This invention is directed to the development of an animal model to study infection by species-specific pathogens having a limited host range, preferably human-specific pathogens where the limited host range includes humans, comprised of crossing an animal transgenic for the pathogen with an animal transgenic for the tissue-specific expression of species-specific receptor(s) of the pathogen that restrict infection to the cells of the host species. For example, where the human-specific pathogen is a virus, the animal resulting from such a cross is transgenic both for the infectious provirus and for the human receptor or co-receptors that restrict infection to human cells. Accordingly, the transgenic animal of this invention has a sustained productive viral infection and does not develop a virus-specific immune response, thereby resulting in an extremely useful model to investigate the factors modulating in vivo replication of human pathogens, the pathophysiological effect of pathogen replication and production, and the effectiveness of novel therapies and vaccines modifying or inhibiting the course of pathogenesis.
The development of therapeutic agents for human use is extremely expensive and time intensive. For many human diseases, the therapeutic drug and vaccine development processes have been greatly facilitated by the development of animal models that mimic or approximate human pathophysiological disease processes as well as normal human physiological processes. However, many human diseases are caused by human-specific pathogens that infect only human cells. For example, human immunodeficiency virus type 1(HIV-1), the causative agent of AIDS, can only be propagated in cells from humans and certain primates, such as chimpanzees. Since humans cannot be studied in a systematic fashion, and chimpanzees are on the endangered species list and are difficult and costly to maintain in a laboratory setting, there are no available animal models that mimic the human pathophysiological disease process of AIDS.
A pathogen, such as a virus, may be species-specific if initial infection into host cells is mediated by the interaction of species-specific receptors on the cell surface with the pathogen. There may be other barriers to productive infection in a non-host species, such as regulatory blocks preventing efficient viral replication even after initial infection into the cell. HIV-1 entry into human cells is mediated by CD4 acting in concert with one of several members of the chemokine receptor superfamily such as CXCR4 in the case of T-tropic strains of HIV-1 and CCR5 in the case of M-tropic strains of HIV-1. Mice, commonly used as animal models for human disease, are not infectible with HIV-1 because HIV-1 penetration into mouse cells is prevented by the inability of the envelope protein, gp120, to bind to the mouse homologues of these human receptor molecules (Atchison, R. E., et al., Nature 274:1924-1926, 1996). This inability of HIV to enter murine cells means that HIV cannot initially attach and enter the cells in order to replicate, nor can HIV reinfect cells to maintain a productive HIV infection.
Two basic approaches have been used to bypass the limited host range of HIV, although neither approach has been entirely satisfactory. One approach used has been to introduce a full-length infectious HIV provirus into the germ line of mice as a transgene (Klotman P. E., et al., Curr Top. Microbiol. Immunol. 206:197-222, 1996). The infectious proviral clone used to construct these transgenic mice, NL4-3, was a hybrid construct that was derived by fusing the 5xe2x80x2 half of proviral DNA from the NY5 isolate with the 3xe2x80x2 half of proviral DNA from the LAV isolate of HIV (Adachi A., et al., J. Virol. 59:284-291, 1986). The initial description of these transgenic mice reported that the PBMCs of seven founder mice that transmitted intact copies of the HIV proviral DNA to their progeny did not produce infectious HIV, limiting their usefulness as an in vivo system for studying the pathophysiology of HIV infection (Leonard J. M., et al., Science 242:1665-1670, 1988). However, HIV could be recovered by coculture from the skin, spleen and lymph nodes of the progeny of one founder mouse and these mice displayed a phenotype of growth failure and lymphoid hyperplasia and died within a month of birth. Another group used the full-length NL4-3 provirus to produce six transgenic mouse lines and, although HIV RNA was not detected in their tissues by Northern blot analysis, their macrophages contained low levels of HIV RNA that could be increased by in vitro treatment with macrophage activators (Dickie P., et al., AIDS Res. Hum. Recro. 12:1103-1116, 1996).
In an attempt to increase HIV gene expression in mice transgenic for the NL4-3 construct, different heterologous promoter/enhancer sequences with increased transcriptional activity in mouse cells were introduced into the NL4-3 vector. Transgenic mice constructed using a NL4-3 proviral construct where two NFKB binding sites in the. NL4-3 LTR were replaced with two copies of the murine leukemia virus core enhancer displayed increased HIV RNA expression in lymph nodes, spleen and muscle (Dickie, P., et al., J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 13:101-116, 1996). Infectious HIV-1 could be isolated from their splenocytes and several lines of evidence indicated that this virus was produced by B cells (Dickie P., et al., AIDS Res. Hum. Recro. 12:1103-1116, 1996; Dickie, P., et al., J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 13:101-116, 1996). Increased HIV gene expression was also observed in transgenic mice generated with constructs where the NL4-3 vector was placed under the transcriptional control of either the mouse mammary tumor virus promoter (Jolicoeur, P., et al., J. Virol. 66:3904-3908, 1992) or the CD4 gene enhancer/promoter (Hanna Z., et al., J. Virol.72:121-132, 1998). However, these mice do not produce infectious HIV virions due to the engineered deletion of the 3xe2x80x2-end LTR in the construct. Other attempts using non-infectious constructs included the construction of transgenic mice using HIV-1 deletion mutants such as the NL4-3Agag/pol construct (Dickie, P., et al., Virology 185:109-119, 1991), or mice transgenic for individual HIV genes such as env placed under the control of a tissue-specific promoter (Berrada, F., et al., J. Virol. 69:6770-6778, 1995). Although many of these mice displayed pathological changes that were associated with transgene expression, the alterations introduced into the regulation of viral gene expression may have compromised the physiological relevance of these transgenic mice to HIV-1-infected individuals. Furthermore, even in transgenic mice described above that produce infections, the virus encoded by the HIV-1 provirus cannot reinfect cells in these mice because of the inability of HIV-1 to attach and enter mouse cells, thereby rendering such a model useless for studying factors that inhibit sustained productive HIV infection.
Another approach involves the development of transgenic mice which are transgenic for the HIV-1 co-receptors required for initial entry of the virus into cell. (Browning, et al., Proc. Natl. Acad. Sci. USA 94:14637-14641, 1997). Peripheral blood mononuclear cells and splenocytes isolated from mice transgenic for human CD4 and CCR5 (hu-CD4/CCR5 TG mice) expressed human CD4 and CCR5 and were infectible with selected M-tropic HIV isolates. After in vivo inoculation, HIV-infected cells were detected by DNA PCR in the spleen and lymph nodes of these transgenic mice, but HIV could not be cultured from these cells despite repeated inoculations with high doses of HIV-1. This indicated that although transgenic expression of human CD4 and CCR5 permitted attachment and entry of HIV into mouse cells, sustained HIV infection was prevented by other blocks to HIV replication present in the mouse cells and/or development of an HIV-specific immune response that was rapidly eliminating HIV infected cells. This significantly limited the usefulness of this model for studying HIV-1 infection.
Another limitation of previously described models is that all of the mice transgenic for proviral HIV-1 described above were generated using proviral DNA constructs derived from T-cell line tropic, laboratory-adapted HIV-1 isolates (Klotman P. E., et al., Curr Top. Microbiol. Immunol. 206:197-222, 1996). The terms xe2x80x9cT-cell line tropicxe2x80x9d and xe2x80x9cmonocyte-tropicxe2x80x9d refer to the restricted capacity of various HIV-1 isolates to infect T cell lines and monocytes, respectively. T-cell line tropic isolates are capable of infecting T cell lines but not monocytes, and monocyte-tropic isolates are able to infect monocytes/macrophages but not T cell lines. (Schuitemaker, H., et al., J. Virol. 66:1354-1360, 1991). The basis for such divergent cellular tropism is due to the fact that T-cell tropic lines of HIV-1 must interact, after binding to CD4, with CXCR4 (Feng, Y., et al., Science 272:872-877, 1996), while monocyte-tropic isolates utilize CCR5 as a coreceptor (Berger, E. A., et al., AIDS 11:53-516, 1997; Deng, H., et al., Nature 381, 661-667, 1996; Dragic, T., et al., Nature 381:667-673, 1996; Choe, H., et al., Cell 85:1135-1148, 1996; Alkhatib, G., et al., Science 272:1955-1958, 1996).
Monocyte-tropic HIV-1 strains are the primary isolate detected in individuals during the initial stages of HIV infection (Schuitemaker, H., et al., J. Virol. 66:1354-1360, 1992; Mosier, D., et al., Immunol. Today 15:332-339, 1994), and a large body of the literature suggests that M-tropic HIV-1 isolates are important in the initial establishment of infection (e.g., Van""t Wout A. B., et al., J. Clin. Invest 94:2060-2067, 1994), a suggestion further bolstered by the demonstration that individuals homozygous for a 32 base pair deletion in the CCR5 gene do not become infected with HIV despite multiple exposures to M-tropic and T-cell line tropic HIV-1 and that their mononuclear cells are resistant to in vitro HIV infection (Liu, R., et al., Cell 86:367-377, 1996; Samson, M., et al., Nature 382:722-725, 1996). Thus, investigation of the in vivo behavior of M-tropic HIV-1 isolates is critical for understanding of the pathophysiology of HIV-1 infection.
Accordingly, there is a need to develop an animal model to study infection by human-specific or species-specific pathogens, where such a model maintains a productive viral infection and does not develop a virus-specific immune response.
The invention is directed to the development of animal models to study infection by species-specific pathogens, including infection by human-specific pathogens such as HIV or hepatitis. As such, the invention provides a self-contained system for examining sustained pathogen replication. The invention will allow researchers to use convenient and well studied small laboratory animals, including rats or mice, to study infection by pathogens having limited host ranges not normally including the selected laboratory animal, and to evaluate vaccines against such pathogens and therapeutics directed to diseases caused by or associated with such pathogens. Where the pathogen is xe2x80x9chuman-specificxe2x80x9d, the limited host range includes (or may be limited exclusively to) humans but excludes the selected laboratory animal. Where the pathogen is xe2x80x9cspecies-specificxe2x80x9d, the limited host-range includes (or may be limited exclusively to) a particular species but excludes the selected laboratory animal.
Accordingly, the present invention provides for a transgenic non-human animal comprising a transgene for a species-specific pathogen and a transgene for at least one receptor restricting infection of the pathogen to a member of the host species. In addition, the invention provides a method for creating a transgenic non-human animal, comprising the steps of (i) isolating a fertilized oocyte from a female non-human donor animal; (ii) transferring a transgene for a species-specific pathogen into the fertilized oocyte; (iii) transferring the fertilized oocyte comprising the transgene for a species-specific pathogen into the uterus of a female non-human surrogate animal; (iv) maintaining said female non-human surrogate animal such that said female non-human surrogate animal gives birth to a transgenic non-human animal derived from the fertilized oocyte wherein said transgenic non-human animal comprises the transgene for the species-specific pathogen; (v) isolating a fertilized oocyte from a second female non-human donor animal; (vi) transferring at least one transgene for a receptor restricting infection of the pathogen to the host species into the fertilized oocyte; (vii) transferring the fertilized oocyte containing at least one transgene for a receptor restricting infection of the pathogen to the host species into the uterus of a second female non-human surrogate animal; (viii) maintaining said second female non-human surrogate animal such that said second female non-human surrogate animal gives birth to a transgenic non-human animal derived from the fertilized oocyte wherein said transgenic non-human animal comprises at least one transgene for a receptor restricting infection of the pathogen to the host species; and (ix) crossing the transgenic non-human animal comprising the transgene for the species-specific pathogen with the transgenic non-human animal comprising at least one transgene for a receptor restricting infection of the pathogen to the host species, such that the cross produces a transgenic non-human animal comprising a transgene for the species-specific pathogen and at least one transgene for a receptor restricting infection of the pathogen to the host species. The present invention provides that the host species may be human, such that the resulting cross produces a transgenic non-human animal comprising a transgene for a human-specific pathogen and at least one transgene for a receptor restricting infection of the pathogen to humans.
Also provided by the present invention is a method for screening an agent for the ability to inhibit infection by a species-specific virus, comprising administering the agent to be screened to a non-human animal comprising a transgene for a species-specific pathogen and a transgene for at least one receptor restricting infection of the pathogen to the host species, assaying viral levels of the transgenic animal before and after administration of the agent being screened, and analyzing the effect of the administered agent on the viral levels of the transgenic animal. The host species may be human. The invention further provides for an agent identified by the above method, where said agent is a peptide, protein, nucleic acid, ribonucleic acid, antisense RNA, antibody, drug or compound. Additional objects of the present invention will be apparent from the description which follows.