Self-contained system for sustained viral replication

Details Self-contained system for sustained viral replication Self-contained system for sustained viral replication

1999-12-14

2003-05-13

Priebe, Scott D. (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Method of using a transgenic nonhuman animal in an in vivo...

C800S018000, C800S008000, C800S009000, C800S011000

Reexamination Certificate

active

06563014

ABSTRACT:

FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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 5′ half of proviral DNA from the NY5 isolate with the 3′ 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 3′-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. T

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