WO2008070385A2 - Utilisations d'un modèle murin d'une infection par le vih 1 - Google Patents

Utilisations d'un modèle murin d'une infection par le vih 1 Download PDF

Info

Publication number
WO2008070385A2
WO2008070385A2 PCT/US2007/083736 US2007083736W WO2008070385A2 WO 2008070385 A2 WO2008070385 A2 WO 2008070385A2 US 2007083736 W US2007083736 W US 2007083736W WO 2008070385 A2 WO2008070385 A2 WO 2008070385A2
Authority
WO
WIPO (PCT)
Prior art keywords
hiv
infection
rodent
ecohiv
mice
Prior art date
Application number
PCT/US2007/083736
Other languages
English (en)
Other versions
WO2008070385A3 (fr
Inventor
Mary Jane Potash
David J. Volsky
Original Assignee
The Trustees Of Columbia University In The City Of New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Trustees Of Columbia University In The City Of New York filed Critical The Trustees Of Columbia University In The City Of New York
Publication of WO2008070385A2 publication Critical patent/WO2008070385A2/fr
Publication of WO2008070385A3 publication Critical patent/WO2008070385A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16045—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6045—RNA rev transcr viruses

Definitions

  • HIV-I human immunodeficiency virus
  • HIV-2 HIV-I is the more virulent type, and naturally infects human beings, as well as a small number of non-human primates.
  • HIV-2 infectiousness seems to increase.
  • the duration of this increased infectiousness is shorter.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 the invention.
  • the complete or partial coding region of gpl20 is replaced by the coding region for ecotropic murine leukemia virus gp80.
  • 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.
  • 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.
  • the rodent host is a mouse.
  • any rodent may be used as a model in the invention including, but not necessarily limited to, all species of mouse and rat.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the heterologous viral envelope-coding region replaces a complete or partial coding region of HIV-I gpl 20.
  • the heterologous viral envelope-coding region is an ecotropic murine leukemia virus gp80 coding region.
  • the construct is EcoNDK or EcoHIV.
  • 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.
  • the rodent is a mouse or rat.
  • 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) dete ⁇ nining 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.
  • the determining comprises measuring the level of HIV-I infection before and after administration of the candidate or compound.
  • the rodent is a rat or mouse.
  • the heterologous viral envelope-coding region replaces a complete or partial coding region of HIV-I gpl20.
  • the heterologous viral envelope-coding region is an ecotropic murine leukemia virus gp80 coding region.
  • the construct is EcoNDK or EcoHIV.
  • 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.
  • the antiretroviral drug is an HIV-I reverse transcription inhibitor.
  • the antiviral compound or agent comprises an antiviral drug, a vaccine, or a microbicide.
  • the antiviral compound or agent is an HIV-I reverse transcription inhibitor.
  • the antiretroviral drug is 2', 3' dideoxycytidine (ddC) or abacavir, while in other embodiments, the inhibitor is 2 ⁇ 3' dideoxycytidine (ddC) or abacavir.
  • 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.
  • 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.
  • the sample comprises blood, serum, urine, tissue samples, or a combination thereof
  • 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.
  • 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.
  • 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.
  • 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 ⁇ - 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.
  • 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.
  • RPS9 RT-PCR of mouse ribosomal small protein 9 RNA, used for standardization of samples. The figure shows ethidium bromide staining.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIGS. 10A-B depict a scheme for insertion of gpl20 regions into EcoHIV.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g 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.
  • 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.
  • 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.
  • 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.
  • HIV-I pathogenesis 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.
  • 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/ ⁇ /, J. Virol, 64, 5270-5276, 1990).
  • the inventors constructed HIV-I species with receptors for mouse cells.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • ddC dideoxycytidine
  • 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.
  • 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.
  • 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.
  • 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.
  • a vector of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, or Mokola.
  • VSV vesicular stomatitis virus
  • rabies rabies
  • Ebola or Mokola
  • the complete or partial coding region of gpl20 is replaced by the coding region for ecotropic murine leukemia virus gp80.
  • 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.
  • 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.
  • 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.
  • 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.
  • replication competent EcoHIV is produced by rodent cells infected in tissue culture.
  • the rodent can be infected with the replication competent EcoHIV by intraperitoneal or intravenous injection.
  • the rodent can be infected with isogenic rodent cells expressing the replication competent EcoHIV.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the term “ameliorating disease” refers to reducing HIV-I infection associated symptoms or pathology or AIDS associated symptoms or pathology.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the heterologous viral envelope-coding region replaces a complete or partial coding region of HIV-I gpl20.
  • the heterologous viral envelope-coding region is an ecotropic murine leukemia virus gp80 coding region.
  • the construct is EcoNDK or EcoHIV.
  • 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.
  • 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.
  • the invention provides a method for testing candidate compositions to treat or prevent HIV infection.
  • 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.
  • 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.
  • 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.
  • biological samples include blood, serum, sputum, lacrimal secretions, semen, urine, vaginal secretions, and tissue samples (such as splenic, kidney, lung, or brain tissue).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the agent can be administered to a subject once (e.g., as a single injection or deposition).
  • 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.
  • the agent can be co-administrated with another therapeutic, such as a microbicide, an antibiotic, a chemotherapeutic compound, or a combination thereof.
  • the effective amount of an agent administered to the subject can comprise the total amount of an agent administered over the entire dosage regimen.
  • the agents can be administered to a subject by any means suitable for delivering the agents to cells of the subject.
  • 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.
  • Antiviral agents and/or compounds 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.
  • 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.
  • injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Solid formulations can be used for enteral (oral) administration. They can be formulated as, e.g., pills, tablets, powders or capsules.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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
  • 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.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active agent.
  • the therapeutic and pharmaceutical compositions comprising an antiviral agent or compound may take the form of tablets or lozenges formulated in a conventional manner.
  • 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.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • 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.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.
  • 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.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through nasal sprays or using suppositories.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a delivery reagent e.g., a recombinant plasmid or viral vector
  • 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.
  • 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.
  • 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.
  • 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.
  • EcoHIV a Chimeric HIV-I Construct that Targets Rodent Cells
  • 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.
  • the coding region for ecotropic murine leukemia virus gp80, that targets the virus to rodent, but not to human cells was inserted.
  • EcoHIV was constructed based upon the full-length infectious HIV-I molecular clone, NL4-3, and the full-length infectious ecotropic MLV clone, NCAC.
  • 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.
  • 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 confii ⁇ ned 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.
  • 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 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.
  • 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.
  • 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).
  • [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, ⁇ globin was amplified in parallel to standardize DNA input.
  • 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.
  • 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.
  • WTA Whole Transcriptome Amplification
  • NuGEN Technologies Inc. San Carlos, CA
  • 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 was conducted as described (Nitkiewicz et al., Journal of NeuroVirology, 10:400-408, 2004).
  • Solid phase-radioimmunoassays 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).
  • 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.
  • human CEM cells were not susceptible to infection by the EcoHIV.
  • 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.
  • EcoHIV infected cells formed large syncytia, indicating that gp80 is properly cleaved to fusion competent proteins.
  • 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 .
  • 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.
  • 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.
  • FIG. 2C 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 .
  • 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.
  • 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.
  • 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 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
  • 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.
  • FIG. 3 shows that they may be useful as cell-specific molecular markers of HIV-I neuropathogenesis.
  • 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.
  • 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.
  • spleen cells infected in the mouse produced progeny virus that could spread infection in culture (FIG. 5).
  • 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.
  • mice #15 and 6 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.
  • 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.
  • FIG. 8 provides preliminary data that EcoHIV can induce impairment of immune function in infected mice.
  • 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.
  • mice 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).
  • 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.
  • NDK 9 had lower levels of EcoNDK in brain and spleen and showed significant increases in the expression of IL- 1 ⁇ 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.
  • 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).
  • EcoNDK a chimeric virus based upon an African Clade D HIV-I and MLV is also replication competent.
  • 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.
  • the cellular genes activated by exposure to EcoNDK have also been found to be activated by exposure of human cells to HIV-I .
  • STAT-I expression in neurons as shown in the brain of EcoNDK infected mouse #8, has also been observed in macaques suffering SIV encephalitis.
  • 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.
  • 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.
  • EcoHIV and EcoNDK are less hazardous than are HIV-I and SIV.
  • 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.
  • 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.
  • 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.
  • 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.
  • EcoHIV 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.
  • 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.
  • 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.
  • 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.
  • mice were injected with 2x10 6 pg p24 EcoNDK prepared as described (Potash et al, (2005) Proc. Natl. Acad. ScL USA 102, 3760).
  • RNALater Qiagen, Valencia, CA
  • 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 ⁇ 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).
  • 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.
  • 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.
  • mice [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.
  • 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).
  • 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.
  • speed when an early event in HIV-I replication was targeted, the animal experiment took four days; evaluation can be completed in several hours.
  • safety due to the envelope gene inserted, human cells are not susceptible to EcoHIV and EcoNDK.
  • applicability chimeric viruses can be constructed using HIV-I that present the greatest human risk for assay of drug efficacy in animals.
  • 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.
  • EcoHrV7NL4-3 is a chimeric human immunodeficiency virus type 1 (HIV-I) that can productively infect mice.
  • HIV-I human immunodeficiency virus type 1
  • 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).
  • VRC 4306 was kindly provided by Dr. G. Nabel, NIAID-NIH.
  • Adult female C57BL6/J mice were immunized by four intramuscular injections of 100 ⁇ g 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].
  • 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.
  • 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].
  • 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.
  • 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.
  • 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).

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un produit de construction VIH 1 chimérique, ÉcoVIH, pouvant se répliquer dans une cellule de rongeur. L'invention concerne également un modèle de rongeur commode et sain d'infection par le VIH 1 et du SIDA. Un modèle de rongeur et d'infection par le VIH 1 est également proposé, lequel est utile pour le criblage de médicaments anti-viraux candidats. En outre, l'invention propose un procédé pour mettre à l'essai des compositions immunogéniques ou des interventions pharmaceutiques efficaces dans la prévention d'une infection, la réduction d'une charge virale ou la réduction des symptômes de la maladie chez un sujet.
PCT/US2007/083736 2006-11-08 2007-11-06 Utilisations d'un modèle murin d'une infection par le vih 1 WO2008070385A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US85769006P 2006-11-08 2006-11-08
US60/857,690 2006-11-08

Publications (2)

Publication Number Publication Date
WO2008070385A2 true WO2008070385A2 (fr) 2008-06-12
WO2008070385A3 WO2008070385A3 (fr) 2008-10-30

Family

ID=39492955

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/083736 WO2008070385A2 (fr) 2006-11-08 2007-11-06 Utilisations d'un modèle murin d'une infection par le vih 1

Country Status (1)

Country Link
WO (1) WO2008070385A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113143974A (zh) * 2021-03-03 2021-07-23 武汉科技大学 一种hiv感染致神经损伤动物模型的建立方法及其用途
CN116286993A (zh) * 2023-03-01 2023-06-23 武汉科技大学 一种HIV-tat致神经损伤模型的建立方法及其用途

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050241009A1 (en) * 2004-04-21 2005-10-27 Potash Mary J Development of a murine model of HIV-1 infection on the basis of construction of EcoHIV, a chimeric, molecular clone of human immunodeficiency virus type 1 and ecotropic moloney murine leukemia virus competent to infect murine cells and mice

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050241009A1 (en) * 2004-04-21 2005-10-27 Potash Mary J Development of a murine model of HIV-1 infection on the basis of construction of EcoHIV, a chimeric, molecular clone of human immunodeficiency virus type 1 and ecotropic moloney murine leukemia virus competent to infect murine cells and mice

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
POTASH M.-J. ET AL.: 'A mouse model for study of systemic HIV-1 infection, antiviral immune responses, and neuroinvasiveness' vol. 102, no. 10, March 2005, pages 3760 - 3765 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113143974A (zh) * 2021-03-03 2021-07-23 武汉科技大学 一种hiv感染致神经损伤动物模型的建立方法及其用途
CN113143974B (zh) * 2021-03-03 2023-06-06 武汉科技大学 一种hiv感染致神经损伤动物模型的建立方法及其用途
CN116286993A (zh) * 2023-03-01 2023-06-23 武汉科技大学 一种HIV-tat致神经损伤模型的建立方法及其用途

Also Published As

Publication number Publication date
WO2008070385A3 (fr) 2008-10-30

Similar Documents

Publication Publication Date Title
Parolin et al. Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes
JP4190579B2 (ja) 非分裂細胞への核酸運搬のためのベクターおよび使用方法
Donahue et al. Viral genetic determinants of T-cell killing and immunodeficiency disease induction by the feline leukemia virus FeLV-FAIDS
Gorry et al. Restricted HIV-1 infection of human astrocytes: potential role of nef in the regulation of virus replication
US20040224404A1 (en) Conditionally replicating viral vectors and their use
US5883081A (en) Isolation of novel HIV-2 proviruses
JPH08500965A (ja) ハイブリッドウイルス発現ベクター，その使用および新規アッセイ
US6323019B1 (en) Design of novel highly efficient HIV based packaging systems for gene therapy
Reddy et al. Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants
EP0871757B1 (fr) Replication conditionnelle de vecteurs viraux et leur utilisation
US20050241009A1 (en) Development of a murine model of HIV-1 infection on the basis of construction of EcoHIV, a chimeric, molecular clone of human immunodeficiency virus type 1 and ecotropic moloney murine leukemia virus competent to infect murine cells and mice
WO2008070385A2 (fr) Utilisations d'un modèle murin d'une infection par le vih 1
Das et al. HIV-1 evolves into a nonsyncytium-inducing virus upon prolonged culture in vitro
Kishi et al. Naturally occurring accessory gene mutations lead to persistent human immunodeficiency virus type 1 infection of CD4-positive T cells
AU766264B2 (en) Viral chimeras comprised of caev and hiv-1 genetic elements
Junker et al. Intracellular expression of human immunodeficiency virus type 1 (HIV-1) protease variants inhibits replication of wild-type and protease inhibitor-resistant HIV-1 strains in human T-cell lines
JPH05509232A (ja) 複製効率を増強するためのＨＩＶｎｅｆ遺伝子を含有する発現ベクター
Contag et al. Simian immunodeficiency virus (SIV) from Old World monkeys
US6063374A (en) Recombinant HIV and modified packaging cells and method for using
Anderson Molecular analysis of simian immunodeficiency virus pathogenicity: Role of LTR and envelope sequences
Kräusslich Genetic analysis and gene expression of human immunodeficiency virus
WO1999000490A2 (fr) Vaccin comportant le virus attenue de l'immunodeficience humaine
Raymond Control of acute HIV-1 infection: Intracellular immunization with Herpesvirus saimiri transforming proteins, Tip and StpC and the effect of opiate abuse
Edmonson Development of a simian immunodeficiency virus (SIV) pathogen
El‐Farrash et al. In Vitro Modification of Human Immunodeficiency Virus Type 1 (HIV‐1) Infectivity by the U937 Cells

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07871380

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07871380

Country of ref document: EP

Kind code of ref document: A2