U.S. patent application number 10/399920 was filed with the patent office on 2004-04-15 for mutational profiles in hiv-1 reverse transcriptase correlated with phenotypic drug resistance.
Invention is credited to Dehertogh, Pascale Alfons Rosa, Hertogs, Kurt, Larder, Brendan, Wang, Dechao.
Application Number | 20040073378 10/399920 |
Document ID | / |
Family ID | 26934586 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040073378 |
Kind Code |
A1 |
Dehertogh, Pascale Alfons Rosa ;
et al. |
April 15, 2004 |
Mutational profiles in hiv-1 reverse transcriptase correlated with
phenotypic drug resistance
Abstract
The invention provides novel mutations, mutation combinations or
mutational profiles of HIV-1 reverse transcriptase and/or protease
genes correlated with phenotypic resistance to HIV drugs. More
particularly, the present invention relates to the use of genotypic
characterization of a target population of HIV and the subsequent
correlation of this information to phenotypic interpretation in
order to correlate virus mutational profiles with drug resistance.
The invention also relates to methods of utilizing the mutational
profiles of the invention in databases, drug development, i.e.,
drug design, and drug modification, therapy and treatment design,
clinical management and diagnostic analysis.
Inventors: |
Dehertogh, Pascale Alfons Rosa;
(Puurs, BE) ; Hertogs, Kurt; (Antwerpen, BE)
; Larder, Brendan; (Cambridge, GB) ; Wang,
Dechao; (Cambridge, GB) |
Correspondence
Address: |
PHILIP S. JOHNSON
JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
26934586 |
Appl. No.: |
10/399920 |
Filed: |
April 16, 2003 |
PCT Filed: |
October 22, 2001 |
PCT NO: |
PCT/EP01/12338 |
Current U.S.
Class: |
702/20 |
Current CPC
Class: |
G16B 20/00 20190201;
G16B 20/20 20190201; G16B 50/00 20190201; G16B 20/50 20190201 |
Class at
Publication: |
702/020 |
International
Class: |
G06F 019/00; G01N
033/48; G01N 033/50 |
Claims
1. A computer system comprising at least one database chosen from:
(i) a database correlating the presence of at least one mutation in
a human immunodeficiency virus (HIV) reverse transcriptase and
resistance of at least one strain of HIV to a reverse transcriptase
inhibitor, comprising at least one set of records chosen from: a
set of records corresponding to a correlation between at least one
mutation chosen from 44D, 77L, 115F, 118I, 184V, 208Y, 210W, 211K,
214F, 215F, 215Y, 219E, 219N, and 219Q, and resistance to d4T; a
record corresponding to a correlation between mutation 184I and
resistance towards lamivudine; a set of records corresponding to a
correlation between at least one mutation chosen from 115F and 184V
and resistance towards abacavir; a record corresponding to a
combination of 62V, 75T, 77L, 116Y and 151M and resistance towards
all nucleoside analogues; a set of records corresponding to a
correlation between at least one mutation chosen from 101H, 101P,
103H, 103S, 103T, 106M, 181S, and 190Q and resistance towards
nevirapine; a set of records corresponding to a correlation between
at least one mutation chosen from 101H, 101P, 103H, 103N, 103S,
103T, 106M, 181C, 181S and 190Q and resistance towards delavirdine;
a set of records corresponding to a correlation between at least
one mutation chosen from 101H, 101P, 103H, 103S, 103T, 106M, 181S,
190Q and 236L and resistance towards efavirenz; a record
corresponding to a combination of 184V and 41L and 215Y, wherein
the 184V resistance mutation reverses the effect of 41L and 215Y
mutations on zidovudine; a record corresponding to a 236L mutation,
which increases the sensitivity towards nevirapine; (ii) a database
correlating the presence of at least one mutation in a human
immunodeficiency virus (HIV) protease and resistance of at least
one strain of HIV to a protease inhibitor, comprising a set of
records corresponding to a correlation between at least mutation
selected from 54L, 54M, 54V and any mutation at codon 84 and
resistance towards a protease inhibitor.
2. A computer system according to claim 1 wherein the mutation at
codon 84 is selected from 84A, 84C and 84L.
3. A computer system according to any one of claims 1 to 2 wherein
the protease inhibitor is selected from amprenavir, saquinavir,
nelfinavir, ritonavir and indinavir.
4. A computer system according to any one of claims 1 to 3 wherein
the at least one mutation in the HIV protease is combined with at
least one mutation in the HIV protease at codon 10 and/or codon
90.
5. A computer system according to any one of claims 1 to 4 wherein
the at least one mutation in the HIV protease is combined with at
least one mutation in the HIV protease selected from 10I, 20R, 20T,
24I, 33F, 33I, 33L, 36I, 46L, 71T, 71V, 77I, 77V, 82I, 82V or
90M.
6. A method of evaluating the effectiveness of d4T as an antiviral
therapy of an HIV-infected patient comprising: (a) collecting a
sample from an HIV-infected patient; (b) determining whether the
sample comprises a nucleic acid encoding a HIV reverse
transcriptase having at least one mutation chosen from 44D, 77L,
115F, 118I, 184V, 208Y, 210W, 211K, 214F, 215F, 215Y, 219E, 219N,
and 219Q; (c) using the presence of said at least one mutation of
step b) to evaluate the effectiveness of said antiviral
therapy.
7. A method of evaluating the effectiveness of lamivudine as an
antiviral therapy of an HIV-infected patient comprising: (a)
collecting a sample from an HIV-infected patient; (b) determining
whether the sample comprises a nucleic acid encoding a HIV reverse
transcriptase having at least the mutation 184I; (c) using the
presence of said at least one mutation of step b) to evaluate the
effectiveness of said antiviral therapy.
8. A method of evaluating the effectiveness of abacavir as an
antiviral therapy of an HIV-infected patient comprising: (a)
collecting a sample from an HIV-infected patient; (b) determining
whether the sample comprises a nucleic acid encoding a HIV reverse
transcriptase having at least one mutation chosen from 115F and
184V; (c) using the presence of said at least one mutation of step
b) to evaluate the effectiveness of said antiviral therapy.
9. A method of evaluating the effectiveness of a nucleoside
analogue as an antiviral therapy of an HIV-infected patient
comprising: (a) collecting a sample from an HIV-infected patient;
(b) determining whether the sample comprises a nucleic acid
encoding a HIV reverse transcriptase having at least one mutation
chosen from 62V, 75T, 77L, 116Y and 151M; (c) using the presence of
said at least one mutation of step b) to evaluate the effectiveness
of said antiviral therapy.
10. A method of evaluating the effectiveness of nevirapine as an
antiviral therapy of an HIV-infected patient comprising: (a)
collecting a sample from an HIV-infected patient; (b) determining
whether the sample comprises a nucleic acid encoding a HIV reverse
transcriptase having at least one mutation chosen from 101H, 101P,
103H, 103S, 103T, 106M, 181S, 190Q and 236L; (c) using the presence
of said at least one mutation of step b) to evaluate the
effectiveness of said antiviral therapy.
11. A method of evaluating the effectiveness of delavirdine as an
antiviral therapy of an HIV-infected patient comprising: (a)
collecting a sample from an HIV-infected patient; (b) determining
whether the sample comprises a nucleic acid encoding a HIV reverse
transcriptase having at least one mutation chosen from 101H, 101P,
103H, 103N, 103S, 103T, 106M, 181C, 181S, 190Q and 236L; (c) using
the presence of said at least one mutation of step b) to evaluate
the effectiveness of said antiviral therapy.
12. A method of evaluating the effectiveness of efavirenz as an
antiviral therapy of an HIV-infected patient comprising: (a)
collecting a sample from an HIV-infected patient; (b) determining
whether the sample comprises a nucleic acid encoding a HIV reverse
transcriptase having at least one mutation chosen from 101H, 101P,
103H, 103S, 103T, 106M, 181S, 190Q and 236L; (c) using the presence
of said at least one mutation of step b) to evaluate the
effectiveness of said antiviral therapy.
13. A method of evaluating the effectiveness of zidovudine as an
antiviral therapy of an HIV-infected patient comprising: (a)
collecting a sample from an HIV-infected patient; (b) determining
whether the sample comprises a nucleic acid encoding a HIV reverse
transcriptase having at least one mutation chosen from 184V, 41L
and 215Y; (c) using the presence of said at least one mutation of
step b) to evaluate the effectiveness of said antiviral
therapy.
14. A method of evaluating the effectiveness of a protease
inhibitor as an antiviral therapy of an HIV-infected patient
comprising: (a) collecting a sample from an HIV-infected patient;
(b) determining whether the sample comprises a nucleic acid
encoding a HIV protease having at least one mutation chosen from
54L, 54M, 54V and any mutation at codon 84; (c) using the presence
of said at least one mutation of step b) to evaluate the
effectiveness of said antiviral therapy.
15. A method according to claim 14 wherein the mutation at codon 84
is selected from 84A, 84C and 84L.
16. A method according to claim 14 or 15 wherein the at least one
mutation in the HIV protease is combined with at least one mutation
in the HIV protease at codon 10 and/or codon 90.
17. A method according to any one of claims 14 to 16 wherein the at
least one mutation in the HIV protease is combined with at least
one mutation in the HIV protease selected from 10I, 20R, 20T, 24I,
33F, 33I, 33L, 36I, 46L, 71T, 71V, 77I, 77V, 82I, 82V or 90M.
18. A method of identifying a drug effective against drug resistant
strains of HIV, comprising: i) providing a HIV protease containing
at least one mutation chosen from 54L, 54M, 54V or any mutation at
codon 84; ii) determining a phenotypic response of said drug to
said HIV protease; and iii) using said phenotypic response to
determine the effectiveness of said drug.
19. A drug identified using the method as claimed in claim 18.
20. The method of claim 18 wherein said phenotypic response is
determined using a recombinant virus assay.
21. A method of designing a therapy with d4T for treating a patient
infected with HIV comprising: (a) collecting a sample from an
HIV-infected patient; (b) determining whether the sample comprises
a nucleic acid encoding a HIV reverse transcriptase having at least
one mutation chosen from 44D, 77L, 115F, 118I, 184V, 208Y, 210W,
211K, 214F, 215F, 215Y, 219E, 219N, and 219Q; (c) using the
presence of said at least one mutation of step b) to design the
antiviral therapy.
22. A method of designing a therapy with lamivudine for treating a
patient infected with HIV comprising: (a) collecting a sample from
an HIV-infected patient; (b) determining whether the sample
comprises a nucleic acid encoding a HIV reverse transcriptase
having at least the mutation 184I; (c) using the presence of said
at least one mutation of step b) to design the antiviral
therapy.
23. A method of designing a therapy with abacavir for treating a
patient infected with HIV comprising: (a) collecting a sample from
an HIV-infected patient; (b) determining whether the sample
comprises a nucleic acid encoding a HIV reverse transcriptase
having at least one mutation chosen from 115F and 184V; (c) using
the presence of said at least one mutation of step b) to design the
antiviral therapy.
24. A method of designing a therapy with a nucleoside analogue for
treating a patient infected with HIV comprising: (a) collecting a
sample from an HIV-infected patient; (b) determining whether the
sample comprises a nucleic acid encoding a HIV reverse
transcriptase having at least one mutation chosen from 62V, 75T,
77L, 116Y and 151M; (c) using the presence of said at least one
mutation of step b) to design the antiviral therapy.
25. A method of designing a therapy with nevirapine for treating a
patient infected with HIV comprising: (a) collecting a sample from
an HIV-infected patient; (b) determining whether the sample
comprises a nucleic acid encoding a HIV reverse transcriptase
having at least one mutation chosen from 101H, 101P, 103H, 103S,
103T, 106M, 181S, 190Q and 236L; (c) using the presence of said at
least one mutation of step b) to design the antiviral therapy.
26. A method of designing a therapy with delavirdine for treating a
patient infected with HIV comprising: (a) collecting a sample from
an HIV-infected patient; (b) determining whether the sample
comprises a nucleic acid encoding a HIV reverse transcriptase
having at least one mutation chosen from 101H, 101P, 103H, 103N,
103S, 103T, 106M, 181C, 181S, 190Q and 236L; (c) using the presence
of said at least one mutation of step b) to design the antiviral
therapy.
27. A method of designing a therapy with efavirenz for treating a
patient infected with HIV comprising: (a) collecting a sample from
an HIV-infected patient; (b) determining whether the sample
comprises a nucleic acid encoding a HIV reverse transcriptase
having at least one mutation chosen from 101H, 101P, 103H, 103S,
103T, 106M, 181S, 190Q and 236L; (c) using the presence of said at
least one mutation of step b) to design the antiviral therapy.
28. A method of designing a therapy with zidovudine for treating a
patient infected with HIV comprising: (a) collecting a sample from
an HIV-infected patient; (b) determining whether the sample
comprises a nucleic acid encoding a HIV reverse transcriptase
having at least one mutation chosen from 184V, 41L and 215Y; (c)
using the presence of said at least one mutation of step b) to
design the antiviral therapy.
29. A method of designing a therapy with a protease inhibitor for
treating a patient infected with HIV comprising: (a) collecting a
sample from an HIV-infected patient; (b) determining whether the
sample comprises a nucleic acid encoding a HIV protease having at
least one mutation chosen from 54L, 54M, 54V and any mutation at
codon 84; (c) using the presence of said at least one mutation of
step b) to design the antiviral therapy.
30. A method according to claim 29 wherein the mutation at codon 84
is selected from 84A, 84C and 84L.
31. A method according to claim 29 or 30 wherein the at least one
mutation in the HIV protease is combined with at least one mutation
in the HIV protease at codon 10 and/or codon 90.
32. A method according to any one of claims 29 to 31 wherein the at
least one mutation in the HIV protease is combined with at least
one mutation in the HIV protease selected from 10I, 20R, 20T, 24I,
33F, 33I, 33L, 36I, 46L, 71T, 71V, 77I, 77V, 82I, 82V or 90M.
Description
[0001] The present invention is directed to the field of nucleic
acid diagnostics and the identification of base variation in target
nucleic acid sequences. More particularly, the present invention
relates to the use of such genotypic characterization of a target
population of HIV and the subsequent association, i.e.,
correlation, of this information to phenotypic interpretation in
order to correlate virus mutational profiles with drug resistance.
The invention also relates to methods of utilizing the mutational
profiles of the invention in drug development, i.e., drug design,
drug modification, and drug development, therapy and treatment
design, clinical management and diagnostic analysis.
[0002] Retroviral inhibitors may block viral replication in various
ways. For example, Nucleoside Reverse Transcriptase Inhibitors
(NRTIs), compete with the natural nucleoside triphosphates for
incorporation into elongating viral DNA by reverse transcriptase.
Chemical modifications that distinguish these compounds from
natural nucleosides result in DNA chain termination events. NRTIs
that are currently available include zidovudine (ZDV), didanosine
(ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC) and
abacavir (ABC).
[0003] Nucleotide reverse transcriptase inhibitors (NtRTIs) have
the same mode of action as NRTIs, but they differ in that they are
already monophosphorylated and therefore they require fewer
metabolic steps. Adefovir (bis-POM-PMEA) and bis-POC PMPA belong to
this category of treatments.
[0004] Non-Nucleoside Reverse Transcriptase inhibitor (NNRTIs) are
a group of structurally diverse compounds which inhibit HIV reverse
transcriptase by noncompetitive binding to or close to the active
site of the viral reverse transcriptase enzyme, thereby inhibiting
its activity. Available compounds in this group include nevirapine
(NVP), delavirdine (DLV) and efavirenz.
[0005] Protease Inhibitors (PIs) are peptidomimetic and bind to the
active site of the viral protease enzyme, thereby inhibiting the
cleavage of precursor polyproteins necessary to produce the
structural and enzymatic components of infectious virions. PIs that
are currently available include saquinavir (SQV), ritonavir (RTV),
indinavir (IDV) nelfinavir (NFV), amprenavir (APV) and ABT-378
(lopinavir).
[0006] The options for antiretroviral therapy have improved
considerably as new agents have become available. Current
guidelines for antiretroviral therapy recommend a triple
combination therapy regimen for initial treatment, such as one PI
and 2 NRTIs or one NNRTI and 2 NRTIs. These combination regimens
show potent antiretroviral activity and are referred to as HAART
(highly active antiviral therapy).
[0007] Additionally, the development and standardization of plasma
HIV-1 RNA quantification assays has led to the use of viral load
measurements as a key therapy response monitoring tool. The goal of
antiretroviral therapy is to reduce plasma viremia to below the
limit of detection on a long-term basis. However, in a significant
number of patients, maximal suppression of virus replication is not
achieved and for those in whom this goal is reached, a significant
number experience viral load rebound. Viral load data provide no
information on the cause of the failure.
[0008] Why therapies fail may be due to a number of factors,
including insufficient antiviral activity of the regimen,
individual variations in drug metabolism and pharmacodynamics,
difficulties in adhering to dosing regimen, requirements for
treatment interruption due to toxicity, and viral drug resistance.
Moreover, drug resistance may develop in a patient treated with
sub-optimal antiretroviral therapy or a patient may be infected
with drug-resistant HIV-1. Although drug resistance may not be the
primary reason for therapy failure, in many cases any situation
which permits viral replication in the presence of an inhibitor
sets the stage for selection of resistant variants.
[0009] Viral drug resistance can be defined as any change in the
virus that improves replication in the presence of an inhibitor.
HIV-1 drug resistance was first described in 1989 and involved
patients that had been treated with zidovudine monotherapy, which
represented the only treatment option at that time. See Larder, B.
A., et al., Science 243, 1731-1734 (1989). Emergence of resistance
is almost always being observed during the course of treatment of
patients with single antiretroviral drugs. Similarly, in vitro
passage of viral cultures through several rounds of replication in
the presence of antiretroviral compounds leads to the selection of
viruses whose replication cycle is no longer susceptible to the
compounds used. Resistance development has also been observed with
the introduction of dual NRTI combination therapy as well as during
the administering of the more potent NNRTIs and PIs. Individual
antiretroviral agents differ in the rate at which resistance
develops: selection for resistant variants may occur within weeks
of treatment or resistance may emerge after a longer treatment
period.
[0010] Extensive genetic analysis of resistant viral isolates
generated through in vivo or in vitro selection has revealed that
resistance is generally caused by mutations altering the nucleotide
sequence at some specific site(s) of the viral genome. The
mutational patterns that have been observed and reported for HIV-1
and that are correlated with drug resistance are very diverse: some
antiretroviral agents require only one single genetic change, while
others require multiple mutations for resistance to appear. A
summary of mutations in the HIV genome correlated with drug
resistance has been compiled. See Schinazi, R. F., Larder, B. A.
& Meliors, J. W. 1997. Int. Antiviral News. 5, 129-142 (1997).
Additionally, an electronic listing with mutations has also become
available at http://hiv-web.lanl.gov, www.hivb.stanford.edu, and
http://www.hivresistanceweb.com.
[0011] It should be noted that the degree of susceptibility of a
genetic variant to an antiretroviral compound is expressed herein
relative to the wild-type virus (HIV III/LAI reference sequence) as
found, for example, in GenBank, the sequence of which is hereby
incorporated by reference. Susceptibilities are generally expressed
as ratios of IC.sub.50 or IC.sub.90 values (the IC.sub.50 or
IC.sub.90 value being the drug concentration at which 50% or 90%
respectively of the viral population is inhibited from
replicating). Additionally, the genetic mutation is normally
written as in reference to the wild type virus, i.e., K101N refers
to replacement of a Lysine at codon 101 with a Asparagine. However,
the mutations of the invention do not depend on the wild-type
example listed in order to be within the practice of the invention.
For example, the mutation 101N, refers to an Asparagine at the 101
codon regardless of the whether there was a Lysine at 101 prior to
mutation.
[0012] Of course, as antiretroviral drugs are administered for
longer periods of time, mostly in combination with each other, and
as new antiretrovirals are being developed and added to the present
drugs, new resistance-correlated genetic variants are being
discovered. Of particular import is that the combination of
antiretroviral agents can influence resistance characteristics. For
example, different NNRTI resistance-correlated mutations were
selected on NNRTI-zidovudine combination therapy and different NRTI
resistance-correlated mutations were selected in dual NRTI
combination therapy. In the latter case, the result is high-level
multi-drug resistance to all NRTIs.
[0013] Moreover, once viral resistance has developed, salvage
therapy options may be severely restricted due to cross-resistance
within each drug class. Recently, interest has been focused on the
characterization of alterations in viral drug susceptibility for
better clinical management. This is as important for initial
treatment as for when a therapy change is called for in order to
minimize the emergence of resistance and improve the long-term
prognosis of the patient. The choice of therapy regimen will be
supported by knowledge of the resistance profile of the circulating
virus population. Additionally, therapy combinations will have a
greater chance of being effective if they include agents that have
a demonstrated potential of suppressing a particular virus
population.
[0014] To achieve these and other advantages, and in accordance
with the purpose of the invention as embodied and broadly described
herein, the present invention, in one aspect, provides a computer
system comprising a database correlating the presence of at least
one mutation in an HIV reverse transcriptase and the resistance of
at least one strain of HIV to a reverse transcriptase inhibitor
(RTI); and/or a database correlating the presence of at least one
mutation in an HIV protease and the resistance of at least one
strain of HIV to a protease inhibitor (PI). More specifically, the
database comprises a set of records corresponding to a correlation
between a mutation and drug resistance.
[0015] In a further embodiment, the invention provides for a method
of identifying drugs effective against NNRTI or NRTI resistant
strains of HIV, the method comprising the steps of: providing at
least one strain of HIV comprising HIV reverse transcriptase
containing at least one mutation described herein, determining the
phenotypic response of the drug to the strain of HIV, and using the
phenotypic response to determine the effectiveness of the drug. In
an even further embodiment, the invention provides a method of
identifying drugs effective against protease inhibitor (PI)
resistant strains of HIV, wherein the strain of HIV comprises HIV
protease containing at least one mutation described herein,
determining the phenotypic response of said drug to said strain of
HIV, and using the phenotypic response to determine the
effectiveness of the drug. In another embodiment, the invention
provides for the drug identified using the methods of the
invention.
[0016] The invention also provides for a method of designing
therapy for treating patients infected with HIV comprising:
collecting a sample from an HIV-infected patient; determining
whether the sample comprises at least one nucleic acid encoding HIV
reverse transcriptase having at least one mutation described herein
or HIV protease having at least one mutation described herein; and
using the presence of the nucleic acid to design a therapy for the
patient.
[0017] The invention also includes isolated HIV reverse
transcriptase complexes resistant to at least one NNRTI or at least
one NRTI comprising at least one mutation described above and an
isolated HIV protease complex resistant to a PI comprising at least
one mutation described above.
[0018] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1: Nucleoside analogue susceptibility of MDR
patient-derived recombinant HIV variants. Recombinant viruses were
produced from patient plasma samples as described in Example 2 and
tested for susceptibility to (a) d4T, (b) ddC and (c) ddl. The mean
fold increase in IC.sub.50 values (Mean fold resistance) relative
to wild-type controls are shown for groups of viruses with
different genotypes, i.e., the codon 151-M multi-drug resistance
cluster (n=27), viruses with 69D/N (n=195), or 75M (n=43) in a
background of AZT and 3TC resistance mutations and codon 69
insertion mutants (n=45) in a background of AZT resistance
mutations. Error bars indicate standard errors. Note that the total
number (n=310) is higher than the 302 MDR samples described because
a small minority were 69D/N and 75M double mutants and are
represented in both groups.
[0020] FIG. 2: Therapy histories of three patients whose HIV-1
isolates developed codon 69 insertions. Nucleoside analogue
therapies (AZT, 3TC, ddC, ddI or d4T) are shown as horizontal bars,
indicating the time period in which each patient (1, 2 or 3)
received a particular treatment. The time point at which plasma
samples were obtained for genotypic and phenotypic analysis are
shown by the arrows together with the specific codon 69 insertion
detected. Any other therapies besides nucleosides that these
patients may have been receiving are not indicated on this
figure.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The invention, in one aspect, provides novel mutations or
mutational profiles of HIV-1 reverse transcriptase and/or protease
genes correlated with phenotypic resistance to anti-HIV drugs. More
particularly, the present invention also relates to the use of
genotypic characterization of a target population of HIV and the
subsequent correlation of this information to phenotypic
interpretation in order to correlate virus mutational profiles with
drug resistance. The invention also relates to methods of utilizing
the mutational profiles of the invention in databases, drug
development, i.e., drug design, and drug modification, therapy and
treatment design, clinical management and diagnostic analysis.
[0022] The present invention concerns a computer system
comprising:
[0023] at least one database correlating the presence of at least
one mutation in a human immunodeficiency virus (HIV) reverse
transcriptase and resistance of at least one strain of HIV to a
reverse transcriptase inhibitor, comprising:
[0024] at least one set of records corresponding to a correlation
between at least one mutation chosen from 44D, 77L, 115F, 118I,
184V, 208Y, 210W, 211K, 214F, 215F, 215Y, 219E, 219N, and 219Q, and
resistance to d4T,
[0025] a record corresponding to a correlation between mutation
184I and resistance towards lamivudine,
[0026] at least one set of records corresponding to a correlation
between at least one mutation chosen from 115F and 184V and
resistance towards abacavir.
[0027] a combination of 62V, 75T, 77L, 116Y and 151M and resistance
towards all nucleoside analogues
[0028] at least one set of records corresponding to a correlation
between at least one mutation chosen from 101H, 101P, 103H, 103S,
103T, 106M, 181S, and 190Q and resistance towards nevirapine,
[0029] at least one set of records corresponding to a correlation
between at least one mutation chosen from 101H, 101P, 103H, 103N,
103S, 103T, 106M, 181C, 181S, 190Q and 236L and resistance towards
delavirdine,
[0030] at least one set of records corresponding to a correlation
between at least one mutation chosen from 101H, 101P, 103H, 103S,
103T, 106M, 181S, 190Q and 236L and resistance towards
efavirenz,
[0031] a combination of 184V and 41L and 215Y, wherein the 184V
resistance mutation reverses the effect of 41L and 215Y mutations
on zidovudine
[0032] a 236L mutation, which increases the sensitivity towards
nevirapine
[0033] at least one database correlating the presence of at least
one mutation in a human immunodeficiency virus (HIV) protease and
resistance of at least one strain of HIV to a protease inhibitor,
comprising:
[0034] at least one set of records corresponding to a correlation
between at least mutation selected from 54L, 54M, 54V, 84A, 84C and
84L and resistance towards a protease inhibitors selected from
amprenavir, saquinavir, nelfinavir, ritonavir, indinavir.
[0035] The present invention further concerns a method of
evaluating the effectiveness of an antiviral therapy of an
HIV-infected patient comprising:
[0036] (a) collecting a sample from an HIV-infected patient;
[0037] (b) determining whether the sample comprises at least one
nucleic acid encoding HIV having at least one mutation selected
from:
[0038] i) a first nucleic acid encoding a HIV reverse transcriptase
having at least one mutation chosen from
[0039] 44D, 77L, 115F, 118I, 184V, 208Y, 210W, 211K, 214F, 215F,
215Y, 219E, 219N, and 219Q, and resistance to d4T,
[0040] 184I and resistance towards lamivudine,
[0041] 115F and 184V and resistance towards abacavir.
[0042] 62V, 75T, 77L, 116Y and 151M and resistance towards all
nucleoside analogues
[0043] 101H, 101P, 103H, 103S, 103T, 106M, 181S, and 190Q and
resistance towards nevirapine,
[0044] 101H, 101P, 103H, 103N, 103S, 103T, 106M, 181C, 181S, 190Q
and 236L and resistance towards delavirdine,
[0045] 101H, 101P, 103H, 103S, 103T, 106M, 181S, 190Q and 236L and
resistance towards efavirenz,
[0046] 184V and 41L and 215Y, wherein the 184V resistance mutation
reverses the effect of 41L and 215Y mutations on zidovudine
[0047] 236L mutation, which increases the sensitivity towards
nevirapine
[0048] ii) a second nucleic acid encoding a HIV protease having at
least one mutation selected from: 54L, 54M, 54V, 84A, 84C and 84L
and resistance towards a protease inhibitors selected from
amprenavir, saquinavir, nelfinavir, ritonavir, indinavir.
[0049] (c) using the presence of said at least one nucleic acid to
evaluate the effectiveness of said antiviral therapy.
[0050] The present invention further concerns a method of
identifying a drug effective against drug resistant strains of HIV,
comprising:
[0051] i) providing at least one strain of HIV comprising:
[0052] a) at least one HIV reverse transciptase containing at least
one mutation chosen from:
[0053] 44D, 77L, 115F, 118I, 184V, 208Y, 210W, 211K, 214F, 215F,
215Y, 219E, 219N, and 219Q, and resistance to d4T,
[0054] 184I and resistance towards lamivudine,
[0055] 115F and 184V and resistance towards abacavir.
[0056] 62V, 75T, 77L, 116Y and 151M and resistance towards all
nucleoside analogues
[0057] 101H, 101P, 103H, 103S, 103T, 106M, 181S, and 190Q and
resistance towards nevirapine,
[0058] 101H, 101P, 103H, 103N, 103S, 103T, 106M, 181C, 181S, 190Q
and 236L and resistance towards delavirdine,
[0059] 101H, 101P, 103H, 103S, 103T, 106M, 181S, 190Q and 236L and
resistance towards efavirenz,
[0060] 184V and 41L and 215Y, wherein the 184V resistance mutation
reverses the effect of 41L and 215Y mutations on zidovudine
[0061] 236L mutation, which increases the sensitivity towards
nevirapine
[0062] b) at least one strain of HIV comprising HIV protease
containing at least one mutation chosen from: 54L, 54M, 54V, 84A,
84C and 84L and resistance towards a protease inhibitors selected
from amprenavir, saquinavir, nelfinavir, ritonavir, indinavir.
[0063] ii) determining a phenotypic response of said drug to said
strain of HIV; and
[0064] iii) using said phenotypic response to determine the
effectiveness of said drug.
[0065] The instant invention provides methods to identify drug
effective for treating HIV infected individuals. The present
invention further provides for phenotyping methods for assessing
HIV therapy of an individual.
[0066] The instant invention further provides a method of designing
a therapy for treating a patient infected with HIV comprising:
[0067] i) collecting a sample from an HIV-infected patient;
[0068] ii) determining whether the sample comprises
[0069] a) at least one nucleic acid encoding HIV reverse
transcriptase having at least one mutation chosen from:
[0070] 44D, 77L, 115F, 118I, 184V, 208Y, 210W, 211K, 214F, 215F,
215Y, 219E, 219N, and 219Q, and resistance to d4T,
[0071] 184I and resistance towards lamivudine,
[0072] 115F and 184V and resistance towards abacavir.
[0073] 62V, 75T, 77L, 116Y and 151M and resistance towards all
nucleoside analogues
[0074] 101H, 101P, 103H, 103S, 103T, 106M, 181S, and 190Q and
resistance towards nevirapine,
[0075] 101H, 101P, 103H, 103N, 103S, 103T, 106M, 181C, 181S, 190Q
and 236L and resistance towards delavirdine,
[0076] 101H, 101P, 103H, 103S, 103T, 106M, 181S, 190Q and 236L and
resistance towards efavirenz,
[0077] 184V and 41L and 215Y, wherein the 184V resistance mutation
reverses the effect of 41L and 215Y mutations on zidovudine
[0078] 236L mutation, which increases the sensitivity towards
nevirapine
[0079] b) at least one nucleic acid encoding HIV protease having at
least one mutation chosen from 54L, 54M, 54V, 84A, 84C and 84L and
resistance towards a protease inhibitors selected from amprenavir,
saquinavir, nelfinavir, ritonavir, indinavir.
[0080] iii) using the presence of said at least one nucleic acid to
design the therapy for said patient.
[0081] Preferentially at least an additional mutation is present in
the HIV protease nucleic acid selected from 10I, 20R, 20T, 24I,
33F, 33I, 33L, 36I, 46L, 71T, 71V, 77I, 77V, 82I, 82V or 90M. More
preferential, HIV protease nucleic acid having a mutation at codon
54 comprises at least an additional mutation selected from codon 10
and 90 and confers resistance to a protease inhibitor selected from
Amprenavir, Indinavir, Nelfinavir, Ritonavir and Saquinavir. In one
embodiment the instant invention provides for a chimaeric HIV virus
comprising at least one mutation of the instant invention
[0082] Not to be limited as to theory, the invention may utilize a
combinational approach involving genotypic and phenotypic
resistance testing to correlate mutations with resistance
phenotypes. Without the specific combination of the technologies
mentioned above, this correlation between mutation and resistance
would not have been detected. In addition to the observation of
these genotypic and phenotypic profiles in isolates from routine
clinical practice, site-directed mutants were generated to confirm
that these mutations actually form the basis of this pattern of
drug resistance.
[0083] Resistance of HIV to antiretroviral drugs may be determined
at the genotypic level by identifying mutations in the HIV-1 genome
and by inferring the resistance of HIV-1 to antiretroviral drugs
through searching for mutational patterns known to correlate with
resistance. Alternatively, resistance of HIV to antiretroviral
drugs may be determined at the phenotypic level by culturing the
virus in the presence of the inhibitors, and by measuring to what
extent the drug inhibits viral replication. In this case, one
measures the effect of all mutational interactions, the effects of
genetic changes as yet unknown or not previously identified, the
effect of the background genotype, etc., on the phenotype. Assays
for detection of mutations in HIV-1 may be based on polymerase
chain reaction (PCR) amplification of viral genomic sequences.
These amplified sequences are then analyzed using either
hybridization or sequencing techniques. Hybridization-based assays
include primer-specific PCR, which makes use of synthetic
oligonucleotides designed to allow selective priming of DNA
synthesis. See Larder, B. A., et al., AIDS 5, 137-144 (1991);
Richman, D. D., et al., J. Infect. Dis. 164, 1075-1081 (1991);
Gingeras, T. R., et al., J. Infect. Dis. 164, 1066-1074 (1991).
Only when primer sequences match the target sequence (wild-type or
mutant) at the 3' end, is amplification of target sequences
possible and DNA fragments are produced. Knowledge of the primer
sequences allows one to infer the sequence of the viral isolate
under investigation, but only for the region covered by the primer
sequences. Other hybridization-based assays include differential
hybridization (Eastman, P. S., et al., J. Acg. Imm. Def. Syndr.
Human Retrovirol. 9, 264-273 (1995); Holodniy, M., et al., J.
Virol. 69, 3510-3516 (1995); Eastman, P. S., et al., J. Clin.
Micro. 33, 2777-2780 (1995).); Line Probe Assay (LiPAJ HIV-11 RT,
Innogenetics) (Stuyver, L., et al., Antimicrob. Agents Chemotherap.
41, 284-291 (1997).); and GENECHIP.RTM. technology (Affymetrix)
(D'Aquila, R. T. Clin. Diagnost. Virol. 3, 299-316 (1995); Fodor,
S. P. A. et al., Nature 364, 555-556 (1993); Fodor, S. P. A. Nature
227, 393-395 (1997). DNA sequencing assays, on the other hand,
provides information on all nucleotides of the sequenced region.
Target sequences are amplified by PCR. Sequencing results may be
reported as amino acid changes at positions in the protease gene
and the reverse transcriptase gene compared to the wild-type
reference sequence. The changes included in the genotyping report
may be limited to mutations at positions known to manifest drug
resistance-associated polymorphisms. Polymorphisms at positions not
associated with drug resistance are not required.
[0084] Phenotyping assays measure the ability of a replicating
virus to grow in the presence of specific inhibitors compared to a
wild-type sensitive reference virus. Consequently, these assays
directly measure the degree of viral resistance or susceptibility
to specific inhibitors. Applicable phenotyping assays include but
are not limited to: the PBMC (peripheral blood mononuclear cells)
p24 Antigen Assay, which was the first standardized assay for
determination of viral drug resistance in clinical HIV-1 isolates
(Japour, A. J., et al., Antimicrob. Agents Chemother. 37, 1095-1101
(1993); Kusumi, K. et al., J. Virol. 66, 875-885 (1992); and the
Recombinant Virus Assays (RVAs) which was first described as an
alternative means of assessing phenotypic resistance to
RT-inhibitors (Kellam, P. & Larder, B. A., Antimicrob. Agents
Chemother. 38, 23-30 (1994); and Pauwels, R., et al., 2nd
International Workshop on HIV Drug Resistance and Treatment
Strategies, Lake Maggiore, Italy. Abstr. 51(1998).
[0085] As is the case with the genotyping assays, the recombinant
virus assay starts with the amplification of viral target sequences
by means of PCR. The amplicons are incorporated into a proviral
laboratory clone with sequences homologous to those present in the
amplicon deleted. This generates a stock of chimeric viruses. The
viruses are tested for their ability to grow in the presence of
different concentrations of drugs. Results are obtained by
calculating IC.sub.50 values for each inhibitor and by reporting
the results as IC.sub.50 values, expressed in .mu.M concentrations,
or by computing the ratio of the IC.sub.50 values found for the
chimeric virus to the IC.sub.50 values found for a wild type
susceptible laboratory virus tested in parallel. In the latter
case, resistance is expressed as "fold-resistance" compared to a
wild-type susceptible HIV-1 strain. In order to meet the need for
high-volume testing and a short turn-around time for an individual
test, the latest generation of phenotyping assays has undergone
further modifications. The use of reporter gene systems for
susceptibility testing allows the implementation of laboratory
automation and standardization. See Pauwels, et al., J. Virol.
Methods 20, 309-321 (1998); Paulous, S., et al., International
Workshop on HIV Drug Resistance, Treatment Strategies and
Eradication, St. Petersburg, Fla., USA. Abstr. 46 (1997); and
Deeks, S. G., et al., 2nd International Workshop on HIV Drug
Resistance and Treatment Strategies, Lake Maggiore, Italy. Abstr.
53 (1998).
[0086] The Antivirogram.RTM. assay (Virco) (WO 97/27480) is based
on homologous recombination of patient derived HIV-1 gag/PR/RT
sequences into a proviral HIV-1 clone correspondingly deleted for
the gag/PR/RT sequences. See Pauwels, et al., J. Virol. Methods 20,
309-321 (1998). A similar assay (Phenosense ViroLogic) is based on
enzymatic ligation of patient-derived PR/RT sequences into a
correspondingly deleted proviral vector carrying an indicator gene,
luciferase, inserted in the deleted HIV-1 envelope gene. See Deeks,
S. G., et al., 2nd International Workshop on HIV Drug Resistance
and Treatment Strategies, Lake Maggiore, Italy. Abstr. 53 (1998).
Hertogs et al. Antimicrob. Agents Chemother. 44(3) 568-573 (2000)
the disclosures of which are herein incorporated by reference.
[0087] To summarize, the development of high-throughput phenotyping
and genotyping assays has allowed the establishment of a database
containing the phenotypic resistance data and the genotypic
sequences of over 30,000 clinical isolates. Correlative data
analysis and mutational cluster analysis of the database enables a
search for mutational patterns with accompanying resistance. An
example of which is virtual phenotyping (see PCT/EP01/04445).
[0088] In one embodiment, a neural network to accurately predict
the development of therapeutic agent resistance or sensitivity
based upon genotypic and phenotypic information and to accurately
define the genetic basis of therapeutic agent resistance can be
used. (see U.S. patent application Ser. No. 09/589,167 filed Jun.
8, 2000, PCT/EP01/06360, the disclosure of which is expressly
incorporated herein by reference in its entirety).
[0089] Table 1 below lists some of the most commonly occurring
resistance-correlated mutations appearing in clinical isolates
after treatment with antiretroviral drugs.
1TABLE 1 Examples of commonly occurring resistance-correlated
mutations appearing in clinical isolates after treatment with
antiretroviral drugs. Protease Inhibitors Primary D30N Nelfinavir
Mutations: M46L, V82A Indinavir G48V, L90M Saquinavir V82A
Ritonavir I50V Amprenavir Secondary L101/F/R/V, K20R/M, L24I, V32I,
L33F, M36I, M46I, Mutations: I47V, I54V/L, L63P, A71V/T, G73S,
V771, V82A/F/T/S, I84V, N88D, L90M Compensatory In a PI-resistant
mutational background, mutations at gag Mutations cleavage site(s)
may partially restore viral replicative Efficiency Reverse
Transcriptase Inhibitors NRTI M41L, K65R, D67N, T69D, K70R, L74V,
V75T/M, Mutations: M184V, L210W, T215Y/F, K219Q/E MDR A62V, V751,
F77L, F116Y, Q151 M Mutations: T69S with associated insertions of 1
to 3 amino acids between codons 68 and 70 of RT NNRTI A98G, L100I,
K101E, K103N/T, V106A, V108I, Mutations: V179D/E, Y181 C/l,
Y188C/L/H, G190A, P225H, P236L Reversal M184I/V decreases the
effect of zidovudine resistance Mutations: mutations M41 L and
T215Y. L74V decreases the effect of zidovudine resistance mutation
T215Y. K65R in mutational background (D67N, K70R, T215Y and K219Q)
decreases zidovudine resistance. Y181C decreases the effect of
zidovudine resistance mutation T215Y.
[0090] The invention contemplates resistance-correlated mutations
to any type of HIV treatment therapy including but not limited to
mutations conferring resistance to Protease Inhibitors and Reverse
Transcriptase Inhibitors (NRTIs, NtRTIs, and NNRTIs) in addition to
Multi-Drug Resistant Mutations.
[0091] In one embodiment, the invention contemplates mutations
conferring resistance to Protease Inhibitors (PIs). Table 1 lists
two categories of mutations for all PIs: primary and secondary
mutations. Primary mutations may be the major contributor to the
development of resistance to a particular drug. Secondary mutations
appear either later during the course of therapy and also lead to
resistance, or are already present as natural polymorphisms in a
PI-naive viral isolate. A great number of secondary mutations
enhance resistance to several PI-inhibitors simultaneously. This
may lead to broad cross-resistance to this class of inhibitors,
although subtle different phenotypic effects of those secondary
mutations may exist.
[0092] Not to be limited as to theory, mutations occurring in the
protease gene may impair cleaving efficiency of the polyprotein by
the protease. Compensatory mutations have been found at the gag
cleavage sites that allow more efficient cleaving of the sites by
proteases that have mutated. Several studies of clinical isolates
from protease-treated patients who have acquired PI
resistance-correlated mutations have shown mutations at gag p7/p1
and/or p1/p6 sites significantly raised the replicative efficiency
of the mutant viruses.
[0093] Other mutations within the practice of the invention may
confer resistance to NRTIs and NNRTIs. For example, the mutations
typically conferring resistance to the NRTI zidovudine are M41L,
D67N, K70R, L210W, T215Y and K219Q. Multiple mutations in HIV-1
reverse transcriptase also may confer high-level resistance to
zidovudine and other NRTIs. Multiple mutations, when present, may
act synergistically, and susceptibility decreases as the number of
resistance-correlated mutations increases. For example, mutations
correlated with resistance to didanosine are L74V, K65R. Resistance
to lamivudine is also correlated with the emergence of mutations
M184V and M184I that confer very high resistance levels in addition
to low-level resistance to didanosine, and zalcitabine. A low-level
resistance to lamivudine may also be present in the absence of the
184 mutation while resistance to abacavir is correlated with
mutations K65R, L74V, Y115F and M184V.
[0094] Another embodiment of the invention relates to multi-drug
resistance mutations (MDR) and particularly MDRs to NRTIs. For
example, the RT mutational constellation A62V, V75T, F77L, F116Y
and Q151M together causes resistance to all nucleoside
analogues.
[0095] Mutations conferring resistance to Non-Nucleoside Reverse
Transcriptase inhibitor (NNRTIs) are also contemplated by the
invention. For example, resistance-correlated mutations for
nevirapine are A98G, L100I, K103N, V106A, V108I, Y181C/I, Y188C and
G190A. These mutations are K103/N/T, Y181C and P236L for
delavirdine and for resistance to efavirenz, the mutations are
L100I, K101E, K103N, V108I, V179D. Y181C and Y188L.
[0096] Another aspect of the invention concerns reversal mutations.
For example, the M184V lamivudine resistance mutation decreases the
effect of zidovudine resistance mutations M41L and T215Y, while the
L74V didanosine resistance mutation decreases the effect of
zidovudine resistance mutation T215Y. Whether the described
reversal effects are phenotypically significant or not, however,
may depend on the combinations of mutations that are present.
[0097] In another embodiment, mutations may increase sensitivity to
inhibitors. For example, the delavirdine mutation, P236L increases
sensitivity of this mutant to inhibition by nevirapine and the
lamivudine-resistance mutation M184V causes increased
susceptibility to adefovir and to PMPA above the non-mutant
sequence. This increased sensitivity seems to be reflected in an
enhanced treatment outcome.
[0098] Novel mutations of HIV-1 reverse transcriptase (Table 2)
within the practice of the invention, and their correlated
phenotypic drug resistance, include but are not limited to those
shown in Table 2.
2TABLE 2 Novel RT Mutations and the Correlated Drug Resistance
Reverse Transcriptase Mutation Resistant to: 41L d4T 44D d4T 62V
d4T 67N d4T 69D d4T 69N d4T 69SXX d4T 70R d4T 75A d4T 75I d4T 75M
d4T 75T d4T 77L d4T 115F d4T 116Y d4T 118I d4T 151M d4T 184V d4T
208Y d4T 210W d4T 211K d4T 214F d4T 215F d4T 219E d4T 219N d4T 219Q
d4T 215Y d4T
[0099] The existence of a single mutation or any combination of the
mutations in Table 2 may confer resistance to d4T or one or more
other treatments from the correlated class. Furthermore, if tools
are used, such as those described herein, one may take the
identified mutation and the correlated-resistance to a class of
treatment. Therefore, the invention also provides that the listed
mutations and new combination of mutations, armed with the
correlated class of drug, can be used to predict new resistance
phenotypes such as resistance to additional PIs, NRTIs, NNRTIs, or
MDR resistance. Additionally, the existence of a combination of
mutations may confer the same or a different drug resistance
profile.
[0100] The present invention is also drawn to methods of using the
correlations of the invention. In one embodiment, the invention
provides for a database comprising the correlation between: the
presence of at least one mutation in HIV reverse transcriptase and
the resistance of at least one strain of HIV to a reverse
transcriptase inhibitor (RTI); or the presence of at least one
mutation in HIV protease and the resistance of at least one strain
of HIV to a protease inhibitor (PI).
[0101] In a further embodiment, the database may assist a physician
in developing a treatment program or in determining the appropriate
HIV therapy or combination therapy. For example, the
VirtualPhenotype.RTM. assay system is a diagnostic tool for
monitoring HIV-1 drug resistance. The system can be used for
studying resistance development in clinical trials of
anti-HIV-drugs, for improved clinical management of HIV-1 infected
patients and for studying epidemiological aspects of drug
resistance. It allows for a rapid determination of the drug
sensitivity of the HIV-1 population circulating in the plasma of
patients who have been exposed to antiretroviral drugs or who have
been infected with drug resistant HIV-1 strains.
[0102] The invention also provides for a method of monitoring HIV-1
drug resistance using a method such as the one used in the
VirtualPhenotype.RTM., which combines in one test the determination
of the genetic sequence of patient-derived HIV-1 genetic material
and the interpretation of sequence variations found in the patient
HIV strain with respect to the possible existence of antiviral drug
resistance. In one embodiment, mutations associated with resistance
to the different nucleoside reverse transcriptase inhibitors
zidovudine, (AZT), didanosine (ddI), zalcitabine (ddC), stavudine
(d4T), lamivudine (3TC) and abacavir, the nucleotide reverse
transcriptase inhibitor adefovir (PMEA), the non-nucleoside reverse
transcriptase inhibitors nevirapine, delavirdine and efavirenz, and
the protease inhibitors saquinavir, ritonavir, indinavir and
nelfinavir, are evaluated.
[0103] The methods of monitoring HIV-1 drug resistance, may also be
used in combination with phenotypic drug resistance testing of
viral isolates. For example, in one embodiment, a phenotypic test
is utilized that is based upon the construction of chimeric HIV-1
strains composed of the protease (PR) and reverse transcriptase
(RT) gene sequences which are isolated and amplified from the
patient viral RNA. These strains may subsequently be recombined
inside CD4+ T cells with a standard laboratory isogenic (HXB2)
HIV-1 DNA construct from which the PR/RT gene sequences were
deleted. The recombinant strains may then be grown in the presence
of the above-mentioned antiviral drugs and the susceptibility of
the viral isolates may be expressed as fold-change value of the
IC50 of the drug on the patient isolates over the IC50 of the drug
on a wild-type laboratory reference strain.
[0104] In one embodiment, the sample to be tested is prepared from
a patient and the genotypic assay is performed through automated
population-based full-sequence analysis (ABI). Therefore, the
sequencing method used may provide information on all nucleotides
of the sequenced region. Sequencing results may be reported as
amino acid changes at positions in the protease gene and the
reverse transcriptase gene compared to the wild-type reference
sequence. The changes included in the genotyping report may be
limited to mutations at positions known to manifest drug
resistance-associated polymorphisms. Polymorphisms at positions not
associated with drug resistance are not required.
[0105] In an even further embodiment, a report may be generated
that shows the region of the patient virus that has been sequenced,
the mutations detected by the test, and/or an interpretation of the
evidence obtained. The interpretation may include the
antiretroviral drugs, the drug(s) for which a known
resistance-associated mutation has been identified and/or to what
extent the observed mutations are indicative of resistance to the
drugs.
[0106] Knowledge of correlated geno- and phenotypes, together with
knowledge of the catalytic site on the viral target for new
compounds may also be utilized to tailor the construction of new
molecules and the implementation of new (combination) treatments
for HIV.
[0107] In another embodiment, the invention is drawn to a method of
evaluating the effectiveness of antiretroviral therapy of an
HIV-infected patient comprising: collecting a sample from an
HIV-infected patient; and determining whether the sample comprises
at least one nucleic acid encoding HIV reverse transcriptase having
at least one mutation or HIV protease having at least one mutation.
The sample may be a plasma sample, blood cells, or other tissue.
Further, the invention has the potential to ameliorate HIV
genotypic resistance diagnostics and can, in principle, lead to a
better therapy and, under certain conditions, even be life
saving.
[0108] In a further embodiment, the invention provides for a method
of identifying or designing drugs effective against NNRTI or NRTI
resistant HIV, the method comprising the steps of: providing at
least one strain of HIV comprising a nucleic acid encoding HIV
reverse transcriptase containing at least one mutation, and
determining the phenotypic response of the HIV strain to a drug. In
an even further embodiment, the invention provides a method of
identifying drugs effective against PI resistant strains of HIV,
wherein the strain of HIV comprises HIV protease containing at
least one mutation, and determining the phenotypic response of said
strain of HIV to said drug. The invention is also useful for
interpretation of resistance of HIV isolates. It can also be used
in full sequence analysis of HIV. In addition, the invention has
applications for hybridization-based HIV analyses or in drug
design, development, testing and marketing. In a further
embodiment, the invention includes the drugs designed by the
methods of the invention.
[0109] The invention also provides for a method of designing
therapy for treating patients infected with HIV comprising
correlating the presence of HIV reverse transcriptase having at
least one mutation described above with resistance to at least one
NNRTI or at least one NRTI, or correlating the presence of HIV
protease having at least one mutation with resistance to at least
one PI.
[0110] The identification of the comparative mutations of the
invention may lead to improved antiretroviral drug treatment
programs. As outlined above, there is ample evidence demonstrating
that poor virologic response to drug therapy may be correlated with
the existence of genotypic and/or phenotypic viral resistance to
one, several, or in the worst case, all available antiretroviral
drugs. As a consequence, resistance testing using the correlations
of the invention may be used as a tool for identifying those drugs
that no longer contribute towards decreasing the plasma viral
load.
EXAMPLES
Example 1
The Identification of Mutational Patterns in HIV-1 Reverse
Transcriptase and the Correlated Phenotypic Resistance
[0111] Plasma samples were obtained from HIV-1-infected individuals
from routine clinical practice in Europe and the US and were
shipped to the laboratory on dry ice and stored at -70.quadrature.C
until analysis. Phenotypic analysis was performed using the
recombinant virus assay. See Kellam, P., and B. A. Larder.
Antimicrob Agents Chemother 38:23-30 (1994); Hertogs, K., et al.,
Agents Chemother. 42:269-276 (1998); Briefly, protease (PR) and
reverse transcriptase (RT) coding sequences were amplified from
patient-derived viral RNA with HIV-1 specific primers. After
homologous recombination of amplicons into a PR-RT deleted proviral
clone, the resulting recombinant viruses were harvested, titrated
and used for in vitro susceptibility testing to antiretroviral
drugs. The results of this analysis were expressed as
fold-resistance values, reflecting the fold-increase in mean
IC.sub.50 (.mu.M) of a particular drug when tested with
patient-derived recombinant virus isolates, relative to the mean
IC.sub.50 (.mu.M) of the same drug obtained when tested with a
reference wild-type virus isolate (IIIB/LAI).
[0112] Genotypic analysis was performed by automated
population-based full-sequence analysis (ABI). Results of the
genotypic analysis are reported as amino acid changes at positions
along the reverse transcriptase gene compared to the wild-type
(HXB2) reference sequence. Cluster analysis by
VirtualPhenotype.RTM. interpretational allowed detection of the
occurrence of mutational pattern in the database containing the
genetic sequences of the clinical isolates and linkage with the
corresponding resistance profiles of the same isolates. (See PCT
EP01/04445)
[0113] For the modeling studies, mutations were generated in the RT
gene of HXB2, a wild-type laboratory HIV-1 strain, using the
QuikChange.sup.J Site-Directed Mutagenesis Kit, STRATAGENE.sup.7,
Stratagene Cloning systems, La Jolla, Calif., USA.
[0114] Analysis of the Clinical Isolates
[0115] Table 3 reports the frequency of mutations 44D/A, 118I,
184V, 215Y, and 41L in RT in clinical isolates with various levels
of phenotypic resistance to zidovudine (ZDV) and lamivudine (3TC).
The mutant isolates described here were drawn from a pool of
clinical isolates.
[0116] Table 3 reports the frequency of mutations 44D/A, 118I,
184V, 215Y, and 41L in RT in clinical isolates with various levels
of phenotypic resistance to zidovudine (ZDV) and lamivudine (3TC).
The mutant isolates described here were drawn from a pool of
clinical isolates.
3TABLE 3 Frequency of ZDV and 3TC Resistance-correlated Mutations
in Clinical Isolates Susceptible or Resistant to ZDV and/or 3TC
Compared to a Sample of Fully Susceptible Isolates Frequency (%) of
mutations ZDV 3TC resistance- resistance- correlated correlated
mutations mutations No. of Resistance Class.sup.a 41L 215Y 184V
44D/A 1181 samples ZDV (<4), 3TC (<4) 4.5 4.8 0 1.3 3.1 314
ZDV (<4), 3TC (>10) 18.3 18.8 90 1.3 6.3 240 ZDV (>10),
3TC (<4) 59.5 68.9 0 14.9 18.9 74 ZDV (>10), 3TC 77 72.2 4
30.2 39.7 126 (4, <50) ZDV (>10), 3TC (50) 77.5 66.9 84.1
28.5 37.8 151 .sup.aResistance (in parentheses) is expressed as the
fold increase in the mean IC.sub.50 of the drug relative to the
mean IC.sub.50 of the same drug for a wild-type reference
laboratory HIV-1 strain.
[0117] Isolates that are susceptible (WT) to both ZDV and 3TC
(n=195): the frequency of any of the six mutations listed above was
low.
[0118] Isolates that are resistant to ZDV (>10-fold, n=220):
Table 3 shows that the ZDV resistance-correlated mutations 215Y,
41L and 70R were high in frequency in this category and throughout
all 3TC resistance categories. Mutation 184V was the predominant
mutation in the high-resistance 3TC class (>50-fold), whereas
184V was rare in the intermediate 3TC resistance group and absent
in the low-level resistance group and the 3TC susceptible group.
The mutations 44D/A and 118I were present in all 3TC resistance
categories.
[0119] Isolates that are resistant to 3TC (>10-fold, n=295):
Table 3 shows that the frequency of the high-level 3TC
resistance-correlated mutation, 184V, was high in all ZDV
resistance categories (low, intermediate and high) and was the
predominant mutation in the ZDV susceptible and
intermediate-resistance group. As the resistance to ZDV increased,
so did the frequency of the ZDV resistance-correlated mutations
41L, 70R and 215Y, while the frequency of mutation 184V decreased.
Mutations 44D/A and 118I also substantially increased in frequency
as resistance to ZDV increased.
[0120] The results thus far show that low and intermediate
resistance to 3TC was not related to the presence of mutation 184V.
Indeed, this mutation was practically absent in these classes.
Table 3 further indicates that mutations 44D/A and 118I were
present in high frequencies only in the presence of ZDV resistance
mutations 215Y, 41L and 70R. In the isolates that were susceptible
to ZDV, the frequency of ZDV resistance-correlated mutations was
low and 44A/D and 118I were also rare, even though 3TC resistance
was greater than 10-fold. In this group the high frequency of 184V
accounted for the resistance to 3TC.
[0121] Analysis of the Mutants Generated by Site-Directed
Mutagenesis
[0122] Table 4 shows the codon changes introduced into a wild-type
HXB2 background together with the fold-resistance values obtained
when the different mutants were tested in the drug susceptibility
assay. All six mutants carrying mutation 184V were highly resistant
to 3TC. Two of them carried both 44D/A and 118I, while all but one
(SDM23) carried ZDV resistance-correlated mutations.
4TABLE 4 3TC and ZDV resistance-correlated mutations and phenotypic
resistance in mutants with site-directed mutations 3TC resistance
profile AZT resistance profile Fold Fold Mutant Mutation(s)
resistance.sup.a n.sup.b Mutations resistance SDM05 4 (0.3).sup.c
41L, 210W, 211K, 214F, 215Y 64 (15.0) 5 SDM18 2 (0.6) 41L, 67N,
210W, 211K, 214F, 215Y 45 (13.4) 3 SDM19 4 (0.7) 41L, 67N, 69D,
210W, 211K, 214F, 215Y 46 (18.2) 2 SDM28 44D 1 (0.1) 2 (0.3) 6
SDM31 44D 22 (2.5) 41L, 67N, 210W, 211K, 214F, 215Y 48 (11.8) 4
SDM32 44D 8 (2.2) 41L, 67N, 69D, 210W, 211K, 214F, 215Y 49 (5.9) 6
SDM29 1181 2 (0.2) 2 (0.4) 6 SDM33 1181 7 (1.0) 41L, 67N, 210W,
211K, 214F, 215Y 49 (8.0) 6 SDM34 1181 32 (3.9) 41L, 67N, 69D,
210W, 211K, 214F, 215Y 34 (14.4) 5 SDM30 44D, 1181 3 (0.3) 1 (0.4)
6 SDM35 44D, 1181 14 (1.4) 41L, 67N, 210W, 211K, 214F, 215Y 49
(9.6) 5 SDM36 44D, 1181 15 (2.1) 41L, 67N, 69D, 210W, 211K, 214D,
215Y 49 (10.6) 5 SDM22 184V 78 (16.3) 41L, 210W, 211K, 214F, 215Y 7
(0.9) 5 SDM23 184V 82 (13.6) 2 (0.5) 6 SDM24 184V 85 (14.2)
69S-S-S, 210W, 211K, 214D, 215Y 27 (16.2) 5 SDM26 184V 72 (13.8)
41L, 67N, 210W, 211K, 214F, 215Y 25 (1.4) 5 SDM38 184V, 44D, 1181
82 (13.5) 41L, 67N, 210W, 211K, 214F, 215Y 20 (4.1) 6 SDM39 184V,
44D, 1181 84 (13.9) 41L, 67N, 69D, 210W, 211K, 214F, 215Y 21 (5.3)
5 .sup.aFold increase in the mean IC.sub.50 of the drug relative to
the mean IC.sub.50 of the same drug for a wild-type reference
laboratory HIV-1 strain. .sup.bn, number of replicate tests run for
each phenotype drug resistance determination. .sup.cStandard errors
are indicated in parentheses.
[0123] All of the mutants followed the predicted ZDV resistance or
susceptibility pattern. At the same time, three mutants were
generated with a change at codon 44, three with a change at codon
118 and three with a change at both codons 44 and 118. Within each
of these three groups two mutants also carried changes at positions
correlated with resistance to ZDV, whereas one mutant remained
wild-type at those codons. The drug resistance values listed in
Table 4 clearly show that the presence of mutations at codon 44 and
118, singly or together, can cause intermediate resistance to 3TC
(8 to 32-fold), distinguishable from the high resistance to 3TC
(>62-fold) caused by mutation 184V. Moreover, the intermediate
resistance to 3TC was only observed when mutations at positions 44
and/or 118 occurred in a ZDV-resistant background (41L, 67N, 210W,
215Y) while resistance caused by mutation 184V was obviously not
related to ZDV resistance.
[0124] Relationship Between the Presence of Changes at RT Positions
44 or 118 in Clinical Samples and Antiretroviral Therapy
[0125] As can be deduced from Table 4, changes at position 44 and
118 may occur in virus samples with or without the M184V
substitution, but they appeared at higher incidence in samples with
ZDV resistance. It was therefore of interest to look at the
antiretroviral treatments administered to patients with
HIV-isolates that contained 44D or 118I. We identified a subset of
86 samples with 44D and 88 samples with 118I originating from
patients for whom antiretroviral histories were available. Although
it was not possible to draw conclusions regarding the incidence of
changes at 44 or 118 from this subset according to treatment
history, as this was not a randomized study, this analysis
nevertheless shed some light on the conditions that may lead to
mutations at these positions.
[0126] For the 44D subset, {fraction (50/86)} of the samples
originated from patients who were receiving lamivudine at the
sample date and 5 patients in this subset had never received 3TC
prior and up to the sample date. All 5 patients had received
zidovudine/didanosine at some time and all HIV-isolates were
wild-type at position 184. The zidovudine treatment experience was
extensive, as expected for historical reasons. All except one
patient had received zidovudine in combination with other NRTI's
and {fraction (70/86)} had also received zidovudine monotherapy in
the past. The one patient reported to be zidovudine naive had
received stavudine. This sample contained 41L and 215Y.
[0127] Results for 118I subset were similar in that 55/88 samples
originated from patients who were on lamivudine at the sample date
and 2 patients had never received lamivudine (both had received
zidovudine plus didanosine). Most patients, 83/88, had received
zidovudine in combination with other NRTI's, and 70 had also
received zidovudine monotherapy. The 5/88 zidovudine naive patients
had received stavudine. For a few patients, consecutive samples
showing the evolution of 44D or 118I were available.
[0128] These results indicate that mutations E44D/A and V118I in
HIV-1 RT confer a low to intermediate level of resistance to 3TC
when they occur in clinical isolates possessing a ZDV-resistant
background. The cluster analysis of genotypically and
phenotypically characterized clinical isolates and the results from
the site-directed mutagenesis experiment confirm that indeed
mutations at codons 44 and 118 are correlated with low and
intermediate level of resistance to 3TC, with the restriction that
ZDV resistance-correlated mutations be present. Additionally, the
analysis of the clinical samples for which therapy histories were
available and in which prior ZDV exposure was shown to be
extensive, confirmed the results obtained from our large clinical
data set in that mutations 44D/A and 118I appeared in the context
of ZDV mutations.
[0129] Mutations 44D/A and 118I each are capable of independently
generating resistance to 3TC. The experiment with site-directed
mutagenesis does not indicate the existence of synergistic effects
between the two mutants with respect to their phenotypic effect on
3TC resistance.
Example 2
Determining the Genetic Basis of HIV-1 Multi-Nucleoside
Resistance
[0130] 892 HIV-1 samples were surveyed in our resistance database
from patients failing therapy using a standardized
recombinant-based phenotypic assay and by DNA sequence analysis.
Multi-nucleoside resistance was correlated with complex mutational
patterns in the RT coding region. Plasma samples were obtained from
patients who had received antiretroviral therapy. Selection was on
the basis of a viral load >1000 HIV-1 RNA copies/ml and for the
purpose of this study, patients with this level of plasma HIV-1
were considered to be failing therapy.
[0131] Viral RNA was extracted from 200 .mu.L patient plasma using
the QIAAMP.RTM. Viral RNA Extraction Kit (Qiagen, Hilden, Germany),
according to the manufacturers instructions. cDNA encompassing part
of the pol gene was produced using Expand.TM. reverse transcriptase
(Boehringer Mannheim) as described previously. See Hertogs K., et
al., Antimicrob. Agents Chemother. 42: 269-276 (1998). A 2.2 kb
fragment encoding the protease and RT regions was then amplified by
nested polymerase chain reaction (PCR) using PCR primers and
conditions as described. Id. This genetic material was subsequently
used in both phenotyping and genotyping experiments.
[0132] MT-4 cells (Harada S., et al, Science 229: 563-566 (1985).)
were co-transfected with pol gene PCR fragments and the protease-RT
deleted HIV-1 molecular clone, pGEM3.quadrature.PRT, as described.
See Hertogs K., et al., Antimicrob. Agents Chemother. 42: 269-276
(1998). This resulted in viable recombinant viruses containing
protease/RT from the donor PCR fragment. Phenotypic susceptibility
to nucleoside analogues was determined using an MT-4 cell viral
cytopathic effect (CPE) protection assay as described. Id. Fold
resistance values were derived by dividing the mean IC.sub.50 for a
patient's recombinant virus by the mean IC.sub.50 for wild-type
control virus (strain HXB2-D). The PCR products obtained from
patient plasma samples were genotyped by dideoxynucleotide-based
sequence analysis. Samples were sequenced using the BigDye.TM.
terminator kit (Applied Biosystems, Inc. (ABI)) and resolved on an
ABI 377 DNA sequencer.
[0133] Mutations in the RT coding region were created by
site-directed mutagenesis of a wild-type HXB2-D EcoRI-PstI
restriction enzyme fragment, encompassing the HIV-1 pol gene and
cloned into pGEM3 (Promega). Single and multiple nucleotide changes
were introduced into RT using the ExSite.TM. mutagenesis kit
(Strategene). All mutant clones were verified by DNA sequence
analysis of the entire RT. PCR fragments were prepared from the
mutated clones and the altered RT coding regions were transferred
into the HIV-1 HXB2-D genetic background by homologous
recombination as described above. The susceptibility of these
recombinant viruses to nucleoside analogues was subsequently
determined by the MT-4 cell CPE protection assay. Id.
[0134] Phenotypic Susceptibility Analysis
[0135] The recombinant virus assay (Antivirogram.RTM.) was used to
determine simultaneously the susceptibility of the samples to AZT,
3TC, d4T ddI and ddC. From this analysis, 302 samples were
identified with four-fold or greater increases in IC.sub.50
(relative to a wild-type control virus) to at least four of these
inhibitors. Thus, a substantial number of MDR viruses were present
in the sample population.
[0136] Genotypic analysis of Multi-Nucleoside Resistant Samples
[0137] Genotypic analysis was performed on all 892 samples by
dideoxy-nucleotide sequencing. Complex patterns of multiple
mutations were seen in the RT coding regions of the MDR samples.
These included combinations of AZT and 3TC resistance mutations
(particularly 41L, 67N, 210W and 215Y with 184V/I) plus mutations
at codons 69 (T69A/N) and/or 75 (V75M). This analysis highlighted
the incidence of the codon 151 mutational cluster in the MDR group.
In addition, a novel family of amino acid insertions and
rearrangements between codons 67 and 70 were also prevalent in the
MDR group. These two patterns of mutations were correlated with
high-level phenotypic multi-nucleoside resistance (FIG. 2), 27
samples having the codon 151 cluster and 45 samples having
insertions and rearrangements (typically a T69S substitution,
followed by insertion of two amino acids). The mean fold increases
in IC.sub.50 to d4T, ddI and ddC for these different groups are
shown in FIG. 2. This analysis indicated that codon 69 insertion
mutants had a high degree of d4T and ddC resistance (>10-fold),
which was also seen with the codon 151 cluster. However, samples
with AZT and 3TC resistance mutations plus T69A/N or V175M showed
only modest levels of resistance to these drugs (FIG. 2). Not
surprisingly, all four groups shown in FIG. 2 were highly resistant
to AZT and 3TC (mean fold increase in AZT IC.sub.50 of >500-fold
and >30-fold for 3TC). This was because many MDR samples
contained mutations conferring AZT resistance (e.g., 41L, 67N, 210W
and 215Y) and 3TC resistance (Met184V/I).
[0138] Spectrum of Different Insertions Seen in the RT Codon 67 to
70 Region
[0139] The extensive variety of insertions in the, codon 67 to 70
region of RT is summarized in Table 3. The largest group (n=16) had
a T69S substitution followed by insertion of two S residues. The
next largest group (n=10) also had a T69S substitution but in this
case a different insertion of S-G. Samples with a number of
different double amino acids inserted after 69Ser were also
identified. In addition, insertions of two or three amino acids
between codons 68 and 69 were also seen. The positions of these
insertions were based on the fact that T69 and L70 were contiguous.
In some samples there were rarely observed substitutions at codon
67 (A67G/S/G), rather than the common 67N AZT resistance mutation.
In two samples deletion of codon 70 was observed (after insertion
of three residues between codons 68 and 69), and a single
substitution of T69S without an insertion was seen in four samples
(Table 3). The inserted residues did not show any obvious patterns
in terms of codon usage. For example, the S-S insertions were
rarely direct repeats of the S69 codon, suggesting that simple
reiterations of S69 could not account for the appearance of these
insertions in the RT.
[0140] Patients' Therapy Patterns in Relation to Codon 69
Insertions
[0141] The codon 69 insertions were always present in a background
of AZT resistance mutations, especially T215Y/F. This may not be
surprising as therapy histories from many of the patients whose
samples were analyzed in this study revealed a common pattern of
AZT therapy, followed by combination therapy with nucleosides and
protease inhibitors (data not shown). FIG. 3 shows typical
treatment patterns for three patients, indicating the time samples
were obtained for virological analysis. It was not possible from
these histories to determine precisely the nucleoside analog(s)
responsible for selecting codon 69 insertions. Sequential samples
from patient 1 revealed an interesting transition of 69S-[S-S] to
69S-[S-G] during a period of 3TC/d4T combination therapy.
[0142] Susceptibility Analysis of HIV-1 Variants Constructed by
Site-Directed Mutagenesis
[0143] To investigate the significance of the observed mutational
patterns correlated with MDR virus we constructed a series of
viruses by site-directed mutagenesis with specific changes in a
defined genetic background (HXB2-D). T69A or V75M in a background
of AZT mutations conferred little or no resistance to 3TC, d4T, ddl
or ddC. Variants were also constructed with 69S-[S-S], either alone
or together with two AZT resistance mutations (210W and 215Y). In
addition, the potential role of A62V, a substitution also
frequently correlated with the insertions was investigated by
adding this mutation to a background of 69S-[S-S] plus 210W/215Y.
Susceptibility data for six nucleoside analogues are summarized in
Table 4. These data showed that the 69S-[S-S] insertion alone did
not confer multi-nucleoside resistance. In fact, this virus only
had a significant decrease in susceptibility to 3TC. By contrast,
the variants with the insert plus AZT resistance mutations had
decreased susceptibility to AZT, 3TC, d4T, ddC and abacavir
(4-[(2-amino-6-cyclopro-
pyl-amino)-9H-purin-9-yl]-2-cyclopentene-1-methanol, 1592U89),
confirming that the 69 insertions plus AZT mutations conferred the
MDR phenotype.
Example 3
Use of Neural Networks to Define the Genetic Basis of HIV-1
Resistance to d4T
[0144] Three neural network models (the 9RT, 26RT and 60RT models)
were developed to investigate how mutation patterns influence d4T
resistance. The 9 RT model was based on the nine mutations listed
in the Stanford sequence database (http://www.hivb.stanford.edu)
associated with d4T resistance (62V, 69D, 69N, 69SXX, 751, 75T,
77L, 116Y, and 151M). The other models were based on adding either
the next 17 or 51 most frequent RT mutations present in d4T
resistant samples. Thus, the 26 RT mutation model included the 9 RT
mutation model plus the 17 most frequent mutations in d4T resistant
samples. These 17 mutations were 41L, 44D, 67N, 70R, 75A, 75M,
115F, 118I, 184V, 208Y, 210W, 214F, 215F, 215Y, 219E, 219N, and
219Q. The 60 RT mutation model consisted of the 26 RT mutation
model, plus the 34 next most frequent mutations in d4T resistant
samples. These 34 mutations were 20R, 35I, 39A, 43E, 60I, 65R,
122K, 123E, 135T, 162C, 177E, 196E, 200A, 207E, 211K, 228H, 272A,
277K, 286A, 293V, 297K, 329L, 356K, 357T, 358K, 359S, 360T, 371V,
375V, 376A, 386I, 390R, 399D, and 400A. In order to discover which
mutations had contributed to this improved prediction, improved
sample IS9-26 and IS9-60 were identified by comparing the
phenotypic outputs of the 9-model and 26-model, and the 9-model and
60-model on the test set. The corresponding genotypes of the
improved samples were collected and analyzed, all extra mutations
contained in the improved samples were screened out, and the
frequency of each mutation found in IS9-26 and IS9-60 was
calculated and compared with that of the mutation being found in
the whole samples. All mutations with higher difference of two
frequencies were identified and considered to play a role in
conferring resistance to d4T. In this example, the threshold
frequency was set to 9%. The following mutations were identified
from the 9- and 26-models: 41L (44%-79%), 44D (13%-26%), 67N
(36%-56%), 70R (21%-30%), 118I (21%-36%), 210W (34%-65%), and 215Y
(44%-81%). The following mutations were identified from the 9- and
60-models: 41L (44%-73%), 67N (36%-56%), 181I (21%-32%), 210W
(34%-62%), 211K (49%-59%), and 215Y (44%-74%). In conclusion, these
results show that at least 17 RT mutations (the 8 identified here
plus the 9 identified above from the Stanford Database) may confer
d4T resistance, including AZT resistance mutations. The results
also identified 10 other mutations that may also confer resistance:
184V (36%-42%), 214F (88%-94%), 75A (0.7%-0.6%), 75M (4%-8%), 115F
(1%-0.2%), 208Y (13%-21%), 215F (9%-11%), 219E (5%-4%), 219N
(4%-11%), and 219Q (12%-16%).
Example 4
[0145] Overview of the mean fold increase in resistance and the
effect of the 101 mutations in the HIV reverse transcriptase gene
sequence. The fold increase in resistance is calculated from the
increase in the mean IC.sub.50 of the drug relative to the mean
IC.sub.50 of the same drug for a wild-type reference laboratory
HIV-1 strain. The effect of the mutations was investigated on
Nevirapine, Delavirdine and Efavirenz. The results are displayed as
mean fold increase and the standard deviation thereof.
5TABLE 5 Overview of the reverse transcriptase mutations at
position 101 and their corresponding influence on the resistance
towards reverse transcriptase inhibitors. The data are expressed as
fold increase in resistance compared to a laboratory reference
strain (WT). Genotype Nevirapine Delavirdine Efavirenz WT 1.3 1.8
0.9 n 20 19 20 Stdev 0.5 0.6 0.8 101E 4.9 7.7 2.4 n 5 5 5 Stdev 5.8
20.4 2.7 101Q 1.9 1.2 0.9 n 5 5 5 Stdev 2.4 1.5 1.0 101P 53.4 162.4
84.2 n 20 17 18 Stdev 14.3 77.8 81.4 101H 24.0 45.5 3.5 n 23 23 23
Stdev 14.8 65.4 3.1
Example 5
[0146] Overview of the mean fold increase in resistance and the
effect of the 103 mutations in the HIV reverse transcriptase gene
sequence. The fold increase in resistance is calculated from the
increase in the mean IC.sub.50 of the drug relative to the mean
IC.sub.50 of the same drug for a wild-type reference laboratory
HIV-1 strain. The results are displayed as mean fold increase and
the standard deviation thereof.
6TABLE 6 Overview of the reverse transcriptase mutation at position
103 and their corresponding influence on the resistance towards
reverse transcriptase inhibitors. The data are expressed as mean
fold increase in resistance compared to a laboratory reference
strain (WT). Genotype Nevirapine Delavirdine Efavirenz WT 1.3 1.8
0.9 n 20 19 20 Stdev 0.5 0.6 0.8 103N 48.1 176.1 486.8 n 10 10 10
Stdev 13.8 83.8 153.3 103R 1.1 1.6 0.6 n 5 5 5 Stdev 2.8 2.1 1.3
103T 43.7 84.5 2.8 n 4 4 4 Stdev 32.0 61.7 1.2 103H 69.4 191.6 52.7
n 5 5 5 Stdev 16.5 105.2 23.4 103S 45.4 18.3 8.4 n 19 15 18 Stdev
9.0 9.6 3.8
Example 6
[0147] Overview of the mean fold increase in resistance and the
effect of the 181 mutations in the HIV reverse transcriptase gene
sequence. The fold increase in resistance is calculated from the
increase in the mean IC.sub.50 of the drug relative to the mean
IC.sub.50 of the same drug for a wild-type reference laboratory
HIV-1 strain. The results are displayed as mean fold increase and
the standard deviation thereof.
7TABLE 7 Overview of the reverse transcriptase mutation at position
181 and their corresponding influence on the resistance towards
reverse transcriptase inhibitors. The data are expressed as fold
increase in resistance compared to a laboratory reference strain
(WT). Genotype Nevirapine Delavirdine Efavirenz WT 1.3 1.8 0.9 n 20
19 20 Stdev 0.5 0.6 0.8 181C 56.0 82.5 2.0 n 11 11 11 Stdev 8.4
59.2 0.8 181I 63.8 81.7 2.0 n 5 5 5 Stdev 20.1 57.9 1.0 181S 82.8
147.9 8.7 n 5 5 4 Stdev 21.7 64.4 3.5
Example 7
[0148] Overview of the mean fold increase in resistance and the
effect of the 190 mutations in the HIV reverse transcriptase gene
sequence. The fold increase in resistance is calculated from the
increase in the mean IC.sub.50 of the drug relative to the mean
IC.sub.50 of the same drug for a wild-type reference laboratory
HIV-1 strain. The results are displayed as mean fold increase and
the standard deviation thereof.
8TABLE 8 Overview of the reverse transcriptase mutation at position
190 and their corresponding influence on the resistance towards
reverse transcriptase inhibitors. The data are expressed as fold
increase in resistance compared to a laboratory reference strain
(WT). Genotype Nevirapine Delavirdine Efavirenz WT 1.3 1.8 0.9 n 20
19 20 Stdev 0.5 0.6 0.8 190A 60.7 2.3 24.8 n 4 4 4 Stdev 12.6 0.7
11.3 190S 75.9 1.9 483.6 n 6 6 6 Stdev 21.8 3.4 231.1 190E 40.0
131.1 385.3 n 24 22 24 Stdev 7.3 61.2 22.0 190Q 52.7 34.0 401.1 n
22 21 22 Stdev 8.8 58.9 22.5
Example 8
Fold Increase in Resistance of Protease Inhibitors by the 84A
Mutation (in a Background)
[0149]
9TABLE 9 Overview of the mean fold increase in resistance and the
effect of the 84A mutation in the HIV protease sequence. The fold
increase in resistance is calculated from the increase in the mean
IC.sub.50 of the drug relative to the mean IC.sub.50 of the same
drug for a wild-type reference laboratory HIV-1 strain. # Genotype
Indinavir Ritonavir Nelfinavir Saquinavir Amprenavir 1 WT 0.9 0.6
0.7 0.9 1 n 6 6 6 6 6 Stdev 0.5 0.4 0.2 0.2 0.4 2 10I 0.7 1.15 1
0.9 1.1 n 4 4 4 4 4 Stdev 0.2 0.7 0.6 0.3 0.4 3 46I 0.6 0.8 1.5 0.6
1.2 n 5 5 5 5 5 Stdev 0.6 0.8 0.9 0.2 0.2 4 46I + 84V 2.6 4.6 5.6
2.5 2.3 n 2 2 2 2 2 Stdev 2.1 1.6 4.5 2.4 3.0 5 46I + 84A 26.8 51.8
61.9 38.1 7.7 n 5 5 5 5 5 Stdev 20.9 39.1 18.1 11 25.3 6 10I + 46I
2.9 1.5 0.8 0.5 5.6 n 5 5 5 5 5 Stdev 3.7 0.3 0.7 0.3 2.9 7 10I +
46I + 84V 2.4 11.9 3.1 4.1 4.5 n 4 4 4 4 4 Stdev 1.5 7.1 3.1 2.7
4.1 8 10I + 46I + 84A 33.4 77.3 43.9 48.2 17 n 5 5 5 5 5 Stdev 29.9
38.3 25 9.6 31.1 9 10L/I + 46I + 84A 44.5 114 56.2 46.4 24.9 n 5 5
5 5 5 Stdev 22.9 44.4 12.5 9.5 8.2 10 10I + 46I + 84A 26 44.5 36.9
42.8 11 n 4 5 5 5 5 Stdev 11 20 24.7 9.7 5 11 46I + 71V 3.7 1.4 1
0.6 2.2 n 5 5 5 5 4 Stdev 2.3 0.8 0.1 0.5 1.8 12 46I + 71V + 84V/I
3.1 1.8 0.7 0.6 2.2 n 1 1 1 1 1 Stdev 13 46I + 71V + 84A 56.5 102.2
59.9 47.7 23 n 6 6 6 6 5 Stdev 22.8 28 21.3 7.1 12.6 14 10F + 46I +
71V 22.2 4.1 45.2 1.9 4.2 n 5 5 5 5 4 Stdev 1.9 2.1 16.1 1.1 2.2 15
10I + 46I + 71V + 84V 50.5 45.6 57.1 15.9 17.8 n 14 14 14 14 12
Stdev 26.0 29.8 17.4 8.6 11.3 16 10I + 46I + 71V + 84A 45.9 114.9
54.9 48.2 15.2 n 5 5 5 5 5 Stdev 28.3 42.9 21.8 13.1 2.5 17 10V +
46I + 71T + 84A 57.9 91.9 51.2 48.2 39.8 n 5 5 5 5 5 Stdev 21.2
66.8 22.2 7.9 24.7 18 10I + 46I + 71V + 77I 2.2 1.65 6.1 1.3 0.9 n
6 6 5 6 6 Stdev 2.6 0.7 1.8 0.8 0.4 19 10I + 46I + 71V + 77I + 84V
2.2 3.1 2.8 2.5 1.4 n 3 3 3 3 3 Stdev 5.2 2.1 8.0 2.1 0.5 20 10I +
46I + 71V + 77I + 84A 57.9 121.3 68.6 47.1 15 n 5 5 5 5 4 Stdev
22.2 71.4 22.1 6.1 6.6 21 10I + 33L/I + 46I + 71V + 77V/I + 84A
31.4 38.6 63.1 44.7 10.9 n 5 5 6 6 5 Stdev 16 14.9 13.5 6 5.5
[0150] This table exemplifies that 84A mutation in a background of
PR mutations confers an additional effect towards the protease
inhibitors investigated. This indicates that even in a complex
background of mutations the 84A mutation has an effect. The 84A
mutation displays a different resistance profile compared with 84V,
and teaches that not only the presence but also the exact identity
of the mutation is important.
Example 9
Fold Increase in Resistance of Protease Inhibitors by the 84L
Mutation (in a Background)
[0151]
10TABLE 10 Overview of the mean fold increase in resistance and the
effect of the 84L mutation in the HIV-1 protease sequence. The fold
increase in resistance is calculated from the increase in the mean
IC50 of the drug relative to the mean IC50 of the same drug for a
wild-type reference laboratory HIV-1 strain. # Genotype Indinavir
Ritonavir Nelfinavir Saquinavir Amprenavir 1 10I + 36I + 71V 1.4
1.2 1.5 1.6 1.0 n 6 6 6 6 6 Stdev 0.5 0.5 0.6 0.8 0.6 2 10I + 36I +
71I + 84V 1.0 1.5 0.8 0.8 1.1 n 1 1 1 1 1 Stdev 3 10I + 36I + 71I +
84L 3.7 0.8 39.4 10.2 0.3 n 4 5 5 5 5 Stdev 3.4 0.9 17.4 6.5
0.2
[0152] Genotyping of the sample displays that the 84L mutation is
linked to protease resistance as compared to the result obtained
from the 84V mutation in the same background.
Example 10
Fold Increase in Resistance of Protease Inhibitors by the 84C
Mutation (and in a Background)
[0153]
11TABLE 11 Overview of the mean fold increase in resistance and the
effect of the 84C mutation in the HIV protease sequence The fold
increase in resistance is calculated from the increase in the mean
IC50 of the drug relative to the mean IC50 of the same drug for a
wild-type reference laboratory HIV-1 strain. # Genotype Indinavir
Ritonavir Nelfinavir Saquinavir Amprenavir 1 WT 0.9 0.6 0.7 0.9 1.0
n 6 6 6 6 6 Stdev 0.5 0.4 0.2 0.2 0.4 2 84V 0.7 2.4 0.9 0.7 2.0 n 9
9 9 9 2 Stdev 0.9 2.0 0.8 0.6 0.9 3 84C 2.7 2.9 51.2 26.4 5.9 n 5 5
5 4 5 Stdev 1.4 3.2 16.2 6.8 3.1 4 10I 0.7 1.2 1.0 0.9 1.1 n 4 4 4
4 4 Stdev 0.2 0.7 0.6 0.3 0.4 5 10I + 84V 1.8 6.5 2.0 2.7 1.2 n 3 3
3 3 3 Stdev 1.1 2.9 3.1 1.3 0.3 6 10I + 84C 4.3 5.4 61.9 37.1 3.4 n
7 7 7 7 5 Stdev 3.2 3.8 18.0 16.5 1.5 7 77I 0.4 0.4 0.9 0.3 0.6 n 3
3 3 3 3 Stdev 0.1 0.3 0.3 0.1 0.3 8 77I + 84V 2.5 5.0 2.8 3.5 3.3 n
1 1 1 1 1 Stdev 9 77I + 84C 3.1 2.8 61.9 18.1 2.2 n 5 4 5 5 5 Stdev
1.7 1.3 24.1 13.3 1.2 10 77I + 84C 3.2 3.2 38.2 18.5 4.9 n 5 6 6 5
6 Stdev 2.9 1.4 22.3 12.1 2.5 11 10I + 24I + 36I + 46I + 71V + 84C
7.5 47 51.7 46.4 12.8
[0154] The unique 84C mutation confers resistance towards protease
inhibitors. The appearance of the 84C mutation is indicative of but
not limited to Nelfinvir resistance.
Example 11
Fold Increase in Resistance of Protease Inhibitors by the 54M
Mutation (in a Background)
[0155]
12TABLE 12 Influence of the 54M mutation in a background of
different protease mutations and their corresponding effect on
protease inhibitor resistance. The effect for the investigated
compounds is expressed as a mean fold change in IC50 towards the
mean IC50 determined for a wild type laboratory HIV strain.
Indinavir Ritonavir Nelfinavir Saquinavir Fold Fold Fold Fold
Amprenavir change in change in change in change in Fold change
Virco ID Genotype IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 in
IC.sub.50 V021667 10I 0.7 1.2 1.0 0.9 1.1 V053832 10I + 33F 0.4 2.9
1.6 0.5 0.6 V052977 10I + 33F + 54M 1.8 35.5 3.8 0.5 12.2 V048878
10I + 33F + 54M + 71V + 77I + 90M 5.2 17.2 7.3 4.0 13.7 V048879 10I
+ 33F + 54M + 71V + 77I + 90M 6.5 74.0 45.7 51.8 14.1 V052978 10I +
33F + 54M + 71V + 90M 5.4 17.9 29.1 9.9 19.3 V055199 10I + 33F +
54M + 90M 2.3 18.4 11.7 5.6 38.7 V052953 10I + 33F + 77I 0.8 1.1
0.9 1.2 1.5 V048880 10I + 33F + 77I + 90M 1.3 3.6 4.9 2.7 3.8
V048877 10I + 33F + 77I + 90M 2.9 20.5 9.4 1.5 7.9 V052979 10I +
54M 1.8 20.0 3.8 1.4 10.4 V052982 10I + 54M + 71V + 77I + 90M 2.4
13.4 10.1 4.2 3.1 V052981 10I + 54M + 71V + 90M 3.2 9.4 8.7 5.4 8.9
V048869 10I + 54M + 77I + 90M 7.1 20.0 28.5 5.1 9.3 V052980 10I +
54M + 90M 1.3 4.1 7.0 1.4 1.4 V052942 10I + 71V 0.6 0.6 0.9 0.8 0.5
V052949 10I + 71V + 77I + 90M 2.7 2.3 8.6 2.1 0.6 V052948 10I + 71V
+ 90M 8.6 10.5 9.0 4.8 2.0 V030496 10I + 77I 0.4 0.5 0.7 0.3 0.5
V052947 10I + 77I + 90M 2.9 6.9 8.2 3.9 1.2 V052943 10I + 90M 0.5
1.8 2.2 1.1 0.4 V052944 10I + 90M 0.5 1.5 1.4 0.7 0.7 V052950 33F
0.6 1.5 1.0 1.2 0.7 V048872 33F + 54M + 77I + 90M 3.2 8.7 7.4 5.2
30.3 V048873 33F + 54M + 77I + 90M 1.7 11.4 7.7 3.1 37.5 V052955
33F + 71V + 90M 5.3 77.9 36.6 20.4 2.7 V052951 33F + 77I 0.4 0.3
5.2 0.4 0.3 V052954 33F + 77I + 90M 1.7 2.2 5.9 1.7 2.1 V052940 71V
0.7 0.4 0.6 0.8 0.3 V052946 71V + 90M 0.9 2.0 3.0 1.4 0.5 V19263
77I 0.4 0.4 0.9 0.3 0.6 V052945 77I + 90M 0.6 1.9 3.0 1.4 0.3
V20160 WT 0.9 0.6 0.7 0.9 1.0
Example 12
Fold Increase in Resistance of Protease Inhibitors by the 54L
Mutation (in a Background)
[0156]
13TABLE 13 Influence of 54L mutation in a background of different
protease mutations and their corresponding effect on protease
inhibitor resistance. The effect for the investigated compounds is
expressed as a mean fold change in IC.sub.50 towards the mean
IC.sub.50 determined for a wild type laboratory HIV strain.
Ritonavir Nelfinavir Saquinavir Indinavir Fold Fold Fold Amprenavir
Fold change change in change in change in Fold change Virco ID
Genotype in IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 in IC.sub.50
V021667 10I 0.7 1.2 1.0 0.9 1.1 V053832 10I + 33F 0.4 2.9 1.6 0.5
0.6 V048875 10I + 33F + 54L + 71V + 77I + 90M 3.7 25.1 12.5 12.0
24.6 V048874 10I + 33F + 54L + 71V + 77I + 90M 2.1 21.3 14.5 4.6
32.1 V052969 10I + 33F + 54L + 90M 1.3 4.8 3.4 1.4 10.4 V052953 10I
+ 33F + 77I 0.8 1.1 0.9 1.2 1.5 V048880 10I + 33F + 77I + 90M 1.3
3.6 4.9 2.7 3.8 V048877 10I + 33F + 77I + 90M 2.9 20.5 9.4 1.5 7.9
V052971 10I + 54L 0.7 1.5 1.0 0.7 2.6 V052976 10I + 54L + 71V + 77I
+ 90M 1.7 6.5 8.9 1.8 2.0 V048868 10I + 54L + 71V + 77I + 90M 6.2
9.7 15.6 16.5 4.1 V052975 10I + 54L + 71V + 90M 10.2 30.5 41.3 24.2
6.2 V052973 10I + 54L + 77I 0.8 1.4 2.8 0.8 2.1 V052974 10I + 54L +
90M 1.7 3.6 2.6 1.1 1.2 V052942 10I + 71V 0.6 0.6 0.9 0.8 0.5
V052949 10I + 71V + 77I + 90M 2.7 2.3 8.6 2.1 0.6 V052948 10I + 71V
+ 90M 8.6 10.5 9.0 4.8 2.0 V030496 10I + 77I 0.4 0.5 0.7 0.3 0.5
V052947 10I + 77I + 90M 2.9 6.9 8.2 3.9 1.2 V052943 10I + 90M 0.5
1.8 2.2 1.1 0.4 V052944 10I + 90M 0.5 1.5 1.4 0.7 0.7 V052968 10L/I
+ 33F/L + 54L + 77I 1.3 4.7 3.5 1.5 2.9 V052950 33F 0.6 1.5 1.0 1.2
0.7 V052955 33F + 71V + 90M 5.3 77.9 36.6 20.4 2.7 V052951 33F +
77I 0.4 0.3 5.2 0.4 0.3 V052954 33F + 77I + 90M 1.7 2.2 5.9 1.7 2.1
V052970 54L 0.6 2.0 1.6 0.7 0.8 V053854 54L + 71V 1.7 2.3 5.9 0.9
5.0 V052972 54L + 77I 0.8 2.4 3.0 1.0 1.1 V052940 71V 0.7 0.4 0.6
0.8 0.3 V052946 71V + 90M 0.9 2.0 3.0 1.4 0.5 V19263 77I 0.4 0.4
0.9 0.3 0.6 V052945 77I + 90M 0.6 1.9 3.0 1.4 0.3 V20160 WT 0.9 0.6
0.7 0.9 1.0
Example 13
Fold Increase in Resistance of Protease Inhibitors by the 54V
Mutation (in a Background)
[0157]
14TABLE 14 Influence of 54V on protease resistance in a background
of different mutations and their corresponding effect on drug
resistance. The effect for the investigated compounds is expressed
as a mean fold change in IC50 towards the mean IC50 determined for
a wild type laboratory HIV strain. Indinavir Ritonavir Nelfinavir
Saquinavir Fold Fold Fold Fold Amprenavir change in change in
change in change in Fold change Virco ID Genotype IC.sub.50
IC.sub.50 IC.sub.50 IC.sub.50 in IC.sub.50 V021667 10I 0.7 1.2 1.0
0.9 1.1 V053832 10I + 33F 0.4 2.9 1.6 0.5 0.6 214846 10I + 33F +
54V + 71V + 77I + 90M 13.8 57.6 43.5 27.8 4.2 V052960 10I + 33F +
54V + 71V + 90M 9.9 70.9 40.7 21.8 6.4 V052959 10I + 33F + 54V +
77I + 90M 12.8 28.5 7.5 1.0 7.0 V052958 10I + 33F + 54V + 77I + 90M
16.3 16.6 4.8 1.0 7.8 V052957 10I + 33F + 54V + 90M 1.4 13.9 6.9
1.6 5.2 V052953 10I + 33F + 77I 0.8 1.1 0.9 1.2 1.5 V048880 10I +
33F + 77I + 90M 1.3 3.6 4.9 2.7 3.8 V048877 10I + 33F + 77I + 90M
2.9 20.5 9.4 1.5 7.9 V052962 10I + 54V 0.8 1.0 0.6 0.9 0.5 V052967
10I + 54V + 71V + 77I + 90M 0.4 0.7 0.8 0.6 0.4 V052966 10I + 54V +
71V + 90M 7.7 44.5 55.4 28.6 1.5 V052965 10I + 54V + 77I + 90M 8.2
12.5 15.1 6.3 0.7 V052942 10I + 71V 0.6 0.6 0.9 0.8 0.5 V052949 10I
+ 71V + 77I + 90M 2.7 2.3 8.6 2.1 0.6 V052948 10I + 71V + 90M 8.6
10.5 9.0 4.8 2.0 V030496 10I + 77I 0.4 0.5 0.7 0.3 0.5 V052947 10I
+ 77I + 90M 2.9 6.9 8.2 3.9 1.2 V052943 10I + 90M 0.5 1.8 2.2 1.1
0.4 V052944 10I + 90M 0.5 1.5 1.4 0.7 0.7 V052950 33F 0.6 1.5 1.0
1.2 0.7 V053831 33F + 54V 5.4 129.7 5.9 1.0 7.5 V053358 33F + 54V +
77I + 90M 1.2 4.9 2.1 1.3 1.0 V048876 33F + 54V + 77I + 90M 0.6 5.6
1.6 1.5 1.1 V052955 33F + 71V + 90M 5.3 77.9 36.6 20.4 2.7 V052951
33F + 77I 0.4 0.3 5.2 0.4 0.3 V052954 33F + 77I + 90M 1.7 2.2 5.9
1.7 2.1 V052961 54V 0.7 1.2 1.0 1.0 0.5 V052964 54V + 71V + 90M 0.7
7.1 3.3 1.4 0.3 V052963 54V + 77I + 90M 1.2 2.0 3.2 1.3 0.5 V052940
71V 0.7 0.4 0.6 0.8 0.3 V052946 71V + 90M 0.9 2.0 3.0 1.4 0.5
V19263 77I 0.4 0.4 0.9 0.3 0.6 V052945 77I + 90M 0.6 1.9 3.0 1.4
0.3 V20160 WT 0.9 0.6 0.7 0.9 1.0
[0158] All references, patents, and patent applications cited
herein are incorporated by reference in their entirety.
[0159] It will be apparent to those skilled in the art that various
modifications and variations can be made in the compositions and
methods of the present invention without departing from the spirit
or scope of the invention. Thus, it is intended that the present
description cover the modifications and variations of this
invention provided that they come within the scope of the appended
claims and their equivalents.
Sequence CWU 1
1
2 1 99 PRT Human immunodeficiency virus 1 Pro Gln Val Thr Leu Trp
Gln Arg Pro Leu Val Thr Ile Lys Ile Gly 1 5 10 15 Gly Gln Leu Lys
Glu Ala Leu Leu Asp Thr Gly Ala Asp Asp Thr Val 20 25 30 Leu Glu
Glu Met Ser Leu Pro Gly Arg Trp Lys Pro Lys Met Ile Gly 35 40 45
Gly Ile Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Gln Ile Leu Ile 50
55 60 Glu Ile Cys Gly His Lys Ala Ile Gly Thr Val Leu Val Gly Pro
Thr 65 70 75 80 Pro Val Asn Ile Ile Gly Arg Asn Leu Leu Thr Gln Ile
Gly Cys Thr 85 90 95 Leu Asn Phe 2 560 PRT Human immunodeficiency
virus 2 Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly
Met 1 5 10 15 Asp Gly Pro Lys Val Lys Gln Trp Pro Leu Thr Glu Glu
Lys Ile Lys 20 25 30 Ala Leu Val Glu Ile Cys Thr Glu Met Glu Lys
Glu Gly Lys Ile Ser 35 40 45 Lys Ile Gly Pro Glu Asn Pro Tyr Asn
Thr Pro Val Phe Ala Ile Lys 50 55 60 Lys Lys Asp Ser Thr Lys Trp
Arg Lys Leu Val Asp Phe Arg Glu Leu 65 70 75 80 Asn Lys Arg Thr Gln
Asp Phe Trp Glu Val Gln Leu Gly Ile Pro His 85 90 95 Pro Ala Gly
Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp Val Gly 100 105 110 Asp
Ala Tyr Phe Ser Val Pro Leu Asp Glu Asp Phe Arg Lys Tyr Thr 115 120
125 Ala Phe Thr Ile Pro Ser Ile Asn Asn Glu Thr Pro Gly Ile Arg Tyr
130 135 140 Gln Tyr Asn Val Leu Pro Gln Gly Trp Lys Gly Ser Pro Ala
Ile Phe 145 150 155 160 Gln Ser Ser Met Thr Lys Ile Leu Glu Pro Phe
Arg Lys Gln Asn Pro 165 170 175 Asp Ile Val Ile Tyr Gln Tyr Met Asp
Asp Leu Tyr Val Gly Ser Asp 180 185 190 Leu Glu Ile Gly Gln His Arg
Thr Lys Ile Glu Glu Leu Arg Gln His 195 200 205 Leu Leu Arg Trp Gly
Leu Thr Thr Pro Asp Lys Lys His Gln Lys Glu 210 215 220 Pro Pro Phe
Leu Trp Met Gly Tyr Glu Leu His Pro Asp Lys Trp Thr 225 230 235 240
Val Gln Pro Ile Val Leu Pro Glu Lys Asp Ser Trp Thr Val Asn Asp 245
250 255 Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr
Pro 260 265 270 Gly Ile Lys Val Arg Gln Leu Cys Lys Leu Leu Arg Gly
Thr Lys Ala 275 280 285 Leu Thr Glu Val Ile Pro Leu Thr Glu Glu Ala
Glu Leu Glu Leu Ala 290 295 300 Glu Asn Arg Glu Ile Leu Lys Glu Pro
Val His Gly Val Tyr Tyr Asp 305 310 315 320 Pro Ser Lys Asp Leu Ile
Ala Glu Ile Gln Lys Gln Gly Gln Gly Gln 325 330 335 Trp Thr Tyr Gln
Ile Tyr Gln Glu Pro Phe Lys Asn Leu Lys Thr Gly 340 345 350 Lys Tyr
Ala Arg Met Arg Gly Ala His Thr Asn Asp Val Lys Gln Leu 355 360 365
Thr Glu Ala Val Gln Lys Ile Thr Thr Glu Ser Ile Val Ile Trp Gly 370
375 380 Lys Thr Pro Lys Phe Lys Leu Pro Ile Gln Lys Glu Thr Trp Glu
Thr 385 390 395 400 Trp Trp Thr Glu Tyr Trp Gln Ala Thr Trp Ile Pro
Glu Trp Glu Phe 405 410 415 Val Asn Thr Pro Pro Leu Val Lys Leu Trp
Tyr Gln Leu Glu Lys Glu 420 425 430 Pro Ile Val Gly Ala Glu Thr Phe
Tyr Val Asp Gly Ala Ala Asn Arg 435 440 445 Glu Thr Lys Leu Gly Lys
Ala Gly Tyr Val Thr Asn Arg Gly Arg Gln 450 455 460 Lys Val Val Thr
Leu Thr Asp Thr Thr Asn Gln Lys Thr Glu Leu Gln 465 470 475 480 Ala
Ile Tyr Leu Ala Leu Gln Asp Ser Gly Leu Glu Val Asn Ile Val 485 490
495 Thr Asp Ser Gln Tyr Ala Leu Gly Ile Ile Gln Ala Gln Pro Asp Gln
500 505 510 Ser Glu Ser Glu Leu Val Asn Gln Ile Ile Glu Gln Leu Ile
Lys Lys 515 520 525 Glu Lys Val Tyr Leu Ala Trp Val Pro Ala His Lys
Gly Ile Gly Gly 530 535 540 Asn Glu Gln Val Asp Lys Leu Val Ser Ala
Gly Ile Arg Lys Val Leu 545 550 555 560
* * * * *
References