Note: Descriptions are shown in the official language in which they were submitted.
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CELL-BASED ASSAY FOR IMMUNODEFICIENCY VIRUS INFECTIVITY
AND SENSITIVITY
The present invention relates genetically modified cells; to an assay and
methods and the usage thereof to measure the infectivity and viral
resistantlsensitivity of isolate from peripheral blood mononuclear cells
(PBMC)
and plasma of an immunodeficiency virus. The present invention has utility in
determining the HN 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.
$ackground of the Invention
There is currently no cast 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 SN, respectively). These include assays that
detect the enzymatic activity of the reverse transcriptase (RT) protein, ELISA
based assays for the detection ofHN/SIV core antigen (HN-1 p24 or HN-21SIV
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 HIVISIV
infection. In this approach, mammalian cells are ,genetically modified to
express
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a reporter gene such as ~3-galactosidase ((3-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),
S microscopy (~i-gal), or the utilization of radioisotopes (CAT). The firefly
luciferase gene, under control of the HIV-l LTR promoter; has been used as a
reporter gene for HIV-I infection. Luciferase is very sensitive marker gene
for
a
HIV-1 infection, since expression of a relatively few number of luciferase
molecules can result in appreciable activity Ieve'.ls using standard
luciferase
detection assays.
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 CCRS
chemokine
co-receptor on the surface of such cell lines. The failure to detect infection
of
I S 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 Iirnitations.
Figure 1 is a schematic block diagram illustrating a generalized sequence
of steps in creating an assay for detecting and analyzing primary HIV.
Figures 2A-2E are schematics illustrating the; construction of various gene
transfer expression plasmids of the present invention.
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Figure 3 is a schematic illustrating the production of lentiviral transduction
vectors for the delivery of marker genes of the preser,~t invention. A gene
transfer
plasmid representatively including those shown in Figure 2 are separately
transfected into a host cell together with viral based packaging and envelope
plasmids.
Figures 4A and 4$ are graphs illustrating the relationship between the
concentration of vector and infectious units as determined with (3-gal, GFP
and
luciferase activity.
Figure 5 is a graph illustrating a nearly linear relationship between HIV-1
I O infectious units and luciferase activity for a cell line: of the present
invention.
Figure 6 is a graph illustrating the relationship between infectious virus
units and luciferase activity for viruses: TIVI, WIIbII, KIWE and YLJ2; using
a
cell Iine of the present invention.
Figure 7 is a graph illustrating a correlation between infectious virus units
and luciferase activity.
Figures 8A-8C 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.
Figures 9A-9C are graphs illustrating drug sensitivity to AZT for 100, 500,
and 2500 virus infectious units respectively, according to the present
invention.
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Figure 10 is a schematic illustrating the construction of a Tat transduction
plasmid.
Figures 11 A and 11 B are graphs illustrating the viral amplification two
days following infection with equal quantities of YU~2 HIV {a) for infectivity
and
S (b) p24 antigen.
Summary of t a Inventi~i
The present invention pertains to a cell-based assay for analyzing primary
HIV including an immortalized cell line that expresses the CCRS, CXCR4 and
CD4 receptors and a marker gene. The CCRS 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 irnmunodeficiency virus
infection
sensitive clonal cells, the method including selecting a cell line expressing
CCRS,
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.
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 fox determining
immunodeficiency
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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 drug
candidate. The
present invention affords the ability to test virus derived from blood plasma
as
well as cell culture.
5 D~S~ription of the Inventi~
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 CCRS in an immortalized cell
line significantly enhances the detection of primary IEiIV-1. The ability to
detect
primary isolates of HN-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 carnprise primary HIV-l; (2) the analysis of a significantly
greater
proportion of virus; and (3) high throughput testing, via miniaturization and
sampling of small sample volumes.
Most prior art molecular clones of HN-1 leave been derived by tissue
culture methods that select for viruses that do not require CCRS 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
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a macrophage by binding the CCRS 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 NL~43 (W. Paxton et aI. 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 IEiIV-i 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 CCRS chemokine co-receptor. Most HTV-2 and SIV
viruses also require the CCRS. Several groups l;uave produced cell lines that
express CD4, CXCR4 and CCRS 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, i 997).
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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
tlSSUeS.
As used herein, "immunodeficiency virus" is defined as various strains and
stocks of HIV-1, HIV-2, SIV and lentiviruses.
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.
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.
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."
By making genetic modifications to a CCRS 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:
Figure 1 is a schematic block diagram illustrating a generalized sequence
of steps in creating an assay for measuring HIV-1 ding 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 expressiion under the regulation
of an
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immunodeficiency virus 10. Preferably, the marker gene vector is placed under
the control of HIV-1 or HIV-2 long terminal rf;peats (LTRs) and the Rev
responsive element (RRE). The marker genes illustratively including (3-gal,
luciferase, GFP, CAT and other fluorescent proteins. Preferably, the marker
gene
S is Iuciferase. CD4 positive cells are then transduce;d with the vector in
order to
confirm appropriate marker gene expression from the transduction vectors 20.
Preferably, the cells are CD4, CCRS, CXCR4 positive. It is appreciated that an
amplicon gene is readily substituted far a marker gene to induce amplification
of
viral stocks (not shown). Preferably, the amplicon ;gene is Tat.
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 1 S
is
positive for CCRS, CD4 and CXCR4 receptors and optionally other receptors
illustratively including CCR3, CCR2B and 'i'-lymphocyte expressed 7
transmembrane domain receptor. Preferably, the origin cell Iine is HeLa. More
preferably, the cell line is JS3 (Oregon Health Sciences University) or a cell
line
that naturally expresses CD4, CCRS and CXCR4. T:he cells of immortalized cell
line 1S are then tested for sensitivity to HN-1 infectiion 2S. Expansion of
highly
sensitive cells to HN-1 infection 3S. The clone of 4~0 is used to transduce SO
the
highly sensitive immortalized cell line of 3S. Preferably, the receptor is
selected
to create a cell which is highly sensitive to iinfectiou 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
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gene product in the absence of HIV-1 60. 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.
Infection of the clones expressing low background levels of at least two
markers such as ~i-gaI and luciferase with HIV-1 confirms the relationship
between infectious viral units and marker gene (luci.ferase) activity f0,
although
in practice expression of a single marker gene is operative herein. Clones
that
relate infectious virus units, such as ~i-gal, with a second marker gene
activity such
as luciferase f nd 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 durin;; 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.
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.
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The present invention pertains to cell lines that are capable of detecting
sensitivity of a given strain of HN 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
5 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 i.s well
suited for
10 determining specific anti-retroviral drugs suited t:o contain replication
of the
various HIV stxains 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.
The present invention also provides a method! for detection of primary HN
from plasma, including noninfectious HIV-1 found in plasma. A loss of viral
infectivity is due in part to a loss of env or envblocking. VSV-G serves to
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mediate infection of HN-1 particles and thus, in the absence of VSV-G the
virus
remains noninfectious, whereas in its presence infe~~tivity is complemented.
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.
The methods and indicator cell lines of the present invention are operative
to analyze drug sensitivity of primary HIV which has been purifed and taken
directly from infected host plasma.
To directly analyze the relationship between luciferase expression and
infectious virus units, a series of gene transfer plasm.ids are constructed to
express
luciferase, (3-galactosidase ((3-gal), and green fluorescence protein (GFP),
respectively. (3-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).
Figures 2A through 2E illustrate the different gene transfer expression
plasrnids
that are constructed. The (i-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 HIV 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 iuciferase.
The J53BL cell line or its functional equivalents are operative in
accordance with the present invention: It is appreciated that the nucleic acid
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sequences coding for CCRS, CXCR4, CD4, lucii:erase, (3-galactosidase, GFP,
CAT, Tat and the J53 cell Iine 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 T>NA sequences which encode
substantially the same receptor amino acid sequences, cell line amino acid
sequences, marker gene sequences and amplicon :>equences 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
functionally 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 CCRS, CXCR4, CD4, luciferase, (3-
galactosidasc; GFP, CAT, Tat and J53 sequences iincluding altered sequences in
which functionally equivalent amino acid residues are substituted for residues
within the sequence resulting in a silent change. Fc>r 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
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other members of a class to which the amino acid belongs. For example, non-
polar {hydrophobic) amino acids include alanine, Ieucine, isoleucine, valine,
proline, phenylalanine, tryptophan and rnethionine;. Polar neutral amino acids
include glycine, serine, threonine, cysteine, tyrosine, asparagine and
glutamine.
S 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 l;he delivery of marker
genes.
In order to generate vector stocks for tra~nsduction with the different
reporter genes, the (3-gal, GFP and luciferase gene transfer plasmids,
including
those containing the HIV-2 LTR (shown in Figure 2), are separately transfected
1 S into cultures of 293T cell together with a lentiviral-based packaging
plasmid
(pCMV-GP1), and the pCMV-VSV-G env plasmid {Figure 3). Forty-eight hours
later, the vector-containing culture supernatants arf; 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
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expression. Two days after infection of the HIV-HeLa cells with the different
vector stocks, (3-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 luminomet~er. Figures 4A and 4B show
the relationship between concentration (HIV-1 p24 antigen, Coulter Inc.) of
the
vector stocks and infectious units as determined with (3-gal and GFP, {virus
infectious units) or luciferase activity.
Example 2 - Generation of (3-gal, luciferase and GF'P
indicator cell lines to quantify HIV/STV infection.
The following pairs of vector stocks (derived. as described above) are used
to co-transduce cultures of HeLa-CD4 cells: (a) pluf+ p(3-gal; (b) pluf +
pLTR2-
~i-gal, (c) pluf + pGFP, (d) pluf + pLTR2-GFP, (e) pLTR2-luf + phi-gal, (f)
pLTR2-luf + pLTR-2~3-gal, (g) pLTR2-luf + pGFP, (h) pLTR2-luf + pLTR2-GFP.
Three days later, the cells are biologically cloned b;y limiting dilution in
48 well
plates. Wells containing clonal cells {confirrried after initial plating by
microscopy) are expanded into replica cultures. One. replica culture set is
infected
with HIV-ilSG3 and analyzed for marker gene expression (HIV-1 infection
provided Tat and Rev to activate marker gene expression) as described above.
Expression positive cells cultures are identified, expanded and cryopreserved.
Since the expression of relatively few mol~;cules of luciferase produces
substantial luciferase activity levels, 36 non-HIV-1 infected, Iuciferase
expression-
positive clonal cultures (derived from HeLa-CD4 cells transduced with pluf+
p~3-
gal) are analyzed for luciferase activity to determine basal background
expression
levels. The HeLa-CD4 cells being obtained from the AIDS Research and
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Reference Reagent Repository of NIbI. Of the 3< clones analyzed, luciferase
activity ranged from IS to 250 units. Analysis for (3-gal expression in
response
to HIV-1 infection indicated approximately 70% of the clones expressed both ~i-
gal and luciferase. The two clones {referred to as H:eLa-~3-gal-lufl, and HeLa-
(3-
5 gal-luf2) that exhibited the lowest background levels of luciferase
expression and
are positive for (3-gal expression are used to directly analyze the
relationship
between HIV-I 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-(3-gal-lufl;
and
10 HeLa-(3-gal-luf2. After 48 hours, one set of cultures is analyzed for
luciferase
activity and the other was analyzed for [3-gal. Figure 5 shows the
relationship
between HIV-1 infectious units (~i-gal positive cells) and luciferase activity
for the
HeLa-(3-gal-lufl cell line. The HeLa-[i-gal-luf2 cell line gave nearly
identical
results with slightly higher luciferase activity levels at the lower virus
inoculums.
15 Between approximately 10 and I0,000 virus infectious units. A near-linear
relationship to luciferase activity is shown in Fil;ure 5. The linear range of
detection using the luciferase marker in Figure 5 is approximately 3 orders of
magnitude, and as few as 10-20 infected cells out c~f 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, IIJ such that
A=n{IC~'~" + 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
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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 ~i-gal and luciferase reporter genes.
The present invention utilizes a combination of a reporter assay system for
sensitively and rapidly quantifying infectious HN-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-CCRS positive J53 cell clone (Dr.
David
Kabat, Oregon Health Sciences University, Portland,'Oregon) with the pluf and
p(3-gal expression vectors as described above. The pluf and phi-gal transduced
J53
cells (termed J53-[igal/Iuf) are infected with six different virus isolates
(using four
five-fold serial dilutions) that were unable to efficiently infect other CD4,
CXCR4
1 S 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
YIJ2 clone, included as a control, are highly infectious in the J53(3-gal/luf
cell
line.
To assess the relationship between infectious virus units and luciferase
activity in the J53 ~i-gal/luf cell Iine, four serial five-i:old dilutions of
the following
viruses are prepared and analyzed: TIVI, WIMI, KTWE and YU2. Between
approximately 100 to 10,000 infectious units, the dsata show a linear
relationship
with luciferase activity (Figure 6). Background levels of luciferase are
between
100 and 1 S0. The J53~i-gal/luf cell line represents a transduced population
of cells
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since integration of the transduction vector into the genome of the J53 cells
can
occur differently in each cell.
To minimize luciferase background levels of nori 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(3-gal/luf cell line as
described above and characterized for luf and (3-l;al 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-C 13, is. confirmed to
express
both luciferase and ~i-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. Eaclh stock is analyzed for HIV-
1
p24 antigen concentration, SI and NSI phenotype, and infectivity in HeLa-CD4
{MAGI), HeLa-CD4-CCRS (P4), H9 CD4-CCRS (HiS), and HeLa-CD4-CCRS
(JS3-C13) cells. These results are summarized in Table 2.
These results show that the J53-C13 cell line is sensitive to primary HIV-1.
Importantly, the JS3-C13 cell line is sensitive to HIV-1 infection to a degree
similar to PBMC. To confirm the importance of the CCRS 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 CCRS. JC11 is transduced to express b-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 bothb-gal and
designated
J11-C5, which is capable of expressing both b-gal and luciferase, is selected
for
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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-Ca3 and J11-CS cell lines.
The
results show a marked reduction in virus titer in the J11-CS cell line,
indicating
S ' that the CCRS co-receptor is necessary for efficient infection/detection
of primary
virus isolates.
To analyze the relationship between luciferase and ~i-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-C 13 cells. After two days
the
number of ~i-gal positive cells and luciferase activity is determined. Figure
7
shows a strong correlation (r=0.92) between (3-gal positivity (infectious
virus
units) and luciferase activity over 2 orders of rnag~nitude.
Example 4 - Evaluation of primary HIV-1 isolates for drug
sensitivity/resistance using the J53~3-l;al/luf {J51-C13) cell line.
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 SARO 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.Nevarip:ine (NVP), respectively. Two
days after infection the cells are lysed and the claxii:ied lysates are
examined for
luciferase activity using standard methods (Prome~;a). Figure 8 shows the
effect
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of different concentrations of drug on virus replication relative to non-drug
treated
viruses - as determined by luciferase activity as an indicator.
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 SO% 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.
Figure 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.
The amplification of primary virus from infected individuals is required
for phenotypic resistance in assays that test wholle 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-C I3
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 Figure 10. To eliminate Tat transactivation
of the lentivirus vector LTR; Tat is constructed into a self deleting U3
transduction vector, Figure 10. Three days after transductian, single cells
are
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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~.O1. After 40 hrs. virus production
5 is measured by HIV-1 p24 antigen ELISA and the highest HIV-1 producing Iines
from each are selected for further analysis. T:he highest HIV-1 producer,
designated J53-CMVtat 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
10 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/CCRS/CXCR4 + Tat expressing
cell line to capture and amplify prirnaxy virus.
The J53tat cell line is compared with PBMC for primary virus
15 amplification. PBMC and J53tat are each infected with 2.SE5 infective
particles
- of YLJ2. Two days later the concentration of progeny virus is analyzed for
infectivity in J53BL indicator cells as shown in Fil;ure 1 I(a) and by p24
antigen
ELISA, as shown in Figure I I(b). The J53tat cell lane amplifies primary virus
to
higher titers and more rapidly than PBMC. Since i;he parental J53BL cell line
is
20 highly sensitive to primary virus, Tat facilitates the rapid generation of
high titered
primary virus stocks for resistance testing without sc;lection of longer term
culture,
such as PBMC culture for virus amplification.
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Example 7 -Detection of drug resistancelsensitivity
that effect various stages of virus life c;ycle.
The JS3tat cell line is used to produce virus arid thereby enable viral
testing
of drug candidates that affect various stages of the vinzs life cycle: Thus,
viral
S 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 JS3tat 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 JS3BL cell line in the
presence of the same drug concentrations. Table 6 slhows that YU2 is sensitive
to
protease inhibitor, with increasing concentrations c;ausing greater
inhibition.
Example 8 - Detection of noninfectious cultured vimzs.
To test how to recover noninfectious virus, a molecular clone is generated
1 S to produce env minus HIV-1 (pSG3-envy. 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 ultracentrifugedl for 1.S hours at 115,000g
at
4°C. The pellet is resuspended in 100 ul DME;M. The infectivity is then
determined using JS3BL cells. The infectivity is determined to be 7.SE4.
Without
mixing of VSV-G the infectivity is 0.
YU2 virus containing wild-type envelope is pelleted through sucrose by
ultracentrifugation to strip away the gp 120 glycopr~atein (SU). The
resuspended
(100 ul) virus is mixed with and without VSG-G (1:1) and repelleted by
ultracentrifugation (ISO,OOOg, 2 hours, 4°C). The pellets are
resuspended in 100
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22
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 vines.
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. UVithout 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./
CCRS/CXCR4 or CD4/CCRS/CXCR.4 + Tat
expressing cell line to capture and amplify primary virus.
Plasma from patients infected with HIV-1 is tested fox 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 ~i-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.
HIV is incubated with J53BL cell line for four hours to allow binding
and entry into J53BL cells, reverse transcription proceeds and the viral eDNA
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
III
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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 purif ed 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.
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 modiifications are intended
to fall
within the scope of the appended claims.
All references cited herein are intended to be incorporated to the same
extent as if each reference was individually incorporated by reference.
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Table 1. Efficient HIV-1 infection of a C:D4
CCRS expressing HeLa cell line.
HIV-I isolatesp24 pgiml PBMC,'b HeLa-CD4-
CCRS
KEWI 2.64E+05 4.58E+p4 2.30E+05
WIMI 1.31E+05. 1.15E+04 8.10E+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+04
a HTV-1 isolates were derived by coculture (7-10 days) of HIV-1
infected patient PBMC with PHA stimulate;d normal donor BPMC.
b Virus titer - determined by endpoint dilution titration in PBMC,
calculated by the Spea~iman-Karber Formula (TC117 50/ml)
c Virus titer - determined by counting the :number of virus infected
(blue) HeLa-CD4-CCRS-[3-gal indicator cells (J53-C16).
SUBSTITUTE SWEET (RULE 26)
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SUBSTITUTE SHEET (RILE .26)
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Table 3. RT resistance confemin~ mutations
YU2 SAlltO LEMI
M41 L
AZT D6'7N D67N
K70R
T215Y
K219Q
3TC M 184V
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Table 4. CCRS facilitates infection
of primary isolates of HIV-1
Virus Stock J53-C13 titer JI1-CS titer
DBP D-7 6.3()E+04 Neg.
DBP d140 1.20E+04 4.OOE+01
HVH D-7 6.10E+p4 l.lOE+04
HVH D140 6.80E+04 1.00E+03
SHL D-7 6.OOE+04 Neg.
SHL D140 7.70E+04 Neg.
TED D 127 I .30E+OS Neg.
TED D2-11 3.20E+04 Neg.
XHB2 1.75E+OS Neg.
YLJ2 4.85E+05 7.SOE+04
89.6 1.40E+05 Neg.
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.
SUBSTITUTE SHEET (RULE 26)
i;,
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Table S. Traps Tat expression enhances HIV-1 production '
Virus JC53 JC53-CMVtat
Exp 1. Exp. 2 Ex:p. 1 Exp. 2
YU2 1240 920 6430 4910
KEWI 3120 2760 9:190 2590
Nos. represent pg of p24 antigen per ml.
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Table,6. Number of colony formed in the
presence of protease Tnhibitor Indinavir
INDINAViR
0 MM 0.008 MM 0.04 0.2 MM I .0 MM
DrIM
0.2 M.O.I> 1000 > 1000 34:3 99 0
0.04 M.O.I198 I05 2 2 0
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Table 7
Conditions Recovery (%)
No sucrose, no VSV-G 0.006
Sucrose, no VSV-G .
Sucrose, VSV-G 20
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i,
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Table 8. Detection/IsoIation of HIV-1 from human
plasma using J53BL cells
Plasma virus TCIU/PBMC TCIU/J53 BL
LEMI 3.47 x 103/m 4.40 x 103/ml
ALPI 3.47 x 1031m 1.20 x 104/m1
GADA 7.81 x 1031mI 1.20 x 104/m1
TCIU = tissue culture infectious units
SUBS'1~T'UTE SH~E'1' (RiIL~ 2Ei)
