Note: Descriptions are shown in the official language in which they were submitted.
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TRANSGENIC RABBITS EXPRESSING CD4 AND CHEMOKINE RECEPTOR
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. provisional
application number 60/050,480 filed June 23, 1997, which is
hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Entry of HIV into target cells requires cell-surface
CD4 and additional host cell cofactors, such as CCRS, for
primary macrophage-tropic strains of HIV (Deng et al. Nature
381:661-666 (1996)) and CXCR4 for T cell tropic isolates (Feng
et al. Science 272:872-877 (1996)).
In the HIV field, two groups recently reported
efforts to develop transgenic rabbits as models of HIV
disease, both involving introduction of human CD4 (Dunn, C. S.
et al., J. Gen. Virol. 76(Pt 6):1327-1336 (1995); Gillespie,
F. P. et al., Mol. Cell Biol. 13:2952-2958 (1993); Leno, M. et
al., Virolocrv 213:450-454 (1995)). These studies confirmed
that rabbit PBMC expressing human CD4 were more susceptible to
infection by HIV-1 than were their normal counterparts.
The references discussed herein are provided solely
for their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention.
SUMMARY OF THE INVENTION
One aspect of the invention is a transgenic rabbit
or rabbit cell expressing a human chemokine receptor and human
CD4.
A further aspect of the invention is a method of
generating a transgenic rabbit or rabbit cell comprising
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(a) introducing a transgene comprising a human chemokine
receptor into a fertilized rabbit pronucleus;
(b) introducing a transgene comprising human CD4 into a
fertilized rabbit pronucleus;
(c) implanting the product of (a) into the oviduct of a
pseudopregnant rabbit;
{d) implanting the product of (b) into the oviduct of a
pseudopregnant rabbit;
(e) obtaining litters of pups from the product of (c) and the
product of (d), wherein at least one pup from each litter is
transgenic for the transgene of (a) or (b); and
(f) breeding the transgenic pups of each litter of (e) to each
other to obtain a rabbit transgenic for both transgenes.
A further aspect of the invention is a method of
generating a transgenic rabbit or rabbit cell comprising
(a) introducing a transgene comprising a human chemokine
receptor and a transgene comprising human CD4 into a
fertilized rabbit pronucleus;
(b) implanting the product of (a) into the oviduct of a
pseudopregnant rabbit; and
(c) obtaining a litter of pups from the product of (b),
wherein at least one pup from the litter is transgenic for
both transgenes.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Distinct coreceptor utilization maps to
individual amino acids within the V3 hypervariable loop of
gp120. Coreceptor utilization assessed by the
transfection-infection assay was determined for a panel of
matched chimeric HIV-1 variants with similar V3 regions in the
backbone of NL4-3. Cellular tropism (monocyte-derived
macrophages and HeLa cells) and syncytium-inducing (SI) versus
non-syncytium-inducing (NSI) phenotype determined by MT-2
co-cultivation assay are described in detail elsewhere and are
summarized here for reference. All HIV-1 variants grew well
in PBMC.
Figure 2. Enhanced HIV-1 infection of CHO cells in
the presence of human chromosome 12. Parental CHO and CHO-12
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(carrying chromosome 12) cells were transiently transfected
with vectors encoding human CD4 and human CCR5 were analyzed
for intracellular expression of p24 after culturing with or
' without HIV-1 BaL. Cells positive for both CD4 and p24
(indicated by the box marker) were markedly increased in the
' CHO-12 cells.
Figure 3. Transfected rabbit SIRC cells are
permissive for infection by specific steps in the HIV viral
life cycle. In Figure 3A, the SIRC cells were transfected
with plasmids encoding CD4 with or without HIV-1 Nef and the
relative cell surface expression of CD4 was measured by FACS.
SIRC cells, NIH-3T3 cells, and HeLa cells were co-transfected
by an LTR reporter (CAT) construct with and without a plasmid
encoding HIV-1 Tat, and the results shown in Figure 3B. SIRC
cells, NIH-3T3 cells, and HeLa cells also were co-transfected
by a Rev-dependent reporter (CAT) construct with and without a
plasmid encoding HIV-1 Rev, and the results are shown in
Figure 3C.
Figure 4. Total cellular mRNA was extracted from
HeLa and SIRC cell lines expressing human CD4 and human CCRS,
which cell lines were cultured in the presence or absence of
HIV-1 YU-2, and the mRNA analyzed for the presence of
unspliced (9KB) and partially spliced (4KB)viral mRNA species.
The results are shown in Figure 4.
Figure 5. Transfected rabbit SIRC cells are
permissive for infection by HIV-1. The indicated cell lines
were stained for intracellular p24 and assessed by FACS after
culturing with or without HIV-1 BaL.
Figure 6. Marked cytopathic effects and syncytium
formation are evident in transfected rabbit cell cultures
exposed to HIV-1. Cells expressing human CD4 without CCR5
' showed no histologic effects upon culturing with HIV-1 BaL,
while cells expressing both CD4 and CCR5 were destroyed and
formed frequent multinucleated giant cells upon infection with
BaL.
Figure 7. HeLa cells, SIRC cells, and NIH-3T3 cells
were inoculated with various strains of HIV-1 (BaL, YU-2, JR-
CSF and NL4-3) and the culture supernatants were tested for
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p24 content by an ELISA. Open bars represent cells encoding
human CD4 and solid bars represent cells encoding both CD4 and
CCRS.
Figure 8. The BaL and YU-2 culture supernatants
from Figure 7 were serially transferred onto PHA-blasted human
PBMC and the p24 content measured by an ELISA. As shown in
Figure 8, SIRC-CD4-CCR5 cells produced functional virions.
Figure 9. Primary rabbit peripheral blood
lymphocytes were isolated and transfected with HIV clone YU-2.
Culture supernatants were harvested and used to inoculate PHA-
blasted human PBMC. The p24 content was measured by an ELISA,
and the results shown in Figure 9.
DETAILED DESCRIPTION
Generally, the nomenclature used hereafter and the
laboratory procedures in cell culture, molecular genetics, and
nucleic acid chemistry and hybridization described below are
those well known and commonly employed in the art. Standard
techniques are used for recombinant nucleic acid methods,
polynucleotide synthesis, cell culture, and transgene
incorporation (e. g., electroporation, microinjection,
lipofection). Generally enzymatic reactions, oligonucleotide
synthesis, and purification steps are performed according to
the manufacturer's specifications. The techniques and
procedures are generally performed according to conventional
methods in the art and various general references which are
provided throughout this document. The procedures therein are
believed to be well known in the art and are provided for the
convenience of the reader. All the information contained
therein is incorporated herein by reference.
Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Although any methods and materials similar
or equivalent to those described herein can be used in the
practice or testing of the present invention, the preferred
methods and materials are described. For purposes of the
present invention, the following terms are defined below.
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The term "corresponds to" is used herein to mean
that a polynucleotide sequence is homologous (i.e., is
identical, not strictly evolutionarily related) to all or a
. portion of a reference polynucleotide sequence, or that a
5 polypeptide sequence is identical to a reference polypeptide
. sequence. In contradistinction, the term "complementary to"
is used herein to mean that the complementary sequence is
homologous to all or a portion of a reference polynucleotide
sequence. For illustration, the nucleotide sequence "TATAC"
corresponds to a reference sequence "TATAC" and is
complementary to a reference sequence "GTATA".
The terms "substantially corresponds to",
"substantially homologous", or "substantial identity" as used
herein denotes a characteristic of a nucleic acid sequence,
wherein a nucleic acid sequence has at least about 70 percent
sequence identity as compared to a reference sequence,
typically at least about 85 percent sequence identity, and
preferably at least about 95 percent sequence identity as
compared to a reference sequence. The percentage of sequence
identity is calculated excluding small deletions or additions
which total less than 25 percent of the reference sequence.
The reference sequence may be a subset of a larger sequence,
such as a portion of a gene or flanking sequence, or a
repetitive portion of a chromosome. However, the reference
sequence is at least 18 nucleotides long, typically at least
about 30 nucleotides long, and preferably at least about 50 to
100 nucleotides long. "Substantially complementary" as used
herein refers to a sequence that is complementary to a
sequence that substantially corresponds to a reference
sequence.
As used herein, a "heterologous gene" or
"heterologous CD4" is defined in relation to the transgenic
nonhuman organism producing such a gene product. A
heterologous polypeptide, also referred to as a xenogeneic -
polypeptide, is defined as a polypeptide having an amino acid
sequence or an encoding DNA sequence corresponding to that of
a cognate gene found in an organism not consisting of the
transgenic nonhuman animal. Thus, a transgenic mouse
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harboring a human CD4 gene can be described as harboring a
heterologous lymphocyte transduction gene. A transgene
containing various gene segments encoding a heterologous
protein sequence may be readily identified, e.g. by
hybridization or DNA sequencing, as being from a species of
organism other than the transgenic animal. For example,
expression of human CD4 amino acid sequences may be detected
in the transgenic nonhuman animals of the invention with
antibodies specific for human CD4 epitopes encoded by human
CD4 gene segments. A cognate heterologous gene refers to a
corresponding gene from another species; thus, if murine CD4
is the reference, human CD4 is a cognate heterologous gene (as
is porcine, ovine, or rat CD4, along with CD4 genes from other
species).
Oligonucleotides can be synthesized, for example, on
an Applied Bio Systems oligonucleotide synthesizer according
to specifications provided by the manufacturer.
In general, transgenic rabbits can be produced using
the techniques of, for example Snyder et al. (Mol. Rep. Dev.
40:419-28 (1995)), Fan et al. (Proc. Natl. Acad. Sci. U.S.A.
91:8724-8728 (1994)), and Taylor et al. {Frontiers in
Bioscience 2:d298-308 (1997)). Specific pathogen-free {SPF)
New Zealand White rabbits are preferably used. They are
preferably housed in an SPF barrier facility for at least 2
weeks before use. Typically, embryos from 5-6 female donors
are collected for microinjection to enable 2-3 recipients to
be implanted, and approximately 6 microinjection days are
planned per construct to achieve at least 3 founder animals.
The gestation period is 30 days and the average litter size
from implanted recipients is typically 4-6 pups (the normal
nontransgenic litter size is 8 pups). In the fifth week the
pups are screened with DNA isolated from an ear biopsy. In a
typical experiment approximately 10% of the recipients pups
are transgene-positive. They are typically ready to mate -
after 5 months.
In some embodiments, transgenic rabbits are
generated as follows. For superovulation, 50 units of
pregnant mare serum (PMS) is injected i.v. to each of 5
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potential embryo donors at 5 months of age, and 4 days later
50 units of human chromic gonadotropin are injected i.v. At
that time, the embryo donors are mated with a proven fertile
male. On the following morning, the donors are euthanized
with sodium pentobarbital, and the oviducts are flushed with
sterile culture medium to recover embryos: an average of 15-
20 embryos per donor are obtained. Fertilized embryos are
microinjected with a DNA solution containing the construct of
interest, then they are incubated for 2-3 h to monitor
survival. The microinjected embryos are implanted through the
infundibulum into the oviducts of a recipient that had been
mated with a vasectomized male in the previous day.
Typically, 2-3 recipients are prepared per experiment for
implanting 10-20 embryos in each oviduct, and at least 8
recipients (usually requiring 4 or more microinjection days,
depending upon embryo yield) are planned per construct.
Implanted recipients are placed on a warm blanket until they
have recovered from anesthesia. They are monitored daily
during subsequent housing: in the fourth week, each pregnant
recipient is provided with a nesting box having breeder
bedding. Compared to the mouse, rabbit embryos have twice the
diameter, a tougher and thicker cell membrane layer, a
slightly more granular cytosol, and similar-sized pronuclei.
Thus, preferably, twofold greater DNA concentrations are used
at higher purity for rabbit embryo microinjection than for the
mouse.
To expand each line, founders are mated with
nontransgenic animals or transgenic animals carrying other
transgenes. Males allow a more rapid expansion of the line to
F1 pups than females; therefore, F1 males are mated to several
nontransgenic females whenever possible to increase the number
of hemizygotes. To establish homozygous F2 lines for study,
hemizygous F1 males are cross-bred with F1 females. Candidate
F2 rabbits are tested by backcrossing against nontransgenic
animals. Proven homozygous F2 males and females are mated to
maintain an F2 line.
Embryos from independent transgenic rabbit lines are
typically frozen for long-term storage and 2-3 males and one
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female are maintained as breeder stock for each construct
line. At least 150 embryos are collected and placed in 1.5 M
glycerol in microtubes. The tubes are cooled quickly to -7°C,
cooled further to -35°C at 0.5°C/min, then plunged into liquid
nitrogen. Embryos are stored in two separate liquid nitrogen
tanks.
Rabbits are quite sensitive to noise, excessive room
activity, and improper handling; and breeding efficiency, as
well as recovery from surgery, can be reduced significantly in
animals harboring pathogens. When rabbits are stressed, they
readily absorb or abort fetuses and often ignore newborn pups.
The use of SPF rabbits in barrier facility having restricted
access, a nursery for pregnant and nursing females, and a
temporary holding room to quarantine recently shipped rabbits
while their health status is checked minimizes these problems.
Genotyping is performed on DNA extracted from ear
punch samples as described above. Since typically up to 25%
of transgene-positive founders fail to express the desired
protein, preferably 3 independent founder animals (FO) for
each transgene construct are obtained in order to achieve 2
expressors. These transgene-positive FO animals are mated at
sexual maturity directly with human CD4 transgenic rabbits to
produce F1 animals carrying both transgenes at an expected
rate of 25% of the pups (based on Mendelian transmission).
Transmission of the CD4 gene in the stable transgenic rabbit
line is monitored by flow cytometry of PBMC isolated from
peripheral blood samples obtained by ear venipuncture.
Transgene expression patterns are assessed by flow
cytometry and immunohistochemistry with a thorough survey of
hematolymphoid (thymus, lymph nodes, spleen, peripheral blood
lymphocytes and monocytes, peritoneal macrophages) and
non-hematopoietic (all major organs, including the central
nervous system) tissues; anti-CCR5 and anti-CXCR4 monoclonal
antibodies for this purpose are now available from several
sources. Animal lines with tissue-appropriate expression are
expanded for studies with HIV-1.
In some embodiments of the invention, rabbit cells
expressing human CD4 and human CCR5 or CXCR4 are provided.
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These cells can be engineered by conventional transfection to
express human CD4 and human CCRS or CXCR4 or may be
freshly-isolated primary rabbit lymphocytes from the
transgenic rabbits of the invention. The transgene DNA used
for the construction of transfected cells or transgenic
animals may comprise cDNA or human genomic DNA. If cDNA, a
transgene is typically provided as a construct in which the
transgene is placed under the control of a heterologous
promoter with other appropriate elements for expression, such
an enhancer sequences. The coding sequences for CD4, CCRS,
CCR3, CXCR4, and other chemokine receptors are well
characterized in the art. Expression constructs are well
known in the art and are exemplified in the Experimental
Examples. If genomic DNA, typically the transgene is provided
as part of a genomic DNA clone, such as in a P1 or BAC clone.
Such clones typically provide the transgene under the control
of regulatory elements native to the transgene.
The CD4 and chemokine receptor transgene of choice
can be separately used to generate transgenic rabbits, from
which progeny transgenic rabbits can be mated to generate
rabbits doubly transgenic, i.e., transgenic for both CD4 and a
chemokine receptor such as CCRS. In some embodiments, both
transgenes are introduced to the same embryo or host cell,
either as parts of separate DNA molecules or as part of the
same DNA molecule.
Viral gene expression and production of infectious
virions in these cells can be, for example, measured as
follows. Cultures are inoculated with several doses of
infectious virus (quantitated by conventional endpoint
dilution/TCID50 analysis) of representative strains (e. g.,
BaL, NL4-3, YU-2, ADA) and successful infection/expression is
quantitated simultaneously by: (1) intracellular staining for
p24 expression and FAGS to determine the proportion of
infected cells at each dose; (2) conventional quantitative -
ELISA of secreted p24 to measure gene expression; and (3)
quantitation of infectious virion production by harvesting
supernatants and performing reinfection endpoint analyses on
both the human and rabbit cell lines. These experiments
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provide quantitative comparisons of the permissivity of these
cells for the replication of HIV-1, which is a key determinant
of viral spread in vivo.
Tat and Rev functions are assessed directly and
5 quantitatively in rabbit cells and compared with these
functions in human and murine cells. Rev function is assessed
by an established S1 nuclease assay in which Rev-defective
proviruses are introduced into the target cells in the
presence or absence of wild type Rev; the spliced and
10 unspliced transcripts are then measured by nuclease protection
using HIV-1-specific DNA probes (Malim, M. et al., Mol. Cell.
Biol. 13:6180-6189 (1993)). Tat function is assessed by an
established method involving cotransfection of an HIV-1-LTR
construct (linked to a reporter gene) with or without a Tat
expression vector (Newstein, M. et al., J Virol. 64:4565-4567
(1990)). The downregulation of cell surface CD4 by Nef in
primary human cells is measured using FACS in transfection
studies. In addition, the recognized infectivity enhancement
activity of Nef is measured in rabbit cells through the use of
Nef-defective viruses and trans-complementation by Nef
protein. It may also be of interest to assess other auxiliary
functions within these cellular contexts, such as the cell
cycle arrest induced by Vpr (assessed by transfection studies
employing fluorescent probes of DNA content).
In some embodiments of the invention, transgenic
rabbits are provided that express human CD4 and selected human
chemokine receptors (CCR5 and CXCR4, alone and in combination)
selectively in tissues that reflect the expression pattern in
humans.
To achieve a tissue distribution of expression
representing that found in nature for human chemokine
receptors, we plan to use genomic constructs containing the
appropriate endogenous signals for expression. Preferably,
bacteriophage P1 or BAC human genomic clones are used to
encompass the necessary elements. Candidate clones are
obtained from a commercial vendor (for example, Genome
Systems, Inc.,) and analyzed by a combination of conventional
restriction mapping, PCR and Southern hybridization to
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identify suitable clones. One some embodiments, a CCR5 clone
that contains the CCR3 gene as well (also lying within the
3p21 chromosomal region), is used since this receptor has been
implicated in tropism of HIV-1 for the central nervous system.
The increasing availability of several anti-CCRS and
anti-CXCR4 antibodies now makes it possible to assess surface
expression of native, untagged proteins expressed from these
constructs.
The permissivity of primary immune cells from
transgenic rabbits to infection and cytopathicity by various
strains of HIV-1 in vitro can be evaluated as follows, as can
the infection pattern and pathogenicity upon inoculation of
these animals with various strains of HIV-1 in vivo. For
example, PBMC are isolated from CCR5+/CD4+ animals,
CXCR4+/CD4+, CD4+, and non-transgenic animals by venipuncture
and conventional purification using density centrifugation
with Ficoll-Hypaque; initial work will focus on the highest
expressor lines for each transgene. Both blasted (treated
with phytohemagglutin in for 24 hrs.) and non-activated cells
are cultured in recombinant human interleukin-2 (IL-2). For
some studies mixed mononuclear cell populations are used, and
in other experiments cells first are separated into
subpopulations enriched for CD4+ lymphocytes (by established
methods with magnetic beads coated with anti-rabbit CD4
antibodies), CD8+ lymphocytes (by magnetic beads coated with
anti-rabbit CD4 antibodies), or monocytes (by adherence to
plastic). Peritoneal monocyte-derived macrophages are
harvested for infection studies by intraperitoneal injection
of thioglycollate followed several days later by lavaging of
the abdominal cavity with sterile saline. Thymocytes are
harvested by surgical isolation of the thymus followed by
mechanical dispersion of the cells and density gradient
centrifugation. Following each method of cell extraction,
flow cytometry is performed with anti-CD4, anti-CD8 or
anti-CDllb (macrophage) antibodies (and others as needed) to
determine the purity of the cell fractions.
All virus stocks used for in vitro infections are
preferably titrated by endpoint dilution on human activated
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PBMC. Viral infection and spread are preferably monitored by
several methods such as (1) visual inspection of the cultures
via microscope for cytopathic effects and syncytium formation;
(2) quantitative ELISA of secreted p24 antigen in culture
medium harvested serially (every 2 days) over a 21-day period
(may be narrowed or expanded as needed); and (3) intracellular
staining for p24 antigen after fixing and permeabilizing the
cells. Cells are also preferably monitored for downregulation
of surface CD4 expression (rabbit and human CD4), which
typically accompanies intracellular expression of viral gp160,
Nef and other proteins. In other experiments viruses are
serially passaged on transgenic rabbit PBMC to verify that
viral infectivity is preserved. Experiments will also be
performed to test the efficacy of selected human chemokines at
various concentrations (MIP-la, MIP-1 and RANTES for CCRS;
SDF-1 for CXCR4) to suppress viral spread in these cultures,
since this may represent another important characteristic and
disease determinant during typical human infections. A range
of virus strains is used in these studies.
One of ordinary skill in the art would appreciate
that the initial choices of viruses to be used in vivo is
driven by the results of these studies in cell culture, and
that a representative CCR5-dependent virus and a
representative CXCR4-dependent virus are used in the early
studies.
In vivo infections are followed in several ways.
Seroconversion is monitored by serial collection of small
peripheral blood samples (50-200 ~1, every two weeks
initially) and analysis using commercially available HIV-1
antibody assays adapted for rabbit serum by use of
anti-rabbit-IgG antisera as the secondary antibody.
Successful infection is expected to result in the development
of a detectable humoral response to HIV-1 within 3-4 weeks.
Detection of proviral DNA in lysed PBMCs and tissues is
performed using DNA PCR of the highly conserved HIV-1 gag p26
segment and quantified using serial dilution, or if greater
precision is required, by inclusion of an internal DNA
template competitor. The PCR conditions for HIV gag p26
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amplification have been determined to detect reliably the
presence of 10 DNA copies per reaction. The small volumes of
plasma available from rabbits precludes the use of
standardized HIV-1 RNA load assays in their approved format.
Nevertheless, the Chiron HIV-1 RNA bDNA assay is being adapted
for use with small volumes (50 ~1 and 200 ~1) to facilitate
studies of human infants and small animals. An ultrasensitive
HIV-1 RNA QC-PCR assay, adapted from previously reported
assays (Piatak, M. J. et al., Science 259:1749-1754 (1993);
Piatak, M. J. et al. Biotechniques 14:70-81 (1993)), is also
available if HIV-1 RNA levels fall below the limit of
detection of standardized assays. Methods of RNA extraction
based on RNA binding to activated silica are used with rabbit
tissues to assure purification of viral RNA from contaminating
substances that may inhibit PCR. Infectious virus from HIV-1
infected rabbits is obtained by cocultivation of primary PBMCs
from infected rabbits with uninfected human donor cells
readily available from the Irwin Memorial Blood Bank in San
Francisco. HIV-1 cocultivation is performed quantitatively or
qualitatively as needed. In addition, virus is transferred
from one animal to another as definitive evidence of
productive infection in vivo.
In addition to monitoring viral replication and
spread, the effects of viral infection on the animals can also
be assessed in several ways. First, the potential loss of CD4
cells in the periphery is measured by flow cytometry to
measure absolute and relative levels of CD4+ and CD8+ T
lymphocytes. Second, general hematopoietic parameters are
followed to detect common cytopenias associated with HIV
disease. Third, representative animals demonstrating robust
infections with detectable changes in peripheral lymphocyte
counts are sacrificed and subjected to thorough postmortem
examinations and immuno-histochemistry of major hematolymphoid
organs (e.g., thymus, spleen, bone marrow and lymph nodes) -
with anti-HIV antibodies to detect viral infection and
disruptions in tissue architecture or cell morphology. These
studies together provide a substantial knowledge base about
the course of HIV infection in these animals, the degree of
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viral production and spread, and the extent of disease
pathogenesis.
As will be evident to one of skill in the art, the
transgenic rabbits and cells of the invention are especially
useful as animal models of HIV infection and in the screening
of anti-HIV pharmaceuticals.
The following examples are provided for illustration
only and are not intended to limit the claims in any way.
EXPERIMENTAL EXAMPLES
EXAMPLE 1. Structure/function analyses of HIV coreceptors
A transient transfection/infection assay system was
developed that is useful for distinguishing between
permissivity and nonpermissivity to infection by HIV-1. Using
this assay we found that the murine form of CCRS (Boring, L.
et al., J. Biol. Chem. 271:7551-7558 (1996)) exhibited
virtually no detectable capacity to support infection by
macrophage-tropic HIV-1, defining as least one key basis for
the failure of transgenic mice expressing human CD4 to serve
as permissive hosts for HIV (see for example, (Lores, P. et
al., AIDS Res and Human Retroviruses. 8:2063-2071 (1992)). To
identify the critical elements lacking in the nonfunctional
murine form of CCR5, chimeric human/mouse CCR5 receptors were
prepared and evaluated for HIV-1 coreceptor function.
Extensive experiments with selective substitutions
demonstrated that multiple elements distributed throughout the
extracellular segments contribute to viral entry. Similar
observations recently have been reported by others (Bieniasz,
P. et al., EMBO J. (1997)). Further studies with chimeric
receptors revealed that viral coreceptor activity is
dissociable from ligand-dependent signaling responses
(Atchison, R. E. et al., Science 274:1924-1926 (1996)).
Additional studies involved mutating human CCR5 within the
highly conserved aspartate-arginine-tyrosine (DRY) sequence -
that is thought to be critical for G-protein coupling.
Despite its failure to induce chemotaxis or generate second
messengers, this mutant remained a potent coreceptor for HIV-1
internalization. Similar findings were reported recently by
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others (Farzan, M. et al., J. Biol. Chem. 272:6854-6857
(1997)). These findings indicate that signal transduction is
not a component of the viral entry mechanism.
5 EXAMPLE 2 Viral determinants of tropism and coreceptor
utilization
CCRS mediates viral entry into macrophages, whereas
CXCR4 mediates entry into many CD4-positive transformed T-cell
lines. Although virtually all primary HIV-1 isolates
10 replicate in primary CD4-positive T-lymphocytes,~certain
variants ("macrophage-tropic ) fail to infect transformed
T-cell lines, whereas other strains ("T-cell tropic )
replicate well in these cell lines but not in macrophages.
Changes in cellular tropism by HIV-1 strains seem to be a key
15 event in the pathogenesis of HIV-1 disease (Gouilleux, F. et
al., EMBO J. 14:2005-2013 (1995); Koot, M. et al., J. Infect.
Dis. 173:349-354 (1996); Richman, D. D. et al., J. Infect.
Dis. 169:968-974 (1994); Schuitemaker, H. et al., J. Virol.
66:1354-1360 (1992); Tersmette, M. et al., J. Virol.
62:2026-2032 (1988); Tersmette, M. et al., J. Virol.
63:2118-2125 (1989)). Amino acids in the hypervariable V3
region of the HIV-1 envelope glycoprotein 120 (gp120) have
been shown to influence these effects (Cann, A. J. et al., J.
Virol. 66:305-309 (1992); Chesebro, B. et al., J. Virol.
70:9055-9059 (1996); De Jong, J.J. et al., J. Virol.
66:6777-6780 (1992); Fouchier, R.A.M. et al., J. Virol.
66:3183-3187 (1992); Freed, E.O. et al., J. Biol. Chem.
270:23883-23886 (1995); Hwang, S.S. et al., Science 71:71-74
(1991); Stamatatos, L. et al., J. Virol. 67:5635-5639 (1993)).
Two recent reports using chimeric viruses indicated that the
gp120 V3 loop can influence the ability of HIV-1 variants to
use different chemokine receptors (Choe, H. et al., Cell
85:1135-1148 (1996); Cocchi, F. et al., Nature Medicine
2:1244-1247 (1996)). Concurrently we evaluated in our -
transfection-infection system chimeric viruses containing the
V1-V3 region derived from NL4-3 in the backbone of JR-CSF
[JR-CSF(NL4-3)] or the V3 loop of JR-CSF in the backbone of
NL4-3 [NL4-3(JR-CSF)]. Both chimeric viruses selectively used
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the chemokine receptor for cell entry according to the origin
of their V3 loop inserts: JR-CSF(NL4-3) preferentially used
CXCR4 rather than CCR5, whereas NL4-3(JR-CSF) used
preferentially CCR5 and not CXCR4. Hence, these data
substantiate the important role of the V3 loop in determining
coreceptor use by a given HIV-1 strain. To investigate the
role of individual amino acids within the V3 loop, we studied
the infectivity pattern of a panel of recombinant HIV-1
variants containing highly related V3 loop sequences from two
primary isolates with either T-cell or macrophage (JR-CSF)
tropism. These sequences were created by site-directed
mutagenesis of individual amino acids in the V3 loop within
the genomic background of NL4-3. These variants differ in
their ability to infect primary monocyte-derived macrophages
and HeLa cells stably expressing CD4 and in their capacity to
induce syncytia upon infection of MT-2 cells (Fig 1). This
analysis revealed that all of these macrophage-tropic variants
used CCRS, whereas the T-cell-tropic variants used CXCR4, and
the dual-tropic variants effectively utilized either receptor
(Fig. 1). Thus, the cellular tropism of HIV-1 variants with
an isogenic background other than the V3 region is clearly
linked to selective use of coreceptors. This analysis also
demonstrated that the same V3 amino acids dictating cellular
tropism also control the engagement of either CXCR4 or CCRS.
For example, in variants 126 and 134, position 13 regulated
the reciprocal use of these two coreceptors, thereby
determining whether the variant targeted either macrophages or
T cells. These data show that both the coreceptor preference
and the cellular tropism of HIV-1 are linked to the same
positions in the V3 loop. These findings support the
hypothesis that genetic adaptation to additional coreceptors
may be responsible for the phenotypic evolution of HIV-1 in
vivo (Weiss, R.A., Science 272:1885-1886 (1996)). By this
mechanism, the evolution of multiple quasispecies in vivo that
use different chemokine receptors for cell entry potentially
leads to infection of other cell types and the concomitant
acceleration of HIV-1 disease.
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EXAMPLE 3 Cross-species restrictions to the HIV replication
cycle
Native rabbit T cells are partially susceptible to
infection by high-titer HIV-1 stocks in vitro and in vivo
(Filice, G. et al., Nature 335:366-369 (1988); Gordon, M.R. et
al., Annals of the New York Academy of Sciences 616:270-280
(1990); Reina, S. et al., J. Virol. 67(9):5367-5374 (1993);
Kulaga, H. et al., Proc. Natl. Acad. Sci. U.S.A.
85:4455-4459(1988)). Furthermore, at least a modest degree of
enhancement of this susceptibility was observed upon
introduction of human CD4 into various rabbit cell lines
(Yamamura, Y. et al., Intl. Immunol. 3:1183-1187 (1991);
Hague, B.F. et al., Proc. Natl. Acad. Sci. U.S.A. 89:7963-7
(1992)). We sought to determine whether or not human
chemokine receptors can significantly increase the
susceptibility of rabbit cells to entry by HIV-1, and whether
other post-entry restrictions are manifested in this
background. We performed several experiments to determine
whether or not rabbit cells are permissive for specific steps
in the HIV viral life cycle. First, a transfectable rabbit
epithelial cell line (SIRC) was transfected by conventional
methods with plasmids encoding human CD4 with or without HIV-1
Nef, and the relative cell surface expression of CD4 was
measured by fluorescence-activated cell sorting (FACS); as
observed previously in human cells, the presence of HIV-1 Nef
markedly attenuated the surface expression of human CD4 in
rabbit cells as shown qualitatively by FACS profiles (Fig. 3A,
left) and by statistical analysis of the FACS histograms (Fig.
3A, right). Therefore, rabbit cells are permissive for the
downregulation activity of HIV-1 Nef, which is one its key
recognized functions. Second, the activity of HIV-1 Tat in
promoting expression of viral genes via the HIV-1 LTR was
tested in SIRC cells by co-transfection of an LTR reporter
(CAT) construct with and without a plasmid encoding HIV-1 Tat.
While Tat only modestly augmented LTR activity in murine (NIH-
3T3) cells compared to its robust activity in human (HeLa)
cells, Tat-dependent expression of the reporter gene was
readily detected in rabbit cells (Fig. 3B). Therefore, rabbit
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cells (but not mouse cells) support HIV-1 Tat function.
Third, the activity of HIV-1 Rev in suppressing splicing of
viral transcripts was tested in SIRC cells by co-transfection
of a Rev-dependent reporter (CAT) construct with and without a
plasmid encoding HIV-1 Rev. Again, while Rev~functioned only
modestly in murine (NIH-3T3) cells compared to its robust
activity in human (HeLa) cells, its anti-splicing activity was
readily detected in rabbit cells (Fig. 3C). Therefore, rabbit
cells (but not mouse cells) support HIV-1 Rev function.
l0 Further analysis of the permissivity of rabbit cells for HIV
infection and for Rev function per se was performed using
stable transfectants of the SIRC and HeLa cell lines
expressing human CD4 and human CCRS. Total cellular RNA
extracted from cells cultured in the presence or absence of
the HIV-1 strain YU-2 was analyzed by Northern blotting for
the presence of unspliced (9kB) and partially spliced (4kB)
viral mRNA species. Both types of Rev-dependent mRNA species
were detected in the human and rabbit cell lines following
inoculation with YU-2 (Fig.4), indicating that rabbit cells
expressing the human proteins required for HIV-1 entry are
permissive for entry, reverse transcription, gene expression,
and appropriate splicing of viral RNA transcripts. To
determine further the permissivity of rabbit cells for HIV
infection and replication, rabbit derived SIRC-CD4 and
SIRC-CD4-CCRS cells were cultured in the presence or absence
HIV-1 BaL, and subjected to several analyses. First, after
three days in culture the cells were fixed, permeabilized and
immunostained with anti-p24 antibody. Virtually all of the
SIRC-CD4-CCR5 cells exposed to BaL expressed viral p24, while
essentially none of the SIRC-CD4 cells did (Fig. 5). These
observations reveal that the introduction of human CCR5 into
these cells in the presence of CD4 confers upon them
substantial permissivity for HIV entry, and the abundance of
viral antigens implies that these cells are also permissive
for subsequent viral gene transcription. Second, these
cultures were examined microscopically following routine
histochemical (hematoxylin and eosin) staining. The
SIRC-CD4-CCRS cells, but not the SIRC-CD4 cells, exhibited
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marked cytopathicity and syncytium formation upon exposure to
BaL (Fig. 6); these observations further support the
conclusion that active viral entry and subsequent viral gene
transcription are occurring in these cells and require both
CCRS and CD4. Third, paired cell lines were inoculated with
various strains of HIV-1 and the culture supernatants were
tested for HIV-1 p24 content by conventional ELISA. Three
CCR5-dependent virus strains (BaL, YU-2 and JR-CSF) were all
found to replicate and cause significant secretion of viral
p24 upon inoculation of the rabbit (SIRC) or human (HeLa), but
not mouse (NIH-3T3), transfectants expressing human CCRS/human
CD4 {Fig. 7); only the human line supported replication of the
CXCR4-dependent strain NL4-3 due to the absence of CXCR4 in
the rabbit transfectants (Fig.7). That these supernatants
contained infectious virions was readily demonstrated by
serial transfer onto PHA-blasted human PBMC, which revealed
the clear production of functional virions by SIRC-CD4-CCRS
cells (Fig.8). Finally, to verify that these observations in
transfected cell lines are pertinent to primary cells from
rabbits, primary peripheral blood lymphocytes from a rabbit
were isolated by density gradient centrifugation with the aid
of Ficoll-Hypaque and were transfected by electroporation with
a proviral plasmid representing the HIV clone YU-2. Culture
supernatants from these transfected cells were harvested and
used to inoculate PHA-blasted human PBMC. ELISA assay of the
human PBMC culture mediate demonstrated that the supernatants
from the primary rabbit cell transfectants contained
replication-competent virus (Fig.9), confirming that native
rabbit cells are indeed permissive for HIV-1 replication if
one bypasses the normal cellular entry mechanism. Moreover,
the earlier data demonstrate that this normal entry mechanism
also can be fully reconstituted in rabbit cells. Together,
this extensive investigation demonstrates that rabbits
reconstituted with human CD4 and human chemokine receptors as
HIV coreceptors should recapitulate an in vivo system that is
permissive for viral replication and spread.
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EXAMPLE 4 Trans~enesis with rabbits
A. Two initial constructs. encoding human CCR5
have been prepared and microinjected into rabbit embryos.
Both are cDNA constructs (constructs #1 and #2) in established
5 vectors intended to promote broad tissue expression: one
utilizes the murine major histocompatibility complex (MHC)
class I promoter region, which drives expression in many
hematolymphoid and other tissues; the second utilizes a
promoter/enhancer derived from cytomegalovirus (CMV), which
10 also drives relatively unselective tissue expression. More
specifically, construct 1 contains promoter region from murine
Major Histocompatibility Complex (MHC) class I genes upstream
of the human CCRS cDNA and a rabbit [3-globin polyadenylation
sequence downstream of the CCR5 cDNA. The CCR5 sequence
15 encodes an epitope-tagged variant of human CCRS. Construct 2
contains promoter/enhancer region from cytomegalovirus
upstream of the human CCR5 cDNA and a rabbit (3-globin
polyadenylation sequence downstream of the CCRS cDNA. CCRS
from this construct is not epitope-tagged.
20 Thus far 19 pups from one construct have been
screened by PCR-based methods and by Southern blotting, and 2
independent transgene-positive founders have been identified
and confirmed. One CCR5-founder animal has been mated with a
CD4-positive animal.
Four promising antibodies were identified that show
clear and specific positive staining of cells expressing human
CCR5.
B. Use of a Pl clone to create transctenic mice and rabbits
Three Pl clones from Genome Systems Inc. (St. Louis,
MO) were obtained which were originally screened for hCCR2B.
The positive clones were identified by Genome Systems control
numbers 2425, 2426 and 2427. A number of C-C chemokine
receptors are located at the p21 locus of human chromosome 3,-
and CCR2B and CCRS have been shown to be approximately 20 kb
apart (Raport et al., J. Biol. Chem. 271:17101 (1996)). The
three P1 clones were tested for human CCRS by PCR analysis and
for length of the 3' UT region by PCR on all three clones
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using vector specific primers (T7 and Sp6) and primers from
the 3' UT region of the hCCRS cDNA. The PCR showed that clone
2426 and 2427 contained hCCR5 and greater than 3 kb of 3' UT
of hCCR5 in each clone. Some preliminary Southerns have been
done on the 2426 clone showing an insert of approximately 80
kb by pulsed field gel electrophoresis. An Miu I fragment of
2426 which contains the entire insert plus 2 kb of 5' vector
sequence and 5 kb of 3' vector sequence has been isolated and
injected into mice, as described by Linton et al., (Linton et
al., J. Clin. Invest. 92(6):3029-3037 (1993)).
Although the present invention has been described in
some detail by way of illustration for purposes of clarity of
understanding, it will be apparent that certain changes and
modifications may be practiced within the scope of the claims.
Such modifications and variations which may be apparent to a
person skilled in the art are intended to be within the scope
of this invention.
All publications and patent applications herein are
incorporated by reference in their entirety for all purposes
to the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
