Title:  Method of using mouse model for evaluation of HIV vaccines

United States Patent:  6,248,721

Appl. No.:  848760

The present invention provides animals and methods for the evaluation of vaccines. In particular, the present invention provides humanized animal models for the evaluation of vaccines designed to confer immunity against human pathogens, including vaccines directed against the human immunodeficiency virus. The present invention further relates to HIV vaccines. In particular, the present invention provides attenuated replication-competent HIV vaccines and replication-defective HIV vaccines. In addition, the invention provides modified Leishmania cells expressing HIV proteins.

SUMMARY OF THE INVENTION

The present invention provides novel humanized animal models that permit the identification of immune-modulating genes and combinations thereof useful for the treatment of human tumors. In addition, the present invention provides methods of treating subjects having a tumor with one or more immune-modulating genes and provides tumor cell vaccines comprising tumor cells modified to express immune-modulating genes. The novel animals of the present invention provide a means of evaluating vaccines, including cancer vaccines and vaccines directed against human pathogens (e.g., HIV, malaria, Leishmania, etc.). In addition, the invention provides HIV vaccines including live attenuated HIV vaccines and HIV DNA vaccines.

Accordingly, the present invention provides an immunodeficient mouse comprising human T lymphocytes expressing the CD45 antigen wherein at least 5% of the human T lymphocytes expressing the CD45 antigen represent immature naive T lymphocytes. The invention is not limited by the nature of the immunodeficient mouse strain employed. In a preferred embodiment, the immunodeficient mouse is a SCID/beige mouse.

In another preferred embodiment, the immunodeficient mouse comprising human T lymphocytes further comprising human tumor cells. The invention is not limited by the nature of the human tumor cells employed. The human tumor cells may be established tumor cells, primary tumors cells or tumor cells (established or primary) modified to express one or more immune-modulating genes, genes encoding cell cycle regulators and genes encoding inducers of apoptosis.

In another embodiment, the present invention provides a SCID/beige mouse comprising human immune cells. The invention is not limited by the nature of the human immune cells, these cells may be human PBLs, splenocytes, cells isolated from lymph nodes and/or peritoneal lavage. In a preferred embodiment, the SCID/beige mouse comprising human immune cells further comprising human tumor cells. The invention is not limited by the nature of the human tumor cells employed. The human tumor cells may be established tumor cells, primary tumors cells or tumor cells (established or primary) modified to express one or more immune-modulating genes, genes encoding cell cycle regulators and genes encoding inducers of apoptosis. In a preferred embodiment, the tumor cells are derived from central nervous system cells, most preferably glioblastoma cells. In another preferred embodiment, the tumor cells are malignant melanoma cells.

The present invention further provides a method comprising: a) providing: i) a SCID/beige mouse; ii) human tumor cells; iii) human peripheral blood lymphocytes; b) introducing a first dose of the tumor cells into said mouse; c) reconstituting the mouse containing said tumor cells with the lymphocytes; and d) monitoring the reconstituted mouse for the growth of the tumor cells. The invention is not limited by the nature of the human tumor cells employed. The human tumor cells may be established tumor cells, primary tumors cells or tumor cells (established or primary) modified to express one or more immune-modulating genes, genes encoding cell cycle regulators and genes encoding inducers of apoptosis. In a preferred embodiment, the tumor cells are derived from central nervous system cells, most preferably glioblastoma cells. In another preferred embodiment, the tumor cells are malignant melanoma cells.

In a preferred embodiment, the method further comprises identifying at least one immune modulating gene (or gene encoding a cell cycle regulator or inducer of apoptosis) whose expression prevents the growth of the introduced tumor cells in the reconstituted mouse. In another preferred embodiment, the method comprises, following the reconstitution, the additional step of vaccinating the reconstituted mouse with a second dose of tumor cells. In a preferred embodiment, the first dose of tumor cells comprises unmodified tumor cells and the second dose of tumor cells comprises irradiated tumor cells. In a particularly preferred embodiment, the irradiated tumor cells express at least one immune-modulating gene (or gene encoding a cell cycle regulator or inducer of apoptosis).

In one embodiment of the methods of the present invention, the tumor cells and the lymphocytes come from the same donor. In another embodiment, the tumor cells and the lymphocytes come from different donors.

The present invention further provides a method comprising: a) providing: i) a SCID/beige mouse; ii) irradiated and unirradiated human tumor cells; iii) human peripheral blood lymphocytes; b) reconstituting said mouse with the lymphocytes; c) vaccinating the mouse with the irradiated tumor cells; d) introducing the unirradiated tumor cells into the vaccinated mouse; and e) monitoring the vaccinated mouse for the growth of the unirradiated tumor cells. The invention is not limited by the nature of the irradiated tumor cells. The irradiated tumor cells may be established tumor cells, primary tumors cells or tumor cells (established or primary) modified to express one or more immune-modulating genes, genes encoding cell cycle regulators and genes encoding inducers of apoptosis. In a preferred embodiment, the irradiated and modified tumor cells are derived from central nervous system cells, most preferably glioblastoma cells. In another preferred embodiment, the irradiated and modified tumor cells are malignant melanoma cells.

In a preferred embodiment, the method further comprises identifying at least one immune modulating gene (or gene encoding a cell cycle regulator or inducer of apoptosis) whose expression prevents the growth of said unirradiated tumor cells in said vaccinated mouse.

The present invention also provides a tumor cell vaccine comprising a tumor cell expressing B7-2 and at least one additional immune modulator or a cell cycle regulator or inducer of apoptosis. The vaccines of the present invention are not limited by the nature of the immune modulator or a cell cycle regulator or inducer of apoptosis employed. In a preferred embodiment, the additional immune modulator is a cytokine. The invention is not limited by the nature of the cytokine employed. In a preferred embodiment, the cytokine is selected from the group consisting of interleukin 2, interleukin 4, interleukin 6, interleukin 7, interleukin 12, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, interferon-gamma, tumor necrosis factor-alpha.

The present invention provides a method of treating a tumor comprising: a) providing: i) a subject having a tumor of the central nervous system; ii) an expression vector encoding the human B7-2 protein and at least one additional immune modulator or a cell cycle regulator or inducer of apoptosis; b) transferring the expression vector into the tumor under conditions such that the B7-2 protein and the immune-modulator (and/or a cell cycle regulator or inducer of apoptosis) are expressed by at least a portion of the tumor. In a preferred embodiment, the method further comprises, prior to transfer of the expression vector, the step of removing at least a portion of the tumor from the subject and following the transfer of said expression vector, irradiating the tumor cells expressing the B7-2 protein and the immune-modulator (and/or a cell cycle regulator or inducer of apoptosis) and introducing the irradiated tumor cells back into the subject to create an immunized subject. In another embodiment, the method further comprises introducing at least one additional dose of irradiated tumor cells expressing the B7-2 protein and the immune-modulator (and/or a cell cycle regulator or inducer of apoptosis) into the immunized subject.

The invention further provides a method comprising: a) providing: i) a SCID/beige mouse comprising human immune cells; ii) an injectable preparation comprising at least one component derived from a human pathogen; iii) a composition comprising an infectious human pathogen; b) injecting the mouse with the injectable preparation to produce an injected mouse; c) exposing the injected mouse to the composition; and d) monitoring the exposed mouse for the presence of infection of the human immune cells by the infectious human pathogen. In a preferred embodiment, the injecting of step b) is repeated at least once prior to exposing the mouse to the composition. The injectable preparation comprises a candidate vaccine; a candidate vaccine may comprise proteins or peptides or nucleic acids and may be immunogenic. However, as this method offers a means to evaluate candidate vaccines, some candidate vaccines are expected to be immunogenic (i.e., capable of invoking an immune response directed against the pathogen from which the protein, peptide or nucleic acid was derived) and some are expected to not be immunogenic (and would therefore be rejected as a vaccine for use in subject such as a human). In a preferred embodiment, the injectable preparation is immunogenic. The injectable preparation may comprise pharmacological carriers and excipients such as aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Further, the injectable preparation may comprise as adjuvant (e.g., alum, complete or incomplete Freund's, SAF-1).

The invention is not limited by the nature of the human pathogen. In a preferred embodiment, the human pathogen is selected from the group comprising human immunodeficiency virus, Leishmania, Mycobacterium (e.g., M. tuberculosis), and Plasmodium (the causative agent of malaria).

The invention is not limited by the nature of the component of the human pathogen present in the injectable composition. In a preferred embodiment, the component of the human pathogen comprises at least a portion of a protein derived from the pathogen; the component may be a complete protein molecule or a peptide derived from the complete protein (and may be produced by peptide synthesis, recombinant DNA methodologies or protease digestion of the intact protein). In another preferred embodiment, the component of the human pathogen comprises DNA. In a particularly preferred embodiment, the DNA comprises proviral DNA encoding a human immunodeficiency virus genome capable of expressing viral structural genes but incapable of being packaged into viral particles. In a preferred embodiment, the DNA comprises plasmid DNA selected from the group consisting of pHP-1, pHP-2 and pHP-3.

In another embodiment, the injectable preparation comprises an attenuated replication-competent human immunodeficiency virus (i.e., capable of producing only a limited infection and preferably incapable of producing the pathological changes associated with infection by a non-attenuated HIV). The invention is not limited by the nature of the change which renders the virus attenuated. In a preferred embodiment, the attenuated virus comprises a mutated tat gene; preferably, the genome of the attenuated virus cannot express a functional Tat protein. In another preferred embodiment, the genome of attenuated virus genome further comprises a mutated nef gene; preferably, the genome of the attenuated virus cannot express either a functional Tat protein or Nef protein. In a preferred embodiment, the attenuate virus is selected from the group consisting of HIV.sub.CMV/tat-B, HIV.sub.CMV/AD8/nef-B/tat-B, HIV.sub.NL43/AD8/tat-B/nef-B and HIV.sub.NL43/nef-B/tat-B.

In another embodiment, the injectable preparation comprises attenuated Leishmania cells. The invention is not limited by the nature of the change which renders the Leishmania cell attenuated. In a preferred embodiment, the attenuated Leishmania cell comprises heterologous DNA encoding a cysteine protease gene. The cysteine protease gene may be a leishmanial cysteine protease gene or a cysteine protease gene from another organism. When a leishmanial cysteine protease gene is employed it is preferably placed in operable combination with promoters and optionally enhancers functional in a Leishmania cell. As the leishmanial cysteine protease gene is not present in its native configuration (i.e., chromosomal configuration), it comprises heterologous DNA. Most preferably, the leishmanial cysteine protease gene is present on an expression vector such as pALT-Neo or pX. As shown herein, the overexpression of cysteine protease in Leishmania cells attenuates the Leishmania cell and provides an effective vaccine for the prevention of leishmaniasis. In another preferred embodiment, the heterologous DNA further comprises DNA encoding a human immunodeficiency virus gene selected from the group consisting of the env gene, the gag gene, the pol gene, the tat gene, the rev gene, the vif gene, the vpu gene, the vpr gene and the nef gene. Leishmania cells infect macrophages and therefore, Leishmania cells expressing HIV proteins provide a means to present HIV antigens to a host in the context of an APC (i.e., provides an HIV vaccine).

The invention is not limited by the nature of the monitoring employed to detect the presence (or absence) of infection of the human immune cells in the SCID/bg mouse comprising human immune cells (e.g., a hu-PBL-SCID/bg mouse). Serum collected from the mouse following vaccination and challenge with the pathogen may be examined for the presence of human immunoglobulin directed against the pathogen (evidence of humoral immunity). Cell mediated immunity directed against the pathogen may be examined by a variety of means known to the art, including but not limited to in vitro ELISPOT analysis of .gamma.-IFN production by lymphocytes (e.g., splenocytes) isolated from the vaccinated and challenged mice (using pathogen-infected PBLs as stimulators). Further, the mouse may be examined for the presence of infection in the sites expected to be infected by the pathogen employed for the challenge (e.g., immunostaining of lymphocytes collected from mice challenged with HIV to detect the presence of HIV proteins in and/or on lymphocytes and/or macrophages).

The present invention also provides a method comprising: a) providing: i) a SCID/beige mouse comprising human immune cells; ii) an injectable preparation comprising one or more components derived from a human immunodeficiency virus (HIV); iii) a composition comprising non-attenuated human immunodeficiency virus; b) injecting the mouse with the injectable preparation to produce an injected mouse; c) exposing the injected mouse to the composition; and d) monitoring the exposed mouse for the presence of infection of the human immune cells by the non-attenuated human immunodeficiency virus. The invention is not limited by the non-attenuated virus employed; any virus capable of causing an non-attenuated infection may be employed. Preferably, the non-attenuated virus is a virus isolated from a patient with AIDS (i.e., a clinical isolate) or is a virulent laboratory strain. Particularly preferred non-attenuated HIV strains include the NL4-3 strain, the ADA strain and HIV-1 primary isolates covering the different HIV clades (e.g., 92RW008, 92BR003, 92HT593, etc. available from the NIH AIDS Research and Reference Reagent Program of National Institutes of Health, Bethesda, Md.).

In a preferred embodiment, the injecting of step b) is repeated at least once prior to exposing the mouse to the composition. The injectable preparation comprises a candidate vaccine; a candidate vaccine may comprise proteins or peptides or nucleic acids and may be immunogenic. However, as this method offers a means to evaluate candidate vaccines, some candidate vaccines are expected to be immunogenic (i.e., capable of invoking an immune response directed against the pathogen from which the protein, peptide or nucleic acid was derived) and some are expected to not be immunogenic (and would therefore be rejected as a vaccine for use in subject such as a human). In a preferred embodiment, the injectable preparation is immunogenic. The injectable preparation may comprise pharmacological carriers, excipients and adjuvants as discussed above.

The invention is not limited by the nature of the component of the HIV present in the injectable composition. In a preferred embodiment, the component of the HIV comprises at least a portion of a protein derived from a HIV; the component may be a complete protein molecule or a peptide derived from the complete protein (and may be produced by peptide synthesis, recombinant DNA methodologies or protease digestion of the intact protein). In another preferred embodiment, the component of the HIV comprises DNA derived from HIV. In a particularly preferred embodiment, the DNA comprises proviral DNA encoding a HIV genome capable of expressing viral structural genes but incapable of being packaged into viral particles. In a preferred embodiment, the DNA comprises plasmid DNA selected from the group consisting of pHP-1, pHP-2 and pHP-3.

In another embodiment, the injectable preparation comprises an attenuated replication-competent HIV (i.e., capable of producing only a limited infection and preferably incapable of producing the pathological changes associated with infection by a non-attenuated HIV). The invention is not limited by the nature of the change which renders the virus attenuated. In a preferred embodiment, the attenuated virus comprises a mutated tat gene; preferably, the genome of the attenuated virus cannot express a functional Tat protein. In another preferred embodiment, the genome of attenuated virus genome further comprises a mutated nef gene; preferably, the genome of the attenuated virus cannot express either a functional Tat protein or Nef protein. In a preferred embodiment, the attenuate virus is selected from the group consisting of HIV.sub.CMV/tat-B, HIV.sub.CMV/AD8/nef-B/tat-B, HIV.sub.NL43/AD8/tat-B/nef-B and HIV.sub.NL43/nef-B/tat-B.

In another embodiment, the injectable preparation comprises attenuated Leishmania cells. The invention is not limited by the nature of the change which renders the Leishmania cell attenuated. In a preferred embodiment, the attenuated Leishmania cell comprises heterologous DNA encoding a cysteine protease gene as described above. In another preferred embodiment, the heterologous DNA further comprises DNA encoding a HIV gene selected from the group consisting of the env gene, the gag gene, the pol gene, the tat gene, the rev gene, the vif gene, the vpu gene, the vpr gene and the nef gene.

The invention is not limited by the nature of the monitoring employed to detect the presence (or absence) of infection of the human immune cells in the SCID/bg mouse comprising human immune cells (e.g., a hu-PBL-SCID/bg mouse) as described above.

The invention also provides an attenuated human immunodeficiency virus wherein the genome of the virus comprises a mutated tat gene and a mutated nef gene. The invention is not limited by the nature of the mutation in the tat and nef genes; insertions, deletions, substitutions may be employed to mutate the tat and nef genes. Preferably, the genome of the attenuated virus comprising a mutated tat gene and a mutated nef gene cannot express a functional Tat protein or a functional Nef protein. In a preferred embodiment, the attenuated virus is selected from the group consisting of HIV.sub.CMV/tat-B, HIV.sub.CMV/AD8/nef-B/tat-B, HIV.sub.NL43/AD8/tat-B/nef-B and HIV.sub.NL43/nef-B/tat-B.

The invention further provides a DNA construct comprising the provirus of a replication-defective human immunodeficiency virus, wherein the DNA construct is selected from the group consisting of pHP-1, pHP-2 and pHP-3.

The invention also provides a method comprising: a) providing: i) a SCID/beige mouse; ii) human fetal hematopoietic tissue comprising human immune cells (e.g., fetal liver, bone marrow, thymus, lymph node); iii) an injectable preparation comprising one or more components derived from a human immunodeficiency virus vaccine; iv) a composition comprising non-attenuated human immunodeficiency virus; b) inserting the hematopoietic tissue under the kidney capsule of the mouse to provide a transplanted mouse; c) injecting the transplanted mouse with the vaccine to produce an injected mouse; d) exposing the injected mouse to the composition; and e) monitoring the exposed mouse for the presence of infection of the human immune cells by the non-attenuated human immunodeficiency virus. The invention is not limited by the non-attenuated virus employed; any virus capable of causing an non-attenuated infection may be employed. Preferably, the non-attenuated virus is a virus isolated from a patient with AIDS (i.e., a clinical isolate) or is a virulent laboratory strain. Particularly preferred non-attenuated HIV strains include the NL4-3 strain, the ADA strain and HIV-1 primary isolates covering the different HIV clades (e.g., 92RW008, 92BR003, 92HT593, etc. available from the NIH AIDS Research and Reference Reagent Program of National Institutes of Health, Bethesda, Md.).

In a preferred embodiment, the injecting of step b) is repeated at least once prior to exposing the mouse to the composition. The injectable preparation comprises a candidate vaccine; a candidate vaccine may comprise proteins or peptides or nucleic acids and may be immunogenic. However, as this method offers a means to evaluate candidate vaccines, some candidate vaccines are expected to be immunogenic (i.e., capable of invoking an immune response directed against the pathogen from which the protein, peptide or nucleic acid was derived) and some are expected to not be immunogenic (and would therefore be rejected as a vaccine for use in subject such as a human). In a preferred embodiment, the injectable preparation is immunogenic. The injectable preparation may comprise pharmacological carriers, excipients and adjuvants as discussed above.

The invention is not limited by the nature of the component of the HIV present in the injectable composition. In a preferred embodiment, the component of the HIV comprises at least a portion of a protein derived from a HIV; the component may be a complete protein molecule or a peptide derived from the complete protein (and may be produced by peptide synthesis, recombinant DNA methodologies or protease digestion of the intact protein). In another preferred embodiment, the component of the HIV comprises DNA derived from HIV. In a particularly preferred embodiment, the DNA comprises proviral DNA encoding a HIV genome capable of expressing viral structural genes but incapable of being packaged into viral particles. In a preferred embodiment, the DNA comprises plasmid DNA selected from the group consisting of pHP-1, pHP-2 and pHP-3.

In another embodiment, the injectable preparation comprises an attenuated replication-competent HIV (i.e., capable of producing only a limited infection and preferably incapable of producing the pathological changes associated with infection by a non-attenuated HIV). The invention is not limited by the nature of the change which renders the virus attenuated. In a preferred embodiment, the attenuated virus comprises a mutated tat gene; preferably, the genome of the attenuated virus cannot express a functional Tat protein. In another preferred embodiment, the genome of attenuated virus genome further comprises a mutated nef gene; preferably, the genome of the attenuated virus cannot express either a functional Tat protein or Nef protein. In a preferred embodiment, the attenuate virus is selected from the group consisting of HIV.sub.CMV/tat-B, HIV.sub.CMV/AD8/nef-B/tat-B, HIV.sub.NL43/AD8/tat-B/nef-B and HIV.sub.NL43/nef-B/tat-B.

In another embodiment, the injectable preparation comprises attenuated Leishmania cells. The invention is not limited by the nature of the change which renders the Leishmania cell attenuated. In a preferred embodiment, the attenuated Leishmania cell comprises heterologous DNA encoding a cysteine protease gene as described above. In another preferred embodiment, the heterologous DNA further comprises DNA encoding a HIV gene selected from the group consisting of the env gene, the gag gene, the pol gene, the tat gene, the rev gene, the vif gene, the vpu gene, the vpr gene and the nef gene.

The invention is not limited by the nature of the monitoring employed to detect the presence (or absence) of infection of the human immune cells in the SCID/bg mouse comprising human immune cells (e.g., a hu-PBL-SCID/bg mouse) as described above.

The invention also provides an attenuated human immunodeficiency virus wherein the genome of the virus comprises a mutated tat gene and a mutated nef gene. The invention is not limited by the nature of the mutation in the tat and nef genes; insertions, deletions, substitutions may be employed to mutate the tat and nef genes. Preferably, the genome of the attenuated virus comprising a mutated tat gene and a mutated nef gene cannot express a functional Tat protein or a functional Nef protein. In a preferred embodiment, the attenuated virus is selected from the group consisting of HIV.sub.CMV/tat-B, HIV.sub.CMV/AD8/nef-B/tat-B, HIV.sub.NL43/AD8/tat-B/nef-B and HIV.sub.NL43/nef-B/tat-B.

DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic showing the pLSN, pLSNB70, pLSNGM1, pLSN-BG9 and pLSN-GFP retroviral constructs.

FIGS. 2A-E provide flow cytometry histograms for wild type D54MG cells (2A), B7-2-transduced D54MG cells (2B), GM-CSF-transduced D54MG cells (2C), B7-2 and GM-CSF-transduced D54MG cells (2D), and GFP-transduced D54MG cells (2E). For FIGS. 2A-2D, the histograms on the left represent D54MG cells stained with isotype matched control antibodies while the histograms on the right represent staining with monoclonal anti-human B7-2 antibodies. For FIG. 2E, the histogram on the left represents unstained wild type D54MG cells while the histogram on the right represents unstained GFP-transduced D54MG.

FIGS. 3A and 3B show the inhibition of growth of B7-2-transduced D54MG cells compared to unmodified D54MG cells in Hu-PBL-SCID/bg mice. In FIG. 3A, all mice were reconstituted with PBLs while in FIG. 3B half the mice from both groups were left unreconstituted.

FIGS. 4A and 4B show the inhibition of growth of GM-CSF-transduced D54MG cells compared to unmodified D54MG cells in Hu-PBL-SCID/bg mice. FIGS. 4A and 4B represent data from two separate experiments.

FIGS. 5A and 5B show the inhibition of unmodified D54MG cell challenges in Hu-PBL-SCID/nod mice (5A) and Hu-PBL-SCID/bg mice (5B) vaccinated with irradiated D54MG-B7-2/GM-CSF cells.

FIGS. 6A and 6B show the percentage of CD45+ lymphocytes in reconstituted SCID/bg and SCID/nod mice, respectively.

FIGS. 7A and 7B show RT activity in infected PBLs from LR and HR seronegative individuals using a macrophage tropic HIV at an moi of 0.2 (7A) or at an moi of 0.001-0.0001 (7B), respectively.

FIGS. 8A-8E demonstrate the in vivo infection of hu-PBL-SCID/bg mice. FIGS. 8A-8D show immunostaining for HIV-l infected cells in peritoneal lavage (8A and 8C) and splenocytes (8B and 8D) of mice reconstituted with PBLs from a LR (8C and 8D) or HR individual (8A and 8B). FIG. 8E shows the percentage of HIV-1 infected cells in the peripheral blood, spleen and peritoneal lavage of hu-PBL-SCID/bg mice reconstituted with PBLs from a LR or HR individual and challenged with T cell or macrophagic tropic HIV-1.

FIG. 9 shows an ELISPOT analysis of HIV-1-specific IFN-.gamma. production in PBLs of HR and LR uninfected individuals.

FIGS. 10A-10E demonstrate the HIV-1 infection of human PBLs in hu-PBL-SCID/bg mice treated or untreated with anti-CD8 antibodies. FIGS. 10A-10D show FACS analysis of splenocytes derived from the reconstituted mice (10A and 10C no anti-CD8 treatment; 10B and 10D after anti-CD8 treatment). FIG. 10E shows the percentage of HIV-1 infected cells in the spleen and peritoneal lavage of hu-PBL-SCID/bg mice reconstituted with PBLs from a LR or HR individual, with or without anti-CD8 treatment, and infected with HIV.sub.NLAD8.

FIGS. 11A-11F provide schematics showing the organization of the HIV-1 genome (11A) and the organization of the HIV.sub.NL4-3 virus, (11B) HIV.sub.NL43/AD8/tat-B/nef-B (11C), HIV.sub.NL43/nef-B/tat-B (11D), HIV.sub.CMV/nef-B/tat-B (11E) and HIV.sub.CMV/AD8/nef-B/tat-B (11F) clones.

FIGS. 12A-B provide schematics showing a portion of the wild type HIV-1 sequence as well as the tat-B (FIG. 12A; wild-type sequence provided in SEQ ID NO:26) and nef-B mutations (FIG. 12B; wild-type sequence provided in SEQ ID NOS:27 and 28).

DEFINITIONS

To facilitate understanding of the invention, a number of terms are defined below.

As used herein, "immunodeficient mouse" refers to a mouse or mouse strain which is deficient in immune system function, including at least a deficiency in function or presence of mature B and T lymphocytes. Examples of immunodeficient mouse strains include, but are not limited to the C.B-17-SCID-nod, C.B-17scid/scid and C.B-17-SCID-beige strains. Particularly preferred immunodeficient mice have a severe combined immunodeficiency characterized by a lack of mature T, B and natural killer (NK) lymphocytes (e.g., the C.B-17-SCID-beige mouse strain).

As used herein, "human T lymphocytes" refers to T lymphocytes of human origin. When present in a mouse reconstituted with human blood cells, the human T lymphocytes may be obtained from a variety of sources in the reconstituted mouse including blood (i.e., peripheral blood lymphocytes or PBLs), lymph nodes, spleen, peritoneal lavage, etc.

Human T lymphocytes are identified by the presence of certain markers or cell surface proteins including CD3, CD4, CD8, T cell antigen receptor (TCR) and CD45. The presence of these markers on a lymphocyte may be determined by standard immunocytological means such as incubation (or staining) of a cell suspension containing lymphocytes with antibodies specific for these markers; the antibodies may be directly labelled (e.g. with a fluorophore such as fluorescein, phycoerythrin, Texas Red, etc.) or the presence of the antibody bound to the surface of a lymphocyte may be detected using a secondary antibody (i.e., an antibody directed at the first antibody or a component thereof) that is labelled. The stained lymphocytes may then be analyzed using a fluorescence microscope or a FACS (fluorescence-activated cell sorter) analysis. "Mature human T lymphocytes" express either CD4 or CD8, CD3 and a TCR. "Immature human T lymphocytes" express both CD4 and CD8 (i.e., they are CD4+8+); these cells are also referred to as progenitor T cells. "Immature naive human T lymphocytes" are immature T lymphocytes that have not been activated (i.e., they have not engaged antigen specific for their TCR or been stimulated by a nonspecific mitogen) and are said to be naive. "Immature naive T lymphocytes" includes CD4+8+ T cells as well as CD45RA+ T cells.

A mouse comprising CD45+ T lymphocytes wherein at least 5% of the human CD45+ T cells represent immature naive T lymphocytes is a mouse in which 5% or more of the CD45+ T cells are either CD4+8+ or CD45RA+ or the sum of the % of CD45+ T cells that are CD4+8+ and CD45RA+ is at least 5%.

CD45 proteins are found on the surface all hematopoietic cells, except for erythrocytes [The Leukocyte Antigen Facts Book, Barclay et al. (1993), Academic Press, London, UK, pp. 202-204]. Different isoforms of CD45 are found on different lymphoid cell types; CD45RO is found on activated and memory T cells, whereas CD45RA is found on naive T cells.

The term "SCID/beige mouse" refers to the C.B-17-SCID-beige mouse strain. The terms SCID/beige, SCID-beige and SCID/bg are used interchangeably herein.

The term "human tumor cells" refers to tumor cells of human origin; a tumor cell is a neoplastic or cancerous cell. Tumor cells may be "established tumor cells," i.e., those which can be maintained indefinitely in tissue culture or may be "primary tumor cells," i.e., tumor cells freshly isolated or explanted from a patient. The term "primary tumor cells" encompasses primary tumor cells maintained in tissue culture for less than or equal to 5 passages.

The term "human immune cell" refers to cells of the immune system (e.g., T, B and NK lymphocytes, antigen presenting cells) that are of human origin.

The term "human peripheral blood lymphocytes" refers to nucleated, non-erythroid cells derived from the blood of a human. The terms peripheral blood lymphocytes (PBLs) and peripheral blood mononuclear cells (PBMCs) are used herein interchangeably.

The term "central nervous system cells" refers to cells derived from the central nervous system (i.e., cells derived from the brain and spinal cord).

A mouse "reconstituted with human peripheral blood lymphocytes" is a mouse in which human PBLs have been introduced (e.g., by intraperitoneal injection) and persist for a period of at least 4 weeks.

A "SCID/beige mouse comprising human immune cells" is a SCID/bg mouse that has been reconstituted with human PBLs or another source of human immune cells (e.g., human fetal hematopoietic tissues) and thus, contains human immune cells. SCID/beige mouse comprising human immune cells whose serum contains at least 100 .mu.g/ml of human immunoglobulin (Ig) are preferentially employed for the evaluation of vaccines.

An "injectable preparation" is a preparation suitable for injection into an animal (e.g., a mouse).

An "immune-modulating gene" is a gene encoding a protein that modulates the immune response. Examples of immune-modulating genes include but are not limited to cytokines, costimulatory molecule and chemotactins. The product of an immune-modulating gene is said to be an "immune modulator."A "cytokine" is a hormone-like protein, typically of low molecular weight, that regulates the intensity and duration of the immune response and is involved in cell to cell communication. Examples of cytokines include but are not limited to the interleukins (e.g., interleukin 2, interleukin 4, interleukin 6, interleukin 7, interleukin 12), granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, interferon-gamma (IFN-.gamma.) and tumor necrosis factor-alpha (TNF-.alpha.).

The term "cell cycle regulator" refers to any protein whose activity modulates progression of the cell cycle. Particularly preferred cell cycle regulators are those that block cell cycle progression. Examples include but are not limited to the HIV vpr gene product, p21, inhibitors of mammalian cyclins, etc.

The term "inducer of apoptosis" refers to any protein whose activity induces apoptosis in a cell. Inducers of apoptosis include but are not limited to apoptin (the product of the chicken anemia virus VP3 gene), BAX, BAD, a BCL-X derivative and the HIV vpr gene product.

The term "expression vector" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

Transcriptional control signals in eucaryotes comprise "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription [Maniatis, et al., Science 236:1237 (1987)]. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types [for review see Voss, et al., Trends Biochem. Sci., 11:287 (1986) and Maniatis, et al., supra (1987)]. For example, the SV40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been widely used for the expression of proteins in mammalian cells [Dijkema, et al., EMBO J. 4:761 (1985)]. Two other examples of promoter/enhancer elements active in a broad range of mammalian cell types are those from the human elongation factor 1.alpha. gene [Uetsuki et al., J. Biol. Chem., 264:5791 (1989); Kim et al., Gene 91:217 (1990); and Mizushima and Nagata, Nuc. Acids. Res., 18:5322 (1990)] and the long terminal repeats of the Rous sarcoma virus [Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 (1982)] and the human cytomegalovirus [Boshart et al., Cell 41:521 (1985)].

The term "promoter/enhancer" denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (for example, the long terminal repeats of retroviruses contain both promoter and enhancer functions). The enhancer/promoter may be "endogenous" or "exogenous" or "heterologous." An endogenous enhancer/promoter is one which is naturally linked with a given gene in the genome. An exogenous (heterologous) enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques).

The presence of "splicing signals" on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site [Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp. 16.7-16.8]. A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cells requires signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term "poly A site" or "poly A sequence" as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an expression vector may be "heterologous" or "endogenous." An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene. A commonly used heterologous poly A signal is the SV40 poly A signal. The SV40 poly A signal is contained on a 237 bp BamHI/BclI restriction fragment and directs both termination and polyadenylation [Sambrook, supra, at 16.6-16.7]. This 237 bp fragment is contained within a 671 bp BamHI/PstI restriction fragment.

The term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectant" refers to a cell which has stably integrated foreign DNA into the genomic DNA.

The term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectant" refers to a cell which has stably integrated foreign or exogenous DNA into the genomic DNA of the transfected cell.

The terms "selectable marker" or "selectable gene product" as used herein refer to the use of a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. Selectable markers may be "dominant"; a dominant selectable marker encodes an enzymatic activity which can be detected in any mammalian cell line. Examples of dominant selectable markers include the bacterial aminoglycoside 3' phosphotransferase gene (also referred to as the neo gene) which confers resistance to the drug G418 in mammalian cells, the bacterial hygromycin G phosphotransferase (hyg) gene which confers resistance to the antibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyl transferase gene (also referred to as the gpt gene) which confers the ability to grow in the presence of mycophenolic acid. Other selectable markers are not dominant in that their use must be in conjunction with a cell line that lacks the relevant enzyme activity. Examples of non-dominant selectable markers include the thymidine kinase (tk) gene which is used in conjunction with TK^- cell lines, the carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) gene which is used in conjunction with CAD-deficient cells and the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) gene which is used in conjunction with HPRT^- cell lines. A review of the use of selectable markers in mammalian cell lines is provided in Sambrook et al., supra at pp. 16.9-16.15. It is noted that some selectable markers can be amplified and therefore can be used as amplifiable markers (e.g., the CAD gene).

Claim 1 of 5 Claims

I claim:

1. A method for evaluating a response of human immune cells to a human immunodeficiency virus, said method comprising:

a) providing:

i) a SCID/beige mouse reconstituted with said human immune cells;

ii) an injectable preparation comprising one or more components from said human immunodeficiency virus;

iii) a composition comprising non-attenuated human immunodeficiency virus;

b) injecting said mouse with said injectable preparation to produce an injected mouse;

c) exposing said injected mouse to said composition; and

d) monitoring said exposed mouse for an immune response of said human immune cells to said non-attenuated human immunodeficiency virus;

wherein said component of said human immunodeficiency virus comprises DNA, said DNA comprising proviral DNA encoding a human immunodeficiency virus genome capable of expressing viral structural genes but incapable of being packaged into viral particles, and wherein said DNA comprises plasmid DNA selected from the group consisting of pHP-1, pHP-2 and pHP-3.

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