U.S. patent application number 13/672761 was filed with the patent office on 2013-09-19 for novel hiv-1 reverse transcriptase codon deletion and its use in the management and treatment of hiv infections.
This patent application is currently assigned to EMORY UNIVERSITY. The applicant listed for this patent is EMORY UNIVERSITY. Invention is credited to Raymond F. Schinazi.
Application Number | 20130244964 13/672761 |
Document ID | / |
Family ID | 39745528 |
Filed Date | 2013-09-19 |
United States Patent
Application |
20130244964 |
Kind Code |
A1 |
Schinazi; Raymond F. |
September 19, 2013 |
NOVEL HIV-1 REVERSE TRANSCRIPTASE CODON DELETION AND ITS USE IN THE
MANAGEMENT AND TREATMENT OF HIV INFECTIONS
Abstract
The present invention provides an isolated HIV-1 mutant and
isolated nucleic acid molecules comprising HIV-RT coding sequences
harboring a novel mutation in the S68 codon, and in particular,
deletions of the S68 codon. This novel deletion reduces the
sensitivity of HIV to various nucleoside reverse transcriptase
inhibitors. Methods of using this mutation for selecting effective
antiretroviral agents in vitro and in vivo, methods for monitoring
infection progression in HIV-infected individuals and methods for
avoiding the emergence of and/or to treat individuals infected with
HIV comprising mutations, including deletions, at the S68 codon of
HIV-RT are provided.
Inventors: |
Schinazi; Raymond F.;
(Atlanta, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EMORY UNIVERSITY |
Atlanta |
GA |
US |
|
|
Assignee: |
EMORY UNIVERSITY
Atlanta
GA
|
Family ID: |
39745528 |
Appl. No.: |
13/672761 |
Filed: |
November 9, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12595358 |
Oct 21, 2009 |
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PCT/US2008/004666 |
Apr 10, 2008 |
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13672761 |
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60922838 |
Apr 10, 2007 |
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Current U.S.
Class: |
514/45 ;
514/230.5; 514/263.23; 514/263.4; 514/274; 514/49; 514/50;
514/81 |
Current CPC
Class: |
Y10S 435/911 20130101;
C12Q 1/703 20130101; G16B 20/00 20190201; G16B 50/00 20190201; C12N
9/1276 20130101 |
Class at
Publication: |
514/45 ; 514/50;
514/274; 514/230.5; 514/263.4; 514/49; 514/263.23; 514/81 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under Grant
No. 5R37-AI-41980 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1-10. (canceled)
11. A method of treating an HIV-infected individual with an
effective antiretroviral therapy, comprising: (i) collecting
lymphocytic cells from an HIV-infected individual; (ii) determining
whether the cells comprise nucleic acid encoding HIV-RT sequences
comprising a codon 68 deletion, wherein if HIV-RT sequences
comprising a codon 68 deletion are present, an antiretroviral
therapy is selected which inhibits production of HIV-RT codon 68
deletion variant RNA in the cells; and (iii) administering to the
individual the selected antiretroviral therapy.
12. The method of claim 11, wherein if the HIV-infected individual
was undergoing an antiretroviral treatment prior to step (i), the
treatment is altered based on the determination step (ii).
13. (canceled)
14. The method of claim 12, wherein the alteration of treatment
comprises administering at least one antiretroviral agent that
reduces or eliminates RNA production by the HIV variant having a
codon 68 deletion.
15. The method of claim 14, wherein the codon 68 deletion removes
AGT of codon 68 or GTA spanning codons 68 and 69.
16. The method of claim 14, wherein the at least one antiretroviral
agent is selected from the group consisting of a protease
inhibitor, a non-nucleoside reverse transcriptase inhibitor, an HIV
fusion inhibitor, an HIV integrase inhibitor, an RNAse H inhibitor,
a CD4 binding inhibitor, a CXCR4 binding inhibitor and a CCR5
binding inhibitor.
17. The method of claim 14, wherein the at least one antiretroviral
agent is selected from the group consisting of AZT, DDI, DFDOC,
D4T, DOT and DDC.
18. The method of claim 11, wherein the absence of or decreasing
concentrations of detectable HIV sequences correlates positively
with the assessment that the antiretroviral agent is
therapeutically effective in treating the codon 68 mutant HIV.
19-42. (canceled)
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/595,358, which is a national stage entry of
PCT/US2008/004666, filed Apr. 10, 2008, which claims priority from
U.S. Provisional Patent Application No. 60/922,838, filed Apr. 10,
2007, the contents of which are hereby incorporated by reference in
their entireties.
BACKGROUND OF THE INVENTION
[0003] In 1983, the etiological cause of AIDS was determined to be
the human immunodeficiency virus (HIV). In 1985, it was reported
that the synthetic nucleoside 3'-azido-3'-deoxythymidine
(Zidovudine, AZT, ZDV) inhibits the replication of human
immunodeficiency virus by inhibiting in its 5'-triphosphate form
the HIV-1 reverse transcriptase (HIV-RT). HIV-RT is active early in
the viral replication cycle and is necessary for continued viral
replication. Currently, a total eight synthetic nucleosides have
been approved by the US FDA. These are: AZT (mentioned above),
2',3'-dideoxyinosine (Videx, DDI), 2',3'-dideoxycytidine (DDC),
2',3'-dideoxy-2',3'-didehydrothymidine (stavudine, D4T),
cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane
(emtricitabine, FTC),
(-)-cis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane
(Lamivudine, 3TC),
(1S,4R)-4-[2-amino-6-(cyclopropyl-amino)-9H-purin-9-yl]-2-cyclopent-
ene-1-methanol succinate (abacavir, ABC), and the acyclic
nucleotide
9-[(R)-2-[[bisaisopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]a-
denine fumarate (tenofovir-DF, TDF). All nucleoside reverse
transcriptase inhibitors (NRTI) require phosphorylation to their
triphosphate (TP) forms, while metabolic activation of tenofovir
requires phosphorylation to its 12 diphosphate (tenofovir-DP). Such
so-called NRTI mimic natural nucleosides in the cell. After
cellular phosphorylation to the 5'-triphosphate by cellular
kinases, these synthetic nucleosides can be incorporated into a
growing strand of viral DNA, causing chain termination due to the
absence of the 3'-hydroxyl group found in natural nucleosides that
are used in the DNA chain elongation reaction catalyzed by HIV-RT.
NRTI therapies in HIV treatment are reviewed in Schinazi et al.,
Antiviral Research 71:322-334 (2006)).
[0004] HIV shows high genetic variability in part as a result of
its fast viral replication cycle coupled with the high mutation
rate of and active recombinogenic characteristics of HIV-RT,
especially during viral replication in single cells co-infected by
multiple different strains of HIV. Drug-resistant variants of HIV
can emerge after treatment with an antiviral agent. Drug resistance
most typically occurs by mutation of a gene that encodes for an
enzyme used in viral replication, and most typically in the case of
HIV, reverse transcriptase, protease, or DNA polymerase. NRTI
treatment of HIV-1 infected individuals often leads to the
emergence of mutations in the reverse transcriptase (RT). Less
frequently seen are codon insertions or deletions, either which add
or subtract three nucleotides and leave other codons in the correct
coding frame. Codon insertions (ins) and deletions (del) have been
associated with multi-drug resistance (MDR) in clinical samples
obtained from HIV-1 infected individuals treated with
antiretroviral agents (67del, 69del, 69ins, 70del).
[0005] The .beta.3-.beta.4 hairpin loop of the finger domain of RT
is thought to be directly involved in the interaction of the enzyme
with its substrates (the template-primer complex and the dNTP)
(Tamalet et al., Virol. 270:310-316 (2000)). Genetic rearrangements
in the .beta.3-.beta.4 loop have been found in patients extensively
treated with anti-HIV drugs and experiencing therapeutic failure
(Tamalet, supra; Winters et al., J. Virol. 74(22):10707-10713
(2000)).
[0006] The efficacy of a drug against HIV infection can be
prolonged, augmented, or restored by administering the compound in
combination or alternation with a second, and in particular a
third, antiviral compound that induces a different mutation from
that caused by the principle drug. Alternatively, the
pharmacokinetics, biodistribution, or other parameter of the drug
can be altered by such combination or alternation therapy, although
this is not recommended for HIV infections. In general, combination
therapy is typically preferred over alternation therapy because it
induces multiple simultaneous pressures on the virus. One cannot
predict, however, what mutations will be induced in the HIV-1
genome by a given new drug, whether the mutation is permanent or
transient, or how an infected cell with a mutated HIV-1 sequence
will respond to therapy with other agents in combination or
alternation. This is exacerbated by the fact that there is a
paucity of data on the kinetics of drug resistance in long-term
cell cultures treated with modern antiretroviral agents.
[0007] HIV-1 variants resistant to AZT, DDI, 3TC, D4T, DDC, ABC or
TDF have been isolated from patients receiving long term
monotherapy with these drugs (Larder et al., Science 1989;
243:1731-4; St. Clair et al., Science 1991; 253:1557-9; and
Fitzgibbon et al., Antimicrob. Agents Chemother. 1992; 36:153-7;
Schinazi, et al., Intl. Antiviral News 2000; 8:65-92). Mounting
clinical evidence indicates that AZT and 3TC resistance is a
predictor of poor clinical outcome in both children and adults. The
rapid development of HIV-1 resistance to non-nucleoside reverse
transcriptase inhibitors (NNRTI) has also been reported both in
cell culture and in human clinical trials (Nunberg et al. J. Virol.
1991; 65(9):4887-92; Richman et al., Proc Natl Acad Sci (USA) 1991;
88:11241-5; Mellors et al., Mol. Pharm. 1992; 41:446-51; Richman D
D and the ACTG 164/168 Study Team. Second International HIV-1 Drug
Resistance Workshop. (Noordwijk, the Netherlands. 1993); and Saag
et al., N Engl J Med 1993; 329:1065-1072). In the case of the NNRTI
L'697,661, drug-resistant HIV-1 emerged within 2-6 weeks of
initiating therapy in association with the return of viremia to
pretreatment levels. (Saag et al., supra). Breakthrough viremia
associated with the appearance of drug-resistant strains has also
been noted with other classes of HIV-1 inhibitors, including
protease, fusion and integrase inhibitors. This experience has led
to the realization that the potential for HIV-1 drug resistance
must be assessed early on in the preclinical evaluation of all new
therapies for HIV-1.
[0008] The emergence of resistant HIV strains during viral therapy
has presented a major challenge to delay, prevent or attenuate the
onset of resistance. Common resistance mutations, including
thymidine associated mutations (TAM), K65R and M184V are
problematic in HIV drug development. Mutations observed to emerge
following exposure to various NRTI are summarized in Schinazi et
al., supra, 2006 (see Table 1). Novel NRTI are under pre-clinical
development that are good substrates for cellular kinases, have
high bioavailability (especially oral), reduced toxicity and
significant levels of activity against the commonly found
NRTI-resistant HIV-1 mutants, such as D67N, K70R, T215Y, K219Q,
K65R and M184V (Chu et al., J. Med. Chem. 48:3949-3952 (2005)).
[0009] 2',3'-Dideoxy-2',3'-didehydro-5-fluoro-cytidine (D4FC, DFC;
dexelvucitabine) is a known NRTI compound (see, e.g., EP 0 409 227
A2, U.S. Pat. Nos. 5,703,058 and 5,905,070). Treatment with
.beta.-L-D4FC rapidly selects for a mutation at codon 184
(methionine to valine) of the reverse transcriptase region of the
virus, resulting in a high level of resistance to 3TC and FTC.
.beta.-D-D4FC, in contrast, is not significantly cross-resistant to
AZT, DDC, DDI, D4T, 3TC, (-)-FTC or .beta.-L-D4FC. .beta.-D-D4FC
treatment selects for HIV-1 variants having mutations at codons
163L, K65R, K70N, K70E, or R172K of the HIV-RT region of the virus
(see also Hammond et al., Antimicrob. Agents Chemother.
49(9):3930-3932 (2005)). Thus, .beta.-D-D4FC can be used generally
as salvage therapy for any HIV-infected individual that exhibits
resistance to other anti-HIV agents whose drug resistance patterns
correlate with mutations at codons different from those selected by
3-D-D4FC treatment. Based on this, methods for treating HIV have
been reported that involve administering .beta.-D-D4FC or its
pharmaceutically acceptable salt or prodrug in combination or
alternation with a drug that selects for variants having one or
more mutations in HIV-1 at a location other than codons 163L, K65R,
K70N, K70E, or R172K (U.S. Pat. No. 7,115,584, and Hammond et
al.).
[0010] Current treatments for HIV infection are most often those
referred to as "highly active antiretroviral therapy" or HAART and
involve administering combinations ("cocktails") comprising at
least three drugs--two NRTI in combination with either a protease
inhibitor or a NNRTI. Results of studies on the emergence of drug
resistance and correlations between antiviral drugs and mutation
patterns present in selected HIV variant genes are useful in
directing resistance testing of viruses from HIV-infected
individuals treated with antiviral agents such as NRTI and in
choosing combinations of nucleoside analogs for treatment and
prevention of drug resistant HIV. Characterization of these
mutations is key in determining potential cross-resistance and in
HIV treatment management. It is thus desirable to understand more
about NRTI resistance patterns and how they correlate with HIV
genotypes and mutations in essential HIV genes, such as HIV-RT.
SUMMARY OF THE INVENTION
[0011] The present invention addresses the problems above by
identifying a novel deletion in HIV-1 RT of the S68 codon
("S68del"; which may alternatively be a deletion of the AGT codon
68 trinucleotide, or of the adjacent +1 frameshift trinucleotide
GTA) revealed during the selection of virus with dexelvucitabine
(DFC) in primary human lymphocytes. The novel S68 deletion and the
distinct multi-drug resistant phenotype it imparts on HIV may be an
important variable in NRTI multidrug resistance, management of
HIV-infected persons and improved treatment strategies.
[0012] The S68 deletion was investigated phenotypically against
selected antiviral agents for resistance and demonstrated
resistance to several clinically important NRTI. The S68del
produced greater than 30-fold increased resistance to DFC,
lamivudine, emtricitabine, tenofovir, abacavir and amdoxovir. As
expected, the S68del demonstrated no resistance to NNRTI and
protease inhibitors.
[0013] Codon 68 mutants, and S68del in particular, are expected to
precede immunologic decline of an infected individual over time.
Once the codon 68 mutation has been detected in plasma HIV RNA or
lymphocytes of an HIV-infected individual, a specific therapeutic
regimen is considered. In cases in which the HIV-infected
individual is already undergoing antiretroviral therapy, an
alteration in the therapeutic regimen is preferably considered.
[0014] In certain embodiments, the invention thus provides a
nucleic acid molecule comprising sequences encoding part or all of
HIV-1 RT, the HIV-RT sequences comprising a codon 68 mutation,
provided that when the codon 68 mutation is an S68 substitution, it
occurs alone or in combination with a mutation other than a K65R
mutation. The invention also provides a nucleic acid molecule
comprising sequences encoding part or all of HIV-1 RT, the HIV-RT
sequences comprising a codon 68 mutation, wherein the codon 68
mutation is the only mutation in the HIV-RT sequences. The
invention further provides a nucleic acid molecule comprising
sequences encoding part or all of HIV-1 RT, the HIV-RT sequences
comprising a codon 68 deletion wherein the S68 deletion removes the
codon 68 AGT trinucleotide, or wherein the S68 deletion removes the
GTA trinucleotide spanning codons 68 and 69. Preferably, isolated
nucleic acid molecules of the invention comprise a minimum of nine,
preferably of 10-25 or more nucleotides so that they may be used as
selective primers, e.g., in nucleic acid amplification methods, or
as probes in nucleic acid hybridization techniques.
[0015] The invention also provides an isolated HIV-1 or HIV-2
comprising any of the previously described nucleic acids.
[0016] The present invention also provides a method of evaluating
the effectiveness of an antiretroviral agent or preventing or
treating HIV infection of cells, comprising: (i) treating cells
with an antiretroviral agent; (ii) infecting cells with an HIV-1
(or HIV-2) having a codon 68 mutation in the reverse transcriptase
coding sequence, provided that when the codon 68 mutation is an S68
substitution, it is not in combination with a K65R mutation; and
(iii) determining the effect of the agent on viral replication;
wherein steps (i) and (ii) can be performed in any order.
[0017] In one embodiment, the invention provides a method of
selecting an effective antiretroviral therapy for an HIV-infected
person, the method comprising: (i) collecting a plasma sample from
an HIV-infected person who is being treated with an antiretroviral
agent; and (ii) determining whether the plasma sample comprises
nucleic acid encoding HIV-RT sequences comprising a codon 68
mutation, provided that when the codon 68 mutation is an S68
substitution, it is not in combination with a K65R mutation. In
certain embodiments, the codon 68 mutation is determined by a
method comprising polymerase chain reaction. In certain of such
embodiments, the polymerase chain reaction is nested. In other such
embodiments, the polymerase chain reaction is real-time. In further
embodiments, the method comprising polymerase chain reaction
utilizes primers SK38: ATA ATC CAC CTA TCC CAG TAG GAG AAA T (SEQ
ID NO: 1) and SK39: TTT GGT CCT TGT CTT ATG TCC AGA ATG C (SEQ ID
NO: 2).
[0018] It may be desirable after detecting the codon 68, e.g.,
S68del, mutation to alter the course of the person's current
treatment regimen to include one or more antiretroviral agents that
are effective in inhibiting the replication of an HIV mutant
comprising an S68 mutation, e.g. S68del.
[0019] In another embodiment, the invention provides a method of
selecting an effective antiretroviral therapy for an HIV-infected
individual, comprising: (i) collecting lymphocytic cells from an
HIV-infected individual; and (ii) determining whether the cells
comprise nucleic acid encoding HIV-RT sequences comprising a codon
68 mutation, wherein if HIV-RT sequences comprising a codon 68
mutation are present, an antiretroviral therapy is selected which
inhibits production of HIV-RT codon 68 mutant variant RNA in the
cells, provided that when the codon 68 mutation is an S68
substitution, it is not in combination with a K65R mutation.
[0020] In certain embodiments, the invention provides a method of
selecting an effective antiretroviral therapy for an HIV-infected
individual, comprising: (i) collecting lymphocytic cells from an
HIV-infected individual; and (ii) determining whether the cells
comprise nucleic acid encoding HIV-RT sequences comprising a codon
68 mutation, wherein if HIV-RT sequences comprising a codon 68
mutation are present, an antiretroviral therapy is selected which
inhibits production of HIV-RT codon 68 mutant variant RNA in the
cells, provided that when the codon 68 mutation is an S68
substitution, it is not in combination with a K65R mutation,
wherein if the HIV-infected individual was undergoing an
antiretroviral treatment prior to step (i), the treatment is
altered based on the determination step (ii).
[0021] In further embodiments, the invention provides a method for
evaluating the effectiveness to an HIV-infected individual of a
selected antiretroviral agent or therapy, the method comprising:
(i) collecting a sample from an HIV-infected individual; and (ii)
determining whether the sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at codon 68, e.g., S68del,
in which the presence of the mutation correlates positively with
refractoriness of the individual to the selected antiretroviral
therapy and, if the therapy remains unchanged, to accelerated
immunologic decline of the HIV-infected individual compared to
HIV-infected individuals who do not have the mutation.
[0022] In any of the above methods, the alteration of treatment may
comprise administering at least one antiretroviral agent that
reduces or eliminates RNA production by the HIV variant having a
codon 68 mutation. In certain embodiments, the at least one
antiretroviral agent is selected from a protease inhibitor, a
non-nucleoside reverse transcriptase inhibitor, an HIV fusion
inhibitor, an HIV integrase inhibitor, an RNAse H inhibitor, a CD4
binding inhibitor, a CXCR4 binding inhibitor and a CCR5 binding
inhibitor. In certain embodiments, the at least one antiretroviral
agent is selected from: AZT, DDI, DFDOC, D4T, DOT and DDC.
[0023] In any of the above methods, the absence of, or decreasing
concentrations of, detectable HIV sequence correlates positively
with the assessment that the antiretroviral agent is
therapeutically effective in treating a codon 68, e.g., S68del,
mutation. Moreover, in this method, the presence of, or increasing
concentrations of, detectable HIV sequence correlates positively
with the assessment that the antiretroviral agent is
therapeutically ineffective and that a resistant virus has
developed.
[0024] In another embodiment, the invention provides methods for
evaluating the effectiveness to an HIV-infected individual of
treatment with an antiretroviral agent or therapy prone to
emergence of a codon 68 mutation, the method comprising (i)
collecting a sample from an HIV-infected individual before
treatment with a selected antiretroviral agent prone to emergence
of a codon 68 mutation; (ii) collecting a sample from the
HIV-infected individual after treatment with the selected
antiretroviral agent; (iii) amplifying separately HIV-encoding
nucleic acid in the samples from (i) and (ii) with HIV primers;
(iv) comparing the HIV nucleic acid copy number in samples (i) and
(ii), wherein a ratio of HIV nucleic acid copy number in samples
(i) and (ii) of greater than about 2.5 to 1, 4 to 1, 5 to 1, 10 to
1 or more, correlates positively with the assessment that the
selected antiretroviral agent has not selected an HIV-RT codon 68
mutation, e.g., S68del, and remains therapeutically effective. In
certain embodiments, such methods may additionally or optionally
(e.g., in step (iii)) comprise the use of HIV primers that can
distinguish between the presence and absence of a codon 68
mutation, e.g., S68del in HIV-RT.
[0025] In certain other embodiments, the invention provides methods
for evaluating the effectiveness to an HIV-infected individual of
treatment with an antiretroviral agent or therapy prone to
emergence of a codon 68 mutation, the method comprising: (i)
collecting at least one sample from an HIV-infected individual at
separate time intervals; (ii) amplifying HIV-encoding nucleic acid
in the separate samples using HIV primers; (iii) measuring HIV
nucleic acid copy numbers using the amplification products of step
(ii); and (iv) comparing the HIV nucleic acid copy numbers in the
samples collected during the course of the selected treatment;
whereby a statistically significant decline in HIV nucleic acid
copy numbers detected over the course of the treatment correlates
positively with the assessment that the selected antiretroviral
agent has not selected an HIV-RT codon 68 mutation, e.g., S68del,
and remains therapeutically effective. In certain embodiments, such
methods may additionally or optionally (e.g., in step (ii))
comprise the use of HIV primers that can distinguish between the
presence and absence of a codon 68 mutation, e.g., S68del in
HIV-RT.
[0026] In any of the above products or methods of the invention,
the HIV-RT codon 68 mutation may be an S68 deletion that removes
AGT or that removes the GTA trinucleotide spanning codons 68 and
69.
[0027] In certain embodiments of the present invention, the HIV
specific primers used in the methods of the invention can
distinguish between the presence and absence of a HIV reverse
transcriptase codon 68 mutation, and more particularly, of the
S68del mutation. Examples of such primers include, without
limitation, SK38 Primer: ATA ATC CAC CTA TCC CAG TAG GAG AAA T (SEQ
ID NO: 1) and SK39 Primer: TTT GGT CCT TGT CTT ATG TCC AGA ATG C
(SEQ ID NO: 2). The presence of amplified product may also be
detected with the SK19 probe: ATC CTG GGA TTA AAT AAA ATA GTA AGA
ATG TAT AG (SEQ ID NO: 3). Other HIV specific primers may easily be
designed by those of skill in the art that can detect and
differentiate codon 68 mutations, including those that distinguish
between S68 substitutions and the codon 68 deletion referred to
herein as "S68del".
[0028] The present invention also provides methods for treating a
person infected with HIV comprising the step of administering over
time an antiviral agent that does not select for an HIVvariant
having a codon 68 mutation in the HIV-RT coding sequence. The codon
68 mutation is an S68 deletion that removes AGT or that removes the
GTA trinucleotide spanning codons 68 and 69. In certain
embodiments, the antiretroviral agent is one that is effective at
inhibiting viral replication of an HIV-1 mutant comprising an S68
mutation, e.g., an S68 deletion so that viral RNA production is
reduced or eliminated. In further embodiments, the antiretroviral
agent is selected from an HIV protease inhibitor such as
Lopinavir.RTM.; an HIV fusion inhibitor such as a CD4 binding
inhibitor, a CXCR4 binding inhibitor or a CCR5 binding inhibitor
such as Maraviroc; an HIV integrase inhibitor such as Raltegravir;
an RNAseH inhibitor; an NNRTI such as Sustiva.RTM.. In certain
embodiments, the antiretroviral agent is an NRTI that inhibits
replication of an HIV-1 S68 mutant at concentrations that are no
more than 5-fold (and preferably no more than 2.5-fold) higher than
the concentration of the agent required to inhibit viral
replication of wild-type HIV-1. In certain preferred embodiments,
the antiretroviral agent is selected from: AZT, DDI, DFDOC, D4T,
DOT or DDC. In yet other preferred embodiments, the antiretroviral
agent is AZT. In yet other preferred embodiments, the
antiretroviral agent is DDI.
[0029] In other embodiments, the invention provides a kit
comprising at least one pair of primers designed to detect the
presence of a codon 68 mutation in HIV-RT coding sequences. In
further embodiments, the kit may be designed to detect the codon 68
deletion that removes the AGT trinucleotide encoding S68 or the GTA
trinucleotide spanning codons 68 and 69. The kit may comprise at
least one primer is selected from SK38 and SK39. The kit may
further comprise a nucleic acid probe comprising or consisting
essentially of the following nucleic acid sequence (SK19): ATC CTG
GGA TTA AAT AAA ATA GTA AGA ATG TAT AG (SEQ ID NO: 3).
[0030] In certain embodiments, the invention provides a nucleic
acid product of priming with primers SK38 (SEQ ID NO: 1) and SK39
(SEQ ID NO: 2), wherein the nucleic acid product comprises
sequences encoding HIV-1 RT, the HIV-RT sequences comprising a
codon 68 mutation, provided that when the codon 68 mutation is an
S68 substitution, it is not in combination with a K65R
mutation.
[0031] In other embodiments, the invention provides a nucleic acid
product of priming with primers SK38 (SEQ ID NO: 1) and SK39 (SEQ
ID NO: 2), wherein the nucleic acid product comprises sequences
encoding HIV-1 RT, the HIV-RT sequences comprising a codon 68
mutation, wherein the codon 68 mutation is an S68 deletion that
removes AGT or that removes the GTA trinucleotide spanning codons
68 and 69.
[0032] In certain embodiments, the invention provides any of the
above nucleic acids or nucleic acid products attached to a solid
support. In further embodiments, the invention provides an array
comprising any of the above nucleic acids or nucleic acid
products.
[0033] In certain embodiments, the invention provides use of an
antiretroviral agent to produce a composition useful in treating a
subject infected with HIV-1, wherein the antiretroviral agent is
one which does not select for an HIV-1 variant comprising a codon
68 mutation in the HIV-RT coding sequence. In further embodiments,
the codon 68 mutation is an S68 deletion that removes AGT or that
removes the GTA trinucleotide spanning codons 68 and 69. In certain
embodiments, the antiretroviral agent is selected from: a protease
inhibitor, a non-nucleoside reverse transcriptase inhibitor, an HIV
fusion inhibitor, an HIV integrase inhibitor, an RNAse H inhibitor,
a CD4 binding inhibitor, a CXCR4 binding inhibitor and a CCR5
binding inhibitor. In further embodiments, the antiretroviral agent
is selected from: AZT, DDI, DFDOC, D4T, DOT and DDC.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 is a graph showing percent inhibition by DFC
(dexelvucitabine) and the amounts of DFC (in .mu.m) used during
isolation of the S68del virus.
[0035] FIG. 2 is a graph showing the results of drug inhibition of
the S68del virus. Inhibition was measured using the .sup.3H-TTP RT
incorporation assay. Fold increases were calculated relative to
HIV.sub.LAI. FI50--fold increase in 50% effective concentration.
FI90--fold increase in 90% effective concentration.
AZT--3'-azido3'-deoxythymidine; DFC--dexelvucitabine;
DAPD--(-)-beta-D-2,6-diaminopurine dioxolane; 3TC--lamivudine; (-)
FTC--emtricitabine; TDF--tenofovir disoproxil fumarate;
DOT--1-(beta-D-dioxolane)thymine; D4T--stavudine; DDI--didanosine;
DDC--zalcitabine; D-FDOC--2',3'-dideoxy-5-fluoro-oxacytidine;
DXG--(-)-9-(beta-D-dioxolane)guanine.
[0036] FIG. 3 is a graph showing the results of non-nucleoside
reverse transcriptase inhibitor (Sustiva.RTM.) and protease
inhibitor (Lopinavir.RTM.) inhibition of the S68del virus.
Inhibition was measured using the .sup.3H-TTP RT incorporation
assay. Fold increases were calculated relative to HIV.sub.LAI. FI
EC.sub.50--fold increase in 50% effective concentration. FI
EC.sub.90--fold increase in 90% effective concentration.
[0037] FIG. 4 is a graph generated by the HIV-1 Real-Time PCR assay
for quantifying S68del virus levels in human peripheral blood
mononuclear (PBM) cells. Cp--cycle number of crossing point.
[0038] FIG. 5 is a graph showing the results of a heteropolymeric
DNA colorimetric RT assay performed with particle-derived S68del
and M184V RTs. FI50--fold increase in 50% effective
concentration.
[0039] FIG. 6 is a graph showing the results of a heteropolymeric
DNA colorimetric RT assay performed with recombinant S68del,
virally-derived S68del and virally-derived M184V RTs. The
recombinant S68del enzyme was tested in two separate experiments in
duplicate. The standard errors for AZT-TP, (-) FTC-TP and DFC-TP
were 0.1, 2 and 0.2, respectively. TP--triphosphate.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0040] As used herein, a "phenotypic change" in an HIV mutant is
one that confers a statistically significant change in viral
replication rates in the presence of a select antiviral compound or
agent, defined herein to be at least a 2.5-fold, and preferably at
least a 5-fold or higher increase in EC.sub.50 compared to native
virus in a constant cell line. Similarly, a "resistant virus"
refers to a virus that exhibits a 2.5-fold, and more typically,
five- or greater fold increase in EC.sub.50 compared to naive virus
in a constant cell line, including, but not limited to PBM, MT2 or
MT4 cells. The term "resistance" is used in its most general sense
and includes total resistance or partial resistance or decreased
sensitivity to a nucleoside analogue.
[0041] The term "D-D4FC" is used herein interchangeably with the
terms .beta.-D-D4FC and DFC, below. The S68del mutation was
selected in and confers resistance to DFC, and is expected to
confer resistance to certain DFC prodrugs.
[0042] As used herein, the term "prodrug" refers to the 5' and
N.sup.4 acylated, alkylated, or phosphorylated (including mono, di,
and triphosphate esters as well as stabilized phosphates and
phospholipid) derivatives of D-D4FC. In one embodiment, the acyl
group is a carboxylic acid ester in which the non-carbonyl moiety
of the ester group is selected from straight, branched, or cyclic
alkyl, alkoxyalkyl including methoxymethyl, aralkyl including
benzyl, aryloxyalkyl including phenoxymethyl, aryl including phenyl
optionally substituted by halogen, alkyl, alkyl or alkoxy,
sulfonate esters such as alkyl or aralkyl sulphonyl including
methanesulfonyl, trityl or monomethoxytrityl, substituted benzyl,
trialkylsilyl, or diphenylmethylsilyl. Aryl groups in the esters
optimally comprise a phenyl group. The alkyl group can be straight,
branched or cyclic and is preferably C.sub.1 to C.sub.18.
[0043] As used herein, "S68del" refers to a novel deletion of
sequences at codon 68 of HIV-RT, e.g., HIV-1 RT encoding sequences,
which may alternatively be a deletion of the AGT codon 68
trinucleotide, or of the adjacent +1 frameshift trinucleotide
GTA.
[0044] As used herein, "a codon 68 mutation" refers to a codon 68
substitution or S68del, but not a larger deletion that encompasses
S68del.
[0045] The following terms, unless otherwise indicated, shall be
understood to have the following meanings:
[0046] The term "allelic variant" refers to one of two or more
alternative naturally-occurring forms of a gene, wherein each gene
possesses a unique nucleotide sequence. In a preferred embodiment,
different alleles of a given gene have similar or identical
biological properties.
[0047] The term "polynucleotide" or "nucleic acid molecule" refers
to a polymeric form of nucleotides of at least 10 bases in length.
The term includes DNA molecules (e.g., cDNA or genomic or synthetic
DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as
analogs of DNA or RNA containing non-natural nucleotide analogs,
non-native internucleoside bonds, or both. The nucleic acid can be
in any topological conformation. For instance, the nucleic acid can
be single-stranded, double-stranded, triple-stranded, quadruplexed,
partially double-stranded, branched, hairpinned, circular, or in a
padlocked conformation. The term includes single and double
stranded forms of DNA.
[0048] Unless otherwise indicated, a "nucleic acid comprising SEQ
ID NO: X" refers to a nucleic acid, at least a portion of which has
either (i) the sequence of SEQ ID NO: X, or (ii) a sequence
complementary to SEQ ID NO: X. The choice between the two is
dictated by the context. For instance, if the nucleic acid is used
as a probe, the choice between the two is dictated by the
requirement that the probe be complementary to the desired
target.
[0049] An "isolated" or "substantially pure" nucleic acid or
polynucleotide (e.g., an RNA, DNA or a mixed polymer) is one which
is substantially separated from other cellular components that
naturally accompany the native polynucleotide in its natural host
cell, e.g., ribosomes, polymerases, and genomic sequences with
which it is naturally associated. The term embraces a nucleic acid
or polynucleotide that (1) has been removed from its naturally
occurring environment, provided that it is not an unidentified
member of a library which has not been separated from other
members, (2) is not associated with all or a portion of a
polynucleotide in which the "isolated polynucleotide" is found in
nature, (3) is operatively linked to a polynucleotide which it is
not linked to in nature, or (4) does not occur in nature. The term
"isolated" or "substantially pure" also can be used in reference to
recombinant or cloned DNA isolates, chemically synthesized
polynucleotide analogs, or polynucleotide analogs that are
biologically synthesized by heterologous systems.
[0050] However, "isolated" does not necessarily require that the
nucleic acid or polynucleotide so described has itself been
physically removed from its native environment. For instance, an
endogenous nucleic acid sequence in the genome of an organism is
deemed "isolated" herein if a heterologous sequence (i.e., a
sequence that is not naturally adjacent to this endogenous nucleic
acid sequence) is placed adjacent to the endogenous nucleic acid
sequence, such that the expression of this endogenous nucleic acid
sequence is altered. By way of example, a non-native promoter
sequence can be substituted (e.g., by homologous recombination) for
the native promoter of a gene in the genome of a human cell, such
that this gene has an altered expression pattern. This gene would
now become "isolated" because it is separated from at least some of
the sequences that naturally flank it.
[0051] A nucleic acid is also considered "isolated" if it contains
any modifications that do not naturally occur to the corresponding
nucleic acid in a genome. For instance, an endogenous coding
sequence is considered "isolated" if it contains an insertion,
deletion or a point mutation introduced artificially, e.g., by
human intervention. An "isolated nucleic acid" also includes a
nucleic acid integrated into a host cell chromosome at a
heterologous site, a nucleic acid construct present as an episome.
Moreover, an "isolated nucleic acid" can be substantially free of
other cellular material, or substantially free of culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0052] As used herein, the phrase "degenerate variant" of a
reference nucleic acid sequence encompasses nucleic acid sequences
that can be translated, according to the standard genetic code, to
provide an amino acid sequence identical to that translated from
the reference nucleic acid sequence.
[0053] The term "percent sequence identity" or "identical" in the
context of nucleic acid sequences refers to the residues in the two
sequences which are the same when aligned for maximum
correspondence. The length of sequence identity comparison may be
over a stretch of at least about nine nucleotides, usually at least
about 20 nucleotides, more usually at least about 24 nucleotides,
typically at least about 28 nucleotides, more typically at least
about 32 nucleotides, and preferably at least about 36 or more
nucleotides. There are a number of different algorithms known in
the art which can be used to measure nucleotide sequence identity.
For instance, polynucleotide sequences can be compared using FASTA,
Gap or Bestfit, which are programs in Wisconsin Package Version
10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides
alignments and percent sequence identity of the regions of the best
overlap between the query and search sequences (Pearson, 1990). For
instance, percent sequence identity between nucleic acid sequences
can be determined using FASTA with its default parameters (a word
size of 6 and the NOPAM factor for the scoring matrix) or using Gap
with its default parameters as provided in GCG Version 6.1, herein
incorporated by reference.
[0054] The term "substantial homology" or "substantial similarity,"
when referring to a nucleic acid or fragment thereof, indicates
that, when optimally aligned with appropriate nucleotide insertions
or deletions with another nucleic acid (or its complementary
strand), there is nucleotide sequence identity in at least about
50%, more preferably 60% of the nucleotide bases, usually at least
about 70%, more usually at least about 80%, preferably at least
about 90%, and more preferably at least about 95%, 96%, 97%, 98%,
99%, 99.5% or 99.9% of the nucleotide bases, as measured by any
well-known algorithm of sequence identity, such as FASTA, BLAST or
Gap.
[0055] Alternatively, substantial homology or similarity exists
when a nucleic acid or fragment thereof hybridizes to another
nucleic acid, to a strand of another nucleic acid, or to the
complementary strand thereof, under stringent hybridization
conditions. "Stringent hybridization conditions" and "stringent
wash conditions" in the context of nucleic acid hybridization
experiments depend upon a number of different physical parameters.
Nucleic acid hybridization will be affected by such conditions as
salt concentration, temperature, solvents, the base composition of
the hybridizing species, length of the complementary regions, and
the number of nucleotide base mismatches between the hybridizing
nucleic acids, as will be readily appreciated by those skilled in
the art. One having ordinary skill in the art knows how to vary
these parameters to achieve a particular stringency of
hybridization.
[0056] In general, "stringent hybridization" is performed at about
25.degree. C. below the thermal melting point (Tm) for the specific
DNA hybrid under a particular set of conditions. "Stringent
washing" is performed at temperatures about 5.degree. C. lower than
the Tm for the specific DNA hybrid under a particular set of
conditions. The Tm is the temperature at which 50% of the target
sequence hybridizes to a perfectly matched probe. See Sambrook et
al., supra, page 9.51, hereby incorporated by reference. For
purposes herein, "high stringency conditions" are defined for
solution phase hybridization as aqueous hybridization (i.e., free
of formamide) in 6.times.SSC (where 20.times.SSC contains 3.0 M
NaCl and 0.3 M sodium citrate), 1% SDS at 65.degree. C. for 8-12
hours, followed by two washes in 0.2.times.SSC, 0.1% SDS at
65.degree. C. for 20 minutes. It will be appreciated by the skilled
worker that hybridization at 65.degree. C. will occur at different
rates depending on a number of factors including the length and
percent identity of the sequences which are hybridizing.
[0057] The nucleic acids (also referred to as polynucleotides) of
this invention may include both sense and antisense strands of RNA,
cDNA, genomic DNA, and synthetic forms and mixed polymers of the
above. They may be modified chemically or biochemically or may
contain non-natural or derivatized nucleotide bases, as will be
readily appreciated by those of skill in the art. Such
modifications include, for example, labels, methylation,
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as uncharged
linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.), charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), pendent moieties
(e.g., polypeptides), intercalators (e.g., acridine, psoralen,
etc.), chelators, alkylators, and modified linkages (e.g., alpha
anomeric nucleic acids, etc.) Also included are synthetic molecules
that mimic polynucleotides in their ability to bind to a designated
sequence via hydrogen bonding and other chemical interactions. Such
molecules are known in the art and include, for example, those in
which peptide linkages substitute for phosphate linkages in the
backbone of the molecule.
[0058] The term "mutated" when applied to nucleic acid sequences
means that nucleotides in a nucleic acid sequence may be inserted,
deleted or changed compared to a reference nucleic acid sequence. A
single alteration may be made at a locus (a point mutation) or
multiple nucleotides may be inserted, deleted or changed at a
single locus. In addition, one or more alterations may be made at
any number of loci within a nucleic acid sequence. A nucleic acid
sequence may be mutated by any method known in the art including
but not limited to mutagenesis techniques such as "error-prone PCR"
(a process for performing PCR under conditions where the copying
fidelity of the DNA polymerase is low, such that a high rate of
point mutations is obtained along the entire length of the PCR
product. See, e.g., Leung, D. W., et al., Technique, 1, pp. 11-15
(1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic.,
2, pp. 28-33 (1992)); and "oligonucleotide-directed mutagenesis" (a
process which enables the generation of site-specific mutations in
any cloned DNA segment of interest. See, e.g., Reidhaar-Olson, J.
F. & Sauer, R. T., et al., Science, 241, pp. 53-57 (1988)).
[0059] The term "vector" as used herein is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Other vectors include
cosmids, bacterial artificial chromosomes (BAC) and yeast
artificial chromosomes (YAC). Another type of vector is a viral
vector, wherein additional DNA segments may be ligated into the
viral genome (discussed in more detail below). Certain vectors are
capable of autonomous replication in a host cell into which they
are introduced (e.g., vectors having an origin of replication which
functions in the host cell). Other vectors can be integrated into
the genome of a host cell upon introduction into the host cell, and
are thereby replicated along with the host genome. Moreover,
certain preferred vectors are capable of directing the expression
of genes to which they are operatively linked. Such vectors are
referred to herein as "recombinant expression vectors" (or simply,
"expression vectors").
[0060] "Operatively linked" expression control sequences refers to
a linkage in which the expression control sequence is contiguous
with the gene of interest to control the gene of interest, as well
as expression control sequences that act in trans or at a distance
to control the gene of interest.
[0061] The term "expression control sequence" as used herein refers
to polynucleotide sequences which are necessary to affect the
expression of coding sequences to which they are operatively
linked. Expression control sequences are sequences which control
the transcription, post-transcriptional events and translation of
nucleic acid sequences. Expression control sequences include
appropriate transcription initiation, termination, promoter and
enhancer sequences; efficient RNA processing signals such as
splicing and polyadenylation signals; sequences that stabilize
cytoplasmic mRNA; sequences that enhance translation efficiency
(e.g., ribosome binding sites); sequences that enhance protein
stability; and when desired, sequences that enhance protein
secretion. The nature of such control sequences differs depending
upon the host organism; in prokaryotes, such control sequences
generally include promoter, ribosomal binding site, and
transcription termination sequence. The term "control sequences" is
intended to include, at a minimum, all components whose presence is
essential for expression, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences.
[0062] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which a
recombinant vector has been introduced. It should be understood
that such terms are intended to refer not only to the particular
subject cell but to the progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein. A
recombinant host cell may be an isolated cell or cell line grown in
culture or may be a cell which resides in a living tissue or
organism.
[0063] The term "peptide" as used herein refers to a short
polypeptide, e.g., one that is typically less than about 50 amino
acids long and more typically less than about 30 amino acids long.
The term as used herein encompasses analogs and mimetics that mimic
structural and thus biological function.
[0064] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Exemplary methods and materials are described below, although
methods and materials similar or equivalent to those described
herein can also be used in the practice of the present invention
and will be apparent to those of skill in the art. All publications
and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the present specification,
including definitions, will control. The materials, methods, and
examples are illustrative only and not intended to be limiting.
S68del HIV-1 Mutant
[0065] In the present invention, the inventors have identified a
mutant HIV-1 with a novel mutation in the HIV reverse transcriptase
(RT) coding sequences. The novel mutation is a deletion in HIV-1 RT
of the S68 codon ("S68del"). The S68del mutation was revealed
during the selection of virus to dexelvucitabine (DFC) in primary
human lymphocytes. The mutation reduces the sensitivity of HIV to
nucleoside analogues to varying extents. The identification of this
HIV-RT mutant and characterization of its phenotypic properties
(e.g., its resistance and sensitivity profiles) are important for
the development of assays to monitor NRTI treatment regimens and to
screen for agents which can overcome the effects of the
mutation.
[0066] HIV codon 68 mutants of the invention may be in isolated
form such that they have undergone at least one purification step
away from naturally occurring body fluids. Alternatively, the
mutants may be maintained in isolated body fluid. In certain
embodiments, the mutants may be in RNA or DNA form. The present
invention also includes infectious molecular clones and nucleic
acids comprising the genome or parts thereof from an HIV harboring
a codon 68 mutation, such as, e.g., S68del.
[0067] It is possible that an HIV harboring a codon 68 mutation,
such as, e.g., S68del, shows a greater relative fitness compared to
wild-type HIV. Relative fitness can be measured by pairwise growth
competition assays (Hu et al., J. Acquir. Immune Defic. Syndr.
2007; 45:494-500; Winters et al., J. Virol. 2000; 74;
10707-10713).
Nucleic Acids and Vectors, Viruses and Host Cells Comprising
them
[0068] The invention provides a nucleic acid molecule comprising
sequences encoding HIV-1 RT, and in some embodiments, encoding
fragments of HIV-1 RT, the HIV-RT sequences comprising a codon 68
mutation. In certain embodiments, when the codon 68 mutation is an
S68 substitution, it occurs alone or in combination with a mutation
other than a K65R mutation. The invention also provides a nucleic
acid molecule comprising sequences encoding part or all of HIV-1
RT, the HIV-RT sequences comprising a codon 68 mutation, wherein
the codon 68 mutation is the only mutation in the HIV-RT sequences.
The invention further provides a nucleic acid molecule comprising
sequences encoding HIV-1 RT, the HIV-RT sequences comprising a
codon 68 deletion wherein the S68 deletion removes the codon 68 AGT
trinucleotide, or wherein the S68 deletion removes the GTA
trinucleotide spanning codons 68 and 69. Preferably, isolated
nucleic acid molecules of the invention comprise a minimum of nine,
preferably of 10-25 or more nucleotides so that they may be used as
selective primers, e.g., in nucleic acid amplification methods, or
as probes in nucleic acid hybridization techniques.
[0069] The invention also provides vectors comprising any of the
previously described nucleic acids. Such vectors may be RNA or DNA
based, and may be replicative or integrative vectors, expression
vectors (transient or stable), viral vectors and the like.
[0070] The invention also provides an isolated HIV, such as HIV-1
or HIV-2, comprising any of the previously described nucleic
acids.
[0071] The invention also provides a host cell comprising any one
of the nucleic acids or isolated HIV of the invention.
[0072] The present invention also provides HIV specific primers
which may be used in conjunction with the methods and kits of the
invention. In certain embodiments, a primer of the invention can be
used to amplify a region of HIV-RT comprising codon 68 and
preferably, can be used to distinguish between the presence and
absence of an HIV reverse transcriptase codon 68 mutation, and more
particularly, of the S68del mutation. Examples of such primers
include, without limitation, SK38 Primer: ATA ATC CAC CTA TCC CAG
TAG GAG AAA T (SEQ ID NO: 1) and SK39 Primer: TTT GGT CCT TGT CTT
ATG TCC AGA ATG C (SEQ ID NO: 2). The presence of amplified product
may also be detected with the SK19 probe: ATC CTG GGA TTA AAT AAA
ATA GTA AGA ATG TAT AG (SEQ ID NO: 3). The invention thus also
provides a nucleic acid probe that can be used to distinguish
between the presence and absence of an HIV reverse transcriptase
codon 68 mutation, and more particularly, of the S68del mutation.
Other HIV specific primers and probes may easily be designed by
those of skill in the art that can detect and differentiate codon
68 mutations, including those that distinguish between S68
substitutions and the codon 68 deletion referred to herein as
"S68del".
Methods of Evaluating Effectiveness of an Antiretroviral Agent and
of Monitoring the Progression of HIV-Infection
[0073] The present invention provides methods for monitoring the
clinical progression of human immunodeficiency virus (HIV)
infection and its response to selected antiviral therapies. It
involves, in certain embodiments, measuring HIV nucleic acid copy
number in plasma or lymphocytes (e.g., peripheral blood mononuclear
cells) derived from an HIV-infected person. Such measurements are
performed, e.g., using RT (to copy RNA to cDNA where HIV RNA is
being assessed) and quantitative real time polymerase chain
reaction (PCR) assays, to assess an individual's HIV viral load.
Direct measurement of viral loads enables one to evaluate the
therapeutic effect of one or more select antiretroviral agents, and
therapies comprising administering such agents. Genotypic analyses
of HIV nucleic acid sequences and more specifically, of mutations
that emerge during treatment with select antiretroviral agents
allow one to understand how viral genotypic changes correlate with
viral phenotypic characteristics and reveal how emerging mutations
affect viral RNA levels in the presence of select antiretroviral
agents. When a correlation between viral genotype and phenotypic
characteristics is demonstrated, it provides useful methods that
may be used, alone or in combination, for predicting clinical
outcome and thus for improving patient management and care.
[0074] In working examples disclosed herein, a novel mutation of
HIV RT (deletion of AGT at codon S68, "S68del") was selected in a
dexelvucitabine (DFC)-treated primary human lymphocytes after 14
weeks of culture. The S68del mutation became the dominant virus by
week 19 based on population sequence and clonal analysis. The
S68del was investigated phenotypically against selected antiviral
agents and cross resistance determined by drug susceptibility
assays. The S68del correlated with refractoriness not only to DFC
treatment (during which it emerged), but also to numerous other
clinically important NSRI. Codon insertions and deletions have been
associated with multi-drug resistance (MDR) in clinical samples
from antiretroviral treated individuals (e.g., 76del, 69del, 69ins,
70del) but the S68del has never been reported.
[0075] Mutations at codon 68 of HIV-RT, and more particularly, of
S68del, correlate with resistance to certain antiretroviral
therapies, including DFC and lamivudine, emtricitabine, tenofovir,
abacavir and amdoxovir monotherapies. Codon 68 mutants, and S68 del
in particular, are expected to precede immunologic decline over
time, e.g., by one or more, or 2-6 or more months. Once mutation
such as a deletion at codon 68 has been detected in plasma HIV RNA
or lymphocytes of an HIV-infected individual, a specific
therapeutic regimen is considered. In cases in which the
HIV-infected individual is already undergoing antiretroviral
therapy, an alteration in the therapeutic regimen is preferably
considered. For example, adding, subtracting or changing agents to
overcome resistance to the treatment may be considered.
[0076] In certain embodiments of the invention, quantitative
real-time PCR or other real-time sequence detection assays may be
used to detect and monitor the absolute concentrations and relative
proportions of virus with mutations at codon 68 (e.g., S68del) of
HIV-RT, a mutation which correlates with resistance to therapy with
DFC and cross-resistance to a number of other NRTI, including but
not limited to lamivudine, emtricitabine, tenofovir, abacavir and
amdoxovir. When mutation at codon 68 (e.g., S68del) has been
detected in a person undergoing monotherapy with DFC or any other
antiretroviral agent, an alteration in the therapeutic regimen is
preferably considered for effective patient management. Such
alteration may include, for example, combination therapy, e.g.
adding AZT to the DFC or other antiretroviral agent under which the
mutation is detected, or combination therapy with another antiviral
agent that is effective in inhibiting viral replication of an HIV
harboring the codon 68 mutation (e.g., S68del).
[0077] Accordingly, in one particular embodiment, the invention
provides a method for evaluating the effectiveness to an
HIV-infected individual of a selected antiretroviral agent or
therapy, the method comprising: (i) collecting a sample from an
HIV-infected person treated with an antiretroviral agent (the agent
may be a antiretroviral compound or may be a composition comprising
at least one antiretroviral compound); (ii) amplifying (e.g., by
PCR) the HIV-encoding nucleic acid in the sample using HIV specific
primers; and (iii) testing for the presence of HIV specific nucleic
acid sequences in the amplification product of (ii). The sample
from the HIV-infected individual may be derived, e.g., from plasma
or lymphocytic cells, such as PMB cells, of the infected person.
When the sample is plasma, the HIV encoded nucleic acid is
predominantly viral RNA. When the sample is derived from
lymphocytes, the HIV encoded nucleic acid is predominantly proviral
DNA. In certain preferred embodiments, the HIV specific primers
used in (ii) can distinguish between the presence and absence of a
HIV reverse transcriptase codon 68 mutation, and more particularly,
of the S68del mutation. In certain other separate embodiments, step
(iii), and not necessarily step (ii), distinguishes between the
presence and absence of a codon 68 mutation, e.g., S68del.
[0078] In a further embodiment, the invention provides a method for
evaluating the effectiveness to an HIV-infected individual of a
selected antiretroviral agent or therapy, the method comprising:
(i) collecting a sample from an HIV-infected individual; and (ii)
determining whether the sample comprises nucleic acid encoding HIV
reverse transcriptase having a mutation at codon 68, e.g., S68del,
in which the presence of the mutation correlates positively with
refractoriness of the individual to the selected antiretroviral
therapy and, if the therapy remains unchanged, to accelerated
immunologic decline of the HIV-infected individual compared to
HIV-infected individuals who do not have the mutation. The sample
from the HIV-infected individual may be, e.g., plasma or
lymphocytic cells such as PBM cells. When the sample is plasma, the
HIV encoded nucleic acid is predominantly viral RNA. When the
sample is lymphocytic cells, the HIV encoded nucleic acid is
predominantly proviral DNA.
[0079] In any of the above methods, the absence of, or decreasing
concentrations of, detectable HIV sequence correlates positively
with the assessment that the antiretroviral agent is
therapeutically effective in treating a codon 68, e.g., S68del,
mutation. Moreover, in this method, the presence of, or increasing
concentrations of, detectable HIV sequence correlates positively
with the assessment that the antiretroviral agent is
therapeutically ineffective.
[0080] In another embodiment, the invention provides methods for
evaluating the effectiveness to an HIV-infected individual of
treatment with a selected antiretroviral agent, comprising (i)
collecting a sample from an HIV-infected individual before
treatment with a selected antiretroviral agent; (ii) collecting a
sample from the HIV-infected individual after treatment with the
selected antiretroviral agent; (iii) amplifying separately
HIV-encoding nucleic acid in the samples from (i) and (ii) with HIV
primers; (iv) comparing the HIV nucleic acid copy number in samples
(i) and (ii), wherein a ratio of HIV nucleic acid copy number in
samples (i) and (ii) of greater than about 2.5 to 1, 4 to 1, 5 to
1, 10 to 1 or more, correlates positively with the assessment that
the selected antiretroviral agent is therapeutically effective. The
sample from the HIV-infected individual may be, e.g., plasma or
lymphocytic cells such as PBM cells. When the sample is plasma, the
HIV encoded nucleic acid is predominantly viral RNA. When the
sample is lymphocytic cells, the HIV encoded nucleic acid is
predominantly proviral DNA. In certain embodiments, such methods
may additionally or optionally (e.g., in step (iii)) comprise the
use of HIV primers that can distinguish between the presence and
absence of a codon 68 mutation, e.g., S68del in HIV-RT.
[0081] Methods such as those described above may be modified to
further include one or more steps of collecting and analyzing a
sample from an HIV-infected individual so as to monitor the
efficacy of the course of the individual's treatment over time and
to make changes to the person's treatment regimen as needed, based
on correlations derived from measuring and comparing HIV genomic
mutations, and specific nucleic acid levels before and after
treatment with a selected antiretroviral agent or therapy.
[0082] Accordingly, in certain embodiments, the invention provides
methods for evaluating the effectiveness to an HIV-infected
individual of treatment with a selected antiretroviral agent
treatment prone to emergence of a codon 68 mutation, the method
comprising: (i) collecting at least one sample from an HIV-infected
individual at separate time intervals; (ii) amplifying HIV-encoding
nucleic acid in the separate samples using HIV primers; (iii)
measuring HIV nucleic acid copy numbers using the amplification
products of step (ii); and (iv) comparing the HIV nucleic acid copy
numbers in the samples collected during the course of the selected
treatment; whereby a statistically significant decline in HIV
nucleic acid copy numbers detected over the course of the treatment
correlates positively with the assessment that the selected
antiretroviral agent has not selected for a HIV-RT codon 68
mutation, e.g., S8del, and remains therapeutically effective. In
certain embodiments, such methods may additionally or optionally
(e.g., in step (iii)) comprise the use of HIV primers that can
distinguish between the presence and absence of a codon 68
mutation, e.g., S68del in HIV-RT. The sample from the HIV-infected
individual may be, e.g., plasma or lymphocytic cells such as PBM
cells. When the sample is plasma, the HIV encoded nucleic acid is
predominantly viral RNA. When the sample is lymphocytic cells, the
HIV encoded nucleic acid is predominantly proviral DNA.
[0083] In other embodiments of the invention, the methods may be
used to detect mutations at codon 68 of HIV-RT, e.g., S68del, which
correlate with resistance to a selected antiretroviral therapy and
which precede immunologic decline. Accordingly, the present
invention provides methods for evaluating the effectiveness of a
selected antiretroviral therapy to an HIV-infected individual, the
method comprising: (i) collecting a sample from an HIV-infected
individual who is being treated with an antiretroviral agent; and
(ii) determining (for example, using quantitative, real time PCR)
whether the sample comprises nucleic acid encoding HIV-RT having a
mutation at codon 68, e.g., S68del, in which the presence of the
mutation correlates positively with immunologic decline of the
individual within at least one, two, three four, five, six or ten
or more months. Under such circumstances, the HIV-infecting
individual has become, via the mutation, resistant to the selected
antiretroviral agent. It may be desirable after detecting the codon
68, e.g., S68del, mutation to alter the course of the person's
current treatment regimen. The altered treatment regimen may be a
complete exchange of antiretroviral compounds or agents or may
comprise adding one or more additional antiretroviral agents to the
HIV-infected individual's current treatment regimen. For example,
if the individual was being treated with DFC when the mutation
arose, the individual's therapeutic regimen may desirably be
altered, within about a six to twelve month period of the
mutation's occurrence, by either (i) changing to a different
antiretroviral agent, such as zidovudine (AZT) and stopping DFC
treatment; (ii) increasing the dosage of DFC (which is often less
desirable); or (iii) adding another antiretroviral agent, such as
zidovudine (AZT); to the person's therapeutic regimen; or any
combination thereof. The effectiveness of the modification in
therapy may be evaluated, as set forth above, by monitoring HIV
nucleic acid copy numbers after the treatment change. A subsequent
decrease in circulating HIV RNA copy number, for example,
correlates positively with the effectiveness of the new treatment
regimen.
[0084] Because the mutation at the codon 68, e.g., S68del, may
appear first in plasma HIV RNA and only later in lymphocytic cell
proviral DNA, monitoring the time course of appearance of the codon
68 mutation in proviral DNA may be desirable. Accordingly, the
present invention also provides methods for evaluating the
effectiveness to an HIV-infected person of antiretroviral therapy,
the method comprising: (i) collecting lymphocytic cells from an
HIV-infected person who is being treated with an antiretroviral
agent; and (ii) determining whether the cells comprise proviral HIV
DNA comprising a mutation at codon 68 (e.g., S68del), in which the
presence of the mutation correlates positively with immunologic
decline of the individual over time. (The time depends in part on
how much sooner the mutation identified in the individual's plasma
HIV RNA precedes the mutation being detected in proviral DNA, which
may be anywhere from about 1 to about 5, 6, 7, 8, 9, 10 or more
months). Detection of the codon 68, e.g., S68del, mutation in
proviral DNA is an indicator of immunologic decline and alteration
of the person's therapeutic regimen is desirable.
[0085] In a specific embodiment of the invention, a method for
evaluating the effectiveness to an HIV-infected person of DFC
therapy is provided, the method comprising: (i) collecting a sample
(e.g., plasma) from an HIV-infected person who is being treated
with DFC; (ii) amplifying the HIV-encoding RNA in the sample by
converting the RNA to cDNA and amplifying HIV sequences using HIV
primers and PCR, for example; and (iii) testing for the presence of
HIV sequence in the amplification product of (ii), wherein the
absence of detectable HIV sequence correlates positively with the
conclusion that DFC is therapeutically effective and the presence
of detectable HIV sequence correlates positively with the
conclusion that DFC is therapeutically ineffective. In other
embodiments, the sample from the HIV-infected individual is derived
from or comprises lymphocytic cells and step (ii) comprises
amplifying HIV proviral DNA sequences without a required conversion
of RNA to cDNA. In preferred embodiments of the above methods, the
HIV primers used comprise SK38 Primer: ATA ATC CAC CTA TCC CAG TAG
GAG AAA T (SEQ ID NO: 1) and SK39 Primer: TTT GGT CCT TGT CTT ATG
TCC AGA ATG C (SEQ ID NO: 2), and/or the presence of HIV sequence
is detected using, e.g., an enzyme-linked assay (e.g., a
colorimetric or fluorescence based assay). The presence of the
amplified product may also be detected with the SK19 probe: ATC CTG
GGA TTA AAT AAA ATA GTA AGA ATG TAT AG (SEQ ID NO: 3). Similar
methods in which the HIV copy number is measured are also
provided.
[0086] Another specific embodiment of the invention provides a
method for evaluating the effectiveness to an HIV-infected
individual of DFC therapy, the method comprising: (i) collecting a
sample (e.g., plasma) from an HIV-infected individual who is being
treated with DFC; (ii) amplifying the HIV-encoding RNA in the
sample by converting the RNA to cDNA and amplifying HIV sequences
using HIV primers and PCR to produce a PCR amplification product
that comprises a portion of the HIV-RT gene containing codon 68
(e.g. SK38 Primer: ATA ATC CAC CTA TCC CAG TAG GAG AAA T (SEQ ID
NO: 1) and SK39 Primer: TTT GGT CCT TGT CTT ATG TCC AGA ATG C (SEQ
ID NO: 2)); and (iii) measuring the presence or absence of a
mutation at codon 68 of the HIV-RT, wherein the presence of the
mutation correlates positively with immunologic decline of the
HIV-infected individual over time. In other embodiments, the sample
from the HIV-infected individual is derived from or comprises
lymphocytic cells and step (ii) comprises amplifying HIV proviral
DNA sequences without a required conversion of RNA to cDNA. In
preferred embodiments of the above methods, the HIV primers used
comprise SK38 Primer: ATA ATC CAC CTA TCC CAG TAG GAG AAA T (SEQ ID
NO: 1) and SK39 Primer: TTT GGT CCT TGT CTT ATG TCC AGA ATG C (SEQ
ID NO: 2), and/or the presence of HIV sequence is detected using,
e.g., an enzyme-linked assay (e.g., a colorimetric or fluorescence
based assay). The presence of the amplified product may also be
detected with the SK19 probe: ATC CTG GGA TTA AAT AAA ATA GTA AGA
ATG TAT AG (SEQ ID NO: 3). Similar methods in which the HIV copy
number is measured are also provided.
[0087] The presence of the codon 68, e.g., S68del, mutation
indicates that the effectiveness of monotherapy with DFC is likely
to decline either in the presence or the absence of the codon 68
mutation. Combination therapy with DFC (e.g., by adding AZT) or a
switch to other drugs as provided herein is desirable.
Kits
[0088] The present invention also provides a kit for detection of
mutations at codon 68 (e.g., S68del) of HIV-RT encoding
sequences.
[0089] In certain embodiments, the kit comprises a first pair of
PCR primers which bind outside the region of codon 68 and therefore
may be used to amplify a DNA fragment comprising codon 68 (e.g.
SK38 Primer: ATA ATC CAC CTA TCC CAG TAG GAG AAA T (SEQ ID NO: 1)
and SK39 Primer: TTT GGT CCT TGT CTT ATG TCC AGA ATG C (SEQ ID NO:
2)); and at least two pairs of second round primers which may be
used to amplify selectively codon 68, e.g., S68del, sequences. The
kit may include more than two pairs of second primers. Similar
primers may be readily designed by those skilled in the art; the
first pair of primers need only amplify a conveniently-sized DNA
fragment comprising codon 68 of HIV-RT, and one member of the
second pair of primers should bind selectively to codon 68,
preferably having its 3' terminus at the codon of interest in order
to maximize the probability of a perfect match resulting in
amplification. The kit may also include a probe for detection of
the amplified product containing codon 68, such as the SK19 probe:
ATC CTG GGA TTA AAT AAA ATA GTA AGA ATG TAT AG (SEQ ID NO: 3).
Optionally, the kit may include instructions for interpretation
indicting that the presence of the mutant form at the codon 68 of
HIV-RT correlates with reduced efficacy of a particular
antiretroviral therapeutic agent, e.g., that presence of the codon
68 mutant indicates reduced efficacy of monotherapy with DFC and a
number of other NRTI, including but not necessarily limited to
lamivudine, emtricitabine, tenofovir, abacavir and amdoxovir.
[0090] As shown herein, the S68del mutant HIV demonstrated greater
than 30-fold increased resistance to DFC, lamivudine,
emtricitabine, tenofovir, abacavir and amdoxovir. As expected, the
S68del demonstrated no resistance to NNRTI and protease inhibitors.
HIV-RT containing the S68 deletion demonstrated a 5.6-, 2.5- and
10-fold increase in resistance to DFC-TP, AZT-TP and
emtricitabine-TP, respectively.
Viral Nucleic Acid and Protein Analyses
[0091] As detailed above, it is possible to study the quantity
and/or quality (such as screening for mutations) of HIV-specific
DNA or RNA sequences isolated from HIV-infected individuals (e.g.,
plasma samples or lymphocytic cells such as PBM cells) to evaluate
whether a particular antiretroviral agent or therapy is an
effective one. Well-known extraction and purification procedures
are available for the isolation of DNA from a sample. Proviral DNA,
for example, can be isolated from patient samples, such as from
lymphocytic cells (e.g., PBM cells), by digestion of HIV-infected
cells with proteinase K in the presence of EDTA and a detergent
such as SDS, followed by extraction with phenol.
[0092] HIV-specific RNA can be isolated from samples such as plasma
samples or lymphocytic cells, e.g., PBM cells, using the following
methodology. Suitable infected cells are incubated for a period of
time. The cells are recovered by centrifugation. The cells are
resuspended in an RNA extraction buffer followed by digestion using
a proteinase digestion buffer and digestion with proteinase K.
Proteins are removed in the presence of a phenol/chloroform
mixture. RNA can then be recovered following further centrifugation
steps. (Maniatis, T., et al, Molecular Coning, A Laboratory Manual,
2nd Edition, Cold Spring Harbor Laboratory Press, (1989)).
[0093] Although it is possible to use non-amplified nucleic acid,
due to the relative scarcity of nucleic acid in an HIV-1 sample, it
is preferable to amplify it. Nucleic acid may be selectively
amplified using the general technique of polymerase chain reaction
(PCR), which is an in vitro method for producing large amounts of
specific nucleic acid fragment of defined length and sequence from
small amounts of a template.
[0094] A standard PCR comprises standard reactants, using Mg.sup.2+
concentration, oligonucleotide primers and temperature cycling
conditions for amplification of the HIV gene of interest, such as
the HIV-RT gene, using sequence specific primers. The primers are
chosen such that they will amplify the entire RT gene or a selected
sequence which incorporates nucleotides corresponding to a region
of the wild-type DNA sequence of HIV-1 that includes the codon
which is mutated. In a preferred embodiment of the invention,
primers 38K and 39K (SK38 Primer: ATA ATC CAC CTA TCC CAG TAG GAG
AAA T (SEQ ID NO: 1) and SK39 Primer: TTT GGT CCT TGT CTT ATG TCC
AGA ATG C (SEQ ID NO: 2)) are used to amplify the RT gene.
[0095] RNA cannot be amplified directly by PCR. Its corresponding
cDNA must first be synthesized. Synthesis of cDNA is normally
carried out by primed reverse transcription reactions using
primers, such as for example, using oligo-dT primers which
hybridize to polyA tails found at the 3'-end of many eukaryotic RNA
transcripts (PolII). Advantageously, primers are chosen such that
they will simplify the nucleic acid sequence for RT or a selected
sequence which incorporates nucleotides corresponding to the region
of RNA corresponding to the wild-type DNA sequence or to the region
of the mutant DNA sequence corresponding to the 68th codon of the
reverse transcriptase region. This could be achieved by preparing
an oligonucleotide primer which is complementary to a region of the
RNA strand which is upstream of the corresponding sequence of the
wild-type DNA sequence. cDNA prepared by this methodology (see
Maniatis, T., et al., supra.) can then be used in the same way as
for the DNA already discussed.
[0096] The next stage of the methodology is to hybridize to the
nucleic acid an oligonucleotide which is complementary to a region
of the wild-type DNA sequence (or its corresponding RNA) or to a
region of the mutant DNA sequence (or its corresponding RNA).
[0097] Conditions and reagents are then provided to permit
polymerization of the nucleic acid from the 3'-end of the
oligonucleotide primer. Such polymerization reactions are
well-known in the art.
[0098] If the oligonucleotide primer has at its 3'-end a nucleotide
which is complementary to a mutant genotype, that is a genotype
which has a nucleotide change at the 68th codon in the RT region,
then polymerization of the nucleic acid sequence will only occur if
the nucleic acid of the sample is the same as the mutant genotype.
Polymerization of a wild type nucleic acid sequence will not occur
or at least not to a significant extent because of a mis-match of
nucleotides at the 3'-end of the oligonucleotide primer and the
nucleic acid sequence of the sample.
[0099] If the oligonucleotide primer has at its 3'-end of
nucleotide which is complementary to the wild-type genotype, that
is a genotype which has the wild-type nucleotide at the 68th codon
in the RT region, then there will be polymerization of a nucleic
acid sequence which is wild-type at that position. There will be no
polymerization of a nucleic acid which has a mutant nucleotide at
the 3'-position.
[0100] The preferred length of each oligonucleotide is 15-20
nucleotides, but may vary depending on selected hybridization
conditions that are well known to the skilled worker. The
oligonucleotide can be prepared according to methodology well known
to the skilled worker (Koster, H., Drug Research, 30 p 548 (1980);
Koster, H., Tetrahedron Letters, p 1527 (1972); Caruthers,
Tetrahedron Letters, p 719, (1980); Tetrahedron Letters, p 1859,
(1981); Tetrahedron Letters 24, p 245, (1983); Gate. M. Nucleic
Acid Research, 8, p 1081, (1980)) and is generally prepared using
an automated DNA synthesizer such as an Applied Biosystems 381A
synthesizer.
[0101] It is convenient to determine the presence of an
oligonucleotide primer extended product. The means for carrying out
the detection is by using an appropriate label.
[0102] The label may be conveniently attached to the
oligonucleotide primer or to some other molecule which will bind
the primer extended polymerization product.
[0103] The label may be for example an enzyme, radioisotope or
fluorochrome. A preferred label may be biotin which could be
subsequently detected using streptavidin conjugated to an enzyme
such as peroxidase or alkaline phosphatase. The presence of an
oligonucleotide primer extended polymerization product can be
detected for example by running the polymerization reaction on an
agarose gel and observing a specific DNA fragment labeled with
ethidium bromide, or Southern blotted and autoradiographed to
detect the presence or absence of bands corresponding to
polymerized product. If a predominant band is present which
corresponds only to the labeled oligonucleotide then this indicates
that polymerization has been occurred. If bands are present of the
correct predicted size, this would indicate that polymerization has
occurred.
[0104] For example, DNA isolated from HIV-infected individuals'
plasma samples or PBM cells as described herein is used as a
template for PCR amplification using synthetic oligonucleotide
primers which either match or mis-match with the amplified
sequences. The feasibility of PCR in detecting such mutations has
already been demonstrated. PCR using the Amplification Refractory
Mutation system ("ARMS") (Newton, C. R., et al. Nucleic Acids
Research, 17, p 2503, (1989)) Synthetic oligonucleotide are
produced that anneal to the regions adjacent to an including the
specific mutations such that the 3'-end of the oligonucleotide
either matches of mismatches with a mutant or wild-type sequence.
PCR is carried out which results in the identification of a DNA
fragment (using gel electrophoresis) where a match has occurred or
no fragment where a mismatch occurred.
[0105] DNA is extracted from HIV-1 infected T-cells as described
herein and subjected to "ARMS" PCR analysis using these
primers.
[0106] The presence of a fragment is identified by using an
oligonucleotide primer as described above, i.e., by attempting
polymerization using an oligonucleotide primer which may be labeled
for the amplified DNA fragment under stringent conditions which
only allow hybridization of complementary DNA (the only difference
is that differential hybridization does not have to be performed as
fragments of DNA amplified by the "ARMS" method will be the same
whether derived from mutant or wild-type DNS, so a common
oligonucleotide can be used to detect the presence of these
fragments. The sequence of such an oligonucleotide is derived from
a DNA sequence within the DNA fragment that is conserved amongst
HIV-1 strains).
[0107] The above PCR assay may be adapted to enable direct
detection of mutations associated with D-D4FC resistance in DNA
from PBL samples from infected individuals that have not been
cultured to obtain virus. As this material generally contains
considerably less HIV-1 DNA than that in infected lymphoid cultures
a "double PCR" (or nested set) protocol can be used (Simmonds et
al., J. Virol., 64, 864-872, (1990)) to boost the amount of target
HIV-1 RT DNA signal in the samples. The double PCR overcomes the
problem of limited amplification of a rare template sequence. A
small amount of the pre-amplified material may be used in the
second PCR with primer pairs designed to allow discrimination of
wild type and mutant residues.
[0108] The presence of a codon 68 mutation in RT can also be
determined by quantitative real-time PCR, as described in Example
4.
[0109] It is also possible to detect codon 68 mutations in the
HIV-1 RT RNA isolated from clinical samples using an RNA
amplification system. Using the methodology described by Guatelli
et al. (Proc. Natl. Acad. Sci, (USA), 8/7, 1874-1878, (March 1990))
a target nucleic acid sequence can be replicated (amplified)
exponentially in vitro under isothermal conditions by using three
enzymatic activities essential to retroviral replication: reverse
transcriptase, RNase H and a DNA-dependant RNA polymerase. Such a
methodology may be employed followed by an hybridization step to
distinguish mutant from wild-type nucleotides at discussed
previously.
[0110] The viral RNA or corresponding DNA from an HIV-infected
person may be directly assayed. Alternatively, part or all of the
HIV-RT encoding sequences may be cloned into viral vectors and
amplified to produce larger amounts of viral nucleic acid for
sequencing and other desired analyses.
[0111] According to this aspect of the invention, detection may be
any nucleic acid-based detection means, for example nucleic acid
hybridization techniques or polymerase chain reaction (PCR). The
invention further encompasses the use of different assay formats of
said nucleic acid-based detection means, including restriction
fragment length polymorphism (RFLP), amplified fragment length
polymorphism (AFLP), single-strand chain polymorphism (SSCP),
amplification and mismatch detection (AMD), interspersed repetitive
sequence polymerase chain reaction (IRS-PCR), inverse polymerase
chain reaction (iPCR) and reverse transcription polymerase chain
reaction (RT-PCR), among others.
[0112] Suitable assay means also include nucleic acid hybridization
protocols such as Northern blots and Southern blots.
[0113] In certain embodiments of the invention, the presence of the
S68del mutation may be detected by solid-state nucleic acid
sensors. In specific embodiments, the solid-state sensors are
oligonucleotide microarrays, cDNA microarrays and nucleic acids
bound to any other convenient solid supports, such as beads or
other microspheres. Examples of such sensors are further described
in Sievertzon et al., Expert Rev. Mol. Diagn. 2006; 6:481-492;
Heller, Annu. Rev. Biomed. Eng. 2002; 4:129-153; and Watson et al.,
Curr. Opinion in Biotech. 1998; 9:609-614.
[0114] In one specific embodiment, a line probe assay can be used
to detect the codon 68, e.g., S68del, mutation in samples collected
from HIV-infected individuals. Oligonucleotide probes used to
detect the S68del mutation, for example, are applied to a
nitrocellulose or other suitable membrane. RNA or DNA isolated from
HIV-infected individuals is amplified and labeled, for example, by
biotinylation. The labeled nucleic acid is reverse hybridized to
the probes, and the amount of hybridized nucleic acid is detected.
Details of probe itemization, nitrocellulose strip production and
reverse hybridization have been published previously (Stuyver et
al., J. Clin. Microbiol. 1996; 34:2259-2266; Stuyver et al.,
Antimicrob. Agents Chemother. 1997; 41:284-291; Van Geyt et al., in
Therapies of Viral Hepatitis 1998; 139-145).
[0115] Any of a number of available systems and assays may be used
in conjunction with products and methods of the invention to assess
viral genotypes and associated phenotypes such as antiretroviral
drug susceptibility, including but not limited to certain
commercially available systems (see, e.g., PhotoSense.TM. HIV
(Monogram Biosciences); HIV GenoSure.TM. (LabCorp; see also Baxter
et al., AIDS 2000 14(9):F83-93 (2000) and Durant et al., Lancet
353(9171):2195-2199 (1999); Antivirogram.RTM. (Vireo) and Kellam,
Antimicrob. Agents Chemother. 38:23-30 (1994)).
[0116] In other embodiments, the S68del mutation sequence and drug
resistance profile may be added to HIV genotyping and phenotyping
databases. Samples isolated from an HIV-infected individual may
then be compared to such databases to correlate the viral genotype
and/or viral phenotype of the individual sample to effective
antiretroviral therapies. In particular, such methods comprising
comparison to information stored in a database may be used to
choose effective NRTI treatment (including NRTI in combination with
one or more other NRTI and/or other agents) (see, e.g., Lengauer et
al., Nature Rev. Microbiol. 4:790-797 (2006); Baxter et al., AIDS
2000 14:F83-F93 (2000); and Durant et al., Lancet
353(9171):2195-2199 (1999)).
[0117] Alternatively, the HIV RT protein may be screened directly
or indirectly for the mutation using any of a number of available
protein sequence based techniques. Such techniques include protein
expression based assays, optionally in combination with Western
blotting techniques. In certain embodiments, a denatured form of
the HIV RT protein containing the S68del mutation may be used to
raise antibodies that bind differentially to denatured S68del and
the wild-type RT proteins (or fragments thereof comprising the S68
codon). A variety of protein expression systems are known and
available to the skilled worker. The RT may be expressed in a
baculovirus system, for example. Antibodies having specificity for
an S68del specific epitope that may be engineered include
monoclonal, chimeric, humanized or human antibodies, and also
include any number of antibody fragments and single chain
antibodies. The antibody that binds the S68del and the wild-type
forms of RT differentially can be used in Western blots, ELISAs and
other immunoassays to detect the presence of the S68del HIV
mutation in samples from HIV-infected individuals.
Methods to Avoid Selecting, or to Treat an Individual Harboring,
HIV with a Codon 68 Mutation in HIV-RT
[0118] The invention further provides methods for treating a
subject infected with HIV-1 or HIV-2 comprising the step of
administering over time an antiretroviral agent that does not
select for an HIV-1 mutant having a codon 68 mutation in the HIV
reverse transcriptase coding sequence.
[0119] In one embodiment, the antiretroviral agent administered to
avoid selecting HIV-1 with a codon 68 mutation is a protease
inhibitor. Examples of protease inhibitors include, but are not
limited to, lopinavir, indinavir (Crixivan), nelfinavir
([3S-[2(2S*,3S*),3-alpha,4-a-beta,8a-beta-]]-N-(1,1-dimethylethyl)decahyd-
ro-2-).sub.2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-(phenylthio)bu-
tyl]-3-isoquinolincarboxamide mono-methanesulfonate) (Viracept),
saquinavir (Invirase), or 141W94 (amprenavir;
(S)-tetrahydrofuran-3-yl-N-[(1S,2R)-3-[N-[(4-aminophenyl)sulfonyl]-N-isob-
-utylamino]-1-benzyl-2-hydroxypropyl]carbamate, efavirenz
(S)-6-chloro4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-
-benzoxazin-2-one.), atazanavir sulfate (Reyataz) and Darunavir
(Prezista).
[0120] In another embodiment, the antiretroviral agent administered
to avoid selecting HIV-1 with a codon 68 mutation is an NNRTI.
Examples of NNRTI include, but are not limited to, DMP-266
((S)-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-b-
-enzoxazin-2-one (SUSTIVA, see U.S. Pat. No. 5,519,021);
delavirdine,
(1-[3-(1-methyl-ethyl)amino]-2-pyridinyl-4-[[5-[(methylsulfonyl)amino]-1H-
-indol-2-yl]carbonyl]-, monoethanesulfonate), nevirapine, or
delarvirdine.
[0121] In other embodiments, HIV-infected individuals can be
treated with NRTI against which the S68del mutant does not show
increased resistance. Examples of such NRTI include AZT, DDI,
DFDOC, D4T, DOT and DDC.
[0122] In other embodiments, the antiretroviral agent administered
to avoid selecting HIV-1 with a codon 68 mutation is an HIV fusion
inhibitor, an HIV integrase inhibitor, an RNAse H inhibitor, a CD4
binding inhibitor, a CXCR4 binding inhibitor, or a CCR5 binding
inhibitor.
[0123] The present invention further provides methods for isolating
compounds that are active against an HIV-1 S68del mutant using the
screening methods and the S68del mutant HIV of the invention. A
variety of protocols for characterizing antiretroviral agents and
their effects on viral replication, in vitro and in vivo, such as
those described and exemplified herein, are well known in the
literature. See, e.g., Lennerstrand et al., Antimicrob. Agents
Chemother. 2007 Apr. 2 (Epub ahead of print); Hammond et al.,
Antimicrob. Agents Chemother. 49(9):3930-3932 (2005); Moser et al.,
Antimicrob. Agents Chemother. 49(8):3334-3340 (2005); Parikh et
al., Antimicrob. Agents Chemother. 49(3):1139-1144 (2005); Boyer et
al., J. Virol. 78(18):9987-9997 (2004); Roge et al., Antiviral
Therapy 8:173-182 (2003); Boyer et al., J. Virol. 76(18):9143-9151
(2002); Van Vaerenbergh, Verh. K Acad. Geneeskd. Belg.
63(5):447-473 (2001); Tamalet et al., Virol. 270:310-316 (2000);
and Bazmi et al., Antimicrob. Agents Chemother. 44(7):1783-1788
(2000); each incorporated herein by reference. These or similar
methods may be used to characterize known antiretroviral compounds
and to identify and isolate new antiretroviral compounds that are
useful in the treatment of multidrug resistant HIV-1, such as the
HIV-1 S68del mutant of the invention.
[0124] The dosages for such antiretroviral agents will depend on
such factors as absorption, biodistribution, metabolism and
excretion rates for each drug as well as other factors known to
those of skill in the art. It is to be noted that dosage values
will also vary with the severity of the condition to be alleviated.
It is to be further understood that for any particular subject,
specific dosage regimens and schedules should be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions. Examples of suitable dosage ranges for anti-HIV
compounds, including nucleoside derivatives or protease inhibitors
can be found in the scientific literature and in the Physicians
Desk Reference. Many examples of suitable dosage ranges for other
compounds described herein are also found in public literature or
can be identified using known procedures. These dosage ranges can
be modified as desired to achieve a desired result.
Preparation of Pharmaceutical Compositions
[0125] Any antiretroviral agent described herein can be
administered to the HIV-infected individual as a pharmaceutically
acceptable salt or prodrug in the presence of a pharmaceutically
acceptable carrier or diluent, for any of the indications or modes
of administration as described in detail herein. The active
materials can be administered by any appropriate route, for
example, orally, parenterally, enterally, intravenously,
intradermally, subcutaneously, transdermally, intranasally or
topically, in liquid or solid form.
[0126] The active compound(s) are included in the pharmaceutically
acceptable carrier or diluent in an amount sufficient to deliver to
a HIV-infected individual a therapeutically effective amount of
compound to inhibit viral replication in vivo, especially HIV
replication, without causing serious toxic effects in the treated
individual. By "inhibitory amount" is meant an amount of active
ingredient sufficient to exert an inhibitory effect on viral
replication as measured by, for example, an assay such as the ones
described herein. Preferably, inhibitory effect is at least
2.5-fold, and preferably at least 4-fold, 5-fold, 7-fold, 10-fold
or higher.
[0127] A preferred dose of the compound for all the above-mentioned
conditions will be in the range from about 1 to 75 mg/kg,
preferably 1 to 20 mg/kg, of body weight per day, more generally
0.1 to about 100 mg per kilogram body weight of the recipient per
day. The effective dosage range of the pharmaceutically acceptable
derivatives can be calculated based on the weight of the parent
nucleoside to be delivered. If the derivative exhibits activity in
itself, the effective dosage can be estimated as above using the
weight of the derivative, or by other means known to those skilled
in the art.
[0128] The compounds are conveniently administered in unit any
suitable dosage form, including but not limited to one containing 7
to 3000 mg, preferably 70 to 1400 mg of active ingredient per unit
dosage form. An oral dosage of 50 to 1000 mg is usually
convenient.
[0129] Ideally, the active ingredient should be administered to
achieve peak plasma concentrations of the active compound of from
about 0.02 to 70 micromolar, preferably about 0.5 to 10 mM. This
may be achieved, for example, by the intravenous injection of a 0.1
to 25% solution of the active ingredient, optionally in saline, or
administered as a bolus of the active ingredient.
[0130] The concentration of active compound in the drug composition
will depend on absorption, distribution, metabolism and excretion
rates of the drug as well as other factors known to those of skill
in the art. It is to be noted that dosage values will also vary
with the severity of the condition to be alleviated. It is to be
further understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or practice of the claimed
composition. The active ingredient may be administered at once, or
may be divided into a number of smaller doses to be administered at
varying intervals of time.
[0131] A preferred mode of administration of the active compound is
oral. Oral compositions will generally include an inert diluent or
an edible carrier. They may be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Pharmaceutically compatible bind agents, and/or adjuvant materials
can be included as part of the composition.
[0132] The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring. When the dosage unit form
is a capsule, it can contain, in addition to material of the above
type, a liquid carrier such as a fatty oil. In addition, dosage
unit forms can contain various other materials which modify the
physical form of the dosage unit, for example, coatings of sugar,
shellac, or other enteric agents.
[0133] The compounds can be administered as a component of an
elixir, suspension, syrup, wafer, chewing gum or the like. A syrup
may contain, in addition to the active compounds, sucrose as a
sweetening agent and certain preservatives, dyes and colorings and
flavors.
[0134] The compounds or their pharmaceutically acceptable
derivative or salts thereof can also be mixed with other active
materials that do not impair the desired action, or with materials
that supplement the desired action, such as antibiotics,
antifungals, antiinflammatories, protease inhibitors, or other
nucleoside or non-nucleoside antiviral agents, as discussed in more
detail above. Solutions or suspensions used for parental,
intradermal, subcutaneous, or topical application can include the
following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfate; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. The parental
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic.
[0135] If administered intravenously, preferred carriers are
physiological saline or phosphate buffered saline (PBS).
[0136] Liposomal suspensions (including liposomes targeted to
infected cells with monoclonal antibodies to viral antigens) are
also preferred as pharmaceutically acceptable carriers these may be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811 (which is
incorporated herein by reference in its entirety). For example,
liposome formulations may be prepared by dissolving appropriate
lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl
phosphatidyl choline, arachadoyl phosphatidyl choline, and
cholesterol) in an inorganic solvent that is then evaporated,
leaving behind a thin film of dried lipid on the surface of the
container. An aqueous solution of the active compound or its
monophosphate, diphosphate, and/or triphosphate derivatives is then
introduced into the container. The container is then swirled by
hand to free lipid material from the sides of the container and to
disperse lipid aggregates, thereby forming the liposomal
suspension.
Controlled Release Formulations
[0137] Any antiretroviral agent described herein can be
administered as a controlled release formulation. The field of
biodegradable polymers has developed rapidly since the synthesis
and biodegradability of polylactic acid was reported by Kulkarni et
al., in 1966 (Arch. Surg., 93:839). Examples of other polymers
which have been reported as useful as a matrix material for
delivery devices include polyanhydrides, polyesters such as
polyglycolides and polylactide-co-glycolides, polyamino acids such
as polylysine, polymers and copolymers of polyethylene oxide,
acrylic terminated polyethylene oxide, polyamides, polyurethanes,
polyorthoesters, polyacrylonitriles, and polyphosphazenes. See, for
example, U.S. Pat. Nos. 4,891,225 and 4,906,474 (polyanhydrides),
U.S. Pat. No. 4,767,628 (polylactide, polylactide-co-glycolide
acid), and U.S. Pat. No. 4,530,840, et al. (polylactide,
polyglycolide, and copolymers). See also U.S. Pat. No. 5,626,863
which describes photopolymerizable biodegradable hydrogels as
tissue contacting materials and controlled release carriers
(hydrogels of polymerized and crosslinked macromers comprising
hydrophilic oligomers having biodegradable monomeric or oligomeric
extensions, which are end capped monomers or oligomers capable of
polymerization and crosslinking); and PCT WO 97/05185 directed to
multiblock biodegradable hydrogels for use as controlled release
agents for drug delivery and tissue treatment agents.
[0138] Degradable materials of biological origin are well known,
for example, crosslinked gelatin. Hyaluronic acid has been
crosslinked and used as a degradable swelling polymer for
biomedical applications (U.S. Pat. No. 4,957,744).
[0139] Many dispersion systems are currently in use as, or being
explored for use as, carriers of substances, particularly
biologically active compounds. Dispersion systems used for
pharmaceutical and cosmetic formulations can be categorized as
either suspensions or emulsions. Suspensions are defined as solid
particles ranging in size from a few manometers up to hundreds of
microns, dispersed in a liquid medium using suspending agents.
Solid particles include microspheres, microcapsules, and
nanospheres. Emulsions are defined as dispersions of one liquid in
another, stabilized by an interfacial film of emulsifiers such as
surfactants and lipids. Emulsion formulations include water in oil
and oil in water emulsions, multiple emulsions, microemulsions,
microdroplets, and liposomes. Microdroplets are unilamellar
phospholipid vesicles that consist of a spherical lipid layer with
an oil phase inside, as defined in U.S. Pat. Nos. 4,622,219 and
4,725,442. Liposomes are phospholipid vesicles prepared by mixing
water-insoluble polar lipids with an aqueous solution. The
unfavorable entropy caused by mixing the insoluble lipid in the
water produces a highly ordered assembly of concentric closed
membranes of phospholipid with entrapped aqueous solution.
[0140] U.S. Pat. No. 4,938,763 discloses a method for forming an
implant in situ by dissolving a nonreactive, water insoluble
thermoplastic polymer in a biocompatible, water soluble solvent to
form a liquid, placing the liquid within the body, and allowing the
solvent to dissipate to produce a solid implant. The polymer
solution can be placed in the body via syringe. The implant can
assume the shape of its surrounding cavity. In an alternative
embodiment, the implant is formed from reactive, liquid oligomeric
polymers which contain no solvent and which cure in place to form
solids, usually with the addition of a curing catalyst.
[0141] A number of patents disclose drug delivery systems that can
be used to administer D-D4FC or a nucleotide or other defined
prodrug thereof U.S. Pat. No. 5,749,847 discloses a method for the
delivery of nucleotides into organisms by electrophoration. U.S.
Pat. No. 5,718,921 discloses microspheres comprising polymer and
drug dispersed there within. U.S. Pat. No. 5,629,009 discloses a
delivery system for the controlled release of bioactive factors.
U.S. Pat. No. 5,578,325 discloses nanoparticles and microparticles
of non-linear hydrophilic hydrophobic multiblock copolymers. U.S.
Pat. No. 5,545,409 discloses a delivery system for the controlled
release of bioactive factors. U.S. Pat. No. 5,494,682 discloses
ionically cross-linked polymeric microcapsules.
[0142] U.S. Pat. No. 5,728,402 describes a controlled release
formulation that includes an internal phase which comprises the
active drug, its salt or prodrug, in admixture with a hydrogel
forming agent, and an external phase which comprises a coating
which resists dissolution in the stomach. U.S. Pat. Nos. 5,736,159
and 5,558,879 discloses a controlled release formulation for drugs
with little water solubility in which a passageway is formed in
situ. U.S. Pat. No. 5,567,441 discloses a once-a-day controlled
release formulation. U.S. Pat. No. 5,508,040 discloses a
multiparticulate pulsatile drug delivery system. U.S. Pat. No.
5,472,708 discloses a pulsatile particle based drug delivery
system. U.S. Pat. No. 5,458,888 describes a controlled release
tablet formulation which can be made using a blend having an
internal drug containing phase and an external phase which
comprises a polyethylene glycol polymer which has a weight average
molecular weight of from 3,000 to 10,000. U.S. Pat. No. 5,419,917
discloses methods for the modification of the rate of release of a
drug form a hydrogel which is based on the use of an effective
amount of a pharmaceutically acceptable ionizable compound that is
capable of providing a substantially zero-order release rate of
drug from the hydrogel. U.S. Pat. No. 5,458,888 discloses a
controlled release tablet formulation.
[0143] U.S. Pat. No. 5,641,745 discloses a controlled release
pharmaceutical formulation which comprises the active drug in a
biodegradable polymer to form microspheres or nanospheres. The
biodegradable polymer is suitably poly-D,L-lactide or a blend of
poly-D,L-lactide and poly-D,L-lactide-co-glycolide. U.S. Pat. No.
5,616,345 describes a controlled absorption formulation for once a
day administration that includes the active compound in association
with an organic acid, and a multi-layer membrane surrounding the
core and containing a major proportion of a pharmaceutically
acceptable film-forming, water insoluble synthetic polymer and a
minor proportion of a pharmaceutically acceptable film-forming
water soluble synthetic polymer. U.S. Pat. No. 5,641,515 discloses
a controlled release formulation based on biodegradable
nanoparticles. U.S. Pat. No. 5,637,320 discloses a controlled
absorption formulation for once a day administration. U.S. Pat.
Nos. 5,580,580 and 5,540,938 are directed to formulations and their
use in the treatment of neurological diseases. U.S. Pat. No.
5,533,995 is directed to a passive transdermal device with
controlled drug delivery. U.S. Pat. No. 5,505,962 describes a
controlled release pharmaceutical formulation.
Prodrug Formulations
[0144] Any of antiretroviral agents which are described herein can
be administered as an acylated prodrug or a nucleotide prodrug, as
described in detail below.
[0145] Any of the nucleosides described herein or other compounds
that contain a hydroxyl or amine function can be administered as a
nucleotide prodrug to increase the activity, bioavailability,
stability or otherwise alter the properties of the nucleoside. A
number of nucleotide prodrug ligands are known. In general,
alkylation, acylation or other lipophilic modification of the
hydroxyl group of the compound or of the mono, di or triphosphate
of the nucleoside will increase the stability of the nucleotide.
Examples of substituent groups that can replace one or more
hydrogens on the phosphate moiety or hydroxyl are alkyl, aryl,
steroids, carbohydrates, including sugars, 1,2-diacylglycerol and
alcohols. Many are described in R. Jones and N. Bischofberger,
Antiviral Research, 27 (1995) 1-17. Any of these can be used in
combination with the disclosed nucleosides or other compounds to
achieve a desired effect.
[0146] The active nucleoside or other hydroxyl containing compound
can also be provided as an ether lipid (and particularly a 5'-ether
lipid for a nucleoside), as disclosed in the following references,
which are incorporated by reference herein: Kucera et al., 1990,
AIDS Res. Hum. Retro Viruses. 6:491-501; Piantadosi et al., 1991,
J. Med. Chem. 34:1408.1414; Hosteller et al., 1992, Antimicrob.
Agents Chemother. 36:2025.2029; Hostetler et al., 1990, J. Biol.
Chem. 265:61127.
[0147] Non-limiting examples of U.S. patents that disclose suitable
lipophilic substituents that can be covalently incorporated into
the nucleoside or other hydroxyl or amine containing compound,
preferably at the 5'-OH position of the nucleoside or lipophilic
preparations, include U.S. Pat. Nos. 5,149,794; 5,194,654
5,223,263; 5,256,641; 5,411,947; 5,463,092; 5,543,389; 5,543,390;
5,543,391; and 5,554,728, each of which is incorporated herein by
reference. Foreign patent applications that disclose lipophilic
substituents that can be attached to the nucleosides of the present
invention, or lipophilic preparations, include WO 89/02733, WO
90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO
96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.
[0148] Non-limiting examples of nucleotide prodrugs are described
in the following references: Ho, D. H. W. (1973) Cancer Res. 33,
2816-2820; Holy, A. (1993) In: De Clercq (Ed.), Advances in
Antiviral Drug Design, Vol. I, JAI Press, pp. 179-231; Hong et al.
(1979a) Biochem. Biophys. Res. Commun. 88, 1223-1229; Hong et al.
(1980) J. Med. Chem. 28, 171-177; Hosteller et al. J. Biol. Chem.
265, 6112-6117; Hosteller et al. (1991) J. Biol. Chem. 266,
11714-11717; Hosteller et al. (1994a) Antiviral Res. 24, 59-67;
Hosteller et al. (1994b) Antimicrobial Agents Chemother. 38,
2792-2797; Hunston et al. (1984) J. Med. Chem. 27, 440-444; Ji et
al. (1990) J. Med. Chem. 33 2264-2270; Jones et al. (1984) J. Chem.
Soc. Perkin Trans. I, 1471-1474; Juodka, B. A. and Smart, J. (1974)
Coll. Czech. Chem. Comm. 39, 363-968; Kataoka et al. (1989) Nucleic
Acids Res. Sym. Ser. 21, 1-2; Kataoka, S., and Uchida Heterocycles
32, 1351-1356; Kinchington et al. (1992) Antiviral Chem. Chemother.
3, 107-112; Kodama et al. (1989) Jpn. J. Cancer Res. 80, 679-685;
Korty, M. and Engels, J. (1979) Naunyn-Schmiedeberg's Arch.
Pharmacol. 310, 103-111; Kumar et al. (1990) J. Med. Chem., 33,
2368-2375; LeBec, C., and Huynh-Dinh, T. (1991) Tetrahedron Lett.
32, 6553-6556; Lichtenstein et al. (1960) J. Biol. Chem. 235,
457-465; Luethy et al. (1981) Mitt. Geg. Lebensmittelunters. Hyg.
72, 131-133 (Chem. Abstr. 95, 127093); McGigan, et al. (1989)
Nucleic Acids Res. 17, 6065-6075; McGuigan et al. (1990a)
3'-Antiviral Chem. Chemother. 1 107-113; McGuigan et al. (1990b)
Antiviral Chem. Chemother. 1, 355-360; McGuigan et al. (1990c)
Antiviral Chem. Chemother. 1, 25-33; McGuigan et al. (1991)
Antiviral Res. 15, 255-263; McGuigan et al. (1993b) J. Med. Chem.
36, 1048-1052.
[0149] Alkyl hydrogen phosphate derivatives of the anti-HIV agent
AZT may be less toxic than the parent nucleoside analogue.
Antiviral Chem. Chemother. 5, 271-277; Meyer et al. (1973)
Tetrahedron Lett. 269-272; Nagyvary et al. (1973) Biochem. Biophys.
Res. Commun. 55, 1072-1077; Namane et al. (1992) J. Med. Chem. 35,
3039-3044; Nargeot et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80,
2395-2399; Nelson et al. (1987) J. Am. Chem. Soc. 109, 4058-4064;
Nerbonne et al. (1984) Nature 301, 74-76; Neumann et al. (1989) J.
Am. Chem. Soc. 111, 4270-4277; Ohno et al. (1991) Oncology 48,
451-455; Palomino et al. (1989) J Med. Chem. 32, 22-625; Perkins et
al. (1993) Antiviral Res. 20 (Suppl. I). 84; Piantadosi et al.
(1991) J. Med. Chem. 34, 1408-1414; Pompon et al. (1994). Antiviral
Chem. Chemother. 5, 91-98; Postemark, T. (1974) Annu. Rev.
Pharmacol. 14, 23-33; Prisbe et al. (1986) J. Med. Chem. 29,
671-675; Pucch et al. (1993) Antiviral Res. 22, 155-174; Pugaeva et
al. (1969) Gig. Trf. Prof Zabol. 14, 47-48 (Chem. Abstr. 72, 212);
Robins, R. K. (1984) Pharm. Res. 11-18; Rosowsky et al. (1982) J.
Med. Chem. 25, 171-178; Ross, W. (1961) Biochem. Pharm. 8, 235-240;
Ryu et al. (1982) J. Med. Chem. 25, 1322-1329; Saffhill et al.
(1986) Chem. Biol. Interact. 57, 347-355; Saneyoshi et al. (1980)
Chem. Pharm. Bull. 28, 2915-2923; Sastry et al. (1992) Mol.
Pharmacol. 41, 441-445; Shaw et al. (1994) 9th Annual AAPS Meeting.
San Diego, Calif. (Abstract); Shuto et al. (1987) Tetrahedron Lett.
28, 199-202; Shuto et al. (1988) Pharm. Bull 36, 209-217. An
example of a useful phosphate prodrug group is the
S-acyl-2-thioethyl group, also referred to as "SATE".
[0150] Standard reference works setting forth the general
principles of recombinant DNA technology known to those of skill in
the art include Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, John Wiley & Sons, New York (1998 and Supplements to
2001); Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d
Ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989);
Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODS IN
BIOLOGY AND MEDICINE, CRC Press, Boca Raton (1995); McPherson, Ed.,
DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford
(1991). Standard reference works setting forth the general
principles of retrovirology known to those of skill in the art
include RETROVIRUSES, Coffin, John M.; Hughes, Stephen H.; Varmus,
Harold E., Plainview (NY): Cold Spring Harbor Laboratory Press
(1997) and ANTIRETROVIRAL RESISTANCE IN CLINICAL PRACTICE,
Gerretti, Anna Maria, editor London: Mediscript Ltd. (2006).
Standard reference works setting forth the general principles of
immunology known to those of skill in the art include: Harlow and
Lane ANTIBODIES: A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1999); and Roitt et
al., IMMUNOLOGY, 3d Ed., Mosby-Year Book Europe Limited, London
(1993). Standard reference works setting forth the general
principles of medical physiology and pharmacology known to those of
skill in the art include: Harrison's PRINCIPLES OF INTERNAL
MEDICINE, 14th Ed., (Anthony S. Fauci et al., editors), McGraw-Hill
Companies, Inc., 1998.
[0151] All publications and patents cited are hereby incorporated
by reference in their entirety.
[0152] Throughout this specification and paragraphs, the word
"comprise" or variations such as "comprises" or "comprising" will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers.
[0153] The following are examples which illustrate the compositions
and methods of this invention. These examples should not be
construed as limiting: the examples are included for the purposes
of illustration only. This invention has been described with
reference to its preferred embodiments. Variations and
modifications of the invention, will be obvious to those skilled in
the art from the foregoing detailed description of the invention.
It is intended that all of these variations and modifications be
included within the scope of this invention.
EXAMPLES
Example 1
Discovery of S68del Virus in DFC-Treated Viral Pool
[0154] Compound.
[0155] .beta.-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine
(DFC, D-d4FC, RVT).
[0156] Cells.
[0157] Peripheral blood mononuclear (PBM) cells were separated by
ficoll-hypaque (Histopaque 1077: Sigma) density gradient
centrifugation from Buffy coats obtained from the American Red
Cross (Atlanta, Ga.). Buffy coats were derived from healthy
seronegative donors. Cells were activated with 3 .mu.g/ml
phytohemagglutinin A (Sigma-Aldrich, St. Louis, Mo.) in 500 ml of
RPMI-1640 (Mediatech Inc., Herndon, Va.) containing 100 ml
heat-inactivated fetal bovine serum (Hyclone), 83.3 IU/ml
penicillin, 83.3 .mu.g/ml streptomycin, 1.6 mM L-glutamine
(Mediatech Inc., Herndon, Va.), for 2-3 days prior to use.
[0158] Virus.
[0159] HIV-1/LAI obtained from the Centers for Disease Control and
Prevention (Atlanta, Ga.) was used as the virus for the resistant
pool. A multiplicity of infection (MOI) of 0.1, as determined by a
limiting dilution method in PBM cells, was selected to begin the
infected pool.
[0160] Selection of Resistant Virus.
[0161] Naive PBM cells were treated with DFC at 0.1 .mu.M for one
hour prior to inoculation with HIV-1/LAI. The treated PBM cell
group and a control nontreated PBM cell group were allowed to
infect for 1 hr. IL-2 (26 IU/ml)-supplemented RPMI-1640 was then
added for a final concentration of 1.times.10.sup.6 cells/ml. Virus
was passaged every 6 days with a fresh treatment of DFC, ranging
from 0.1 .mu.M to 6 .mu.M over 52 weeks. RT activity was measured
weekly and used to determine percent inhibition of DFC. Total RNA
was isolated from culture supernatants using the commercial QIAmp
Viral RNA mini kit (Qiagen, Valencia, Calif.). Reverse
transcriptase PCR was performed using Invitrogen Superscript
Reverse Trancriptase III to generate second strand cDNA from viral
RNA using Ambion DECAprime II primers. PCR was performed using
Invitrogen Platinum Taq polymerase (high fidelity). A 1346 bp
fragment of the HIV-1 genome was amplified using forward primer
5'-ttgactcagattggttgcactttaa-3' (SEQ ID NO: 4) and reverse primer
5'-aagaacccatagtaggagcagaaac-3' (SEQ ID NO: 5). The PCR product was
purified using the QIAquick PCR purification kit. The samples were
sequenced in both directions for the HIV-1 RT amino acids 01-300.
Sequencing was performed in parallel between the control virus and
DFC treated virus to determine if there were any mutations created
by the applied drug pressure on weeks when the virus appeared to be
resistant (Table 1 (WT--wild type); FIG. 1).
TABLE-US-00001 TABLE 1 Selection of S68del HIV-1 in DFC-treated
human PBM cells. Mutation Percentages of by Number of clones
isolates DFC Culture population sequenced containing RT (.mu.M)
week sequencing S68.DELTA. K65R WT Total mutations 0 4 WT 0.1 10 WT
1 11 WT 10 10 100% WT 1 12 WT 100% WT 0.1 14 S68.DELTA./WT 5 1 7
72% S68.DELTA., 14% WT, 14% S68N 0.1 15 WT 0.1 16 WT 3 8 11 27%
S68.DELTA., 73% WT 1 17 S68.DELTA./WT 2 5 7 29% S68.DELTA., 71% WT
1 18 S68.DELTA./WT 2 2 4 50% S68.DELTA., 50% WT 1 19 S68.DELTA. 7 7
100% WT 0 20 S68.DELTA. 12 1 1 14 86% S68.DELTA., 7% K65R, 7% WT 1
22 S68.DELTA. 0 23 S68.DELTA. 28 29 94% S68.DELTA., 6% S68.DELTA. +
T69 or T69S 2 24 S68.DELTA. 3 25 S68.DELTA./WT/ WT 3 26
S68.DELTA./WT/ 2 8 10 20% S68.DELTA., 80% WT K65R 3 27
S68.DELTA./WT/ 1 7 8 12% S68.DELTA., 88% WT K65R 6 28 K65R 6 29
K65R 8 8 100% K65R 6 30 K65R 8 8 100% K65R 1.5 36 K65R 6 49 K65R SN
negative 6 52 S68.DELTA./WT 5 2 1 8 63% S68.DELTA. + K65R, 25%
K65R, 12% WT
[0162] Population sequencing of virus during this assay revealed a
disruption of the S68 codon in the reverse transcriptase (RT)
sequence, which may alternatively be a deletion of the AGT codon 68
trinucleotide, or of the adjacent +1 frameshift trinucleotide GTA
(Table 2).
TABLE-US-00002 TABLE 2 Amino acid sequence changes for S68del
mutation. Mutation HIV-1.sub.LAI sequence Sequence Change S68del
67/GAC 68/AGT 69/ACT 67/GAC 69/ACT 70/AAA (SEQ ID NO: 6) (SEQ ID
NO: 7)
[0163] The S68del mutation was first detected by population
sequencing at week 14 as a mix with wild-type (WT) (Table 1). By
week 19, S68del dominated the pool. At week 25, some K65R was
detected as well. In week 28, only K65R was detectible. At week 52,
the pool contained a mixture of S68del and K65R.
[0164] Cloning of the S68del virus demonstrated that S68del can
occur independently or as a mixture with wild-type, K65R, T69A or
T69S. Sometimes the mutations can be found in the same genome. No
other mutation in the reverse transcriptase region was
detected.
[0165] Deletions that occur in the RT region between codons 67 and
69 have been known to occur in combination with T69G or Q151M
mutations (Hu et al., J. Acquir. Immune Defic. Syndr. 2007;
45:494-500; Winters et al., J. Virol. 2000; 74; 10707-10713). The
alignment of published sequences with the S68del sequence as seen
in Table 3 shows that the S68 deletion occurred without any
associated mutations in the .beta.3-.beta.4 loop. The published
sequences were found in clinical samples from patients that had
undergone multiple drug treatments for HIV-1 infection and occurred
with other multiple drug resistant (MDR) mutations (Table 3;
AF271766: Boyer et al., J. Virol. 2004; 78:9987-97; AF311203:
Hammond et al., Antimicrob. Agents Chemother. 2005; 49:3930-2;
DQ394304: Hu et al., J. Acquir. Immune Defic. Syndr. 2007;
45:494-500; AF311157: Tamalet et al., Virology 2000; 270:310-6 and
EF154395: Villena et al., Journal of Virology 2007, 81:4713-4721).
In contrast, the S68del mutation was discovered in vitro in PBM
cells under monotherapy.
TABLE-US-00003 TABLE 3 Alignment of the S68del sequence with
previously published HIV-1 RT deletions. RT region of HIV-1 Virus
Deletion 63 64 65 66 67 68 69 70 71 72 73 S68deletion S68.DELTA. I
K K K D -- T K W R K (SEQ ID NO: 12) AF271766 D67.DELTA., T69G,
K70R I K K K -- s G R W R K (SEQ ID NO: 13) AF311203 K70.DELTA.,
S68N I K K K D N T -- W R K (SEQ ID NO: 14) DQ394304 K70.DELTA.,
S68G I K K K D G T -- W R K (SEQ ID NO: 15) AF311157 T69.DELTA.,
D67S, S68G I K K K S G -- K W R K (SEQ ID NO: 16) EF154395
T69.DELTA., S68G, K70G I K K K D G -- G W R K (SEQ ID NO: 17) LAI I
K K K D S T K W R K (SEQ ID NO: 18) pNL4-3 I K K K D S T K W R K
(SEQ ID NO: 19)
Mutated sequences are in bold and large font.
Example 2
Drug Susceptibility of In Vitro-Selected S68del HIV-1
[0166] Mutations that occur in the HIV-1 RT region between amino
acids 62 and 78 increase NRTI resistance significantly (Hu et al.,
J. Acquir. Immune Defic. Syndr. 2007; 45:494-500). Drug resistance
of the S68del virus isolated at week 23 (HIV.sub.s68.DELTA.-23) was
measured with the 3H-TTP RT incorporation assay in human PBM cells
(Schinazi, et al., Antimicrob. Agents Chemother. 1990;
34:1061-1067; Stuyver et al., Antimicrob. Agents Chemother. 2002;
46:3854-60). HIV.sub.s68.DELTA.-23 was population sequenced to
ensure the dominant population was the deletion at codon 68.
TOPO.RTM. cloning (Invitrogen) performed on HIV.sub.s68.DELTA.-23
indicated that approximately 90% of the population was pure S68del.
The other approximately 10% either had a T69 deletion or S68del
with a mutation T69A. The susceptibility of the S68del virus to
several nucleoside reverse transcriptase inhibitors (NRTI), a
non-nucleoside reverse transcriptase inhibitor (NNRTI) and a
protease inhibitor (PI) was tested. Fold increases were measured
relative to HIV.sub.LAI (Table 4, FIGS. 2 and 3). Data are the
averages of 2-6 independent experiments.
TABLE-US-00004 TABLE 4 Drug susceptibility results for
HIV.sub.s68.DELTA.-23. EC50 EC90 FI EC50 FI EC90 Virus Compound
(.mu.M) (.mu.M) (.mu.M) (.mu.M) S68.DELTA.-23 AZT 0.0038 0.0140 1.4
0.7 DOT 0.66 3.47 3.7 3.3 Sustiva 0.00039 0.0035 3.8 1.6 Lopinavir
0.006 0.019 0.49 0.59 Abacavir 3.9 11.3 47.3 8.7 D4T 0.41 1.5 4.1
4.8 DDI 0.63 4.5 1.2 2.5 DDC 0.22 0.93 2.5 3.4 DFDOC 0.44 1.45 4.8
4.2 DFC 1.1 3.4 34.4 9.2 DAPD 7.9 34.5 38.2 32.4 3TC 0.88 3.3 43.5
31.7 (-)-FTC 0.30 1.1 62.5 48.1 TDF 0.43 1.6 33.7 5.3 DXG 1.6 5.1
6.2 3.7 Drug abbreviations are defined in the description of FIG 2.
FI EC.sub.50 - fold increase in 50% effective concentration. FI
EC.sub.90 - fold increase in 90% effective concentration.
[0167] HIV.sub.s68.DELTA.-23 showed increased resistance (FI
EC.sub.50 greater than 5) against NRTI such as DFC, DXG, DAPD, TDF,
3TC, abacavir and (-)FTC. HIV.sub.s68.DELTA.-23 showed modest
resistance (FI EC.sub.50 between 2.5 and 5) against NRTI such as
D-FDOC, D4T, DDC, and DOT. HIV.sub.s68.DELTA.-23 showed no
resistance (FI EC.sub.50 less than 2.5) against NRTI such as DDI
and AZT. HIV.sub.s68.DELTA.-23 susceptibility to NNRTI
(Sustiva.RTM.) or PI (Lopinavir.RTM.) was not significantly
changed.
Example 3
Construction of S68del HIV-1 by Site-Directed Mutagenesis
[0168] An S68del mutant HIV-1 was reconstructed by site-directed
mutagenesis using the Stratagene Quick II XL Site Directed
Mutagenesis Methodology (Stratagene). An intermediate vector with a
4 kb fragment of pNL4-3 (AF3244930) containing the RT coding region
was cloned into the pCR2.1 vector (Invitrogen). Primers MC0014F:
CAA TAC TCC AGT ATT TGC CAT AAA GAA AAA AGA CAC TAA ATG GAG AAA ATT
AGT AGA TTT CAG AGA AC (SEQ ID NO: 8) and MC0015R: GTT CTC TGA AAT
CTA CTA ATT TTC TCC ATT TAG TGT CTT TTT TCT TTA TGG CAA ATA CTG GAG
TAT TG (SEQ ID NO: 9) were selected according to Stratagene
strategy. Using these primers, the S68del mutation was generated in
the intermediate vector and confirmed by sequencing. The HIV-1
fragment from the intermediate vector was digested with Spe I/Age I
and subcloned into the full-length pNL4-3 vector. The S68del
pNL4-3-based infectious clone was then transfected into HEK293T
cells and supernatant was collected after 4 days. The supernatant
obtained from HEK293T was used to infect fresh PBM cells, after
which the HIV-1 virus pool was passaged to establish an infective
pool.
Example 4
Site-Directed Mutagenesis for Protein Expression
[0169] A site directed S68 deletion mutant was created by digesting
pCR 2.1 (Invitrogen) and pNL4-3 (AF3244930) with restriction
enzymes Eco RI and Spe I. The pCR2.1 was cut into a single band of
3.9 kb, and the pNL4-3 was cut into one 4.2 kb band and one 10.6 kb
band. Nucleic acids were separated by gel electrophoresis in a TAE
gel and extracted from bands using the Qiagen gel extraction kit.
The 4.2 kb band from pNL4-3 containing the HIV-RT encoding
sequences was ligated into the linearized pCR2.1, making the
construct MC002. Site directed mutagenesis was performed on the
MC002 plasmid. Primers used had the S68 codon AGT deleted, thus
introducing the S68 deletion into the pNL4-3 RT background.
Presence of the S68 codon AGT deletion (and no other surrounding
mutations) was confirmed by sequencing in both directions.
[0170] To ligate the RT of pNL4-3 comprising the deletion at S68
codon 68 into protein expression vector pE60 (Invitrogen), MC002
was amplified using forward primer
5'-CGCGCCCATGGTGCCCATTAGTCCTATTGAGACTGTACC-3' (SEQ ID NO: 10) and
reverse primer 5'-GCGCGCAGATCTTAGTACTTTCCTGATTCC AGCACTGAC-3' (SEQ
ID NO: 11). The PCR product and pQE60 were digested with Bgl II and
Nco I. The digested products were separated by gel electrophoresis
nucleic acids in excised gel bands extracted using the Qiagen gel
extraction kit. The amplified PCR product comprising the RT of
pNL4-3 was ligated into the linearized pQE60. The ligation mix was
transformed into chemically competent bacteria (Alpha-select).
Plasmid DNA from selected transformants was extracted using a
mini-prep kit (Qiagen). Plasmid DNA was sequenced in both
directions to confirm the ligation.
Example 5
Quantitative HIV-1 Real-Time PCR Assay for Determining Viral Load
of S68del HIV-1
[0171] The real-time PCR assay for quantifying virus levels in PBM
cells serves as a more sensitive method for measuring drug
resistance of S68del HIV-1 than other standard methods. Results for
this assay performed for pNL4-3 are shown in FIG. 4. SK38 Primer:
ATA ATC CAC CTA TCC CAG TAG GAG AAA T (SEQ ID NO: 1) and SK39
Primer: TTT GGT CCT TGT CTT ATG TCC AGA ATG C (SEQ ID NO: 2) were
used in the real-time PCR assay. The presence of amplified product
was detected with the SK19 probe: ATC CTG GGA TTA AAT AAA ATA GTA
AGA ATG TAT AG (SEQ ID NO: 3). The resistance of S68del virus in
the pNL4-3 background (S68del.sub.pNL4-3) against AZT and DFC was
measured by this assay (Table 5). Four concentrations were tested
in duplicate for each data point.
TABLE-US-00005 TABLE 5 Resistance of S68del.sub.pNL4-3 against AZT
and DFC in human PBM cells measured by quantitative real-time PCR.
S68del in Fold Increase Samples pNL4-3 .mu.M pNL4-3 for S68del AZT
EC50 = 0.13 0.10 AZT FI50 = 0.73 EC90 = 0.33 0.30 AZT FI90 = 0.89
DFC EC50 = 0.21 3.1 DFC FI50 = 14.7 EC90 = 2.3 8.5 DFC FI90 = 3.7
Drug abbreviations are defined in the description of FIG. 2.
EC.sub.50 - 50% effective concentration. EC.sub.90 - 90% effective
concentration. FI50 - fold increase in 50% effective concentration.
FI90 - fold increase in 90% effective concentration.
[0172] S68del.sub.pNL4-3 showed increased resistance against DFC,
but not against AZT. These results confirmed results obtained by
the .sup.3H-TTP RT incorporation assay and the heteropolymeric-DNA
colorimetric RT assay with the in vitro selected S68del virus.
Thus, the results show that the S68del.sub.pNL4-3 virus will be a
useful and valid construct to measure S68del drug resistance
phenotypes. Furthermore, the real-time PCR assay will serve as an
accurate method for measuring drug resistance. Finally, these
results confirm that the deletion of the AGT codon 68
trinucleotide, or deletion of the adjacent +1 frameshift
trinucleotide GTA, is solely responsible for increased resistance
of the virus to growth in the presence of DFC.
Example 6
Enzymatic Characterization of the Recombinant S68del Reverse
Transcriptase by a Heteropolymeric DNA Polymerase Assay
[0173] The principle and performance of the non-radioactive RT
assay has been described (Lennerstrand et al., Antimicrob. Agents
Chemother. 2007; 51:2078-2084). Separate kit components, such as
covalently-linked DNA microtiter plates and tracer solution
(alkaline phosphate (AP)-conjugated anti-BrdU antibody) were
obtained from Cavidi Tech, Uppsala, Sweden. In brief, the 96-well
microtiter plate used consisted of an 18 base heteropolymeric DNA
primer covalently bound to the well. In the RT assay, the DNA
primer is bound to a 50-base DNA template at 50 ng/well (190 nM)
with a 5'-A.sub.12-3' tail with a 5'-(GTCA).sub.5-3' repeat
(Integrated DNA Technologies, USA). The RT assay reaction mixture
(total volume 150 .mu.l/well) contained: Hepes 50 mM, pH 7.3;
MgCl.sub.2 10 mM; Triton X-100 0.5%; bovine serum albumin 0.1
mg/ml; dATP, dGTP, dCTP and 5-bromo-2'-deoxyuridine-5'-triphosphate
(BrdUTP) at 1.0 .mu.M each (where BrdUTP replaces TTP) (Sigma). To
obtain ATP primer unblocking reaction in the assay, the ATP
(Amersham/GE Health Care) was set to physiological concentration
(3.2 mM). However, the ATP was only used in the assay with virus
pellets as sample, not merely for primer unblocking reaction but to
protect degradation of substrate in the crude sample. Furthermore,
the dNTP level including BrdUTP was increased from 1 .mu.M to 4
.mu.M for the assay with the virus pellet samples. Subsequently, in
the assay with recombinant purified RT enzyme, no ATP besides 1
.mu.M dNTP was used.
[0174] The reaction was started with the addition of RT either as
recombinant or crude virus pellet form in a similar activity range
as previous published (Lennerstrand et al., Antimicrob. Agents
Chemother. 2007; 51:2078-2084). The RT reaction mixture was
incubated at 33.degree. C. for 180 min and terminated by NaOH (to
dehybridize the template) and water washing of the plates. The
tracer incubation step with anti-BrdU antibodies-AP-conjugated and
the detection step for color absorbance at 405 nm was performed as
previously described (Lennerstrand et al., Antimicrob. Agents
Chemother. 2007; 51:2078-2084. The NRTI-TP used were AZT-TP (Cavidi
Tech), DFC-TP and (-) FTC-TP. The latter nucleotides were
synthesized from the corresponding nucleoside analog (Ludwig et
al., J. Org. Chem. 1989; 54:631-635).
[0175] The resistance of RT derived from S68del and M184V particles
against AZT, (-)FTC and DFC triphosphates was tested by a
heteropolymeric-DNA colorimetric RT assay with 3.2 mM ATP.
Fold-increases were measured relative to HIV.sub.LAI (FIG. 5). Both
S68del and M184V RTs showed increased resistance to (-)FTC. Only
S68del RT showed increased resistance to DFC (FIG. 5). RT from
virally-derived S68del demonstrated a 5.6-, 2.5- and 10-fold
increase in resistance to DFC-TP, AZT-TP and (-)FTC-TP,
respectively, in the enzymatic assay (FIG. 6).
[0176] The level of resistance to NRTI-TP by the S68del mutants
compared to wild type RT was determined as IC.sub.50 values of RT
activity in the absence of ATP (Table 6). Fold-increased resistance
values were determined by dividing the IC.sub.50 for the mutant by
the IC.sub.50 for respective wild type. The RT activity was linear
during the assay time within the substrate range used, and thus
steady state kinetics were assumed.
TABLE-US-00006 TABLE 6 DFC-TP AZT-TP (--)FTC-TP Reverse IC.sub.50
.+-. Fold- IC.sub.50 .+-. Fold- IC.sub.50 .+-. Fold- Transcriptase
SE.sup.a incr .sup.b SE.sup.a incr .sup.b .+-. SE.sup.a incr .sup.b
Wild type 0.14 .+-. 0.02 1.0 0.27 .+-. 0.02 1.0 3.5 .+-. 0.3 1.0
Recombinant 1.1 .+-. 0.2 7.9 0.7 .+-. 0.1 2.6 27 .+-. 2 7.7 S68del
.sup.aThe IC.sub.50 values are expressed as .mu.M of NRTI-TP. The
IC.sub.50 are averages from at least two separate experiments
conducted in duplicate. The IC.sub.50 values were determined using
seven different concentrations of NRTI-TP adjusted optimally for
each mutant's expected IC.sub.50 value. Standard errors (.+-.SE)
are indicated. .sup.b Fold increase is calculated by dividing the
mutant RT IC.sub.50 by the respective wild type IC.sub.50.
[0177] Enzymatic studies of the S68del RT detected similar
resistance to NRTI-TP with and without ATP. Without being bound by
theory, this result suggests that S68del resistance is not
ATP-dependent and most likely occurs by enhanced substrate
discrimination.
Sequence CWU 1
1
19128DNAArtificial SequencesourceSynthetic primer 1ataatccacc
tatcccagta ggagaaat 28228DNAArtificial SequencesourceSynthetic
primer 2tttggtcctt gtcttatgtc cagaatgc 28335DNAArtificial
SequencesourceSynthetic probe 3atcctgggat taaataaaat agtaagaatg
tatag 35425DNAArtificial SequencesourceSynthetic primer 4ttgactcaga
ttggttgcac tttaa 25525DNAArtificial SequencesourceSynthetic primer
5aagaacccat agtaggagca gaaac 2569DNAHuman immunodeficiency virus
type 1 6gacagtact 979DNAHuman immunodeficiency virus type 1
7gacactaaa 9868DNAArtificial SequencesourceSynthetic primer
8caatactcca gtatttgcca taaagaaaaa agacactaaa tggagaaaat tagtagattt
60cagagaac 68968DNAArtificial SequencesourceSynthetic primer
9gttctctgaa atctactaat tttctccatt tagtgtcttt tttctttatg gcaaatactg
60gagtattg 681039DNAArtificial SequencesourceSynthetic primer
10cgcgcccatg gtgcccatta gtcctattga gactgtacc 391139DNAArtificial
SequencesourceSynthetic primer 11gcgcgcagat cttagtactt tcctgattcc
agcactgac 391210PRTHuman immunodeficiency virus type 1 12Ile Lys
Lys Lys Asp Thr Lys Trp Arg Lys 1 5 101310PRTHuman immunodeficiency
virus type 1 13Ile Lys Lys Lys Ser Gly Arg Trp Arg Lys 1 5
101410PRTHuman immunodeficiency virus type 1 14Ile Lys Lys Lys Asp
Asn Thr Trp Arg Lys 1 5 101510PRTHuman immunodeficiency virus type
1 15Ile Lys Lys Lys Asp Gly Thr Trp Arg Lys 1 5 101610PRTHuman
immunodeficiency virus type 1 16Ile Lys Lys Lys Ser Gly Lys Trp Arg
Lys 1 5 101710PRTHuman immunodeficiency virus type 1 17Ile Lys Lys
Lys Asp Gly Gly Trp Arg Lys 1 5 101811PRTHuman immunodeficiency
virus type 1 18Ile Lys Lys Lys Asp Ser Thr Lys Trp Arg Lys 1 5 10
1911PRTHuman immunodeficiency virus type 1 19Ile Lys Lys Lys Asp
Ser Thr Lys Trp Arg Lys 1 5 10
* * * * *