U.S. patent application number 10/936037 was filed with the patent office on 2005-02-10 for cell-based method and assay for measuring the infectivity and drug sensitivity of immunodeficiency virus.
This patent application is currently assigned to UAB Research Foundation. Invention is credited to Kappes, John C., Wu, Xiaoyun.
Application Number | 20050032046 10/936037 |
Document ID | / |
Family ID | 32993352 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050032046 |
Kind Code |
A1 |
Kappes, John C. ; et
al. |
February 10, 2005 |
Cell-based method and assay for measuring the infectivity and drug
sensitivity of immunodeficiency virus
Abstract
Methods and reagents for the capture of primary HIV are
provided. A cell line expressing CCR5, CXCR4 and CD4 receptors
binds and is infected by primary HIV. The cell line contains a
marker gene sequence, the marker gene sequence expressed in near
linear quantities over at least two orders of magnitude in response
to HIV infection. Primary HIV is amplified to create a primary
virus stock through insertion of an amplicon gene into the receptor
expressing cell line. HIV amplification occurs rapidly and is
operative with noninfectious HIV through amplification in the
presence of an infectivity complement. The present invention is
useful in determining host HIV titer, drug sensitivity, HIV
amplification, gene sequencing and co-receptor utilization.
Inventors: |
Kappes, John C.;
(Birmingham, AL) ; Wu, Xiaoyun; (Birmingham,
AL) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
UAB Research Foundation
Birmingham
AL
|
Family ID: |
32993352 |
Appl. No.: |
10/936037 |
Filed: |
September 8, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10936037 |
Sep 8, 2004 |
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09719340 |
Apr 13, 2001 |
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6797462 |
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09719340 |
Apr 13, 2001 |
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PCT/US99/14104 |
Jun 23, 1999 |
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60090317 |
Jun 23, 1998 |
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Current U.S.
Class: |
435/5 |
Current CPC
Class: |
G01N 33/5091 20130101;
G01N 2333/70514 20130101; G01N 33/56988 20130101; C12Q 1/703
20130101; G01N 2333/715 20130101 |
Class at
Publication: |
435/005 |
International
Class: |
C12Q 001/70 |
Claims
That which is claimed:
1. A method for determining the sensitivity of an HIV virus to an
antiviral compound comprising: a) providing an immortalized cell
characterized as expressing CCR5, CXCR4, and CD4 receptors and
having stably incorporated a marker gene operably linked to a
promoter, wherein expression of the marker gene occurs upon HIV
infection of the immortalized cell; b) providing the immortalized
cell a first composition comprising at least one anti-viral
compound; c) contacting said immortalized cell with a second
composition comprising at least one HIV virus; and, d) assaying for
marker gene expression.
2. The method of claim 1, wherein said marker gene encodes
luciferase, .beta.-galactosidase, green fluorescent protein, or a
polypeptide that infers antibiotic resistance.
3. The method of claim 1, wherein said promoter comprises the HIV-1
LTR or HIV-2 LTR.
4. The method of claim 1, wherein said HIV-1 virus is a primary
HIV-1 virus.
5. The method of claim 1, wherein said anti-viral compound is
selected from the group consisting of AZT, 3TC, Nevaripine, and an
antibody.
6. The method of claim 1, wherein the anti-viral compound affects
early stages of the viral life cycle.
7. The method of claim 6, wherein said anti-viral compound affects
the activity of Reverse Transcriptase, Integrase, or Env.
8. The method of claim 1, wherein said anti-viral compound affects
late stages of the viral life cycle.
9. The method of claim 8, wherein said anti-viral compound affects
the activity of Gag or protease.
10. The method of claim 1, wherein said anti-viral compound is
indinavir.
11. The method of claim 1, wherein expression of the marker gene
quantitates the sensitivity of the HIV virus to the antiviral
compound.
12. The method of claim 6, wherein expression of the marker gene
quantitates the sensitivity of the HIV virus to the antiviral
compound.
13. The method of claim 8, wherein expression of the marker gene
quantitates the sensitivity of the HIV virus to the antiviral
compound.
14. An immortalized cell characterized as expressing CCR5, CXCR4,
and CD4 receptors and having stably incorporated an amplicon gene
operably linked to a promoter.
15. A method for amplifying HIV virus comprising: a) providing an
immortalized cell characterized as expressing CCR5, CXCR4, and CD4
receptors and having stably incorporated an amplicon gene operably
linked to a promoter active in said immortalized cell; and, b)
contacting said immortalized cell with a composition comprising at
least one HIV viral isolate.
16. The method of claim 15, wherein said HIV viral isolate is a
primary HIV-1 viral isolate.
17. The method of claim 15, wherein said promoter is a constitutive
or an inducible promoter.
18. The method of claim 15, wherein said promoter is a CMV
promoter.
19. The method of claim 15, wherein the immortalized cell further
has stably incorporated a marker gene operably linked to a second
promoter, wherein expression of the marker gene occurs upon HIV
infection of the cell.
20. The method of claim 19, wherein said marker gene encodes
luciferase, .beta.-galactosidase, green fluorescent protein, or
polypeptide that infers antibiotic resistance.
21. The method of claim 16, further comprising isolating said HIV
virus.
22. The method of claim 21, further comprising: a) contacting a
second immortalized cell with the isolated HIV virus, wherein said
second immortalized cell is characterized by expressing CCR5,
CXCR4, and CD4 receptors and having stably incorporated a marker
gene operably linked to a second promoter, wherein expression of
the marker gene occurs upon HIV infection of the immortalized cell;
and, b) assaying for marker gene expression.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/719,340, filed Apr. 13, 2001; which was a national stage
filing under 35 U.S.C. .sctn.371 of PCT/US99/14104, filed Jun. 23,
1999, which International Application published by the
International Bureau in English on Dec. 29, 1999; which claims the
benefit of U.S. Provisional Application No. 60/090,317, filed Jun.
23, 1998; all of which are herein incorporated in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates genetically modified cells, to
an assay and methods and the usage thereof to measure the
infectivity and viral resistant/sensitivity of isolate from
peripheral blood mononuclear cells (PBMC) and plasma of an
immunodeficiency virus. The present invention has utility in
determining the HIV co-receptor usage, discovery of new drugs
effective against HIV and monitoring a drug therapy protocol in
order to enhance the effectiveness of drug treatment regimes
against HIV-1 infection.
BACKGROUND OF THE INVENTION
[0003] There is currently no cost effective, "high throughput"
method for analyzing the drug resistant phenotype of primary virus
isolates derived from individuals receiving antiretroviral
treatment. Various in vitro biologic and immunologic techniques
have been developed to detect human and simian immunodeficiency
viruses (HIV and SIV, respectively). These include assays that
detect the enzymatic activity of the reverse transcriptase (RT)
protein, ELISA based assays for the detection of HIV/SIV core
antigen (HIV-1 p24 or HIV-2/SIV p27), direct quantitation of
infectious virus by syncytial focus plaque assays or limiting
dilution titration in susceptible host cells, visualization of
virions by electron microscopy, in situ hybridization, and various
nucleic acid-based assays. Recently, genetic reporter-based assays
have been created to detect HIV/SIV infection. In this approach,
mammalian cells are genetically modified to express a reporter gene
such as .beta.-galactosidase (.beta.-gal), green fluorescent
protein (GFP) or chloramphenicol acetyltransferase (CAT) in
response to infection and Tat protein expression. These detection
systems require enumeration of the number of infection-positive
cells by flow cytometry or fluorescence microscopy (GFP),
microscopy (.beta.-gal), or the utilization of radioisotopes (CAT).
The firefly luciferase gene, under control of the HIV-1 LTR
promoter, has been used as a reporter gene for HIV-1 infection.
Luciferase is very sensitive marker gene for HIV-1 infection, since
expression of a relatively few number of luciferase molecules can
result in appreciable activity levels using standard luciferase
detection assays.
[0004] The sensitive detection of the virus quasispecies that
comprise primary HIV isolates has proved difficult using
immortalized CD4 positive cell lines. At least in part, this has
been due to the lack of expression of the CCR5 chemokine
co-receptor on the surface of such cell lines. The failure to
detect infection of primary virus isolates (T-cell and macrophage
tropic viruses) using immortalized cell lines has greatly impeded
the development of useful approaches for detecting, quantifying and
analyzing HIV infection of primary virus isolates. The present
invention largely overcomes the prior art limitations.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1 is a schematic block diagram illustrating a
generalized sequence of steps in creating an assay for detecting
and analyzing primary HIV.
[0006] FIG. 2 is a schematic illustrating the construction of
various gene transfer expression plasmids of the present
invention.
[0007] FIG. 3 is a schematic illustrating the procedure of
lentiviral transduction vectors for the delivery of marker genes of
the present invention. A gene transfer plasmid representatively
including those shown in FIG. 2 are separately transfected into a
host cell together with viral based packaging and envelope
plasmids.
[0008] FIG. 4 is a graph illustrating the relationship between the
concentration of vector and infectious units as determined with
.beta.-gal, GFP and luciferase activity.
[0009] FIG. 5 is a graph illustrating a nearly linear relationship
between HIV-1 infectious units and luciferase activity for a cell
line of the present invention.
[0010] FIG. 6 is a graph illustrating the relationship between
infection virus units and luciferase activity for viruses: TIVI,
WIMI, KIWE and YU2, using a cell line of the present invention.
[0011] FIG. 7 is a graph illustrating a correlation between
infectious virus units and luciferase activity.
[0012] FIGS. 8(a)-(c) are graphs illustrating the effect that
different concentrations of 3TC, AZT, and Nevaripine, respectively,
have on virus replication relative to non-drug treated viruses as
determined by luciferase activity according to the present
invention.
[0013] FIGS. 9(a)-(c) are graphs illustrating drug sensitivity to
AZT to 100, 500, and 2500 virus infectious units respectively,
according to the present invention.
[0014] FIG. 10 is a schematic illustrating the construction of a
Tat transduction plasmid.
[0015] FIGS. 11(a) and (b) are graphs illustrating the viral
amplification two days following infection with equal quantities of
YU2 HIV(a) for infectivity and (b) p24 antigen.
SUMMARY OF THE INVENTION
[0016] The present invention pertains to a cell-based assay for
analyzing primary HIV including an immortalized cell line that
expresses the CCR5, CXCR4 and CD4 receptors and a marker gene. The
CCR5 or CD4 receptors enable binding and entry of HIV wherein
marker gene expression correlates the magnitude of virus infection.
An immortalized cell line is disclosed capable of allowing
efficient amplification of primary HIV. A method is further
disclosed wherein the cell line contains a gene that can be
expressed in response to infection of the virus. A method is
further disclosed for producing an immunodeficiency virus infection
sensitive clonal cells, the method including selecting a cell line
expression CCR5, CXCR4 and CD4, thereafter transducing the cell
line with a gene vector encoding for a marker gene such as
luciferase such that marker gene expression correlates to the
magnitude of immunodeficiency virus binding by said cell line and
establishing sensitive clonal cells therefrom.
[0017] The present invention finds utility as a method for
detecting, isolating and analyzing primary HIV by infecting a cell
line of the present invention with a quantity of virus and after
some time measuring marker gene expression. Practicing the method
of the present invention for determining immunodeficiency virus
titer and conducting the presence of a drug candidate indicates the
sensitivity of a given strain, type, species or genus of virus to
the given candidate. The present invention affords the ability to
test virus derived from blood plasma as well as cell culture.
DESCRIPTION OF THE INVENTION
[0018] The use of an immortalized cell line to detect and analyze
primary HIV other than PBMC offers numerous advantages which are
exploited to develop a novel assay. Currently, one of the major
limitations is that immortalized cell lines are refractory to
primary HIV. The expression of CCR5 in an immortalized cell line
significantly enhances the detection of primary HIV-1. The ability
to detect primary isolates of HIV-1 with greater sensitivity than
currently possible is an aspect of the present invention. Unlike
previous assays, the present invention provides for: (1) the
sampling and analysis of a representative population of viruses
that comprise primary HIV-1; (2) the analysis of a significantly
greater proportion of virus; and (3) high throughput testing, via
miniaturization and sampling of small sample volumes.
[0019] Most prior art molecular clones of HIV-11 have been derived
by tissue culture methods that select for viruses that do not
require CCR5 co-receptor for infection, herein defined as T-tropic
viruses. Such clones are not able to infect monocytes and
macrophages. The term "T-trophic virus" is intended herein to
define a phenotype of an immunodeficiency virus capable of
infecting a T-cell by binding the CD4 receptor on the T-cell. The
term "macrophage trophic virus" is intended to mean a phenotype of
an immunodeficiency virus capable of infecting a macrophage by
binding the CCR5 co-receptor on the macrophage. This difference in
tropism has been mapped to the viral env gene. The SG3 (S. K. Ghosh
et al. 1993, Virology 194:858-864) and NL43 (W. Paxton et al. 1993,
J. Virol. 67:7229-7237) strains of HIV-1 are derived by extensive
passage in tissue culture. They represent T-cell tropic viruses and
do not infect monocytes and macrophages. These viruses are not
representative of the complex mixtures of viruses that exit in
infected individuals. Primary HIV-1 represent virus that is derived
directly from the blood of an HIV infected individual. Primary HIV
can also be derived by short term culture in vitro culture in
primary peripheral blood mononuclear cells (PBMC). Primary HIV can
also be derived using the cell line of this invention. Such
isolates are complex mixtures and may contain macrophage- and/or
T-tropic viruses. During the natural history/progression of HIV-1
infection there is generally a shift from a population of
macrophage-tropic toward one of T-tropic viruses. T-cell tropic
viruses are able to infect cells that express CD4 and CXCR4, while
macrophage tropic (M-tropic) viruses also require expression of the
CCR5 chemokine co-receptor. Most HIV-2 and SIV viruses also require
the CCR5. Several groups have produced cell lines that express CD4,
CXCR4 and CCR5 in attempts to render them sensitive to infection
with primary HIV-1 (both T-cell and macrophage tropic viruses).
Only recently have such cell lines been derived which appear to be
susceptible to infection with diverse HIV-1 isolates (Platt et al.,
J. Virol. 72:2855, 1988; Overbaugh et al., J. Virol. 71:3932,
1997).
[0020] As used herein, "primary HIV" is defined as HIV derived
directly from an infected host organism from sources such as blood,
plasma, PBMC, CSF and other tissues.
[0021] As used herein, "immunodeficiency virus" is defined as
various strains and stocks of HIV-1, HIV-2, SIV and
lentiviruses.
[0022] As used herein, "minor population" is defined as a titer of
a given viral strain, type or species or genus that constitutes
less than 10% of the total quantity of virus present obtained from
a host culture or organism.
[0023] As used herein, "major population" is defined as the
numerically dominant viral strain, type, species or genus of a
viral titer obtained from a host culture or organism.
[0024] As used herein, "drug sensitivity" is defined as the
effectiveness of a drug to inhibit HIV replication and/or
expression with a host cell, the term is used synonymously with
"drug resistivity."
[0025] By making genetic modifications to a CCR5 or CD4 expressing
cell line, the present invention represents is an efficient method
for analyzing the drug sensitivity properties of primary HIV, such
as HIV-1.
[0026] FIG. 1 is a schematic block diagram illustrating a
generalized sequence of steps in creating an assay for measuring
HIV-1 drug sensitivity according to the present invention. The
creation of a cell based assay of the present invention involves a
series of steps. Initially, a vector is constructed for the purpose
of transducing mammalian cells with a marker gene. Such a marker
gene transduction plasmids bring the marker gene expression under
the regulation of an immunodeficiency virus 10. Preferably, the
marker gene vector is placed under the control of HIV-1 or HIV-2
long terminal repeats (LTRs) and the Rev responsive element (RRE).
The marker genes illustratively including .beta.-gal, luciferase,
GFP, CAT and other fluorescent proteins. Preferably, the marker
gene is luciferase. CD4 positive cells are then transduced with the
vector in order to confirm appropriate marker gene expression from
the transduction vectors 20. Preferably, the cells are CD4, CCR5,
CXCR4 positive. It is appreciated that an amplicon gene is readily
substituted for a marker gene to induce amplification of viral
stocks (not shown). Preferably, the amplicon gene is Tat.
[0027] Clones of the stable CD4 positive cell line are established
30. A stable CD4 positive cell line is selected for low marker
background expression levels 40. Preferably, the marker gene is
luciferase. An immortalized cell line 15 is positive for CCR5, CD4
and CXCR4 receptors and optionally other receptors illustratively
including CCR3, CCR2B and T-lymphocyte expressed 7 transmembrane
domain receptor. Preferably, the origin cell line is HeLa. More
preferably, the cell line is J53 (Oregon Health Sciences
University) or a cell line that naturally expresses CD4, CCR5 and
CXCR4. The cells of immortalized cell line 15 are then tested for
sensitivity to HIV-1 infection 25. Expansion of highly sensitive
cells to HIV-1 infection 35. The clone of 40 is used to transduce
50 the highly sensitive immortalized cell line of 35. Preferably,
the receptor is selected to create a cell which is highly sensitive
to infection by HIV-1 isolates. Clones established from this second
transduction are both highly sensitive to infection with primary
HIV-1 isolates and express low background levels of the marker gene
product in the absence of HIV-160. Those clones which are positive
for the marker gene are identified 70. Such positive clones 70 have
utility to promote HIV production upon transduction with Tat 75.
HIV primary virus stock production is exploited herein to
selectively enrich drug resistant minor HIV strains infecting a
host 85.
[0028] Infection of the clones expressing, low background levels of
at least two markers such as .beta.-gal and luciferase with HIV-1
confirms the relationship between infectious viral units and marker
gene (luciferase) activity 80, although in practice expression of a
single marker gene is operative herein. Clones that relate
infectious virus units, such as .beta.-gal, with a second marker
gene activity such as luciferase find utility in the measurement of
HIV co-receptor utilization (not shown). The cells capable of
expressing marker genes in response to HIV infection are optionally
used to measure viral sensitivity in the presence of a drug 90.
Drug, resistance to various pharmaceutical during viral life cycle
events such as envelop formation 92, reverse transcription 94 and
proteolysis 96 is optionally determined. The measurement of viral
sensitivity finds utility in HIV viral target resistance analysis
98. The resulting composition of clones in a suitable medium is
amenable for use to quantify HIV-1 titer. The present invention
also finds utility in quantifying the drug sensitivity of
particular HIV-1 phenotypes.
[0029] It is appreciated that the present invention is applicable
to use with immunodeficiency viruses other than the representative
HIV-1. By transducing a cell line to express co-receptors adapted
for binding an immunodeficiency virus, a variety of viruses may be
assayed with the present invention.
[0030] The present invention pertains to cell lines that are
capable of detecting sensitivity of a given strain of HIV to
inhibitors that act upon various stages of the virus life cycle by
monitoring the effect various drugs have on early viral life cycle
stages such as reverse transcription, integration and envelop
mediated receptor binding, envelope fusion, as well as the late
life cycle stages complexes such as proteolysis and Gag complex
formation. A method and a kit are provided for monitoring the major
and minor virus populations infecting, a given host. Through the
enrichment and detection of minor drug resistant virus populations
and the sensitivity of those populations to viral inhibitors, the
assay is well suited for determining specific anti-retroviral drugs
suited to contain replication of the various HIV strains infecting
a given host. Such a tailored therapeutic protocol is more
effective in inhibiting viral amplification and/or reduces
pharmacological side effects. In particular, the J53 Tat cell line
is well suited to detect sensitivity of a host's particular viral
infection to inhibit or affect the various stages of the virus life
cycle. The present invention provides more rapid viral
amplification as compared to conventional PBMC cells thereby
allowing more rapid amplification, with fewer cycles of reverse
transcription. Further applications of the present invention
include measurement of HIV attributes of co-receptor utilization,
antibody neutralization, isolation, titration, gene sequencing, and
CTL assays.
[0031] The present invention also provides a method for detection
of primary HIV from plasma, including noninfectious HIV-1 found in
plasma. A loss of viral infectivity is due in part to a loss of env
or env blocking. VSV-G serves to mediate infection of HIV-1
particles and thus, in the absence of VSV-G the virus remains
noninfectious, whereas in its presence infectivity is
complemented.
[0032] It is appreciated that in addition to VSV-G other
infectivity complements are also operative herein including
adenovirus, liposome, monoclonal antibody and other vectors to
complement noninfectious HIV.
[0033] The methods and indicator cell lines of the present
invention are operative to analyze drug sensitivity of primary HIV
which has been purified and taken directly from infected host
plasma.
[0034] To directly analyze the relationship between luciferase
expression and infectious virus units, a series of gene transfer
plasmids are constructed to express luciferase,
.beta.-galactosidase (.beta.-gal), and green fluorescence protein
(GFP), respectively, .beta.-gal, GFP, and luciferase (luf) are
placed under control of the HIV-1 or HIV-2 long terminal repeats
(LTR), and the Rev Responsive Element (RRE). FIG. 2 illustrates the
different gene transfer expression plasmids that are constructed.
The .beta.-gal and GFP markers allow for direct enumeration of the
number of infectious virus units as infected cells by counting
under a microscope. The luciferase marker allows for sensitive and
high throughput quantitation of FHV infection. In the present
invention the requirement of Tat and Rev for marker gene expression
is different from previous work in that it allows for highly
regulated and decreased background level expression of the marker
gene. This is particularly important for luciferase.
[0035] The J53BL cell line or its functional equivalents are
operative in accordance with the present invention. It is
appreciated that the nucleic acid sequences coding for CCR5, CXCR4,
CD4, luciferase, .beta.-galactosidase, GFP, CAT, Tat and the J53
cell line as a whole can be altered by substitutions, additions or
deletions that provide for functionally equivalent cells. As used
herein, "functional equivalency" is defined to mean a nucleic acid
sequence which encodes for a product that performs operationally
within the present invention with at least half the effectiveness
of the product derived from the unaltered nucleic acid sequence of
a receptor, amplicon, marker gene or cell line. Due to the
degeneracy of nucleotide coding sequences, other DNA sequences
which encode substantially the same receptor amino acid sequences,
cell line amino acid sequences, marker gene sequences and amplicon
sequences may be used in the practice of the present invention.
These include but are not limited to nucleotide sequences which are
altered by the substitution of different codons that encode a
funtionally equivalent amino acid residue within the sequence thus
producing a silent change. Likewise, receptor, marker, amplicon and
cell lines proteins or fragments or derivatives thereof of the
present invention include, but are not limited to, those containing
as a primary amino acid sequence all or part of the amino acid
sequence of the sequences for CCR5, CXCR4, CD4, luciferase,
.beta.-galactosidase, GFP, CAT, Tat and J53 sequences including
altered sequences in which functionally equivalent amino acid
residues are substituted for residues within the sequence resulting
in a silent change. For example, one or more amino acid residues
within a sequence are optionally substituted by another amino acid
of a similar polarity which acts as a functional equivalent,
resulting in a silent alteration. Substitutes for an amino acid
within a sequence may be selected from other members of a class to
which the amino acid belongs. For example, non-polar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. Polar neutral amino acids
include glycine, serine, threonine, cysteine, tyrosine, asparagine
and alutamine. The positively charged (basic) amino acids include
arginine, lysine and histidine. Negatively charged (acidic) amino
acids include aspartic acid and glutamic acid. Also included within
the scope of the present invention are proteins or fragments or
derivatives associated with J53BL which are differentially modified
during or after translation by operations such as glycosilation,
proteolytic cleavage and linkage to an antibody or other cellular
ligand.
EXAMPLE 1
Generation of Transduction Vectors for the Delivery of Marker
Genes
[0036] In order to generate vector stocks for transduction with the
different reporter genes, the .beta.-gal, GFP and luciferase gene
transfer plasmids, including those containing, the HIV-2 LTR (shown
in FIG. 2), are separately transfected into cultures of 293T cell
together with a lentiviral-based packaging plasmid (pCMV-GP1), and
the pCMV-VSV-G env plasmid (FIG. 3). Forty-eight hours later, the
vector-containing culture supernatants are harvested, clarified by
low-speed centrifugation, filtered through 0.45 micron filters,
analyzed for HIV-1 p24 core antigen concentration by ELISA,
aliquoted, and cryopreserved as stocks. Four serial five-fold
dilutions (normalized for p24 antigen concentration) of the stocks
are prepared and used to infect replica cultures of HIV-HeLa cell.
The HIV-HeLa cells contained an integrated HIV-1 provirus that is
defective in vpr and env, and produces the Tat and Rev protein for
transactivating marker gene expression. Two days after infection of
the HIV-HeLa cells with the different vector stocks, .beta.-gal and
GFP expression is quantified using a microscope to count the number
of positive cells/well. Luciferase expression is measured using
standard assay methods (Promega) and a luminometer. FIG. 4 shows
the relationship between concentration (HIV-1 p24 antigen, Coulter
Inc.) of the vector stocks and infectious units as determined with
.beta.-gal and GFP, (virus infectious units) or luciferase
activity.
EXAMPLE 2
Generation of .beta.-gal, Luciferase and GFP Indicator Cell Lines
to Quantify HIV/SIV Infection
[0037] The following pairs of vector stocks (derived as described
above) are used to co-transduce cultures of HeLa-CD4 cells: (a)
pluf+p.beta.-gal, (b) pluf+pLTR2-.beta.gal, (c) pluf+pGFP, (d)
pluf+pLTR2-GFP, (e) pLTR-2-luf+p.beta.-gal, (f)
pLTR2-luf+pLTR-2.beta.-ga- l, (g) pLTR2-luf+pGFP, (h)
pLTR2-luf+pLTR-2-GFP. Three days later, the cells are biologically
cloned by limiting dilution in 48 well plates. Wells containing
clonal cells (confirmed after initial plating by microscopy) are
expanded into replica cultures. One replica culture set is infected
with HIV-1/SG3 and analyzed for marker gene expression (HFIV-1
infection provided Tat and Rev to activate marker gene expression)
as described above. Expression positive cells cultures are
identified, expanded and cryopreserved.
[0038] Since the expression of relatively few molecules of
luciferase produces substantial luciferase activity levels, 36
non-HIV-1 infected, luciferase expression-positive clonal cultures
(derived from HeLa-CD4 cells transduced with pluf+p.beta.-gal) are
analyzed for luciferase activity to determine basal background
expression levels. The HeLa-CD4 cells being obtained from the AIDS
Research and Reference Reagent Repository of NIH. Of the 36 clones
analyzed, luciferase activity ranged from 15 to 250 units. Analysis
for .beta.-gal expression in response to HIV-1 infection indicated
approximately 70% of the clones expressed both .beta.-gal and
luciferase. The two clones (referred to as HeLa-.beta.-gal-luf1,
and HeLa-.beta.-gal-luf2) that exhibited the lowest background
levels of luciferase expression and are positive for .beta.-gal
expression are used to directly analyze the relationship between
HIV-1 infectious units and luciferase activity. Serial dilutions of
two different HIV-1 strains (HIV-1/SG3 and HIV-1/NL43) are
normalized for p24 antigen concentration and used to infect replica
cultures of HeLa-.beta.-gal-luf1, and HeLA-.beta.-gal-luf2. After
48 hours, one set of cultures is analyzed for luciferase activity
and the other was analyzed for .beta.-gal. FIG. 5 shows the
relationship between HIV-1 infectious units (.beta.-gal positive
cells) and luciferase activity for the HeLa-.beta.-gal-luf1 cell
line. The HeLa-.beta.-gal-luf2 cell line gave nearly identical
results with slightly higher luciferase activity levels at the
lower virus inoculums. Between approximately 10 and 10,000 virus
infectious units. A near-linear relationship to luciferase activity
is shown in FIG. 5. The linear range of detection using the
luciferase marker in FIG. 5 is approximately 3 orders of magnitude,
and as few as 10-20 infected cells out of approximately 100,000 can
generate a virus-positive (above background) result. As referred to
herein "near linear" is intended to mean an increase in marker
activity, A proportional to an increase in the surrounding virus
infectious unit concentration, IU such that A=n(IU).sup.1.+-.x+b
where n is a real number; x is a real number between 0 and 0.5; b
is the measured background level of marker expression in the
absence of virus; for at least 2 orders of magnitude of IU. This
dynamic range allows for quantitative analysis of virus infection
from approximately 10 to 10000 infectious units, thereby reducing,
the necessity of dilution of virus in order to generate
quantitative data.
EXAMPLE 3
Sensitive Detection of HIV-1 Primary Viruses Using .beta.-gal and
Luciferase Reporter Genes
[0039] The present invention utilizes a combination of a reporter
assay system for sensitively and rapidly quantifying, infectious
HIV-1 over a wide linear range with a cell line which is highly
sensitive to infection with both M-tropic and T-cell tropic
viruses. Transduction of the CD4-CCR5 positive J53 cell clone (Dr.
David Kabat, Oregon Health Sciences University, Portland, Oreg.)
with the pluf and p.beta.-gal expression vectors as described
above. The pluf and p.beta.-gal transduced J53 cells (termed
J53-.beta.gal/luf) are infected with six different virus isolates
(using four five-fold serial dilutions) that were unable to
efficiently infect other CD4, CXCR4 expressing cell lines (P4 or
Hi5) or a CD4, CXCR4 expressing cell line (MAGI) (see Table 2).
Table 1 shows that all viruses, including the macrophage tropic YU2
clone, included as a control, are highly infectious in the
J53.beta.-gal/luf cell line.
[0040] To assess the relationship between infectious virus units
and luciferase activity in the J53.beta.-gal/luf cell line, four
serial five-fold dilutions of the following viruses are prepared
and analyzed: TIVI, WIMI, KIWE and YU2. Between approximately 100
to 10,000 infectious units, the data show a linear relationship
with luciferase activity (FIG. 6). Background levels of luciferase
are between 100 and 150. The J53 .mu.l-gal/luf cell line represents
a transduced population of cells since integration of the
transduction vector into the genome of the J53 cells can occur
differently in each cell.
[0041] To minimize luciferase background levels of non HIV induced
expression and thus maximize sensitivity using luciferase as a
reporter for HIV infection, cultures of single cell clones are
derived from the J53.beta.-gal/luf cell line as described above and
characterized for luf and .beta.-gal expression in response to
HIV-1 infection. Ten clones expressing between 17 and 750 luf
activity are selected for analysis. Clone number 13, termed
J53-C13, is confined to express both luciferase and .beta.-gal, and
is used for subsequent analysis as described below. Stocks of
twenty different HIV-1 isolates are obtained from HIV-1 infected
individuals by standard coculture techniques. Each stock is
analyzed for HIV-1 p24 antigen concentration, SI and NSI phenotype,
and infectivity in HeLa-CD4 (MAGI), HeLa-CD4-CCR5 (P4), H9CD4-CCR5
(Hi5), and HeLa-CD4-CCR5 (J53-C13) cells. These results are
summarized in Table 2.
[0042] These results show that the J53-C13 cell line is sensitive
to primary HIV-1. Importantly, the J53-C13 cell line is sensitive
to HIV-1 infection to a degree similar to PBMC. To confirm the
importance of the CCR5 co-receptor for this level of sensitivity,
the JC11 cell line is analyzed for comparison. JC11 is the parental
cell line to J53-C13. It expresses equal amounts of CD4 and CXCR4
but is negative for CCR5. JC11 is transduced to express .beta.-gal
and luciferase, and positive cells are biologically cloned exactly
as described above for J53-C13. A clone designated J11-C5, which is
capable of expressing both .beta.-gal and designated J11-C5, which
is capable of expressing both .beta.-gal and luciferase, is
selected for comparison with J53-C13). Both cell lines are infected
with primary virus isolates and molecularly cloned virus including
YU2, SG3, and 89.6 (a dual tropic clone). Table 4 shows the titer
of each virus in the J53-C13 and J11-C5 cell lines. The results
show a marked reduction in virus titer in the J11-C5 cell line,
indicating that the CCR5 co-receptor is necessary for efficient
infection/detection of primary virus isolates.
[0043] To analyze the relationship between luciferase and
.beta.-gal expression over a range of different virus
concentrations, 5-fold serial dilutions are prepared from seven
different virus stocks and used to infect J53-C13 cells. After two
days the number of .beta.-gal positive cells and luciferase
activity is determined. FIG. 7 shows a strong, correlation (r=0.92)
between .beta.-gal positivity (infectious virus units) and
luciferase activity over 2 orders of magnitude.
EXAMPLE 4
Evaluation of Primary HIV-1 Isolates for Drug
Sensitivity/Resistance Using the J53.beta.-gal/luf (J51-C13) Cell
Line
[0044] HIV-1 isolates are derived by PBMC coculture from two
different HIV-1 infected patients (LEMI and SARO) receiving
anti-retroviral treatment. The RT sequence of each isolate is
analyzed for nucleic acid sequence using ABI sequencing methods.
Known drug resistance conferring mutations found in the LEMI and
SARO RT sequences are shown in Table 3. The LEMI and SAR0 and YU2
(included as a control) virus stocks are used to infect the J53-C13
reporter cell line in the presence of AZT, 3TC and Nevaripine
(NVP), respectively. Two days after infection the cells are lysed
and the clarified lysates are examined for luciferase activity
using standard methods (Promega). FIG. 8 shows the effect of
different concentrations of drug on virus replication relative to
non-drug treated viruses--as determined by luciferase activity as
an indicator.
[0045] A major problem with existing methods for evaluating HIV-1
drug sensitivity is that differences in virus inoculum can have
significant effects on the IC50 for a given drug. That is, as the
infectious dose of virus is increased, the concentration of drug
that inhibits virus replication by 50% is increased. This factor
has made drug sensitivity testing extremely difficult to
standardize among independent laboratories. J53-C13 cells are
infected with 100, 500, and 2500 infectious units of virus and
analyzed for drug sensitivity as described above. FIG. 9 shows the
results for drug sensitivity to AZT. There is no significant shift
in the IC50 among the different drug concentrations tested.
Analysis of 3TC and Nevaripine showed similar results (data not
shown).
EXAMPLE 5
Generation of a Tat Expressing Cell Line to Rapidly Amplify Virus
Production from Infected Cells
[0046] The amplification of primary virus from infected individuals
is required for phenotypic resistance in assays that test whole
virus. Currently, the only effective means by which this can be
accomplished, is by culture of infected tissue with donor PBMC. The
present invention confirms that the JC53 and the J53-C13 cell lines
are highly sensitive to infection of primary virus isolates. Thus,
these cell lines may be utilized to amplify the primary virus
isolate instead of PBMC. To this end, JC53 cells are transduced
with the HIV Tat gene under control of the CMV, or LTR promoter, as
shown in FIG. 10. To eliminate Tat transactivation of the
lentivirus vector LTR, Tat is constructed into a self-deleting U3
transduction vector, FIG. 10. Three days after transduction, single
cells are cloned and 33 are identified to be Tat expression
positive, 10 containing LTR-2 as a promoter and 23 containing CMV
as a promoter for Tat expression. To identify which of these clones
could most efficiently promote HIV-1 replication, HIV-1 YU2 is used
for infection at an MOI=0.01. After 40 hrs., virus production is
measured by HIV-1 p24 antigen ELISA and the highest HIV-1 producing
lines from each are selected for further analysis. The highest
HIV-1 producer, designated J53-CNV tat is infected with the YU2
clone and the KEWI virus isolate at MOIs of approximately 0.1. As a
control, the JC53 cell line is analyzed in a parallel experiment.
40 hrs. later culture supernatants are analyzed for HIV-1
production by p24 antigen ELISA. The results, shown in Table 5,
indicate that the Tat expressing cell lines causes a 4-6-fold
increase in HIV-1 replication.
EXAMPLE 6
The Use of CD4/CCR5/CXCR4+Tat Expressing Cell Line to Capture and
Amplify Primary Virus
[0047] The J53tat cell line is compared with PBMC for primary virus
amplification. PBMC and J53tat are each infected with 2.5E5
infective particles of YU2. Two days later the concentration of
progeny virus is analyzed for infectivity in J53BL indicator cells
as shown in FIG. 11(a) and by p24 antigen ELISA, as shown in FIG.
11(b). The J53tat cell line amplifies primary virus to higher
titers and more rapidly than PBMC. Since the parental J53BL cell
line is highly sensitive to primary virus, Tat facilitates the
rapid generation of high titered primary virus stocks for
resistance testing without selection of longer term culture, such
as PBMC culture for virus amplification.
EXAMPLE 7
Detection of Drug Resistance/Sensitivity that Effect Various Stages
of Virus Life Cycle
[0048] The J53tat cell line is used to produce virus and thereby
enable viral testing of drug candidates that affect various stages
of the virus life cycle. Thus, viral drug resistance mutations in
early stage targets such as reverse transcriptase (RT), integrase
(IN) and env; and late stage targets such as protease and Gag are
analyzed by the methods of the present invention. The J53tat cells
are infected with HIV YU2 (MOI of either 0.2 or 0.04), and protease
inhibitor (indinavir) is added to the cultures at various
concentrations. Forty hours after infection the culture supernatant
is collected and used to infect the J53BL cell line in the presence
of the same drug concentrations. Table 6 shows that YU2 is
sensitive to protease inhibitor, with increasing concentrations
causing greater inhibition.
EXAMPLE 8
Detection of Noninfectious Cultured Virus
[0049] To test how to recover noninfectious virus, a molecular
clone is generated to produce env minus HIV-1 (pSG3-env). SG3-env
virus, derived by transfection, is mixed (1:2, v:v) with VSV-G
derived from the supernatant of pDm transfected 293T cell cultures.
The mixture is ultracentrifuged for 1.5 hours at 115,000 g at
4.degree. C. The pellet is resuspended in 100 ul DMEM. The
infectivity is then determined using J53BL cells. The infectivity
is determined to be 7.5E4. Without mixing of VSV-G the infectivity
is 0.
[0050] YU2 virus containing wild-type envelope is pelleted through
sucrose by ultracentrifugation to strip away the gp120 glycoprotein
(SU). The resuspended (100 ul) virus is mixed with and without
VSG-G (1:1) and repelleted by ultracentrifugation (150,000 g, 2
hours, 4.degree. C.). The pellets are resuspended in 100 ul DMEM,
and the infectious units are determined using J53BL cell summarized
as in Table 7. Virus pelleted through sucrose is noninfectious.
Virus pelleted through sucrose, mixed with VSV-G and repelleted had
a marked increase in infectivity. The recovery in infectivity is
approximately 20% compared with the original virus stock.
EXAMPLE 9
Detection of Noninfectious Plasma Virus
[0051] Patient plasma (GADA) is mixed with and without VSV-G,
pelleted through sucrose, resuspended in 100 ul DMEM as per Example
10. Infectivity is measured using, J53BL cells. Without VSV-G 1500
infectious particles are detected. With VSV-G 2500 infectious
particles are detected as summarized in Table 7.
EXAMPLE 10
Detection of Plasma Virus Using CD4/CCR5/CXCR4 or
CD4/CCR5/CXCR4+Tat Expressing Cell Line to Capture and Amplify
Primary Virus
[0052] Plasma from patients infected with HIV-1 is tested for the
presence of infectious virus in the plasma towards J53BL cells.
Three serial dilutions of plasma are incubated with J53BL cell line
for 4 hours. Three days later the cells are stained for .beta.-gal
and infectious units are counted by microscopy as summarized in
Table 8.
EXAMPLE 11
Integrated HIV Genome Expansion with Limited Rounds of Reverse
Transcription
[0053] HIV is incubated with J53BL cell line for four hours to
allow binding and entry into J53BL cells, reverse transcription
proceeds and the viral cDNA is integrated into the chromosomes of
J53BL cells. Thereafter, HIV replication is suppressed through
expression of an inhibitor of viral gene expression, such as the
rev inhibitor, rev m10 by conventional techniques. The HIV genome
is expanded as J53BL cells divide and increase in number, without
further rounds of reverse transcription. The increased copy numbers
of the viral genome are purified and sequenced. By relieving the
inhibitory effect on rev, viral gene expression will return to
normal in the expanded cells, and virus can be analyzed.
[0054] Based on the description and examples of the present
invention, it is appreciated that modifications of the present
invention will be apparent to one skilled in the art of the present
invention. Such modifications are intended to fall within the scope
of the appended claims.
[0055] All references cited herein are intended to be incorporated
to the same extent as if each reference was individually
incorporated by reference.
1TABLE 1 Efficient HIV-1 infection of a CD4 CCR5 expressing HeLa
cell line. HIV-1 Isolate.sup.a p24 pg./ml PEMC.sup.b
HeLa-CD.sup.4-CCR5.sup.c KEWI 2.64E+05 4.58E+04 2.30E+05 WIMI
1.31E+05 1.15E+04 8.1 OE+04 SMBA 5.34E+05 4.58E+04 1.60E+05 CHVI
6.50E+04 1.15E+04 7.OOE+04 DECH 2.07E+05 4.58E+04 1.30E+05 TIVI
1.20E+05 1.84E+05 4.20E+05 YU-2 6.20E+05 7.36E+05 5.10E+05
.sup.aHIV-1 isolates were derived by coculture (7-10 days) of HIV-1
infected patient PBMC with PHA stimulated normal donor PBMC.
.sup.bVirus titer - determined by endpoint dilution titration in
PBMC calculated by the Spearman-Karber Formula (TCID 50/mi).
.sup.cVirus titer - determined by counting the number of virus
infected (blue) HeLa-CD4-GCR5-.beta.-gal indicator cells (J53-Cl
6).
[0056]
2TABLE 2 Comparison of HIV-1 isolate infectivity in different cell
types. HeTa-CD4 HeLa-CD4-CCRS H9 CD4-CC5 HeLa-CD4-CCR5. Syncylia
HIV-1 isolate (MAGI cells) (P4 cells) (Hi5 cells) (J53-CI 6) PBMC
phenotype IVED 1563 2.10E+02 2.24E+03 <1.2E+2 2.2E+4 1.84E+05
NSI SHTI 2045 2.00E+O1 5.72E+02 2.88E+03 1.2E+5 4.58E+04 SI RIJA
0676 1.50E+01 1.68E+02 <1.2E+2 4.5E+5 1.84E+05 SI ELRA 1452
1.50E+01 I.00E+02 <1.2E+2 9.SE+5 4.58E+04 NSI JAME 2457 5.00E+00
4.00E+00 <1.2E+2 1.8E+5 4.58E+04 SI WOAL 0263 1.80E+01 5.40E+02
<1.2E+2 8.5E+4 4.58E+04 SI WIMI 2294 2.10E+01 1.52E+02
<1.2E+2 4.SE+-I 1.15E+04 SI DAKE 2205 6.20E+01 3.28E+02
<1.2E+2 1.1E+5 4.58E+04 NSI BARE 1219 1.80E+OI 4.48E+02
<1.2E+2 1.3E+5 4.58E+04 NSI SMBA 1685 2.20E+01 6.40E+01
<1.2E+2 1.IE+5 4.58E+04 SI TIVI 2007 8.90E+01 4.60E+02
<1.2E+2 4.1E+5 1.84E+05 SI SWBA 1427 2.80E+01 4.48E+02
<1.2E+2 9.0E+4 4.58E+04 NSI CHVI 2467 5.00E+01 4.00E+00
<1.2E+2 2.8E+4 1.15+04 SI VIJE 1945 7.80E+01 3.04E+02 2.88E+03
2.1E+5 4.58E+04 NSI DECH 1.20E+01 2.00E+01 <1.2E+2 1.2E+5
4.58E+04 NSI SHCH 1.80E+02 1.76E+04 <1.2E+2 3.6E+5 4.58E+04 NSI
PELE 1256 1.20E+01 2.40E+01 2.88E+03 I.OE+5 1.15E+04 SI KEWI 2431
1.90E+03 4.54E+05 <1.2E+2 2.0E+5 4.58E+04 NSI MADA 2442 2.00E+01
1.40E+D3 <1.2E+2 2.8E+5 4.58E+04 NSI VATE 2328 2.00E+01 3.76E+02
<1.2E+2 4.5E+4 1.15E+O,1 SI SG3-293T 2.20E+05 1.89E+05 1.84E+05
2.0E+5 1.84E+05 SI YU-2-293T 0.00E+00 3.50E+05 4.58E+04 5.9E+4
7.36E+05 NSI
[0057]
3TABLE 3 RT resistance conferring mutations. YU2 SARO LEMI AZT M41
L D67N 3TC D67N K70R T215Y K219Q M1 84V
[0058]
4TABLE 4 CCRS facilitates infection of primary isolates of HIV-1.
Virus Stock J53-C13 liter J11-C5 titer DBP D-7 6.30E+04 Neg. DEP
d-140 1.20E+04 4.00E+01 HVH D-7 6.10E+04 1.10E+04 HVH D140 6.80E+04
1.00E+03 SHL D-7 6.00E+04 Neg. SHL D140 7.70E+04 Neg. TED 0127
1,30C:+05 Neg. TED D211i 3.20E+04 Neg. HXE2 1.75E-05 Neg. YU2
4.85E+05 7.50E+04 89.6 1.40E+05 Ncq. Virus titer was determined by
counting the # of beta-gal positive cells. Results indicate
infection positive cells per ml of stock virus Neg. (negative)
titers were undetectable below 40 infectious units per ml.
[0059]
5TABLE 5 Trans Tat expression enhances HIV-1 production. JC53
JC53-CMVtat Virus Exp. 1 Exp. 2 Exp. 1 Exp. 2 YU2 1240 920 6430
4910 KEWI 3120 2760 9590 7590 Nos. represent pg. of p24 antigen per
ml.
[0060]
6TABLE 6 Number of colony formed in the presence of protease
inhibitor Indinavir. Indinavir 0 .mu.m 0.008 .mu.m 0.04 .mu.m 0.2
.mu.m 1.0 .mu.m 0.2 >1000 >1000 343 99 0 M.O.I. 0.4 198 105 2
2 0 M.O.I.
[0061]
7 TABLE 7 Conditions Recovery (%) No sucrose, No VSV-G 0.006
Sucrose, No VSV-G 0 Sucrose, VSV-G 20%
[0062]
8TABLE 8 Detection/Isolation of HIV-1 from human plasma using J53BL
cells. Plasma virus TCIU/PBMC TCIU/J53 BL LEMI 3.47 .times.
10.sup.3/m 4.40 .times. 10.sup.3/ml ALPI 3.47 .times. 10.sup.3/m
1.20 .times. 10.sup.4/ml GADA 7.81 .times. 10.sup.3/ml 1.20 .times.
10.sup.4/Ml TCIU = tissue culture infectious units
* * * * *