WO1991004327A1

WO1991004327A1 - Transgenic animal model for viral infections

Info

Publication number: WO1991004327A1
Application number: PCT/US1990/005248
Authority: WO
Inventors: Cha-Mer Wei
Original assignee: Tsi Corporation
Priority date: 1989-09-15
Filing date: 1990-09-14
Publication date: 1991-04-04
Also published as: EP0489868A1; CA2066614A1; EP0489868A4; JPH05505096A

Abstract

A transgenic animal model for viral infections, especially human immunodeficiency virus, is constructed by incorporating the genes ncoding essential components for viral infection of the animal into the chromosome of the embryo. Expression of the genes essential for infection and replication of the virus provides a means for assaying for compounds which inhibit infection and replication of the virus. An example of the construction of a transgenic animal expressing the human CD4 receptor, the receptor required for infection of human cells by HIV-1, on the surface of its lymphoid cells demonstrates the effectiveness of the techniques.

Description

TRANSGENIC ANIMAL MODEL FOR VIRAL INFECTIONS

Background of the Invention

This generally relates to animals models for drug screening, and more particularly to a transgenic animal model for screening of antiviral compounds.

In many cases viral infection is species and/or tissue specific due to the virus recognizing specific structures for infection to occur. . Ji example of an extremely species specific virus is human immunodeficiency virus or HIV. AIDS is a fatal disease caused by human immunodeficiency virus type 1 (HIV-1).

The lack of a suitable animal model has been a major stumbling block to the development of effective vaccines and therapeutics for AIDS. The only means for testing potential efficacy of anti-AIDS therapies has been in vitro testing. There is very little known regarding the correlation between in vitro testing and in vivo efficacy but it is believed to vary considerably. There is therefore an urgent need to search for a common laboratory animal to be developed as an AIDS model.

Although models using related viruses have been proposed for a variety of animal species, attempts to infect laboratory animals such as rodents and macaques with HIV-1 have not been successful. The only species besides human that has become reproducibly infected is the chimpanzee. As of 1989, approximately 50 chimpanzees have been infected with HIV using tissue from patients with AIDS, in vitro infected autologous cells, blood products from other infected chimpanzees and cell-free virus. These animals have now been observed for up to two years. Anti-HIV antibodies have been consistently demonstrated in their serum, and HIV has been readily and repeatedly recovered from peripheral blood. However, these animals have not yet developed evidence of AIDS-like disease symptoms.

The availability of adequate numbers of chimpanzees for AIDS research is also a problem. The chimpanzee is an endangered species. Only the animals produced by breeding in domestic colonies are allowed for research uses in the United States. Currently there are about 1,200 chimpanzees residing in biomedical research colonies and 80 in pharmaceutical industry colonies in the United States. Only about 300 chimpanzees are suitable for breeding to produce about 35 animals per year for research use. This number is strongly limiting the po Titial of the chimpanzee to be developed as an important animal model for AIDS. Accordingly, an alternative laboratory animal to replace the chimpanzee as an AIDS model is very important to the progress of AIDS research.

Recently, Kulaga, et al, at the National Institutes of Health have shown that rabbit T-cell lines transformed by human T- lymphotropic virus type I (HTLV-I), herpesvirus or SV40 can be infected by HTV-l. After infection with high titer HIV-1 stock, the rabbit cells demonstrated the production of HTV-l specific mRNA and proteins. The cell-free culture superaatants of infected cells contained infectious virus, as established by successful passage into a susceptible human T-cell line.

More recently, two research groups independently demonstrated that rabbits can be infected with HIV-1. The results have been published by Kulaga, et al., in the J.Exper.Med. (1989) and by Filice, et al., in Nature 335, 366-369 (1988), respectively. However, in both cases, high titers of HIV-1 and human producer cells were needed to achieve infection. It is not known whether the transmission of HTV-l into rabbit cells, as described, uses routes similar to those occurring during infection of human cells by HIV-1.

It is therefore an object of the present invention to provide methods and means for developing transgenic animals that are more susceptible than non-transgenic animals to infection with viruses, especially human immunodeficiency virus type I (HTV-l) infection.

It is a further object of the invention to provide transgenic animals for use in the development of safe, effective antiviral vaccines for protection of individuals not yet infected and the development of drug therapies for those already infected.

It is another object of the invention to provide methods and means for studying the infectivity and replication of viruses, especially HTV-l, as a model for diseases caused by these viruses, including AIDS.

It is still another object of the invention to provide fusion genes that are capable of directing the expression of cell surface receptors for viruses, such as the human T4 receptor or the expression of transcription activators such as human T lymphotropic virus type I (HTLV-I) tax gene, in transgenic animals and in cultured cells.

Summary of the Invention

A transgenic animal model for viral infections, especially human immunodeficiency virus, is constructed by incorporating the genes encoding essential components for viral infection of the animal into the chromosome of the embryo. Expression of the genes essential for infection and replication of the virus provides a means for assaying for compounds which inhibit infection and replication of the virus. In the preferred embodiment, cultured cells or transgenic animals are modified to express one or more of the components required for infection with HIV-1: the cellular receptors, such as the human T4 receptor, the regulatory proteins which control HIV-1 gene expression in infected cells such as tat, vif, and rev, helper gene functions provided by heφesvirus ateles, HTLV-1 or SV40 viruses, and human cellular transcription factors such as NF-kB, Spl or AP-1.

An example of the construction of a transgenic mouse expressing the human CD4 receptor, the receptor required for infection of human cells by HIV-1, on the surface of its lymphoid cells demonstrates the effectiveness of the techniques. Brief Description of the Drawings

Figure 1 is a schematic of the HTLV-1 tax fusion gene to be introduced into animal genomes. The shaded area indicates tissue specific promoter/enhancer such as the CD4 gene promoter, MoMuLV LTR or human ribosomal protein gene promoter. The open area represent HTLV-I tax coding sequence. The SV40 transcri on termination signal is shown in the solid area.

Figure 2 is a schematic of the CD4 genomic clones. The CD4 genomic clones were isolated from human genomic libraries in cosmid p\VE15 and analyzed by BamHI and Notl cleavage mapping. The maps of pCD17A.2, pCD4.1 and pCD2B.l were constructed based on the reference map published by Maddon, et al., Proc. Natl. Acad. Sci. 84, 9155-9159 (1988).

Detailed Description of the Invention The present invention is described with reference to construction of transgenic animals and animal cells capable of being infected with HTV. In particular, the following example demonstrates the construction of vectors for the incorporation and expression of CD4 (the human T4 receptor is essential for binding of the HIV envelope protein) for use as an assay for compounds having anti-

HTV-1 activity. This example can be modified by those skilled in the art using published techniques and commercially available reagents to construct vectors for the incoφoration and expression of other proteins required for development of transgenic cells and animals as assays for antiviral compounds and vaccines for other species specific or tissue specific viruses. As used herein, "antivirals" includes antiviral drugs, vaccines, and other virus-specific inhibitory agents. Experimental Design and Methods.

The essential components for HTV-l infection can be categorized into three groups: (1) cellular receptors and associated proteins responsible for HTV binding and penetration, such as the human T4 receptor; (2) the regulatory proteins which control HIV-1 gene expression in infected cells such as tat (viral transactivator), vif, and rev, and (3) helper gene functions such as those provided by heφesvirus ateles, HTLV-1 or SV40 viruses; and (4) host cell factors responsible for HIV replication such as NF-kB, Spl or AP-1.

(1) Cellular receptors and associated proteins responsible for

HIV binding and penetration. T4 lymphocytes, also known as the helper T-cell lymphocytes, play a major role in the human immune system. These cells express a glycoprotein on the cell surface called CD4. During HIV infection the envelope protein gpl20 of HIV tightly binds to the CD4 receptor to mediate viral entry into the cell. Accordingly, the CD4 receptor is an essential component in HIV pathogenesis which leads to AIDS. Recently, Weiner, et al., suggested that one or more non-CD4 proteins were involved in HTV-1-cellular receptor interactions. However, these proteins have not been cloned or purified.

Animals may express analogs of these proteins, although they are probably less efficient than human proteins for HIV binding and penetration. In order to make the animals more susceptible to infection, genes encoding the human proteins are incoφorated into the animal genomes for expression in the animal, making them more susceptible to HIV infection.

The gene for CD4, shown in Figure 1, has been cloned. When the genes encoding the proteins described by Weiner, et al., are cloned, they can be engineered and tailored using recombinant DNA technology for construction of vectors for use in making transgenic animals.

The vectors containing these genes can be tested for expression in cultured rabbit cells before being introduced into embryos by microinjection techniques. (2) The regulatoi proteins which control HIV-1 gene expression in infected cells.

HTV-l is a genetically complex virus. Several regulatory genes are required for its normal replication cycle. These include tat (transactivator), rev (differential regulator) and vif (infectivity factor). One of reasons HTV primarily infects human cells may be inefficient expressf ^n of these regulatory proteins in non-human host cells.

Transgenic animals expressing one or more HTV regulatory proteins may be more susceptible to HIV infection. Most of these genes are cloned. They can be engineered using recombinant DNA technology to constrict expression vectors for use in making transgenic animals.

(3) Helper gene functions provided by other viruses.

The HTLV-I tax gene is being engineered to form fusion gene constructs for introduction into the animal genome. It has been reported by Schmid, et al., Science 216, 1065-1070 (1982), that HTLV- I transformed human T-cell lines are highly susceptible to HTV-l infection in vitro. Recently the HTLV-I trans-activator (tax) gene product was shown by Ruben, et al., Science 241, 89-92 (1988), to activate the expression of interleukin-2 receptor gene. It was also reported by Zack, et al., Science 240, 1026-1029 (1988), that human peripheral blood leukocytes infected with HTV-l in vitro can be induced to produce large quantities of HTV-l after mitogenic stimulation by non-infectious HTLV-1 virions. Accordingly, a transgenic rabbit carrying the transgene derived from one or more HTLV-I viral genes expressed in T-cells has a good chance of being more susceptible to the HTV infection.

HTV-l infects the HTLV-I transformed rabbit cells, but not non-transformed peripheral blood lymphocytes, even though they have been activated by Con A, phytohemagglutinin or interleukin-2. This result indicates that one or more of HTLV-I gene products render the transformed cells susceptible to the HTV infection. Based on the observations by Ruben, et al., that the trans- activator (tax) of HTLV-I activates the interleukin-2 receptor gene in infected cells in addition to trans-activating all the viral genes, a functional transcription unit containing the tax coding sequence will be constructed. The tax is encoded by two exons in the HTLV-I genome, as described in RNA Tumor Viruses. Weiss, et al., editors, Vol. 2 (Cold F^aring Harbor Laboratory 1984). A continuous and complete tax coding sequence can be constructed by using a portion of the HTLV-I genome and chemically synthesized oligonucleotides. It will be inserted into an expression vector promoted by an appropriate promoter/enhancer and terminated by the SV40 polyadenylation site. Promoters/enhancers include the Moloney murine leukemia virus (MMLV) LTR, ribosomal protein gene, and CD4 gene promoter, shown in Figure 1. If the tax gene is proven not to be responsible for HTV susceptibility, other viral cellular genes can be engineered and introduced into the animal genome. (4) Host cellular factors which regulate HTV genes.

Several human cellular factors are important for HIV gene expression. Griffin, et al. have reported that HIV gene expression in the monocyte lineage is regulated by NF-KB. The same factor also stimulates the HIV enhancer in activated T-cells. Jones, et al., have shown that human Spl transcription factor binds to promoter sequences within the AIDS retrovirus long terminal repeat and activates RNA synthesis five to eight-fold in vitro. These factors can be purified and their cDNA cloned using recombinant DNA technology. Each cDNA can be spliced to appropriate promoter/enhancer to produce tissue specific expression vector. Alternatively, the genomic DNA of NF-KB and Spl can be cloned. Their endogenous promoters/enhancers will be used to drive gene expression. The DNA constructs will be introduced into embryos of animals by microinjection techniques. In the preferred embodiment wherein a transgenic animal which is more susceptible to infection is made, a transgenic rabbit is developed as the HTV infection model, although other animals, especially the mouse, are also useful. The rabbit is inexpensive, abundant and easy to handle. Its biology, immunology and physiology have been studied quite extensively. In certain aspects its immune system 1 "s similarities with the human system.

The observation that common New Zealand rabbits or rabbit T-cell lines with appropriate manipulation can be infected with HTV-l provides a strong scientific base for transgenic rabbits to be more susceptible to HTV-l infection after proper genetic manipulation. These transgenic rabbits will be very valuable tools for the development of AIDS vaccines and therapeutics.

The ideal rabbit model should develop an AID-like disease by mimicking long-term HTV infections including viremia, latency, and disease progressing the immune dysfunction and possible neoplastic diseases. These transgenic rabbits can also be used to study (a) molecular interactions between viral structural and/or regulatory proteins and those of the host (b) the ability of the virus to evade the host immune system, (c) the molecular mechanism by which HIV induces alterations of normal cellular and immune functions and (d) the molecular mechanism of viral persistence, latency, and disease progression. If the transgenic rabbits are susceptible to the HIV-1 infection, but incapable of developing an AIDS-like disease, they are still useful for additional research and as subjects for further genetic manipulation.

Example: Isolation of the human CD4 gene, construction of vectors, and incorporation and expression of the gene in transgenic animals. Recombinant DNA techniques known to those skilled in the art were used throughout the studies. These techniques include cloning, transformation, screening, agarose gel electrophoresis, polyacrylamide gel electrophoresis, restriction enzyme mapping, modification of DNA fragments, preparation of plasmid DNA, Southern blotting and filter hybridization. The detailed protocols can be found in Maniatis, et al. Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, NY 1982), Davis, et al., Advanced Bacterial Genetics (Cold Spring Harbor Laboratory, NY 1980) or in manual! provided by many reagent supply companies.

The DNA sequencing techniques used are also known to those skilled in the art, including Sanger's chain termination method, Sanger, et al., Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977) and Messing, et al., Nucleic Acids Res. 9, 309-321 (1981), for nucleotide sequence determination for confirmation of the DNA constructs at the splicing sites. Many commercial suppliers provide both reagent kits and detailed protocols. Oliogonucleotides used for hybridization probes and DNA sequencing primers are chemically synthesized using an automated DNA synthesizer. This service can be obtained from commercial sources.

Isolation of Human CD4 Receptor Gene Maddon, et al., have cloned human CD4 cDNA and pieces of CD4 genomic DNA, as reported in £gU 42, 93-104 (1985) and Proc. Natl. Acad. Sci. USA 84, 9155-9159 (1987). However, no complete gene in a single clone was isolated.

Their CD4 cDNA was used as a hybridization probe to screen two human genomic libraries cloned in a cosmid vector pWE15, developed by Wahl, et al., Proc. Natl. Acad. Sci. USA 84, 2160-2164 (1987). One library is derived from human placenta DNA, the other from human lymphocyte DNA. Both libraries were obtained from a commercial supplier, Stratagene (La Jolla, California). The clones containing human CD4 genomic DNA isolated from the genomic libraries were confirmed by partial nucleotide sequencing by a chain termination method. The human CD4 genomic clones were analyzed by restriction endonuclease cleavage mapping. Preferably only the complete gene containing the promoter/enhancer, the entire coding sequence and the polyadenylation site is used for embryo microinjection studies. The cosmid clone based on the pWE15 vector has a capacity to accommodate 35 to 40 kb of genomic DNA (24), large e-ough for the entire human CD4 gene which is 28 kb long.

The partial restriction endonuclease cleavage map of the human CD4 gene is shown in Figure 2. Several cosmid clones which encompass various regions of this gene, including one which includes all coding sequences and the promoter, were isolated by screening human DNA genomic libraries (placenta and lymphocyte) with either the cDNA itself or with 36 base pair oligonucleotides directed against the 5' end. The sequences of the oligonucleotides are:

5'- CAAGCCCAGA GCCCTGCCAT TTCTGTGGGC TCAGGT -3'

5'- TTCTGTGGGC TCAGGTCCCT ACTGCTCAGC CCCTTC -3'; and

5'- TGTATCCCCT TTTTTGCCCA GCACCACTTT GTTTCC -3'. Several positive clones were identified by in situ hybridization. They were confirmed by subcloning and partial sequence determination using the chain termination method. They were further analyzed by restriction endonuclease cleavage mapping in combination with Southern blotting analysis using labeled cDNA or oligonucleotides as probes. By comparing with a reference map published by Maddon, et al., it was possible to construct maps for pCD17A , pCD4.1 and pCD2B.l clones. The pCD17A.2 clone covers the entire human CD4 gene including 5 kb of 5' promoter/enhancers. Clone pCD4.1 contains the 3' portion of CD4 gene starting from the second intron. The pCD2B.l clone covers only the first two exons and most of the 5' sequence. The entire insert of pCD17A.2 can be excised intact away from the cosmid vector with Notl digestion. The 40 kb insert generated by Notl was purified and dissolved in microinjection buffer at 3 μg/ml for microinjection into rabbit embryos.

The procedures for microinjection are similar to those published by Knight, et al., Proc. Natl. Acad. Sci. USA 85, 3130-3134 (1988). Adult male and female rabbits are generally used. Rabbit zygote 'onors are injected subcutaneously with 50 international units of mare gonadotropin four days before mating. Immediately after mating, they are injected intravenously with 150 international units of chorionic gonadotropin. Nineteen hours later single-cell zygotes are flushed from rabbit donor oviducts.

The pronuclei obtained using this procedure were injected with the 40 kb human CD4 genomic DNA (3 μg/ml). The injected zygotes were implanted through the fimbrial end of the oviduct of a recipient rabbit which is made pseudo pregnant two days earlier by intravenous injection of 150 international units of chorionic gonadotropin or by mating with a sterile male.

The pregnancy is carried to the term. The live borns are allowed to grow to several weeks of age. The peripheral blood is withdrawn for preparation of lymphocytes. High molecular weight genomic DNA is isolated from lymphocytes and used in Southern blotting analysis. Transgenic rabbits carrying a complete copy of human CD4 gene are tested for evidence of transgene expression, for example, by reacting the transgenic lymphocytes with antibodies against the human CD4 polypeptides and staining the lymphocytes with a fluorescein labeled second antibody against the first antibody. It is expected that the T4 lymphocytes of transgenic rabbits carrying a functional human CD4 gene should express human CD4 receptor molecules on their surface. Infection of transgenic rabbits with HIV-1 is carried out according to Kulaga, et al., J. Exp. Med. (1989). For infection with HTV-l, rabbits are given a single intravenous injection with 5 x 10⁶ A3.01 cells infected with HIV-1 (A3.01 is a human T-cell line derived from a leukemic individual and is highly permissive to HIV-1 infection). The injected cells are near peak infection as determined by monitoring syncytia formation and reverse transcriptase activity in the cell-free culture supematants. Serum samples taken at three week intervals post-infection are tested by EOSA for the presence of antibodies directed against HTV-l proteins using kits from Dupont (Wilmin ^on, DE). In addition, Western blot analyses are used to determine recognition of HTV-l encoded proteins by rabbit antibodies. The presence of HTV-l genome in the host DNA can be also identified by gene amplification using polymerase chain reactions. Oligonucleotide primers designed to anneal to the plus and minus strands of regions in the HTV-l genome can be used to amplify those regions which can be easily characterized.

Modifications and variations of the transgenic animal models for screening of antiviral compounds and vaccines, and method for making, will be obvious to those skilled in the art from the foregoing detailed description of the invention. Such modifications and variations are intended to come within the scope of the appended claims.

Claims

We claim.

1. A transgenic animal model for testing antivirals comprising eukaryotic cells expressing genes incoφorated therein which encode essential proteins for infection of the cell by a selected virus.

2. The transgenic animal model of claim 1 wherein the virus is human immunodeficiency virus.

3. The transgenic animal model of claim 1 wherein the essential protein is selected from the group consisting of cellular receptors and associated proteins responsible for binding and penetration of the virus into the cell, regulatory proteins which control viral gene expression in infected cells, helper gene functions encoded by other viruses, and host cell factors involved in viral replication.

4. The transgenic animal model of claim 2 wherein the essential protein is selected from the group consisting of the CD4 cellular receptor, the tat, vif, and rev regulatory proteins; the functional products of the heφesvirus, HTLV, and SV40 helper virus genes; and cellular factors NF-kB, Spl and AP-1.

5. The transgenic animal model of claim 1 wherein the eukaryotic cells are grown in cell culture.

6. The transgenic animal model of claim 1 wherein the eukaryotic cells are a transgenic animal.

7. The transgenic animal model of claim 6 wherein the animal is a transgenic mouse.

8. The transgenic animal model of claim 6 wherein the animal is a transgenic rabbit.

9. The transgenic animal model of claim 6 wherein the gene encodes CD4 and the CD4 is expressed on the surface of the transgenic animal cells.

10. The transgenic animal model of claim 9 wherein the animal cells are an animal selected from the group consisting of transgenic mice and transgenic rabbits.

11. A method for assaying for compounds inhibiting viral infection or replication comprising providing a transgenic animal model for testing antivirals including eukaryotic cells expressing genes incoφo? *.ed therein which encode essential proteins for infection of the cell by a selected virus.

12. The method of claim 11 wherein the virus is human immunodeficiency virus.

13. The method of claim 11 wherein the essential protein is selected from the group consisting of cellular receptors and associated proteins responsible for binding and penetration of the virus into the cell, regulatory proteins which control viral gene expression in infected cells, and helper gene functions encoded by other viruses, and host cell factors involved in viral replication.

14. The method of claim 11 wherein the essential protein is selected from the group consisting of the CD4 cellular receptor, the tat, vif, and rev regulatory proteins; the functional products of the heφesvirus, HTLV, and SV40 helper virus genes; and cellular factors NF-kB, Spl and AP-1.

15. The method of claim 11 wherein the eukaryotic cells are grown in cell culture, further comprising culturing the cells in the presence of a compound to be tested for inhibition of viral infection or viral replication.

16. The method of claim 11 wherein the eukaryotic cells are a transgenic animal, further comprising administering to the animal a compound to be tested for inhibition of viral infection or viral replication.

17. The method of claim 16 wherein the animal is a transgenic mouse.

18. The method of claim 16 wherein the animal is a transgenic rabbit.

19. The method of claim 16 wherein the gene encodes CD4 and the CD4 is expressed on the surface of cells.

20. The method of claim 19 wherein the cells are transgenic animal cells selected from the group consisting of transge ^τc mouse cells and transgenic rabbit cells.

21. A vector for making a transgenic animal model for testing antivirals comprising a promoter in combination with a gene encoding an essential protein for infection of the cell by a selected virus, wherein the combination is capable of incoφoration into the genome of a eukaryotic cells and expression of the protein encoding gene after incoφoration.

22. The vector of claim 21 wherein the virus is human immunodeficiency virus.

23. The vector of claim 21 wherein the essential protein is selected from the group consisting of cellular receptors and associated proteins responsible for binding and penetration of the virus into the cell, regulatory proteins which control viral gene expression in infected cells, and helper gene functions encoded by other viruses, and host cell factors involved in viral replication.

24. The vector of claim 22 wherein the essential protein is selected from the group consisting of the CD4 cellular receptor, the tat, vif, and rev regulatory proteins; the functional products of the heφesvirus, HTLV, and SV40 helper virus genes; and cellular factors NF-kB, Spl and AP-1.

25. The vector of claim 22 wherein the promoter/enhancers are derived from the human CD4 gene and is in combination with a gene encoding the CD4 protein.