USES OF A MURINE MODEL OF HIV-I INFECTION

[0001] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0002 J This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

[0003] U.S. Application Serial No. 60/564,505, filed April 21, 2004, is incorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

[0004] This invention was made with government support under NIH Grant No. R21 DA- 14934. As such, the United States government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

[0005] AIDS - acquired immunodeficiency syndrome - was first reported in the United States in 1981 and has since become a major worldwide epidemic. AIDS is caused by the human immunodeficiency virus (HIV). By killing or damaging cells of the body's immune system, HIV progressively destroys the body's ability to fight infections and certain cancers. There are two types of HIV, HIV-I and HIV-2. HIV-I is the more virulent type, and naturally infects human beings, as well as a small number of non-human primates. Compared with persons infected with HIV-I , those with HIV-2 are less infectious early in the course of infection. As the disease advances, HIV-2 infectiousness seems to increase. However, compared with HIV-I, the duration of this increased infectiousness is shorter. There is currently no cure for HIV infection or AIDS.

SUMMARY OF THE INVENTION

[0006] The invention is directed to a process for constructing and producing an HIV-I construct capable of infecting rodent cells. This allows for the development of a convenient and safe mouse model of HIV-I infection which can be used for: 1) testing potential routes to HIV- 1 pathogenesis in an animal that is susceptible to HIV-I infection and spread; 2) screening and testing potential antiviral therapies for HIV-I ; 3) screening and testing potential HIV-I vaccines in an immunocompetent host which is susceptible to HIV-I infection; and 4) screening and testing potential microbicides useful to treat HIV-I infection. The infectious HIV-I constructof the invention, EcoHIV, is a molecular virus chimera which was constructed based upon the foil length infectious HIV-I molecular clone, NL4-3, and the full length infectious ecotropic MLV clone, NCAC. A similar virus chimera was also constructed based upon the full- length infectious molecular clone of Clade D HIV-I, NDK. EcoHIV and EcoNDK can be recovered from harvest of the culture medium from mammalian cells transfected with the EcoHIV plasmid. The virus is competent to replicate in primary mouse splenic lymphocytes or primary mouse macrophages, producing HIV-I RNA, HIV-I core antigen p24, and fusogenic viral envelope. EcoHIV was shown to infect conventional immunocompetent mice after intravenous inoculation by detection of viral DNA in spleen, macrophages, and brain cells and by detection of serum antibodies to viral Tat and Gag proteins. EcoNDK was also shown to infect conventional mice.

[0007] In contrast with other strategies used to create animal models of HIV infection, EcoHIV replaces HIV-I envelope glycoprotein with MLV envelope, which provides the virus with receptors competent to interact directly with mouse cells. Other approaches instead modify the mouse itself by grafting human tissue or by introduction of human genes. Additionally, EcoHIV is advantageous because it is designed to be non-infectious to humans and it was shown not to infect human peripheral blood lymphocytes in culture. Thus, EcoHIV provides a less hazardous alternative to using HIV-I or its derivatives.

[0008] The invention provides compositions and methods for use in developing a rodent model of HIV-I infection and AIDS. More specifically, the invention provides compositions and methods for use in developing a murine model of HIV-I infection and AIDS for investigation of viral replication, control and pathogenesis.

[0009] The invention provides a chimeric HIV construct capable of infecting a rodent cell, comprising coding and regulatory regions of the HIV-I genome, and a heterologous viral envelope. In a specific embodiment, the invention provides a chimeric HIV-I construct capable of infecting a rodent cell, comprising coding and regulatory regions of the HIV-I genome, wherein the complete or partial coding region of gpl20 is replaced by the coding region for a heterologous viral envelope. Importantly, a molecular clone of HIV-I of any clade or construction can be used for construction of the chimeric construct of the invention. Similarly, a molecular clone of any heterologous viral envelope that permits infection of rodent cells can be used for construction of the chimeric construct of the invention. In a useful embodiment, the complete or partial coding region of gpl20 is replaced by the coding region for ecotropic murine leukemia virus gp80.

[0010] The invention also provides a method for producing a rodent model of HIV-I infection comprising administering the EcoHIV construct of the invention to a rodent. In another embodiment, the invention provides a rodent model of HIV-I infection and propagation in the rodent host in which at least the somatic cells are susceptible to infection by the EcoHIV construct of the invention and wherein expression of the construct is sufficient to effect phenotypic changes consistent with HIV-I pathology. In one embodiment, the rodent host is a mouse. However, any rodent may be used as a model in the invention including, but not necessarily limited to, all species of mouse and rat.

[0011] The invention additionally provides a rodent model of AIDS in which at least the somatic cells are susceptible to infection by EcoHIV and wherein the virus replicates consistent with the expression of HIV-I during human infection. In yet another embodiment, the invention provides a model of HIV-I infection of an immunocompetent rodent suitable for testing HIV- directed immunogenic compositions or vaccines, or other pharmaceutically veterinarilly suitable compositions for their efficacy in preventing infection in a subject inoculated with or exposed to EcoHIV during mating.

[0012] The invention also provides a model of HIV-I infection of an immunocompetent rodent suitable for testing HIV-I directed immunogenic compositions or vaccines, or other pharmaceutically or veterinarilly suitable compositions for their efficacy in reducing viral load in a subject inoculated with the EcoHIV construct. The invention further provides a model of HIV-I infection of an immunocompetent rodent suitable for testing HIV- 1 directed immunogenic compositions or vaccines, or other pharmaceutically or veterinarilly suitable compositions for their

efficacy in ameliorating disease in a subject inoculated with the EcoHIV construct. In addition, the invention provides a rodent model for treatment of HIV-I infection in which somatic cells are susceptible to infection by the EcoHIV construct and pharmaceutical interventions may be tested for their efficacy in reducing viral load.

[0013] An aspect of the invention provides for a rodent infected by an effective dose of a chimeric HIV-I construct comprising coding and regulatory regions of an HIV-I genome and a heterologous viral envelope gene, wherein expression of the construct generates phenotypic changes in the rodent associated with HIV-I pathology. In one embodiment, the heterologous viral envelope-coding region replaces a complete or partial coding region of HIV-I gpl 20. In another embodiment, the heterologous viral envelope-coding region is an ecotropic murine leukemia virus gp80 coding region. In a further embodiment, the construct is EcoNDK or EcoHIV. In some embodiments, the effective dose is at least about 1 x 10 pg of p24, at least about 1 x 10 ⁷ pg of p24, at least about 1 x 10 ⁸ pg of p24, at least about 1 x 10 ⁹ pg of p24, at least about 1 x 10 ¹⁰ pg of p24, at least about 1 x 10 ¹² pg of p24, or at least about 1 x 10 ¹⁵ pg of p24. In other embodiments, the rodent is a mouse or rat.

[0014] One aspect of the invention provides a method for identifying or testing efficacy of a candidate antiviral compound. The method comprises: (1) administering a candidate anti-viral compound to a rodent infected with an effective dose of a chimeric HIV-I construct comprising coding and regulatory regions of an HIV-I genome and a heterologous viral envelope gene, wherein the chimeric virus replicates in cells of the rodent pathology; and (2) determining whether the rodent exhibits a reduction in HIV infection as compared to a rodent infected with the chimeric HIV-I construct in the absence of the candidate compound, wherein a reduction in the HIV infection in the rodent that received the compound indicates that the compound is an antiviral compound. Another aspect of the invention provides a method for identifying or testing efficacy of a candidate antiviral compound. The method comprises: (1) administering a candidate anti-viral compound to a rodent prior to infection with an effective dose of the chimeric HIV-I construct comprising coding and regulatory regions of an HIV-I genome and a heterologous viral envelope gene, wherein the chimeric virus replicates in cells of the rodent pathology; and (2) determining whether the rodent exhibits a reduction in HIV infection as compared to a rodent infected with the chimeric HIV-I construct in the absence of the candidate compound, wherein a reduction in the HIV infection in the rodent that received the compound indicates that the compound is an antiviral

compound. The invention also provides a method for screening agents to identify an agent that can decrease levels of HIV infection in a subject. The method comprises: (1) administering a candidate agent to a rodent infected with an effective dose of a chimeric HIV- 1 construct comprising coding and regulatory regions of an HIV-I genome and a heterologous viral envelope gene; and (2) deteπnining the level of HIV-I infection in biological samples obtained from the rodent before and after administration of the agent, wherein a decrease in the levels of HIV infection in the rodent after administration indicates that the agent decreases levels of HIV infection. In one embodiment, the determining comprises measuring the level of HIV-I infection before and after administration of the candidate or compound. In another embodiment, the rodent is a rat or mouse. In a further embodiment, the heterologous viral envelope-coding region replaces a complete or partial coding region of HIV-I gpl20. In some embodiments, the heterologous viral envelope- coding region is an ecotropic murine leukemia virus gp80 coding region. In other embodiments, the construct is EcoNDK or EcoHIV. In further embodiments, the effective dose is at least about 1 x 10 pg of p24, at least about 1 x 10 ⁷ pg of p24, at least about 1 x 10 ⁸ pg of p24, at least about 1 x 10 ⁹ pg of p24, at least about 1 x 10 ¹⁰ pg of p24, at least about 1 x 10 ¹² pg of p24, or at least about 1 x 10 ¹⁵ pg of p24. In one embodiment, the antiretroviral drug is an HIV-I reverse transcription inhibitor. In another embodiment, the antiviral compound or agent comprises an antiviral drug, a vaccine, or a microbicide. In some embodiments, the antiviral compound or agent is an HIV-I reverse transcription inhibitor. In a further embodiment, the antiretroviral drug is 2', 3' dideoxycytidine (ddC) or abacavir, while in other embodiments, the inhibitor is 2\ 3' dideoxycytidine (ddC) or abacavir. In some embodiments, the determining comprises quantitating EcoNDK DNA or RNA levels. In further embodiments, the determining comprises quantitating EcoHIV DNA or RNA levels, while in some embodiments, the determining comprises quantitating antibody titres of HIV proteins. In one embodiment, the administering comprises subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; infusion; oral, nasal, or topical delivery; sexual transmission, or a combination of the administering modes described. In another embodiment, the sample comprises blood, serum, urine, tissue samples, or a combination thereof

[0015] Additional aspects of the invention are apparent in view of the description which follows.

DESCRIPTION OF THE FIGURES

[0016] FIG. IA depicts a map of the construction of the chimeric HIV-I, EcoHIV. The virus carries all HIV-I structural and regulatory genes, named above or under bars, except most of the coding region of gpl20. 1405 bp of gpl20 was excised and replaced by the coding region of the MLV ecotropic envelope gene gp80 with its stop codon in place. HIV-I cis- regulatory elements were preserved and expression of the entire construct is driven by the HIV-I LTR.

[0017J FIG. IB is a photograph of a western blot. Cultures were sampled over time for p24 expression by immunoblot, compared to human CEM cells infected by HIV-I or EcoHIV. The amount of p24 detected by Elisa in EcoHIV-infected lymphocyte cultures underestimates the protein detected by Western blot.

[0018] FIGS. 1 C-D are microscopy images. At seven days after infection cells were (FIG. 1C) stained for HIV-I antigens, right panel, uninfected cells were stained in parallel, left panel; or (FIG. ID) co-cultured with a nine-fold excess of uninfected cells and examined for syncytia after two days, right panel, uninfected cells co-cultured in parallel, left panel.

[0019] FIG. 2 shows detection of HIV-I DNA in various compartments of EcoHIV infected mice six weeks after infection (Panel A) Spleen and brain DNA were standardized by β- globin content for quantitation using standard curves as shown; macrophage samples are not standardized because of low amount of DNA obtained. 1-5: infected mice, C: uninfected mouse. Panel B shows viral DNA in CD4-positive but not CD4-negative splenic lymphocytes. 18: infected mouse, C: uninfected mouse. Panel C shows a dose response to EcoHIV infection testing viral DNA in the spleen. 26-29 and 34-37: infected mice, C: uninfected mouse. The table summarizes detection of EcoHIV DNA in various tissues in six independent experimental infections.

[0020] FIG. 3 demonstrates that EcoHIV infected mice show increased expression of MCP-I and complement C3 RNA in the brain. Gene expression was probed by RT-PCR on RNA isolated from brains of mice #6-10 and control animal using mouse-specific primers ; downstream primer was used for first-strand cDNA synthesis; the number of DNA PCR cycles is indicated. HIV-I infected human fetal astrocytes (VI, V2) probed with human C3 primers 7 days pi. served as positive control for C3, Cl, C2; uninfected astrocytes. RPS9: RT-PCR of

mouse ribosomal small protein 9 RNA, used for standardization of samples. The figure shows ethidium bromide staining.

[0021] FIG. 4 shows detection of expressed HIV-I genome in vivo and reactivation of virus in culture. Isolated spleen cells from mice 6 and 8 were either directly tested for the presence of Vif RNA by RT-PCR (Ex vivo) or cultured first for 2 days in the presence of concanavalin A to activate cells and virus and then tested (In vitro). C: uninfected mouse cells; CEMxHIVHIV-infected CEM cells.

[0022] FIG. 5 shows rescue of EcoHIV from spleens of infected animals by serial co- cultivation with uninfected mouse spleen cells. The culture was harvested at the fourth passage, stained with serum from an HIV-I infected person and FITC-labeled anti-human IgG to detect HIV-I antigens.

[0023] FIG. 6 demonstrates that EcoHIV infected mice produce anti-HIV-1 antibodies. A-B: Sera were collected 12 weeks after EcoHIV infection (6 weeks after infection for mouse #15), diluted as indicated in the Figure, and tested for binding HIV-I proteins in solid phase. Antibody binding was detected using radioiodinated anti-mouse Ig. (A) Recombinant HIV-I p55 was bound to wells at 250 ng per well. (B) Recombinant HIV-I Tat was bound to wells at 500 ng per well. Both recombinant proteins were provided by the NIH Aids Research Reagent Program.

[0024] FIG. 7 shows neuropathological findings in brains of mice 6-12 weeks after EcoHIV infection. (A-C), cellular aggregate in the region inferior to basal ganglia from mouse #129-8 as seen at low, medium, and high power (H&E). Note increased vascularity, pyknotic cells, and the possible multinucleated giant cell in the center of the field. (D) region inferior to basal ganglia in control uninfected mouse #129-C. No lesions were found. Medium power (H&E). (E), another, similar aggregate near to that shown in (A-C), inferior to basal ganglia in mouse #129-8, at medium power (H&E). (F), Leptomeningeal infiltrate, with mononuclear cells, mouse #129-1 , medium power (H&E). Mouse #129-1 and mouse #129-8 were evaluated 6 and 12 weeks after EcoHIV inoculation, respectively.

[0025] FIG. 8 shows impaired immune activation in lymphocytes from mice infected by EcoHIV. Spleen cells were harvested from mice infected by two injections of EcoHIV or from uninfected mice and were activated to the expression of the cytokine interferon-gamma by culture with concanavalin A. Interferon-gamma production was detected by flow cytometry. Infected

mouse T-I shows profound impairment of immune activation and infected mouse 2'-2 shows some reduction relative to the uninfected mouse.

[0026] FIG. 9 shows the infectivity and cellular response of EcoNDK, a chimeric virus based upon Clade D HIV-I NDK. Panel A shows EcoNDK viral DNA in spleen and brain three weeks after inoculation; a standard curve of amplification of plasmid DNA is at the right. Panel B shows quantitative RT-PCR of total cellular RNA from brain tissue of infected mice NDK 8 and 9, comparing levels of transcripts to levels found in the control brain. Asterisks indicate differences compared to control at p < 0.05 by t test. Panel C shows immunocytochemical staining for STAT-I in mouse cortical brain sections, arrows indicate examples of more intense staining for STAT-I in infected mouse NDK 8 (right panel) compared to the control brain (left panel). The final magnification as shown is 360x.

[0027] FIGS. 10A-B depict a scheme for insertion of gpl20 regions into EcoHIV.

[0028] FIG. 1 IA is a bar graph depicting rapid infection of mice with different batches of EcoNDK with efficient viral DNA and p24 production in the spleen. One mouse each was inoculated i.p. with 10 ⁷ pg p24 of a different independently prepared batch of EcoNDK and tested for viral DNA burden in spleen cells after five days (solid column). The striped column represents viral p24 core antigen content, a measure of EcoNDK expression in the spleen.

[0029] FIG. 11 B is a western blot depicting the detection of mature p24 protein in macrophages from infected mice. Two intact EcoNDK infected, but not UV-EcoNDK or vehicle treated mice clearly expressed mature p24 in macrophages, indicating that EcoNDK completes its life cycle in mouse cells in vivo.

[0030] FIG. 11C is a bar graph showing induction of antibodies to HIV-I structural and regulatory proteins in long term-infected mice. The peak titers obtained in mice 46, 47, and 49 are shown at 3-5 months after infection, indicating continuing production of viral structural and regulatory proteins several months after primary infection.

[0031] FIG. 12A-B demonstrate EcoHIV DNA and RNA burdens in mice after ddC administration. Real-time PCR was conducted on DNA (FIG. 12A) or cDNA synthesized from total RNA (FIG. 12B) isolated from splenic lymphocytes, wherein data were normalized by amplification of a cellular gene in parallel.

[0032] FIG. 12C is a blot depicting PCR conducted on the DNA extracts used in FIG. 12A, detected after electrophoresis, and transferred by hybridization with a radiolabeled probe. The mean DNA burden in copies per million spleen cells in vehicle-treated treated mice was 5552, in mice treated with 1.2 mg ddC, 897, and in mice treated with 6.0 mg, 213 with p < 0.001 by the Mann- Whitney test comparing either ddC treated group to the control. The mean spliced vz/RNA burden in copies per μg RNA in vehicle-treated treated mice was 144, in mice treated with 1.2 mg ddC, 9.1, and in mice treated with 6.0 mg, 5.5 with p < 0.001 by the Mann- Whitney test comparing either ddC treated group to the control.

[0033] FIG. 13 is a graph depicting HIV-I specific IgG. The left panel (A) shows serum anti-HIV-1 Gag levels over time in mice immunized with the control plasmid pUC19. The right panel (B) shows the responses of mice immunized with VRC 4306, each symbol represents the average titre of an individual mouse. The arrows indicate the times of infection by EcoHIV/NL4-3.

[0034] FIG. 14 is a graph depicting protection against EcoHIV/NL4-3 infection in VRC 4306 immunized mice. The left panel (A) shows the virus burden obtained by real-time PCR in spleen cells from mice immunized with VRC 4306 and pUC19 and infected by EcoHIV/NL4-3. The right panel (B) shows the virus burden in peritoneal macrophages from the same mice. Each symbol represents the average number of viral DNA copies, normalized by amplification of a cellular gene in tissue of an individual mouse. The horizontal line indicates the mean virus burden in each group.

[0035] FIG. 15 is a graph showing the expression of green fluorescence protein in macrophages that was analyzed by flow cytometry as a measurement of viral protein expression in cells from a mouse infected by sexual transmission of EcoHIV.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Studies of HIV-I pathogenesis have been hampered because of lack of a suitable animal model. Because the mouse immune system has been extensively researched, a murine model of HIV-I infection would be ideal and extremely valuable for evaluation of therapies and vaccines. However, prior to the present invention mouse cells were believed to be effectively resistant to HIV-I infection, replication and spread. Multiple blocks to productive infection of

mouse cell lines have been reported (Levy, et al., AIDS retrovirus (ARV-2) clone replicates in transfected human and animal fibroblasts, Science 232:998-1001 (1986); Maddon, et al., The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain, Cell 47:333-348 (1986); Winslow, et al., The blocks to human immunodeficiency virus type 1 Tat and Rev functions in mouse cell lines are independent, J. Virol. 67:2349-2354 (1993)). For example, blocks to HIV- replication have been reported at the level of virus entry, transcription, RNA transport, protein processing, and viron assembly (Bieniasz et al., (2000) J. Virol, 74:9868-9877; Mariani, et al, (200O)J. Virol 74:3859-3870). However, the inventors recently published findings indicating the primary murine cells are susceptible to HIV-I replication in tissue culture (Nitkiewicz et al., Productive infection of primary murine astrocytes, lymphocytes, and macrophages by human immunodeficiency virus type 1 in culture. JNeurovirol. 10:400-408 2004).

[0037] Since primary mouse cells are permissive to HIV-I expression, the principal difficulty in constructing mouse models of HIV-I infection rests in achieving efficient virus binding and entry into murine cells. Previous models employing mice or rats transgenic for the human receptors for HIV-I, CD4 and either CCR5 or CXCR4, failed to exhibit significant susceptibility to HIV-I infection (Sawada et al, J. Exp. Med, 187, 1439-1449, 1998; Browning et al, Proc. Natl. Acad. ScL USA, 94, 14637-14641, 1997). The inventors disclose herein, a different strategy based upon studies of infection in culture by HIV-I enveloped by heterologous proteins (Nitkiewicz et al, Journal ofNeuroVirology, 10:400-408, 2004; Hinkula et al, Cells Tissues Organs, 111, 169-184, 2004; Page e/ α/, J. Virol, 64, 5270-5276, 1990). Rather than endow mice with receptors for HIV-I, the inventors constructed HIV-I species with receptors for mouse cells. As described in the examples, which follow, the inventors converted the host species range of HIV-I from primate to rodent by replacing the coding region of its surface envelope glycoprotein, gpl20, with the envelope-coding region from ecotropic MLV that restricts the replication of the virus to rodents (Albritton et al, Cell, 57, 659-666, 1989). Two such chimeric viruses were constructed, EcoHIV on a backbone of Clade B NL4-3 (Adachi et al, J. Virol, 59, 284-291, 1986) and EcoNDK on a backbone of Clade D NDK (Ellrodt et al, Lancet, 1, 1383-1385, 1984). The chimeric virus replicated in murine lymphocytes but not human lymphocytes in culture. EcoHIV and EcoNDK established systemic infection in mice after one inoculation. Importantly, this experimental infection reproduced several major characteristics of HIV-I infection of human beings including virus targeting to lymphocytes and macrophages, induction of immune responses to viral proteins, neuroinvasiveness, and elevation of expression

of inflammatory and antiviral factors in the brain. These findings indicate that EcoHIV and similar chimeric viruses can serve as important tools for investigation of HIV-I disease and intervention in a versatile and convenient animal host.

[0038] New antiretro viral drugs capable of overcoming natural and acquired drug resistance are needed to control the HIV-I pandemic. Due, in part, to the absence of a small animal model of primary HIV-I infection, efficacy studies of candidate antiretro viral s are conducted only in human beings. The construction of chimeric HIV-I, EcoHIV and EcoNDK, which can infect conventional mice. Here, this model is also utilized to evaluate drug efficacy.

[0039] HIV-I infection continues to spread world-wide and antiretro viral therapy is not widely available outside resource-rich countries (Fauci, (2006) Science 313, 409). In the US, where treatment is common, the prevalence of drug resistant virus can reach 1 1% in treatment naive patients (S. J. Little, (2000) Antivir. Ther. 5, 33). The non-subtype B HIV-I that account for the vast majority of current infections harbor intrinsic resistance variants to most of the drugs in use (L. Vergne et al, (2006) J. Clin. Virol. 36, 43). Safe, new antiretro viral drugs, ideally of low cost and simple administration, are required to control the pandemic.

[0040] One impediment to timely drug discovery and development is the absence of a small animal model of primary HIV-I infection to predict clinical antiviral efficacy. Because of the absence of such a model and the urgency of drug development, no animal studies of HIV-I drug efficacy are commonly conducted prior to trials in human beings. A chimeric HIV-I was constructed that productively infects mice (Potash et al., (2005) Proc. Natl. Acad. ScL USA 102, 3760) with viral DNA, RNA, and p24 core antigen detected within days and viral proteins produced for months after infection (FIG. 1 1). EcoHIV and EcoNDK consist of a subtype B or a subtype D molecular clone in which the HIV-I envelope gene was replaced by the ecotropic murine leukemia virus envelope gene to switch the host range from humans to rodents. They maintain all other HIV-I coding regions and its LTR, permitting evaluation of most viral targets for intervention (FIG. 1 1) (Potash et al., (2005) Proc. Natl. Acad. Sci. USA 102, 3760). The utility of the system for antiviral drug evaluation has been improved by increasing the virus dose to uniformly infect all inoculated mice and reducing the time of evaluation (FIG. 11). Here, this system can be applied to test antiretro viral drug

efficacy in animals by showing that the HIV-I reverse transcription inhibitor 2',3'- dideoxycytidine (ddC) blocks EcoNDK infection in conventional, immunocompetent mice.

[0041] The versatility in inbred and genetically engineered mouse strains, combined with the extensive knowledge of the murine immune system makes the mouse an ideal animal for HIV and AIDS research. The present invention establishes for the first time a useful mouse model of HIV-I infection and AIDS which possesses many advantages over prior animal models of HIV and AIDS. A specific viral construct, EcoHIV, is provided which is capable of infecting rodent cells. The inventors have found that EcoHIV is infectious to normal mouse lymphocytes, producing infectious progeny virus. The virus of the present invention has the advantage of being non-infectious to human cells, making it safe for researchers compared with HIV-I or its derivatives, SHIV. The inventors disclose herein that EcoHIV infects conventional immunocompetent mice and induces antiviral immune responses. EcoHIV infected mice can be used for studies of viral replication, antiviral therapies, vaccines, microbicides, and pathogenesis.

[0042] Accordingly, the invention provides a chimeric HIV construct capable of infecting a rodent cell, comprising coding and regulatory regions of the HIV-I genome, and a heterologous viral envelope. In a specific embodiment, the present invention provides a chimeric viral construct capable of infecting a rodent cell comprising coding and regulatory regions of the HIV-I genome, wherein the complete or partial coding region of gpl20 is replaced by the coding region for a heterologous viral envelope. Importantly, a molecular clone of HIV-I of any clade or construction can be used for construction of the chimeric construct of the invention. A molecular clone of any heterologous viral envelope that permits infection of rodent cells can be used for construction of the chimeric construct of this invention. For example, a vector of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, or Mokola. In one embodiment, the complete or partial coding region of gpl20 is replaced by the coding region for ecotropic murine leukemia virus gp80.

[0043] The nucleic acids used to practice the invention can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems known and used in the art.

[0044] The invention also provides a method for producing a rodent model of HIV-I comprising administering the EcoHIV construct of the invention to a rodent. In one embodiment of the invention, infectious EcoHIV is recovered by transfection of an EcoHIV bacterial plasmid into a mammalian cell line in culture, and harvesting the culture medium from the transfected cells. Many mammalian cell lines can be used for transfection. In an embodiment of the invention, human embryonic kidney cell line 293T is used. The recovered virus is competent to replicate in primary mouse splenic lymphocytes in culture, producing HIV-I RNA, HIV-I core antigen p24 and fusogenic viral envelope.

[0045] In another embodiment of the invention, replication competent EcoHIV is produced by rodent cells infected in tissue culture. In an embodiment of the invention, the rodent can be infected with the replication competent EcoHIV by intraperitoneal or intravenous injection. In another embodiment of the invention, the rodent can be infected with isogenic rodent cells expressing the replication competent EcoHIV. In another embodiment of the invention, female rodents can be infected by mating with infected males. Any rodent may be used as a model in the invention including, but not necessarily limited to, all species of mouse and rat. In one embodiment, a mouse is used as the rodent model.

[0046] In a further embodiment, the invention provides a rodent model of HIV-I infection and propagation in a rodent host in which at least the somatic cells are susceptible to infection by the EcoHIV construct of the invention and wherein expression of the construct is sufficient to effect phenotypic changes consistent with HIV-I pathology. The term "propagation in a rodent host" as used in the invention refers to the capability of the infectious viral construct to go through more than one cycle of replication in rodent cells and rodents. The skilled artisan can readily identify the occurrence of clinical symptoms consistent with HIV-I infection and pathology. In one embodiment, the rodent model is a mouse. However, any rodent may be used as a model in the invention, including but not necessarily limited to, all species of mouse and rat.

[0047] The invention additionally provides a rodent model of AIDS in which at least the somatic cells are susceptible to infection by the EcoHIV construct and wherein expression of the construct is sufficient to effect phenotypic changes consistent with AIDS pathology. The skilled artisan can readily identify the occurrence of clinical symptoms consistent with AIDS pathology.

[0048] In yet another embodiment, the invention provides a model of HIV-I infection of an immunocompetent rodent suitable for testing an HIV-I directed immunogenic composition

or vaccine for its efficacy in preventing infection or reducing viral load in a subject inoculated with EcoHIV. Additionally, the invention provides a model of HIV-I infection of an immunocompetent rodent suitable for testing an HIV-I directed immunogenic composition or vaccine for its efficacy in ameliorating disease symptoms in a subject inoculated with EcoHIV. As used herein, the term "immunogenic composition" refers to a composition comprising an antigenic molecule where administration of the composition to a subject results in the development in the subject of a humoral and/or cellular immune response to the antigenic molecule or cross reacting molecules. As used herein, the term "ameliorating disease" refers to reducing HIV-I infection associated symptoms or pathology or AIDS associated symptoms or pathology. As used herein, the term "preventing disease" refers to preventing the initiation of HIV-I infection or AIDS, preventing HIV-I infection or AIDS, delaying the initiation of HIV-I infection or AIDS, preventing the progression or advancement of HIV-I infection or AIDS, slowing the progression or advancement of HIV-I infection or AIDS, and delaying the progression or advancement of HIV-I infection or AIDS.

[0049] The invention also provides a rodent model for treatment of HIV-I infection in which somatic cells are susceptible to infection by the EcoHIV construct and pharmaceutically acceptable compounds or veterinarilly acceptable compounds can be tested for their efficacy in reducing viral load. As used herein, the terms "pharmaceutically acceptable" or "veterinarilly acceptable" refer to material that may be administered to a subject in a composition without chums any deleterious or otherwise undesirable biological effects.

[0050] An animal model of AIDS treatment is also provided, wherein expression of the construct is sufficient to effect phenotypic changes consistent with HIV-I infection of humans that can be tested for amelioration by pharmaceutically acceptable or veterinary acceptable compounds. In one embodiment, a rodent infected by an effective dose of a chimeric HIV-I construct comprising coding and regulatory regions of an HIV-I genome and a heterologous viral envelope gene, wherein expression of the construct generates phenotypic changes in the rodent associated with HIV-I pathology. In another embodiment, the heterologous viral envelope-coding region replaces a complete or partial coding region of HIV-I gpl20. In a further embodiment, the heterologous viral envelope-coding region is an ecotropic murine leukemia virus gp80 coding region. In some embodiments, the construct is EcoNDK or EcoHIV. In other embodiments, the effective dose is at least about 1 x 10 ⁶ pg of p24, at least

about 1 x 10 ⁷ pg of p24, at least about 1 x 10 ^s pg of p24, at least about 1 x 10 ⁹ pg of p24, at least about 1 x 10 ¹⁰ pg of p24, at least about 1 x 10 ¹² pg of p24, or at least about 1 x 10 ¹⁵ pg of p24.

[0051] The invention provides methods for the screening of drug candidates or leads useful in treating or preventing HIV infection related diseases, such as AIDS. The methods can include binding assays and/or functional assays, and may be performed in vitro, in cell systems, or in animals.

[0052] In one embodiment, the invention provides a method for testing candidate compositions to treat or prevent HIV infection. In another embodiment, the invention provides for methods of testing the efficacy of a candidate anti-viral drug or vaccine, using a rodent infected with an effective dose of a chimeric HIV-I construct of the invention comprising coding and regulatory regions of an HIV-I genome and a heterologous viral envelope gene. The method can encompass: (a) administering the candidate drug or vaccine to a rodent infected with an effective dose of the chimeric HIV-I construct; and (b) determining whether the rodent exhibits a reduction in HIV infection as compared to a rodent described above in the absence of the candidate drug or vaccine. A decrease in the level of HIV infection in the rodent indicates that the drug is efficacious. In another embodiment, the invention provides a method for identifying or testing efficacy of a candidate antiviral compound, wherein the method comprises: (1) administering a candidate anti-viral compound to a rodent prior to infection with an effective dose of the chimeric HIV-I construct comprising coding and regulatory regions of an HIV-I genome and a heterologous viral envelope gene, wherein the chimeric virus replicates in cells of the rodent pathology; and (2) determining whether the rodent exhibits a reduction in HIV infection as compared to a rodent infected with the chimeric HIV-I construct in the absence of the candidate compound, wherein a reduction in the HIV infection in the rodent that received the compound indicates that the compound is an antiviral compound. In a further embodiment, the invention provides a method for screening for agents that decrease levels of HIV infection using a rodent infected with an effective dose of a chimeric HIV-I construct comprising coding and regulatory regions of an HIV-I genome and a heterologous viral envelope gene. The method can entail (a) providing a library of candidate agents; (b) administering a candidate agent to the rodent; (c) obtaining a biological sample from the rodent before and after (b); and (d) measuring the level of HIV-I infection in the sample before and after administration of the agent. A decrease in the levels of HIV infection after administration of the agent indicates

that the agent reduces the levels of HIV infection in the rodent. Non-limiting examples of biological samples include blood, serum, sputum, lacrimal secretions, semen, urine, vaginal secretions, and tissue samples (such as splenic, kidney, lung, or brain tissue).

[0053] Nucleic acids, vectors, capsids, or polypeptides can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g. fluid or gel precipitin reactions, immunodiffusion, Immunoelectrophoresis, .quadrature.adioimmunoassay (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography

[0054] The candidate agent may be of various origin, nature, and composition. It may be any organic or inorganic substance, such as a lipid, peptide, polypeptide, nucleic acid, or a small molecule, in isolated or in mixture with other substances. The candidate agent may be all or part of a combinatorial library of products, for instance. The candidate agent can be an antiviral agent, a vaccine or a microbicide. An antiviral agent is effective to inhibit the formation and/or replication of a virus in a mammal. This includes agents that interfere with either host or viral mechanisms necessary for the formation and/or replication of a virus in a mammal. Such agents can be, for example, an HIV inhibitor, a microbicide, a nucleic acid molecule (such as a DNA vaccine), or an antiviral drag (such as an anti-retroviral drug). Antiviral agents include, but are not limited to, ribavirin, amantadine, Levovirin, and Viramidine. A microbicide can be a compound or substance that plays a role in reducing the infectivity of microbes, such as viruses or bacteria.

[0055] In practicing the screening methods of the invention, a test compound is provided. It can be contacted with a polypeptide of the invention in vitro or administered to a cell of the invention or an animal of the invention in vivo. Compounds can also be screened using the compositions, cells, non-human animals and methods of the invention for their ability to ameliorate HIV infection and HIV related diseases or complications in an animal. Combinatorial chemical libraries are one means to assist in the generation of new chemical

compound leads for, e.g., a compound that can be used to treat or ameliorate HIV infection and HIV related diseases or complications.

[0056] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (see, e.g., Gallop et al. (1994) 37(9): 1233-1250). Preparation and screening of combinatorial chemical libraries are well known to those skilled in the art, see, e.g., U.S. Pat. Nos. 6,004,617; 5,985,356. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37: 487- 493, 1991, Houghton et al. Nature, 354: 84-88, 1991). Other chemistries for generating chemical diversity libraries include, but are not limited to: peptoids (see, e.g., WO 91/19735), encoded peptides (see, e.g., WO 93/20242), random bio-oligomers (see, e.g., WO 92/00091), benzodiazepines (see, e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (see, e.g., Hobbs, Proc. Nat. Acad. Sci. USA 90: 6909-6913, 1993), vinylogous polypeptides (see, e.g., Hagihara, J. Amer. Chem. Soc. 114: 6568, 1992), non-peptidal peptidomimetics with a Beta-D-Glucose scaffolding (see, e.g., Hirschmann, J. Amer. Chem. Soc. 1 14: 9217-9218, 1992), analogous organic syntheses of small compound libraries (see, e.g., Chen, J. Amer. Chem. Soc. 1 16: 2661, 1994), oligocarbamates (see, e.g., Cho, Science 261 : 1303, 1993), and/or peptidyl phosphonates (see, e.g., Campbell, J. Org. Chem. 59: 658, 1994). See also Gordon, J. Med. Chem. 37: 1385, 1994; for nucleic acid libraries, peptide nucleic acid libraries, see, e.g., U.S. Pat. No. 5,539,083; for antibody libraries, see, e.g., Vaughn, Nature Biotechnology 14: 309-314, 1996; for carbohydrate libraries, see, e.g., Liang et al. Science 274: 1520-1522, 1996, U.S. Pat. No. 5,593,853; for small organic molecule libraries, see, e.g., for isoprenoids U.S. Pat. No. 5,569,588; for thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; for pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; for morpholino compounds, U.S. Pat. No. 5,506,337; for benzodiazepines U.S. Pat. No. 5,288,514.

[0057] The following references are incorporated by reference: Parmley and Smith, (1989) Adv. Exp. Med. Biol. 251 :215-218; Scott and Smith, (1990) Science 249:386-390; Fowlkes et al, (1992) BioTechniques 13:422-427; Oldenburg et al., (1992) Proc. Natl. Acad. ScL USA 89:5393-5397; Yu et al., (1994) Cell 76:933-945; Staudt et al., (1988) Science 241 :577-580; Bock et al., (1992) Nature 355:564-566; Tuerk et al., (1992) Proc. Natl. Acad. ScL USA 89:6988-6992; Ellington et al., (1992) Nature 355:850-852; U.S. Patent Nos. 5,096,815; 5,223,409; and 5,198,346, all to Ladner et al.; Rebar et al., (1993) Science 263:671-673; and PCT Pub. WO 94/18318.

[0058] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., U.S. Pat. Nos. 6,045,755; 5,792,431 ; 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif, 9050 Plus, Millipore, Bedford, Mass.). A number of robotic systems have also been developed for solution phase chemistries. These systems include automated workstations, e.g., like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the invention. The nature and implementation of modifications to these devices is known to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, NJ. ; Asinex, Moscow, Ru; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., etc.).

[0059] In one embodiment of the invention, the agent can be administered to a subject once (e.g., as a single injection or deposition). Alternatively, the agent can be administered once or twice daily to a subject in need thereof for a period of from about two to about twenty-eight days, or from about seven to about ten days. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 times per year, or a combination thereof. Furthermore, the agent can be co-administrated with another therapeutic, such as a microbicide, an antibiotic, a chemotherapeutic compound, or a combination thereof. Where a dosage regimen comprises multiple administrations, the effective amount of an agent administered to the subject can comprise the total amount of an agent administered over the entire dosage regimen.

[0060] The agents can be administered to a subject by any means suitable for delivering the agents to cells of the subject. For example, the agents can be administered by methods suitable to transfect cells. Transfection methods for eukaryotic cells are well known in the art, and include direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate precipitation, and transfection mediated by viral vectors.

[0061] Antiviral agents and/or compounds (for example, those identified through the screening methods of the invention) can be formulated and administered to reduce HIV infection and/or the symptoms associated with HIV infection by any means that produces contact of the active ingredient with the agent's site of action in the body of an animal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

[0062] Pharmaceutical and therapeutic compositions comprising an antiviral agent or compound for use in accordance with the invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic and pharmaceutical compositions comprising an antiviral agent or compound of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa (1985), the entire disclosure of which is herein incorporated by reference. For systemic administration, injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions comprising an antiviral agent or compound of the invention can be formulated in liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the therapeutic and pharmaceutical compositions comprising an antiviral agent or compound may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the invention are characterized as being at

least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use.

[0063] The present pharmaceutical formulations can comprise an antiviral agent or compound identified by the screening methods of the invention (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier. The pharmaceutical formulations can also comprise antiviral agents and compounds which are encapsulated by liposomes and a pharmaceutically-acceptable carrier. Useful pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, or hyaluronic acid.

[0064] The pharmaceutical compositions can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTP A-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions can be packaged for use in liquid form, or can be lyophilized.

[0065] For solid pharmaceutical compositions comprising an antiviral agent or compound identified by the screening methods of the invention, conventional nontoxic solid pharmaceutically-acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, or magnesium carbonate.

[0066] Solid formulations can be used for enteral (oral) administration. They can be formulated as, e.g., pills, tablets, powders or capsules. For solid compositions, conventional nontoxic solid carriers can be used which include, e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, or magnesium carbonate. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10% to 95% of active ingredient (e.g., peptide). A non-solid formulation can also be used for enteral administration. The carrier can be

selected from various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, or sesame oil. Suitable pharmaceutical excipients include e.g., starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, or ethanol.

[0067] Nucleic acids, peptides, or polypeptides, when administered orally, can be protected from digestion. This can be accomplished either by complexing the nucleic acid, peptide or polypeptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the nucleic acid, peptide or polypeptide in an appropriately resistant carrier such as a liposome. Means of protecting compounds from digestion are well known in the art, see, e.g., Fix, Pharm Res. 13: 1760-1764, 1996; Samanen, J. Pharm. Pharmacol. 48: 119-135, 1996; U.S. Pat. No. 5,391,377, describing lipid compositions for oral delivery of therapeutic agents (for example, liposomal delivery).

[0068] For oral administration, the therapeutic and pharmaceutical compositions comprising an antiviral agent or compound may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

[0069] Preparations for oral administration may be suitably formulated to give controlled release of the active agent. For buccal administration the therapeutic and pharmaceutical compositions comprising an antiviral agent or compound may take the form of tablets or

lozenges formulated in a conventional manner. For administration by inhalation, the compositions for use according to the invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflate or may be formulated containing a powder mix of the therapeutic agents and a suitable powder base such as lactose or starch.

[0070] The therapeutic and pharmaceutical compositions comprising an antiviral agent or compound may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.

[0071] Suitable enteral administration routes for the methods of the invention include oral, rectal, or intranasal delivery. Suitable parenteral administration routes include intravascular administration (e.g. intravenous bolus injection, intravenous infusion, intraarterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation. The therapeutic and pharmaceutical compositions comprising an antiviral agent or compound identified by the screening methods of the invention can also be administered by injection, infusion, or oral delivery.

[0072] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.

In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the therapeutic and pharmaceutical compositions comprising an antiviral agent or compound identified by the screening methods of the invention are formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing. For oral administration, the therapeutic and pharmaceutical compositions comprising an antiviral agent or compound identified by the screening methods of the invention are formulated into conventional oral administration forms such as capsules, tablets, and tonics.

[0073] Therapeutic and pharmaceutical compositions comprising an antiviral agent or compound identified by the screening methods of the invention can also be formulated as a sustained and/or timed release formulation. Such sustained and/or timed release formulations may be made by sustained release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of which are each incorporated herein by reference. The therapeutic and pharmaceutical compositions comprising an antiviral agent or compound identified by the screening methods of the invention can be used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions. Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, may be readily selected for use with the therapeutic and pharmaceutical compositions comprising an antiviral agent or compound identified by the screening methods of the invention. Single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gelcaps, caplets, or powders, that are adapted for sustained release are encompassed by the invention.

[0074] The antiviral agents can be administered to the subject either as RNA, in conjunction with a delivery reagent, or as a nucleic acid (e.g., a recombinant plasmid or viral vector) comprising sequences which express a gene product of interest. Suitable delivery reagents for administration of the antiviral agents or compounds include the Mirus Transit

TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.

[0075] The dosage administered can be a therapeutically effective amount of a pharmaceutical composition comprising an antiviral agent or compound identified by the screening methods of the invention that is sufficient to ameliorate HIV-I related symptoms in a subject, which can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired.

[0076] Toxicity and therapeutic efficacy of therapeutic compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (The Dose Lethal To 50% Of The Population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapeutic agents which exhibit large therapeutic indices are useful. Therapeutic compositions that exhibit some toxic side effects can be used.

[0077] A therapeutically effective dose of the antiviral agents or compounds identified by the screening methods of the invention depend upon a number of factors known to those or ordinary skill in the art. The dose(s) of the antiviral agents or compounds can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the antiviral agents or compounds to have upon the subject. Exemplary doses can include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram.

[0078] The methods described herein are by no means all-inclusive, and further methods to suit the specific application is understood by the ordinary skilled artisan. Moreover, the effective amount of the therapeutic and pharmaceutical compositions comprising an antiviral

agent or compound identified by the screening methods of the invention can be further approximated through analogy to compounds known to exert the desired effect.

[0079] The practice of aspects of the invention can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). All patents, patent applications and references cited herein are incorporated in their entirety by reference.

[0080] The following examples illustrate the invention, and are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.

EXAMPLES

Example 1 - Materials and Methods

Design of EcoHIV, a Chimeric HIV-I Construct that Targets Rodent Cells

[0081] EcoHIV is designed to carry all the coding and regulatory regions of the HIV-I genome, except for the gene encoding the viral envelope glycoprotein, gpl20 that targets the virus to the CD4 bearing cells. In its place, the coding region for ecotropic murine leukemia virus gp80, that targets the virus to rodent, but not to human cells, was inserted. Specifically, EcoHIV was constructed based upon the full-length infectious HIV-I molecular clone, NL4-3, and the full-length infectious ecotropic MLV clone, NCAC. Referring to the numbering systems ofNL4-3 and NCAC, respectively, a fragment from nucleotide 6310 to 7750, encoding amino acids 31 to 510 of HIV-I gpl20 was excised from NL4-3 and replaced in frame with a fragment from nucleotide 6129 to 8823 of NCAC, encoding amino acids 2 to 697 and the termination codon of gp80. The resulting envelope glycoprotein contains 727 amino acids. The overlapping coding sequences of Vpu, Tat, and Rev as well as the cis Rev response elements of HIV-I are preserved in the final construct. The resulting EcoHIV resides on the bacterial plasmid, pUC 18. Infectious EcoHIV can be recovered by transfection of the bacterial plasmid into a mammalian cell line in culture, for example the human embryonic kidney cell line, 293T, and harvest of the culture medium from transfected cells. The culture medium contains a virus competent to replicate in primary mouse splenic lymphocytes, producing HIV DNA, HIV-I RNA, HIV-I core antigen p24 and fusogenic viral envelope. Viral infection can be maintained by the addition of uninfected splenic lymphocytes to infected cells, a method in which EcoHIV is produced in an infectious form and transmitted in culture.

[0082] EcoHIV is prepared for infection by transfection of plasmid DNA into human embryonic kidney cells in culture, bypassing receptor-mediated entry. In several tissue culture studies, it was confiiτned that EcoHIV is replication competent in mouse lymphocytes but unable to infect either primary or transformed human cells. Infected mouse lymphocytes produced HIV-I Gag protein and mature core antigen p24 as shown by Western blot and immunofluorescent staining.

[0083] EcoNDK was similarly constructed using Clade D NDK as backbone and MLV NCAC to provide the gp80 coding region. Nucleotides 5846-7265, encoding gpl20 were deleted from NDK and nucleotide 6129 to 8823 of NCAC, encoding amino acids 2 to 697 and the termination codon of gp80 were inserted in frame. EcoNDK resides on the bacterial plasmid pUCl 8 and is prepared by transfection of plasmid DNA in culture.

Mice, Cells and Sample Preparation

[0084] All animal studies were conducted with the approval of the St. Luke's-Roosevelt IACUC. Adult female BALB/c, 129P3, and 129X1 mice were purchased from Jackson Labs (Bar Harbor, ME). For inoculation of EcoHIV, mice were anesthetized with isoflurane and a 0.1ml solution of virus stock was injected into the tail vein. Prior to euthanasia, mice were anesthetized for bleeding, then they were subjected to carbon dioxide asphyxiation, spleens, brains, lungs, and kidneys were surgically removed, and peritoneal macrophages were harvested by peritoneal wash. Spleen cell suspensions were prepared and cultures were established as described (Nitkiewicz et al, Journal ofNeuroVirology, 10:400-408, 2004). Cells were fractionated into CD4-bearing and CD4-negative using Dynabeads (Dynal, Oslo, Norway) and fractionation was confirmed by flow cytometry.

[0085J Splenic lymphocytes were infected in culture with 0.5 pg p24 EcoHIV per cell and harvested later for immunoblot or microscopy. Transformed human CEM cells infected with HIV-I or EcoHIV served as controls. For PCR, brain, kidney, or lung tissue was weighed and then homogenized using a disposable pestle. DNA was isolated from cells or tissues using DNAzol (Invitrogen, Carlsbad, CA), precipitated with ethanol, and resuspended in water; RNA was isolated with either Trizol (Invitrogen) or with Rneasy (Qiagen, Valencia, CA).

PCR

[0086J EcoHIV DNA present in 5 x 10 ^s lymphocytes or 1.25 mg brain, kidney, or lung was amplified using primers NE5 (5'-ATGATCTGTAGTGCTGCGCGTTCAACG-S') (SEQ ID NO:1) and Eco3 (S'-GAGCCGGGCGAAGCAGTACTGACCCCTC-S') (SEQ ID NO: 2) that span the joint between HIV-I and MLV, amplification was conducted as described (Chowdhury et al, J. NeuroVirol, 8, 599-610, 2002), and reaction products were detected using the 32P-labeled probe, EcoP3 (5'-GGTTAACCCGCGAGGCCCCCTAATCCCC-S') (SEQ ID NO: 3). The single copy cellular gene, β globin, was amplified in parallel to standardize DNA input. For detection of singly spliced Vif RNA, cDNA was prepared and amplified as described (Chowdhury et al., J. NeuroVirol, 8, 599-610, 2002) using sense primer nt 616-641, antisense primer nt 5071-5091 , and radiolabeled probe nt 5127-5156 with numbering according to the NL4-3 genome.

Quantitative real-time RT-PCR (QPCR)

[0087] For QPCR, RNA was isolated from brain tissue using a modified Trizol protocol optimized to handle the high lipid component of brain homogenates. RNA quality was assessed in the Agilent 2100 bioanalyzer. Twenty ng of total RNA was used for Whole Transcriptome Amplification (WTA), based on Ribo-SPIAT technology from NuGEN Technologies Inc. (San Carlos, CA), to generate cDNA, according to the manufacturer's protocol. The size distribution and linearity of amplification was measured prior to quantitative analysis. Expression of selected cellular genes in the brain was examined using Taqman chemistry with MGB probes and primers selected from the Applied Biosystems Assay on Demand program. The relative efficiency of all assays was compared to glyceraldehyde 3 phosphate dehydrogenase and was within the parameters established for δδ Ct analysis. Following probe and primer optimization all cDNA's were diluted 1 :300 following amplification and Bused as described previously (Kim et al, J. Neuroimmunol, 157:17-26, 2004). Test transcript values were normalized to levels of glyceraldehyde 3 phosphate dehydrogenase and presented as fold change versus the levels in control brain samples.

Fluorescence Microscopy

[0088] Microscopy was conducted as described (Nitkiewicz et al, Journal of NeuroVirology, 10:400-408, 2004) using serum from an AIDS patient and FITC-labeled anti- human Ig (Sigma, St. Louis, MO). Nuclei were stained with propidium iodide. Images were captured using a Zeiss Model Axioplan 2 microscope (Carl Zeiss) with a HAMAMATSU ORCA- ER digital camera (HAMAMATSU Corp).

Immunoblot and Radioimmunoassay

[0089] Immunoblot was conducted as described (Nitkiewicz et al., Journal of NeuroVirology, 10:400-408, 2004). Solid phase-radioimmunoassays (RIA) were constructed by coupling purified recombinant viral proteins (NIH AIDS Research Reagent Repository) to immunolon wells (Dynatech Laboratories, Chantilly, VA) using 250 ng Gag per well or 500 ng Tat per well. Dilutions of mouse sera were added to wells and immunoglobulin (Ig) binding was detected using ¹²⁵ I-labeled anti-mouse Ig (Amersham Biosciences, Piscataway, NJ).

Immunocytochemistry of Brain Sections

[0090] After euthanasia of control and EcoNDK infected mice, a sagital portion of the cerebral hemisphere was fixed in 10% neutral buffered formalin, then each brain was cut into an average of five coronal sections, dehydrated, embedded in paraffin, and sections of 6μ were cut for staining and microscopy. Sections were subjected to basic indirect immunohistochemical staining using a biotin-avidin system as described (Sharer et al, J. Med. Primatol, . 20, 211 -217, 1991) with rabbit polyclonal anti-STATl sc-346 (Santa Cruz Biotechnology, Santa Cruz, CA) binding detected with diaminobenzidine as a chromogen and hematoxylin and eosin counterstaining. Negative control slides were run omitting the primary anti-STAT-1 antibody. Sections were examined in a Zeiss Photomicroscope III and images were captured using a Nikon DNlOO digital camera.

Example 2 - Results

Host Range of EcoHIV in Culture

[0091J To redirect HIV-I to infect the rodent, the ecotropic MLV gp80 gene carrying its own stop codon was inserted in-frame into the NL4-3 env gene, preserving the first 90 coding residues, deleting the subsequent 1440, and resuming HIV-I near the beginning of the gp41 coding region (FIG. IA). The resulting chimeric virus, EcoHIV, contains all the known coding and regulatory regions of the HIV-I genome with the exception of gpl 20; gp41 is unlikely to be expressed because it lacks an in-frame codon for initiation of translation. The biological activity of EcoHIV in culture was tested using several approaches. Mitogen stimulated murine splenic lymphocytes were infected and cells harvested over one week of infection for analysis of the expression of HIV-I p24 by Western blot (FIG. 1 B). Cells of the transformed human T cell line, CEM, were exposed to HIV-I or to EcoHIV as positive and negative controls, respectively. Fully processed p24 increased in amount with time after infection of mouse cells indicating that it was newly synthesized and properly processed by HIV-I protease. In contrast, human CEM cells were not susceptible to infection by the EcoHIV. In similar studies the inventors infected primary mitogen stimulated human lymphocytes or transformed mouse cells with EcoHIV and could detect no p24 production. EcoHIV infected mouse lymphocytes were also examined for the presence of viral antigens by indirect immunofluorescence staining with AIDS patient serum or for the presence of syncytia during co-cultivation with fresh splenic lymphocytes (FIG. IC-D). HIV-I antigens were detected in EcoHIV infected but not in uninfected mouse lymphocytes, at a frequency of approximately 10%, similar to what was observed during infection of mouse lymphocytes by pseudotyped HIV-I (Nitkiewicz et al,

Journal ofNeuroVirology, 10:400-408, 2004), both of which are less efficient infections than the infection of human lymphocytes by HIV-I in culture. Upon co-cultivation EcoHIV infected cells formed large syncytia, indicating that gp80 is properly cleaved to fusion competent proteins. These findings demonstrate that EcoHIV is biologically active, it can productively infect primary murine lymphocytes but not transformed lymphocytes in culture, and that it acquired the host range of ecotropic MLV and is unable to infect human cells.

Evidence of Persistent Productive Infection of Mice by EcoHIV Including

Evidence of Neuroinvasion and Neuropathology and Impaired Immune Function

[0092] Based upon the demonstration of EcoHIV infection of murine lymphocytes in culture, the inventors tested whether EcoHIV can also establish infection in vivo, in conventional mice (FIG. 2). Adult immunocompetent mice were inoculated by a single intravenous injection of 10 ^s pg p24 EcoHIV. Six weeks after infection or mock-infection, mice were euthanized and tissues were collected for analysis (FIG. 2A). Guided by the cell types infected by HIV-I in human beings, the inventors tested viral infection in lymphocytes, macrophages, and brain cells by PCR amplification for a region unique to the EcoHIV genome that spans the joint between HIV-I and MLV. DNA from 5 x 10 ⁵ spleen cells or 1.25 mg brain tissue was run in each reaction. Because only about 5-10 x 10 ⁵ peritoneal macrophages were obtained from each animal, the entire sample was subjected to amplification. Viral DNA was detected in one or more tissues of 4 of the 5 mice injected. The peak virus burden attained in the spleen at three or six weeks after infection, approximately 1 in 1 ,000 cells carrying viral DNA, is similar to the range of 1 in 200 to 1 in 20,000 HIV-I DNA positive cells observed in resting lymphocytes in HIV-I infected human beings (Chun et al, Proc. Natl. Acad. ScL USA, 95, 8869- 8873, 1998). In contrast to EcoHIV infection in culture, the efficiency of EcoHIV infection in vivo is comparable to that of HIV-I .

[0093] The inventors also performed limited investigations of the cell and tissue tropism of EcoHIV in the mouse, testing known infected mice, and the dose response of infection. Splenic lymphocytes were fractionated into CD4-positive and CD4-negative populations prior to DNA PCR for EcoHIV (FIG. 2D). CD4-positive but not CD4-negative lymphocytes carried viral DNA and the DNA burden was higher in CD4-positive cells than in unfractionated cells. In all, 2 out of 3 mice tested carried viral DNA in CD4-positive but not in CD4- negative splenic lymphocytes and none of 4 mice tested carried viral DNA in lungs or kidneys.

The lowest dose of EcoHIV tested, 3 x 10 ⁴ pg, infected 3 out of 4 mice tested at six weeks after infection and raising the dose 10-fold yielded uniform infection in 4 mice (FIG. 2C). The table in FIG. 2 shows a summary of the results of virus detection in different tissues from six independent experiments of inoculation of mice with EcoHIV at doses from 3 x 10 ⁴ pg to 5 x 10 pg p24 per mouse. EcoHIV was most frequently detected in splenic lymphocytes, but in two mice virus was present in the brain but not the spleen; a total of 33 of 43 mice tested carried EcoHIV DNA in one or more tissues. Overall the initiation of spreading infection after a single exposure, replication competence, infectivity of progeny, and tissue distribution of EcoHIV in the mouse reproduce several important features of the natural infection of human beings by HIV-I .

[0094] FIG. 2 shows detection of EcoHIV DNA at 6 weeks post inoculation and summarizes findings of several experiments. FIG. 3 shows changes in inflammatory gene expression in infected mouse brains. FIGS. 4 and 5 show evidence of EcoHIV replication in mice. FIG. 6 documents the production and specificity of antiviral antibodies in infected animals, and FIG. 7 shows neuropathological changes at 6 and 12 weeks. FIG. 8 shows impaired immune function in mice receiving two injections of EcoHIV.

[0095] As shown in FIG. 2, EcoHIV DNA was found in the spleens of the majority of inoculated mice and at a lower frequency in peritoneal macrophages or in the brain. Infection in the brain may be under-represented because the procedures of DNA extraction and PCR amplification from the brain are currently in the process of being optimized. EcoHIV DNA was present in CD4-bearing lymphocytes and the virus burden in the spleen increased with increasing virus dose. As shown in the table summarizing six experiments, detection of EcoHIV DNA in the spleen was clearly the strongest indicator of infection, and by that measure 73% of EcoHIV inoculated mice became persistently infected with the virus. Thus, viral DNA can be reproducibly detected in a large proportion of EcoHIV infected mice, the virus persists for months after infection, and at least in some animals, virus enters the brain. The presence of EcoHIV DNA in lymphocytes, macrophages, and the brain but not in lung or kidney indicates that EcoHIV has a tissue distribution similar to mat of HIV-I in human beings.

Brain pathology

[0096] As one measure of neuropathology, brain tissue RNA from these mice was tested for transcriptional activation of genes coding for molecules implicated in neuropathogenesis, such as MCP-I, C3, and the EFN-induced factor Cig5 (FIG. 3). MCP-I is a marker of HIV- 1- and SIV-associated brain disease, elevated C3 was correlated with neuroinflammation, and IFN-related genes are modulated in brains of SIV infected macaques. C3 and Cig5 are modulated specifically in HIV-I infected human astrocytes in culture, suggesting that they may be useful as cell-specific molecular markers of HIV-I neuropathogenesis. As shown in FIG. 3, MCP-I and C3 RNA were significantly increased in brain tissues from infected mice #6 and #8, as was C3 in cultured HIV-I -infected human fetal astrocytes serving as control. Cig5 expression was reduced in infected mice consistent with the temporal pattern of expression of IFN related genes in HIV-I- infected macrophages and astrocytes observed in vitro. FIG. 7 presents direct neuropathological studies in EcoHIV infected mice and FIG. 9 shows changes in host cell gene expression in mice infected by EcoNDK. EcoHIV-infected mouse #8 had brain lesions (FIG. 7) and altered expression of three molecular markers in brain tissue. Mouse #6 had elevated MCP-I and C3 but no brain lesions, suggesting that pathogenic changes in the brain can potentially be detected in this model by sensitive molecular assays prior to appearance of overt histopathology.

[0097] A preliminary histopathological examination of brain tissues from one experiment was performed. Brain lesions containing cellular infiltrates or aggregates were found in the brain of EcoHrV-infected mouse #1 (6 weeks p.i.) and mouse #8 (12 weeks pi.), but not in brains from control mice or other infected animals. Note a large multinucleated cell in the middle of the lesion in FIG. 7. Similar multinucleated giant cells are the hallmark of HIV-I encephalitis. Thorough examination of these samples is ongoing, including staining for cell types present in the lesions. These results provide the most direct evidence that EcoHIV infection of mice is pathogenic and in some animals, induces a brain disease highly analogous to that observed in HIV-I infected patients. EcoNDK is also neuroinvasive and potentially neuropatho genie, see FIG. 9.

Evidence of HIV-I replication in vivo and humoral response to virus

[0098] In seeking direct evidence for virus expression and production in animals, spleen cells isolated from mice infected for 3 months were tested for Vif RNA by RT-PCR either immediately or after stimulation in culture (FIG. 4). Vif RNA is a singly spliced viral transcript produced during HIV-I replication. Vif RNA signals were detected in directly tested cells from mouse #6 and #9 and culture of mouse #6 cells clearly increased Vif RNA levels

indicating virus reactivation or increased virus expression. Consistent with the expression of viral RNA, spleen cells infected in the mouse produced progeny virus that could spread infection in culture (FIG. 5). These results show that EcoHIV is folly replication competent, that it is expressed at low levels in vivo, and that infected cell activation increases virus expression. This pattern of Eco HIV expression in mice is reminiscent of that in HIV-I infected patients.

[0099J In natural virus infections, antiviral immune responses generally accompany ongoing virus replication. Because immunocompetent mice were used for EcoHIV inoculation, detection of antiviral responses is an indicator of the endogenous production of viral proteins through active EcoHIV infection as well as direct evaluation of the ability of the mouse to mount anti-HIV immune responses to HIV-I antigens presented during infection. Using sera from mice 3 months after infection the humoral response to HIV-I structural protein Gag or regulatory protein Tat was tested by radioimmunoassay (FIG. 6). Serum from mouse #15 was collected six weeks after infection. At 12 weeks after infection, four out of five mice also had antibodies to HIV-I Gag and Tat (FIG. 6) and also carried viral DNA in the spleen; mouse #15 was both seropositive and positive for viral DNA six weeks after infection. Mouse #9 carried no viral DNA in the spleen and was seronegative for antiviral antibodies. Initial studies indicated that the titers of the mouse antibodies ranged from 1 :40 to >1 :320. The presence of an antiviral immune response at 3 months after infection and induction of a response against viral Tat which is not present in the virus particle indicate continuing virus replication in the body as well as the fact that the humoral immune response to virus in mice remains functional. Both observations are important for the feasibility of testing vaccine using the EcoHIV model of mouse infection. The concordance between detection of viral DNA and detection of antiviral antibodies provides another confirmation of active virus replication and spread in EcoHIV infected mice.

Impaired immune function after EcoHIV infection

[0100] FIG. 8 provides preliminary data that EcoHIV can induce impairment of immune function in infected mice. In a pilot study, lymphocytes from mice receiving two injections of EcoHIV failed to respond to immune activation in culture by the production of the key cytokine interferon-gamma. These findings raise the possibility that further modification of the EcoHIV infection protocol may result in immune deficiency in mice.

Infection of mice by a virus chimera based upon an African HIV-I

[0101] To determine the general applicability of this approach to the study of different natural HIV-I species in mice, the inventors constructed EcoNDK with the MLV gp80 gene inserted into NDK, a highly cytopathic Clade D HIV-I (Ellrodt et al, Lancet, 1, 1383 - 1385,1984). EcoNDK contains the gag gene of NDK that contributes to its high virulence in culture (de Mareuil et al, J. Virol, 66, 6797-6801, 1992) and can enhance its activity in the mouse. Three weeks after inoculation with 10 pg p24 EcoNDK three mice were euthanized and spleen and brain were collected. Viral DNA was detected in splenic lymphocytes from each mouse, and two of the three mice also carried viral DNA in the brain (FIG. 4A). NDK8 had about 1 copy of viral DNA per 5,000 lymphocytes, comparable to mice infected by EcoHIV (FIG. 2) and also comparable to HIV-I infected persons (Chun et al, Proc. Natl Acad. Set USA, 95, 8869-8873, 1998). In addition, this is the earliest time that the inventors assayed mouse brain tissue for the presence of chimeric HIV-I and it is clear that the virus can invade the brain by three weeks after exposure.

[0102] Immune deficiency or neurological impairment by HIV-I infection of human beings takes years to develop and indeed the inventors have observed no overt signs of immune dysfunction or marked encephalitis in EcoHIV infected mice, most of which were euthanized six weeks after inoculation. However, molecular markers of cellular abnormalities associated with SIV infection or HIV-I infection in the brain have been described, some of which predict later neurological disease (Zink et al, J. Infect. Dis., 184, 1015-1021, 2001; Conant et al, Proc. Natl. Acad. Set USA, 95, 3117-3121, 1998; Lane et al, MoI Med., 2, 27-37, 1996; Roberts et al., Am. J. Pathol, 162, 2041-2057, 2003). To investigate subtle changes that may occur early in HIV-I infection of the mouse, the inventors determined the expression of complement component C3, IL-I β, IL-6, MCP-I, and STAT-I that are among the factors that influence inflammatory or antiviral responses to HIV-I in the brain. QPCR was conducted, expression normalized to a housekeeping transcript, and the data are reported as fold increase relative to transcript levels in brain tissue of the control mouse (FIG. 4B). NDK 8, which had the highest virus burden in spleen and the brain, showed significant increases in the expression of C3, IL-I β, MCP-I, and STAT-I . Increased expression of C3 was also seen in viral DNA positive brains from two mice infected by EcoHIV. NDK 9 had lower levels of EcoNDK in brain and spleen and showed significant increases in the expression of IL- 1 β and STAT- 1 , but not in C3 and MCP- 1. IL-6, expression was similar in NDK 8, NDK 9, and the control mouse brain. Because STAT-I in NDK 8 was the most highly induced transcript observed, the inventors tested STAT-I protein expression in cortical brain sections of NDK 8 versus the control mouse (FIG. 4C). Increased

expression of STAT-I protein was observed in cytoplasm of neurons in NDK 8 relative to the control but not in other cell types. These findings indicate that the approach to construction of HTV- 1 tropic to mice can be generalized to different HIV-I backbones. Moreover they indicate that in only a few weeks of infection, chimeric HIV-I elicits cellular responses in mouse brain like those seen in HIV-I or SIV infection in the brain (Zink et ah, J. Infect. Dis., 184, 1015-1021, 2001; Conant et al, Proc. Natl. Acad. ScL USA, 95, 3117-3121, 1998; Lane et al, MoI. Med., 2, 27-37, 1996; Roberts et. al, Am. J. Pathol, 162,2041-2057,2003).

[0103] As shown in FIG. 9, EcoNDK, a chimeric virus based upon an African Clade D HIV-I and MLV is also replication competent. Three weeks after inoculation, all mice tested carried EcoNDK DNA in the spleen. Two mice also carried viral DNA in the brain and these mice suffered changes in cellular RNA expression in the brain consistent with the presence of the virus. It is noteworthy that the cellular genes activated by exposure to EcoNDK have also been found to be activated by exposure of human cells to HIV-I . In addition, STAT-I expression in neurons, as shown in the brain of EcoNDK infected mouse #8, has also been observed in macaques suffering SIV encephalitis. These findings indicate the generality of the approach of constructing HIV-I competent to replicate in mice and alter host physiology. Moreover, it shows that the approach can be used to investigate the replication and control of HIV-I of African origin.

Insertion of gpl20 domains into gp80 of EcoHIV

[0104] Because the binding of the HIV-I envelope glycoprotein to human cells can be pathogenic, a strategy was developed for reconstructing EcoHIV to include gpl20 regions V3, VI/V2, or V1-V3 implicated in binding certain cellular co-receptors. The summary of steps for these insertions from HXB-2 into gp80 in EcoHIV is shown in FIG. 10. The insertion point is between amino acid 264 and 265, within the proline-rich region that tolerated insertion of GFP and yielded viable MLV-GFP virus. Several viral clones were obtained, V3 insertion was confirmed by sequencing. Transfection DNA into human embryonic kidney cells yielded viable virus in each case indicating that these insertions can be tolerated. The option for insertion of potentially pathogenic or antigenic domains from HIV-I gpl20 into functional EcoHIV may be valuable for reproducing certain aspects of HIV-I mediated pathogenesis in the mouse model or for induction of anti-gpl20 immune responses in vaccine development.

Discussion

[0105] The mouse model of HIV-I infection introduced here consists of inoculation of conventional mice with a chimeric HIV-I that employs species-specific cellular receptors to enter mouse cells. The infection spreads to multiple organs, induces antiviral immune responses, and alters cellular gene expression in the brain. Because the ecotropic envelope that they carry does not mediate entry into human cells, EcoHIV and EcoNDK are less hazardous than are HIV-I and SIV. These chimeric viruses can be useful for modeling many aspects of HIV-I infection of human beings in a tractable animal host.

[0106] The infectivity of EcoHIV in murine lymphocytes in culture demonstrates that the HIV-I genome tolerates the insertion of the MLV envelope-coding region and that the essential cis-regulatory elements of HIV-I as well as all the coding regions are functional. It also indicates that the ecotropic gp80 associates with the HIV-I core to mediate virus entry, as has been shown in studies analogous to ours in construction of a replication competent chimera of HTLV-I and MLV (Delebecque et al, J. Virol, 76, 7883-7889, 2002). Because HIV-I protease is active at the cell membrane during virion budding (Kaplan et al, Journal of Virology, 68, 6782-6786, 1994) and can cleave MLV pi5 to pi 2 (Kieraan et al, Journal of Virology, 72, 9621- 9627, 1998), fusogenic pl2 is likely to be present at the cell surface to mediate the observed cell fusion that is not generally seen with MLV. This gain of function may facilitate cell-to-cell transmission of the virus in the mouse. In culture, the host range of EcoHIV reproduces that of ecotropic MLV, EcoHIV infects mouse but not human lymphocytes, but only a minority of cells are infected, raising the possibility that EcoHIV targets a sub-population of cells. This proposal is under investigation. In the mouse, ecotropic MLV replicates in T lymphocytes with both envelope and the viral LTR contributing to tropism (Rosen et al, Journal of Virology, 55, 862- 866, 1985; Evans et ah, J. Virol, 61, 1350-1357, 1987) but EcoHIV replicates in macrophages and the brain, as well as in lymphocytes. Neurotropism and neuropathogenesis are common features of lend viruses including HIV-I, in part because of their replication in macrophages (Patrick et al, J. Virol, 76, 7923-7931 , 2002). However, HIV-I gpl20 that targets the virus to human macrophages as well as T cells is absent from EcoHIV. The LTR present in EcoHIV does not influence HIV-I tropism in cell culture (Pomerantz et al, Journal of Virology, 65, 1041-1045, 1991) but it is possible that it affects the EcoHIV host range the inventors observed in the animal. Further research is required to determine the basis for viral infection and expression in different tissues in EcoHIV-infected animals.

[0107] One inoculation of about 10 ⁵ pg of p24 was sufficient to establish EcoHIV infection in more than 75% of the mice tested and viral DNA was detected in the major target cell types of HIV-I. EcoHIV and EcoNDK reached virus burdens in the spleen comparable to HIV-I burdens in resting lymphocytes in human beings (Chun et al, Proc. Natl. Acad. ScL USA, 95, 8869-8873, 1998), indicating that both viruses are significantly infectious under the conditions employed. The extent of infection by EcoHIV was somewhat lower at twelve weeks after infection than at three or six weeks. This could arise from a self-limiting infection or from effective antiviral immunity. The presence of EcoHIV in multiple organs and its transmission from spleen cells in culture indicate that the progeny virus is highly infectious. On the other hand, EcoHIV infected mice consistently produced anti-Gag and anti-Tat antibodies. Therefore the inventors favor the second possibility: that infected mice mount immune responses that control EcoHIV infection, at least temporarily. This host- virus balance is reminiscent of the first years of HIV-I infection in human beings while the immune system is intact and the infection is controlled (Pantaleo et al, Nature Medicine, 10, 806-810, 2004). However, in the absence of therapy the balance later shifts to increased viral replication, loss of immune function, and development of disease in the immune and nervous systems. The inventors are currently investigating the consequences of long-term infection of mice by EcoHIV and EcoNDK to determine whether a similar pathogenic shift takes place.

[0108] Although no overt disease was been detected within six weeks of infection, EcoNDK activated IL- lβ, MCP-I, and STAT-I expression, cellular responses in the brain associated with later disease in HIV-I or SFV infection. IL-I β over-expression has been observed in the brain during SIV-induced neurological disease (Lane et ah, MoI. Med., 2, 27-37, 1996) and can be induced in the brain by viral Tat itself (Philippon et ai, Virology, 205, 519-529, 1994). STAT-I was induced in astrocytes, microglia, and neurons during SIV encephalitis (Roberts et al, Am. J. Pathol., 162, 2041-2057, 2003), its induction was confined to neurons in the brain of EcoNDK infected mice tested three weeks after inoculation. MCP-I can be induced in the brain by viral Tat alone (Pu et al., MoI. Cell NeuroscL, 24,224-237, 2003), it has been detected in the brains of human beings suffering from HIV-I associated dementia (Conant et al, Proc. Natl. Acad. ScL USA, 95, 3117-3121, 1998) and indeed induction of MCP-I in the central nervous system has been described as a predictor of later brain disease in an SIV model of encephalitis (Zink et al., J. Infect. Dis., 184,1015-1021, 2001). EcoNDK infection of mice thus mimics human or monkey infection by primate lentiviruses in activation of cellular gene expression in the brain, possibly as a consequence of expression of HIV-I Tat.

[0109] EcoHIV infection of mice reproduces key characteristics of HIV-I infection of human beings including host cell range, early neuroinvasiveness, systemic immune responses, and induction of inflammatory and antiviral responses in the brain. Further modification of the EcoHIV construct to increase virulence may tip the balance observed during the early weeks of mouse infection from immune response to immune dysfunction. However, it should be clear that the control of EcoHIV infection in the context of antiviral immune responses is an excellent starting point for vaccine studies. EcoHIV can provide a link between the range of experimental models established in mice and the knowledge base of HIV-I molecular genetics to investigate HIV-I infection in a tractable animal host.

Example 3

[0110] Material and Methods

[0111] Mouse treatment and infection. All animal studies were conducted with the approval of the St. Luke's-Roosevelt Institutional Animal Care and Use Committee. Groups of 7 adult 129Xl/SvJ mice were injected intraperitoneally every 12 h for 96 h with either 0.6 ml 2% DMSO in saline (vehicle), 0.6 ml ddC (1 mg/ml) or 3 ml ddC. At 51 h after the first injection, mice were injected with 2x10 ⁶ pg p24 EcoNDK prepared as described (Potash et al, (2005) Proc. Natl. Acad. ScL USA 102, 3760). They were euthanized 48 h after infection, spleens were surgically removed, and a fragment of the spleen was immediately placed in RNALater (Qiagen, Valencia, CA) for RNA extraction. A cell suspension was prepared from the rest of the spleen for DNA extraction.

[0112] Quantitative Real-Time PCR (OPCR) and PCR. DNA was isolated from spleen cells using DNAzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Total RNA was isolated using the RNeasy Lipid Tissue Kit (Qiagen) according to the manufacturer's instructions and cDNA was synthesized using the Superscript System (Invitrogen). Custom QPCR primers incorporating Taqman chemistry were purchased from Applied Biosystems and amplification was conducted in an Applied Biosystems (ABI, Foster City, CA) 7500 instrument according to the manufacturer's instructions. EcoNDK primers employed for DNA detection amplify an 82 bp region located in the gag gene: forward primer: 5'-TGG GAC CAC AGG CTA CAC TAG A-3 (SEQ ID NO: 4), reverse primer: 5'- CAG CCA AAA CTC TTG CTT TAT GG-3' (SEQ ID NO: 5), probe: 5'-TGA TGA CAG CAT GCC AGG GAG TGG-3'(SEQ ID NO: 6). DNA content was standardized by

amplification of murine β globin: forward primer: 5'-GGT TTC CTT CCC CTG GCT AT- 3'(SEQ ID NO: 7), reverse primer: CGC TTC CCC TTT CCT TCT G (SEQ ID NO: 8), probe: 5'-CTG CTC AAC CTT CC-3'(SEQ ID NO: 9). To unambiguously detect newly synthesized EcoNDK RNA, primers were designed to amplify a region in vif generated by splicing: forward primer 5'-AAG AGG CGA GGG GCA GCG A-3'(SEQ ID NO: 10), reverse primer: 5'-TCT TTA CTT TTC TTC TTG GTA CTA CCT TTA TG-3'(SEQ ID NO: 11), probe: 5'-AGT AGT AAT ACA AGA CAA TAG TG-3' (SEQ ID NO: 12). RNA was standardization by amplification of the murine GAPDH transcript using primers and a probe from ABI. Conventional PCR to amplify a region in EcoNDK at the fusion of HIV-I and MLV envelope sequences was conducted as described (Potash et al., (2005) Proc. Natl. Acad. ScL USA 102, 3760).

[0113] Rapid infection of mice with different batches of EcoNDK with efficient viral DNA and p24 production in the spleen. One mouse each was inoculated i.p. with 10 ⁷ pg p24 of a different independently prepared batch of EcoNDK and tested for viral DNA burden in spleen cells after five days as described above. In addition, as a measure of EcoNDK expression in the spleen freshly prepared spleen cells were lysed and viral p24 core antigen content was determined by p24 ELISA (HIV-I /Ag kit, Beckman Coulter, Hialeah, FL).

[0114] Detection of mature p24 protein in macrophages from infected mice. As an alternative test for productive infection, mice were injected with 10 ⁷ pg p24 intact EcoNDK or UV-irradiated inactivated EcoNDK or phosphate buffered saline and 10 days later peritoneal macrophages were isolated and tested by immunoblotting for the presence of HIV- 1 Gag proteins with monoclonal anti-p24 antibody as described (Potash et al., (2005) Proc. Natl Acad. ScL USA 102, 3760). Two intact EcoNDK infected, but not UV-EcoNDK or vehicle treated mice clearly expressed mature p24 in macrophages, indicating that EcoNDK completes its life cycle in mouse cells in vivo.

[0115] Induction of antibodies to HIV-I structural and regulatory proteins in long term- infected mice. Mice were infected with 10 ⁵ pg EcoHIV and serum antibodies to HIV-I Gag, protease, Tat, and Rev were measured by Elisa using recombinant proteins (AIDS Research and Reference Reagent Repository). The peak titers were obtained in mice 46, 47, and 49 at 3-5 months after infection.

[0116] Results and Discussion

[0117] Uniform and efficient primary infection of mice by chimeric HIV-I and induction of humoral immune response to viral structural and regulatory proteins were examined in these studies. In optimization studies, EcoNDK was found to be more infectious in mice than EcoHIV, that there was a virus dose response, and that viral products were easily detected in all infected mice within days of infection.

[0118] Mice were treated in groups of seven either with vehicle, 1.2, or 6.0 mg ddC daily for two days prior to and two days following intraperitoneal injection of EcoNDK. They were then euthanized and quantitative real-time PCR was conducted to measure viral DNA and viral RNA in the spleen (FIG. 12). Spliced RNA was amplified for distinction from the input viral genome. One experiment is shown that is representative of four conducted. Two days after inoculation, EcoNDK DNA and RNA were detectable by real-time PCR in spleens of all mice (FIG. 12A-B). Both doses of ddC significantly inhibited viral DNA synthesis and subsequent RNA synthesis (p < 0.001). Spleens of mice receiving the higher dose had viral DNA burdens 96% lower and mRNA burdens 87% lower than in control mice, consistent with the ability of ddC to terminate proviral DNA synthesis. The results of real-time PCR for viral DNA were confirmed by Southern blot (FIG. 12C). These findings indicate that EcoNDK-infected mice reproduce the sensitivity of HIV-I replication to ddC observed in human beings (R. Yarchoan et al, (1988) Lancet 1, 76).

[0119] Mice are routinely employed to test toxicity of candidate antiretrovirals, the method reported here of primary infection of mice by chimeric HIV-I for the first time permits practical evaluation of antiviral drug efficacy in vivo. There are four advantages to this system. First, speed: when an early event in HIV-I replication was targeted, the animal experiment took four days; evaluation can be completed in several hours. Second, safety: due to the envelope gene inserted, human cells are not susceptible to EcoHIV and EcoNDK. Third, applicability: chimeric viruses can be constructed using HIV-I that present the greatest human risk for assay of drug efficacy in animals. Fourth, cost: because conventional mice and not monkeys or immunodeficient or genetically engineered mice are employed, the cost of conducting studies with sufficient subjects to obtain statistical power is relatively low. The system of chimeric HIV-I infection of mice holds promise for facilitating new antiretroviral development and application.

[0120] All publications referenced herein are hereby incorporated in their entirety. While the foregoing invention has been described in some detail for purposes of clarity and

understanding, one skilled in the art can appreciate, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.

Example 4

[0121] EcoHrV7NL4-3 is a chimeric human immunodeficiency virus type 1 (HIV-I) that can productively infect mice. This study tests the utility of EcoHIV/NL4-3 infection to reveal protective immune responses to an HIV-I vaccine. Immunocompetent mice were first immunized with VRC 4306 which encodes subtype B consensus sequences of gag, pol, and nef and then were infected by EcoHIV/NL4-3. Anti-Gag antibodies were sampled during immunization and infection. The extent of EcoHIV/NL4-3 infection in spleen cells and peritoneal macrophages was determined by quantitative real-time PCR (QPCR). Although antibody titres were not significantly different in control and vaccinated groups, VRC 4306 immunization induced protective responses that significantly reduced virus burden in both lymphocyte and macrophage compartments. These results indicate that EcoHIV/NL4-3 infection can be controlled by HIV-I vaccine induced responses, introducing a small animal model to test vaccine efficacy against HIV-I infection.

[0122] As HIV-I infection continues to spread worldwide the need for a safe and effective vaccine remains unmet. Extraordinary scientific efforts have been expended to identify the means to elicit protective immunity against HIV-I [1,2]. This endeavor is impeded by the absence of immunocompetent experimental animals susceptible to HIV-I. Consequently, vaccine efficacy is tested for the ability to reduce the efficiency of HIV-I transmission and the frequency of new infections only in human beings at risk.

[0123] To permit the use of conventional mice for investigation of HIV-I replication and control, we switched the tropism of HIV-I from human to rodent [3]. The regions encoding gpl20 of subtype B NL4-3 [4] and subtype D NDK [5] were replaced with that of the gp80, the envelope of ecotropic murine leukemia virus (MLV). The resulting infectious chimeric viruses, EcoHIV/NL4-3 and EcoHIV/NDK, encode all HIV-I proteins except gpl20 and can infect conventional mice systemically, replicating in lymphocytes and macrophages, and inducing immune responses. Antiretroviral drugs in clinical use inhibited EcoHIV/NDK infection in mice, demonstrating that HIV-I enzymes retain their native functions in mice [6]. Here we report proof of principle that EcoHIV infection of mice can be used to evaluate

protective efficacy of potential vaccines, in this case VRC 4306, a DNA vaccine encoding subtype B consensus sequences of gag, pol, and nef[7],

[0124] Materials and methods

[0125] VRC 4306 was kindly provided by Dr. G. Nabel, NIAID-NIH. Adult female C57BL6/J mice were immunized by four intramuscular injections of 100 μg VRC 4306 or control plasmid pUC19 at two week intervals with five mice per group. Seven weeks and ten weeks after the first immunization, mice were infected with 5 x 10 ⁶ pg p24 EcoHIV/NL4-3 by intraperitoneal injection of cell-free virus, prepared as described [3]. One week after the challenge EcoHIV/NL4-3 infection, mice were euthanized by carbon dioxide asphyxiation and blood, spleen, and peritoneal macrophages were collected. DNA was isolated from spleen and macrophages on the day of euthanasia using DNAzol. QPCR was conducted as described [6] using primers that amplify sequences with the MLV envelope: forward primer (5'-GGCCAAACCCCGTTCTG-S ') (SEQ ID NO: 13), reverse primer (5'-ACTTAACAGG TTTGGGCTTGGA-3') (SEQ ID NO: 14) and probe (5'-CAGACCAACAGCCACT-S') (SEQ ID NO: 15). Custom primers and probe were obtained from ABI. Data are reported as number of viral DNA copies per 10 ⁶ cells, cell numbers were obtained by amplification of glyceraldehyde phosphate dehydrogenase by QPCR as described [6].

[0126] Elisa was conducted to detect anti-Gag antibodies in mouse sera. Wells were coated with 50 ng HIV-1/LAV Gag p55, the recombinant protein was provided by the NIH AIDS Research and Reference Reagent Repository. Preimmune sera and sera obtained eight weeks and eleven weeks after immunization were tested at 50-fold dilutions. Bound antibody was detected using horseradish peroxidase conjugated anti-mouse IgG and the chromogenic substrate 3,3',5,5'-tetramethylbenzidine, wells were read at OD ₄ so. The statistical significance of differences in virus burden or anti-Gag titres between groups was determined by t Test, the significance of the difference in frequency of anti-Gag positive mice between different groups was also determined by Chi-square Test.

[0127] Results

[0128] This study was conducted to determine whether EcoHIV/NL4-3 infection of mice can be reduced by immune responses induced by a known immunogenic HIV-I vaccine, VRC 4306 [7]. Mice were immunized either with a control plasmid, pUC19, or VRC 4306.

Immunization using HIV-I DNA vaccines is generally most efficient when it is followed by boosting responses through infection with a viral vector of the antigens [8]. In this case, the viral antigenic boost was provided by an initial infection by EcoHIV/NL4-3 itself. The efficacy of responses was tested in a subsequent challenge infection. Serological responses to HIV-I Gag were measured as a simple readout for immunization. Most infected mice mounted immune responses to HIV-I Gag one week after the challenge infection; two of five control mice mounted responses, indicating that EcoHIV/NL4-3 infection itself induces humoral responses, as previously observed [3]. VRC 4306 immunized mice had higher titres than the control group and four out of five mounted responses but the differences did not reach statistical significance (by t Test p=0.07 or by Chi-square test, p=0.197) (FIG. 13 A-B). These results suggest that this DNA vaccine immunization did not induce extensive humoral responses to Gag.

[0129] Immunization with VRC 4306 has been shown to induce CD4 and CD8 T lymphocyte responses to HIV-I Gag [7], suggesting that both T helper cells and cytotoxic effector cells were primed. To investigate whether these potential protective immune responses could control EcoHIV/NL4-3 infection, we measured the viral burdens in VRC 4306 and control immunized mice after the challenge virus infection. To distinguish newly synthesized EcoHIV/NL4-3 DNA from the gag, pol, and nefTMK vaccine, we amplified a region in the chimeric envelope consisting of MLV sequences that is absent from VRC 4306. Viral burdens in splenic lymphocytes and peritoneal macrophages were determined by QPCR amplification, normalized by amplification of a cellular gene (FIG. 14A-B). The EcoHIV/NL4-3 burden was significantly reduced in VRC 4306 immunized compared to control mice; virus detected in both the lymphoid and macrophage compartments was sensitive to the immune responses induced by vaccination. These findings provide clear evidence that the extent of EcoHIV/NL4-3 infection in mice is a legitimate measure for the generation of protective immunity by an HIV-I vaccine.

[0130] Discussion

[0131] The results from this study indicate that EcoHIV/NL4-3 infection of conventional mice can be controlled by immune responses to an HIV-I vaccine, forming a suitable system to test vaccine efficacy. In this model, HIV-I proteins expressed by infected lymphocytes and macrophages assemble to form infectious HIV-I [3], a system closely analogous to the infected human host. Like infected humans, infected mice mount serological responses to

HIV-I proteins. This observation indicates that HIV-I proteins are immunogenic to mice in their native form in infected cells or virions. The DNA vaccine, VRC 4306, has several features that may contribute to induction of protective immune responses that can be recalled by live virus infection [7]. It is a codon optimized vector encoding consensus subtype B Gag- Pol-Nef, thus it carries sequences closely related to EcoHIV/NL4-3 that are optimized for expression. In addition, point mutations were introduced into protease, reverse transcriptase, and integrase to inactivate enzyme function and minimize cytotoxicity, permitting efficient expression of antigens by transduced cells in the mouse.

[0132] In this study, immunization with VRC 4306 DNA alone was sufficient to prime responses to virus infection. EcoHIV/NL4-3 burden was reduced in both spleen cells and macrophages in VRC 4306 immunized mice compared to controls. Using magnetic bead purification of CD4 bearing T lymphocytes from spleens of infected mice, we have previously shown that lymphocytes account for the majority of Eco/NL4-3 infected cells in the spleen [3], however we cannot rule out the possibility that some of the infection observed in unfractionated spleen arises from macrophages. The magnitude of the reduction of virus burden with immunization shown here was greater in macrophages than in spleen cells. We have previously shown that macrophages from infected mice express more viral RNA than do lymphocytes [6], suggesting that more viral antigen may be present in macrophages for targeting by immune effectors.

[0133] In this study, we focused upon providing proof of principle that vaccine induced immune responses control EcoHIV/NL4-3 challenge infection and did not investigate the mechanism of protection. It is likely that cytotoxic T lymphocyte responses directed against the immunogens, Gag, Pol, and/or Nef mediated the reduction in virus burden. This view is based on the observation that mice immunized with VRC 4306 mount both CD4 and CD8 T lymphocyte responses to Gag [7]. In addition, antiviral CD8 responses are believed to be responsible for successful vaccination in the SIV macaque system [9] and for protection in HIV-I exposed uninfected sex workers [10]. Although responses to the MLV envelope may be induced by EcoHIV/NL4-3 infection, they are unlikely to be responsible for the protection observed. Both VRC 4306 and pUC19 immunized mice were only exposed to the MLV envelope by EcoHIV/NL4-3 infection, so responses to this antigen cannot account for the differences in virus burden between groups.

[0134] A recent study by Rollman et al. evaluated vaccine efficacy in HLA-transgenic mice testing several immunization protocols for their ability to induce specific immune responses [H]. In that study, vaccinated mice reduced virus burden in mouse lymphocytes that had been infected by pseudotyped HIV-I in culture and implanted into the peritoneal cavity. It is notable that the best protection against virus challenge in that model was achieved through immunization with DNA alone, the method employed here. Taken together, Rollman et α/.'s investigation of immunization protocols and HLA restricted responses and our study of the efficacy of vaccination to reduce systemic HIV-I infection hold promise for the utility of evaluation of future HIV-I vaccines for protective efficacy in immunocompetent mice.

References

1. McMichael, A. HIV Vaccines. Ann Rev Immunol 2006, 24, 227-255.

2. Letvin, N. Progress and obstacles in the development of an AIDS vaccine. Nat Rev Immunol 2006, 6, 930-939.

3. Potash, M. J., Chao, W., Bentsman, G. et al. A mouse model for study of systemic HIV-I infection, antiviral immune responses, and neuroinvasiveness. Proc. Natl. Acad. Sci. USA 2005, 102, 3760-3765.

4. Adachi, A., Gendelman, H. E., Koenig, S. et al. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 1986, 59, 284-291.

5. Ellrodt, A., Barre-Sinoussi, F., Le Bras, P. et al. Isolation of human Tlymphotropic retrovirus (LAV) from Zairian married couple, one with AIDS, one with prodromes. Lancet 1984, 1(8391), 1383-1385.

6. Hadas, E., Borjabad, A., Chao, W. et al. Testing antiretroviral drug efficacy in conventional mice infected with chimeric HIV-I . AIDS 2007, 21, 905-909.

7. Kong, W.-P., Huang, Y., Yang, Z.-Y., Chakrabarti, B.K., Moodie, Z. & Nabel, GJ. Immunogenicity of multiple gene and clade human immunodeficiency virus type 1 DNA vaccines. J. Virol. 2003, 77, 12764-12772.

8. Girard, M. P., Osmanov, S.K. & Kieny, M. P. A review of vaccine research and development: the human immunodeficiency virus (HIV). Vaccine 2006, 24(19), 4062-4081.1 1

9. Barouch, D., Santra, S., Schmitz, J. et al. Control of Viremia and Prevention of Clinical AIDS in Rhesus Monkeys by Cytokine- Augmented DNA Vaccination. Science 2000, 290, 486-492.

10. Rowland- Jones, S., Dong, T., Fowke, K.R. et al. Cytotoxic T cell responses to multiple conserved epitopes in HIV-resistant prostitutes in Nairobi. J. Clin. Invest. 1998, 102, 1758-1765.

11. Rollman, E., Mathy, N., Brave, A. et al. Evaluation of immunogenicity and efficacy of combined DNA and adjuvanted protein vaccination in a human immunodeficiency virus type 1 /murine leukemia virus pseudotype challenge model. Vaccine 2007, 25, 2145-2154

Example 5

[0135] Heterosexual transmission is the predominant route through which new HIV-I infections are acquired globally. There are currently no small animals that transmit HIV-I through mating, impeding investigation of cell types, pathogenesis, and control of this critical route in the continuing spread of HIV-I.

[0136] EcoHIV/NL4-3 and EcoHIV/NDK are chimeric HIV-I that we constructed to encode ecotropic murine leukemia virus type 1 envelope in place of gpl20, switching the tropism of the virus from human to rodent. Infected mice carry viral RNA in lymphoid tissue, macrophages, and the male reproductive tract. These observations raise the possibility that EcoHIV can be transmitted from infected males to females sexually.

[0137] Based upon the time-course of infection in macrophages, preliminary studies were conducted using males one week after EcoHIV/NL4-3 or EcoHIV/NDK infection; several virus batches were tested. Each infected male was housed with two uninfected females, transmission from a total of nine males was tested. After one week of cohabitation, females were euthanized and spleens and peritoneal macrophages were harvested for assay of virus burden by quantitative PCR amplification of HIV-I gag RNA. As shown in the Table 1, each male transmitted virus through mating and five of the nine males tested transmitted virus to both mated females. Overall, the rate of sexual transmission of chimeric HIV-I in mice was 77%.

[0138] Table 1. Sexual Transmission of HIV through Mating.

[0139] Experiment 4 was conducted with EcoHIV/NL4-3 that encodes green fluorescence protein that is expressed in infected cells but is not encapsidated in virions. The expression of green fluorescence protein in macrophages was analyzed by flow cytometry as a measurement of viral protein expression in infected cells (FIG. 15). Approximately 11% of macrophages from this female expressed viral protein after sexual transmission of EcoHrV7NL4-3. This frequency of infected cells is comparable to the frequency of HIV-I infected T lymphocytes during the last stages of AIDS.

[0140] The high rate of sexual transmission of EcoHIV in mice and high rate of virus expression in cells of infected females recommend this model for evaluation of interventions designed to control or block sexual transmission of HIV-I in humans including microbicides and vaccines.