tested. The pattern of susceptibility to all of the drugs
tested was examined and compared to known patterns of
susceptibility. A pattern of susceptibility to the NNRTIs
was observed for patient RTV-964 in. which there was a
moderate decrease (10 fold) in delavirdine susceptibility
10 and a significiant decrease (750-fold) in nevi.rapine
susceptibility.
Determination of genotype of patient HIV samples
RTV-964 DNA was analyzed by ABI chain terminator automated
I5 sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type Glade B HIV-1 (HIV
Sequence Database Los Alamos, NM). The genotype was
examined for sequences that are different from the control
sequence. Mutations were noted at positions M41L, K43E,
20 D67N, K70R, L74I, V75S, Y181I, R211T, T215Y, D218E, a.nd
K219Q compared to the control sequence. M41L, D67N, K70R,
L74I, V75S, T215Y, and K219Q are associated with NR:TI
resistance. A mutation at R211T is a known polymorphism in
the sequence among different wild-type (drug-sensitive)
25 variants of HIV. Y181I had previously been shown to be
associated with high level resistance to nevirapine. We
examined the mutation, Y181I, using site directed
mutagenesis and in vitro phenotypic susceptibility testing
to correlate the observed changes in genotype w~_th
30 phenotype.
Site directed mutagenesis is used to confirm the role of
specific mutations in phenotypic susceptibility to anl~i-
retroviral drugs in HIV
35 The Y181I mutation was introduced into the resistance test
vector using the mega-primer method for site-directed
mutagenesis (Sakar and Sommar, Ibid). A resistance test
vector containing the Y181I mutation (Y181I -RTV) was then
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tested using the phenotypic assay described earlier and the
results were compared to those determined using a
genetically defined resistance test vector that was wild
type at position 181. We determined the pattern of
phenotypic susceptibility to t=he NNRTIs, delavirdine,
nevirapine and efavirenz, in the Y181I-RTV. On a wild type
background (i.e. Y181I mutation alone) the Y181I-RTV
displayed a moderate loss of susceptibility (20-fold) t:o
delavirdine and a significant loss of susceptibility (740-
fold) to nevirapine compared to a wild type control RTV.
The Y181I- RTV showed wild-type susceptibility (1.4-fold) t:o
efavirenz.
EXAMPLE 9
Using Resistance Test Vectors And Site Directed Mutants '.~o
Correlate Genotypes And Phenotypes Associated With NNR'.CI
Drug Susceptibility And Resistance in HIV: Y188
Preparation of resistant test vectors and phenotypic
analysis of patient 97-300 HIV samples
A resistance test vector was constructed as described in
Example 1 from a patient sample designated 97-300 , Th:is
patient had been previously treated with d4T and 3'TC
(NRTIs), indinavir (a PRI) and efavirenz (an NNRTI).
Isolation of viral RNA and RT/PCR was used to generate a
patient derived segment that ~~omprised viral sequences
coding for all of PR and as 1 - 313 of RT. The PDS was
inserted into an indicator gene viral vector to generate a
resistance test vector designated RTV-300. RTV-300 was then
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
comprised members of the classe~~ known as NRTIs (AZT, 3TC,
d4T, ddI and ddC), NNRTIs (delavirdine, efavirenz and
nevirapine), and PRIs (indinavir, nelfinavir, ritonavir, and
saquinavir). An IC50 was determined for the resistance test
vector pool for each drug tested. The pattern of
susceptibility to all of the drug tested was examined a.nd
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compared to known patterns of susceptibility. A pattern c>f
susceptibility to the NNRTIs was observed for patient RTV-
300 in which there was moderate decrease (25-fold) i_n
delavirdine sisceptibility and a substantial decrea~~e
(greater than 800-fold) in nevirapine susceptibility.
Determination of genotype of patient HIV samples
RTV-300 DNA analyzed by ABI chain terminator automated
sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type Glade B HIV-1_ (H:CV
Sequence Database Los Alamos, NH). The genotype was
examined for sequence that are different from the control
sequence. Mutations were noted a~ positions K32N, M184V and
Y188L compared to the control ~~equence. The mutation at
M184V is associated with 3TC resistance. Y188L h<~d
previously been shown to be associated with high level
resistance to efavirenz. Other mutations at position Y188
(i.e Y188C and Y188H) have been reported to have been
selected for by treatment with several NNRTIs (E-ePseU, E-
EPS, HEPT, Nevirapine, BHAP, U-8720E, TIBO 882913,
Loviride). We examined the mutation, Y188L, using site
directed mutagenesis and in vitro phenotypic susceptibility
testing to correlate the observed changes in genotype with
phenotype.
Site directed mutagenesis is used to confirm the role of
specific mutations in phenotypic susceptibility to
antiretroviral drugs in HIV
The Y188L mutation was introduced into the resistance test
vector using the mega-primer method for site-directed
mutagenesis (Sakar and Sommar, Ibid.): A resistance test
vector containing the Y188L mutation (Y188L-RTV) was then
tested using the phenotypic assay described earlier and t:he
results were compared to those determined using a
genetically defined resistance test vector that was wild
type at position 188. We determined the pattern of
phenotypic susceptibility to the NNRTIs, delavirdine,
nevirapine and efavirenz, in the Y188L-RTV. On a wild type
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background (i.e. Y188L mutation alone) the Y188L-R'TV
displayed a slight loss of susceptibility (9-fold) to
delavirdine and substantial loss of susceptibility (greater
than 800-fold) to nevirapine and a significant loss of
susceptibility (109-fold) to efavirenz compared to a wild
type control RTV. The approximate 100-fold loss of
susceptibility to efavirenz way; not as high as had been
previously reported.
Site directed mutagenesis is used to confirm the role of
specific mutations in pnenotypic susceptibility to
antiretroviral drugs in HIV
The Y188C mutation was introduced into the resistance test
vector using the mega-primer method for site-directed
mutagenesis (Sakar and Sommar, Ibid.). A resistance test
vector containing the Y188C mutation (Y188C-RTV) was then
tested using the phenotypic assay described earlier and t:he
results were compared to those determined using a
genetically defined resistance test vector that was wild
type at position 188. We determined the pattern of
phenotypic susceptibility to the NNRTIs., delavirdine,
nevirapine and efavirenz, in the Y188C-RTV. On a wild t;rpe
background (i.e. Y188C mutation alone) the Y188C-RTV
displayed a slight loss of :susceptibility (3-fold) to
delavirdine and a moderate loss of susceptibility (30-fo:Ld)
to nevirapine and efavirenz (20-fold) compared to a wild
type control RTV.
Site directed mutagenesis is used to confirm the role of
specific mutations in phenotypic susceptibility to
antiretroviral drugs in HIV
The Y188H mutation was introduced into the resistance test
vector using the mega-primer method for site-directed
mutagenesis (Sakar and Sommar, Ibid.).. A resistance test
vector containing the Y188H mutation (Y188H-RTV) was then
tested using the phenotypic assay described earlier and the
results were compared to those- determined ~~sing a
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genetically defined resistance test vector that was wild
type at position 18B. We determined the pattern of
phenotypic susceptibility to tt~e NNRTIs, delavirdine and
nevirapine, in the Y188H-RTV. On a wild type background
(i.e. Y188H mutation alone) the Y188H-RTV displayed a
moderate loss of susceptibility (3.5-fold) to nevirapine
compared to a wild type control RTV. The phenotypic
susceptibility of Y188H to efavirenz was not determined.
EXAMPhE 10
Using Resistance Test Vectors And Site Directed Mutants To
Correlate Genotypes And Phenotypes Associated With NNRTI
Drug Susceptibility And Resistance in HIV: E138 and Y188
Preparation of resistant test vectors and phenotypic
analysis of patient 97-249 HIV samples
A resistance test vector was constructed as described in
Example 1 from a patient sample designated 97-209. This
patient had been previously treated with AZT, ddI, d4T and
3TC (NRTIs), indinavir (a PRIs) and adefovir. 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 as 1 - 313 of RT. The PDS was inserted into an
indicator gene viral vector to generate resistance test
vector designated RTV-209. RTV-209 was then tested in a
phenotypic assay to determine accurately and quantitatively
the level of susceptibility to a panel of anti-retrovi:ral
drugs. This panel of anti-retroviral drugs comprised members
of the classes known as NRTIs (A.ZT, 3TC, d4T, ddI and ddC),
NNRTIs (delavirdine, efavirenz and nevirapine), and P:RIs
(indinavir, nelfinavir, ritonavir, and saquinavir). An I~~50
was determined for the resistance test vector pool for each
drug tested. The pattern of susceptibility to all of the
drug tested. The pattern of susceptibility to all of the
drugs tested was examined and compared to known patterns of
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susceptibility. A pattern of susceptibility to the NNRT=~s
was observed for patient RTV-209 in which there was a
moderate decrease (75-fold) in delavirdine susceptibility
and a substantial decrease (greater than 800-fold) :in
5 nevirapine susceptibility.
Determination of genotype of patient HIV samples
RTV-209 DNA was analyzed by ABI chain terminator automat~sd
sequencing. The nucleotide sequence was compared to the
10 consensus sequence of a wild type Glade B HIV-1 (HIV
Sequence Database Los Alamos, NM). The genotype w,as
examined for sequences that are different from the control
sequence. Mutations were noted at positions A62V, S68G,
V76I, F77L, F116Y, E138A, Q151M, M184V, Y188L and E291D
15 compared to the control sequence. The mutations at A62V,
V75I, F77L, F116Y, Q151M and M184V are associated with NRTI
resistance. A mutation at E138k; had previously been shown
to be associated with resistance to several NNRTIs and a
mutation at Y188L had previously been shown to be associated
20 wiht a decrease in susceptibility to efavirenz. We examined
the mutations Y188L and E1:38A using site directed
mutagenesis and in vitro phenot~rpic susceptibility testing
to correlate the observed changes in genotype with
phenotype.
Site directed mutagenesis is used to .confirm the role of
specific mutations in phenotypic susceptibility to
antiretroviral drugs in HIV
The E138A mutation alone and in combination with Y188L was
introduced into resistance test vectors using the mega
primer method for site-directed mutagenesis (Sakar and
Sommar, Ibid.). Resistance test vectors containing i~he
E138A mutation (E138A-RTV) or the E138 mutation along with
the Y1881 mutation (E138A-Y188L-RTV) were then tested using
the phenotypic assay described earlier and the results wE=re
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compared to those determined using a genetically defined
resistance test vector that was wild type at positions 188
and 138. We determined the pattern of phenotyp:Lc
susceptibility to the NNRTIs, df=_lavirdine, nevirapine and
efavirenz, in the E138A-RTV, Y188L-RTV and E138-Y188L-RTV.
On a wild type background (i.a. E138A mutation alone) the
E138A-RTV displayed wild-type susceptibility to delavirdine
(1.6-fold), nevirapine (1.3-fold) and efavirenz (1.4-fold).
The Y188L-RTV displayed a slight loss of susceptibility
(greater than 800-fold) to nevirapine and a significant loss
of susceptibility (110-fold) to efavirenz. The E138A-Y188L-
RTV displayed a moderate loss of susceptibility (75-fold) to
delavirdine and efavirenz (88-fold) and a substantial loss
of susceptibility to nevirapin~~ (greater than 800-fold)
compared to a wild type control- RTV. The combination of
mutations resulted in an increased effect on delavirdine
susceptibility compared tc the effect observed for each
mutation alone.
EXAMPhE 11
Using Resistance Test Vectors And SiteDirected Mutants To
Correlate Genotypes And Phenotypes Associated With NNRTI
Drug Susceptibility And Resistance in HIV: A98
Preparation of resistant test vectors and phenotypic
analysis of patient 98-675 HIV samples
A resistance test vector was constructed as described in
Example 1 from a patient sample designated 98-675. This
patient had been previously treated with ddI, AZT, and 3TC
(NRTIs), and saquinavir and nelfv~navir (PRIs). 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 as 1 - 313 of RT. The PDS was inserted into an
indicator gene viral vector to generate a resistance test
vector designated RTV-675. RTV-675 was then tested in a
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-17
phenotypic 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, ddI
and ddC), NNRTIs (delavirdine, E_~favirenz and nevirapine),
and PRIs (indinavir, nelfinavir, ritonavir, and saquinavir).
An IC50 was determined for the resistance test vector pool
for each drug tested. The pattern of susceptibility to all
of the drugs tested was examined and compared to known
patterns of susceptibility. A pattern of susceptibility t.o
the NNRTIs was observed for patient RTV-675 in which wilcl-
type susceptibility (2.1-fold) was observed for delavirdine
and a slight decrease (6-fold) in nevirapine susceptibility
was observed.
Determination of genotype of patient HIV samples
RTV-675 DNA was analyzed by ABI chain terminator automatE:d
sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type Glade B HIV-1 (H=fV
Sequence Database Los Alamos, NM). The genotype was
examined for sequences that are different from the control
sequence. Mutations were noted at positions M41L, S48t,
L74V, A98G, M184V and T215Y are associated with NR'rI
resistance. A mutation at A98G had previously been shown v o
be associated with resistance to :nevirapine. We examined t)ze
mutation A98G using site directed mutagenesis and in vitro
phenotypic susceptibility testing to correlate the observed
changes in genotype with phenotype.
Site directed mutagenesis is used to confirm the role of
specific mutations in phenotypic susceptibility to
antiretroviral drugs in HIV
The A98G mutation into the resistance test vector ussng the
mega-primer method for site-directed-mutagenesis (Sakar and
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Sommar, Ibid.). A resistance test vector containing the
A98G mutation (A98G-RTV) was then tested using the
phenotypic assay described earlier and the results were
compared to those determined using a genetically defined
resistance test vector that was wild type at position 98.
We determined the pattern of phenotypic susceptibility to
the NNRTIs, delavirdine, nevirapine and efavirenz, in t:he
A98G-RTV. On a wild type background (i.e. A98G matati_on
alone) the A98G RTV displ<~yed a slight lass of
susceptibility to delavirdine (3-fold), nevirpine (8-fold)
and efavirenz (3-fold) compared to a wild type control RfV.
Example 12 Using Resistance Test Vectors and Site Directed
Mutants to Correlate Genotypes And Phenotypes Associated
with NNRTI Drug Susceptibility and Resistance in HIV: A98
and 6190
Preparation of resistant test vectors and phenotypic
analysis of patient B HIV samples.
A resistant test vector was constructed as described in
Example 1 from a patient sample designated B. The anti-
retroviral treatment this patient received is unknown.
Isolation of viral RNA and RTi'PCR was used to generate a
patient derived segment that comprised viral sequences
coding for all of PR and as 1-313 of RT. The PDS was
inserted into an indicator gene viral vector to generate a
resistant test vector designated RTV-B. Individual clones
of the RTV-B pool were selected and then 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 camprised
members of the classes known as NRTIs (AZT, 3TC, d4T, ddI
and ddC), NNRTIs (delavirdine and nevirapine), and PRIs
(indinavir, nelfinavir, ritonavi_r, and saquinavir). An IC50
was determined for the resistance test vector clone for each
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drug tested. The pattern of sup>ceptibility to all of the
drugs tested was examined and compared to known patterns of
susceptibility. A pattern of susceptibility to the NNRTIs
was observed for patient RTV-B clone 1 in which there was an
increase in susceptibility (0.55-fold) to delaviridine, a
substantial loss of susceptibility (640-fold) to nevirapine
and significant loss of susceptibility (250-fold) to
efavirenz.
Determination of genotype of patient HIV samples
RTV-B clone 1 DNA was analyzed by ABI chain terminator
automated sequencing. The nucleotide sequence was compared
to the consensus sequence of a wild type Glade B HIV-1 (HIV
Sequence Database Los Alamos, NM). The genotype was
examined for sequences that are different from the control
sequence. Mutations were noted at positions M41L, A98~~,
M184V, L210W, 8211?, T215Y, E297P, and G190S compared to the
control sequence. M41L, M184V, L210W and T215Y are
associated with NRTI resistance. A mutation at A98G had
previously been shown to be associated with resistance to
nevirapine. A mutation at pos_Ltion G190A had previously
been shown to be associated witru changes in susceptibility
to nevirapine. Other changes at position 190 (i.e. E, Q,
and T) have also been reported. We examined the mutations
A98G and G190S, using site directed mutagenesis and i_n vitro
phenotypic susceptibility testing to correlate the observed
changes in genotype with phenotype.
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Site directed mutagenesis is used to confirm the role of
specific mutations in phenotypic susceptibility to anti-
viral drugs in HIV
The A98 and G190S mutations were introduced alone or in
5 combination into the resistance test vector using the mega
primer method for site-directed mutagenesis (Sakar and
Sommar, Ibid. ) . Resistance test 'sectors containing the A98G
mutation (A98G-RTV), the G190S mutation (G190S-RTV) and boi~h
mutations (A98G-G190S-RTV) were then tested using the
10 phenotypic assay described earlier and the results were
compared to those determined u~;ing a genetically definE~d
resistance test vector that was wild type at position 98 and
190. We determined the pattern of phenotypic susceptibility
to the NNRTIs, delavirdine, nevirapine and efavirenz, in t)~e
15 three vectors. On a wild type background (i.e. A9BG
mutation alone) the A98G-RTV displayed a slight Loss of
susceptibility to delavirdine (3--fold), nevirapine (8-fold)
and efavirenz (3-fold) compared to a wild type control RTV.
On a wild type background (i.e. G190S mutation alone) the
20 G190S-RTV displayed increased susceptibility (0.5-fold) to
delavirdine, a moderate loss of :susceptibility (75-fold) to
nevirapine and a slight loss of susceptibility (8-fold) to
efavirenz compared to a wild type control RTV. The A98G-
G190S-RTV displayed increased susceptibility (0.8-fold) to
25 delavirdine, but a substantial loss of susceptibility to
both nevirapine (greater than 800-fold) and efavirenz
(greater than 250-fold) compared to a wild type control RfV.
Although only a slight loss of susceptibility to efavire:nz
was observed for the individual mutations, the combination
30 of A98G and G190S resulted ._n a substantial loss of
susceptibility to efavirenz. Likewise, this combination of
mutation resulted in a greater loss of susceptibility to
nevirapine than the sum of the t:wo mutations alone.
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EXAMPhE 13
Using Resistance Test Vectors and Site Directed Mutants
Correlate Genotypes And Phenotypes Associated With NNRTI
Drug Susceptibility And Resistance in HIV: Y181 and A98
Preparation of resistant test vectors and phenotypic
analysis of patient 98-1057 samples
A resistance test vector was constructed as described in
Example 1 from a patient sample designated 98-1057. This
patient had been previously treated with ddI, d4T, AZT, anal
3TC (NRTIs), saquinavir and indinavir (PRIs) and delavirdir,,e
(an NNRTI). Isolation of viral RNA and RT/PCR was used t.o
generate a patient derived segment that comprised viral
sequences coding for all of PR and as 1-313 RT. The PDS was
inserted into an indicator gene viral vector to generate
resistance test vector designated RTV-1057. RTV-1057 was
then 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-retrovira:L drugs
comprised members of the classes known as NRTIs (AZT, 3TC,
d4T, ddI, and ddC), NNRTIs (delavirdine, efavirenz and
nevirapine) and PRIs (indinavir, nelfinavir, ritonavir, and
saquinavir). An IC50 was determined for the resistance test
vector pool for each drug tested. The pattern of
susceptibility to all of the drugs tested was examined and
compared to known patterns of susceptibility. A pattern of
susceptibility to the NNRTIs was observed for patient RT'J-
1057 in which there was a moderate decrease in delavirdine
(35-fold) susceptibility and a significant decrease (610-
fold) in nevirapine susceptibility.
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Determination of genotype of patient HIV samples
RTV-1057 DNA was analyzed by ABI chain terminator automated
sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type Glade B HIV-1 (HIV
Sequence Database, Los Alamos, NM). The genotype was
examined for sequences that are different from the control
sequence. Mutations were noted at positions T39A, M41L,
A62V, D67E, T69SST, A98G, I135T, Y181C, T200I and T21'.~Y
compared to the control sequence M41L, A62V, D67E, T69SST,
and T215Y are associated with NRT:I resistance. Mutations at
positions I135T and T200I are known polymorphisms in the
sequence among different wild-type (drug-sensitive) variants
of HIV. Y181C and A98G have been previously shown to be
associated with resistance to certain NNRTIs. We examined
the mutations Y181C and A98G using site directed mutagenesis
and in vitro phenotypic susceptibility testing to correlate
the observed changes in genotype with phenotype.
Site directed mutagenesis is used to confirm the role of
specific mutations in phenotypic susceptibility to anti
retroviral drugs in HIV
The Y181C and A98G mutations were introduced alone and in
combination into resistance test vectors using the mega-
primer method for site-directed mutagenesis (Sakar and
Sommar, Ibid.). Resistance test vectors containing the
Y181C mutation (Y181C-RTV) and the A98G mutation (A98G-RfV)
and both mutations (Y181C-A98G-RTV) were then tested using
the phenotypic assay described earlier and the results were
compared to those determined using a genetically defined
resistance test vector that was wild type at position 1.81
and 98. We determined the pattern of phenoty~>ic
susceptibility to the NNRTIs, dc:lavirdine, neviraphine and
efavirenz, in the three vectors. On a wild type background
(i.e. Y181C mutation alone) the f181C-RTV displayed moderate
loss of susceptibility (35-~old)_ to delavirdine, a
significant loss of susceptibility (161-fold) to nevirapine
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and a slight loss of susceptibility (3-fold) to efaviren.z
compared to a wild type control R'fV. The A98G-RTV displayed
a slight loss of susceptibility to delavirdine (3-fold),
nevirapine (8-fold) and efavirenz (3-fold) compared to a
wild type control RTV. The Y181C-A98G-RTV displayed
significant loss of suscep~ibility (240-fold) t:o
delavirdine, a substantial loss of susceptibility (greate:r
than 800-fold) to nevirapine and a slight loss of
susceptibility (7-fold) to efavirenz compared to a wild type
control RTV. THese data indicated that the comination of the
two mutations, Y181C and A98G, resulted in a greater loss of
susceptibility to both delavirdine and nevirapine than the
sum of effects observed fer these two mutations
individually.
EXAMPLE 14
Using Resistant Test Vectors and Site Directed Mutants t.o
Correlate Genotypes and Phenotypes Associated with NNR'.rI
Drug Susceptibility and Resistance in HIV: K101 and 6190
Preparation of resistant test vectors and phenotypic
analysis of patients 98-644 and 98-1060 HIV samples
A resistance test vector was constructed as described in
Example 1 from a patient sample designated 98-644. This
patient had been previously treated with d4T (an NNRTI),
indinavir (a PRI and efavirenz (an NNRTI). A second
resistance test vector was constructed as described in
Example 1 from a patient sample designated 98-1060. This
patient had been previously treated with d4T (an NNRTI).
indinavir (a PRI) and efavirnez (an NNRTI) . 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 as 1-313 of RT. The PDS was inserted into an indicator
gene viral vector to generates resistance test vectors
designated RTV-644 and RTV-1060. RTV-644 and RTV-1060 were
then 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
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comprised members of the classes ltnown as NNRTIs (AZT, 3TC',
d4T, ddI, and ddC), NNRTIs (cielavi.rdine and nevirapine), and
PRIs (indinavir, nelfinavir, ritonavir, and saquinavir). An
IC50 was determined for the resistance test vector pool for
each drug tested. The pattern of susceptiblity to all of
the drugs tested was examined and compared to known patterns
of susceptibility. A pattern of susceptibility to the
NNRTIs was observed for patient RTV-644 in which there was
a very slight (2.5-fold) decrease in delavirdine
susceptibility and a significant (600-fold) decrease in
nevirapine susceptibility. A pattern of susceptibility too
the NNRTIs was observed for patient RTV-644 in which there
was a very slight (2.5-fold) decrease in delavirdine
susceptibility and a signigicant (600-fold) decrease :in
nevirapine susceptibility. A pattern of susceptibility too
the NNRTIs was observed for patient RTV-1060 in which wild-
type susceptibility (1.5-fold) to delavirdine was observed.
A significant decrease in efavi.renz susceptibility (900-
fold) and a substantial decrease ~o nevirapine (greater than
S00-fold) susceptibility was observed for RTV-1060.
Determination of genotype of patient HIV samples
RTV-644 and RTV-1060 DNA were analyzed by ABI chain
terminator automated sequencing. The nucleotide sequence
was compared to the consensus of a wild type Glade B HIV-1
(HIV Sequence Database Los Alamos, NM). The genotype was
examined for sequences that are different from the control
sequence. Mutations were noted at positions K101E and G190S
for RTV-644 compared to the control sequence and mutations
were noted at positions K101E, G190S, T200A and T215Y for
RTV-1060 compared to the control sequence. The sequence at
position T215 was a mixture of wild-type and mutation. A
mutation at position K101E had been previously shown to be
associated with resistance to several NNRTIs including high
level resistance to delavi.rdine. A mutation at position
G190A had previously been shown to be associated with
changes in susceptibility to nevirapine. Other changes at
position 190 (i.e. E, Q and T) have also been reported. We
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examined the mutations K101E and c~190S, using site directed
mutagenesis and in vitro phenotypic susceptibility testing
to correlate the observed changes in genotype with
phenotype.
5
Site directed mutagenesis is used to confirm the role of
specific mutations in phenotypic susceptibility t:o
antiretroviral drugs in HIV
The K101E and Gl9oS mutations were introduced alone and in
10 combination into resistance test vectors using the mega
primer method for site-directed mutagenesis (Sakar and
Sommar, Ibid.). Resistance te:~t vectors containing the
K101E mutation (K101E-RTV), the G190S mutation (G190S-RTV)
were then tested using the phenotypic assay described
15 earlier and the results were compared to those determined
using a genetically defined resistance test vector that was
wild type at positions 101 and 190. We determined the
pattern of phenotypic susceptibility to the NNRTI:~,
delavirdine, nevirapine and efavirenz, in all three vectors.
20 On a wild type background ( i . a . K101E mutation alone ) tlZe
K101E-RTV displayed a slight loss of susceptibility (5-fold)
to delavirdine and efavirenz (5-fold) and a moderate loss of
susceptibility (12-fold) to nevirapine compared to a wild
type control RTV. The K101E-G190S-RTV displayed increased
25 susceptibility to delavirdine (0.5-fold), a moderate loss of
susceptibility to nevirapine (75-fold) and a slight loss of
susceptibility (7.6-fold) r_o efavirenz compared to a wild
type control RTV. The K101E-G190S-RTV displayed wild-type
susceptibility (l.4-fold) to del.avirdine and a substantial
30 loss of susceptibility to both nevirapine (greater than 800-
fold) and efavirenz (greater than 250-fold) compared to a
wild type control RTV.
In this example, the combination of mutations, G190S a.nd
35 K101E, displayed a novel phenotypic pattern. The
combination resulted in the reversal of the effect on
delavirdine susceptibility observed for the G190S mutation
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alone and a greater than additive effect on t)ze
susceptibility for both nevirapine and efavirenz.
EXAMPLE 15
Using Resistance Test Vectors And Site Directed Mutants 'To
Correlate Genotypes And Phenotypes Associated With NNRTI
Drug susceptibility And Resistance in HIV: V108I
Preparation of resistant test vectors and phenotypic
analysis of patient 98-652 HIV samples
A resistance test vector was constructed as described in
Example 1 from a patient sample designated 98-652. This
patient had no previous anti-retroviral treatment.
Isolation of viral RNA and RT/F?CR was used to generate a
patient derived segment that comprised viral. sequences
coding for all of PR and as 1 - 313 or RT. The PDS was
inserted into an indicator gene viral vector to generate a
resistance test vector designated RTV-652. RTV-652 was then
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
comprised members of hte classes known. as NRTIs (AZT, 3TC,
d4T, ddI and ddC), NNRTIs (delav:irdine and nevirapine), a,nd
PRIs (indinavir, nelfinavir, ritonavir and saquinavir). An
IC50 was determined for the resistance test vector pool for
each drug tested. The pattern of susceptibility to all of
the drugs tested was examined and compared to known patterns
of susceptibility. A pattern of susceptibility to t:he
NNRTIs was observed for patient: RTV-652 in which increase
susceptibility (0.97-fold) to delavirdine was observed and
a slight decrease (5-fold) in nevirapine susceptibility was
observed.
Determination of genotype of patient HIV samples
RTV-652 DNA was analyzed by ABI chain terminator automal~ed
sequecing. The nucleotide sequence was compared to the
consensus sequence of a wild type Glade B HIV-1 (13IV
Sequence Database Los Alamos, NM1. The genotype was
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examined for sequences that are diffrent from the control
sequence. Mutations were noted at positions M41L, V108I,,
I135T, L210W, R211K and T215D compared to the contro.L
sequence. M41L, L210W and T215D are associated with NRT:I
resistance. Mutations at positions I135T and R211K are known
polymorphisms in the sequence among different wild-type
(drug-sensitive) variants of HIV. V108I is known to b~~
associated with resistance to several NNRTIs. We examined
the mutation V108I using site directed mutagenesis and i:n
vitro phenotypic susceptibility testing to correlate the
observed changes in genotype with phenotype.
Site directed mutagenesis is used to confirm the role of
specific mutations in phenotypic susceptibility to
antiretroviral drugs in HIV
The V108I mutation was introduced into the resistance test
vector using the mega-primer method for site directed
mutagenesis (Sakar and Sommar, Ibid.). A resistance test
vector containing the V108I mutation (V108I-RTV) was then
tested using the phenotypic assay described earlier and th.e
results were compared to those determined using a
genetically defined resistance test vector that was wild
type at position 108. We determined the pattern of
phenotypic susceptibility to the NNRTIs, delaviridine,
nevirapine and efavirenz, in the V108I -RTV. On a wild ty~>e
background (i.e. V108I mutation alone) the V108I -RTV
displayed wild-type susceptibility (1.3-fold) t:o
delaviridine and efavirenz (1.7-i=old) and a slight loss of
susceptibility (3-fold) to nevir_apine compared to a type
control RTV.
EXAMPLE 16
Using Resistance Test Vectors And Site Directed Mutants 'ro
Correlate Genotypes And Phenotypes Associated With NNR'rI
Drug Susceptibility And Resistance in HIV: K103 and K101 and
6190
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Preparation of resistant test vectors and phenotypic
analysis of patient 98-955 HIV samples
A resistance test vector was constructed as described i.n
Example 1 from a patient sample designated 98-955. This
patient had been previously treated with nelfinavir (a PRI).
Isolation of viral RNA and RT/PCR was used to generate a
patient derived segment that comprised viral sequence's
coding for all of PR and as 1 - 313 of RT. The PDS was
inserted into an indicator gene viral vector to generate a
resistance test vectors designated RTV-955. RTV-955 was then
tested in a phenotypic assay tc> 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, ddI and ddC), NNRTIs (delaviridine, efavirenz and
nevirapine), and PRIs (indinavir, nelfinavir, ritonavir, and
saquinavir). An IC50 was determined for the resistance test
vector pool for each drug tested. The pattern of
susceptibility to all of the drugs tested was examined a:nd
compared to known patterns of susceptibility. A pattern of
susceptibility to the NNRTIs was observed for patient RTV-
955 in which there was a slight decrease (4-fold) in
delaviridine susceptibility and a significant decrease (530-
fold) in nevirapine susceptibility.
Determination of genotype of patient HIV samples
RTC-955 DNA was analyzed by ABI chain terminator automated
sequencing. The nucleotide sequence was compared to the
consensus sequence of wild type Glade B HIV-1 (HIV Sequence
Database Los Alamos, NM). The genotype was examined for
sequences that are different from the control sequence.
Mutations were noted at positions K20R, V35I, A62V, D67N,
T69D, V75I, F77L, K101E, K103N, Y115F, F116Y, Q151M, I167V,
Y181C, M184V, G190A, I202V, R211K, F214L, T215V, and K27.9Q
compared to the control sequence. Mutations at positions
K101E, K103N, Y181C, G190A, and F214 L were mixtures of
wild-type and the mutation. A62V, D67N, T69D, V75I, F7'7L,
Y115F, F116Y, Q151M, M184V, T215V and K219Q are associated
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with NRTI resistance. Mutations at. V35I, R211K and F214L are
known polymorphism in the sequence among different wild-type
(drug sensitive) variants of HIV. a mutation at position
K101E had been previously shown to be associated with
resistance to the NNRTIs. A mutation at Y181I had previous7_y
been shown to be associated with high level resistance t:o
nevirapine. a mutation at K103N had previously been shown too
be associated with resistance to the three NNRTIs,
delaviridine and nevirapine and efavirenz. We examined the
mutations K101E, J103N and G1.90A using site directed
mutagenesis and in vitro phenotypic susceptibility testing
to correlate the observed changes in genotype with
phenotype.
Site directed mutagenesis is used to confirm the role of
specific mutations in phenotypic susceptibility to anti-
retroviral drugs in HIV
The K101E, K103N and G190A mutations were introduced alone
and in combination into resistance test vectors using the
mega-primer method for site-directed mutagenesis (Sakar and
Sommar, Ibid.). Resistance test vectors containing the K101E
mutation (K101E-RTV), the K103N mutation (K0103N-RTV), the
6190 mutation (g190A-RTV and two mutations (KlOlE-G190A-RTV)
and (K103N-G190A-RTV) were then tested using the phenotypic
assay described earlier and the results were compared to
those determined using a genetically defined resistance test
vector that was wild type at positions 101, 103 and 190. We
determined the pattern of phenotypic susceptibility to the
NNRTIs, delaviridine, nevirapine, and efavirenz, in all 5
vectors. On a wild type background (i.e. K101E mutation
alone) the K101E-RTV displayed a slight loss (5-fold) os
susceptibility to delavirdine and efavirenz (5-fold) anc~ a
moderate loss of susceptibility (12-fold) to nevirapine (55-
fold) and efavirenz(30-fold) compared to a wild type control
RTV. On a wild type background (i.e. G190A mutation alone)
the G190A -RTV displayed increased susceptibility (8-fo:Ld)
efavirenz compared to a wild type control RTV. The K10:LE-
G190A-RTV displayed wild-type susceptibility (2-fold) to
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delavirdine, substantial loss of: susceptibility (greater
than 800-fold) to nevirapine and a significant loss of
susceptibility (120-fold) to efavirenz compared to a wild
type control RTV. The K103N-6190-RTV displayed a moderate
5 loss of susceptibility (40-fold) to delavirdine, substantial
loss of susceptibility (greater than 800-fold) to nevirapin.e
and a significant loss of susceptibility (215-fold) to
efavirenz compared to a wild type. control RTV. Th.e
introduction of a second mutation to a vector containing th.e
10 G190A resulted in the reversal of the effect on delavirdin.e
susceptibility observed for the G190A mutation alone. The
6190-a mutation displayed an increased susceptibility t.o
delviridine. However, the addition of either K10E or K103N
to the G190A mutation resulted in a slight loss of
15 susceptibility to delavirdine. Furthermore, the combination
of G190A and K101E resulted in a greater than additive
effect on the loss of susceptibility to nevirapine arid
efavirenz. Lastly, these data indicated that the combination
of the two mutations G190A and K103N resulted in a greater
20 loss of susceptibility to both nevirapine and efavirenz than
the sum of effects observed for these two mutations
individually.
EXAMPLE 17
25 Using Test Vectors And Site Directed Mutants To Correlate
Genotypes And Phenotypes Associated With NNRTI Drug
Susceptibility An Resistance in HIV: V106 and V189 and V1l31
and F227
30 Preparation of resistant test vectors and phenotypic
analysis of patient 98-1033 and 98-757 HIV samples
A resistance test vector was constructed as described in
Example 1 from a patient samples designated 98-1033. This
patient had been previously treated with AZT, d$T, 3TC a:nd
35 ddI (NRTI), saquinavir, indinavir and nelfinavir (PRIs and
nevirapine (an NNRTI). a second resistance test vector was
constructed as described in Example 1 from a sample obtained
from the same patient at a different time point and
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designated 98-757. This patient had received an additional
8 weeks of treatment with nevirapine San NNRTI) d4T (a.n
NRTI), and saquinavir and relfin.avir (PRIs). 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 as 1 - 313 of RT. The PDS was inserted into an
indicator gene viral vector to generate resistance test
vectors designated RTV-1033 and RTV-757. RTV-1033 and RTV-
757 were then tested in a phenotypic assay to determine
accurately and quantitatively the level of susceptibility t:o
a panel of anti-retroviral drugs. This panel of anti-
retroviral drugs comprised members of the classes known as
NRTIs (AZT, 3TC, d4T, ddI and ddC), NNRTIs (delavirdine and
nevirapine), and PRIs (indi.navir, nelfinavir, ritonavir, and
saquinavir). An IC50 was determined for the resistance test
vector pool for each drug tested. The pattern of
susceptibility to all of the drugs tested was examined and
compared to known patterns of susceptibility. A pattern of
susceptibility to the NNRTIs was observed for patient RTV-
1033 in which there was a moderate decrease (30-fold) in
delavirdine susceptibility and a substantial decrease
(greater than 800-fold) in nevirapine susceptibility and a
significant decrease (200-fold) i.n efavirenz susceptibility.
A pattern of susceptibility to the NNRTIs was observed for
patient RTV-757 in which there was a slight decrease ( 10-
fold) in delavirdine susceptibility and a substantial
decrease (greater than 800-fold) in nevirapine
susceptibility.
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Determination of Genotype of Patient HIV Samples
R.TV-1033 and RTV-757 DNA were analyzed by ABI chain
terminator automated sequencing. The nucleotide sequence
was compared to the consensus sequence of a wild type Glade
B HIV-1 (HIV Sequence Database Los Alamos, NM). The
genotype was examined for sequences that are different from
the control sequence. Mutations were noted at positions
V35I, D67N, T69D, K70R, V106A, V189L, T200A, I202T, R211K,
T215F, D218E, K219Q, H221Y, F227L, L228H and 8289 for RTV'-
1033 compared to the control sequence. Mutations were noted
at positions V35I, D67N, T69D, K70R, V106A, V108I, L109V,
Y108C, V189L, T200A, I202T, R21.1K, T215F, D218E, K219Q,
H221Y, L228H, L283I and R284K for RTV-757 compared to the
control sequence. The sequences at positions V106A, V108I
and L109V were a mixture of wild--type and mutation. D67D1,
T69D, K70R, T215F and K219Q are associated with NRTI
resistance. Mutations at V35I, 'r200A, R211K and R284K ai:e
known polymorphisms in the sequence among different wild-
type (drug-sensitive) variants of HIV. A mutation at V106A
had previously been shown to be associated with increase
resistance to nevirapine. A mutation at V189I had
previously been shown to be associated with NNRTI resistance
but a mutation to L at this posit_Lon had not been previou sly
reported to be associated with NNRTI resistance. A mutation
at V108I had previously been sruown to be associated wi~~h
increased resistance to both delavirdine and nevirapine. A
mutation at Y181C had also previously been shown to be
associated with increased resistance to both delavirdine a:nd
nevirapine. We examined the mutations V106A, V189L, V181C
and F227L using site directed mutagenesis and in vitro
phenotypic susceptibility testing tc correlate the observed
changes in genotype with phenotype.
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Site directed mutagenesis is used to confirm the role of
specific mutations in phenotypic susceptibility to anti-
retroviral drugs in HIV
The mutations V106A, V189L, V181C an F227L were introduced
alone and in combination into resistance test vectors using
the mega-primer method for site-cirected mutagenesis (Saka r
and Sommar, Ibid.). Resistance test vectors containing t he
V106A mutation (V106A-RTV), the V189L mutation (V189L-RTV),
the V181C mutation (V181C-RTV) and F227L mutation lF2271
RTV) and two mutations (V106A-Y1.81C-RTV) and (V106A-V189L-
RTV) and (V106A-F227-RTV) and (V181C-F227-RTV) and three
mutations, (V106A-Y181C-F227L-RTV) were then tested using
the phenotypic assay described earlier and the results were
compared to those determined using a genetically defined
resistance test vector that was wild type at positions 106,
189, 181 and 227. We determine:i the pattern of phenotypic
susceptibility to the NNRTIs, delavirdine, nevirapine a.nd
efavirenz, in all nine vectors. On a wild type background
(i.e. V106A mutation alone) the V106A-RTV displayed a slight
loss (5-fold) of susceptibility to delavirdine and a
moderate loss of susceptibility (60-fold) to nevirapine and
wild-type susceptibility (1.7-fo:Ld) to efavirenz compared to
a wild type control RTV. On a wild type background (i..e.
V189L mutation alone) the V189-RTV displayed wild type
susceptibility to delavirdine (1.8-fold), nevirapine (1.3-
fold) and efavirenz (1.3-fold) compared to a wild type
control RTV. On a wild type background (i.e. V181C mutation
alone) the Y181C-RTV displayed a significant loss of
susceptibility (100-fold) to delavirdine and a substantial
loss of susceptibility (greater than 800-fold) to nevirapine
and a slight loss of susceptibility (4-fold) to efavirenz
compared to a wild type control RTV. On a wild type
background (i.e. F227L mutation alone) the F227L-RTV
displayed increased susceptibility (0.03-fold) to
delavirdine and efavirenz (0.48-fold) and a slight loss of
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susceptibility (3-fold) to nevirapine compared to a wi7_d
type control RTV. The V106A-Y181C-RTV displayed a
significant loss of susceptibility (100-fold) t:o
delavirdine, a substantial loss of susceptibility (greater
than 800-fold) to nevirapine and slight loss of
susceptibility (4-fold) to efavirenz compared to a wild type
control RTV. The V106A-V189L-RTV displayed a slight loss o f
susceptibility (3-fold) to delavirdine, a moderate loss of
susceptibility (50-fold) to nevirapine and wild-type
susceptibility (1-fold) to efavirenz compared to a wild type
control RTV. The V106A-F227-RTV displayed a slight loss of
susceptibility (3-fold) to delavirdine, a substantial loss
of susceptibility (greater than 800-fold) to nevirapine and
a slight loss of susceptibility (8-fold) to efavirenz
compared to a wild type control RTV. The Y181C-F227L-RTV
displayed increased susceptibility (0.89-fold) to
delavirdine and efavirenz (0.99-fold) and a significant loss
of susceptibility (285-fold) to nevirapine compared to a
wild type control RTV. The V106A-Y181C-F227L-RTV displayed
a moderate loss (50-fold) of su~~ceptibility to delavirdine
and a substantial loss of susceptibility (greater than 800-
fold) to nevirapine and a slight loss of susceptibility (12-
fold) to efavirenz compared to a wild type control P,TV.
EXAMPLE 18
Using Resistance Test Vectors And Site Directed Mutants To
Correlate Genotypes And Phenotypes Associated With NNRTI
Drug Susceptibility And Resistance In HIV: Y188 and L100 and
K103
Preparation of resistance test vectors and phenotypic
analysis of patient 98-1058 HIV samples
A resistance test vector was constructed as described in
Example 1 from a patient sample designated 98-1058. Tlzis
patient had been previously treated with ddI, d4T, AZT, 3'TC,
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ddC and abacavir (NRTIs), indinavir and amprenavir (PRI~~)
and nevirapine (an NNRTI). Isolation of viral RNA and
RT/PCR was used to generate a pat=ient derived segment that
comprised viral sequences coding for all of RP and as 1 -
5 313 of RT. The PDS was inserted into an indicator gene
viral vector to generate a resistance test vector designated
RTV-1058. Individual clones of RTV-1058 were selected and
were then tested in a phenotypic assay to determine
accurately and quantitatively the level of susceptibility to
10 a panel of anti-retroviral drugs. The panel of ant:i-
retroviral drugs comprised members of the classes known as
NRTIs (AZT, 3TC, d4T, ddI and ddC), NNRTIs (delavirdine a:zd
nevirapine), an PRIs (indinavir, nelfinavir, ritonavir, a:nd
saquinavir). An IC50 was determined for the resistance test
15 vector pool for each drug tested. The pattern of
susceptibility to all of the drugs tested was examined and
compared to known patterns of susceptibility. A pattern of
susceptibility to the NNRTIs was observed for clones 4, 5
and 10 from patient RTV-1058. Clone 4 displayed a
20 significant loss of susceptibility (85-fold) for delavirdine
and a substantial loss of susceptibility (greater than 800-
fold) for nevirapine. Clone 5 displayed a substantial loss
of susceptibility (250-fold) to delavirdine and a
significant loss of susceptibility (120-fold) to nevirapine.
25 Clone 10 displayed a substantial loss of susceptibility
(greater than 250-fold) to del~avirdiwe and (greater than
800-fold) to nevirapine.
Determination of genotype of patient HIV samples
30 RTV-1058 DNA was analyzed by ABI chain terminator automai~ed
sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type Glade B HIV-1 (13IV
sequence Database Los Almos, NM). The genotype was examined
for sequences that are different from the control sequen~~e.
35 Mutations were noted at positions M41L, A62V, D6~N, T69SST,
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L74V, L100I, K103N, V118I, I135T, T200S, L210W, R211K arid
T215Y compared to the control seqvaence. L74V and L100I were
mixtures of wild-type and mutation. Clone 4 contained
mutations at positions K103N and Y188L. Clone 5 contained
mutations at positions L100I and K103N. Clone 10 contained
mutations at positions L100I, K103N and Y188L. M41L, A62V,
D67N, T69SST, L79V, L210W and T215Y are associated with NRTI
resistance. Mutations at positions I135T, T200S and R211T
are known polymorphisms in the sequence among different
wild-type (drug-sensitive) variants of HIV. A mutation at
L100I had previously been shown to be associated with
resistance to delavirdine and nevirapine. A mutation at
K103N had previously been shown to be associated with
resistance to delavirdine, nevvArapine and efavirenz. We
examined the mutations, Y188L, '~100I and K103N, using site
directed mutagenesis and in vitro phenotypic susceptibility
testing to correlate the observe=_d changes in genotype with
phenotype.
Site directed mutagenesis is used to confirm the role of
specific mutations in phenotypic suspectibility to anti-
restroviral drugs in HIV
The mutations Y188L, L100I and K103N were introduced alone
and in combinationn into resistance test vectors using 'the
mega-primer method for site-directed mutagenesis (Sakar and
Sommar, Ibid.). Resistance test vectors containing the
Y188L mutation (Y188L-RTV), the L100I mutation (L100I-RTV),
the K103N mutation (K103N-RTV), the two mutations (K103N-
Y188L-RTV) and (L100I-K103N-RT'J), and, the three mutations
(L100I-K103N-Y188L-RTV) were then tested using the
phenotypic assay described earlier and the results were
compared to those determined !sing a genetically defined
resistance test vector that was wild type at positions 188,
100, and 103. We determined the pattern of phenotypic
susceptibility to the NNRTIs, delay-irdine, nevlrapine and
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efavirenz, in all 6 vectors. On a wild type background
(i.e. Y188L mutation alone) the Y1.88L-RTV displayed a slight
loss of susceptibility (9-fold) to delavirdine, a
substantial loss of susceptibility (greater than 800-fold)
to nevirapine and a moderate loss of susceptibility (110-
fold) to efavirenz compared to a wild type control RTV. On
a wild type background (i.e. L100I mutation alone) the
L100I-RTV displayed a moderate loss of susceptibility (30-
fold)to delavirdine and efavirenz (10-fold) and a slight
displayed moderate loss of susceptibility (10-fold) and a
slight loss of susceptibility (3-fold) to nevirapi:ne
compared to a wild type control RTV. On a wild type
background (i.e. K103M mutation alone) the K103N-RTV
displayed moderate loss of to delavirdine susceptibility
(50-fold), nevirapine (55-fold;. and efavirenz (30-fold)
compared to a wild type control RTV. The K103N-Y188L-RTV
displayed substantial loss of susceptibility to delavirdine
(greater than 250-fold), nevirap:ine (greater than 800-fold)
and efavirenz (greater that 25()-fold). compared to a wild
control RTV. The L100I-K103N-RTV displayed substantial loss
of susceptibility (greater that 250-fold) to delavirdine and
efavirenz (greater that 250-fo=.d) and a moderate loss of
susceptibility (70-fold) to nev~irapine compared to a wild
type control RTV. The L100I-K103N-Y188L-RTV displayed
substantial loss of susceptibility to delavirdine (great=er
than 250-fold), nevirapine (greater than 800-fold), and
efavirenz (greater than 250-fold) compared to a wild type
control RTV. Novel combinations resulted in unpredeicted
resistance patterns than were different from those patterns
observed for the each mutation alone.
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