U.S. patent application number 10/681078 was filed with the patent office on 2004-04-08 for means and methods for monitoring antiretroviral therapy and guiding therapeutic decisions in the treatment of hiv/aids.
This patent application is currently assigned to ViroLogic, Inc.. Invention is credited to Whitcomb, Jeannette.
Application Number | 20040067487 10/681078 |
Document ID | / |
Family ID | 26905977 |
Filed Date | 2004-04-08 |
United States Patent
Application |
20040067487 |
Kind Code |
A1 |
Whitcomb, Jeannette |
April 8, 2004 |
Means and methods for monitoring antiretroviral therapy and guiding
therapeutic decisions in the treatment of HIV/AIDS
Abstract
This invention relates to antiviral drug susceptibility and
resistance tests to be used in identifying effective drug regimens
for the treatment of human immunodeficiency virus (HIV) infection
and acquired immunodeficiency syndrome (AIDS) and further relates
to the means and methods of monitoring the clinical progression of
HIV infection and its response to antiretroviral therapy,
particularly nucleoside reverse transcriptase inhibitor therapy
using phenotypic susceptibility assays or genotypic assays.
Inventors: |
Whitcomb, Jeannette; (San
Mateo, CA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS, LLP.
3300 HILLVIEW AVENUE
PALO ALTO
CA
94304
US
|
Assignee: |
ViroLogic, Inc.
|
Family ID: |
26905977 |
Appl. No.: |
10/681078 |
Filed: |
October 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10681078 |
Oct 7, 2003 |
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09881033 |
Jun 12, 2001 |
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6653081 |
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60211245 |
Jun 12, 2000 |
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Current U.S.
Class: |
435/5 |
Current CPC
Class: |
C12Q 1/703 20130101 |
Class at
Publication: |
435/005 |
International
Class: |
C12Q 001/70 |
Claims
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 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 230 alone or in combination with a
mutation at codon 103 or a mutation at codon 181, wherein the
presence of such a mutation correlates with a decrease in
non-nucleoside reverse transcriptase inhibitor susceptibility and
drug-dependent stimulation of viral replication.
2. The method of claim 1, wherein the mutated codon 230 encodes a
leucine (L).
3. The method of claim 1, wherein the mutated codon 103 encodes an
asparagine (N).
4. The method of claim 1, wherein the HIV-infected patient is being
treated with an antiretroviral agent.
5. 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 230 alone or in
combination with a mutation at codon 103 or a mutation at codon 181
and an indicator gene into a host cell; (b) culturing the host cell
from step (a); (c) measuring the expression of the indicator gene
in a target host cell; and (d) comparing the measurement of the
expression of the indicator gene from step (c) with the measurement
of the expression of the indicator gene measured when steps (a)-(c)
are carried out in the absence of the candidate antiretroviral drug
compound; wherein a test concentration of the candidate
antiretroviral drug compound is present at steps (a)-(c); at steps
(b)-(c); or at step (c).
6. A resistance test vector comprising: (i) an HIV patient-derived
segment which comprises reverse transcriptase having a mutation in
at least one of codons 230, 103 or 181, and (ii) and an indicator
gene, wherein the expression of the indicator gene is dependent
upon the patient derived segment.
7. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 230 alone or in combination with at
least one mutation at a codon selected from the group consisting
of: codon 101, codon 103, codon 190, codon 221 and codon 238,
wherein the presence of the mutations correlate with a decrease in
non-nucleoside reverse transcriptase inhibitor susceptibility and
drug-dependent stimulation of viral replication.
8. The method of claim 7, wherein the mutated codon 230 encodes a
leucine (L).
9. The method of claim 7, wherein the mutated codon 101 encodes a
glutamic acid (E), the mutated codon 103 encodes an asparagine (N),
the mutated codon 190 encodes a serine (S), the mutated codon 221
encodes a tyrosine (Y), or the mutated codon 238 encodes a
threonine (T).
10. The method of claim 7, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
11. 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 230 alone or in
combination with a mutation at codon 103, codon 190, codon 221 or
codon 238 and an indicator gene into a host cell; (b) culturing the
host cell from step (a); (c) measuring the expression of the
indicator gene in a target host cell; and (d) comparing the
measurement of the expression of the indicator from step (c) with
the measurement of the expression of the indicator gene measured
when steps (a)-(c) are carried out in the absence of the candidate
antiretroviral drug compound; wherein a test concentration of the
candidate antiretroviral drug compound is present at steps (a)-(c);
at steps (b)-(c); or at step (c).
12. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 230, 101, 103, 190, 221, and/or 238 and an indicator
gene, wherein the expression of the indicator gene is dependent
upon the patient derived segment.
13. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 codons 241 and 103 or 135 in which the
presence of the mutations correlate with a decrease in NNRTI
susceptibility and drug-dependent stimulation of viral
replication.
14. The method of claim 13, wherein the mutated codon 241 encodes a
serine (S).
15. The method of claim 13, wherein the mutated codon 103 encodes
an asparagine (N) and the mutated codon 135 encodes a threonine
(T).
16. The method of claim 13, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
17. 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(s) 241 and 103 or
135 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).
18. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 241, 103 and/or 135 and an indicator gene, wherein the
expression of the indicator gene is dependent upon the patient
derived segment.
19. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 codons 241 and 101, 106 135, 138 or 190 in
which the presence of the mutations correlate with a decrease in
NNRTI susceptibility and drug-dependent stimulation of viral
replication.
20. The method of claim 19, wherein the mutated codon 241 encodes
an isoleucine (I).
21. The method of claim 19, wherein the mutated codon 101 encodes a
glutamic acid (E), the mutated codon 106 encodes a methionine (M),
the mutated codon 135 encodes a threonine (T), the mutated codon
138 encodes an alanine (A) and/or the mutated codon 190 encodes an
alanine (A).
22. The method of claim 19, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
23. 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(s) 241 and 101, 106,
135, 138 or 190 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).
24. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 241, 101, 106, 135, 138 and/or 190 and an indicator gene,
wherein the expression of the indicator gene is dependent upon the
patient derived segment.
25. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 codons 245 and 98, 135, and/or 181 in which
the presence of the mutations correlate with a decrease in NNRTI
susceptibility and drug-dependent stimulation of viral
replication.
26. The method of claim 25, wherein the mutated codon 245 encodes
an glutamic acid (E).
27. The method of claim 25, wherein the mutated codon 98 encodes a
glycine (G), the mutated codon 135 encodes a threonine (T), and/or
the mutated codon 181 encodes a cysteine (C).
28. The method of claim 25, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
29. 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(s) 245 and 98, 135,
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).
30. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 245, 98, 135, and/or 181 and an indicator gene, wherein
the expression of the indicator gene is dependent upon the patient
derived segment.
31. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 codons 245 and 101, 103, 135, and/or 190 in
which the presence of the mutations correlate with a decrease in
NNRTI susceptibility and drug-dependent stimulation of viral
replication.
32. The method of claim 31, wherein the mutated codon 245 encodes
an glutamic acid (E).
33. The method of claim 31, wherein the mutated codon 101 encodes a
glutamic acid (E), the mutated codon 103 encodes an asparagine (N),
the mutated codon 135 encodes a threonine (T) and/or the mutated
codon 190 encodes an alanine (A).
34. The method of claim 31, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
35. 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(s) 245 and 101, 103,
135, and/or 190, 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).
36. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 245, 101, 103, 135, and/or 190 and an indicator gene,
wherein the expression of the indicator gene is dependent upon the
patient derived segment.
37. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 codons 245 and 103, 225, and/or 270 in which
the presence of the mutations correlate with a decrease in NNRTI
susceptibility and drug-dependent stimulation of viral
replication.
38. The method of claim 37, wherein the mutated codon 245 encodes a
glutamic acid (E).
39. The method of claim 37, wherein the mutated codon 103 encodes
an asparagine (N), the mutated codon 225 encodes a histidine (H),
and/or the mutated codon 270 encodes an methionine (M).
40. The method of claim 37, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
41. 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(s) 245 and 103, 225,
and/or 270 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).
42. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 245, 103, 225, and/or 270 and an indicator gene, wherein
the expression of the indicator gene is dependent upon the patient
derived segment.
43. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 codons 245 and 135, and/or 138 in which the
presence of the mutations correlate with a decrease in NNRTI
susceptibility and drug-dependent stimulation of viral
replication.
44. The method of claim 43, wherein the mutated codon 245 encodes a
threonine (T).
45. The method of claim 43, wherein the mutated codon 135 encodes a
threonine (T), and/or the mutated codon 138 encodes an glycine
(G).
46. The method of claim 43, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
47. 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(s) 245 and 135,
and/or 138 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).
48. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 245, 135, and/or 138 and an indicator gene, wherein the
expression of the indicator gene is dependent upon the patient
derived segment.
49. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 codons 245 and 98, 103, 135, 181 and/or 190 in
which the presence of the mutations correlate with a decrease in
NNRTI susceptibility and drug-dependent stimulation of viral
replication.
50. The method of claim 49, wherein the mutated codon 245 encodes a
threonine (T).
51. The method of claim 49, wherein the mutated codon 98 encodes a
glutamic acid (G), the mutated codon 103 encodes an asparagine (N),
the mutated codon 135 encodes a threonine (T), the mutated codon
181 encodes a cysteine (C), and/or the mutated codon 190 encodes an
alanine (A).
52. The method of claim 49, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
53. 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(s) 245 and 98, 103,
135, 181, and/or 190 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).
54. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 245, 98, 103, 135, 181, and/or 190 and an indicator gene,
wherein the expression of the indicator gene is dependent upon the
patient derived segment.
55. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 codons 245 and 103 in which the presence of
the mutations correlate with a decrease in NNRTI susceptibility and
drug-dependent stimulation of viral replication.
56. The method of claim 55, wherein the mutated codon 245 encodes a
threonine (T).
57. The method of claim 55, wherein the mutated codon 103 encodes
an asparagine (N).
58. The method of claim 55, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
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 further comprising a mutation at codon(s) 245 and 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 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).
60. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 245 and 103 and an indicator gene, wherein the expression
of the indicator gene is dependent upon the patient derived
segment.
61. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 codons 245 and 103, 135 and/or 225 in which
the presence of the mutations correlate with a decrease in NNRTI
susceptibility and drug-dependent stimulation of viral
replication.
62. The method of claim 61, wherein the mutated codon 245 encodes a
methionine (M).
63. The method of claim 61, wherein the mutated codon 103 encodes
an asparagine (N), the mutated codon 135 encodes a threonine (T)
and/or the mutated codon 225 encodes a histidine (H).
64. The method of claim 61, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
65. 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(s) 245 and 103, 135
and/or 225 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).
66. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 245, 103, 135, and/or 225 and an indicator gene, wherein
the expression of the indicator gene is dependent upon the patient
derived segment.
67. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 codons 270 and 103 and/or 135 in which the
presence of the mutations correlate with a decrease in NNRTI
susceptibility and drug-dependent stimulation of viral
replication.
68. The method of claim 67, wherein the mutated codon 270 encodes a
serine (S).
69. The method of claim 67, wherein the mutated codon 103 encodes
an asparagine (N) and/or the mutated codon 135 encodes a threonine
(T).
70. The method of claim 67, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
71. 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(s) 270 and 103
and/or 135 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).
72. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 270, 103, and/or 135 and an indicator gene, wherein the
expression of the indicator gene is dependent upon the patient
derived segment.
73. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 230 in which the presence of the
mutation correlates with a decrease in NNRTI susceptibility and
drug-dependent stimulation of viral replication.
74. The method of claim 73, wherein the mutated codon 230 encodes a
leucine (L).
75. The method of claim 73, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
76. 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 230 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).
77. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutations
at codons 230 and an indicator gene, wherein the expression of the
indicator gene is dependent upon the patient derived segment.
78. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 241 in which the presence of the
mutation correlates with a decrease in NNRTI susceptibility and
drug-dependent stimulation of viral replication.
79. The method of claim 78, wherein the mutated codon 241 encodes a
serine (S).
80. The method of claim 78, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
81. 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 241 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).
82. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutation at
codon 241 and an indicator gene, wherein the expression of the
indicator gene is dependent upon the patient derived segment.
83. A method of assessing the effectiveness of non nucleoside
reverse transcriptase 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 270 in which the presence of the
mutation correlates with a decrease in NNRTI susceptibility and
drug-dependent stimulation of viral replication.
84. The method of claim 83, wherein the mutated codon 270 encodes a
serine (S).
85. The method of claim 83, wherein the HIV-infected patient is
being treated with an antiretroviral agent.
86. 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 270 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).
87. A resistance test vector comprising an HIV patient-derived
segment further comprising reverse transcriptase having mutation at
codon 270 and an indicator gene, wherein the expression of the
indicator gene is dependent upon the patient derived segment.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/211,245, filed Jun. 12, 2000, the content of
which is incorporated herein by reference in its entirety.
[0002] Throughout this application, various publications are
referenced by author and date within the text. Full citations for
these publications may be found listed alphabetically at the end of
the specification immediately preceding the claims. All patents,
patent applications and publications cited herein, whether supra or
infra, are hereby incorporated by reference in their entirety. The
disclosures of these publications in their entireties are hereby
incorporated by reference into this application in order to more
fully describe the state of the art as known to those skilled
therein as of the date of the invention described and claimed
herein.
TECHNICAL FIELD
[0003] This invention relates to antiretroviral drug susceptibility
and resistance tests to be used in identifying effective drug
regimens for the treatment of human immunodeficiency virus (HIV)
infection and acquired immunodeficiency syndrome (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 less
susceptible ("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 using phenotypic susceptibility tests
and/or genotypic tests to identify patients whose virus/viruses
exhibit drug-dependent stimulation of replication in the presence
of anti-retroviral agents.
BACKGROUND OF THE INVENTION
[0004] 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 S K, Taylor M E, et
al. (1995) Nature 343, 117-122) (Ho D D, Naumann A U, Perelson A S,
et al. (1995) Nature 373, 123-126). The aim of antiretroviral
therapy is to achieve substantial and prolonged suppression of
viral replication. Achieving sustained viral control is likely to
involve the use of sequential therapies, generally each therapy
comprising combinations of three or more antiretroviral drugs.
Choice of initial and subsequent therapy should, therefore, be made
on a rational basis, with knowledge of resistance and
cross-resistance patterns being vital to guiding those decisions.
The primary rationale of combination therapy relates to synergistic
or additive activity to achieve greater inhibition of viral
replication. The tolerability of drug regimens will remain
critical, however, as therapy will need to be maintained over many
years.
[0005] In an untreated patient, some 10.sup.10 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.sup.4 to 10.sup.5 mutations per day at each position
in the HIV genome. The result is the rapid establishment of
extensive genotypic variation. While some template positions may be
more error prone, (Mansky L M, Temin H M (1995) J Virol 69,
5087-5094) (Schinazi R F, Lloyd R M, Ramanathan C S, et al. (1994)
Antimicrob Agents Chemother 38, 268-274), mathematical modeling
suggests that, at every nucleotide position, mutation may occur
10.sup.4 times per day in infected individuals.
[0006] For antiretroviral drug resistance to occur, the target
enzyme must be modified while preserving its function in the
presence of the inhibitor. Point mutations leading to an amino acid
substitution may result in changes in shape, size, or charge of the
active site, substrate binding site, or surrounding regions of the
enzyme. Mutants resistant to antiretroviral agents have been
detected at low levels before the initiation of therapy (Mohri H,
Singh M K, Ching W T W, et al. (1993) Proc Natl Acad Sci USA 90,
25-29) (Njera I, Richman D D, Olivares I, et al. (1994) AIDS Res
Hum Retroviruses 10, 1479-1488) (Njera i, Holguin A, Quiones-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 J M (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 the dominant quasispecies (Frost S D W, McLean A
R (1994) AIDS 8, 323-332) (Kellam P, Boucher C A B, Tijnagal JMGH
(1994) J Gen Virol 75, 341-351) ultimately leading to drug
resistance and virologic failure in the patient.
[0007] A mutation or mutations that results in virus that can not
only replicate in the presence of drug (i.e. resistant virus) but
could actually replicate more efficiently in the presence of drug
than in the absence of drug (i.e. drug-dependent stimulation of
virus), would present an especially important phenotype to
identify. In this case, a drug could actually accelerate the rate
of destruction to the immune system and progression of disease.
[0008] Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
[0009] Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are
a chemically diverse group of compounds which are potent inhibitors
of HIV-1 Reverse Transcriptase (RT) in 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 phenylacetamide (a-APA) compound (loviride), the
quinoxaline-class inhibitors (HBY-097), the benzodiazepin-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 E A (1996) Antiviral Drug Resistance, ed. D D
Richman, John Wiley & Sons, Ltd). Three NNRTIs: nevirapine
(NVP, Viramune, Boehringer Ingelheim, Ingelheim am Rhein, Germany),
delavirdine (DLV, Rescriptor, Pharmacia & Upjohn, Kalamazoo,
Mich., USA), and efavirenz (EFV, Sustiva, Dupont, Wilmington, Del.,
USA) are licensed for use in the USA.
[0010] High-level resistance to individual compounds appears to
develop rapidly, often within a few weeks of initiating
monotherapy, frequently involving only single-point mutations, and
in many cases leading to considerable cross-resistance to other
NNRTIs. Most mutations reported occur in the codon groups 100-108
and 181-190 which encode for the two b-sheets adjacent to the
catalytic site of the RT enzyme (Kohlstaedt L A, Wang J, Friedman J
M, et al. (1992) Science 256, 1783-90). The NNRTI 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, or disrupting the orientation of conserved aspartic acid
side chains essential for catalytic activity (D'Aquilla R T. (1994)
Clin Lab Med 14, 393-423) (Arnold E., Ding J., Hughes S H, et al.
(1995) Curr Opin Struct Biol 5, 27-38).
[0011] Mutations conferring reduced susceptibility to nevirapine
have been described at HIV RT codons 98, 100, 103, 106, 108, 181,
188 and 190 (Richman D D, Havlir D, Corbeil J. (1994) J Virol 68,
1660-1666). The most frequently selected variant during nevirapine
monotherapy is a Tyr.sup.181aCys (Y181C) mutation, which results in
a 100-fold reduction in sensitivity to this agent, and 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 Y181C mutation, but maintains activity in the
presence of mutations HIV RT at codons 100 and 103, and in vitro
selects for a unique mutation, GLU.sup.138aLys (E138K), in the
region where it most closely interacts with RT (Richman, D D, Ibid)
(Richman D D, Shih C-K, Lowy I, et al. (1991) Proc Natl Acad Sci
USA 88, 11241-11245).
[0012] Resistance to loviride when used as monotherapy develops in
most patients by week 24. It has been mapped to a range of HIV RT
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 detected at 24 weeks (Staszewski S, Miller V,
Rehmet S, et al. (1996) AIDS 10, F1-7).
[0013] Although the K101E, K103N, and Y181C, mutations also confer
cross-resistance to BHAPs, (Balzarini J, Karlsson A, Perez-Perez
M-J, et al. (1992) Virology 192, 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 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 238 during combination therapy with
zidovudine.
[0014] While HBY-097 may initially select for a mutation at HIV RT
codon 190 in vitro, further passage consistently selects for
mutations at HIV RT codons 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).
[0015] Mutation at codon 181 has been reported to antagonize
zidovudine resistance due to the typical 41 and 215 codon
mutations, (Zhang D, Caliendo A M, Eron J J, 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 B A, Kellam P, Kemp S D (1993) Nature 365, 451-453)
treatment with this triple combination does provide superior
immunological and virological responses than treatment with
zidovudine plus didanosine alone over a 48-week period in patients
with CD4 cell counts <350/mm 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 F E, 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 D D, Ibid) (Nunberg J H, Schleif
W A, Boots E J, et al. (1990) J Virol 65, 4887-4892) (DeJong M D,
Loewenthl M, Boucher C A B, et al. (1994) J Infect Dis 169,
1346-1350) (Cheeseman S H, Havlir D, McLaughlin M M, et al. (1995)
J Acquir Immune Defic Syndr 8, 141-151. However, the 181 mutant is
not being observed during treatment with this combination, and the
most common mutation occurs at codon 190 (Richman D D, Ibid). This
suggests that the codon 181 mutation, which is antagonistic to
zidovudine resistance in vitro, is not compatible, or not preferred
in vivo, with selection favoring other mutations which allow for
reduced susceptibility to nevirapine concomitant with zidovudine
resistance.
[0016] 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 the appearance of drug-resistant virus. A
`second generation` NNRTI, the pyridinone derivative L-702,019,
demonstrated only a 3-fold change in IC.sub.50 between wild-type
and codon 181 mutant HIV-1, and required multiple mutations to
engender high-level resistance (Goldman M E, O'Brien J A, Ruffing T
L, et al. (1993) Antimicrob Agents Chemother 37, 947-949).
[0017] Similarly, Efavirenz (EFZ) was introduced as a second
generation NNRTI relatively recently. Efavirenz has a unique
profile in that it retains activity against viruses containing the
common RT mutation, Y181C. In vitro, efavirenz selects for
mutations at codons 100, 101, 103, 108, 179, 181, and 188. This is
similar to the in vivo resistance profile, which includes mutations
at codons 100, 103, 108, 190 and 225, (and possibly 101, 179, 181
and 188). (Winslow D L, Garber S, Reid C, it al. Fourth
International Antiviral Therapy 1977; 1(Suppl.1): 6. Conference on
HIV Drug Resistance Sardinia, Italy, (1995) (Winslow D L, Garber S,
Reid C, et al. Antiviral Therapy 1997; 1(suppl.1):6) (Young S D,
Britcher S F, Tran L O, et al. Antimicrobial Agents &
Chemotherapy 1995; 39.2602-2609.) (Bacheler L T, Anton E, Jeffrey
S, et. al. Antiviral Therapy 1998; 3(Suppl.1): 15-16) (Bacheler L
T, Weislow O, Snyder S & Hanna G. 12.sup.th World AIDS
Conference, 1998, Geneva, Switzerland, Abstract 41213.)
[0018] 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 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. (NNRTIs) 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 (NNRTIs). It is
also an object of this invention to provide the means and
compositions for evaluating HIV antiretroviral drug resistance and
susceptibility. Still another object of this invention is to
provide a means of determining whether a candidate anti-retroviral
drug will cause increased or stimulated viral replication. This and
other objects of this invention will be apparent from the
specification as a whole.
SUMMARY OF THE INVENTION
[0019] The present invention relates to methods, using phenotypic
and genotypic methods to monitor 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 or 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 of a mutation at codon 230 either alone or in combination
with a mutation at codon 103 or 181 of HIV RT in NNRTI inhibitor
treated patients, in which the presence of the mutations correlates
with decreased susceptibility to delavirdine, nevirapine and
efavirenz, and with drug-dependent stimulation of viral replication
in the presence of delavirdine, nevirapine or efavirenz. The
mutations were found in plasma HIV RNA after a period of time
following initiation of therapy. The development of the mutation at
codon 230, in addition to the mutation at codon 103 or 181 in HIV
RT, was found to be an indicator of the development of resistance,
and ultimately of immunological decline. Resistance test vectors
containing the single site mutation at codon 230 (M230L), and M230L
in combination with a mutation at either 103 (K103N) or 181 (Y181C)
in reverse transcriptase were constructed using site directed
mutagenesis (Sarkar G, Sommer S S. (1990). Biotechniques
8:404-407). These mutations were observed to be associated with
decreased susceptibility to the NNRTI and, in some combinations,
drug-dependent stimulation of viral replication.
[0020] This invention is based in part on the discovery of a
mutation at codon 230 in combination with mutations at codons 101,
103, 190, 221 and 238 of HIV RT in NNRTI treated patients, in which
the presence of the mutations correlates with a decrease in
susceptibility to delavirdine, nevirapine and efavirenz, and with
drug-dependent stimulation of viral replication in the presence of
delavirdine, nevirapine or efavirenz.
[0021] This invention is based in part on the discovery of a
mutation at codon 241 of RT that was discovered to occur in
NNRTI-treated patients. The presence of the mutation at 241, in
addition to other NNRTI-resistance mutations (these mutations may
include previously described NNRTI-resistance mutations such as:
K101E, K103N, V106M, I135T, E138A and G190A) correlates with
decreased susceptibility to delavirdine, nevirapine and efavirenz.
Resistance test vectors containing patient sequences with these
mutations exhibited reduced susceptibility to delavirdine,
nevirapine and efavirenz as well as drug dependent stimulation of
replication in the presence of all three drugs.
[0022] This invention is based in part on the discovery of
mutations at codon 245 of RT that was discovered to occur in
NNRTI-treated patients. The presence of the mutation at 245, in
addition to other NNRTI-resistance mutations (which may include
previously described NNRTI-resistance mutations such as: A98G,
K101E, K103N, I135T, E138A, Y181C, G190A and P225H) correlates with
decreased susceptibility to delavirdine, nevirapine and efavirenz.
Resistance test vectors containing patient sequences with these
mutations exhibited reduced susceptibility to delavirdine,
nevirapine and efavirenz as well as drug dependent stimulation of
replication in the presence of all three drugs. Resistance test
vectors containing a single site mutation at codon 245 (V245E or
T), and as well as test vectors containing V245E or T in
combination with mutations at 103 (K103N) and 135 (I135T) in RT
were constructed using site directed mutagenesis. While V245E alone
had no effect on susceptibility to the NNRTI, The triple
combination of mutations (K103N, I135T and 245 E or T) was observed
to be associated with decreased susceptibility to the NNRTI and
drug-dependent stimulation of viral replication.
[0023] This invention is based in part on the discovery of a
mutation at codon 270 of RT that was discovered to occur in
NNRTI-treated patients. The presence of the mutation at 270 in
addition to other NNRTI-resistance mutations (which may include
previously described NNRTI-resistance mutations such as: K103N,
I135T and P225H) correlates with decreased susceptibility to
delavirdine, nevirapine and efavirenz, and drug-dependent
stimulation of viral replication. This invention is based in part
on the discovery of a patient-derived segment containing multiple
mutations at HIV RT codons 35, 67, 69, 70, 106, 189, 200, 202, 208,
211, 215, 218, 219, 221, 227, 228, 283, 284, 286, 293 and 297 of RT
that was discovered in an NNRTI-treated patient. Resistance test
vectors containing patient sequences with these mutations exhibited
reduced susceptibility to delavirdine, nevirapine and efavirenz as
well as drug dependent stimulation of replication in the presence
of all three drugs. Site-directed reversion of specific mutations
demonstrated that many of the mutations play a role in the
drug-dependent stimulation of viral replication, but that none of
the mutations is sufficient on it's own to cause such an effect.
Specifically, mutations at 106, 189, 227, 283, 284 and 286 are
observed to modulate the resistance and stimulation of viral
replication seen with this.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1. Resistance Test Vector. Schematic representations of
the HIV-1 genome (top panel) and the resistance test vector (bottom
panel), comprising a patient derived segment and an indicator gene
viral vector.
[0025] FIG. 2. Two Cell Assay. Schematic Representation of the
Assay. A resistance test vector is generated by cloning the
patient-derived segment (PDS) into an indicator gene viral vector.
Using defective PR and RT sequences, it was shown that luciferase
activity is dependent on functional PR and RT. The resistance test
vector is 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 segment (PDS). The particles are then harvested and
used to infect fresh cells. The assay is performed in the absence
of drug and in the presence of drug over a wide range of
concentrations. Protease PR inhibitors are added to the cells
following transfection, and are thus present during particle
maturation. In contrast, RT inhibitors are added to the cells at
the time of, or prior to, viral particle infection. The amount of
luciferase is determined and the percentage (%) inhibition is
calculated at the different drug concentrations tested.
[0026] FIG. 3. Examples of phenotypic drug susceptibility profiles.
Data are analyzed by plotting the percent inhibition of luciferase
activity vs. log.sub.10 drug concentration. This plot is used to
calculate the drug concentration that are required to inhibit virus
replication by 50% (IC.sub.50) and by 95% (IC.sub.95). Shifts in
the inhibition curves towards higher drug concentrations are
interpreted as evidence of decreased drug susceptibility, ("drug
resistance"). Three typical curves for the NNRTI's delavirdine
(DLV), nevirapine (NVP) and efavirenz (EFV) are shown. A reduction
in drug susceptibility (or "increased resistance") manifests as a
shift in the drug susceptibility curve toward higher drug
concentrations (to the right) as compared to a baseline
(pre-treatment) sample, or as compared to a drug susceptible virus
control, such as PNL4-3 or HXB-2.
[0027] FIG. 4. Examples of phenotypic drug susceptibility profiles
showing drug-dependent stimulation of viral replication. Data are
analyzed by plotting the percent inhibition of luciferase activity
vs. log.sub.10 drug concentration. This plot is used to calculate
the drug concentration that are required to inhibit virus
replication by 50% (IC.sub.50) and by 95% (IC95). In these graphs,
stimulation of viral replication manifests as percent inhibition
less than zero (i.e. negative inhibition). Three typical curves
showing drug-dependent stimulation of viral replication in the
presence of the NNRTI's delavirdine (DLV), nevirapine (NVP) and
efavirenz (EFV) are shown. The results obtained from the
patient-derived or site-directed mutant resistance test vectors are
compared to results obtained from a drug susceptible virus control,
such as PNL4-3 or HXB-2.
[0028] FIG. 5. Table of patient viruses containing mutations at HIV
RT codon position 230, as described in Examples 3, 4, and 5.
[0029] FIG. 6. Reduced susceptibility and drug-dependent
stimulation of viral replication of three site-directed mutants:
K103N, M230L, and K103N combined with K230L, as described in
Example 4. The results from each site-directed mutant are compared
to each other, as well as to those results obtained from a drug
susceptible virus control, such as PNL4-3, or HXB-2. In these
graphs, stimulation of viral replication manifests as percent
inhibition less than zero (i.e. negative inhibition).
[0030] FIG. 7. Reduced susceptibility and drug-dependent
stimulation of viral replication for three site-directed mutants:
Y181C, M230L, and Y181C combined with K230L, as described in
Example 3. The results from each site-directed mutant for each
NNRTI (delavirdine, efavirenz, and nevirapine) are compared to each
other, as well as to those results obtained from a drug susceptible
virus control, such as PNL4-3, or HXB-2. In these graphs,
stimulation of viral replication manifests as percent inhibition
less than zero (i.e. negative inhibition).
[0031] FIG. 8. Reverse mutagenesis of HIV RT codon positions 241
and 277 in Patient 11073, as described in Example 6. The top panel
of the figure shows the graphs from the patient sample, which
contains the V241S mutation. In these graphs, stimulation of viral
replication manifests as percent inhibition less than zero (i.e.
negative inhibition) for all three NNRTIs (delavirdine, efavirenz,
and nevirapine). The bottom panel shows the results of
site-directed reversion of the mutation to 241V. In this graph,
there is no longer negative inhibition (stimulation of viral
replication) in any of the three NNRTIs (delavirdine, efavirenz, or
nevirapine).
[0032] FIG. 9. Susceptibility curves of two time-points separated
by 32 weeks in the course of therapy of Patient 014451, as
described in Example 7. The top panel of the figure shows
susceptibility curves from patient sample 014459, (week 0). The
bottom panel shows susceptibility curves from patient sample
014451, (week 32), which show both reduced susceptibility as well
as drug-dependent stimulation of viral replication in the presence
of all three NNRTIs, which coincides with the emergence of HIV RT
mutations at codons 101, 106, and 190.
[0033] FIG. 10. Table of patient viruses containing mutations at
HIV RT codon position 245, as described in Examples 8, 9, 10 and
11.
[0034] FIG. 11. Reverse mutagenesis of HIV RT codon positions 245,
270, 277, 292, 293, and 297 in Patient 010829, as described in
Example 11. The results from each mutant are compared to results
obtained from a drug susceptible virus control, such as PNL4-3, or
HXB-2. The top panel of the figure shows the graphs from the
patient sample, containing the mutations at codon positions 245,
270, 277, 292, 293, and 297. In these graphs, stimulation of viral
replication manifests as percent inhibition less than zero (i.e.
negative inhibition) for all three NNRTIs (delavirdine, efavirenz,
and nevirapine). The second panel of graphs shows the site-directed
reversion of the mutation at codon 245, followed by the reversion
of the mutation at codon 270. Both single reverse mutants retain
profiles consistent with drug-dependent stimulation of viral
replication (negative inhibition) in the presence of any of the
NNRTIs (delavirdine, efavirenz, or nevirapine). The bottom panel
shows the reversion of HIV-RT mutations at codons 245, 270, 277,
292, 293, and 297. In this final panel of graphs, there is no
longer negative inhibition (stimulation of viral replication) in
any of the three NNRTIs (delavirdine, efavirenz, or nevirapine),
although this site-directed mutant retains reduced drug
susceptibility (drug resistance).
[0035] FIG. 12. Reduced susceptibility and drug-dependent
stimulation of viral replication in of a site-directed mutants with
HIV RT mutations K103N, 135T, and V245T, as described in Example
13. The results from each mutant are compared to results obtained
from a drug susceptible virus control, such as PNL4-3, or HXB-2.
Although mutations at each HIV RT codon position alone did not
result in drug-dependent stimulation, the triple mutant exhibits
drug-dependent stimulation for all three NNRTIs (delavirdine,
efavirenz, or nevirapine), In these graphs, stimulation of viral
replication manifests as percent inhibition less than zero (i.e.
negative inhibition).
[0036] FIG. 13. Reverse mutagenesis of HIV RT codon position 270 in
Patient 13522 as described in Example 12. The results from each
mutant are compared to results obtained from a drug susceptible
virus control, such as PNL4-3, or HXB-2. The top panel of the
figure shows the graphs from the patient sample, containing the
1270S mutation. In these graphs, stimulation of viral replication
manifests as percent inhibition less than zero (i.e. negative
inhibition) for all three NNRTIs (delavirdine, efavirenz, and
nevirapine). The second panel of graphs shows the site-directed
reversion of the mutation at codon 270. Although no longer
exhibiting negative inhibition (stimulation of viral replication)
for any of the three NNRTIs (delavirdine, efavirenz, or
nevirapine), the site-directed mutant retains reduced drug
susceptibility (drug resistance) to all three NNRTIs.
[0037] FIG. 14. Table of clinical history of twelve viral samples
from Patient 1033, as described in Example 14. Data include: drug
regimen, duration of drug regimen, viral load, phenotypic
fold-change in susceptibility values relative to a drug sensitive
virus control, such as PNL4-3, or HXB-2, and percent negative
inhibition for those samples exhibiting drug-dependent stimulation
of virus production.
[0038] FIG. 15. Table of HIV-RT amino acid mutations for twelve
viral samples from Patient 1033, as described in Example 14.
[0039] FIG. 16. Effect of specific mutations on 1033-3 virus as
discussed in Example 14, as determined by site-directed
mutagenesis. The results from each mutant are compared to results
obtained from a drug susceptible virus control, such as PNL4-3, or
HXB-2. In these graphs, stimulation of viral replication manifests
as percent inhibition less than zero (i.e. negative
inhibition).
[0040] FIG. 17. Effect of M184V mutation on stimulation phenotype
as described in Example 14, as determined by site-directed
mutagenesis. Reversion of the mutation at HIV RT codon 184 results
in increasing levels of drug-dependent stimulation of viral
replication, as manifested by percent inhibition less than zer0
(i.e. negative inhibition).
[0041] FIG. 18. Patient 014451, reduced susceptibility to NNRTI an
drug dependent stimulation of viral replication associated with
mutations at codon 101, codon 106 and codon 190 (Example 7).
[0042] FIG. 19. Effect of M184V mutation on drug-dependent
stimulation of viral replication: RTV-309
DETAILED DESCRIPTION OF THE INVENTION
[0043] 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 (NNRTI) antiretroviral therapy, and to the
detection of variants of HIV that exhibit drug-dependent
stimulation of replication in the presence of one or more
NNRTIs.
[0044] In one embodiment, the present invention provides for a
method of assessing the effectiveness of antiretroviral therapy of
a patient comprising (i) collecting a biological sample from an
HIV-infected patient; and (ii) determining whether the biological
sample comprises nucleic acid encoding HIV RT having a mutation at
one or more positions codon in the RT. The mutation(s) correlate
positively with changes in phenotypic
susceptibility/resistance.
[0045] 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 230 and 103 or 181. Using a phenotypic
susceptibility assay, this invention established that mutations at
codon 230, either alone or in combination with a mutation at codon
103 or 181 of HIV RT, correlate with a decreased susceptibility to
delavirdine, nevirapine and efavirenz. Patient derived resistance
test vectors containing the mutation M230L, either alone or in
combination with other NNRTI-resistance mutations show reductions
in susceptibility that range from 10-fold to >450-fold for
delavirdine, from 5-fold to >250-fold for efavirenz, and from
10-fold to >600-fold for nevirapine. The percent stimulation of
viral replication in patient derived resistance test vectors
containing mutations at codon 230 in HIV RT ranges from 0% to
.about.100% for all three NNRTIs. Site-directed resistance test
vectors containing mutations at 230, either alone or in combination
with 103 or 181 were constructed. The mutation at 230 alone causes
reduced susceptibility to delavirdine (58-fold), nevirapine
(40-fold) and efav-renz (23-fold), and drug-dependent stimulation
of replication in the presence of nevirapine (.about.50%) and
delavirdine (.about.50%). The combination of mutations at 230 and
103 causes reduced susceptibility to delavirdine (>250-fold),
nevirapine (>600-fold) and efavirenz (>470-fold) and
drug-dependent stimulation of replication in the presence of
delavirdine (.about.100%) nevirapine (.about.70%), and efaviraenz
(.about.40%). The combination of mutations at 230 and 181 causes
reduced susceptibility to delavirdine (>250-fold), nevirapine
(>800-fold) and efavirenz (25-fold) but no drug-dependent
stimulation of replication in the presence of delavirdine,
nevirapine, or efaviraenz.
[0046] 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 241, 103 and 135 or at codon 241 in combination
with mutations at 101, 106, 135, 138 and 190. Using a phenotypic
susceptibility assay, this invention established that mutations at
codon 241, in combination with a mutation at codon 103, or
mutations at 101, 106 and 190 of HIV RT are correlated with
decreased susceptibility (increased resistance) to delavirdine,
nevirapine and efavirenz. Patient-derived resistance test vectors
containing mutations at 241 in addition to other NNRTI-resistance
mutations (e.g. 101, 103, 106, 135, 138 and 190) displayed
reductions in susceptibility ranging from 41-fold to >250-fold
for delavirdine, and showed high level reductions in susceptibility
to nevirapine (>600-fold) and efavirenz (>470-fold).
Patient-derived resistance test vectors containing mutations at 241
in combination with other NNRTI-resistance mutations (e.g. 101,
103, 106, 135, 138 and 190) displayed drug-dependent stimulation of
viral replication ranging from 70-100% for all three NNRTIs.
[0047] In another 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 245, 103 and 135 or having a mutation at codon
245 in combination with additional mutations that could include 98,
101, 103, 135, 138, 181, 190 and/or 225. Using a phenotypic
susceptibility assay, this invention established that a mutation at
codon 245 in combination with mutations at codons 103 and 135, or a
mutation at codon 245 with additional mutations that may include
98, 101, 103, 135, 138, 181, 190 and 225 of HIV RT correlated with
decreased susceptibility to delavirdine, nevirapine and efavirenz.
Patient-derived resistance test vectors containing mutations at 245
and additional NNRTI-resistance mutations as described above
exhibited reductions in susceptibility ranging from 20-fold to
>250-fold for delavirdine, from 8-fold to >600-fold for
nevirapine and from 5-fold to >470-fold for efavirenz.
Patient-derived resistance test vectors containing mutations at 245
and additional NNRTI-resistance mutations as described above
exhibited drug-dependent stimulation in viral replication ranging
from 20-100% for all three NNRTIs. Site-directed resistance test
vectors were constructed containing mutations at 245, 103 and 135
and in various combinations as described above. Mutations at HIV RT
codon 245 (V245E or V245T) alone cause no significant reduction in
susceptibility to delavirdine, nevirapine or efavirenz. The
combination of mutations at 245 (V245E) with 103 (K103N) and 135
(I135T) causes reduced susceptibility to delavirdine (169-fold),
nevirapine (244-fold) and efavirenz (93-fold) and drug-dependent
stimulation of replication in the presence of delavirdine
(.about.20%) and nevirapine (.about.15%) but not efavirenz. The
combination of mutations at 245 (V245T) with 103 (K103N) and 135
(1135T) causes reduced susceptibility to delavirdine
(>250-fold), nevirapine (544-fold) and efavirenz (174-fold) and
drug-dependent stimulation of replication in the presence of
delavirdine (.about.50%), nevirapine (.about.40%), and efavirenz
(.about.25%).
[0048] 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 270 and additional mutations at codons 103 and
135, with or without mutation at codon 225. Using a phenotypic
susceptibility assay, this invention established that mutation at
codon 270 in combination with mutations at codon 103 and 135 with
or without mutation at codon 225 of HIV RT are correlated with a
decreased susceptibility to delavirdine, nevirapine, and efavirenz,
and drug-dependent stimulation of replication in the presence of
delavirdine, nevirapine and efavirenz. Patient-derived resistance
test vectors containing mutations at 270 in addition to other
NNRTI-resistance mutations (e.g. 103, 135, with or without 225)
displayed high-level reductions in susceptibility to all three
NNRTIs and drug-dependent Stimulations in viral replication ranging
from 80-110% for all three NNRTIs.
[0049] Under the foregoing circumstances, the phenotypic
susceptibility/resistance profile and genotypic profile of the HIV
virus infecting the patient has been altered, reflecting some
change in the response to the antiretroviral agent. In the case of
NNRTI antiretroviral therapy, the HIV virus infecting the patient
may be resistant to any combination of the three NNRTIs described
herein. Furthermore, the virus may be found to replicate more
efficiently in the presence of one or more drug(s) than in the
absence of those drugs. 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 patient's
therapeutic regimen. For example, if the patient was being treated
with efavirenz (DMP-266) when the 230 and 103 mutation arose, the
patient's therapeutic regimen may desirably be altered by
eliminating NNRTI antiretroviral agents, such as delavirdine,
efavirenz or nevirapine; and/or (ii) 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
regimen.
[0050] The phrase "correlates positively," as used herein,
indicates that a particular result renders a particular conclusion
more likely than other conclusions.
[0051] 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) transcribing the
HIV-encoding RNA in the biological sample to cDNA; (iii) amplifying
a patient-derived segment (PDS) using HIV PCR primers that result
in a product comprising the RT gene; (iv) performing sequencing
reactions using primers that yield sequences comprising wild type
or mutant amino acids at codons 230, 103 and 181; and (v)
determining the presence or absence of mutations at codons 230, 103
and 181 from the sequences.
[0052] 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 biological sample from an HIV-infected patient; (ii)
transcribing the HIV-encoding RNA in the biological sample to cDNA;
(iii) amplifying a patient-derived segment (PDS) using HIV PCR
primers that result in a product comprising the RT gene; (iv)
performing sequencing reactions using primers that yield sequences
comprising wild type or mutant amino acids at codons 230, 101, 103,
190, 221, and 238; and (v) determining the presence or absence of
mutations at codons 230, 101, 103, 190, 221, and 238 from the
sequences.
[0053] This invention also provides for a method of assessing the
effectiveness of non nucleoside reverse transcriptase
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 230
alone or in combination with a mutation at codon 103 or a mutation
at codon 181, wherein the presence of such a mutation correlates
with a decrease in non-nucleoside reverse transcriptase inhibitor
susceptibility and drug-dependent stimulation of viral
replication.
[0054] This invention also provides for 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 230 alone or in combination with a mutation at codon 103
or a mutation at codon 181 and an indicator gene into a host cell;
(b) culturing the host cell from step (a); (c) measuring the
expression of the indicator gene in a target host cell; and (d)
comparing the measurement of the expression of the indicator gene
from step (c) with the measurement of the expression of the
indicator gene measured when steps (a)-(c) are carried out in the
absence of the candidate antiretroviral drug compound; wherein a
test concentration of the candidate antiretroviral drug compound is
present at steps (a)-(c); at steps (b)-(c); or at step (c) and
wherein a decrease in expression of the indicator gene measured in
the persence of the candidate antiretroviral drug compound is
indicative of the biological effectiveness of the compound.
[0055] This invention provides for a resistance test vector
comprising: (i) an HIV patient-derived segment which comprises
reverse transcriptase having a mutation in at least one of codons
230, 103 or 181, and (ii) and an indicator gene, wherein the
expression of the indicator gene is dependent upon the patient
derived segment.
[0056] This invention also provides for a method of assessing the
effectiveness of non nucleoside reverse transcriptase
antiretroviral therapy of an HIV-infected patient comprising:
[0057] (a) collecting a biological sample from an HIV-infected
patient; and
[0058] (b) evaluating whether the biological sample comprises
nucleic acid encoding HIV reverse transcriptase having a mutation
at codon 230 alone or in combination with at least one mutation at
a codon selected from the group consisting of: codon 101, codon
103, codon 190, codon 221 and codon 238, wherein the presence of
the mutations correlate with a decrease in non-nucleoside reverse
transcriptase inhibitor susceptibility and drug-dependent
stimulation of viral replication.
[0059] 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 biological sample from an HIV-infected patient; (ii)
transcribing the HIV-encoding RNA in the biological sample to cDNA;
(iii) amplifying a patient-derived segment (PDS) using HIV PCR
primers that result in a product comprising the RT gene; (iv)
performing sequencing reactions using primers that yield sequences
comprising wild type or mutant amino acids at codons 241, 103 and
135; and (v) determining the presence or absence of mutations at
codons 241, 103 and 135 from the sequences.
[0060] 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 biological sample from an HIV-infected patient; (ii)
transcribing the HIV-encoding RNA in the biological sample to cDNA;
(iii) amplifying a patient-derived segment (PDS) using HIV PCR
primers that result in a product comprising the RT gene; (iv)
performing sequencing reactions using primers that yield sequences
comprising wild type or mutant amino acids at codons 241, 101, 106,
135, 138, and 190; and (v) determining the presence or absence of
mutations at codons 241, 101, 106, 135, 138, and 190 from the
sequences.
[0061] 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 biological sample from an HIV-infected patient; (ii)
transcribing the HIV-encoding RNA in the biological sample to cDNA;
(iii) amplifying a patient-derived segment (PDS) using HIV PCR
primers that result in a product comprising the RT gene; (iv)
performing sequencing reactions using primers that yield sequences
comprising wild type or mutant amino acids at codons 245, 98, 135
and 181; and (v) determining the presence or absence of mutations
at codons 245, 98, 135 and 181 from the sequences.
[0062] 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 biological sample from an HIV-infected patient; (ii)
transcribing the HIV-encoding RNA in the biological sample to cDNA;
(iii) amplifying a patient-derived segment (PDS) using HIV PCR
primers that result in a product comprising the RT gene; (iv)
performing sequencing reactions using primers that yield sequences
comprising wild type or mutant amino acids at codons 245, 101, 103,
135 and 190; and (v) determining the presence or absence of
mutations at codons 245, 101, 103, 135, and 190 from the
sequences.
[0063] 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 biological sample from an HIV-infected patient; (ii)
transcribing the HIV-encoding RNA in the biological sample to cDNA;
(iii) amplifying a patient-derived segment (PDS) using HIV PCR
primers that result in a product comprising the RT gene; (iv)
performing sequencing reactions using primers that yield sequences
comprising wild type or mutant amino acids at codons 245, 103, 225,
and 270; and (v) determining the presence or absence of mutations
at codons 245, 103, 225 and 270 from the sequences.
[0064] 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 biological sample from an HIV-infected patient; (ii)
transcribing the HIV-encoding RNA in the biological sample to cDNA;
(iii) amplifying a patient-derived segment (PDS) using HIV PCR
primers that result in a product comprising the RT gene; (iv)
performing sequencing reactions using primers that yield sequences
comprising wild type or mutant amino acids at codons 245, 135 and
138; and (v) determining the presence or absence of mutations at
codons 245, 135 and 138 from the sequences.
[0065] 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 biological sample from an HIV-infected patient; (ii)
transcribing the HIV-encoding RNA in the biological sample to cDNA;
(iii) amplifying a patient-derived segment (PDS) using HIV PCR
primers that result in a product comprising the RT gene; (iv)
performing sequencing reactions using primers that yield sequences
comprising wild type or mutant amino acids at codons 245, 98, 103,
135, 181, and 190; and (v) determining the presence or absence of
mutations at codons 245, 98, 103, 135, 181, and 190 from the
sequences.
[0066] 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 biological sample from an HIV-infected patient; (ii)
transcribing the HIV-encoding RNA in the biological sample to cDNA;
(iii) amplifying a patient-derived segment (PDS) using HIV PCR
primers that result in a product comprising the RT gene; (iv)
performing sequencing reactions using primers that yield sequences
comprising wild type or mutant amino acids at codons 245, and 103;
and (v) determining the presence or absence of mutations at codons
245, and 103 from the sequences.
[0067] 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 biological sample from an HIV-infected patient; (ii)
transcribing the HIV-encoding RNA in the biological sample to cDNA;
(iii) amplifying a patient-derived segment (PDS) using HIV PCR
primers that result in a product comprising the RT gene; (iv)
performing sequencing reactions using primers that yield sequences
comprising wild type or mutant amino acids at codons 245, 103, 135
and 225; and (v) determining the presence or absence of mutations
at codons 245, 103, 135 and 225 from the sequences.
[0068] 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 biological sample from an HIV-infected patient; (ii)
transcribing the HIV-encoding RNA in the biological sample to cDNA;
(iii) amplifying a patient-derived segment (PDS) using HIV PCR
primers that result in a product comprising the RT gene; (iv)
performing sequencing reactions using primers that yield sequences
comprising wild type or mutant amino acids at codons 270, 103 and
135; and (v) determining the presence or absence of mutations at
codons 270, 103 and 135 from the sequences.
[0069] The presence of the mutations at codons 230 and either 103
or 181 of HIV RT indicates that the effectiveness of the current or
prospective NNRTI therapy has been diminished. As shown by this
invention, a mutation at codon 230, in combination with mutations
at either 103 or 181, reduces drug susceptibility, and results in
drug-dependent stimulation of viral replication. Using the methods
of this invention, change in the NNRTI therapy would be
indicated.
[0070] Similarly, using the means and methods of this invention the
presence of mutations at codons 230, 101, 103, 190, 221, and 238 of
the HIV RT gene indicates that the effectiveness of the current or
prospective NNRTI therapy has been diminished. As shown by this
invention, mutations at codons 230, 101, 103, 190, 221 and 238,
reduce drug susceptibility, and result in drug-dependent
stimulation of viral replication. Using the methods of this
invention, change in the NNRTI therapy would be indicated.
[0071] Similarly, using the means and methods of this invention the
presence of mutations at codons 241, 103, and 135 of the HIV RT
gene indicates that the effectiveness of the current or prospective
NNRTI therapy has been diminished. As shown by this invention,
mutations at codons 241, 103, and 135 reduce drug susceptibility,
and result in drug-dependent stimulation of viral replication.
Using the methods of this invention, change in the NNRTI therapy
would be indicated.
[0072] Similarly, using the means and methods of this invention the
presence of mutations at codons 241, 101, 106, 135, 138, and 190 of
the HIV RT indicates that the effectiveness of the current or
prospective NNRTI therapy has been diminished. As shown by this
invention, mutations at codons 241, 101, 106, 135, 138, and 190
reduce drug susceptibility, and result in drug-dependent
stimulation of viral replication. Using the methods of this
invention, change in the NNRTI therapy would be indicated.
[0073] Similarly, using the means and methods of this invention the
presence of mutations at codons 245, 98, 135, and 181 of the HIV RT
indicates that the effectiveness of the current or prospective
NNRTI therapy has been diminished. As shown by this invention,
mutations at codons 245, 98, 135, and 181 reduce drug
susceptibility, and result in drug-dependent stimulation of viral
replication. Using the methods of this invention, change in the
NNRTI therapy would be indicated.
[0074] Similarly, using the means and methods of this invention the
presence of mutations at codons 245, 101, 103, 135, and 190 of the
HIV RT indicates that the effectiveness of the current or
prospective NNRTI therapy has been diminished. As shown by this
invention, mutations at codons 245, 101, 103, 135, and 190 reduce
drug susceptibility, and result in drug-dependent stimulation of
viral replication. Using the methods of this invention, change in
the NNRTI therapy would be indicated.
[0075] Similarly, using the means and methods of this invention the
presence of mutations at codons 245, 103, 225, and 270 of the HIV
RT indicates that the effectiveness of the current or prospective
NNRTI therapy has been diminished. As shown by this invention,
mutations at codons 245, 103, 225, and 270 reduce drug
susceptibility, and result in drug-dependent stimulation of viral
replication. Using the methods of this invention, change in the
NNRTI therapy would be indicated.
[0076] Similarly, using the means and methods of this invention the
presence of mutations at codons 245, 135, and 138 of the HIV RT
indicates that the effectiveness of the current or prospective
NNRTI therapy has been diminished. As shown by this invention,
mutations at codons 245, 135, and 138 reduce drug susceptibility,
and result in drug-dependent stimulation of viral replication.
Using the methods of this invention, change in the NNRTI therapy
would be indicated.
[0077] Similarly, using the means and methods of this invention the
presence of mutations at codons 245, 98, 103, 135, 181, and 190 of
the HIV RT indicates that the effectiveness of the current or
prospective NNRTI therapy has been diminished. As shown by this
invention, mutations at codons 245, 98, 103, 135, 181, and 190
reduce drug susceptibility, and result in drug-dependent
stimulation of viral replication. Using the methods of this
invention, change in the NNRTI therapy would be indicated.
[0078] Similarly, using the means and methods of this invention the
presence of mutations at codons 245 and 103 of the HIV RT indicates
that the effectiveness of the current or prospective NNRTI therapy
has been diminished. As shown by this invention, mutations at
codons 245 and 103 reduce drug susceptibility, and result in
drug-dependent stimulation of viral replication. Using the methods
of this invention, change in the NNRTI therapy would be
indicated.
[0079] Similarly, using the means and methods of this invention the
presence of mutations at codons 245, 103, 135, and 225 of the HIV
RT indicates that the effectiveness of the current or prospective
NNRTI therapy has been diminished. As shown by this invention,
mutations at codons 245, 103, 135, and 225 reduce drug
susceptibility, and result in drug-dependent stimulation of viral
replication. Using the methods of this invention, change in the
NNRTI therapy would be indicated.
[0080] Similarly, using the means and methods of this invention the
presence of mutations at codons 270, 103, and 135 of the HIV RT
indicates that the effectiveness of the current or prospective
NNRTI therapy has been diminished. As shown by this invention,
mutations at codons 270, 103, and 135 reduce drug susceptibility,
and result in drug-dependent stimulation of viral replication.
Using the methods of this invention, change in the NNRTI therapy
would be indicated.
[0081] Another preferred, non-limiting, specific embodiment of the
invention is as follows: A method of evaluating the effectiveness
of antiretroviral therapy of an HIV-infected patient comprising:
(i) collecting a biological sample from an HIV-infected patient;
and (ii) determining whether the biological sample comprises
nucleic acid encoding the HIV RT gene, and having a mutation at
codon 230 and either codon 103 or 181. Using the phenotypic
susceptibility assay, it was observed that the presence of
mutations at codons 230 and either 103 and 181 correlates
positively with decreased susceptibility to delavirdine,
nevirapine, and efavirenz and results in drug-dependent stimulation
of viral replication. In another specific embodiment, the mutated
codon 230 of HIV RT encodes leucine (L), while the mutated codon
103 encodes an asparagine (N). In a still further specific
embodiment, the mutated codon 230 or HIV RT encodes leucine (L) and
the mutated codon at 181 encodes a cysteine (C).
[0082] Yet another preferred, non-limiting, specific embodiment of
the invention is as follows: A method of evaluating the
effectiveness of antiretroviral therapy of an HIV-infected patient
comprising: (i) collecting a biological sample from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding the HIV RT gene, and having
mutations at codons 230, 101, 103, 190, 221, and 238. Using the
phenotypic susceptibility assay, it was observed that the presence
of mutations at codons 230, 101, 103, 190, 221, and 238 correlates
positively with decreased susceptibility to delavirdine,
nevirapine, and efavirenz and results in drug-dependent stimulation
of viral replication. In another specific embodiment, the mutated
codon 230 of HIV RT encodes a leucine (L), the mutated codon 101
encodes a glutamic acid (E), the mutated codon 103 encodes an
asparagine (N), the mutated codon 190 encodes a serine (S), the
mutated codon 221 encodes a tyrosine (Y), and the mutated codon 238
encodes a threonine (T).
[0083] Yet another preferred, non-limiting, specific embodiment of
the invention is as follows: A method of evaluating the
effectiveness of antiretroviral therapy of an HIV-infected patient
comprising: (i) collecting a biological sample from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding the HIV RT gene, and having
mutations at codons 241, 103, and 135. Using the phenotypic
susceptibility assay, it was observed that the presence of
mutations at codons 241, 103, and 135 correlates positively with
decreased susceptibility to delavirdine, nevirapine, and efavirenz
and results in drug-dependent stimulation of viral replication. In
another specific embodiment, the mutated codon 241 of HIV RT
encodes a serine (S), the mutated codon 103 encodes an asparagine
(N), and the mutated codon 135 encodes a threonine (T).
[0084] Yet another preferred, non-limiting, specific embodiment of
the invention is as follows: A method of evaluating the
effectiveness of antiretroviral therapy of an HIV-infected patient
comprising: (i) collecting a biological sample from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding the HIV RT gene, and having
mutations at codons 241, 101, 106, 135, 138, and 190. Using the
phenotypic susceptibility assay, it was observed that the presence
of mutations at codons 241, 101, 106, 135, 138, and 190 correlates
positively with decreased susceptibility to delavirdine,
nevirapine, and efavirenz and results in drug-dependent stimulation
of viral replication. In another specific embodiment, the mutated
codon 241 of HIV RT encodes an isoleucine (I), the mutated codon
101 encodes a glutamic acid (E), the mutated codon 106 encodes an
methionine (M), the mutated codon 135 encodes a threonine (T), the
mutated codon 138 encodes an alanine (A), and the mutated codon 190
encodes an alanine (A).
[0085] Yet another preferred, non-limiting, specific embodiment of
the invention is as follows: A method of evaluating the
effectiveness of antiretroviral therapy of an HIV-infected patient
comprising: (i) collecting a biological sample from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding the HIV RT gene, and having
mutations at codons 245, 98, 135, and 181. Using the phenotypic
susceptibility assay, it was observed that the presence of
mutations at codons 245, 98, 135, and 181 correlates positively
with decreased susceptibility to delavirdine, nevirapine, and
efavirenz and results in drug-dependent stimulation of viral
replication. In another specific embodiment, the mutated codon 245
of HIV RT encodes a glutamic acid (E), the mutated codon 98 encodes
a Glycine (G), the mutated codon 135 encodes a threonine (T), and
the mutated codon 181 encodes a cysteine (C).
[0086] Yet another preferred, non-limiting, specific embodiment of
the invention is as follows: A method of evaluating the
effectiveness of antiretroviral therapy of an HIV-infected patient
comprising: (i) collecting a biological sample from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding the HIV RT gene, and having
mutations at codons 245, 101, 103, 135, and 190. Using the
phenotypic susceptibility assay, it was observed that the presence
of mutations at codons 245, 101, 103, 135, and 190 correlates
positively with decreased susceptibility to delavirdine,
nevirapine, and efavirenz and results in drug-dependent stimulation
of viral replication. In another specific embodiment, the mutated
codon 245 of HIV RT encodes a glutamic acid (E), the mutated codon
101 encodes a glutamic acid (E), the mutated codon 103 encodes
asparagine (N), the mutated codon 135 encodes a threonine (T), and
the mutated codon 190 encodes an alanine (A).
[0087] Yet another preferred, non-limiting, specific embodiment of
the invention is as follows: A method of evaluating the
effectiveness of antiretroviral therapy of an HIV-infected patient
comprising: (i) collecting a biological sample from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding the HIV RT gene, and having
mutations at codons 245, 103, 225, and 270. Using the phenotypic
susceptibility assay, it was observed that the presence of
mutations at codons 245, 103, 225, and 270 correlates positively
with decreased susceptibility to delavirdine, nevirapine, and
efavirenz and results in drug-dependent stimulation of viral
replication. In another specific embodiment, the mutated codon 245
of HIV RT encodes a glutamic acid (E), the mutated codon 103
encodes asparagine (N), the mutated codon 225 encodes a histidine
(H), and the mutated codon 270 encodes a methionine (M).
[0088] Yet another preferred, non-limiting, specific embodiment of
the invention is as follows: A method of evaluating the
effectiveness of antiretroviral therapy of an HIV-infected patient
comprising: (i) collecting a biological sample from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding the HIV RT gene, and having
mutations at codons 245, 135, and 138. Using the phenotypic
susceptibility assay, it was observed that the presence of
mutations at codons 245, 135, and 138 correlates positively with
decreased susceptibility to delavirdine, nevirapine, and efavirenz
and results in drug-dependent stimulation of viral replication. In
another specific embodiment, the mutated codon 245 of HIV RT
encodes a threonine (T), the mutated codon 135 encodes a threonine
(T), and the mutated codon 138 encodes an glycine (G).
[0089] Yet another preferred, non-limiting, specific embodiment of
the invention is as follows: A method of evaluating the
effectiveness of antiretroviral therapy of an HIV-infected patient
comprising: (i) collecting a biological sample from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding the HIV RT gene, and having
mutations at codons 245, 98, 103, 135, 181, and 190. Using the
phenotypic susceptibility assay, it was observed that the presence
of mutations at codons 245, 98, 103, 135, 181, and 190 correlates
positively with decreased susceptibility to delavirdine,
nevirapine, and efavirenz and results in drug-dependent stimulation
of viral replication. In another specific embodiment, the mutated
codon 245 of HIV RT encodes a threonine (T), the mutated codon 98
encodes a glutamic acid (G), the mutated codon 103 encodes a
asparagine (N), the mutated codon 135 encodes a threonine (T), the
mutated codon 181 encodes a cysteine (C), and the mutated codon 190
encodes an alanine (A).
[0090] Yet another preferred, non-limiting, specific embodiment of
the invention is as follows: A method of evaluating the
effectiveness of antiretroviral therapy of an HIV-infected patient
comprising: (i) collecting a biological sample from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding the HIV RT gene, and having
mutations at codons 245 and 103. Using the phenotypic
susceptibility assay, it was observed that the presence of
mutations at codons 245 and 103 correlates positively with
decreased susceptibility to delavirdine, nevirapine, and efavirenz
and results in drug-dependent stimulation of viral replication. In
another specific embodiment, the mutated codon 245 of HIV RT
encodes a threonine (T), and the mutated codon 103 encodes a
asparagine (N).
[0091] Yet another preferred, non-limiting, specific embodiment of
the invention is as follows: A method of evaluating the
effectiveness of antiretroviral therapy of an HIV-infected patient
comprising: (i) collecting a biological sample from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding the HIV RT gene, and having
mutations at codons 245, 103, 135, and 225. Using the phenotypic
susceptibility assay, it was observed that the presence of
mutations at codons 245, 103, 135, and 225 correlates positively
with decreased susceptibility to delavirdine, nevirapine, and
efavirenz and results in drug-dependent stimulation of viral
replication. In another specific embodiment, the mutated codon 245
of HIV RT encodes a methionine (M), the mutated codon 103 encodes
an asparagine (N), the mutated codon 135 encodes a threonine (T),
and the mutated codon 225 encodes a histidine (H).
[0092] Yet another preferred, non-limiting, specific embodiment of
the invention is as follows: A method of evaluating the
effectiveness of antiretroviral therapy of an HIV-infected patient
comprising: (i) collecting a biological sample from an HIV-infected
patient; and (ii) determining whether the biological sample
comprises nucleic acid encoding the HIV RT gene, and having
mutations at codons 270, 103, and 135. Using the phenotypic
susceptibility assay, it was observed that the presence of
mutations at codons 270, 103, and 135 correlates positively with
decreased susceptibility to delavirdine, nevirapine, and efavirenz
and results in drug-dependent stimulation of viral replication. In
another specific embodiment, the mutated codon 270 of HIV RT
encodes a serine (S), the mutated codon 103 encodes asparagine (N),
and the mutated codon 135 encodes a threonine (T).
[0093] 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 particularly,
the invention describes the resistance test vector comprising the
HIV reverse transcriptase having mutations at codons 230 and either
103 or 181 for drug screening. The invention also describes the
resistance test vector comprising the HIV reverse transcriptase
having mutations at codons 230 and 101, 103, 190, 221, and 238. The
invention also describes the resistance test vector comprising the
HIV reverse transcriptase having mutations at codons 241, 103, and
135. The invention also describes the resistance test vector
comprising the HIV reverse transcriptase having mutations at codons
241, 101, 106, 190, 135, and 138. The invention also describes the
resistance test vector comprising the HIV reverse transcriptase
having mutations at codons 245, 98, 135, and 181. The invention
also describes the resistance test vector comprising the HIV
reverse transcriptase having mutations at codons 245, 101, 103,
135, and 190. The invention also describes the resistance test
vector comprising the HIV reverse transcriptase having mutations at
codons 245, 103, 225, and 270. The invention also describes the
resistance test vector comprising the HIV reverse transcriptase
having mutations at codons 245, 135 and 138. The invention also
describes the resistance test vector comprising the HIV reverse
transcriptase having mutations at codons 245, 98, 103, 135, 181,
and 190. The invention also describes the resistance test vector
comprising the HIV reverse transcriptase having mutations at codons
245 and 103. The invention also describes the resistance test
vector comprising the HIV reverse transcriptase having mutations at
codons 245, 103, 135, and 225. The invention also describes the
resistance test vector comprising the HIV reverse transcriptase
having mutations at codors 270, 103 and 135.
[0094] The structure, life cycle and genetic elements of the
viruses which could be tested in the drug susceptibility and
resistance test of this invention would be known to one of ordinary
skill in the art. It is useful to the practice of this invention,
for example, to understand the life cycle of a retrovirus, as well
as the viral genes required for retrovirus rescue and infectivity.
Retrovirally infected cells shed a membrane virus containing a
diploid RNA genome. The virus, studded with an envelope
glycoprotein (which serves to determine the host range of
infectivity), attaches to a cellular receptor in the plasma
membrane of the cell to be infected. After receptor binding, the
virus is internalized and uncoated as it passes through the
cytoplasm of the host cell. Either on its way to the nucleus or in
the nucleus, the reverse transcriptase molecules resident in the
viral core drive the synthesis of the double-stranded DNA provirus,
a synthesis that is primed by the binding of a tRNA molecule to the
genomic viral RNA. The double-stranded DNA provirus is subsequently
integrated in the genome of the host cell, where it can serve as a
transcriptional template for both mRNAs encoding viral proteins and
virion genomic RNA, which will be packaged into viral core
particles. On their way out of the infected cell, core particles
move through the cytoplasm, attach to the inside of the plasma
membrane of the newly infected cell, and bud, taking with them
tracts of membrane containing the virally encoded envelope
glycoprotein gene product. This cycle of infection--reverse
transcription, transcription, translation, virion assembly, and
budding--repeats itself over and over again as infection
spreads.
[0095] The viral RNA and, as a result, the proviral DNA encode
several cis-acting elements that are vital to the successful
completion of the viral lifecycle. The virion RNA carries the viral
promoter at its 3' end. Replicative acrobatics place the viral
promoter at the 5' end of the proviral genome as the genome is
reverse transcribed. Just 3' to the 5' retroviral LTR lies the
viral packaging site. The retroviral lifecycle requires the
presence of virally encoded transacting factors. The
viral-RNA-dependent DNA polymerase (pol)-reverse transcriptase is
also contained within the viral core and is vital to the viral life
cycle in that it is responsible for the conversion of the genomic
RNA to the integrative intermediate proviral DNA. The viral
envelope glycoprotein, env, is required for viral attachment to the
uninfected cell and for viral spread. There are also
transcriptional trans-activating factors, so called
transactivators, that can serve to modulate the level of
transcription of the integrated parental provirus. Typically,
replication-competent (non-defective) viruses are self-contained in
that they encode all of these trans-acting factors. Their defective
counterparts are not self-contained.
[0096] In the case of a DNA virus, such as a hepadnavirus,
understanding the life cycle and viral genes required for infection
is useful to the practice of this invention. The process of HBV
entry has not been well defined. Replication of HBV uses an RNA
intermediate template. In the infected cell the first step in
replication is the conversion of the asymmetric relaxed circle DNA
(rc-DNA) to covalently closed circle DNA (cccDNA). This process,
which occurs within the nucleus of infected liver cells, involves
completion of the DNA positive-strand synthesis and ligation of the
DNA ends. In the second step, the cccDNA is transcribed by the host
RNA polymerase to generate a 3.5 kB RNA template (the pregenome).
This pregenome is complexed with protein in the viral core. The
third step involves the synthesis of the first negative-sense DNA
strand by copying the pregenomic RNA using the virally encoded p
protein reverse transcriptase. The P protein also serves as the
minus strand DNA primer. Finally, the synthesis of the second
positive-sense DNA strand occurs by copying the first DNA strand,
using the P protein DNA polymerase activity and an oligomer of
viral RNA as primer. The pregenome also transcribes mRNA for the
major structural core proteins.
[0097] The following flow chart illustrates certain of the various
vectors and host cells which may be used in this invention. It is
not intended to be all inclusive.
[0098] Flow chart
[0099] Vectors 1
[0100] Host Cells
[0101] Packaging Host Cell--transfected with packaging expression
vectors
[0102] Resistance Test Vector Host Cell--a packaging host cell
transfected with a resistance test vector
[0103] Target Host Cell--a host cell to be infected by a resistance
test vector viral particle produced by the resistance test vector
host cell
[0104] Resistance Test Vector
[0105] "Resistance test vector" means one or more vectors which
taken together contain DNA or RNA comprising a patient-derived
segment and an indicator gene. In the case where the resistance
test vector comprises more than one vector the patient-derived
segment may be contained in one vector and the indicator gene in a
different vector. Such a resistance test vector comprising more
than one vector is referred to herein as a resistance test vector
system for purposes of clarity but is nevertheless understood to be
a resistance test vector. The DNA or RNA of a resistance test
vector may thus be contained in one or more DNA or RNA molecules.
In one embodiment, the resistance test vector is made by insertion
of a patient-derived segment into an indicator gene viral vector.
In another embodiment, the resistance test vector is made by
insertion of a patient-derived segment into a packaging vector
while the indicator gene is contained in a second vector, for
example an indicator gene viral vector. As used herein,
"patient-derived segment" refers to one or more viral segments
obtained directly from a patient using various means, for example,
molecular cloning or polymerase chain reaction (PCR) amplification
of a population of patient-derived segments using viral DNA or
complementary DNA (cDNA) prepared from viral RNA, present in the
cells (e.g. peripheral blood mononuclear cells, PBMC), serum or
other bodily fluids of infected patients. When a viral segment is
"obtained directly" from a patient it is obtained without passage
of the virus through culture, or if the virus is cultured, then by
a minimum number of passages to essentially eliminate the selection
of mutations in culture. The term "viral segment" refers to any
functional viral sequence or viral gene encoding a gene product
(e.g., a protein) that is the target of an anti-viral drug. The
term "functional viral sequence" as used herein refers to any
nucleic acid sequence (DNA or RNA) with functional activity such as
enhancers, promoters, polyadenylation sites, sites of action of
trans-acting factors, such as tar and RRE, packaging sequences,
integration sequences, or splicing sequences. If a drug were to
target more than one functional viral sequence or viral gene
product then patient-derived segments corresponding to each said
viral gene would be inserted in the resistance test vector. In the
case of combination therapy where two or more anti-virals targeting
two different functional viral sequences or viral gene products are
being evaluated, patient-derived segments corresponding to each
functional viral sequence or viral gene product would be inserted
in the resistance test vector. The patient-derived segments are
inserted into unique restriction sites or specified locations,
called patient sequence acceptor sites, in the indicator gene viral
vector or for example, a packaging vector depending on the
particular construction being used as described herein.
[0106] As used herein, "patient-derived segment" encompasses
segments derived from human and various animal species. Such
species include, but are not limited to chimpanzees, horses,
cattles, cats and dogs.
[0107] Patient-derived segments can also be incorporated into
resistance test vectors using any of several alternative cloning
techniques. For example, cloning via the introduction of class II
restriction sites into both the plasmid backbone and the
patient-derived segments or by uracil DNA glycosylase primer
cloning (refs).
[0108] The patient-derived segment may be obtained by any method of
molecular cloning or gene amplification, or modifications thereof,
by introducing patient sequence acceptor sites, as described below,
at the ends of the patient-derived segment to be introduced into
the resistance test vector. For example, in a gene amplification
method such as PCR, restriction sites corresponding to the
patient-sequence acceptor sites can be incorporated at the ends of
the primers used in the PCR reaction. Similarly, in a molecular
cloning method such as cDNA cloning, said restriction sites can be
incorporated at the ends of the primers used for first or second
strand cDNA synthesis, or in a method such as primer-repair of DNA,
whether cloned or uncloned DNA, said restriction sites can be
incorporated into the primers used for the repair reaction. The
patient sequence acceptor sites and primers are designed to improve
the representation of patient-derived segments. Sets of resistance
test vectors having designed patient sequence acceptor sites
provide representation of patient-derived segments that would be
underrepresented in one resistance test vector alone.
[0109] Resistance test vectors are prepared by modifying an
indicator gene viral vector (described below) by introducing
patient sequence acceptor sites, amplifying or cloning
patient-derived segments and inserting the amplified or cloned
sequences precisely into indicator gene viral vectors at the
patient sequence acceptor sites. The resistance test vectors are
constructed from indicator gene viral vectors which are in turn
derived from genomic viral vectors or subgenomic viral vectors and
an indicator gene cassette, each of which is described below.
Resistance test vectors are then introduced into a host cell.
Alternatively, a resistance test vector (also referred to as a
resistance test vector system) is prepared by introducing patient
sequence acceptor sites into a packaging vector, amplifying or
cloning patient-derived segments and inserting the amplified or
cloned sequences precisely into the packaging vector at the patient
sequence acceptor sites and co-transfecting this packaging vector
with an indicator gene viral vector.
[0110] In one preferred embodiment, the resistance test vector may
be introduced into packaging host cells together with packaging
expression vectors, as defined below, to produce resistance test
vector viral particles that are used in drug resistance and
susceptibility tests that are referred to herein as a
"particle-based test." In an alternative preferred embodiment, the
resistance test vector may be introduced into a host cell in the
absence of packaging expression vectors to carry out a drug
resistance and susceptibility test that is referred to herein as a
"non-particle-based test." As used herein a "packaging expression
vector" provides the factors, such as packaging proteins (e.g.
structural proteins such as core and envelope polypeptides),
transacting factors, or genes required by replication-defective
retrovirus or hepadnavirus. In such a situation, a
replication-competent viral genome is enfeebled in a manner such
that it cannot replicate on its own. This means that, although the
packaging expression vector can produce the trans-acting or missing
genes required to rescue a defective viral genome present in a cell
containing the enfeebled genome, the enfeebled genome cannot rescue
itself.
[0111] Indicator or Indicator Gene
[0112] "Indicator or indicator gene" refers to a nucleic acid
encoding a protein, DNA or RNA structure that either directly or
through a reaction gives rise to a measurable or noticeable aspect,
e.g. a color or light of a measurable wavelength or in the case of
DNA or RNA used as an indicator a change or generation of a
specific DNA or RNA structure. Preferred examples of an indicator
gene is the E. coli lacZ gene which encodes beta-galactosidase, the
luc gene which encodes luciferase either from, for example,
Photonis pyralis (the firefly) or Renilla reniformis (the sea
pansy), the E. coli phoA gene which encodes alkaline phosphatase,
green fluorescent protein and the bacterial CAT gene which encodes
chloramphenicol acetyltransferase. Additional preferred examples of
an indicator gene are secreted proteins or cell surface proteins
that are readily measured by assay, such as radioimmunoassay (RIA),
or fluorescent activated cell sorting (FACS), including, for
example, growth factors, cytokines and cell surface antigens (e.g.
growth hormone, Il-2 or CD4, respectively). "Indicator gene" is
understood to also include a selection gene, also referred to as a
selectable marker. Examples of suitable selectable markers for
mammalian cells are dihydrofolate reductase (DHFR), thymidine
kinase, hygromycin, neomycin, zeocin or E. coli gpt. In the case of
the foregoing examples of indicator genes, the indicator gene and
the patient-derived segment are discrete, i.e. distinct and
separate genes. In some cases a patient-derived segment may also be
used as an indicator gene. In one such embodiment in which the
patient-derived segment corresponds to more than one viral gene
which is the target of an anti-viral, one of said viral genes may
also serve as the indicator gene. For example, a viral protease
gene may serve as an indicator gene by virtue of its ability to
cleave a chromogenic substrate or its ability to activate an
inactive zymogen which in turn cleaves a chromogenic substrate,
giving rise in each case to a color reaction. In all of the above
examples of indicator genes, the indicator gene may be either
"functional" or "non-functional" but in each case the expression of
the indicator gene in the target cell is ultimately dependent upon
the action of the patient-derived segment.
[0113] Functional Indicator Gene
[0114] In the case of a "functional indicator gene" the indicator
gene may be capable of being expressed in a "packaging host
cell/resistance test vector host cell" as defined below,
independent of the patient-derived segment, however the functional
indicator gene could not be expressed in the target host cell, as
defined below, without the production of functional resistance test
vector particles and their effective infection of the target host
cell. In one embodiment of a functional indicator gene, the
indicator gene cassette, comprising control elements and a gene
encoding an indicator protein, is inserted into the indicator gene
viral vector with the same or opposite transcriptional orientation
as the native or foreign enhancer/promoter of the viral vector. One
example of a functional indicator gene in the case of HIV or HBV,
places the indicator gene and its promoter (a CMV IE
enhancer/promoter) in the same or opposite transcriptional
orientation as the HIV-LTR or HBV enhancer-promoter, respectively,
or the CMV IE enhancer/promoter associated with the viral
vector.
[0115] Non-Functional Indicator Gene
[0116] Alternatively the indicator gene, may be "non-functional" in
that the indicator gene is not efficiently expressed in a packaging
host cell transfected with the resistance test vector, which is
then referred to a resistance test vector host cell, until it is
converted into a functional indicator gene through the action of
one or more of the patient-derived segment products. An indicator
gene is rendered non-functional through genetic manipulation
according to this invention.
[0117] 1. Permuted Promoter In one embodiment an indicator gene is
rendered non-functional due to the location of the promoter, in
that, although the promoter is in the same transcriptional
orientation as the indicator gene, it follows rather than precedes
the indicator gene coding sequence. This misplaced promoter is
referred to as a "permuted promoter." In addition to the permuted
promoter the orientation of the non-functional indicator gene is
opposite to that of the native or foreign promoter/enhancer of the
viral vector. Thus the coding sequence of the non-functional
indicator gene can neither be transcribed by the permuted promoter
nor by the viral promoters. The non-functional indicator gene and
its permuted promoter is rendered functional by the action of one
or more of the viral proteins. One example of a non-functional
indicator gene with a permuted promoter in the case of HIV, places
a T7 phage RNA polymerase promoter (herein referred to as T7
promoter) promoter in the 5' LTR in the same transcriptional
orientation as the indicator gene. The indicator gene cannot be
transcribed by the T7 promoter as the indicator gene cassette is
positioned upstream of the T7 promoter. The non-functional
indicator gene in the resistance test vector is converted into a
functional indicator gene by reverse transcriptase upon infection
of the target cells, resulting from the repositioning of the T7
promoter, by copying from the 5' LTR to the 3' LTR, relative to the
indicator gene coding region. Following the integration of the
repaired indicator gene into the target cell chromosome by HIV
integrase, a nuclear T7 RNA polymerase expressed by the target cell
transcribes the indicator gene. One example of a non-functional
indicator gene with a permuted promoter in the case of HBV, places
an enhancer-promoter region downstream or 3' of the indicator gene
both having the same transcriptional orientation. The indicator
gene cannot be transcribed by the enhancer-promoter as the
indicator gene cassette is positioned upstream. The non-functional
indicator gene in the resistance test vector is converted into a
functional indicator gene by reverse transcription and
circularization of the HBV indicator gene viral vector by the
repositioning of the enhancer-promoter upstream relative to the
indicator gene coding region.
[0118] A permuted promoter may be any eukaryotic or prokaryotic
promoter which can be transcribed in the target host cell.
Preferably the promoter will be small in size to enable insertion
in the viral genome without disturbing viral replication. More
preferably, a promoter that is small in size and is capable of
transcription by a single subunit RNA polymerase introduced into
the target host cell, such as a bacteriophage promoter, will be
used. Examples of such bacteriophage promoters and their cognate
RNA polymerases include those of phages T7, T3 and Sp6. A nuclear
localization sequence (NLS) may be attached to the RNA polymerase
to localize expression of the RNA polymerase to the nucleus where
they may be needed to transcribed the repaired indicator gene. Such
an NLS may be obtained from any nuclear-transported protein such as
the SV40 T antigen. If a phage RNA polymerase is employed, an
internal ribosome entry site (IRES) such as the EMC virus 5'
untranslated region (UTR) may be added in front of the indicator
gene, for translation of the transcripts which are generally
uncapped. In the case of HIV, the permuted promoter itself can be
introduced at any position within the 5' LTR that is copied to the
3' LTR during reverse transcription so long as LTR function is not
disrupted, preferably within the U5 and R portions of the LTR, and
most preferably outside of functionally important and highly
conserved regions of U5 and R. In the case of HBV, the permuted
promoter can be placed at any position that does not disrupt the
cis acting elements that are necessary for HBV DNA replication.
Blocking sequences may be added at the ends of the resistance test
vector should there be inappropriate expression of the
non-functional indicator gene due to transfection artifacts (DNA
concatenation). In the HIV example of the permuted T7 promoter
given above, such a blocking sequence may consist of a T7
transcriptional terminator, positioned to block readthrough
transcription resulting from DNA concatenation, but not
transcription resulting from repositioning of the permuted T7
promoter from the 5' LTR to the 3' LTR during reverse
transcription.
[0119] 2. Permuted Coding Region In a second embodiment, an
indicator gene is rendered non-functional due to the relative
location of the 5' and 3' coding regions of the indicator gene, in
that, the 3' coding region precedes rather than follows the 5'
coding region. This misplaced coding region is referred to as a
"permuted coding region." The orientation of the non-functional
indicator gene may be the same or opposite to that of the native or
foreign promoter/enhancer of the viral vector, as mRNA coding for a
functional indicator gene will be produced in the event of either
orientation. The non-functional indicator gene and its permuted
coding region is rendered functional by the action of one or more
of the patient-derived segment products. A second example of a
non-functional indicator gene with a permuted coding region in the
case of HIV, places a 5' indicator gene coding region with an
associated promoter in the 3' LTR U3 region and a 3' indicator gene
coding region in an upstream location of the HIV genome, with each
coding region having the same transcriptional orientation as the
viral LTRs. In both examples, the 5' and 3' coding regions may also
have associated splice donor and acceptor sequences, respectively,
which may be heterologous or artificial splicing signals. The
indicator gene cannot be functionally transcribed either by the
associated promoter or viral promoters, as the permuted coding
region prevents the formation of functionally spliced transcripts.
The non-functional indicator gene in the resistance test vector is
converted into a functional indicator gene by reverse transcriptase
upon infection of the target cells, resulting from the
repositioning of the 5' and 3' indicator gene coding regions
relative to one another, by copying of the 3' LTR to the 5' LTR.
Following transcription by the promoter associated with the 5'
coding region, RNA splicing can join the 5' and 3' coding regions
to produce a functional indicator gene product. One example of a
non-functional indicator gene with a permuted coding region in the
case of HBV, places a 3' indicator gene coding region upstream or
5' of the enhancer-promoter and the 5' coding region of the
indicator gene. The transcriptional orientation of the indicator
gene 5' and 3' coding regions are identical to one another, and the
same as that of the indicator gene viral vector. However, as the
indicator gene 5' and 3' coding regions are permuted in the
resistance test vectors (i.e., the 5' coding region is downstream
of the 3' coding region), no mRNA is transcribed which can be
spliced to generate a functional indicator gene coding region.
Following reverse transcription and circularization of the
indicator gene viral vector, the indicator gene 3' coding region is
positioned downstream or 3' to the enhancer-promoter and 5' coding
regions thus permitting the transcription of mRNA which can be
spliced to generate a functional indicator gene coding region.
[0120] 3. Inverted Intron In a third embodiment, the indicator gene
is rendered non-functional through use of an "inverted intron,"
i.e. an intron inserted into the coding sequence of the indicator
gene with a transcriptional orientation opposite to that of the
indicator gene. The overall transcriptional orientation of the
indicator gene cassette including its own, linked promoter, is
opposite to that of the viral control elements, while the
orientation of the artificial intron is the same as the viral
control elements. Transcription of the indicator gene by its own
linked promoter does not lead to the production of functional
transcripts as the inverted intron cannot be spliced in this
orientation. Transcription of the indicator gene by the viral
control elements does, however, lead to the removal of the inverted
intron by RNA splicing, although the indicator gene is still not
functionally expressed as the resulting transcript has an antisense
orientation. Following the reverse transcription of this transcript
and integration of the resultant retroviral DNA, or the
circularization of hepadnavirus DNA, the indicator gene can be
functionally transcribed using its own linked promoter as the
inverted intron has been previously removed. In this case, the
indicator gene itself may contain its own functional promoter with
the entire transcriptional unit oriented opposite to the viral
control elements. Thus the non-functional indicator gene is in the
wrong orientation to be transcribed by the viral control elements
and it cannot be functionally transcribed by its own promoter, as
the inverted intron cannot be properly excised by splicing.
However, in the case of a retrovirus and HIV specifically and
hepadnaviruses, and HBV specifically, transcription by the viral
promoters (HIV LTR or HBV enhancer-promoter) results in the removal
of the inverted intron by splicing. As a consequence of reverse
transcription of the resulting spliced transcript and the
integration of the resulting provirus into the host cell chromosome
or circularization of the HBV vector, the indicator gene can now be
functionally transcribed by its own promoter. The inverted intron,
consisting of a splice donor and acceptor site to remove the
intron, is preferably located in the coding region of the indicator
gene in order to disrupt translation of the indicator gene. The
splice donor and acceptor may be any splice donor and acceptor. A
preferred splice donor-receptor is the CMV IE splice donor and the
splice acceptor of the second exon of the human alpha globin gene
("intron A").
[0121] Indicator Gene Viral Vector--Construction
[0122] As used herein, "indicator gene viral vector" refers to a
vector(s) comprising an indicator gene and its control elements and
one or more viral genes. The indicator gene viral vector is
assembled from an indicator gene cassette and a "viral vector,"
defined below. The indicator gene viral vector may additionally
include an enhancer, splicing signals, polyadenylation sequences,
transcriptional terminators, or other regulatory sequences.
Additionally the indicator gene viral vector may be functional or
nonfunctional. In the event that the viral segments which are the
target of the anti-viral drug are not included in the indicator
gene viral vector they are provided in a second vector. An
"indicator gene cassette" comprises an indicator gene and control
elements. "Viral vector" refers to a vector comprising some or all
of the following: viral genes encoding a gene product, control
sequences, viral packaging sequences, and in the case of a
retrovirus, integration sequences. The viral vector may
additionally include one or more viral segments one or more of
which may be the target of an anti-viral drug. Two examples of a
viral vector which contain viral genes are referred to herein as an
"genomic viral vector" and a "subgenomic viral vector." A "genomic
viral vector" is a vector which may comprise a deletion of a one or
more viral genes to render the virus replication incompetent, but
which otherwise preserves the mRNA expression and processing
characteristics of the complete virus. In one embodiment for an HIV
drug susceptibility and resistance test, the genomic viral vector
comprises the HIV gag-pol, vif, vpr, tat, rev, vpu, and nef genes
(some, most or all of env may be deleted). A "subgenomic viral
vector" refers to a vector comprising the coding region of one or
more viral genes which may encode the proteins that are the
target(s) of the anti-viral drug. In the case of HIV, a preferred
embodiment is a subgenomic viral vector comprising the HIV gag-pol
gene. In the case of HBV a preferred embodiment is a subgenomic
viral vector comprising the HBV P gene. In the case of HIV, two
examples of proviral clones used for viral vector construction are:
HXB2 (Fisher et al., (1986) Nature, 320, 367-371) and NL4-3,
(Adachi et al., (1986) J. Virol., 59, 284-291). In the case of HBV,
a large number of full length genomic sequences have been
characterized and could be used for construction of HBV viral
vectors: GenBank Nos. M54923, M38636, J02203 and X59795. The viral
coding genes may be under the control of a native enhancer/promoter
or a foreign viral or cellular enhancer/promoter. A preferred
embodiment for an HIV drug susceptibility and resistance test, is
to place the genomic or subgenomic viral coding regions under the
control of the native enhancer/promoter of the HIV-LTR U3 region or
the CMV immediate-early (1E) enhancer/promoter. A preferred
embodiment for an HBV drug susceptibility and resistance test, is
to place the genomic or subgenomic viral coding regions under the
control of the CMV immediate-early (1E) enhancer/promoter. In the
case of an indicator gene viral vector that contains one or more
viral genes which are the targets or encode proteins which are the
targets of an anti-viral drug(s) then said vector contains the
patient sequence acceptor sites. The patient-derived segments are
inserted in the patient sequence acceptor site in the indicator
gene viral vector which is then referred to as the resistance test
vector, as described above.
[0123] "Patient sequence acceptor sites" are sites in a vector for
insertion of patient-derived segments and said sites may be: 1)
unique restriction sites introduced by site-directed mutagenesis
into a vector; 2) naturally occurring unique restriction sites in
the vector; or 3) selected sites into which a patient-derived
segment may be inserted using alternative cloning methods (e.g. UDG
cloning). In one embodiment the patient sequence acceptor site is
introduced into the indicator gene viral vector. The patient
sequence acceptor sites are preferably located within or near the
coding region of the viral protein which is the target of the
anti-viral drug. The viral sequences used for the introduction of
patient sequence acceptor sites are preferably chosen so that no
change, or a conservative change, is made in the amino acid coding
sequence found at that position. Preferably the patient sequence
acceptor sites are located within a relatively conserved region of
the viral genome to facilitate introduction of the patient-derived
segments. Alternatively, the patient sequence acceptor sites are
located between functionally important genes or regulatory
sequences. Patient-sequence acceptor sites may be located at or
near regions in the viral genome that are relatively conserved to
permit priming by the primer used to introduce the corresponding
restriction site into the patient-derived segment. To improve the
representation of patient-derived segments further, such primers
may be designed as degenerate pools to accommodate viral sequence
heterogeneity, or may incorporate residues such as deoxyinosine (I)
which have multiple base-pairing capabilities. Sets of resistance
test vectors having patient sequence acceptor sites that define the
same or overlapping restriction site intervals may be used together
in the drug resistance and susceptibility tests to provide
representation of patient-derived segments that contain internal
restriction sites identical to a given patient sequence acceptor
site, and would thus be underrepresented in either resistance test
vector alone.
[0124] Host Cells
[0125] The resistance test vector is introduced into a host cell.
Suitable host cells are mammalian cells. Preferred host cells are
derived from human tissues and cells which are the principle
targets of viral infection. In the case of HIV these include human
cells such as human T cells, monocytes, macrophage, dendritic
cells, Langerhans cells, hematopoeitic stem cells or precursor
cells, and other cells. In the case of HBV, suitable host cells
include hepatoma cell lines (HepG2, Huh 7), primary human
hepatocytes, mammalian cells which can be infected by pseudotyped
HBV, and other cells. Human derived host cells will assure that the
anti-viral drug will enter the cell efficiently and be converted by
the cellular enzymatic machinery into the metabolically relevant
form of the anti-viral inhibitor. Host cells are referred to herein
as a "packaging host cells," "resistance test vector host cells,"
or "target host cells." A "packaging host cell" refers to a host
cell that provides the trans-acting factors and viral packaging
proteins required by the replication defective viral vectors used
herein, such as the resistance test vectors, to produce resistance
test vector viral particles. The packaging proteins may be provided
for by the expression of viral genes contained within the
resistance test vector itself, a packaging expression vector(s), or
both. A packaging host cell is a host cell which is transfected
with one or more packaging expression vectors and when transfected
with a resistance test vector is then referred to herein as a
"resistance test vector host cell" and is sometimes referred to as
a packaging host cell/resistance test vector host cell. Preferred
host cells for use as packaging host cells for HIV include 293
human embryonic kidney cells (293, Graham, F. L. et al., J. Gen
Virol. 36: 59, 1977), BOSC23 (Pear et al., Proc. Natl. Acad. Sci.
90, 8392, 1993), tsa54 and tsa201 cell lines (Heinzel et al., J.
Virol. 62, 3738, 1988), for HBV HepG2 (Galle and Theilmann, L.
Arzheim.-Forschy Drug Res. (1990) 40, 1380-1382). (Huh, Ueda, K et
al. Virology *1989) 169, 213-216). A "target host cell" refers to a
cell to be infected by resistance test vector viral particles
produced by the resistance test vector host cell in which
expression or inhibition of the indicator gene takes place.
Preferred host cells for use as target host cells include human T
cell leukemia cell lines including Jurkat (ATCC T1B-152), H9 (ATCC
HTB-176), CEM (ATCC CCL-119), HUT78 (ATCC T1B-161), and derivatives
thereof.
[0126] This invention is illustrated in the Experimental Details
section which follows. These sections are set forth to aid in an
understanding of the invention but are not intended to, and should
not be construed to, limit in any way the invention as set forth in
the claims which follow thereafter.
EXPERIMENTAL DETAILS
Example 1
Phenotypic Drug Susceptibility and Resistance Test Using Resistance
Test Vectors
[0127] Phenotypic drug susceptibility and resistance tests are
carried out using the means and methods described in PCT
International Application No. PCT/US97/01609, filed Jan. 29, 1997
which is hereby incorporated by reference.
[0128] In these experiments patient-derived segment (PDS)
corresponding to the HIV protease(PR) and reverse transcriptase
coding regions were either patient-derived segments amplified by
the reverse transcription-polymeras- e chain reaction method
(RT-PCR) using viral RNA isolated from viral particles present in
the serum of HIV-infected individuals or were mutants of wild type
HIV-1 made by site directed mutagenesis of a parental clone of
resistance test vector DNA. Isolation of viral RNA was performed
using standard procedures (e.g. RNAgents Total RNA Isolation
System, Promega, Madison Wis. or RNAzol, Tel-Test, Friendswood,
Tex.). The RT-PCR protocol was divided into two steps. A retroviral
reverse transcriptase [e.g. Moloney MuLV reverse transcriptase
(Roche Molecular Systems, Inc., Branchburg, N.J.), or avian
myeloblastosis virus (AMV) reverse transcriptase, (Boehringer
Mannheim, Indianapolis, Ind.)] was used to transcribe viral RNA
into cDNA. The cDNA was then amplified using a thermostable DNA
polymerase [e.g. Taq (Roche Molecular Systems, Inc., Branchburg,
N.J.), Tth (Roche Molecular Systems, Inc., Branchburg, N.J.),
PrimeZyme (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, Ind.) OR
GeneAmp XL PCR kit (Tth+Vent), (Roche Molecular Systems, Inc.,
Branchburg, N.J.)].
[0129] The primers, ApaI primer (PDSApa) and AgeI primer (PDSAge),
used to amplify the "test" patient-derived segments contained
sequences resulting in ApaI and AgeI recognition sites being
introduced into the 5' and 3' termini of the PCR product,
respectively as described in PCT International Application No.
PCT/US97/01609, filed Jan. 29, 1997.
[0130] Resistance test vectors incorporating the "test"
patient-derived segments were constructed as described in PCT
International Application No. PCT/US97/01609, filed Jan. 29, 1997,
using an amplified DNA product of 1.5 kB prepared by RT-PCR using
viral RNA as a template 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 comprises a representative
sample of the HIV viral quasi-species present in the serum of a
given patient, many (>100) independent E. coli transformants
obtained in the construction of a given resistance test vector were
pooled and used for the preparation of plasmid DNA.
[0131] A packaging expression vector encoding an amphotrophic MuLV
4070A env gene product enables production in a resistance test
vector host cell of resistance test vector viral particles which
can efficiently infect human target cells. 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 resistance test vector to enable
production in the resistance test vector host cell of infectious
pseudotyped resistance test vector viral particles.
[0132] Resistance tests performed with resistance test vectors were
carried out using packaging host and target host cells consisting
of the human embryonic kidney cell line 293 (Cell Culture Facility,
UC San Francisco, SF, CA) or the Jurkat leukemic T-cell line
(Arthur Weiss, UC San Francisco, SF, CA).
[0133] Resistance tests were carried out with resistance test
vectors using two host cell types. Resistance test vector viral
particles were produced by a first host cell (the resistance test
vector host cell) that was prepared by transfecting a packaging
host cell with the resistance test vector and the packaging
expression vector. The resistance test vector viral particles were
then used to infect a second host cell (the target host cell) in
which the expression of the indicator gene is measured.
[0134] The resistance test vectors containing a functional
luciferase gene cassette were constructed and host cells 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, (NRTIs) non-nucleoside reverse transcriptase inhibitors
(NRTIs) 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 PRIs, were
used to infect target host cells grown either in the absence of
NRTIs or NNRTIs, or in the presence of increasing concentrations of
the drug. The amount of luciferase activity produced in infected
target host cells in the presence of varying concentrations of drug
was compared to the amount of luciferase produced in infected
target host cells in the absence of drug. "Drug resistance" was
measured as the amount of drug required to inhibit by 50% the
luciferase activity detected in the absence of drug (inhibitory
concentration 50%, IC50). The IC50 values were determined by
plotting percent drug inhibition vs. log.sub.10 drug concentration.
Stimulation of viral replication was measured as the percent
increase in luciferase activity detected when infection occurs in
the presence of drug as compared to in the absence of drug.
[0135] Host cells were seeded in 10-cm-diameter dishes and were
transfected several days after plating with resistance test 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 transfection. Cell
culture media containing resistance test vector viral particles was
harvested one to four days after transfection and was passed
through a 0.45-mm filter before being used to infect target host
cells, or being stored at -80.degree. C. HIV capsid protein (p24)
levels in the harvested cell culture media were determined by an
EIA method as described by the manufacturer (SAIC; 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. One 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:
% luciferase inhibition=1-(RLUluc [drug] RLUluc).times.100
[0136] where RLUluc [drug] is the Relative Light Unit of luciferase
activity in infected cells in the presence of drug, and RLUluc is
the Relative Light Unit of luciferase activity in infected cells in
the absence of drug. IC50 values were obtained from the sigmoidal
curves that were generated from the data by plotting the percent
inhibition of luciferase activity vs. the log.sub.10 drug
concentration. The drug inhibition curves are shown in FIG. 3. The
percent luciferase stimulation is the negative of the % inhibition
calculated using the formula above. Curves showing drug-dependent
stimulation of viral replication are shown in FIG. 4.
Example 2
Correlating Phenotypic Susceptibility and Genotypic Analysis
[0137] Phenotypic Susceptibility Analysis of Patient HIV
Samples
[0138] 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
reverse transcriptase inhibitors (NRTIs), non-nucleoslde reverse
transcriptase inhibitors (NNPTIs), and protease inhibitors (PRIs).
The panel of drugs can be expanded as new drugs or new drug targets
become available. An IC50 is determined for each resistance test
vector pool for each drug tested. The pattern of susceptibility to
all of the drugs tested is examined and compared to known patterns
of susceptibility. The effect of drug on viral replication (i.e.
luciferase activity) is further analyzed for any evidence of
drug-dependent stimulation of viral replication, which would appear
as a negative percent inhibition in the drug susceptibility graph.
A patient sample may be further examined for genotypic changes
correlated with the pattern of susceptibility observed.
[0139] Genotypic Analysis of Patient HIV Samples
[0140] Resistance test vector DNAs, either pools or clones, are
analyzed by any of the genotyping methods known to one of ordinaryt
standing in the art (see PCT International Application No.
PCT/US97/01609, filed Jan. 29, 1997). In one embodiment of the
invention, patient HIV sample sequences are determined using viral
RNA purification, RT/PCR and ABI chain terminator automated
sequencing. The sequence that is determined is compared to control
sequences present in the database or is compared to a sample from
the patient prior to initiation of therapy, if available. The
genotype is examined for sequences that are different from the
control or pre-treatment sequence and correlated to the observed
phenotype.
[0141] Phenotypic Susceptibility Analysis of site directed
Mutants
[0142] 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 drugs 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 (Sarkar G, Sommer
S S. (1990). Biotechniques 8:404-407). A resistance test vector
containing the specific mutation or group of mutations is then
tested using the phenotypic susceptibility assay described above
and the susceptibility profile is compared to that of a genetically
defined wild-type (drug susceptible) resistance test vector which
lacks the specific mutations. Observed changes in the pattern of
phenotypic susceptibility to the antiretroviral drugs tested are
attributed to the specific mutations introduced into the resistance
test vector.
Example 3
Using Resistance Test Vectors and Site Directed Mutants to
Correlate Genotypes and Phenotypes Associated with NNRTI Drug
Susceptibility and Resistance, and Drug-Dependent Stimulation of
Replication in HIV: M230L+Y181C
[0143] Preparation of resistant test vectors and phenotypic
analysis of patient ARG-014 HIV samples Resistance test vectors
were constructed as described in Example 1 from virus samples
obtained from an individual patient at three separate time points
over a 16-week period. This patient had been previously treated
with two NRTIs (didanosine and lamivudine) and a PRI indinavir. At
the time the first sample (designated 98-773) was obtained, the
patient began taking a new anti-viral regimen including an NRTI
(abacavir) an NNRTI (nevirapine) and two PRI's (nelfinavir and
saquinavir) (PRIs). The second sample (98-1046) was obtained 8
weeks later and a third sample (98-887) was obtained 16 weeks
later. Viral load measurements showed that the patient experienced
a transient reduction in viral load (at week 4) followed by a
return to baseline viral load (.about.60,000 copies/ml) at weeks 8
and 16. Isolation of viral RNA and RT/PCR was used to generate
patient derived segments (PDSs) that comprised viral sequences
coding for all of PR and aa 1-313 of RT.
[0144] The PDS were inserted into an indicator gene viral vector to
generate resistance test vectors designated RTV-773, RTV-1046 and
RTV-887. These RTVs 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 comprised members of the classes known as NRTIs (zidovudine,
lamivudine, Stavudine, didanosine, zalcitabine, and abacavir),
NNRTIs (delavirdine, efavirenz and nevirapine), and PRIs
(amprenavir, 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 samples RTV-1046 and RTV-887, in which there
was a moderate decrease (5-fold) in efavirenz susceptibility and a
significant decrease in nevirapine (>600-fold) and delavirdine
(>250-fold) susceptibility.
[0145] Determination of Genotype of Patient HIV Samples
[0146] RTV-773, RTV-1046 and RTV-887 DNA were analyzed by ABI chain
terminator automated sequencing. The nucleotide sequence was
compared to the consensus sequence of a wild type clade B HIV-1
(HIV Sequence Database Los Alamos, N. Mex.). The genotype was
examined for sequences that are different from the control
sequence. In RTV-773 (baseline sample) mutations were noted at HIV
RE codons 41, 74, 184, 210, 211, 215, 228, and 296 compared to the
control sequence. Mutations M41L, L74I, M184V, L210W and T215Y are
associated with NRTI resistance. The mutations R211K and T296S are
known sequence polymorphisms found among different wild-type
(drug-sensitive) variants of HIV. In RTV-1046 and RTV-887 two
additional mutations appeared at 181 and 230. The mutation Y181C
has been previously shown to be associated with resistance to the
NNRTIs nevirapine and delavirdine but not efavirenz. We examined
the mutation, M230L, alone, and in combination with Y181C, using
site directed mutagenesis and in vitro phenotypic susceptibility
testing to correlate the observed changes in genotype with
phenotype.
[0147] Site directed mutagenesis used to confirm the role of
specific mutations in phenotypic susceptibility to anti-retroviral
drugs in HIV
[0148] The M230L mutation was introduced into a wild-type (drug
sensitive) resistance test vector using the mega-primer method for
site-directed mutagenesis (Sakar and Sommar, Ibid.). The resulting
resistance test vector containing the M230L mutation (M230L-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 230.
We determined the pattern of phenotypic susceptibility to the
NNRTIs, delavirdine, nevirapine and efavirenz, in the M230L-RTV. On
a wild type background (i.e. M230L mutation alone) the M230L-RTV
displayed a moderate loss of susceptibility to efavirenz (23-fold),
nevirapine (39-fold) and delavirdine (58-fold) compared to a wild
type control RTV. The M230L-RTV showed a drug-dependent stimulation
of viral replication in the presence of delavirdine (.about.50%)
and nevirapine (.about.50%) but not efavirenz. A resistance test
vector containing the M230L mutation and the Y181C mutation
(Y181C/M230L-RTV) was constructed and 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 181 and 230. We determined the pattern
of phenotypic susceptibility to the NNRTIs, delavirdine, nevirapine
and efavirenz, in the Y181C/M230L-RTV. The Y181C/M230L-RTV
displayed a moderate reduction in susceptibility to efavirenz
(25-fold), and high-level reductions in susceptibility to
nevirapine (>600-fold) and delavirdine (>250-fold) compared
to a wild type control RTV. The Y181C/M230L-RTV showed no
drug-dependent stimulation of viral replication in the presence of
any of the NNRTIs.
Example 4
Using Resistance Test Vectors and Site Directed Mutants to
Correlate Genotypes and Phenotypes Associated with NNRTI Drug
Susceptibility and Resistance and Drug-Dependent Stimulation of
Replication in HIV: M230L+K103N
[0149] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient CCTG-2165 HIV Samples
[0150] Resistance test vectors were constructed as described in
Example 1 from virus samples obtained from an individual patient at
two separate time points over an 15-week period. This patient had
been previously treated with two NRTIs (stavudine, laminvudine and
a PRI nelfinavir). At the time the first sample (designated
99-2-009089) was obtained, the patient began taking a new
anti-viral regimen including two NRI's (zidovudine, lamivudine and
an NNRTI). A second sample (99-2-009839) was obtained 4 weeks when
viral loads were undetectable, later and a third sample
(99-2-010835) was obtained 15 weeks after baseline. A brief
cessation of therapy was noted (8 days) between the second (wk 4)
and third (wk 15) visits. Viral load measurements showed that the
patient experienced a transient reduction in viral load (at week
4), followed by a return to baseline viral load (.about.2,000
copies/ml) at week 15. Isolation of viral RNA and RT/PCR was used
to generate patient derived segments (PDS) that comprised viral
sequences coding for all of PR and aa 1-313 of RT from the baseline
and week 15 samples. The PDS were inserted into an indicator gene
viral vector to generate resistance test vectors designated
RTV-009089 and RTV-010835. These RTVs 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 comprised members of the classes
known as NRTIs zidovudine, lamivudine, stavudine, didanosine,
zalcitabine, and abacavir) NNRTIs (delavirdine, efavirenz and
nevirapine), and PRIs (amprenavir, indinavir, nelfinavir,
ritonavir, and saquinavir). An IC50 was determined for each
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 sample RTV-010835 in which
there was a significant decrease in efavirenz (270-fold),
nevirapine (>600-fold) and delavirdine (>250-fold)
susceptibility.
[0151] Determination of Genotype of Patient HIV Samples
[0152] RTV-009089, and RTV-010835 DNA were analyzed by ABI chain
terminator automated sequencing. The nucleotide sequence was
compared to the consensus sequence of a wild type clade B HIV-1
(HIV Sequence Database Los Alamos, N. Mex.). The genotype was
examined for sequences that are different from the control
sequence. In RTV-009089 mutations were noted at positions 49, 102,
122, 169, 178, 184, 195, 200, 211, 245, 250, 275, 276, 277, 286,
and 294 compared to the control sequence. In RTV-010835 additional
mutations appeared at codons 103 and 230. The mutation K103N has
been previously shown to be associated with resistance to the
NNRTIs nevirapine, delavirdine and efavirenz. We examined the
mutation, M230L, alone, and in combination with K103N, using site
directed mutagenesis and in vitro phenotypic susceptibility testing
to correlate the observed changes in genotype with phenotype.
[0153] Site Directed Mutagenesis is Used to Confirm the Role of
Specific Mutations in Phenotypic Susceptibility to Anti-Retroviral
Drugs in HIV
[0154] The M230L mutation was tested alone and is described in
Example 3 above. A resistance test vector containing the M230L
mutation and the K103N mutation (K103N/M230L-RTV) was constructed
and 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 103
and 230. We determined the pattern of phenotypic susceptibility to
the NNRTIs, delavirdine, nevirapine and efavirenz, in the
K103N/M230L-RTV. The K103N/M230L-RTV displayed high-level
reductions in susceptibility to nevirapine (>600-fold),
efavirenz (>470-fold) and delavirdine (>250-fold) compared to
a wild type control RTV. The K103N/M230L-RTV also showed
significant drug-dependent stimulation of viral replication in the
presence of nevirapine (.about.70%), efavirenz (.about.40%) and
delavirdine (.about.100%). A resistance test vector containing the
M230L, K103N and the M184V mutations (K103N/M230L/M184V-RTV) was
constructed and 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 103, 184 and 230. The addition of the M184V mutation onto
the K103N, M230L backbone results in a reversal of the
drug-dependent stimulation of viral replication.
Example 5
Using Resistance Test Vectors to Correlate Genotypes and Phenotypes
Associated with NNRTI Drug Susceptibility and Resistance and
Drug-Dependent Stimulation of Replication in HIV:
M230L+K101E+K103N+G190S
[0155] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient CCTG-1025 HIV Samples
[0156] Resistance test vectors were constructed as described in
Example 1 from virus samples obtained from an individual patient at
two separate time points over a 48-week period. This patient had
been previously treated with four NRTIs (zidovudine, stavudine,
didanosine, lamivudine, and a PRI nelfinavir. At the time the first
sample (designated AA2919) was obtained, the patient began taking a
new anti-viral regimen including two NRTIs (stavudine and abacavir)
and one PRI (indinavir). The viral load in this patient fell below
detectable and remained undetectable for an additional 48 weeks.
Efavirenz was added to the regimen after 9 weeks. A second sample
(00-2-012090) was obtained 48 weeks after the initiation of study
when the viral load increased to 455. Isolation of viral RNA and
RT/PCR was used to generate patient derived segments (PDS) that
comprised viral sequences coding for all of PR and aa 1-313 of RT
from all three samples. The PDS were inserted into an indicator
gene viral vector to generate resistance test vectors designated
RTV-2919 and RTV-012090. These RTVs 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 comprised members of the classes
known as NRTIs (zidovudine, lamivudine, stavudine, didanosine,
zalcitabine, and abacavir) NNRTIs (delavirdine, efavirenz and
nevirapine), and PRIs (amprenavir, indinavir, nelfinavir,
ritonavir, and saquinavir). An IC50 was determined for each
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-012090 in which there was a
significant decrease in susceptibility to efavirenz (>450-fold),
nevirapine (>600-fold) and delavirdine (144-fold) susceptibility
and significant drug-dependent stimulation of viral replication in
the presence of nevirapine (.about.80%), delavirdine (.about.30%)
and efavirenz (.about.40%).
[0157] Determination of Genotype of Patient HIV Samples
[0158] RTV-2919 and RTV-012090 DNA were analyzed by ABI chain
terminator automated sequencing. The nucleotide sequence was
compared to the consensus sequence of a wild type clade B HIV-1
(HIV Sequence Database Los Alamos, N. Mex.). The genotype was
examined for sequences that are different from the control
sequence. In RTV-2919 mutations were noted at HIV RT codons 67, 86,
102, 118, 122, 158, 162, 174, 177, 211, 214, 215, 272, 275, 276,
277, 278, 281, 286 and 293 compared to the control sequence. In
RTV-012090 additional mutations appeared at codons 101, 103, 190
and 230. The mutations K101E, K103N and G190S have been previously
shown to be associated with resistance to the NNRTIs nevirapine,
delavirdine and efavirenz. In this patient sample the appearance of
the mutations K101E, K103N, G190S and M230L are correlated with the
significant drug-dependent stimulation of viral replication in
response to all three NNRTIs.
Example 6
Using Resistance Test Vectors to Correlate Genotypes and Phenotypes
Associated with NNRTI Drug Susceptibility and Resistance and
Drug-Dependent Stimulation of Replication in HIV:
V241S+K103N+1135T
[0159] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient 011073 HIV Samples
[0160] A resistance test vector was constructed as described in
Example 1 from a virus sample obtained from patient 011073. The
prior and current treatment regimens are unknown. Isolation of
viral RNA and RT/PCR was used to generate a patient derived segment
(PDS) that comprised viral sequences coding for all of PR and aa
1-313 of RT. The PDS was inserted into an indicator gene viral
vector to generate a resistance test vector designated RTV-011073.
RTV-011073 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 classes known as NRTIs (zidovudine,
lamivudine, stavudine, didanosine, zalcitabine and abacavir),
NNRTIs (delavirdine, efavirenz and nevirapine), and PRIs
(amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir). An
IC50 was determined for each 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-011073 in which there was a sigificant
decrease in efavirenz (>450-fold), nevirapine (>600-fold) and
delavirdine (>250-fold) susceptibility and a significant
drug-dependent stimulation of viral replication in the presence of
nevirapine (.about.70%), delavirdine (.about.90%) and efavirenz
(.about.80%).
[0161] Determination of Genotype of Patient HIV Samples
[0162] RTV-011073 DNA was analyzed by ABI chain terminator
automated sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type clade B HIV-1 (HIV Sequence
Database Los Alamos, N. Mex.). The genotype was examined for
sequences that are different from the control sequence. In
RTV-011073 mutations were noted at HIV RT codons 20, 67, 69, 70,
102, 103, 118, I135T, 162, 211, 214, 218, 219, 241 and 277 compared
to the control sequence.
[0163] Site Directed Mutagenesis Used to Confirm the Role of
Specific Mutations in Phenotypic Susceptibility to Anti-Retroviral
Drugs in HIV
[0164] A resistance test vector containing all of the mutations
present in RTV-011073 except for the V241S and the T277K was
constructed and tested using the phenotypic assay described
earlier. The results were compared to those determined using the
parent RTV-011073. We determined the pattern of phenotypic
susceptibility to the NNRTIs, delavirdine, nevirapine and
efavirenz, in the reverted vector RTV-011073/241V/277T. The
RTV-011073/241V/277T displayed high-level reductions in
susceptibility to nevirapine (126-fold), efavirenz (54-fold) and
delavirdine (50-fold) compared to a wild type control RTV. The
RTV-011073/241V/277T showed no drug-dependent stimulation of viral
replication in the presence of any NNRTI tested. A resistance test
vector containing all of the mutations present in RTV-011073 except
for the V241S was constructed and tested using the phenotypic assay
described earlier. The results were compared to those determined
using the parent RTV-011073. We determined the pattern of
phenotypic susceptibility to the NNRTIs, delavirdine, nevirapine
and efavirenz, in the reverted vector RTV-011073/241V. The
RTV-011073/241V displayed high-level reductions in susceptibility
to nevirapine (146-fold), efavirenz (54-fold) and delavirdine
(59-fold) compared to a wild type control RTV. The RTV-011073/241V
showed no drug-dependent stimulation of viral replication in the
presence of any NNRTI tested. In this patient sample the
correlation of drug-dependent stimulation of viral replication with
the mutation at V241S is very striking. A resistance test vector
containing a single mutation at V241S (RTV-V241S) was constructed
and tested in the phenotypic assay described earlier. The results
were compared to those determined using a wild type control RTV. We
determined the pattern of phenotypic susceptibility to the NNRTIs,
delavirdine, nevirapine and efavirenz, in the RTV-V241S. The
RTV-V241S displayed small reductions in susceptibility to
nevirapine (9-fold), efavirenz (3-fold) and delavirdine (6-fold)
compared to a wild type control RTV. The RTV-V241S showed
drug-dependent stimulation of viral replication in the presence of
nevirapine (.about.30%), delavirdine (.about.25%) but no
drug-dependent stimulation of viral replication in the presence of
efavirenz.
Example Example 7
Using Resistance Test Vectors to Correlate Genotypes and Phenotypes
Associated with NNRTI Drug Susceptibility and Resistance and
Drug-Dependent Stimulation of Replication in HIV:
V241S+K101E+V106M+1135T- +E138A+G190A
[0165] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient 014451 HIV Samples
[0166] Resistance test vectors were constructed as described in
Example 1 from virus samples obtained from an individual patient at
two separate time points separated by a 32-week period. The prior
and current treatment regimens are unknown. Isolation of viral RNA
and RT/PCR was used to generate patient derived segments (PDS) that
comprised viral sequences coding for all of PR and aa 1-313 of RT
from all three samples. The PDS were inserted into an indicator
gene viral vector to generate resistance test vectors designated
RTV-014459 and RTV-014451. These RTVs 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 comprised members of the classes
known as NRTIs zidovudine, lamivudine, stavudine, didanosine,
zalcitabine, and abacavir) NNRTIs (delavirdine, efavirenz and
nevirapine), and PRIs (amprenavir, indinavir, nelfinavir,
ritonavir, and saquinavir). An IC50 was determined for each
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-014451 in which there was a
significant decrease in efavirenz (>450-fold), nevirapine
(>600-fold) and delavirdine (41-fold) susceptibility and a
significant drug-dependent stimulation of viral replication in the
presence of nevirapine (.about.100%), delavirdine (.about.90%) and
efavirenz (.about.70%).
[0167] Determination of Genotype of Patient HIV Samples
[0168] RTV-014459 and RTV-014459 DNA were analyzed by ABI chain
terminator automated sequencing. The nucleotide sequence was
compared to the consensus sequence of a wild type clade B HIV-1
(HIV Sequence Database Los Alamos, N. Mex.). The genotype was
examined for sequences that are different from the control
sequence. In RTV-014459 mutations were noted at HIV RT 35I, 41, 49,
83, 102, 123, 135, 138, 174, 177, 178, 184, 215, 241, 257, 261,
277, 286, 293 and 297 compared to the control NL4-3 sequence. In
RTV-014451 additional mutations appeared at codons 101, 106 and
190. The mutations K101E, V106M and G190A have been previously
shown to be associated with resistance to the NNRTIs nevirapine,
delavirdine and efavirenz, but not with stimulation of viral
replication. In this patient sample, the appearance of the
mutations K101E, V106M and G190A on the specific genetic backbone
present in this virus is correlated with the significant
drug-dependent stimulation of viral replication in response to all
three NNRTIs. The results shown in Example 6 above suggest that the
mutation V241I may contribute to the stimulation of viral
replication seen in this virus.
Example 8
Using Resistance Test Vectors to Correlate Genotypes and Phenotypes
Associated with NNRTI Drug Susceptibility and Resistance and
Drug-Dependent Stimulation of Replication in HIV: V245E in
combination with multiple other mutations
[0169] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient 005738 HIV Samples
[0170] A resistance test vector was constructed as described in
Example 1 from a virus sample obtained from patient 005738. The
prior and current treatment regimens are unknown. Isolation of
viral RNA and RT/PCR was used to generate a patient derived segment
(PDS) that comprised viral sequences coding for all of PR and aa
1-313 of RT from both samples. The PDS were inserted into an
indicator gene viral vector to generate a resistance test vector
designated RTV-005738. RTV-005738 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 classes known as
NRTIs zidovudine, lamivudine, stavudine, didanosine, zalcitabine,
and abacavir) NNRTIs (delavirdine, efavirenz and nevirapine), and
PRIs (amprenavir, indinavir, nelfinavir, ritonavir, and
saquinavir). An IC50 was determined for each 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 sample RTV-005738 in which there was a
moderate decrease in efavirenz (17-fold), nevirapine (>600-fold)
and delavirdine (63-fold) susceptibility and a significant
drug-dependent stimulation of viral replication in the presence of
nevirapine (.about.20%), delavirdine (.about.40%) and efavirenz
(.about.20%).
[0171] Determination of Genotype of Patient HIV Samples
[0172] RTV-005738 DNA was analyzed by ABI chain terminator
automated sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type lade B HIV-1 (HIV Sequence
Database Los Alamos, N. Mex.). The genotype was examined for
sequences that are different from the control sequence. In
RTV-005738 mutations were noted at positions D67N, K70R, R83K,
A98G, Q102K, K122P, 1135T, 1142V, C162S, K173Q, I178L, Y181C,
G196E, 1202V, T215F, D218E, K219Q, V245E, A272P, R277K, V293I and
E297Q compared to the control sequence.
[0173] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient 007130 HIV Samples
[0174] A resistance test vector was constructed as described in
Example 1 from a virus sample obtained from patient 007130. The
prior and current treatment regimens are unknown. Isolation of
viral RNA and RT/PCR was used to generate a patient derived segment
(PDS) that comprised viral sequences coding for all of PR and aa
1-313 of RT. The PDS were inserted into an indicator gene viral
vector to generate a resistance test vector designated RTV-007130.
RTV-007130 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 classes known as NRTIs zidovudine,
lamivudine, stavudine, didanosine, zalcitabine, and abacavir)
NNRTIs (delavirdine, efavirenz and nevirapine), and PRIs
(amprenavir, 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 RTV-007130 in which there was a significant
decrease in efavirenz (>450-fold), nevirapine (>600-fold) and
delavirdine (>250-fold) susceptibility and a significant
drug-dependent stimulation of viral replication in the presence of
nevirapine (.about.50%), delavirdine (.about.30%) and efavirenz
(.about.40%).
[0175] Determination of Genotype of Patient HIV Samples
[0176] RTV-007130 DNA was analyzed by ABI chain terminator
automated sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type clade B HIV-1 (HIV Sequence
Database Los Alamos, N. Mex.). The genotype was examined for
sequences that are different from the control sequence. In
RTV-007130 mutations were noted at codons 67, 101, 102, 103, 122,
135, 162, 174, 184, 190, 208, 221, 245, 272, 277, 283, 293 compared
to the control sequence.
Example 9
Using Resistance Test Vectors to Correlate Genotypes and Phenotypes
Associated with NNRTI Drug Susceptibility and Resistance and
Drug-Dependent Stimulation of Replication in HIV: V245T in
combination with Multiple Other Mutations
[0177] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient 006782 HIV Samples
[0178] A resistance test vector was constructed as described in
Example 1 from a virus sample obtained from patient 006782. The
prior and current treatment regimens are unknown. Isolation of
viral RNA and RT/PCR was used to generate a patient derived segment
(PDS) that comprised viral sequences coding for all of PR and aa
1-313 of RT. The PDS were inserted into an indicator gene viral
vector to generate a resistance test vector designated RTV-006782.
RTV-006782 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 classes known as NRTIs zidovudine,
lamivudine, stavudine, didanosine, zalcitabine, and abacavir)
NNRTIs (delavirdine, efavirenz and nevirapine), and PRIs
(amprenavir, 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 RTV-007130 in which there was a small decrease
in efavirenz (5-fold), nevirapine. (8-fold) and delavirdine
(18.5-fold) susceptibility and a significant drug-dependent
stimulation of viral replication in the presence of nevirapine
(.about.60%), delavirdine (.about.70%) and efavirenz
(.about.25%).
[0179] Determination of Genotype of Patient HIV Samples
[0180] RTV-006782 DNA was analyzed by ABI chain terminator
automated sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type clade B HIV-1 (HIV Sequence
Database Los Alamos, N. Mex.). The genotype was examined for
sequences that are different from the control sequence. In
RTV-006782 mutations were noted at HIV RT codons Q102K, D123E,
1135T, E138A, C162S, G196E, 1202V, V245T, R277K, T286A, V293I,
P294T and E297K compared to the control sequence.
[0181] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient 012123 HIV Samples
[0182] A resistance test vector was constructed as described in
Example 1 from a virus sample obtained from patient 012123. The
prior and current treatment regimens are unknown. Isolation of
viral RNA and RT/PCR was used to generate a patient derived segment
(PDS) that comprised viral sequences coding for all of PR and aa
1-313 of RT. The PDS were inserted into an indicator gene viral
vector to generate a resistance test vector designated RTV-012123.
RTV-012123 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 classes known as NRTIs zidovudine,
lamivudine, stavudine, didanosine, zalcitabine, and abacavir)
NNRTIs (delavirdine, efavirenz and nevirapine), and PRIs
(amprenavir, 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 RTV-012123 in which there was a significant
decrease in efavirenz (>450-fold), nevirapine (>600-fold) and
delavirdine (>250-fold) susceptibility, and a significant
drug-dependent stimulation of viral replication in the presence of
nevirapine (.about.20%), delavirdine (.about.80%) and efavirenz
(.about.70%).
[0183] Determination of Genotype of Patient HIV Samples
[0184] RTV-012123 DNA was analyzed by ABI chain terminator
automated sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type clade B HIV-1 (HIV Sequence
Database Los Alamos, N. Mex.). The genotype was examined for
sequences that are different from the control sequence. In
RTV-012123 mutations were noted at HIV RT codons 20, 39, 41, 44,
60, 67, 98, 102, 103, 118, 122, 135, 142, 162, 173, 181, 190, 196,
202, 210, 215, 221, 245, 272, 277, 293 and 297 compared to the
control sequence.
[0185] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient 014397 HIV Samples
[0186] A resistance test vector was constructed as described in
Example 1 from a virus sample obtained from patient 014397. The
prior and current treatment regimens are unknown. Isolation of
viral RNA and RT/PCR was used to generate a patient derived segment
(PDS) that comprised viral sequences coding for all of PR and aa
1-313 of RT. The PDS were inserted into an indicator gene viral
vector to generate a resistance test vector designated RTV-014397.
RTV-014397 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 classes known as NRTIs zidovudine,
lamivudine, stavudine, didanosine, zalcitabine, and abacavir)
NNRTIs (delavirdine, efavirenz and nevirapine), and PRIs
(amprenavir, 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 RTV-014397 in which there was a significant
decrease in efavirenz (>450-fold), nevirapine (>600-fold) and
delavirdine (>250-fold) susceptibility and a significant
drug-dependent stimulation of viral replication in the presence of
nevirapine (.about.100%), delavirdine (.about.100%) and efavirenz
(.about.70%).
[0187] Determination of Genotype of Patient HIV Samples
[0188] RTV-014397 DNA was analyzed by ABI chain terminator
automated sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type clade B HIV-1 (HIV Sequence
Database Los Alamos, N. Mex.). The genotype was examined for
sequences that are different from the control sequence. In
RTV-014397 mutations were noted at HIV RT codons 31, 102, 103, 123,
162, 166, 177, 211, 215, 228, 245, 272 and 277 compared to the
control sequence.
Example 10
Using Resistance Test Vectors to Correlate Genotypes and Phenotypes
Associated with NNRTI Drug Susceptibility and Resistance and
Drug-Dependent Stimulation of Replication in HIV: V245M in
combination with Multiple Other Mutations
[0189] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient 013415 HIV Samples
[0190] A resistance test vector was constructed as described in
Example 1 from a virus sample obtained from patient 013415. The
prior and current treatment regimens are unknown. Isolation of
viral RNA and RT/PCR was used to generate a patient derived segment
(PDS) that comprised viral sequences coding for all of PR and aa
1-313 of RT. The PDS were inserted into an indicator gene viral
vector to generate a resistance test vector designated RTV-013415.
RTV-013415 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 classes known as NRTIs zidovudine,
lamivudine, stavudine, didanosine, zalcitabine, and abacavir)
NNRTIs (delavirdine, efavirenz and nevirapine), and PRIs
(amprenavir, 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 RTV-013415 in which there was a significant
decrease in efavirenz (>450-fold), nevirapine (>600-fold) and
delavirdine (>250-fold) susceptibility and a significant
drug-dependent stimulation of viral replication in the presence of
nevirapine (.about.20%), delavirdine (.about.30%) and efavirenz
(.about.20%).
[0191] Determination of Genotype of Patient HIV Samples
[0192] RTV-013415 DNA was analyzed by ABI chain terminator
automated sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type clade B HIV-1 (HIV Sequence
Database Los Alamos, N. Mex.). The genotype was examined for
sequences that are different from the control sequence. In
RTV-013415 mutations were noted at HIV RT codons 35, 102, 103, 122,
123, 135, 162, 177, 200, 207, 211, 225, 245, 264, 277 and 290
compared to the control sequence.
Example 11
Using Resistance Test Vectors to Correlate Genotypes and Phenotypes
Associated with NNRTI Drug Susceptibility and Resistance and
Drug-Dependent Stimulation of Replication in HIV:
K103N+V245E+1270M
[0193] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient 10829 HIV Samples
[0194] A resistance test vector was constructed as described in
Example 1 from a virus sample obtained from patient 10829. The
prior and current treatment regimens are unknown. Isolation of
viral RNA and RT/PCR was used to generate a patient derived segment
(PDS) that comprised viral sequences coding for all of PR and aa
1-313 of RT. The PDS were inserted into an indicator gene viral
vector to generate a resistance test vector designated RTV-010829.
RTV-010829 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 classes known as NRTIs zidovudine,
lamivudine, stavudine, didanosine, zalcitabine, and abacavir)
NNRTIs (delavirdine, efavirenz and nevirapine), and PRIs
(amprenavir, indinavir, nelfinavir, ritonavir, and saauinavir). 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-011073 in which there was a sigificant
decrease in efavirenz (>450-fold), nevirapine (>600-fold) and
delavirdine (>250-fold) susceptibility and a significant
drug-dependent stimulation of viral replication in the presence of
nevirapine (.about.100%), delavirdine (.about.100%) and efavirenz
(.about.80%).
[0195] Determination of Genotype of Patient HIV Samples
[0196] RTV-010829 DNA was analyzed by ABI chain terminator
automated sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type lade B HIV-1 (HIV Sequence
Database Los Alamos, N. Mex.). The genotype was examined for
sequences that are different from the control sequence. In
RTV-010829 mutations were noted at HIV codons 31, 35, 90, 102, 103,
122, 162, 196, 211, 214, 215, 228, 245, 270, 276, 277, 292, 293 and
297 compared to the control sequence.
[0197] Site Directed Revese-Mutagenesis Used to Confirm the Role of
Specific Mutations in Phenotypic Susceptibility to Anti-Retroviral
Drugs in HIV
[0198] A resistance test vector containing all of the mutations
present in RTV-010829 except for the V245E mutation
(RTV-010829/245V) was constructed and tested using the phenotypic
assay described earlier. The results were compared to those
determined using the parent RTV-010829. We determined the pattern
of phenotypic susceptibility to the NNRTIs, delavirdine, nevirapine
and efavirenz, in the reverted vector RTV-010829/245V. The
RTV-010829/245V displayed high-level reductions in susceptibility
to nevirapine (505-fold), efavirenz (189-fold) and delavirdine
(182-fold) compared to a wild type control RTV. The RTV-010829/245V
showed drug-dependent stimulation of viral replication in the
presence of nevirapine (.about.80%), efavirenz (.about.60%) and
delavirdine (.about.60%). A resistance test vector containing all
of the mutations present in RTV-010829 except for the 1270M
(RTV-010829/270I) was constructed and tested using the phenotypic
assay described earlier. The results were compared to those
determined using the parent RTV-010829. We determined the pattern
of phenotypic susceptibility to the NNRTIs, delavirdine, nevirapine
and efavirenz, in the reverted vector RTV-010829/270I. The
RTV-010829/270I displayed high-level reductions in susceptibility
to nevirapine (341-fold), efavirenz (145-fold) and delavirdine
(247-fold) compared to a wild type control RTV. The RTV-010829/245V
showed drug-dependent stimulation of viral replication in the
presence of nevirapine (.about.30%), efavirenz (.about.20%) and
delavirdine (.about.40%). A resistance test vector containing all
of the mutations present in RTV-010829 except for V245E, I270M,
V276I, R277K, V292I, V293I and E297K (RTV-010829/245V-297E) was
constructed and tested using the phenotypic assay described
earlier. The results were compared to those determined using the
parent RTV-010829. We determined the pattern of phenotypic
susceptibility to the NNRTIs, delavirdine, nevirapine and
efavirenz, in the reverted vector RTV-010829/245V-297E. The
RTV-010829/245V-297E displayed high-level reductions in
susceptibility to nevirapine (233-fold), efavirenz (76-fold) and
delavirdine (198-fold) compared to a wild type control RTV. The
RTV-010829/245V-297E showed no drug-dependent stimulation of viral
replication in the presence of nevirapine, efavirenz, or
delavirdine. In this patient sample, drug-dependent stimulation of
viral replication is modulated by the presence of the mutations
V245E and 1270M. Drug-dependent stimulation of viral replication
can be completely abrogated by reversion of mutations V245E, I270M,
V276I, R277K, V292I, V293I and E297K.
Example 12
Using Resistance Test Vectors to Correlate Genotypes and Phenotypes
Associated with NNRTI Drug Susceptibility and Resistance and
Drug-Dependent Stimulation of Replication in HIV:
K103N+1135T+1270S
[0199] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient 013522 HIV Samples
[0200] A resistance test vector was constructed as described in
Example 1 from a virus sample obtained from patient 013522. The
prior and current treatment regimens are unknown. Isolation of
viral RNA and RT/PCR was used to generate a patient derived segment
(PDS) that comprised viral sequences coding for all of PR and aa
1-313 of RT from all three samples. The PDS were inserted into an
indicator gene viral vector to generate a resistance test vector
designated RTV-013522. RTV-013522 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 classes known as
NRTIs (zidovudine, lamivudine, stavudine, didanosine, zalcitabine
and abacavir), NNRTIs (delavirdine, efavirenz and nevirapine), and
PRIs (amprenavir, indinavir, nelfinavir, ritonavir, and
saquinavir). An IC50 was determined for each 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-013522 in which there was a sigificant
decrease in efavirenz (>450-fold), nevirapine (>600-fold) and
delavirdine (>250-fold) susceptibility and a significant
drug-dependent stimulation of viral replication in the presence of
nevirapine (.about.90%), delavirdine (.about.110%) and efavirenz
(.about.100%).
[0201] Determination of Genotype of Patient HIV Samples
[0202] RTV-013522 DNA was analyzed by ABI chain terminator
automated sequencing. The nucleotide sequence was compared to the
consensus sequence of a wild type clade B HIV-1 (HIV Sequence
Database Los Alamos, N. Mex.). The genotype was examined for
sequences that are different from the control sequence. In
RTV-013522 mutations were noted at HIV RT codons 35, 60, 68, 102,
103, 135, 162, K166R, 184, 196, 204, 211, 248, 250, 270, 286, 293
and 297 compared to the control sequence.
[0203] Site Directed Revese-Mutagenesis is Used to Confirm the Role
of Specific Mutations in Phenotypic Susceptibility to
Anti-Retroviral Drugs in HIV
[0204] A resistance test vector containing all of the mutations
present in RTV-010829 except for the 1270S (RTV-013522/270I) was
constructed and tested using the phenotypic assay described
earlier. The results were compared to those determined using the
parent RTV-013522. We determined the pattern of phenotypic
susceptibility to the NNRTIs, delavirdine, nevirapine and
efavirenz, in the reverted vector RTV-013522/270I. The
RTV-013522/270I displayed moderate reductions in nevirapine
(14-fold) and efavirenz (8-fold) susceptibility and a significant
reduction in delavirdine (40-fold) susceptibility compared to a
wild type control RTV. The RTV-013522/270I showed no drug-dependent
stimulation of viral replication in the presence of any of the
NNRTI tested. In this patient sample the correlation of
drug-dependent stimulation of viral replication with the mutation
at 1270S is very striking. A resistance test vector containing a
single mutation at 1270S (RTV-1270S) was constructed and tested in
the phenotypic assay described earlier. The results were compared
to those determined using a wild type control RTV. We determined
the pattern of phenotypic susceptibility to the NNRTIs,
delavirdine, nevirapine and efavirenz, in the RTV-V241S. The
RTV-1270S exhibited small reductions in susceptibility to
nevirapine (15-fold), efavirenz (4-fold) and delavirdine (13-fold)
compared to a wild type control RTV. The RTV-1270S showed
drug-dependent stimulation of viral replication in the presence of
nevirapine (.about.60%), delavirdine (.about.60%) and efavirenz
(.about.40%).
Example 13
Site Directed Mutagenesis Used to Confirm the Role of Specific
Mutations in Phenotypic Susceptibility to Anti-Retroviral Drugs in
HIV
[0205] V245E
[0206] A resistance test vector containing a single mutation,
V245E, (RTV-V245E) was constructed and tested in the phenotypic
assay described earlier. The results were compared to those
determined using a genetically defined resistance test vector that
was wild type at position 245. We determined the pattern of
phehotypic susceptibility to the NNRTIs, delavirdine, nevirapine
and efavirenz, in RTV-V245E. The RTV-V245E displayed no reductions
in susceptibility to nevirapine, efavirenz or delavirdine compared
to a wild type control RTV. The RTV-V245E showed no drug-dependent
stimulation of viral replication in the presence of nevirapine,
efavirenz, or delavirdine.
[0207] V245T
[0208] A resistance test vector containing a single mutation,
V245T, (RTV-V245T) was constructed and tested in the phenotypic
assay described earlier. The results were compared to those
determined using a genetically defined resistance test vector that
was wild type at position 245. We determined the pattern of
phenotypic susceptibility to the NNRTIs, delavirdine, nevirapine
and efavirenz, in RTV-V245E. The RTV-V245T displayed no reductions
in susceptibility to nevirapine, efavirenz or delavirdine compared
to a wild type control RTV. The RTV-V245T showed no drug-dependent
stimulation of viral replication in the presence of nevirapine,
efavirenz, or delavirdine.
[0209] I135T
[0210] A resistance test vector containing a single mutation,
I135T, (RTV-I135T) was constructed and tested in the phenotypic
assay described earlier. The results were compared to those
determined using a genetically defined resistance test vector that
was wild type at position 135. We determined the pattern of
phenotypic susceptibility to the NNRTIs, delavirdine, nevirapine
and efavirenz, in RTV-I135T. The RTV-I135T displayed no reductions
in susceptibility to nevirapine, efavirenz or delavirdine compared
to a wild type control RTV. The RTV-I135T showed no drug-dependent
stimulation of viral replication in the presence of nevirapine,
efavirenz, or delavirdine.
[0211] K103N
[0212] A resistance test vector containing a single mutation,
K103N, (RTV-K103N) was constructed and tested in the phenotypic
assay described earlier. The results were compared to those
determined using a genetically defined resistance test vector that
was wild type at positions 103. We determined the pattern of
phenotypic susceptibility to the NNRTIs, delavirdine, nevirapine
and efavirenz, in RTV-K103N. The RTV-K103N displayed reductions in
susceptibility to nevirapine (67-fold), efavirenz (29-fold) and
delavirdine (87-fold) compared to a wild type control RTV. The
RTV-K103N exhibited no drug-dependent stimulation of viral
replication in the presence of nevirapine, efavirenz, or
delavirdine.
[0213] K103N+I135T
[0214] A resistance test vector containing two mutations, K103N and
I135T (RTV-K103N/I135T) was constructed and tested in the
phenotypic assay described earlier. The results were compared to
those determined using a genetically defined resistance test vector
that was wild type at position 103 and 135. We determined the
pattern of phenotypic susceptibility to the NNRTIs, delavirdine,
nevirapine and efavirenz, in RTV-K103N/I135T. The RTV-K103N/I135T
displayed reductions in susceptibility to nevirapine (171-fold),
efavirenz (80-fold) and delavirdine (121-fold) compared to a wild
type control RTV. The RTV-K103N/I135T exhibited no drug-dependent
stimulation of viral replication in the presence of nevirapine,
efavirenz, or delavirdine.
[0215] I135T+V245E
[0216] A resistance test vector containing two mutations, I135T and
V245E (RTV-I135T/V245E) was constructed and tested in the
phenotypic assay described earlier. The results were compared to
those determined using a genetically defined resistance test vector
that was wild type at positions 135 and 245. We determined the
pattern of phenotypic susceptibility to the NNRTIs, delavirdine,
nevirapine and efavirenz, in RTV-I135T/V245E. The RTV-I135T/V245E
displayed minor reductions in susceptibility to nevirapine
(2.5-fold), efavirenz (2-fold) and delavirdine (2-fold) compared to
a wild type control RTV. The RTV-I135T/V245E exhibited no
drug-dependent stimulation of viral replication in the presence of
nevirapine, efavirenz, or delavirdine.
[0217] I135T+V245T
[0218] A resistance test vector containing two mutations, I135T and
V245T (RTV-I135T/V245T) was constructed and tested in the
phenotypic assay described earlier. The results were compared to
those determined using a genetically defined resistance test vector
that was wild type at positions 135 and 245. We determined the
pattern of phenotypic susceptibility to the NNRTIs, delavirdine,
nevirapine and efavirenz, in RTV-I135T/V245T. The RTV-I135T/V245T
displayed minor reductions in susceptibility to nevirapine
(4-fold), efavirenz (2.4-fold) and delavirdine (2.5-fold) compared
to a wild type control RTV. The RTV-I135T/V245T exhibited no
drug-dependent stimulation of viral replication in the presence of
efavirenz and only minor stimulation of viral replication in the
presence of nevirapine (.about.10%) or delavirdine
(.about.10%).
[0219] K103N+1135T+V245E
[0220] A resistance test vector containing three mutations, K103N,
I135T and V245E (RTV-K103N/I135T/V245E) was constructed and tested
in the phenotypic assay described earlier. The results were
compared to those determined using a genetically defined resistance
test vector that was wild type at positions 103, 135 and 245. We
determined the pattern of phenotypic susceptibility to the NNRTIs,
delavirdine, nevirapine and efavirenz, in RTV-K103N. The
RTV-K103N/I135T/V245E displayed reductions in susceptibility to
nevirapine (244-fold), efavirenz (93-fold) and delavirdine
(169-fold) compared to a wild type control RTV. The
RTV-K103N/I135T/V245E showed no drug-dependent stimulation of viral
replication in the presence of efavirenz but showed moderate levels
of viral stimulation of replication in the presence of nevirapine
(.about.15%) and delavirdine (.about.20%).
[0221] K103N+I135T+V245T
[0222] A resistance test vector containing three mutations, K103N,
I135T and V245T (RTV-K103N/I135T/V245T) was constructed and tested
in the phenotypic assay described earlier. The results were
compared to those determined using a genetically defined resistance
test vector that was wild type at positions 103, 135 and 245. We
determined the pattern of phenotypic susceptibility to the NNRTIs,
delavirdine, nevirapine and efavirenz, in RTV-K103N. The
RTV-K103N/I135T/V245T displayed significant reductions in
susceptibility to nevirapine (594-fold), efavirenz (174-fold) and
delavirdine (>250-fold) compared to a wild type control RTV. The
RTV-K103N/I135T/V245T showed drug-dependent stimulation of viral
replication in the presence of efavirenz (.about.25%) nevirapine
(.about.40%) and delavirdine (.about.50%).
Example 14
Using Resistance Test Vectors to Correlate Genotypes and Phenotypes
Associated with NNRTI Drug Susceptibility and Resistance and
Drug-Dependent Stimulation of Replication in HIV: Multiple
Mutations Can Modulate Both Susceptibility and Stimulation
Profiles
[0223] Preparation of Resistant Test Vectors and Phenotypic
Analysis of Patient 97-309 HIV Samples
[0224] Resistance test vectors were constructed as described in
Example 1 from virus samples obtained from an individual patient at
thirteen separate time points over a 21/2-year period. This patient
had been previously treated with two NRTIs (zidovudine and
lamivudine) AZT, 3TC (NRTIs), and a PRI (indinavir) for a period of
approximately 2 years at the time the first (97-309) and second
(98-754) samples were obtained. The patient began taking a new
anti-viral regimen including an NRTI (stavudine), an NNRTI
(nevirapine), and two PRIs (nelfinavir and saquinavir) at the time
the third sample (98-1032) was obtained. Additional samples were
obtained 4, 5 and 10 weeks later (00-2-011658, 00-2-011659 and
98-1033, respectively). The patient then stopped taking nevirapine,
and additional samples were obtained after 12, 16, 24 and 36 weeks
(99-2-008973, 98-757, 99-2-8980 and 98-1080, respectively). The
patient then switched therapy again to zidovudine, lamivudine and
indinavir, and additional samples were obtained after 5 months
(AA1264) and 10 months (99-2-006174). Viral load measurements,
patient therapy, and dates of draw are shown in FIG. 14. Following
initiation of d4T, nevirapine, nelfinavir and saquinavir, the
patient experienced a transient reduction in viral load followed by
a return to baseline viral load (.about.1,200,000 copies/ml).
Removal of nevirapine from the regimen led to a reduction in viral
load to <300,000 copies/ml (a 4-fold reduction). Isolation of
viral RNA and RT/PCR was used to generate patient derived segments
that comprised viral sequences coding for all of PR and aa 1-313 of
RT from all twelve samples. The PDS were inserted into an indicator
gene viral vector to generate resistance test vectors designated
RTV-309, RTV-754, RTV-1032, RTV-011658, RTV-011659, RTV-1033,
RTV-008973, RTV-757, RTV-008980, RTV1080, RTV1264 and RTV-006174.
These RTVs were 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 classes known as NRTIs zidovudine,
lamivudine, stavudine, didanosine, zalcitabine, and abacavir)
NNRTIs (delavirdine, efavirenz and nevirapine), and PRIs
(amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir). An
IC50 was determined for each 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, RTV-008973, RTV-757, RTV-008980 and
RTV-1080 in which there were reductions in nevirapine
(>600-fold), delavirdine (from 3 to 18-fold) and efavirenz (from
16 to 300 fold) susceptibility. Furthermore, RTV-1033, RTV-008973,
RTV-757, RTV-008980 and RTV-1080 showed drug-dependent stimulation
of viral replication in response to nevirapine (40 to 250%)
delavirdine (20 to 180%) and efavirenz (30 to 170%). The actual
fold change in susceptibility and the percent stimulation of viral
replication for each RTV is shown in FIG. 14a.
[0225] Determination of Genotype of Patient HIV Samples
[0226] RTV-309, RTV-754, RTV-1032, RTV-011658, RTV-011659,
RTV-1033, RTV-008973, RTV-757, RTV-008980, RTV1080, RTV1264 and
RTV-006174 DNA were analyzed by ABI chain terminator automated
sequencing. The nucleotide sequence was compared to the consensus
sequence of a wild type clade B HIV-1 (HIV Sequence Database Los
Alamos, N. Mex.). The genotype was examined for sequences that are
different from the control sequence. Mutations were observed at the
positions listed in FIG. 14b.
[0227] Site Directed Mutagenesis is Used to Confirm the Role of
Specific Mutations in Phenotypic Susceptibility to Anti-Retroviral
Drugs in HIV
[0228] RTV-1033 displayed the most dramatic drug-dependent
stimulation of viral replication of all of the samples tested from
this patient. A single clone (RTV-1033-3) was obtained from the
patient-derived RTV pool that had mutations and phenotypic patterns
of NNRTI susceptibility and drug-dependent stimulation of viral
replication characteristic of the RTV-1033 pool. The RTV-1033-3 had
the following mutations present in the revere transcriptase: V35I,
D67N, T69D, K70R, V106A, D123G, V189L, T200A, I202T, H208Y, R211K,
T215F, D218E, K219Q, H221Y, L228H, L283I, R284K, T286A, V293I and
E297K. RTV-1033-3 showed dramatic reductions in susceptibility to
delavirdine (27-fold), efavirenz (>450-fold) and nevirapine
(>600-fold) and dramatic drug-dependent stimulation of viral
replication in the presence of delavirdine (190%), efavirenz (180%)
and nevirapine (200%).
[0229] V106A
[0230] A resistance test vector containing all of the mutations
present in RTV-1033-3 except for V106A (RTV-1033-3/106V) was
constructed and tested using the phenotypic assay described
earlier. The results were compared to those determined using the
parent RTV-1033-3. We determined the pattern of phenotypic
susceptibility to the NNRTIs, delavirdine, nevirapine and
efavirenz, in the reverted vector, RTV-1033-3/106V. The
RTV-1033-3/106V displayed a moderate reduction in nevirapine
(9-fold) susceptibility, no reduction in efavirenz susceptibility
and a significant increase in delavirdine (20-fold) susceptibility
(hyper-susceptibility) compared to a wild type control RTV. The
RTV-1033-3/106V showed no drug-dependent stimulation of viral
replication in the presence of delavirdine but showed low levels of
drug-dependent stimulation of viral replication in the presence of
efavirenz (.about.20%) and nevirapine (.about.40%).
[0231] F227L
[0232] A resistance test vector containing all of the mutations
present in RTV-1033-3 except for F227L (RTV-1033-3/227F) was
constructed and tested using the phenotypic assay described
earlier. The results were compared to those determined using the
parent RTV-1033-3. We determined the pattern of phenotypic
susceptibility to the NNRTIs, delavirdine, nevirapine and
efavirenz, in the reverted vector, RTV-1033-3/227F. The
RTV-1033-3/227F displayed significant reductions in nevirapine
(>600-fold), efavirenz (18-fold) and delavirdine (72-fold)
susceptibility compared to a wild type control RTV. The
RTV-1033-3/227F showed drug-dependent stimulation of viral
replication in the presence of delavirdine (.about.100%), efavirenz
(.about.100%) and nevirapine (.about.120%).
[0233] V106A and F227L
[0234] A resistance test vector containing all of the mutations
present in RTV-1033-3 except for V106A and F227L
(RTV-1033-3/106V/227F) was constructed and tested using the
phenotypic assay described earlier. The results were compared to
those determined using the parent RTV-1033-3. We determined the
pattern of phenotypic susceptibility to the NNRTIs, delavirdine,
nevirapine and efavirenz, in the reverted vector,
RTV-1033-3/106V/227F. The RTV-1033-3/106V/227F displayed moderate
reductions in nevirapine (13-fold), efavirenz (6-fold) and
delavirdine (6-fold) susceptibility compared to a wild type control
RTV. The RTV-1033-3/106V/227F showed drug-dependent stimulation of
viral replication in the presence of delavirdine (.about.80%),
efavirenz (.about.80%) and nevirapine (.about.110%).
[0235] V106A, V189L and F227L
[0236] A resistance test vector containing all of the mutations
present in RTV-1033-3 except for V106A, V189L and F227L
(RTV-1033-3/106V/189V/227F) was constructed and tested using the
phenotypic assay described earlier. The results were compared to
those determined using the parent RTV-1033-3. We determined the
pattern of phenotypic susceptibility to the NNRTIs, delavirdine,
nevirapine and efavirenz, in the reverted vector,
RTV-1033-3/106V/189V/227F. The RTV-1033-3/106V/189V/227F displayed
small reductions in nevirapine (2.8-fold), efavirenz (2-fold) and
delavirdine (2.7-fold) susceptibility compared to a wild type
control RTV. The RTV-1033-3/106V/189V/227F showed drug-dependent
stimulation of viral replication in the presence of delavirdine
(.about.35%), efavirenz (.about.30%) and nevirapine
(.about.40%).
[0237] V106A, F227L and T286A
[0238] A resistance test vector containing all of the mutations
present in RTV-1033-3 except for V106A, F227L and T286A
(RTV-1033-3/106V/227F/286T) was constructed and tested using the
phenotypic assay described earlier. The results were compared to
those determined using the parent RTV-1033-3. We determined the
pattern of phenotypic susceptibility to the NNRTIs, delavirdine,
nevirapine and efavirenz, in the reverted vector,
RTV-1033-3/106V/227F/286T. The RTV-1033-3/106V/227F/286T displayed
moderate reductions in nevirapine (6-fold), efavirenz (5-fold) and
delavirdine (3-fold) susceptibility compared to a wild type control
RTV. The RTV-1033-3/106V/227F/286T showed drug-dependent
stimulation of viral replication in the presence of delavirdine
(.about.60%), efavirenz (.about.50%) and nevirapine
(.about.70%).
[0239] L283I. R284K and T286A
[0240] A resistance test vector containing all of the mutations
present in RTV-1033-3 except for L283I, R284K and T286A
(RTV-1033-3/283L/284R/286T) was constructed and tested using the
phenotypic assay described earlier. The results were compared to
those determined using the parent RTV-1033-3. We determined the
pattern of phenotypic susceptibility to the NNRTIs, delavirdine,
nevirapine and efavirenz, in the reverted vector,
RTV-1033-3/283L/284R/286T. The RTV-1033-3/283L/284R/286T displayed
significant reductions in nevirapine (>600-fold), efavirenz
(85-fold) and delavirdine (7-fold) susceptibility compared to a
wild type control RTV. The RTV-1033-3/283L/284R/286T showed
drug-dependent stimulation of viral replication in the presence of
delavirdine (.about.40%), efavirenz (.about.30%) and nevirapine
(.about.80%).
[0241] L283I. R284K, T286A. V293I and E297K
[0242] A resistance test vector containing all of the mutations
present in RTV-1033-3 except for L283I, R284K, T286A, V293I and
E297K (RTV-1033-3/283L/284R/286T/293V/297E) was constructed and
tested using the phenotypic assay described earlier. The results
were compared to those determined using the parent RTV-1033-3. We
determined the pattern of phenotypic susceptibility to the NNRTIs,
delavirdine, nevirapine and efavirenz, in the reverted vector,
RTV-1033-3/283L/284R/286T/293V/297E. The
RTV-1033-3/283L/284R/286T/293V/297E displayed significant
reductions in nevirapine (144-fold), efavirenz (5-fold) and
delavirdine (16-fold) susceptibility compared to a wild type
control RTV. The RTV-1033-3/283L/284R/286T/293V/297E showed no
drug-dependent stimulation of viral replication in the presence of
delavirdine, efavirenz or nevirapine.
[0243] Y181C
[0244] A resistance test vector containing all of the mutations
present in RTV-1033-3, in addition to Y181C (RTV-1033-3/181C) was
constructed and tested using the phenotypic assay described
earlier. The results were compared to those determined using the
parent RTV-1033-3. We determined the pattern of phenotypic
susceptibility to the NNRTIs, delavirdine, nevirapine and
efavirenz, in the mutated vector, RTV-1033-3/181C. The
RTV-1033-3/181C displayed a significant reduction in nevirapine
(>600-fold), efavirenz (>450-fold) and delavirdine
(>250-fold) susceptibility compared to a wild type control RTV.
The RTV-1033-3/181C showed no drug-dependent stimulation of viral
replication in the presence of nevirapine but showed drug-dependent
stimulation of viral replication in the presence of efavirenz
(.about.100%) and nevirapine (.about.100%).
[0245] M184V
[0246] A resistance test vector containing all of the mutations
present in RTV-1033-3 in addition to M184V (RTV-1033-3/184V) was
constructed and tested using the phenotypic assay described
earlier. The results were compared to those determined using the
parent RTV-1033-3. We determined the pattern of phenotypic
susceptibility to the NNRTIs, delavirdine, nevirapine and
efavirenz, in the mutated vector, RTV-1033-3/184V. The
RTV-1033-3/184V displayed a significant reduction in nevirapine
(>600-fold), efavirenz (28-fold) and a moderate reduction in
delavirdine (5-fold) susceptibility compared to a wild type control
RTV. The RTV-1033-3/184V showed drug-dependent stimulation of viral
replication in the presence of delavirdine (100%), efavirenz
(.about.80%) and nevirapine (.about.100%).
[0247] Clone 309-1
[0248] RTV-309 was derived from a virus sample from patient 1033
before the patient had received any NNRTI inhibitors. RTV-309
exhibited a wild type pattern of susceptibility to delavirdine and
efavirenz and a small reduction in susceptibility to nevirapine
(3-fold) and no drug-dependent stimulation of viral replication in
the presence of NNRTIs. A single clone (RTV-309-3) was obtained
from the patient-derived RTV pool that had mutations and phenotypic
patterns of NNRTI susceptibility and drug-dependent stimulation of
viral replication characteristic of the RTV-1033 pool. The
RTV-309-3 had the following mutations present in the revere
transcriptase: V35I, D67N, T69D, K70R, L109I, M184V, V189L, T200A,
I202T, H208Y, R211K, T215F, D218E, K219Q, H221Y, L228H, L283I,
R284K, T286A and E297K. RTV-309-3 exhibited a wild type pattern of
susceptibility to delavirdine and efavirenz and a small reduction
in susceptibility to nevirapine (3-fold) and no drug-dependent
stimulation of viral replication in the presence of NNRTIs.
[0249] M184V
[0250] A resistance test vector containing all of the mutations
present in RTV-309-3 except for the M184V (RTV-309-3/184M) was
constructed and tested using the phenotypic assay described
earlier. The results were compared to those determined using the
parent RTV-309-3. We determined the pattern of phenotypic
susceptibility to the NNRTIs, delavirdine, nevirapine and
efavirenz, in the reverted vector, RTV-309-3/184M. The
RTV-309-3/184M displayed a moderate reduction in delavirdine
(4-fold), efavirenz (5-fold) and nevirapine (9-fold) susceptibility
compared to a wild type control RTV. The RTV-309-3/184M showed
drug-dependent stimulation of viral replication in the presence of
delavirdine (.about.50%), efavirenz (.about.60%) and nevirapine
(.about.80%).
[0251] 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/stimulate said mutant.
* * * * *