U.S. patent application number 11/337344 was filed with the patent office on 2006-05-25 for polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acouired immunodeficiency syndrome.
This patent application is currently assigned to The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Mark Holodniy, David A. Katzenstein, Michael J. Kozal, Thomas C. Merigan.
Application Number | 20060110726 11/337344 |
Document ID | / |
Family ID | 25382403 |
Filed Date | 2006-05-25 |
United States Patent
Application |
20060110726 |
Kind Code |
A1 |
Kozal; Michael J. ; et
al. |
May 25, 2006 |
Polymerase chain reaction assays for monitoring antiviral therapy
and making therapeutic decisions in the treatment of acouired
immunodeficiency syndrome
Abstract
The present invention relates to methods of monitoring, via
polymerase chain reaction, the clinical progression of human
immunodeficiency virus infection and its response to antiretroviral
therapy. According to the invention, polymerase chain reaction
assays may be used to predict immunological decline and to
identify, at an early stage, patients whose infection has become
resistant to a particular antiretroviral drug regimen.
Inventors: |
Kozal; Michael J.; (Menlo
Park, CA) ; Merigan; Thomas C.; (Portola Valley,
CA) ; Katzenstein; David A.; (Menlo Park, CA)
; Holodniy; Mark; (Mountain View, CA) |
Correspondence
Address: |
HOWREY LLP
C/O IP DOCKETING DEPARTMENT
2941 FAIRVIEW PARK DRIVE, SUITE 200
FALLS CHURCH
VA
22042-7195
US
|
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University
|
Family ID: |
25382403 |
Appl. No.: |
11/337344 |
Filed: |
January 23, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10321162 |
Dec 16, 2002 |
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11337344 |
Jan 23, 2006 |
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09782971 |
Feb 13, 2001 |
6503705 |
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10321162 |
Dec 16, 2002 |
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09399082 |
Sep 17, 1999 |
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09782971 |
Feb 13, 2001 |
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08470885 |
Jun 6, 1995 |
5968730 |
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09399082 |
Sep 17, 1999 |
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07883327 |
May 14, 1992 |
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08470885 |
Jun 6, 1995 |
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Current U.S.
Class: |
435/5 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/703 20130101; C12Q 2600/106 20130101 |
Class at
Publication: |
435/005 ;
435/006 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A kit for determining the presence of a mutation at a codon of
interest of the HIV reverse transcriptase enzyme, comprising a
first pair of polymerase chain reaction (PCR) primers for
amplifying by PCR a DNA fragment containing the codon of interest,
and at least two pairs of second primers for amplifying DNA
encoding wild type and mutant forms at the codon of interest.
2. The kit of claim 1 wherein the codon of interest is codon
215.
3. The kit of claim 1 wherein the codon of interest is codon
74.
4. The kit of claim 1 wherein the two pairs of second primers
comprise primer B (5'-GGATGGAAAGGATCACC-3') (SEQ ID NO: 3) and 3M
(3'-AAGTGTGGTCTGTTTTTTGTA-5')(SEQ ID NO: 5), and primers B and 3W
(3'-TGGTGTGGTCTGTTTTTTGTA-5) (SEQ ID NO: 4).
Description
1. INTRODUCTION
[0001] The present invention relates to methods of monitoring, via
polymerase chain reaction, the clinical progression of human
immunodeficiency virus infection and its response to antiretroviral
therapy. According to the invention, polymerase chain reaction
assays may be used to predict immunological decline and to
identify, at an early stage, patients whose infection has become
resistant to a particular antiretroviral drug regimen.
2. BACKGROUND OF THE INVENTION
[0002] Human immunodeficiency virus (HIV) isolated from patients
treated with zidovudine (AZT) may demonstrate markedly reduced in
vitro susceptibility to AZT (Larder et al., 1989, Science
243:1731-1734; Rooke et al., 1989, AIDS 3:411-415; Land et al.,
1990, J. Infect. Dis. 161:326-329; Boucher et al., 1990, Lancet
336:585-590; Japour et al., 1991, Proc. Natl. Acad. Sci.
88:3092-96; Tudor-Williams et al., 1992, Lancet 339:15-19). This
reduced susceptibility has been related to the duration of therapy
with AZT and the severity of HIV disease at the time AZT therapy is
begun (Richman et al., 1990, J. AIDS 3:743-756). Nucleotide
sequence analysis of AZT-resistant HIV strains has revealed a
number of mutations in the reverse transcriptase (RT) gene
associated with decreased AZT susceptibility (Larder et al., 1989,
Science 246:1155-1158; Larder et al., 1991, AIDS USA 89:1934-1938;
St. Clair et al., 1991, Science 253:1557-1559; Richman et al.,
1991, J. Infect. Dis. 164:1075-1081). Molecular cloning experiments
have confirmed that these mutations in the RT gene confer AZT
resistance (Larder et al., 1989, Science 246:1155-1158; Larder et
al., 1991, AIDS 5:137-144; Kellam et al., 1992, Proc. Natl. Acad.
Sci. USA 89:1934-1938; St. Clair et al., 1991, Science
253:1557-1559). Of these mutations the one at codon 215 resulting
in a single amino acid substitution (Thr.fwdarw.Tyr or Phe) has
been shown to be the most common mutation and to have the greatest
impact on in vitro susceptibility to AZT (Larder et al., 1991, AIDS
5:137-144; Richman et al., 1991, J. Infect. Dis. 164:1075-1081;
Boucher et al., 1992, J. Inf. Dis. 165:105-110).
[0003] Several studies have addressed the relationship between in
vitro AZT resistance, mutations in the RT gene and clinical
disease. Richman and coworkers studied 32 patients with different
stages of HIV disease and demonstrated that the development of in
vitro AZT resistance was related to the duration of therapy with
AZT and to the severity of disease at the time AZT was begun
(Richman et al., 1990, J. AIDS 3:743-746). Boucher and coworkers
studied HIV P24-antigenemic patients treated with AZT for 2 years.
They observed that at 6 months, seven patients with a mutation at
codon 215 had a weak, non-statistically significant trend toward
lower CD4 counts compared to nine patients who were wild type at
codon 215 (Boucher et al., 1990, Lancet 336:585-590). After 2 years
nearly all patents had the mutation. Tudor-Williams and coworkers
studied HIV isolates from 19 symptomatic children treated with AZT
for 9-39 months and showed that in vitro AZT resistance was
associated with poor clinical outcome (Tudor-Williams et al., 1992,
Lancet 339:15-19). However, adult studies have not shown a precise
correlation between the development of in vitro resistance and
progression of HIV disease.
3. SUMMARY OF THE INVENTION
[0004] The present invention relates to methods of monitoring, via
polymerase chain reaction (PCR), the clinical progression of human
immunodeficiency virus (HIV) infection and its response to
antiviral therapy. It is based, in part, on the discovery that
plasma HIV RNA copy number, as measured using PCR, may be used as a
sensitive marker of the circulating HIV viral load to assess the
therapeutic effect of antiretroviral compounds. In working examples
described herein, an increase in plasma HIV RNA copy number was
found to correlate with disease progression, and successful
antiretroviral therapy was found to correlate with a decline in
plasma HIV RNA copy number.
[0005] The invention is also based, in part, on the discovery that
genetic changes in HIV which confer resistance to antiretroviral
therapy may be rapidly determined directly from patient peripheral
blood mononuclear cells (PBMC) and/or plasma HIV RNA using a
"nested" PCR procedure. In working examples disclosed herein, a
mutation at codon 215 of HIV reverse transcriptase (RT) was found
to occur in AZT-treated patients which correlated with
refractoriness to AZT treatment. The mutation was found in plasma
HIV RNA one to eight months before it was detectable in PBMC. The
development of the condon 215 mutation in HIV RT was found to be a
harbinger of immunological decline, which occurred between six and
twelve months after the mutation was detectable in plasma HIV
RNA.
[0006] In particular embodiments of the invention, PCR assay may be
used to monitor the clinical progression of HIV infection in
patients receiving antiretroviral therapy. An increase in plasma
HIV copy number detected by such an assay would correlate with
refractoriness to treatment. If a patient being treated with an
antiretroviral therapeutic agent exhibits an increase in plasma HIV
RNA copy number, a physician should consider altering the patients
treatment regimen. It should be noted that the present invention
offers the advantage of being more sensitive in measuring HIV virus
than standard methods which measure plasma p24 antigen or
infectious virus detectable by culture techniques.
[0007] In further embodiments of the invention, PCR assay may be
used to detect mutations at codon 215 of HIV RT which correlate
with resistance to antiretroviral therapy and which precede
immunologic decline by 6-12 months. Once mutation at codon 215 has
been detected in a patient undergoing antiretroviral therapy, an
alteration in the therapeutic regimen must be considered. The speed
at which a modified regimen should be instituted may depend on
whether the mutation is present in plasma HIV RNA or PBMC. If the
mutation is present in PBMC, a rapid alteration in therapy may be
warranted.
[0008] In patients suffering from HIV infection, opportunistic
infections arising as a result of a compromised immune system can
be rapidly fatal. It is therefore extremely important to strive to
avoid deterioration of the immune system in these patients. Because
the present invention enables the early prediction of immunological
decline, it allows alteration of a patient's therapeutic regimen so
as to avoid opportunistic infections, and therefore may be used to
promote survival and improve the quality of life of HIV-infected
patients.
4. DESCRIPTION OF THE FIGURES
[0009] FIG. 1. Human immunodeficiency virus RNA copy number in 200
.mu.l of plasma from 72 subjects as determined by cDNA gene
amplification. Of 39 patients who were not currently receiving
antiretroviral therapy, 20 had a CD4 count<200/mm.sup.3 (HIV
copy number 1,369.+-.707) and 19 had a CD4 count>200/mm.sup.3
(HIV copy number 44.+-.10). Of 33 subjects who were currently on
AZT, 14 had a CD4 count<200/mm.sup.3 (HIV copy number 295.+-.5)
and 19 had a CD4 count>200/ml.sup.3 (HIV copy number 16.+-.5).
Mean copy number (open circles) of subjects not on therapy was
690.+-.360 as compared to 134.+-.219 for patients currently on AZT
(P<0.05, independent sample t test).
[0010] FIG. 2. Human immunodeficiency virus RNA copy number in
plasma from 27 subjects before (pre) and 1 mo after (post)
dideoxynucleoside therapy. (.smallcircle.) AZT; (.circle-solid.)
AZT+ddI; and (.tangle-solidup.) ddI alone. Mean copy number
decreased from 540.+-.175 to 77.+-.35 after therapy (P<0.05
paired t test).
[0011] FIG. 3. Human immunodeficiency virus RNA copy number in
plasma from 9 subjects with two samples obtained before initiation
of therapy (pre 1 and pre 2) and two samples obtained 1 and 2 mo
after commencing therapy (post 1 and post 2). (.tangle-solidup.)
ddI alone; (.circle-solid.) ddI+AZT.
[0012] FIG. 4. Serial CD4 counts in PBMC (cells/.mu.l) of 17
patients in which HIV reverse transcriptase carried a mutation at
codon 215 (top) and of 21 patients in which HIV reverse
transcriptase was wild type at codon 215 (bottom).
[0013] FIG. 5. CD4 cell counts in PBMC from serial time points in
37 patients. .smallcircle.=wild type sequence in serum specimen,
=mutant sequence in serum, .uparw.=wild type sequence in PBMC,
.dwnarw.=mutant sequence in PBMC; A: 16 patients mutant at codon
215 in both serum HIV RNA and PBMC (proviral DNA). B: 10 patients
mutant at codon 215 in serum HIV RNA but wild type in their PBMC.
C: 11 patients whom remained wild type at codon 215 in their serum
HIV RNA and PBMC.
[0014] FIG. 6. Relationship of PBMC to serum genotypes in the 38
patients at study endpoint.
[0015] FIG. 7. Nucleotide sequences of SK38, SK39, and SK19.
5. DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention relates to methods of monitoring, via
PCR, the clinical progression of HIV infection in patients
receiving antiretroviral therapy. For purposes of clarity and not
by way of limitation, the detailed description of the invention is
divided into the following subsections: [0017] (i) PCR assay of
plasma HIV RNA; [0018] (ii) PCR assay of peripheral blood
mononuclear cells; [0019] (iii) PCR assay for mutation at codon 215
of HIV reverse transcriptase; and [0020] (iv) utility of the
invention.
[0021] It should be noted that heparin appears to have an
inhibitory effect on gene amplification via PCR. It is therefore
desireable to avoid using heparin as an anticoagulant of patient
blood samples. If herapin has been used in a sample, the sample may
be purified of heparin, for example, by collecting virus by
ultracentrifugation.
[0022] 5.1 PCR Assay of Plasma HIV RNA
[0023] According to the invention, it is desireable to avoid
degradation of RNA in plasma samples prior to measurement of HIV
RNA copy number. Therefore, in preferred embodiments of the
invention, guanidinium is added to plasma or serum samples prior to
storage at a concentration of about 2.5M and samples are kept
frozen at -70.degree. C., with no samples stored for longer than
about 3 months. Serum may be used interchangeably with plasma
according to the invention.
[0024] RNA may be extracted from plasma using standard techniques,
such as those set forth in Chomczynski and Sacchi, 1987, Ann.
Biochem. 162:156-159. For example, 200 .mu.l of clarified plasma to
which 200 .mu.l of 5M guanidinium thiocyanate had previously been
added may be extracted with phenol/chloroform and precipitated with
isopropanol. The resulting pellet may then be washed in 75 percent
ethanol, dried, and brought up into solution in
diethylpyrocarbonate-treated glass distilled water.
[0025] From plasma RNA, HIV RNA may be transcribed to cDNA using a
suitable reverse transcriptase (for example, Moloney murine
leukemia virus reverse transcriptase) using standard techiques,
such as for example, those set forth in Kawasaki, 1990, in "PCR
Protocols: A Guide to Methods and Applications," Innis et al.,
eds., Academic Press, Berkeley, Calif. pp. 21-27. Any suitable
primer for amplification of HIV genomic RNA sequences may be used,
including, but not limited to, the oligomers SK38, SK39, and SK19
(FIG. 7) described in Kellog et al., 1990, in "PCR Protocols: A
Guide To Methods and Applications," Innis et al., etds., Academic
Press, Berkeley, Ca. pp. 337-347. In a preferred embodiment of the
invention, HIV cDNA may be amplified as follows: to a 100 .mu.l
reaction mixture, cDNA prepared as described supra may be added,
together with 50 pmol of primers SK38 and SK39, 10 mM of each dNTP,
10 mM Tris (pH 8.3), 2.5 mM MgCl.sub.2, 50 mM KCl, and 2.5 U of
recombiTaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, Conn.). The
mixture may then be overlaid with 50 .mu.l of mineral oil, and
tubes containing the reaction may be placed in a DNA thermal cycler
(e.g. Perkin-Elmer Cetus) for about 30 cycles of amplification with
the following program: 95.degree. C./30 seconds, 55.degree. C./30
seconds, and 72.degree. C./60 seconds for denaturation, annealing,
and extension, respectively. Negative and positive controls which
include both high and low copy number HIV RNA and DNA may be added
at each step.
[0026] It is important that the number of cycles not exceed 35, and
preferably, only about 30 cycles of amplification are used in the
PCR. Using a greater humber of cycles may detract from the
sensivity of the assay.
[0027] The copy number of HIV RNA may then be measured by methods
known to the skilled artisan. For example, the number of copies of
HIV RNA in a patient sample may be quantiated by hybridizing the
product of the above PCR with a detectably labeled probe that is
complementary to HIV sequence. The amount of signal generated by
probe hybridized to PCR product may then be compared to the amount
of signal generated by probe hybridized to a known copy number of
HIV. Probe may be detectably labeled by an enzyme, a radioisotope,
a fluorescent compound, a chromogenic compound, or any other
detectably labeled compound.
[0028] In a preferred, nonlimiting embodiment of the invention, at
least one of the PCR primers may be biotinylated, probe may be
labeled with horseradish peroxidase (HRP), and copy number may be
evaluated by an enzyme-linked affinity assay as follows. 96-well
microplates (Maxisorp; Nunc, Naperville, Ill.) may be coated with
100 .mu.l of a 0.1 mg/ml solution of avidin (Sigma Chemical Co.,
St. Louis, Mo.) in 50 mM Na.sub.2CO.sub.3 (pH 9.6) overnight at
room temperature. Wells may then be washed twice with PBS, and then
filled with 300 .mu.l of a blocking solution containing 5.times.
Denhardt's solution, 1% gelatin (Sigma), 250 .mu.l/ml sheared
herring sperm DNA (Promega Biotec, Madison, Wis.) at least
overninght at 4.degree. C. Immediately before use, the blocking
solution may be aspirated from each well and 5 .mu.l of PCR product
prepared as described supra (using at least one biotinylated
primer) may be added to each well together with 65 .mu.l of a
hybridization solution containing 5.times. saline sodium phosphate
EDTA, 5.times. Denhardt's solution; and 1 pmol of HRP-labeled SK19
HIV gaq-specific probe. Because HIV primer was biotinylated, HIV
amplified sequences should selectively adhere to the avidin-coated
wells, so that a capture and hybridization reaction may be carried
out for 1 hour at 42.degree. C. Each well may then be washed about
20 times with PBS containing 0.05% Tween-20, for example, using a
Biomek.TM. 1000 Automated Workstation (Beckman Instruments, Inc.,
Palo Alto, Calif.). The HRP substrate O-phenylenediamine (Sigma)
may then be prepared at 0.6 mg/ml in 0.1 M citrate buffer (pH 5.5)
containing 0.03% hydrogen peroxide, and 150 .mu.l of this solution
may be added to each well. After about 10 minutes the reaction may
be stopped with 1N H.sub.2SO.sub.4 and the optical density of each
well measured at 490 nm, for example by the Biomek 1000. A lower
level of positivity had been defined as an absorbance of 0.135.
This cutoff value was calculated from the mean absorbance obtained
from a group of seronegative samples plus three standard
deviations. Copy number from subject samples may be determined from
the absorbances obtained from a dilution series of an RNA gag gene
construct of known copy number (Holodniy et al., 1991, J. Infect.
Dis. 163:862-866).
[0029] In an alternate preferred, specific embodiment, RNA
collected from plasma may be reverse-transcribed by using 500 ng of
primer A (5'-TTCCCATTAGTCCTATT-3') and 5 units of MuLV RT (Bethesda
Research Labs) in 10 .mu.l of amplification buffer (25 mM kCL, 50
mM Tris HCl pH 8.3, 0.1 mg/ml bovine serum albumin, 1.45 mM each of
dATP, dGTP, dCTP and dTTP, 1.5 mM MgCl.sub.2, and 2.5 U of RNasin
(Promega)) for 10 min. at room temperature, then 30 minutes at
42.degree. C. followed by heat inactivation at 95.degree. C. for 5
min. This cDNA may then be amplified by PCR using 250 ng of primer
NE1 (5'-TCATTGACAGTCCAGCT-3') in a reaction mixture (100 .mu.l)
containing the same buffer as above with 0.25 mM of each dNTP and
2.5 U of Amplitaq DNA polymerase, using about 30 cycles of
94.degree. C. for 1 min., 45.degree. C. for 1 min, and 72.degree.
C. for 2 min, to generate a 768 bp region of the HIV pol gene.
[0030] 5.2 PCR Assay of Peripheral Blood Mononuclear Cells
[0031] Peripheral blood mononuclear cells (PBMCs) may be used fresh
or following cryopreservation (e.g. at -190.degree. C.). DNA may be
prepared from PBMCs using standard techniques for use in detection
of HIV proviral DNA. Any suitable HIV primer oligonucleotide(s) may
be used in PCR to detect HIV provirus.
[0032] In a preferred, nonlimiting embodiment of the invention,
cryopreserved (-190.degree. C.) PBMC may be treated with a lysis
buffer (for example, 0.45 percent Tween-20, 10 mM Tris HCl pH 8.0,
2.5 mM MgCl.sub.2, 50 mM KCl, and 0.1 mg/ml proteinase K) for about
two hours at 56.degree. C. and then heat inactivated at 95.degree.
C. for 10 minutes. Approximately 1 .mu.g of DNA (20 .mu.l of the
PBMC lysate) may be used in the initial PCR amplification with
primers A (5'-TTCCCATTAGTCCTATT-3') and NE1
(5'-TCATTGACAGTCCAGCT-3') with reaction conditions as set forth in
Larder et al., 1991, AIDS 5:137-144 to generate a 768 bp region of
the HIV pol gene.
[0033] 5.3 PCR Assay for Mutation at Codon 215 of HIV Reverse
Transcriptase
[0034] To analyse the changes in codon 215 of the HIV pol gene, a
"double" or "nested" PCR procedure was performed using the primers,
reagents, and reaction conditions described by Larder et al., 1991,
AIDS 5:137-144. Five .mu.l of the 768 bp product generated by PCR
with primers A (5'-TTCCCATTAGTCCTATT-3') and NE1
(5'-TCATTGACAGTCCAGCT-3') and either plasma HIV RNA or PBMC DNA may
be used in a second series of nested PCR amplifications using
primers that detect wild-type sequence or sequence mutated at codon
215. In preferred, non-limiting embodiments of the invention, the
following primers may be used: to detect wild-type sequence primers
B (5'-GGATGGAAAGGATCACC-3') and 3W (3'-TGGTGTGGTCTGTTTTTTGTA-5')
and to detect mutants at codon 215, primers B (supra) and 3M
(3'-AAGTGTGGTCTGTTTTTTGTA-5'). PCR may then be performed as
follows. About 1 .mu.l of template may be used per PCR reaction in
100 .mu.l containing 25 mM KCl, 50 mM Tris HCl pH 8.3, 0.1 mg/ml
bovine serum albumin (BSA), 0.2 mM each of dATP, dGTP, dCTP and
dTTP, 0.25 .mu.l of each oligonucleotide primer, and 1.5 mM
MgCl.sub.2. Reaction mixtures may be heated at 100.degree. C. for
two minutes prior to addition of Taq DNA polymerase (2.5 U,
Perkin-Elmer Cetus, Conn.), overlaid with 100 .mu.l of light
mineral oil, and subjected to 30 cycles consisting of a
denaturation step (1 minute, 94.degree. C.), primer annealing (30
seconds, 45.degree. C.) and DNA synthesis (30 seconds, 72.degree.
C.) using, for example, a Perkin Elmer Cetus DNA thermal cycler.
Ten .mu.l of PCR product from each set of "nested" PCR reactions
may then be analyzed to determine the presence and intensity of the
products. For example, PCR reactions may be analyzed on a 3.0
percent agarose gel with ethidium bromide staining; a portion of a
patient sample subjected to "nested" PCR using primers B and 3W may
be run in a lane next to another portion of the same patient sample
subjected to "nested" PCR using primers B and 3M. A 210 bp PCR
product would be expected; if the patient sample contained HIV RT
having the codon 215 mutation, the lane carrying primer B/3M PCR
product should exhibit a band that is more intense than any
corresponding band in the primer B/3W lane. If the patient sample
contained only wild type HIV RT, the band in the primer B/3W lane
should be more intense than any corresponding band in the primer
B/3M lane. Alternatively, if the patient sample contained a mixture
of wild type and mutant HIV RT, bands of similar intensities should
be in both lanes.
[0035] 5.4 Utility of the Invention
[0036] The present invention relates to methods of monitoring, via
PCR, the clinical progression of HIV infection in patients
receiving antiretroviral therapy. Techniques described in Sections
5.1 through 5.3 supra, may be used as set forth below.
[0037] In one particular embodiment, the present invention provides
for a method of evaluating the effectiveness of antiretroviral
therapy of a patient comprising (i) collecting a plasma sample from
an HIV-infected patient who is being treated with an antiretroviral
agent; (ii) amplifying the HIV-encoding nucleic acid in the plasma
sample using HIV primers in about 30 cycles of PCR; and (iii)
testing for the presence of HIV sequence in the product of the PCR;
in which the absence of detectable HIV sequence correlates
positively with the conclusion that the antiretroviral agent is
therapeutically effective and the presence of detectable HIV
sequence correlates positively with the conclusion that the
antiretroviral agent is therapeutically ineffective. In further,
related, embodiments, the presence of detectable HIV sequence
correlates positively with an absolute CD4 count of less than 200
cells/mm.sup.3, and the absence of detectable HIV sequence
correlates positively with a CD4 count greater than 200
cells/mm.sup.3. The phrase "correlates positively," as used herein,
indicates that a particular result renders a particular conclusion
more likely than other conclusions.
[0038] In another particular embodiment, the present invention
provides for a method of evaluating the effectiveness of
antiretroviral therapy of a patient comprising (i) collecting a
plasma sample from an HIV-infected patient who is being treated
with an antiretroviral agent; (ii) amplifying the HIV-encoding
nucleic acid in the plasma sample using HIV primers in about 30
cycles of PCR; and (iii) measuring the HIV RNA copy number using
the product of the PCR, in which an HIV RNA copy number greater
than about 500 correlates positively with the conclusion that the
antiretroviral agent is therapeutically ineffective, and an HIV RNA
copy number less than about 200 correlates positively with the
conclusion that the antiretroviral agent is therapeutically
effective.
[0039] In a further embodiment, the present invention provides for
a method of evaluating the effectiveness of antiretroviral therapy
of a patient comprising (i) collecting one pre-treatment plasma
sample from an HIV-infected patient who is about to be treated with
an antiretroviral agent; (ii) collecting a post-treatment plasma
sample from the HIV-infected patient after the patient has been
treated with the antiretroviral agent; (iii) amplifying the
HIV-encoding nucleic acid in the pre-treatment and post-treatment
plasma samples using HIV primers in about 30 cycles of PCR; (iv)
measuring the HIV RNA copy number using the products of the PCRs of
step (iii); and (v) comparing the HIV RNA copy number in
pre-treatment and post-treatment plasma samples, in which a ratio
of HIV RNA copy number in pre-treatment and post-treatment plasma
samples of greater than about 4 to 1 correlates positively with the
conclusion that the antiretroviral agent is therapeutically
effective.
[0040] In additional embodiments of the invention, PCR assay may be
used to detect mutations at codon 215 of HIV RT which correlate
with resistance to antiretroviral therapy and which precede
immunologic decline by 6-12 months. Accordingly, the present
invention provides for a method of evaluating the effectiveness of
antiretroviral therapy of a patient comprising (i) collecting a
plasma sample from an HIV-infected patient who is being treated
with an antiretroviral agent; and (ii) determining (for example,
using "nested" PCR) whether the plasma sample comprises nucleic
acid encoding HIV RT having a mutation at codon 215, in which the
presence of the mutation correlates positively with immunologic
decline of the patient within a six to twelve month period. Under
such circumstances, the HIV virus infecting the patient has become,
via the mutation, resistant to the antiretroviral agent. It
therefore maybe desirable after detecting the mutation, to either
increase the dosage of antiretroviral agent, change to another
antiretroviral agent, or add one or more additional antiretroviral
agents to the patient's therapeutic regimen. For example, if the
patient was being treated with zidovudine (AZT) when the mutation
arose, the patient'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 dideoxyinosine (ddI) and stopping AZT treatment;
or
[0041] (ii) increasing the dosage of AZT; or (iii) adding another
antiretroviral agent, such as ddI, to the patient's therapeutic
regimen. The effectiveness of the modification in therapy may be
evaluated, as set forth above, by monitoring the HIV RNA copy
number. A decrease in HIV RNA copy number correlates positively
with the effectiveness of a treatment regimen.
[0042] Because the mutation at the 215 codon appears first in
plasma HIV RNA and later in PBMC proviral DNA, once the mutation is
detected in proviral DNA, the treatment regimen is desirably
modified with haste in order to avoid immune decline. Accordingly,
the present invention provides for a method of evaluating the
effectiveness of antiretroviral therapy of a patient comprising (i)
collecting PBMC from an HIV-infected patient who is being treated
with an antiretroviral agent; and (ii) determining whether the
PBMC-comprise proviral HIV DNA which comprises a mutation at codon
215, in which the presence of the mutation correlates positively
with immunologic decline of the patient within a 4-11 month period
(because, as discussed in Section 7, infra, a mutation in serum HIV
RNA was found to precede the mutation in proviral DNA by 1-8
months). Once the mutation is detected in proviral DNA, immune
decline becomes even more imminent, and alteration of the patient's
therapeutic regimen is desirable.
[0043] When immune decline is heralded by the increase in HIV RNA
copy number and/or the presence of the mutation at codon 215, in
addition to altering the patient's antiretroviral therapy, it may
also be desirable to treat the patient prophylactically for
opportunistic infections, using antifungal, antibiotic, and/or
antiparasitic medications.
[0044] Antiretroviral agent, as used herein, includes any known
antiretroviral agent including, but not limited to,
dideoxynucleosides. In preferred embodiments of the invention the
antiretroviral agent is AZT. Resistance to certain antiretroviral
agents, including AZT, is associated with a mutation at codon 215.
Resistance to other antiretroviral agents, such as ddI, is
associated with a mutation at codon 74 The present invention
provides for analogous techniques in which the effectiveness of
antiretroviral therapy is monitored by determining whether plasma
HIV RNA or PBMC contain a mutation at codon 74 of HIV RT, in which
a mutation at that locus may augur immunological decline and may
warrant a modification of antiretroviral therapy.
[0045] One preferred, non-limiting, specific embodiment of the
invention is as follows: A method of evaluating the effectiveness
of AZT therapy of a patient comprising (i) collecting a plasma
sample from an HIV-infected patient who is being treated with AZT;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA to cDNA and amplifying HIV sequences using HIV
primers in about 30 cycles of PCR; and (iii) testing for the
presence of HIV sequence in the product of the PCR, in which the
absence of detectable HIV sequence correlates positively with the
conclusion that AZT is therapeutically effective and the presence
of detectable HIV sequence correlates positively with the
conclusion that AZT is therapeutically ineffective. In most
preferred embodiments, the HIV primers used comprise NE1 (supra),
SK38 and/or SK39 (supra), and/or the presence of HIV sequence is
detected using an enzyme-linked assay (e.g., a horseradish
peroxidase based assay). Similar embodiments in which the HIV copy
number is measured are also provided for.
[0046] Another preferred, non-limiting, specific embodiment of the
invention is as follows: A method of evaluating the effectiveness
of AZT therapy of a patient comprising (i) collecting a plasma
sample from an HIV-infected patient who is being treated with AZT;
(ii) amplifying the HIV-encoding RNA in the plasma sample by
converting the RNA to cDNA and amplifying HIV sequences using HIV
primers that result in a PCR product that comprises that portion of
the RT gene that contains the 215 codon (e.g. primer NE1, supra);
(iii) performing "nested" PCR using primers that result in PCR
products that reflect the presence of wild type (e.g. primers B and
3W, supra) or 215 codon mutant (e.g. primers B and 3M, supra); and
(iv) determining, via the products of "nested" PCR, the presence or
absence of a mutation at codon 215 of the HIV RT, in which the
presence of the mutation correlates positively with immunologic
decline of the patient within a six to twelve month period. An
analogous method may be used in which the patient sample is PBMC,
and the presence of a mutation is proviral DNA is determined.
6. Example: Reduction in Plasma Human Immunodeficiency Virus
Ribonucleic Acid After Dideoxynucleoside Therapy as Determined by
the Polymerase Chain Reaction
[0047] 6.1 Materials and Methods
[0048] 6.1.1 Patients
[0049] After informed consent was obtained, whole blood samples
were collected by venipuncture in the presence of
acid-citrate-dextrose as an anticoagulant. A single plasma sample
was collected from 39 HIV antibody-positive subjects who were not
receiving antiretroviral therapy at the time of collection and from
33 HIV antibody-positive subjects who were currently on and had
received AZT for a minimum of 3 mo.
[0050] Two plasma samples were collected from an additional 27
subjects before and 1 month. after initiation of dideoxynucleoside
therapy. 18 of these subjects received 500 mg/d of AZT orally.
Seven subjects received a combination of zidovudine (150-600 mg/d)
and 2',3'-dideoxyinosine (ddI)(134-500 mg/d). Two patients received
500 mg/d of ddI alone (see Table I for individual subject
characteristics). Finally, nine of these subjects had two plasma
samples taken 1-3 wk. before initiating antiretroviral therapy and
two plasma samples taken 1 and 2 mo. after commencing therapy.
Plasma was separated within 4 h. by centrifugation at 500 g for 10
min. A second centrifugation was performed on the plasma at 500 g
for 30 min. to remove any cellular material. 200 .mu.l of plasma
was then mixed with 200 .mu.l of a solution containing 5 M
guanidinium thiocyanate, vortexed briefly, and stored at
-70.degree. C. until further use. All samples were assayed within 3
mo. of collection. To decrease variance, all specimens to be
compared from the same subject were run in the same assay.
[0051] 6.1.2 Extraction of RNA from Plasma
[0052] RNA was extracted from plasma by the method described in
Chomczynski et al., 1987, Ann. Biochem. 162:156-159. Briefly, 200
.mu.l of clarified plasma to which 200 .mu.l of 5 M guanidinium
thiocyanate had previously been added was extracted with
phenol/chloroform and precipitated with isopropanol. The resulting
pellet was then washed in 75% ethanol, dried, and brought up in
diethylpyrocarbonate treated, glass distilled water.
[0053] 6.1.3 Reverse Transcription and Amplification of cDNA
[0054] HIV RNA was transcribed to cDNA using Moloney murine
leukemia virus reverse transcriptase (Bethesda Research
Laboratories, Gaithersburg, Md.) by the method described in
Kawasaki, 1990, "In PCR Protocols: A Guide to Methods and
Applications" pp. 21-27, M. A. Innis, D. H. Gelfand, J. J. Sninsky,
and T. J. White, eds. Academic Press, Berkeley, Calif. Oligomers
used for amplification included SK38, SK39, and SK19 (Kellog et
al., 1990, "In PCR Protocols: A Guide to Methods and Applications,"
pp. 337-348, M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J.
White, eds. Academic Press, Berkeley, Calif.). Biotinylation of
SK38 and horseradish peroxidase (HRP) labeling of probe SK19 were
prepared as described in Levenson et al., 1990, "In PCR Protocols:
A Guide to Methods and Applications," pp. 99-112 M. S. Innis, D. H.
Gelfand, J. J. Sninsky, and T. J. White, eds. Academic Press,
Berkeley, Calif. Amplification of HIV cDNA was carried out as
follows: to a 100-.mu.l reaction mixture was added the cDNA, 50
pmol of primers SK38 and SDK39, 10 mM of each dNTP, 10 mM Tris (pH
8.3), 2.5 mM MgCl.sub.2, 50 mM KCl, and 2.5 U of recombiTaq DNA
polymerase (Perkin-Elmer Cetus, Norwalk, Conn.). The mixture was
then overlaid with 50 .mu.l of mineral oil. Tubes were placed in a
DNA thermal cycler (Perkin-Elmer Cetus) for 30 cycles of
amplification with the following program: 95.degree. C./30 s,
55.degree. C./30s, and 72.degree. C./60 s for denaturation,
annealing, and extension, respectively. Negative and positive
controls which included both high and low copy number HIV RNA and
DNA were added at each step.
[0055] 6.1.4 Enzyme-Linked Affinity Assay.
[0056] To detect and quantitate PCR product, 96-well microplates
(Maxisorp; Nunc, Naperville, Ill.) were coated with 100 .mu.l of a
0.1 mg/ml solution of avidin (Sigma Chemical Co., St. Louis, Mo.)
in 50 mM Na.sub.2CO.sub.3 (pH 9.6) overnight at room temperature.
Wells were then washed twice with PBS. Wells were then filled with
300 .mu.l of a blocking solution containing 5.times. Denhardt's
solution, 1% gelatin (Sigma), 250 .mu.l/ml sheared herring sperm
DNA (Promega Biotec, Madison, Wis.) at least overnight at 4.degree.
C. Immediately before use, the blocking solution was aspirated from
each well and 5 .mu.l PCR product and 65 .mu.l of a hybridization
solution, containing 5.times. saline sodium phosphate EDTA,
5.times. Denhardt's solution, and 1 pmol of HRP-labeled SK19 HIV
gag specific probe was added to each well. A capture and
hybridization reaction was then carried out in the well for 1 h. at
42.degree. C. The 96-well microplate was then placed in a
Biomek.TM. 100 Automated Workstation (Beckman Instruments, Inc.,
Palo Alto, Calif.) where wells were washed 20 times with BPS
containing 0.05% Tween-20. The HRP substrate O-phenylenediamine
(Sigma) was prepared at 0.6 mg/ml in 0.1 M citrate buffer (pH 5.5)
containing 0.03% hydrogen peroxide. 150 .mu.l of this substrate
solution was added to each well. After 10 min. the reaction was
stopped with 1 N H.sub.2SO.sub.4 and the optical density of each
well measured at 490 nm by the Biomek 1000. A lower level of
positivity had been defined as an absorbance of 0.135. This cutoff
value was calculated from the mean absorbance obtained from a group
of seronegative samples plus three standard deviations. Copy number
from subject samples were determined from the absorbances obtained
from a dilution series of an RNA gag gene construct of known copy
number described in Holodniy et al., 1991, J. Infect. Dis.
163:862-866. The lower level of sensitivity in this assay was 40
copies of HIV gag gene RNA.
[0057] 6.1.5 Plasma HIV Culture and P24 Antigen Assay
[0058] Quantitative HIV plasma microculture was performed according
to the method described in Ho et al., 1989, N. Engl. J. Med.
321:1621-1625. P24 antigen was detected by an antigen capture assay
by a method provided by the supplier (Abbott Laboratories, North
Chicago, Ill.).
[0059] 6.1.6 Statistical Analysis
[0060] Sample optical density was converted to copy number and
analyses performed on samples expressed as RNA copy number/200
.mu.l of plasma. A t test of independent samples was used in
analysis of subject who did not receive antiretroviral therapy
compared to subjects who were receiving AZT. A t test of paired
samples was used to analyze paired plasma data and CD4 counts from
subjects pre- and post-therapy. All t tests were two tailed. A
Fisher's exact test or chi square test were used for analysis of
proportion where appropriate. Statistical significance was defined
as P<0.05.
[0061] 6.2 Results
[0062] 72 subjects were evaluated in a cross-sectional study of HIV
disease to determine plasma HIV RNA copy number by PCR. The results
are presented in FIG. 1. 39 subjects who were not currently
receiving antiretroviral therapy and 33 subjects who were receiving
AZT were evaluated. Untreated subjects were more likely to have a
positive signal than treated subjects (32 of 39 vs. 16 of 33,
respectively, P 0.008, chi square). In the 39 subjects who were not
currently receiving therapy, the mean plasma HIV RNA copy number
was 690.+-.360 (mean.+-.SEM) per 200 .mu.l of plasma, while the 33
subjects who had been receiving AZT therapy had a mean copy number
of 134.+-.219 (P<0.05). Mean CD4 count for each group was
316.+-.45 and 300.+-.37, respectively (P=NS).
[0063] Subgroups were then analyzed with respect to CD4 count.
Among those with <200 CD4 cells, untreated subjects were more
likely to have positive signal than treated subjects (18 of 19 vs.
9 of 14, P<0.04, Fisher's exact test). Among those with >200
CD4 cells, 14 of 20 untreated subjects vs. 7 of 19 treated subjects
had a detectable signal (P=NS, Fisher's exact test). Untreated
subjects with CD4 count<200/mm.sup.3 had a mean RNA copy number
of 1,369.+-.707 and mean CD4 count of 73.+-.17; untreated subjects
with CD4>200/mm.sup.3 had a mean RNA copy number of 44.+-.10 and
mean CD4 count of 547.+-.45; treated subjects with CD4 counts
<200/mm.sup.3 had a mean RNA copy number of 295.+-.5 and mean
CD4 count of 115.+-.13; and treated subjects with CD4
counts>200/mm.sup.3 had calculated mean RNA copy number of
16.+-.5 (which is below the level of detection of this assay and
would be interpreted as negative) and mean CD4 count of
437.+-.41.
[0064] 27 additional subjects were then evaluated before and 1 mo
after initiation of dideoxynucleoside therapy. Clinical parameters
of the subjects are presented in Table I. PCR results are presented
in FIG. 2. Results show that plasma HIV RNA copy number fell from
540.+-.175 to 77.+-.35 after therapy (P<0.05, paired t test).
Mean CD4 count increased from 399.+-.24 to 442.+-.25 after 4 wk of
therapy (P<0.006, paired t test). TABLE-US-00001 TABLE 1
Clinical Parameters and PCR Analysis of Plasma HIV RNA from 27
Patients Patient Antiviral No. treatment Pre/post CD4 Pre/post HIV
RNA 1* AZT.dagger-dbl. 647/561 106/90 2* AZT 541/651 130/0 3 AZT
840/874 550/0 4 AZT 432/462 100/77 5* AZT 379/415 87/57 6* AZT
428/408 40/42 7 AZT 422/345 94/94 8 AZT 420/402 0/0 9 AZT 432/ 532
93/65 10 AZT 430/430 105/52 11 AZT 429/404 123/50 12 ddI +
AZT.sup..sctn. 280/220 526/0 13 AZT 323/320 78/45 14 AZT 320/456
95/95 15 AZT 353/387 301/0 16 ddI + AZT.sup..sctn. 309/399 300/0 17
ddI + AZT.sup..sctn. 337/398 260/0 18 ddI.sup..parallel. 328/310
966/0 19 ddI + AZT.sup. 383/491 245/0 20 AZT 404/413 0/0 21 AZT
270/450 958/72 22 ddI + AZT.sup. 292/344 60/0 23 AZT 320/ 295
2769/437 24 ddi.sup..parallel. 222/370 3944/0 25 AZT 568/732
2014/925 26 DDI + AZT** 367/473 217/0 27 DDI + AZT** 310/399 439/0
*Remote history of AZT use. " AZT dose 500 mg/d unless otherwise
stated. .sup..sctn.AZT 300 mg/d + ddI 334 mg/d. .sup..parallel.ddi
500 mg/d. .sup. AZT 600 mg/d + ddI 500 mg/d. **AZT 150 mg/d + ddI
134 mg/d.
[0065] Finally, 9 of the 27 subjects had two samples taken before
initiation of therapy and two samples taken 1 and 2 months after
commencing therapy. The results are presented in FIG. 3. When two
pretherapy time points were analyzed for constancy of signal,
results show that mean copy number for each pretherapy time point
was 945.+-.377 and 643.+-.392.
[0066] Two subjects had a second pretherapy sample which was
negative. When both pretherapy copy number values were compared to
posttherapy values, plasma HIV RNA copy number fell from 794.+-.274
to <40 (which is below the lower level of detection in this
assay) after 1 and 2 mo of therapy (P<0.05, paired t test). Mean
CD4 count increased from 314.+-.165 to 378.+-.25 (P<0.05, paired
t test).
[0067] Plasma culture was performed on fresh material obtained from
the initial pretreatment sample for 23 of 27 of these patients.
Only 7 of 23 were plasma virus positive by culture (from 1 to 100
tissue culture infective does/ml). All 23 of these patients were
positive by PCR (>40 copies/200 .mu.l). In addition, a p24
antigen test was performed on all 27 pretreatment samples. Only 2
of 27 had detectable p24 antigen present (>30 pg/ml).
[0068] 6.3 Discussion
[0069] The results presented here demonstrate that plasma HIV RNA
can be detected and quantified by copy number in the majority of
patients infected with HIV. In addition, plasma HIV RNA copy number
may be used as a marker of circulating HIV viral load to assess
treatment effect of antiretroviral compounds including
dideoxynucleoside compounds. We initially conducted a survey to
determine whether treatment or degree of immunologic impairment,
based on CD4 count, affected plasma HIV RNA copy number. Untreated
patients as a group had higher copy numbers than treated patients.
Untreated patients with <200 CD4 cells/mm.sup.3 had a higher
mean copy number than patients with >200 CD4 cells/mm.sup.3.
Likewise, treated patients with <200 CD4 cells/mm.sup.3/had
higher copy numbers than patients with >200 CD4 cells/mm.sup.3,
indicating that patients with more advanced HIV disease have higher
circulating copy numbers than asymptomatic patients, and that the
antiretroviral benefit seen in patients with higher CD4 counts may
be waning.
[0070] To assess the short-term impact of antiretroviral therapy on
patients, 27 patients were evaluated before and 1 mo after
initiation of AZT, ddI, or combination therapy. As CD4 counts
increased after 1 mo of therapy, HIV RNA copy number fell
significantly. However, the response of individual subjects was
variable. 16 of 27 subjects had a marked decrease in copy number
and 11 of 27 did not. Because the majority of subjects received AZT
alone, it was not possible to assess any differences between AZT,
ddI, or combination regimens.
[0071] Finally, nine subjects had two baseline time points taken in
the 3 wk before treatment, followed by two monthly samples
posttreatment. Pretreatment signal was constant in 7 of 9 subjects,
and 2 subjects had discordant samples, i.e., one was positive and
one was negative. This could be related to real changes in
circulating HIV RNA, or introduced during sample collection,
handling, or the assay. However, pretherapy and posttherapy samples
were run in the same assay and so were subject to all of the same
reaction conditions. When sample positivity was considered in
relation to therapy, 16 of 18 pretherapy samples had a positive
signal vs. 0 of 18 posttherapy samples (P<0.001, chi square)
showing suppression of HIV RNA copy number with treatment.
[0072] Currently there is no standard method to assess circulating
viral load in all HIV-infected patients. Plasma viremia, measured
by quantitative microculture, can identify and quantify infectious
virus in 50-100% of patients, principally those with advanced
stages of HIV disease, low CD4 counts, and p24 antigenemia (Ho et
al., 1989, N. Engl. J. Med. 321:1621-1625; Coombs et al., 1989, J.
Virol. Methods 26:23-21; Ehrnst et al., 1988, N. Engl. J. Med.
324:961-964). Many patients with >200 CD4 cells/mm.sup.3 do not
have detectable infectious plasma viremia. This may be due to an
absence of circulating infectious virus, virus which is neutralized
by specific antibody, or the insensitivity of culture techniques.
The results presented here indicate that the majority of patients
with >200 CD4 cells/mm.sup.3 do not have plasma p24 antigen or
infectious virus detectable by culture techniques. In the studies
described herein, it appears that virus undetectable by culture
methods was detectable by PCR methods.
[0073] Attempts have been made to assess HIV viral load in patients
by molecular techniques, mainly by quantitative PCR of HIV proviral
DNA in circulating mononuclear cells or cell-free virion-associated
RNA in plasma. Published data suggest that the number of cells
infected with HIV increases with advancing disease and that HIV
proviral DNA content increases as well. We and others have shown a
decrease in HIV proviral DNA with dideoxynucleoside therapy over
time (Aoki et al., 1990, AIDS Res. Hum. Retroviruses 6:1331-1339).
This was not the case in another published small series (McElrath
et al., 1991, J. Clin. Invest. 87:27-30).
[0074] We have shown that HIV RNA could be quantified in serum and
that copy number increased with disease progression (Holodniy et
al., 1991, J. Infect. Dis. 163:862-866). Plasma HIV RNA has been
shown to be present before and after seroconversion with
quantitative decreases occurring after seroconversion (Hewlett et
al., 1988, J. Clin. Immunoassay 11:161-164). The recent report by
Daar et al. (Daar et al., 1991, N. Engl. J. Med. 324:961-964),
showed a decrease in both plasma viremia and proviral DNA from
PBMC, coinciding with seroconversion after acute infection. In one
report, plasma HIV RNA levels fell with passive immunoglobulin
therapy, suggesting a therapy-based response in circulating HIV RNA
load (Karpas et al., 1990, Proc. Natl. Acad. Sci. USA
87:7613-7617). Ottman and colleagues have been successful in
detecting HIV RNA in plasma from 95% of patients evaluated (Ottman
et al., 1991, J. Virol. Methods 31:273-284). They also studied a
group of patients who were receiving AZT to determine whether there
was any therapeutic impact on HIV RNA signal. 24 of 25 patients who
were receiving AZT had detectable signal. However, methodological
differences in that study vs. the present study may have
contributed to the differences noted between them. First, ottman et
al. used an ultracentrifugation step to sediment virus, enhancing
virion-associated RNA recovery. Second, 40 cycles of amplification
after reverse transcription were performed, which would certainly
increase the sensitivity of such an assay to successfully detect
HIV RNA in virtually all patients. Although sensitivity is
increased with increased cycle number, thus detecting signal in
virtually all patients, the ability to show the quantitative
changes demonstrated here with 30 cycles of amplification is
lost.
[0075] We have previously shown in serum that quantitative serum
cultures were negative in the majority of patients with >200 CD4
cells/mm.sup.3 (Holodniy et al. 1991, J. Infect. Dis. 163:862-866).
In the current study, 23 plasma samples were evaluated by culture
and PCR. All had detectable plasma HIV RNA by PCR<but only seven
were plasma HIV-culture positive. Other published experience
comparing plasma HIV culture and PCR of HIV RNA from plasma is
lacking. Ottman et al. tested only two patients, both of whom were
positive in both assays. Coyle et al. reported that 14 of 20
patients had positive plasma cultures and 12 of 20 patients had
detectable HIV RNA in plasma, but no information was given
regarding concordance or discordance of samples (Coyle et al.,
1990, Clin. Res. 38:778a (Abstr.)).
[0076] The type of plasma sample and method of processing and
storage were found to be very important. The type of anticoagulant
used for sample collection can affect detection of plasma RNA. We
have previously shown that plasma collected in the present of
herapin does not allow detection of signal because of an inhibitory
effect of heparin on gene amplification (Holodniy et al., 1991, J.
Clin. Microbiol. 29:676-679). Although Coyle et al. (1990, Clin.
Res. 38:778a (Abstr.)) found detectable signal from plasma
collected in the presence of herapin, an ultracentrifugation step
preceding RNA analysis lead to removal of most of the heparin from
the enzyme-mediated assay system. However, no comparison
experiments among anticoagulants were performed to demonstrate any
attenuation of signal obtained in the presence of heparin.
[0077] Because of our concern for RNA degradation during specimen
storage and freeze thawing, we decided to store fresh plasma at
-70.degree. C. in the presence of guanidinium and process samples
within 3 mo of collection. Samples were stored in guanidinium-for
RNAase inhibition. Preliminary data from our laboratory would
suggest that plasma HIV RNA signal decays with time in the absence
of this RNA stabilizer.
[0078] In summary, we have shown that plasma HIV RNA copy number
can be quantitated by PCR and does decrease with dideoxynucleoside
therapy. The nonisotopic, microplatebased format presented here
makes it possible to process multiple patient samples with
replicates in a single amplification and assay run.
7. Example: Relationship of a Mutation in the HIV Reverse
Transcriptase Gene to Decline in CD4 Lymphocyte Numbers in Long
Term AZT Recipients
[0079] 7.1 Materials and Methods
[0080] 7.1.1 Study Population
[0081] Cryopreserved PBMC and serum from 40 participants in AIDS
Clinical Trial Group (ACTG) protocols 019 (30 patients) and 016 (10
patients) at Stanford University Medical Center AIDS Clinical Trial
Unit were used in this study. Patients at enrollment in these
studies were AZT naive, had >200 CD4 cells/.mu.l and had few
(016) or no symptoms (019) referable to HIV infection. They were
subsequently treated with AZT for 2 to 4 years. The most common
dosage was 500 mg per day. Approximately one third of patients
received either 1200 mg or 1500 mg per day during the initial part
of their therapy, but were changed to 500 mg per day when lower
doses were found to be as effective but less toxic than higher
doses (Fischl et al., 1990, N. Engl. J. Med. 323:1009-1014;
Volberding et al., 1990, N. Engl. J. Med. 322:941-949). All samples
were obtained from the patients while they were on the protocols
and thus no patient developed an AIDS defining diagnosis.
[0082] 7.1.2 CD4 Cell Counts
[0083] CD4 cell counts were obtained approximately every three
months on each patient. All counts were performed at Stanford's
ACTG-qualified cytofluorometry lab. Samples were stained with
monoclonal antibodies to CD3, CD4, and CD8. The absolute CD4 count
was calculated by multiplying the percent CD4 by the total
lymphocyte count.
[0084] 7.1.3 PBMC Preparation
[0085] Cryopreserved (-190.degree. C.) PBMC were treated with a
lysis buffer (0.45% Tween-20, 10 mM Tris HCl pH 8.0, 2.5 mM
MgCl.sub.2, 50 mM KCl, and 0.1 mg/ml proteinase K) for 2 hours at
56.degree. C. and then heat inactivated at 95.degree. C. for 10
min. Approximately lug of DNA (20 .mu.l of the PBMC lysate) was
used in the initial PCR amplification with primers
A(5'-TTCCCATTAGTCCTATT-3') and NE1(5'-TCATTGACAGTCCAGCT-3') with
reaction conditions as described in Larder et al., 1991, AIDS
5:137-144 to generate a 768 bp region of the HIV pol gene.
[0086] 7.1.4 Serum HIV RNA Preparation
[0087] Cryopreserved (-70.degree. C.) serum was thawed and then 350
.mu.l of sera was added to 350 .mu.l of solution D (Chomczynski et
al., 1987, Anal. Biochem. 162:156-159) (guanadinium
thiocyanate+2-mercaptoethanol) and vortexed. RNA was then extracted
with phenol and chloroform and precipitated with ethanol as
described in Chomczynski et al., 1987, Anal. Biochem.
162:156-15918. HIV RNA was then reverse transcribed to cDNA by
using 500 ng of primer A and 5 units of murine leukemia virus
(MuLV) reverse transcriptase (Bethesda Research Labs) in 10 .mu.l
of amplification buffer (25 mmol/L KCl, 50 mmol/L Tris HCl Ph 8.3,
0.1 mg/ml bovine serum albumin, 1.45 mmol/L each of dATP, dGTP,
dCTP and dTTP, 1.5 mmol/L MgCl.sub.2, 2.5 units of RNasin
(Promega)) for 10 min at room temperature, then 30 min at
42.degree. C. followed by heat inactivation at 95.degree. C. for 5
min. This cDNA was then amplified by PCR using 250 ng of primer NE1
in a reaction mixture (100 .mu.l) containing the same buffer as
above with 0.25 mmol/L of each dNTP and 2.5 units of AmpliTaq DNA
polymerase (Perkin-Elmer Cetus). This reaction mixture underwent 30
cycles of 94.degree. C. for 1 min, 45.degree. C. for 1 min and
72.degree. C. for 2 min to generate a 768 bp region of the HIV pol
gene.
[0088] 7.1.5 PCR Analysis of HIV Reverse Transcriptase Gene
[0089] To analyze the changes in codon 215 of the HIV pol gene, a
"double" PCR procedure was performed using the primers, reagents,
and reaction conditions described in Larder et al., 1991, AIDS
5:137-144. Five .mu.l of the 768 bp product generated by PCR with
primers A and NE1 was used in a second series of nested PCR
amplifications using primer B and 3W to determine if a wild type
sequence was present, or B and 3M to determine if a mutant sequence
was present (primer sequences as set forth supra and in Larder et
al., 1991, AIDS 5:137-144). Samples were run with negative,
positive and reaction mixture controls. Ten .mu.l of PCR product
from each of the second set of PCR reactions were analyzed on a
3.0% agarose gel with ethidium bromide staining. PCR products were
considered to have a mutant or wild type sequence by the method
described by Boucher et al. (1990, Lancet 336:585-590; 1992, J.
Infect. Dis. 165:105-110) and Larder et al., 1991, AIDS 5:137-144:
a sample was considered to contain the wild type sequence at codon
215 if amplification with the primers B and 3W resulted in a 210 bp
PCR product of highest intensity; a sample was considered to
contain a mutant sequence at codon 215 if amplification with the
primers B and 3M resulted in a 210 bp PCR product of highest
intensity. The sample was considered to have a mixture of wild type
and mutant sequences if amplification occurred with both primers 3M
and 3W resulting in PCR products of similar intensity. If a mixture
was detected by PCR then that sample was included in the mutant
group in our statistical analysis.
[0090] 7.1.6 AZT Sensitivity Assay
[0091] Patient PBMC were cocultured with mitogen-stimulated PBMC
from healthy HIV-seronegative donors. Supernatants from these
cultures were collected and frozen when the HIV P24 antigen
concentration exceeded 10,000 pg/ml. 30-100 TCID.sub.50 (50% tissue
culture infectious dose) of virus stock was used to infect one
million donor PBMC pretreated with different concentrations of AZT
(0.0 uM, 0.005 uM, 0.05 uM, 0.5 uM, 5.0 uM). After 7 days, P24
antigen was measured in the cell free supernatant from the cultures
with and without zidovudine. The concentration of AZT required to
inhibit P24 production by 90% (IC.sub.90) as compared to the drug
free cultures was determined by nonlinear regression analysis (Chou
et al., 1984, Adv. Enzyme Regulation 22:27-55). In this assay, the
IC.sub.90s from AZT-naive patients ranged from 0.002 .mu.M to 0.038
.mu.M AZT.
[0092] 7.1.7 Statistical Analysis
[0093] All comparisons between the patients with mutant and wild
type strains were performed using the student's t-test. The
calculations on the IC.sub.90s determined by the zidovudine
sensitivity assay were performed using the log.sub.10 transformed
IC.sub.90 (i.e. geometric means were used rather than arithmetic
means).
[0094] 7.2 Results
[0095] 7.2.1 PCR Analysis of Codon 215 in PBMC
[0096] Proviral DNA was detected by nested PCR in the PBMC of 38 of
40 patients after a mean 34 month treatment period. The two
patients in whom proviral DNA could not be detected had high CD4
counts at the time their PBMC were analyzed (729 and 676
cells/.mu.l). PCR amplification of the PBMC from 17 of 38 patients
(45%) yielded a 210 bp product with the mutant primer, indicating
the presence of a mutation at codon 215 (Thr to Tyr or Phe). The
PBMC from 21 of 38 patients (55%) demonstrated amplification
product only with the wild type primer (a 210 bp product)
indicating the presence of Thr at codon 215.
[0097] The mean length of therapy and starting CD4 counts for the
two groups were similar (Table 2). However, the 17 patients with a
mutation at codon 215 of HIV RT in PBMC proviral DNA had a 50%
decrease in their absolute CD4 count between the time they began
therapy (378 cells/.mu.l) and the end of the study (189
cells/.mu.l). The 21 patients with a wild type sequence at codon
215 experienced a mean 11% increase in their absolute CD4 count
between the time they began therapy (397 cells/.mu.l) and the end
of the study (424 cells/.mu.l). The post-treatment CD4 percentages
of the two groups of patients were also significantly different
(25% in patients with wild-type sequence vs 14% in patients with a
mutation in RT at codon 215) (Table 2). The CD4 counts at each time
point for each patient are shown in FIG. 4. TABLE-US-00002 TABLE 2
CORRELATION OF PATIENT CD4 COUNT CHANGES WITH PCR ANALYSIS OF CODON
215 OF HIV REVERSE TRANSCRIPTASE IN PBMC Wildtype Mutant p Number
of patients 21 17 Months of AZT 33 .+-. 8 35 .+-. 7 0.4 Starting
CD4 measurements Absolute CD4 (cells/.mu.l) 397 .+-. 124 378 .+-.
96 >0.5 CD4 % 26 .+-. 8 25 .+-. 6 >0.5 CD4 measurements at a
time of PCR analysis Absolute CD4 (cells/.mu.l) 424 .+-. 210 189
.+-. 98 <0.0001 CD4 % 25 .+-. 9 14 .+-. 6 0.0001
[0098] 7.2.2 PCR Analysis of Codon 215 in HIV RNA from Serum
[0099] Serial PBMC samples from earlier time points were available
on 8/40 patients; however, serial serum samples from earlier time
points were available on 37/40 patients. In these 37 patients, 135
serum samples were tested for the presence of a codon 215 mutation.
In 87% of these samples (117), reverse transcribed cDNA could be
detected by PCR. Fifteen of the 18 sera that were negative by PCR
had been previously subjected to multiple freeze-thaws and
therefore could be falsely negative. As all patients were
AZT-naive, they were assumed to be wild type at codon 215 at the
start of AZT therapy.
[0100] Twenty-six of the 37 patients developed a mutation in their
HIV RNA. This included the 16 who were also mutant in their PBMC at
the end of the study period (FIG. 5A), and ten patients who were
wild type in their PBMC but mutant in serum HIV RNA at the end of
the study period (FIG. 5B). The time preceding the occurrence of
the 215 mutation in their serum ranged from 2 to 44 months of
therapy. Among these 26 patients, the mean CD4 count at the start
of therapy was 398.+-.139 cells/.mu.l and their mean CD4 count at
the time of first detection of a codon 215 mutation in their serum
was 444.+-.206 cells/.mu.l. Nineteen of the 26 patients with a
codon 215 mutation in their serum had follow-up CD4 counts at least
12 months after the mutation was first detected. In these 19
patients, there was a mean decrease of 100.+-.116 CD4 cells/.mu.ul
(25% decline) at six months and a mean decrease of 170.+-.121 CD4
cells/.mu.l (40% decline) at 12 months.
[0101] The 11 patients who remained wild type in their serum over
the entire 34 month period of zidovudine therapy had an increase of
7.+-.92 CD4 cells/.mu.l (2% increase), (FIG. 5C). The mean CD4
count at the start of therapy for the patients who later developed
a mutant in their serum was 398.+-.139 cells/.mu.l and this was not
significantly different than the starting CD4 counts for those
patients who remained wild type (397.+-.115 cells/.mu.l, p>0.5).
The average length of therapy for both groups was 34 months.
[0102] 7.2.3 Serum Virus Compared to PBMC Provirus
[0103] At the final evaluation of the 38 patients after a mean 34
months of zidovudine, 11 patients were wild type in both serum and
PBMC, 17 were mutant in both serum and PBMC, and 10 patients had a
mutation in their serum but remained wild type in their PBMC FIG.
6. Eight of the 17 patients with a mutation in proviral DNA at the
end of the study period had at least one PBMC sample available from
an earlier time point. In these eight patients, a mutation in serum
HIV RNA preceded the mutation in proviral DNA by 1-8 months. The
findings in these 8 patients and in the 10 patients who were wild
type in their PBMC but mutant in their serum shows that detection
of the serum mutation precedes detection of the mutation in PBMC.
In no instance, did a mutation in patient's PBMC precede its
appearance in serum.
[0104] 7.2.4 AZT Sensitivity Results Determined by Cell Culture
[0105] In vitro AZT susceptibility testing was performed on 17 of
38 patients using a different aliquot of the same post-treatment
PBMC that were used for the PCR analysis. The geometric mean of the
IC.sub.90s of eight patients with the wild type form at 215 was
0.04 .mu.m AZT (range: 0.02-0.28 .mu.m); the geometric mean
IC.sub.90 of nine patients with a mutation at codon 215 was 0.41
.mu.m AZT (range: 0.03-8.0 .mu.M; p=0.002).
[0106] 7.3 Discussion
[0107] As an increasing number of HIV infected individuals are
offered early treatment with AZT, the significance of drug
resistant virus has become an important question. In the present
study we found a strong correlation between the presence of a
mutation at codon 215, which is linked to AZT resistance and an
accelerated decline in CD4 cell number. The patients we studied all
began taking AZT when their CD4 cell numbers were relatively high
and before the onset of AIDS. We observed that the 17 patients with
a mutation at codon 215 in proviral DNA in their PBMC experienced a
mean 50% decrease in their CD4 count between the time that they
began therapy and the time that their cells were analyzed for
mutations. The 21 patients who were wild type at codon 215 in their
proviral DNA at the end of treatment experienced a mean 11%
increase in their CD4 count during the same time period.
[0108] Patient cells were only available during the last year of
the study. However, by extracting and reverse transcribing HIV RNA
from patient's serum specimens we were able to detect codon 215
mutation at earlier time points. The patients in our study with and
without a mutation in serum HIV had similar starting CD4 counts
(397.+-.115 vs. 398.+-.139, p>0.5) and similar lengths of
therapy (34 months in both groups). Yet we found that those
patients who develop a mutation in HIV RNA had a subsequent 40%
decline in their CD4 cells over the next 12 months. The patients
who remained wild type in their serum had a 2% increase in their
CD4 cells over 34 months of therapy.
[0109] These results show that genetic changes in the virus which
confer drug resistance can be rapidly determined directly form
patient PBMC and HIV RNA in patients serum using a nested PCR
procedure. By using PCR we were able to detect viral nucleic acid
in 90% of PBMC samples and 87% of serum samples. Techniques which
require culturing HIV from PBMC or serum may select HIV
subpopulations with greater tropism for certain cells (Kusumi et
al., 1992, J. Virol. 66:875-885; Meyerhans et al., 1989, Cell
58:901-910). This may complicate the analysis of the clinical
significance of AZT resistance detected by phenotypic assays.
[0110] Earlier clinical studies focused on AZT resistance in
patients with initially low CD4 cell counts or who were at high
likelihood of disease progression. Furthermore, these studies
tested HIV isolates which had been passaged in culture. In
contrast, in this study we did not select patients at high
likelihood for disease progression but instead we included all
patients who remained on AZT for at least 2 years and who had high
CD4 counts at the beginning of the study; codon 215 mutations in
serum virus occurred early in treatment. The mean CD4 count at the
first appearance of the mutation was higher than the CD4 count at
the start of therapy (444 vs. 398 cells/.mu.l). This suggests that
mutation of the reverse transcriptase gene is not dependent upon
low CD4+ T cells. On the other hand, we also found that a large
percentage of patients remained wild type at codon 215 and
phenotypically sensitive to AZT despite almost 3 years of therapy.
This may be because our patients were less advanced in their
disease or that by using PCR instead of coculture we were able to
include patients whose virus might not have grown in culture. These
results also suggest that the PBMC may not be the initial source of
mutant virus, as evidenced in 18 of our patients where the serum
HIV RNA mutation preceded that in PBMC by many months. The source
of the mutant HIV detected in serum may be cells in lymphatic,
central nervous system or reticuloendothial sites.
[0111] The significance of specific mutations in the RT gene with
respect to AZT resistance has been defined in patient isolates as
well as through molecular cloning experiments (Larder et al., 1989,
Science 246:1155-1158; Larder et al., 1991, AIDS 5:137-144; Kellam
et al., 1992, 89:1934-1938; St. Clair et al., 1991, Science
253:1557-1559). Of the four mutations first reported to be
associated with AZT resistance (codons 67, 70, 215, 219), the
mutation at codon 215 has been shown to be the most commonly
occurring and to have the greatest impact on susceptibility. This
impact on AZT susceptibility will vary depending on whether or not
additional mutations are present (Larder et al., 1989, Science
246:1155-1158; Larder et al., 1991, AIDS 5:137-144; Kellam et al.,
1992, 89:1934-1938; St. Clair et al., 1991, Science 253:1557-1559;
Richman et al., 1991, J. Infect. Dis. 164:1075-1081; Boucher et
al., 1992, J. Infect. Dis. 165:105-110). Recent sequencing studies
of clinical isolates suggest that there are additional mutations in
the RT gene that may contribute to AZT resistance (Japour et al.,
1991, Proc. Natl. Acad. Sci. 88:3092-96; Kellam et al., 1992, Proc.
Natl. Acad. Sci. USA 89:1934-1938; St. Clair et al., 1991, Science
253:1557-1559). However, the occurrence of the two consecutive
nucleotide changes necessary for the amino acid change at codon 215
may be the most important requirement for the development of
resistance (Kellam et al., 1992, Proc. Natl. Acad. Sci. U.S.A.
89:1934-1938; Richman et al., 1991, J. Infect. Dis. 164:1075-1081;
Boucher et al., 1992, J. Infect. Dis. 165:105-110). In this study,
a subset of 17 patients were tested using a cell culture assay
which confirmed that the viruses with a mutation at codon 215 had
reduced susceptibility to AZT.
[0112] The patients with resistant or sensitive virus in our study
had similar CD4 counts at the start of AZT therapy and received AZT
for a similar period of time. Therefore, the development of
resistance and a mutation at codon 215 could not be attributed to
any known pretreatment characteristic. None of our patients
developed AIDS during our study period and the patients who
developed a mutation in their serum HIV RT did so at a relatively
high CD4 count. Thus, advanced stage of HIV disease could not
explain why some patients developed a mutation while others did
not. Additional characteristics of the patient or virus may explain
why one HIV strain develops a mutation and another does not. It has
been stated that syncytium-inducing, T-cell tropic isolates in
HIV-infected individuals contribute to the CD4 cell decline
(Tersmette et al., 1989, Lancet 1:983-985). If an HIV isolate can
maintain a high level of replicative events despite the presence of
AZT, this virus would have a much greater likelihood of mutation.
Treatment with AZT may select both syncytium-inducing and drug
resistant virus. Selection of more virulent HIV population under
prolonged AZT pressure may explain why some patients experienced a
CD4 cell decline in the months after the RT mutation arose.
[0113] The present report shows a strong association between the
presence of a HIV RT mutation and declining CD4 counts in AZT
treated patients. Furthermore, it demonstrates that a HIV mutation
known to cause AZT resistance can be detected prior to a decline in
CD4 cell number.
[0114] Various publications are cited herein that are hereby
incorporated by reference in their entirety.
Sequence CWU 1
1
8 1 17 DNA Artificial Sequence primer 1 ttcccattag tcctatt 17 2 17
DNA Artificial Sequence primer 2 tcattgacag tccagct 17 3 17 DNA
Artificial Sequence primer 3 ggatggaaag gatcacc 17 4 21 DNA
Artificial Sequence primer 4 atgttttttg tctggtgtgg t 21 5 21 DNA
Artificial Sequence primer 5 atgttttttg tctggtgtga a 21 6 28 DNA
Artificial Sequence oligonucleotide 6 ataatccacc tatcccagta
ggagaaat 28 7 28 DNA Artificial Sequence oligonucleotide 7
tttggtcctt gtcttatgtc cagaatgc 28 8 41 DNA Artificial Sequence
probe 8 atcctgggat taaataaaat agtaagaatg tatagcccta c 41
* * * * *