CA2295059A1 - Transgenic rabbits expressing cd4 and chemokine receptor - Google Patents

Abstract

The invention provides transgenic rabbits and transgenic rabbit cells expessing CD4 and a human chemokine receptor such as CXR4 or CCR5. The invention also provides methods of generating the transgenic rabbit. The double transgene is either generated by introducing the two transgenic entities into a fertilized pronucleus or by breeding two transgenic rabbits each ahving one of the transgenic entities. The rabbit may be infected with HIV.

Description

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 (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 (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 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.

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 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 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 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.

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 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 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 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 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 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 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 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.

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 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 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.

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 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.

Claims (15)

CLAIMS:

1. A transgenic rabbit or rabbit cell expressing a human chemokine receptor and human CD4.

2. The transgenic rabbit or rabbit cell of claim 1, wherein the chemokine receptor is CCR5.

3. The transgenic rabbit or rabbit cell of claim 1, wherein the chemokine receptor is CXCR4.

4. The transgenic rabbit or rabbit cell of claim 1, wherein the rabbit or cell is infectable by a human immunodeficiency virus.

5. The transgenic rabbit or rabbit cell of claim 4, wherein the virus is HIV-1.

6. The transgenic rabbit or rabbit cell of claim 4, wherein the virus replicates in the rabbit or cell.

7. The transgenic rabbit or rabbit cell of claim 1, wherein the chemokine receptor and/or the CD4 are encoded by genomic DNA.

8. A method of generating a transgenic rabbit or rabbit cell comprising (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 a litter 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.

9. The method of claim 8 wherein at least one transgene is provided as a P1 clone.

10. The method of claim 8 wherein at least one transgene expressed under the control of regulatory elements native to the transgene.

11. The method of claim 8, wherein at least one transgene comprises genomic DNA.

12. 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.

13. The method of claim 12 wherein at least one transgene is provided as a P1 clone.

14. The method of claim 12 wherein at least one transgene is expressed under the control of regulatory elements native to the transgene.

15. The method of claim 12, wherein at least one transgene comprises genomic DNA.