WO2007050102A2

WO2007050102A2 - Dendritic cell based vaccines

Info

Publication number: WO2007050102A2
Application number: PCT/US2005/042996
Authority: WO
Inventors: Jonathan Serody; Nancy L. Davis; Robert E. Johnston; Timothy Moran
Original assignee: University Of North Carolina At Chapel Hill
Priority date: 2004-11-29
Filing date: 2005-11-29
Publication date: 2007-05-03
Also published as: WO2007050102A3; WO2007050102A9

Abstract

The present invention provides an isolated human dendritic cell comprising a recombinant alphavirus vector RNA that comprises a heterologous nucleic acid sequence encoding an antigen. In particular embodiments, the alphavirus vector RNA is a Venezuelan Equine Encephalitis virus (VEE) vector RNA. In other embodiments, the antigen is an infectious disease antigen or a cancer antigen. Also provided are compositions and pharmaceutical formulations comprising the inventive dendritic cells, as well as methods of inducing an immune response against an infectious agent or a cancer and methods of treating an infectious disease or cancer.

Description

DENDRITIC CELL BASEDVACCINES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 60/631 ,531 , filed November 29, 2004, the content of which is herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made in part, with federal government support under Grant No. P50 CA 58223-11 from the National Cancer Institute. The United States government has certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to the use of alphavirus vectors to produce dendritic cell-based vaccines.

BACKGROUND OF THE INVENTION There is significant interest in the use of dendritic cell (DC) vaccines as treatments for patients with malignancies and chronic infectious diseases (Paczesny et al., (2003) Semin. Cancer Biol. 13:439; Gandhi et al., (2002) Annu Rev. Med. 53:149). Following activation by inflammatory cytokines or microbial products, DCs possess several characteristics that are implicated in the efficient stimulation of tumor-specific T lymphocytes, including enhanced homing to lymphoid tissues, high level expression of MHC class I- and Il molecules in conjunction with costimulatory molecules, and secretion of immunostimulatory cytokines (Banchereau et al., (1998) Nature 392:245). The ability of DCs to prime tumor-specific T cell responses has been demonstrated in various animal models (Flamand et al, 1994) Eur. J. Immunol. 24:605; Mayordomo et al., (1995) Nat. Med. 1 :1297; Celluzzi et al., (1996) J. Exp. Med. 183:283). These studies have led to several clinical trials evaluating the efficacy of DCs loaded ex vivo with tumor-associated antigens (TAAs) to initiate protective immune responses in cancer patients (Stift et al., (2003) J. Clin. Oncol. 21 :135; Kono et al., (2002) Clin. Cancer Res. 8:3394; Murphy et al., (1999) Prostate 38:37; Fong et al., (2001 ) J. Immunol. 166:4254; Dees et al., (2004) Cancer Immunol. Immunother, 53:777; Chang et al. (2002) Clin. Cancer Res. 8:1021 ; Banchereau et al., (2001 ) Cancer Res. 61 :6451 ). Multiple techniques have been employed for loading DCs with TAAs including pulsing with MHC class I- and/or ll-restricted peptides (Thurner et al., (1999) J. Exp. Med. 190:1669; Nestle et al., (1998) Nat. Med. 4:328; Schuler-Thurner et al., (2002) J. Exp. Med. 195:1279), incubation with tumor cell lysates (Nestle et al., (1998) Nat. Med. 4:328), and electroporation with tumor cell RNA (Heiser et al., (2002) J. Clin. Invest. 109:409). Unfortunately, induction of measurable and durable anti-tumor T cell responses have been infrequent in most clinical trials, suggesting that the stimulatory capacity of current DC vaccines is inadequate (O'Neill et al., (2004) Blood 104:2235). Therefore, alternative strategies for inducing optimal DC maturation and antigen presentation are warranted. Viral vectors that encode TAAs can provide an alternative method for delivering antigens to DCs. Delivery of an entire TAA rather than TAA-derived peptides allows processing and presentation of multiple epitopes on both MHC class I and Il molecules, resulting in a broader CD8⁺ T cell response and incorporation of CD4⁺ T cell help (Yang et al., (2000) J. Immunol. 164:4204; zum Buschenfelde et al., (2001 ) J. Immunol. 167:1712). In contrast to MHC-restricted peptide vaccines, viral vectors can be used to transduce DCs of all MHC haplotypes. Viral vectors can induce DC maturation through both TLR- dependent and -independent pathways, resulting in up-regulation of costimulatory molecules and secretion of Th1 -inducing cytokines (Alexopoulou et al., (2001 ) Nature 413:732; Diebold et al., (2003) Nature 424:324). Additionally, viral vectors may provide stimuli that are required for overcoming tolerance against TAAs, specifically through the down-regulation of T_REG activity (Yang et al., (2004) Nat. Immunol. 5:508).

Several viral vectors have been utilized for transducing human DCs with TAAs (Butterfield et al., (1998) J. Immunol. 161 :5607; Dyall et al., (2001 ) Blood 97:114; Drexler et al., (1999) Cancer Res. 59:4955; Efferson et al., (2003) J. Virol. 77:7411 ). While some of these vectors have entered clinical trials (Di Nicola et al., (2004) Clin. Cancer Res. 10:5381), their widespread use is hampered by inefficient transduction efficiencies, interference with DC function, and induction of anti-vector responses due to pre-existing immunity (Jenne et al., (2001 ) Trends Immunol. 22:102).

Accordingly, there is a need in the art for improved DC-based vaccines for infectious disease agents and cancer.

SUMMARY OF THE INVENTION

Recognizing the limitations of prior DC delivery systems, the inventors have evaluated the use of an alphavirus-derived vector for transduction of human DCs. While it has been shown that an alphavirus-derived vector can infect murine DCs in vivo (MacDonald et al., (2000) J. Virol. 74:914), their capacity to transduce human DCs is unknown. The inventors have demonstrated that an alphavirus vector can infect human DCs. Further, alphavirus-transduced DCs can efficiently process and present alphavirus- encoded antigens, leading to robust proliferation of antigen-specific T cells and acquisition of effector function.

Accordingly, as one aspect the invention provides an isolated dendritic cell (e.g., human dendritic cell), wherein the human dendritic cell comprises a recombinant Venezuelan Equine Encephalitis virus (VEE) vector RNA that comprises a heterologous nucleic acid sequence encoding an antigen. In representative embodiments, the human dendritic cell is an immature dendritic cell.

As further aspects, the invention provides compositions and pharmaceutical formulations comprising the dendritic cell of the invention in a pharmaceutically acceptable carrier.

As another aspect, the invention provides a method of inducing an immune response against an infectious agent in a subject, the method comprising: administering a dendritic cell, composition or pharmaceutical formulation of the invention in an immunogenically effective amount to the subject.

Further provided is a method of treating an infectious disease in a subject, the method comprising: administering a dendritic cell, composition or pharmaceutical formulation of the invention in a treatment effective amount to the subject.

As yet another aspect, the invention provides a method of inducing an anti-cancer immune response in a subject, the method comprising: administering a dendritic cell, composition of pharmaceutical formulation of the invention in an immunogenically effective amount to the subject.

As still a further aspect, the invention provides a method of treating cancer in a subject, the method comprising: administering a human dendritic cell, composition or pharmaceutical formulation of the invention in a treatment effective amount to the subject.

Also encompassed by the invention is a method of inducing an immune response against an infectious agent in a subject, the method comprising: introducing a VEE particle into an immature human dendritic cell, wherein the VEE particle comprises a heterologous nucleic acid sequence encoding an infectious disease antigen; and administering the modified human dendritic cell in an immunogenically effective amount to the subject.

As a further aspect, the invention provides a method of treating an infectious disease in a subject, the method comprising: introducing a VEE particle into an immature human dendritic cell, wherein the VEE particle comprises a heterologous nucleic acid sequence encoding an infectious disease antigen; and administering the modified human dendritic cell in a treatment effective amount to the subject.

As still a further aspect, the invention provides a method of inducing an anti-cancer immune response in a subject, the method comprising: introducing a VEE particle into an immature human dendritic cell, wherein the VEE particle comprises a heterologous nucleic acid sequence encoding a cancer antigen; and administering the modified human dendritic cell in an immunogenically effective amount to the subject.

The invention still further provides a method of treating cancer in a subject, the method comprising: introducing a VEE particle into an immature human dendritic cell, wherein the VEE particle comprises a heterologous nucleic acid sequence encoding a cancer antigen; and administering the modified human dendritic cell in a treatment effective amount to the subject. These and other aspects of the invention are set forth in more detail in the description of the invention that follows.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: VRPs can efficiently transduce immature human DCs. A)

Human immature monocyte-derived DCs were infected with GFP-VRPs for 1 hour (MOI = 10). Cells were analyzed for GFP expression at 12 hours postinfection (hpi) by fluorescent microscopy. The white arrow indicates possible apoptosis of infected DCs. S) Infectivity of immature or LPS-matured DCs (n = 3 donors) was quantified by FACS. DCs were infected for 1 hour (MOI = 10) and GFP expression was evaluated at 6-24 hpi as indicated. Symbols represent mean percentage of GFP-positive DCs +/- standard error of the mean (SEM). C) DCs were infected with GFP-VRP as described above, harvested at 24 hpi, stained with PE-conjugated antibodies specific for CD11c, HLA-DR or CD14, and analyzed by two-color FACS.

Figure 2: VRP infection of immature DCs is dependent upon the MOI, length of infection and cell density during infection. A) Immature DCs (n = 3 donors) were infected for 1 hour with GFP-VRPs at increasing MOIs. The percentage of GFP-positive cells was determined at 12 hpi by FACS. β)

Immature DCs (n = 3 donors) were infected with GFP-VRPs (MOI = 20) for 1 hour at 0.5 x 10⁶ DC/ml, or for 2 hours at 1 x 10⁶ DC/ml. The percentage of GFP-positive cells was determined at 12 hpi by FACS. Graphs represent the mean percentage of GFP-positive cells +/- SEM. *p = 0.002, Student's t test.

Figure 3: VRP-infected DCs remain predominantly viable during the first 24 hours following infection. Immature DCs were infected for 2 hours with GFP-VRP (MOI = 20). At various times post-infection, viability of infected (GFP-positive) DCs and uninfected (GFP-negative) DCs was determined as described in Example 1. Graphs represent the mean percentage of viable cells from two experiments. Figure 4: VRP infection induces maturation of immature DCs.

Immature DCs were either mock-infected (gray histogram), infected for 2 hours with GFP-VRPs at an MOI of 20 (heavy line), treated with TNF-α at 20 ng/ml (thin line), or treated with LPS at 100 ng/ml (dashed line). DCs were harvested at 12 or 24 hpi and stained with the indicated PE-conjugated specific antibodies. Staining with isotype control antibodies was negative. The numbers indicate the median PE fluorescence intensity. The median costimulatory/maturation marker expression in VRP-infected DC cultures includes both GFP-positive and -negative cells. Data is representative of four experiments.

Figure 5: VRP-infected DCs, but not fully matured DCs, secrete high levels of proinflammatory cytokines. Supernatants from immature DCs that were either mock-infected (Mock-DC) or GFP-VRP-infected at an MOI of 20 (VRP-DC) were harvested and analyzed for specific cytokines by CBA (TN F-σ, IL-6, IL-12p70 or IL-8) or ELISA (IFN-σ). Supernatants from DCs that had been previously matured by 24 hours of LPS treatment (LPS-DC) or 48 hours of TNF- a treatment (TNF-DC) were also analyzed. The mean cytokine concentration +/- SEM from three donors is shown. Data is representative of two experiments. *p < 0.05 (Student's t test) when compared to Mock-DC, TNF-DC or LPS-DC.

Figure 6: VRP-transduced DCs stimulate greater expansion of antigen-specific CD8⁺ CTL compared to TNF-cr-matured DCs pulsed with peptide. Immature DCs were transduced with either FMP-VRPs or irrelevant GFP-VRPs for 2 hours (MOI = 20). DCs were washed and cocultured with autologous nonadherent PBMCs at various respondeπstimulator ratios in the presence of IL-2 and IL-7 for 7 days. A) Expansion of FMP-specific CD8⁺ T cells was determined by tetramer analysis. Baseline indicates the percentage of FMP-specific T cells before stimulation. The stimulatory capacity of VRP- infected DCs was compared to TNF-σ-matured DCs (TNF-DC) that had been pulsed with FMP peptide (10 μg/ml) for 2 hours or left untreated

(respondeπstimulator ratio = 20:1 ). Numbers represent the percentage of FMP- specific cells of total CD8⁺ T cells, β) Mean percent of FMP-specific CD8⁺ T cells on day 7 of stimulation from three experiments. *p < 0.05 (Student's t test). C) Percent of FMP-specific CD8⁺ T cells on day 7 of stimulation at various respondeπstimulator ratios. One of two similar experiments is shown. D) PBMCs that had been stimulated with FMP-VRP-infected DCs for 7 days were assayed for effector function in a standard ⁵¹Cr-release assay. Labeled FMP peptide-pulsed (open circles) or unpulsed T2 cells (solid triangles) were incubated with effector cells for 4 hours and specific lysis was calculated as described in Example 1. Lysis of the NK-sensitive cell line K562 was similar to that found using unpulsed T2 cells indicating that the enhanced lytic activity using FMP-pulsed T2 cells was not due to NK-mediated lysis. The graphs represent the mean of triplicate wells +/- SEM. One of two similar experiments is shown.

DETAILED DESCRIPTION OF THE INVENTION

Consistent generation of tumor-specific T cell responses in patients treated with DC-based vaccines has remained elusive. The shortcomings of current vaccines are due in part to inefficient antigen loading of DC, as well as unsatisfactory induction of DC maturation and proinflammatory cytokine secretion (Mclroy et al., (2003) Cancer Immunol. Immunother. 52:583). The present inventors have demonstrated that an alphavirus-derived vector can efficiently transduce human immature DCs in vitro, leading to DC maturation and secretion of proinflammatory cytokines. Furthermore, transduced DCs processed and presented an alphavirus-encoded antigen and stimulated significantly greater expansion of antigen-specific T cells in comparison to peptide-pulsed DCs matured with TNF-σ. Several vectors have been used for transducing human DCs with TAAs

(Butterfield et al., (1998) J. Immunol. 161 :5607; Dyall et al., (2001 ) Blood 97:114; Drexler et al., (1999) Cancer Res. 59:4955; Efferson et al., (2003) J. Virol. 77:741 ). However, the potential clinical use of these vectors is hindered by poor transduction efficiencies, inhibition of DC maturation, questionable safety, and induction of detrimental anti-vector immune responses. The use of alphavirus- derived vectors for DC transduction successfully addresses many of these concerns. For example, VEE-derived vectors have an outstanding safety record in thousands of animal experiments including both rodents and primates (Pushko et al., (1997) Virology 239:389; David et al., (2000) J. Virol. 74:371 ). Further, because VEE is only endemic to specific subtropical regions, pre-existing immunity to VEE is unlikely to be present in the majority of patients.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

All publications, patent applications, patents, and other references mentioned herein or in attachments hereto are incorporated by reference in their entirety.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

I. Definitions.

As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. As used herein, the term "polypeptide" encompasses both peptides and proteins.

A "polypeptide of interest" as used herein is a polypeptide that is desirably expressed in a subject, e.g., because of its biological and/or antigenic properties, and includes reporter polypeptides, therapeutic polypeptides, enzymes, growth factors, immunomodulatory polypeptides, and immunogenic polypeptides.

As used herein, an "isolated" nucleic acid means a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the nucleic acid.

Likewise, an "isolated" polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.

As used herein, an "isolated cell" is a cell that has been removed from a subject or is derived from a cell that has been removed from a subject, and optionally has been enriched or purified from a tissue or organ (e.g., blood, spleen, skin, bone marrow).

The term "nucleic acid" as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA and chimeras of RNA and DNA. The nucleic acid may be double-stranded or single- stranded. Where single-stranded, the nucleic acid may be a sense strand or an antisense strand. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. The term "heterologous nucleic acid" is a well-known term of art and would be readily understood by one of skill in the art to be a nucleic acid that is foreign to the nucleic acid carrier (e.g., viral or plasmid delivery vector).

The heterologous nucleic acid can be associated with appropriate expression control sequences, e.g., transcription/translation control signals and polyadenylation signals.

It will be appreciated that a variety of promoter/enhancer elements can be used depending on the level and tissue-specific expression desired. The promoter can be constitutive or inducible (e.g., the metalothionein promoter or a hormone inducible promoter), depending on the pattern of expression desired. The promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the promoter is not found in the virus into which the promoter is introduced. The promoter is generally chosen so that it will function in the target cell(s) of interest. In particular embodiments, the heterologous nucleotide sequence is operably associated with a promoter that provides high level expression of the heterologous nucleotide sequence, e.g., an alphavirus subgenomic 26S promoter (preferably, a VEE, Sindbis, Girdwood or TR339 26S subgenomic promoter). Inducible expression control elements can be used in those applications in which it is desirable to provide regulation over expression of the heterologous nucleic acid sequence. Inducible promoters/enhancer elements include tissue- preferred and tissue-specific promoter/enhancer elements, which further includes, but is not limited to, muscle preferred or specific (including cardiac, skeletal and/or smooth muscle), neural tissue preferred or specific (including brain-specific), eye preferred or specific (including retina and cornea), liver preferred or specific, bone marrow preferred or specific, pancreatic preferred or specific, spleen preferred or specific, skin (epidermal) preferred or specific, DC preferred or specific, and lung preferred or specific promoter/enhancer elements. Other inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements, examples of which include but are not limited to a Tet on/off element, a RU486-inducible promoter, an ecdysone-inducible promoter, a rapamycin-inducible promoter, and a metalothionein promoter. Moreover, specific initiation signals are generally required for efficient translation of inserted polypeptide coding sequences. These translational control sequences, which can include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic. In embodiments of the invention wherein there are two or more heterologous nucleic acids to be transcribed, the transcriptional units can be operatively associated with separate promoters or with a single upstream promoter and one or more downstream internal ribosome entry site (IRES) sequences (e.g., the picornavirus EMC IRES sequence).

In embodiments of the invention in which the heterologous nucleic acid sequence is transcribed and then translated in the target cells, specific initiation signals are generally required for efficient translation of inserted polypeptide coding sequences. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic. As used herein, the terms "express," "expresses," "expressed" or

"expression," and the like, with respect to a nucleic acid sequence (e.g., RNA or DNA) indicates that the nucleic acid sequence is transcribed and, optionally, translated. Thus, a nucleic acid sequence can express a polypeptide of interest or a functional untranslated RNA.

By the terms "treat," "treats," "treating" or "treatment of," and the like, it is intended that the severity of the subject's condition is reduced or at least partially improved or modified and that some alleviation, mitigation or decrease in at least one clinical symptom is achieved. The terms "treat," "treats," "treating," or "treatment of and the like also include prophylactic treatment of the subject {e.g., to prevent the onset of infection or cancer). As used herein, the terms "prevent," "prevents," and "prevention" (and grammatical equivalents thereof) are not meant to imply complete abolition of disease and encompasses any type of prophylactic treatment that reduces the incidence of the condition, delays the onset and/or progression of the condition, and/or reduces the symptoms associated with the condition.

The terms "vaccination" or "immunization" are well-understood in the art, and are used interchangeably herein unless otherwise indicated. For example, the terms vaccination or immunization can be understood to be a process that increases an organism's immune reaction to antigen and therefore to resist or overcome infection or cancer. In the case of the present invention, vaccination or immunization against an infectious agent or cancer will increase the organism's immune response and resistance to the infectious agent or cancer.

As used herein, a "cancer antigen" includes without limitation naturally occurring cancer cell antigens and modified forms thereof that induce an immune response in a subject, and further includes antigens associated with cancer cells and antigens that are specific to cancer cells and modified forms of the foregoing that induce an immune response in a subject. Cancer cell antigens further encompass naturally occurring tumor cell antigens. As used herein a "tumor cell antigen" includes naturally occurring tumor cell antigens and modified forms thereof that induce an immune response in a subject, and also includes antigens associated with tumors, and antigens that are specific for tumors and modified forms of the foregoing that induce an immune response in a subject. In particular embodiments, the cancer cell antigen is displayed on the outside of the cell or is found in an interior compartment of the cell. In other embodiments, the cancer antigen is secreted by the cell (e.g., antibodies produced by malignant B cells, e.g., in lymphoma). The term "cancer antigen" further encompasses antigens that correspond to proteins that are correlated with the induction of cancer such as oncogenic virus antigens (e.g., human papilloma virus antigens).

Further, the recitation of a "Her2/neu antigen" (or any other specified cancer antigen) includes without limitation any naturally occurring Her2/neu antigen (or other specified cancer antigen), and modified forms thereof that induce an immune response in a subject. Any suitable Her2/neu antigen can be used with the present invention, including the full-length protein and fragments thereof. For example, the antigen can comprise one or multiple epitopes, and can further comprise 6, 10, 15, 20, 30, 40, 50, 75, 100, 250, 500 or more consecutive amino acids of the Her2/neu protein. In particular embodiments, the Her2/neu antigen is as described in U.S. Patent Publication No. 20040241686. In other embodiments, the Her2/neu antigen comprises an epitope as described in U.S. Patent Publication No. 20040121946. In further embodiments, the Her2/neu antigen comprises the all or substantially all (e.g., all but about 1 , 2, 3, 5, 10, 15, 20, 25 or 50 amino acids) extracellular domain of the protein, optionally without the transmembrane and/or cytoplasmic portions, see, e.g., U.S. Patent No. 6,333,169. In other embodiments, the Her2/neu antigen comprises at least about 6, 10, 15, 20, 30, 40, 50, 75, 100, 250, 500 or more consecutive amino acids of the extracellular portion of the Her2/neu protein, optionally without the transmembrane or cytoplasmic portions. As will be appreciated by those skilled in the art, the Her2/neu antigen can be presented in the form of a fusion protein. For example, a fusion protein comprising the Her2/neu antigen and a cytokine (e.g., IL-12, IL-2 or GM-CSF) can be delivered (see, e.g., DeIa Cruz et al., (2003) Vaccine 21 : 1317). Also encompassed by the present invention are modified forms of any of the foregoing that induce an immune response in a subject.

As used herein, an "infectious disease antigen" includes without limitation naturally occurring antigens from an infectious agent and modified forms thereof that induce an immune response in the subject.

The term "alphavirus" has its conventional meaning in the art, and includes Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Encephalitis virus (WEE), Sindbis virus, South African Arbovirus No. 86 (S.A.AR86), Girdwood S.A. virus, Ockelbo virus, Semliki Forest virus, Middelburg virus, Chikungunya virus, O'Nyong-Nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzlagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, Buggy Creek virus, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an alphavirus.

Preferred alphaviruses for use in the present invention are Sindbis virus (including strains such as strain TR339), VEE, S.A.AR86 virus, Girdwood S.A. virus, and Ockeibo virus, and chimeric viruses thereof. The complete genomic sequences, as well as the sequences of the various structural and non-structural proteins are known in the art for numerous alphaviruses and include without limitation: Sindbis virus genomic sequence (GenBank Accession Nos. J02363, NCBI Accession No. NC_001547), S.A.AR86 genomic sequence (GenBank Accession No. U38305), VEE genomic sequence (GenBank Accession No. L04653, NCBI Accession No. NC_001449), Girdwood S.A genomic sequence (GenBank Accession No. U38304), Semliki Forest virus genomic sequence (GenBank Accession No. X04129, NCBI Accession No. NC_003215), and the TR339 genomic sequence (Klimstra et al., (1988) J. Virol. 72:7357; McKnight et al.,(1996) J. Virol. 70:1981).

The term "viral structural protein(s)" as used herein refers to one or more of the proteins that are constituents of a functional virus particle. The alphavirus structural proteins include the capsid protein, E1 glycoprotein, E2 glycoprotein, E3 protein and 6K protein. The alphavirus particle comprises the alphavirus structural proteins assembled to form an enveloped nucleocapsid structure. As known in the art, alphavirus structural subunits consisting of a single viral protein, capsid, associate with themselves and with the RNA genome to form the icosahedral nucleocapsid, which is then surrounded by a lipid envelope covered with a regular array of transmembranal protein spikes, each of which consists of a heterodimeric complex of two glycoproteins, E1 and E2 (See Paredes et al., (1993) Proc. Natl. Acad. ScL USA 90, 9095-99; Paredes et al., (1993) Virology 187, 324-32; Pedersen et al., (1974) J. Virol. 14:40). An "alphavirus vector RNA" (and similar terms) include recombinant (e.g., containing a heterologous nucleic acid sequence) and other modified forms (e.g., one or more attenuating mutations, deletions, insertions and/or other modifications) derived from the wild-type alphavirus genomic RNA. In particular embodiments, the alphavirus vector RNA can be a propagation-incompetent, but replication- competent, replicon as described herein. The wild-type alphavirus genome is a single-stranded, messenger-sense RNA, modified at the 5'-end with a methylated cap, and at the 3'-end with a variable-length poly (A) tract. The viral genome is divided into two regions: the first encodes the nonstructural or replicase proteins (nsP1-nsP4) and the second encodes the viral structural proteins (Strauss and Strauss, Microbiological Rev. (1994) 58:491-562). In other particular embodiments, the alphavirus vector RNA is a double-promoter molecule (as described herein). The alphavirus vector RNA can optionally comprise an alphavirus packaging signal. An "infectious" alphavirus particle is one that can introduce the alphavirus genomic RNA or vector RNA into a permissive cell, typically by viral transduction. Upon introduction into the target cell, the genomic or vector RNA serves as a template for RNA transcription (i.e., gene expression). The "infectious" alphavirus particle may be "replication-competent" (i.e., can transcribe and replicate the genomic or vector RNA) and "propagation- competent" (i.e., results in a productive infection in which new alphavirus particles are produced). In embodiments of the invention, the "infectious" alphavirus particle is a replicon particle that can introduce the vector RNA (i.e., replicon RNA) into a host cell, is "replication-competent" to replicate the replicon RNA, but is "propagation-defective" or "propagation-incompetent" in that it is unable to produce new alphavirus replicon particles in the absence of helper sequences that complement the deletions or other mutations in the replicon (i.e., provide the structural proteins that are not provided by the replicon).

A "replicating" or "replication-competent" alphavirus particle refers to the ability to replicate the viral genomic RNA or vector RNA. Similarly, a "replicating" or "replication-competent" alphavirus vector RNA refers to the ability of the vector RNA to self-replicate. Generally, a "replication-competent" alphavirus particle or vector RNA will comprise sufficient alphavirus non-structural protein coding sequences (i.e., nsP1 through nsP4 coding sequences) to produce functional alphavirus non-structural proteins.

II. Dendritic Cell (DO-Based Vaccines. The present inventors have discovered that alphavirus (e.g., VEE) vectors can be used to deliver antigens, such as infectious agent antigens and cancer associated antigens, to human DC ex vivo to provide DC-based vaccines. Accordingly, as one aspect, the invention provides a human DC, wherein said human DC comprises a recombinant alphavirus vector RNA that comprises a heterologous nucleic acid sequence encoding an antigen. The DC can optionally comprise an alphavirus particle comprising the alphavirus vector RNA. In particular embodiments, the alphavirus particle and/or alphavirus vector RNA is a VEE particle and/or VEE vector RNA.

The invention also provides a human DC, wherein an alphavirus vector RNA, an alphavirus particle comprising an alphavirus vector RNA, or a nucleic acid encoding the alphavirus vector RNA or alphavirus particle has been introduced into the DC ex vivo, wherein the alphavirus vector RNA comprises a heterologous nucleic acid sequence encoding an antigen. In particular embodiments, the alphavirus particle and/or alphavirus vector RNA is a VEE particle and/or VEE vector RNA.

According to the foregoing embodiments, an alphavirus particle and/or alphavirus vector RNA can be introduced into the DC by any method known in the art. For example, the DC can be contacted with an alphavirus particle and the alphavirus vector RNA, and optionally the alphavirus particle, introduced into the cell. Alternatively, the alphavirus vector RNA can be directly introduced into the cell (e.g., by transfection such as by electroporation or lipofection). As a further alternative, a nucleic acid encoding the alphavirus vector RNA or alphavirus particle is introduced into the cell. The nucleic acid encoding the alphavirus vector RNA or alphavirus particle can be any suitable nucleic acid, including non-alphavirus vectors. To illustrate, a DNA such as a DNA virus vector can be introduced into the DC and the alphavirus vector RNA or alphavirus particle expressed therefrom. Optionally, one or more nucleic acids (e.g., one or more DNA molecules such as DNA virus vectors) are introduced into the DC and the alphavirus particle expressed therefrom.

The term "dendritic cell" as used herein includes immature DC, as that term is understood by those skilled in the art. Thus, in representative embodiments, the DC is an immature DC. In some embodiments, an "immature" DC can be characterized by the expression of cell surface markers. For example, a typical phenotype of an immature DC is CDHc⁺, HLA-DR⁺, CD86⁺, CD14^", CD40^", and CD80^". In particular embodiments, introduction of the alphavirus vector into an immature DC induces maturation. In some embodiments, the DC is monocyte-derived. In other embodiments, the DC is derived from a CD34⁺ bone marrow progenitor cell. Suitable DCs can also be isolated from skin (e.g., Langerhans cells [epidermal DC] and interstitial DC) and spleen (which contains several DC populations), and optionally allowed or induced to mature. Dendritic cells can be obtained from a subject (e.g., a cancer patient or a person at risk for cancer), the alphavirus vector RNA, alphavirus particle or nucleic acid encoding the alphavirus vector RNA or alphavirus particle introduced into the DC ex vivo, and then introduced back into the subject. Alternatively, a cell from which a DC can be derived (e.g., monocytes, CD34⁺ bone marrow progenitor cells) is obtained from the subject, DC are derived therefrom, and then modified and administered to the subject as described above. Alternatively, the cell (a DC cell or precursor thereof) can be obtained from a donor subject, modified, and then introduced into a recipient subject. If the DC is not from (or derived from) the recipient, it is desirable that the DC is HLA compatible with the recipient.

One particular method of deriving human DCs is from monocytes. Blood can be obtained from a human subject, peripheral blood mononuclear cells (PBMCs) isolated, cultured in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin 4 (IL-4) for a suitable time. For example, cells can be harvested after approximately 4-14 days as immature DCs, or can be induced to mature, e.g., after further incubation in the presence of lipopolysaccharide (LPS) and/or tumor necrosis factor-α (TNF-σ) for from about 12 or 24 hours to about 1 , 2, 3 or 4 days. Optimized conditions for introducing the alphavirus vector into the DCs can be determined by those skilled in the art based on the teachings provided herein. In particular embodiments, the alphavirus particles are introduced into DCs at an MOI of about 5, 10, 15 or 20 to about 40, 50, 75, 100 or even higher for a period of about 0.5 to about 5 hours, optionally about 1 to 4 hours. The DC can be present at any suitable concentration, e.g., at about 0.05 x 10⁶ to about 10 x 10⁶ cells/ml, 0.1 x 10⁶ to about 5 x 10⁶ cells, or about 0.5 x 10⁶ to about 2.5 x 10⁶ cells/ml.

Any suitable alphavirus (e.g., VEE) vector known in the art can be used in connection with the present invention. The vector can be a live non-attenuated alphavirus vector, an attenuated alphavirus vector and/or a propagation- defective alphavirus vector (e.g., an alphavirus replicon vector). Attenuated alphavirus vectors and alphavirus replicon vectors are described in more detail below. In particular embodiments, the alphavirus vector is a VEE, Girdwood or Sindbis vector.

The heterologous nucleic acid sequence can encode any antigen of interest, including antigens that induce an immune response against an infectious agent or against a cancer cell antigen (e.g., tumor cell antigen) or protein that correlates with the induction of cancer (e.g., an antigen from an oncogenic virus). The antigen can be a naturally-occurring antigen or can be a modified form thereof that induces an immune response in the subject. A modified form of a naturally occurring antigen can be selected to induce the desired immune response with reduced pathogenicity relative to the native antigen. Suitable antigens are described in more detail below. The invention also provides compositions and pharmaceutical formulations comprising a plurality of the DC of the invention, optionally in an immunogenically effective amount.

III. Antigens. The heterologous nucleic acid can express any antigen of interest known in the art, including infectious disease antigens and cancer antigens (including tumor antigens), and can be administered in any suitable form. Any suitable cancer antigen can be delivered according to the present invention. Cancer antigens (including tumor antigens) include without limitation naturally occurring cancer cell antigens and modified forms thereof that induce an immune response in a subject, and further includes antigens associated with cancer cells and antigens that are specific to cancer cells and modified forms of the foregoing that induce an immune response in a subject. The term cancer antigen further encompasses antigens that correspond to proteins that are correlated with the induction of cancer such as oncogenic virus antigens (e.g., human papilloma virus antigens). Exemplary cancer antigens include, without limitation, HER2/neu and BRCA1 antigens for breast cancer, MART-1/MelanA, gp100, tyrosinase, TRP-1 , TRP-2, NY-ESO-1 , CDK-4, β-catenin, MUM-1 , Caspase-8, KIAA0205, SART-1 , PRAME, and p15 antigens, members of the MAGE family, the BAGE family (such as BAGE-1 ), the DAGE/PRAME family (such as DAGE-1 ), the GAGE family, the RAGE family (such as RAGE-1 ), the SMAGE family, NAG, TAG-72, CA125, mutated proto-oncogenes such as p21 ras, mutated tumor suppressor genes such as p53, tumor associated viral antigens (e.g., HPV16 E7), the SSX family, HOM-MEL-55, NY-COL-2, HOM-HD- 397, HOM-RCC-1.14, HOM-HD-21 , HOM-NSCLC-11 , HOM-MEL-2.4, HOM- TES-11 , RCC-3.1.3, NY-ESO-1 , and the SCP family. Members of the MAGE family include, but are not limited to, MAGE-1 , MAGE-2, MAGE-3, MAGE-4 and MAGE-11. Members of the GAGE family include, but are not limited to, GAGE- 1 , GAGE-6. See, e.g., review by Van den Eynde and van der Bruggen (1997) in Curr. Opin. Immunol. 9: 684-693, Sahin et al. (1997) in Curr. OpIn. Immunol. 9: 709-716, and Shawler et al. (1997), the entire contents of which are incorporated by reference herein for their teachings of cancer antigens.

The cancer antigen can also be, but is not limited to, human epithelial cell mucin (Muc-1 ; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), MUC-2, MUC-3, MUC-18, the Ha-ras oncogene product, carcino-embryonic antigen (CEA), the raf oncogene product, CA-125, GD2, GD3, GM2, TF, sTn, gp75, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostatic serum antigen (PSA), prostate-specific membrane antigen (PSMA), alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras oncogene product, £-HCG, gp43, HSP-70 , p17 mel, HSP-70, gp43, HMW, HOJ-1 , melanoma gangliosides, TAG-72, mutated proto-oncogenes such as p21 ras, mutated tumor suppressor genes such as p53, estrogen receptor, milk fat globulin, telomerases, nuclear matrix proteins, prostatic acid phosphatase, protein MZ2-E, polymorphic epithelial mucin (PEM), folate-binding-protein LK26, truncated epidermal growth factor receptor (EGFR), Thomsen-Friedenreich (T) antigen, GM-2 and GD-2 gangliosides, polymorphic epithelial mucin, folate- binding protein LK26, human chorionic gonadotropin (HCG), pancreatic oncofetal antigen, cancer antigens 15-3,19-9, 549, 195, squamous cell carcinoma antigen (SCCA), ovarian cancer antigen (OCA), pancreas cancer associated antigen (PaA), mutant K-ras proteins, mutant p53, and chimeric protein P210_BCR-ABL-

The cancer antigen can also be an antibody produced by a B cell tumor (e.g., B cell lymphoma; B cell leukemia; myeloma; hairy cell leukemia), a fragment of such an antibody, which contains an epitope of the idiotype of the antibody, a malignant B cell antigen receptor, a malignant B cell immunoglobulin idiotype, a variable region of an immunoglobulin, a hypervariable region or complementarity determining region (CDR) of a variable region of an immunoglobulin, a malignant T cell receptor (TCR), a variable region of a TCR and/or a hypervariable region of a TCR. In one embodiment, the cancer antigen of this invention can be a single chain antibody (scFv), comprising linked VH, and VL domains, which retains the conformation and specific binding activity of the native idiotype of the antibody.

Also encompassed by the present invention are modified forms of the cancer antigens described above which induce an immune response in a subject, and which optionally have reduced pathogenicity as compared with the naturally occurring antigen.

The antigens that can be used in accordance with the present invention are in no way limited to the cancer antigens listed herein. Other cancer antigens can be identified, isolated and cloned by methods known in the art such as those disclosed in U.S. Pat. No. 4,514,506, the entire contents of which are incorporated by reference herein.

The cancer to be treated or immunized against (i.e., prophylactic treatment) can be a tumor-forming cancers and further can be, but is not limited to, B cell lymphoma, T cell lymphoma, myeloma, leukemia, hematopoietic neoplasias, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non- Hodgkins lymphoma, Hodgkins lymphoma, uterine cancer, adenocarcinoma, breast cancer, pancreatic cancer, colon cancer, lung cancer, renal cancer, bladder cancer, liver cancer, prostate cancer, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcimona, gastric cancer, brain cancer, angiosarcoma, hemangiosarcoma, head and neck cancer, thyroid carcinoma, soft tissue sarcoma, bone sarcoma, testicular cancer, uterine cancer, cervical cancer, gastrointestinal cancer, and any other cancer now known or later identified (see, e.g., Rosenberg (1996) Ann. Rev. Med. 47:481- 491 , the entire contents of which are incorporated by reference herein).

An infectious disease antigen according to the present invention includes without limitation naturally occurring antigens from an infectious agent and modified forms thereof that induce an immune response in the subject. The antigen can be any antigen suitable for inducing an immune response in a subject against an infectious disease, including but not limited to microbial, bacterial, protozoal, parasitic and viral diseases. Such infectious disease antigens can include, but are not limited to, antigens from Hepadnaviridae including hepatitis A, B, C, D, E, F, G, etc.; Flaviviridae including human hepatitis C virus (HCV), yellow fever virus and dengue viruses; Retroviridae including human immunodeficiency viruses (HIV), simian immunodeficiency virus (SIV), and human T lymphotropic viruses (HTLV1 and HTLV2); Herpesviridae including herpes simplex viruses (HSV-1 and HSV-2), Epstein Barr virus (EBV), cytomegalovirus, varicella-zoster virus (VZV), human herpes virus 6 (HHV-6) human herpes virus 8 (HHV-8), and herpes B virus; Papovaviridae including human papilloma viruses; Rhabdoviridae including rabies virus; Paramyxoviridae including respiratory syncytial virus; Reoviridae including rotaviruses; Bunyaviridae including hantaviruses; Filoviridae including Ebola virus; Adenoviridae; Parvoviridae including parvovirus B 19; Arenaviridae including Lassa virus; Orthomyxoviridae including influenza viruses; Poxviridae including Orf virus, molluscum contageosum virus, smallpox virus and Monkey pox virus; Coronaviridae including corona viruses such as the severe acute respiratory syndrome (SARS) virus; and Picornaviridae including polioviruses; mixoviruses; orbiviruses; picodnaviruses; encephalomyocarditis virus (EMV); Parainfluenza viruses (e.g., human parainfluenza virus type 2), Coxsackieviruses, Echoviruses, Rubeola virus, Rubella virus, human papillomaviruses, Canine distemper virus, Canine contagious hepatitis virus, Feline calicivirus, Feline rhinotracheitis virus, TGE virus (swine), Foot and mouth disease virus, simian virus 5, human metapneuomovirus, enteroviruses, and any other pathogenic virus now known or later identified (see, e.g., Fundamental Virology, Fields et al., Eds., 3^rd ed., Lippincott-Raven, New York, 1996, the entire contents of which are incorporated by reference herein for the teachings of pathogenic viruses). As further examples, the antigen can be an orthomyxovirus antigen (e.g., an influenza virus antigen, such as the influenza virus hemagglutinin (HA) surface protein, influenza neuraminidase protein, the influenza virus nucleoprotein (NP) antigen or inactivated influenza virions, or an equine influenza virus antigen), or a lentivirus antigen (e.g., an equine infectious anemia virus antigen, a SIV antigen, or a HIV antigen, such as, e.g., HIV or SIV gp120, gp160, gp41 , or matrix/capsid protein, or the gag, pol or env gene products. The antigen may also be an arenavirus antigen (e.g., Lassa fever virus antigen, such as the Lassa fever virus nucleocapsid protein gene and the Lassa fever envelope glycoprotein gene), a Picornavirus antigen (e.g., a Foot and Mouth Disease virus antigen), a poxvirus antigen (e.g., a vaccinia antigen, such as the vaccinia L1 or L8 genes), an Orbivirus antigen (e.g., an African horse sickness virus antigen), a flavivirus antigen (e.g., a yellow fever virus antigen, a West Nile virus antigen, or a Japanese encephalitis virus antigen), a filovirus antigen (e.g., an Ebola virus antigen, or a Marburg virus antigen, such as NP and GP genes), a bunyavirus antigen (e.g., RVFV, CCHF, and SFS antigens), a norovirus antigen (e.g., a Norwalk virus antigen), or a coronavirus antigen (e.g., an infectious human coronavirus antigen, such as the human coronavirus envelope glycoprotein gene, or a porcine transmissible gastroenteritis virus antigen, or an avian infectious bronchitis virus antigen). The antigen may further be a polio antigen, herpes antigen (e.g., CMV, EBV, HSV antigens), mumps antigen, measles antigen, rubella antigen, diptheria toxin or other diptheria antigen, pertussis antigen, hepatitis (e.g., hepatitis A or hepatitis B) antigen (e.g., HBsAg, HBcAg, HBeAg), or any other vaccine antigen known in the art. The antigen can be an antigen from a pathogenic microorganism, which can include but is not limited to, Rickettsia, Chlamydia, Mycobacteria, Clostridia, Corynebacteria, Mycoplasma, Ureaplasma, Legionella, Shigella, Salmonella, pathogenic Escherichia co// species, Bordatella, Neisseria, Treponema, Bacillus, Haemophilus, Moraxella, Vibrio, Staphylococcus spp., Streptococcus spp.,

Campylobacter spp., Borrelia spp., Leptospira spp., Erlichia spp., Klebsiella spp., Pseudomonas spp., Helicobacter spp., and any other pathogenic microorganism now known or later identified (see, e.g., Microbiology, Davis et al, Eds., 4^th ed., Lippincott, New York, 1990, the entire contents of which are incorporated herein by reference for the teachings of pathogenic microorganisms).

Specific examples of microorganisms from which the antigen of this invention can be obtained include, but are not limited to, Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Treponema pallidum, Bacillus anthracis, Salmonella typhi, Vibrio cholera, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter jejuni, Clostridium difficile, Clostridium botulinum, Mycobacterium tuberculosis, Borrelia burgdorferi, Haemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenza, and enterotoxic Escherichia coli.

The antigen can further be an antigen from a pathogenic protozoa, including, but not limited to, Plasmodium species (e.g., malaria antigens), Babeosis species, Schistosoma species, Trypanosoma species, Pneumocystis carnii, Toxoplasma species, Leishmania species, and any other protozoan pathogen now known or later identified.

The antigen can also be an antigen from pathogenic yeast or fungi, including, but not limited to, Aspergillus species, Candida species, Cryptococcus species, Histoplasma species, Coccidioides species, and any other pathogenic fungus now known or later identified.

Other specific examples of various antigens include, but are not limited to, the influenza virus nucleoprotein (residues 218-226; Fu et al. (1997) J. Virol. 71 : 2715-2721 ), antigens from Sendai virus and lymphocytic choriomeningitis virus (An et al. (1997) J. Virol. 71 : 2292-2302), the B1 protein of hepatitis C virus (Bruna-Romero et al. (1997) Hepatology 25: 470-477), gp 160 of HIV (Achour et al. (1996) J. Virol. 70: 6741-6750), amino acids 252-260 of the circumsporozoite protein of Plasmodium berghei (Allsopp et al. (1996) Eur. J. Immunol. 26: 1951- 1958), the influenza A virus nucleoprotein (residues 366-374; Nomura et al. (1996) J. Immunol. Methods 193: 4149), the listeriolysin O protein of Listeria monocytogenes (residues 91-99; An et al. (1996) Infect. Immun. 64: 1685-1693), the E6 protein (residues 131-140; Gao et al. (1995) J. Immunol. 155: 5519-5526) and E7 protein (residues 21-28 and 48-55; Bauer et al. (1995) Scand. J. Immunol. 42: 317-323) of human papillomavirus type 16, the M2 protein of respiratory syncytial virus (residues 82-90 and 81-95; Hsu et al. (1995) Immunology 85: 347-350), the herpes simplex virus type 1 ribonucleotide reductase (Salvucci et al. (1995) J. Gen. Virol. 69: 1122-1131), the rotavirus VP7 protein (Franco et al. (1993) J. Gen. Virol. 74: 2579-2586), P. falciparum antigens (causing malaria) and hepatitis B surface antigen (Gilbert et al. (1997) Nature Biotech. 15: 1280-1283).

The antigen can also be an antigen from chronic or latent infective agents, which typically persist because they fail to elicit a strong immune response in the subject. Illustrative latent or chronic infective agents include, but are not limited to hepatitis B, hepatitis C, Epstein-Barr Virus, herpes viruses, human immunodeficiency virus, and human papilloma viruses.

The DC, compositions, pharmaceutical formulations and methods of the invention can be used to produce an immune response to, and optionally to treat, any infectious disease agent including but not limited to those identified above. In particular embodiments, the antigen is an HlV antigen, SIV virus antigen, human papilloma virus antigen, influenza virus antigen, or a modified form of any of the foregoing.

Also encompassed by the present invention are modified forms of the infectious disease antigens described above which induce an immune response against the infectious agent, and which optionally have reduced pathogenicity as compared with the naturally occurring antigen. IV. Alphavirus Vectors.

Alphavirus vectors are known in the art, and any suitable alphavirus vector can be used to deliver the heterologous nucleic acid encoding the antigen to the DC including live, propagation-competent alphavirus vectors, attenuated alphavirus vectors, and/or propagation-incompetent, replicating, alphavirus vectors such as an alphavirus replicon vector (as described below). The alphavirus vector can be in the form of an alphavirus particle or can be delivered as an alphavirus vector RNA. Further, as discussed above, a nucleic acid encoding an alphavirus vector RNA or one or more nucleic acids encoding an alphavirus particle can be introduced into the DC.

Alphavirus vectors, including replicon vectors, are described in U.S. Patent No. 5,505,947 to Johnston et al.; U.S. Patent No. 5,792,462 to Johnston et al.; U.S. Patent No. 6,156,558; U.S. Patent No. 6,521 ,325; U.S. Patent No. 6,531 ,135; U.S. Patent No. 6,541 ,010; and Pushko et al. (1997) Virol. 239:389- 401 ; U.S. Patent No. 5,814,482 to Dubensky et al.; U.S. Patent No. 5,843,723 to Dubensky et al.; U.S. Patent No. 5,789,245 to Dubensky et al.; U.S. Patent No. 5,739,026 to Garoff et al.; the disclosures of which are incorporated herein by reference in their entireties. In embodiments of the invention, the alphavirus vector is a VEE vector, Sindbis vector or Girdwood vector, including VEE, Sindbis or Girdwood particles and VEE, Sindbis or Girdwood vector RNAs.

Specific types of alphavirus vectors are discussed in more detail below.

A. Double Promoter Vectors.

In embodiments of the invention, the alphavirus vector is an alphavirus double promoter vector (e.g., an alphavirus particle, alphavirus vector RNA or a nucleic acid encoding either of the foregoing)! A double promoter vector is typically a replication and propagation competent virus or vector RNA that retains the sequences encoding the alphavirus structural proteins sufficient to produce an alphavirus particle. Double promoter vectors are described in United States Patent No. 5,185,440, 5,505,947 and 5,639,650, the disclosures of which are incorporated in their entireties by reference. Illustrative alphavirus for constructing the double promoter vectors include Sindbis (e.g., TR339), Girdwood and VEE viruses. In addition, the double promoter vector may contain one or more attenuating mutations. Attenuating mutations are described in more detail hereinbelow.

In representative embodiments, the double promoter vector is constructed so as to contain a second subgenomic promoter (Ae., 26S promoter) inserted 3' to the viral RNA encoding the structural proteins or between nsP4 and the native 26S promoter. The heterologous nucleic acid can be inserted between the second subgenomic promoter, so as to be operatively associated therewith, and the 3' UTR of the virus genome. Heterologous nucleic acid sequences of less than 3 kilobases, less than 2 kilobases, or less than 1 kilobase, can be inserted into the double promoter vector.

B. Replicon Vectors.

In other representative embodiments, the alphavirus vector is an alphavirus replicon vector (e.g., an alphavirus replicon particle, alphavirus replicon RNA or a nucleic acid encoding either of the foregoing), which are infectious, propagation- defective, replicating virus vectors. Replicon vectors are described in more detail in WO 96/37616 to Johnston et al.; U.S. Patent No. 5,505,947 to Johnston et al.; U.S. Patent No. 5,792,462 to Johnston et al.; U.S. Patent No. 6,156,558; U.S. Patent No. 6,521 ,325; U.S. Patent No. 6,531 ,135; U.S. Patent No. 6,541 ,010; and Pushko et al. (1997) Virol. 239:389-401. Illustrative alphaviruses for constructing the replicon vectors according to the present invention are Sindbis (e.g., TR339), Girdwood, and VEE.

In general, in the replicon system, the viral genome contains the viral sequences necessary for viral replication (e.g., the nsp1-4 genes), but is modified so that it is defective for expression of at least one viral structural protein required for production of new viral particles. RNA transcribed from this vector contains sufficient viral sequences (e.g., the viral nonstructural genes) for RNA replication and transcription. Thus, if the transcribed RNA is introduced into susceptible cells, it will be replicated and translated to give the replication proteins. These proteins will transcribe the recombinant genomic RNA, and optionally a transgene (if present). The autonomously replicating RNA (Ae., replicon) can only be packaged into virus particles if the defective or alphavirus structural protein genes that are deleted from or defective in the replicon are provided by one or more helper molecules, which are provided to the helper cell, or by a stably transformed packaging cell.

In some embodiments, the helper molecules do not contain the viral nonstructural genes for replication, but these functions are provided in trans by the replicon molecule. The transcriptase functions translated from the replicon molecule transcribe the structural protein genes on the helper molecule, resulting in the synthesis of viral structural proteins and packaging of the replicon into virus-like particles. Generally, the helper molecules do not contain a functional alphavirus packaging signal. As the alphavirus packaging or encapsidation signal is located within the nonstructural genes, the absence of these sequences in the helper molecules precludes their incorporation into virus particles. Typically, the replicon molecule comprises an alphavirus packaging signal.

Accordingly, the replicon molecule is "propagation defective" or "propagation incompetent," as described herein. Typically, the resulting alphavirus particles and vector RNAs are propagation defective inasmuch as the alphavirus replicon particle or replicon RNA does not encode all of the alphavirus structural proteins required for encapsidation, at least a portion of at least one of the required structural proteins being deleted therefrom, such that the replicon RNA initiates only an abortive infection; no new viral particles are produced, and there is no spread of the infection to other cells. Alternatively, the replicon RNA may comprise one or more mutations within the structural protein coding sequences or promoter driving expression of the structural protein coding sequences, which interfere(s) with the production of a functional structural protein(s).

The replicon molecule is self-replicating. Accordingly, the replicon molecule comprises sufficient coding sequences for the alphavirus nonstructural polyprotein so as to support self-replication. In embodiments of the invention, the replicon encodes the alphavirus nsP1 , nsP2, nsP3 and nsP4 proteins.

In particular embodiments, the replicon RNA does not encode one or more of the capsid, E1 or E2 alphavirus structural proteins. By "do(es) not encode" one or more structural proteins, it is intended that the replicon molecule does not encode a functional form of the one or more structural proteins and, thus, a complementing sequence must be provided by a helper or packaging cell to produce new virus particles. In embodiments of the invention, the replicon RNA does not encode any of the alphavirus structural proteins.

The replicon may not encode the structural protein(s) because the coding sequence is partially or entirely deleted from the replicon molecule. Alternatively, the coding sequence is otherwise mutated so that the replicon does not express the functional protein. In embodiments of the invention, the replicon lacks all or substantially all of the coding sequence of the structural protein(s) that is not encoded by the replicon, e.g., so as to minimize recombination events with the helper sequences. In particular embodiments, the replicon RNA can encode at least one, but not all, of the alphavirus structural proteins. For example, the alphavirus capsid protein may be encoded by the replicon molecule. Alternatively, one or both of the alphavirus glycoproteins may be encoded by the replicon molecule. As a further alternative, the replicon may encode the capsid protein and either the E1 or E2 glycoprotein.

In other embodiments, none of the alphavirus structural proteins are encoded by the replicon RNA. For example, all or substantially all of the sequences encoding the structural proteins (e.g., E1 , E2 and capsid) may be deleted from the replicon RNA. Replicon vectors that do not encode the alphavirus capsid protein can nonetheless comprise a capsid translational enhancer region operably associated with the heterologous nucleic acid sequence, or the sequences encoding the non-structural proteins and/or encoding the other alphavirus glycoproteins (e.g., E1 and/or E2 glycoproteins) so as to enhance expression thereof. See, e.g., PCT Application No. PCT/U S01/27644; U.S. Patent No.

6,224,879 to Sjoberg et al., Smerdou et al., (1999) J. Virology 73:1092; Frolov et al., (1996) J. Virology 70:1182; and Heise et al. (2000) J. Virol. 74:9294-9299 (the disclosures of which are incorporated herein in their entireties).

C. Attenuating Mutations.

The alphavirus particle or alphavirus vector RNA can further comprise , attenuating mutations. The phrases "attenuating mutation" and "attenuating amino acid," as used herein, mean a nucleotide sequence containing a mutation, or an amino acid encoded by a nucleotide sequence containing a mutation, which mutation results in a decreased probability of causing disease in its host (i.e., reduction in virulence), in accordance with standard terminology in the art. See, e.g., B. Davis et al., MICROBIOLOGY 132 (3d ed. 1980). The phrase "attenuating mutation" excludes mutations or combinations of mutations that would be lethal to the virus.

Appropriate attenuating mutations will be dependent upon the alphavirus used, and will be known to those skilled in the art. Exemplary attenuating mutations include, but are not limited to, those described in United States Patent No. 5,505,947 to Johnston et al., U.S. Patent No. 5,185,440 to Johnston et al., U.S. Patent No. 5,643,576 to Davis et al., U.S. Patent No. 5,792,462 to Johnston et al., and U.S. Patent No. 5,639,650 to Johnston et al., the disclosures of which are incorporated herein in their entirety by reference.

When the alphavirus structural proteins are from VEE, suitable attenuating mutations include but are not limited to codons at E2 amino acid position 76 which specify an attenuating amino acid, preferably lysine, arginine, or histidine as E2 amino acid 76; codons at E2 amino acid position 120 which specify an attenuating amino acid, preferably lysine as E2 amino acid 120; codons at E2 amino acid position 209 which specify an attenuating amino acid, preferably lysine, arginine or histidine as E2 amino acid 209; codons at E1 amino acid 272 which specify an attenuating amino acid, preferably threonine or serine as E1 amino acid 272; codons at E1 amino acid 81 which specify an attenuating amino acid, preferably isoleucine or leucine as E1 amino acid 81 ; codons at E1 amino acid 253 which specify an attenuating amino acid, preferably serine or threonine as E1 amino acid 253; or the deletion of E3 amino acids 56-59, or a combination of the deletion of E3 amino acids 56-59 together with codons at E1 amino acid 253 which specify an attenuating mutation, as provided above, or any combination of the foregoing attenuating mutations.

Another suitable attenuating mutation is an attenuating mutation at nucleotide 3 of the VEE genomic RNA, i.e., the third nucleotide following the 5' methylated cap (see, e.g., U.S. Patent No. 5,643,576 describing a G->C mutation at nt 3). The mutation may be a G -> A, U or C, but is preferably a G-> A mutation. When the alphavirus structural and/or non-structural proteins are from S.A.AR86, exemplary attenuating mutations in the structural and non-structural proteins include, but are not limited to, codons at nsP1 amino acid position 538 which specify an attenuating amino acid, preferably isoleucine as nsP1 amino acid 538; codons at E2 amino acid position 304 which specify an attenuating amino acid, preferably threonine as E2 amino acid 304; codons at E2 amino acid position 314 which specify an attenuating amino acid, preferably lysine as E2 amino acid 314; codons at E2 amino acid 372 which specify an attenuating amino acid, preferably leucine, at E2 amino acid residue 372; codons at E2 amino acid position 376 which specify an attenuating amino acid, preferably alanine as E2 amino acid 376; in combination, codons at E2 amino acid residues 304, 314, 372 and 376 which specify attenuating amino acids, as described above; codons at nsP2 amino acid position 96 which specify an attenuating amino acid, preferably glycine as nsP2 amino acid 96; and codons at nsP2 amino acid position 372 which specify an attenuating amino acid, preferably valine as nsP2 amino acid 372; in combination, codons at nsP2 amino acid residues 96 and 372 which encode attenuating amino acids at nsP2 amino acid residues 96 and 372, as described above; codons at nsP2 amino acid residue 529 which specify an attenuating amino acid, preferably leucine, at nsP2 amino acid residue 529; codons at nsP2 amino acid residue 571 which specify an attenuating amino acid, preferably asparagine, at nsP2 amino acid residue 571 ; codons at nsP2 amino acid residue 682 which specify an attenuating amino acid, preferably arginine, at nsP2 amino acid residue 682; codons at nsP2 amino acid residue 804 which specify an attenuating amino acid, preferably arginine, at nsP2 amino acid residue 804; codons at nsp3 amino acid residue 22 which specify an attenuating amino acid, preferably arginine, at nsP3 amino acid residue 22; and in combination, codons at nsP2 amino acid residues 529, 571 , 682 and 804 and at nsP3 amino acid residue 22 which specify attenuating amino acids, as described above, or any combination of the foregoing attenuating mutations. Other illustrative attenuating mutations include those described in PCT

Application No. PCT/US01/27644 (the disclosure of which is incorporated herein in its entirety). For example, the attenuating mutation may be an attenuating mutation at amino acid position 537 of the S.A.AR86 nsP3 protein, more preferably a substitution mutation at this position (see, e.g., Table 1 below), still more preferably a nonsense mutation that results in substitution of a termination codon. Translational termination (i.e., stop) codons are known in the art, and include the "opal" (UGA), "amber" (UAG) and "ochre" (UAA) termination codons. In embodiments of the invention, the attenuating mutation results in a Cys-^opal substitution at S.A.AR85 nsP3 amino acid position 537.

Further exemplary attenuating mutations include an attenuating insertion mutation following amino acid 385 of the S.A.AR86 nsP3 protein. Preferably, the insertion comprises an insertion of at least 2, 4, 6, 8, 10, 12, 14, 16 or 20 amino acids. In embodiments of the invention, the inserted amino acid sequence is rich in serine and threonine residues (e.g., comprises at least 2, 4, 6, or 8 such sites) that serve as a substrate for phosphorylation by serine/threonine kinases.

In some embodiments, the attenuating mutation comprises an insertion of the amino acid sequence Ile-Thr-Ser-Met-Asp-Ser-Trp-Ser-Ser-Gly-Pro-Ser-Ser- Leu-Glu-lle-Val-Asp (SEQ ID NO:1 ) following amino acid 385 of nsP3 (i.e., the first amino acid is designated as amino acid 386 in nsP3). In other embodiments of the invention, the insertion mutation comprises insertion of a fragment of SEQ ID NO:1 that results in an attenuated phenotype. Preferably, the fragment comprises at least 4, 6, 8, 10, 12,14 or 16 contiguous amino acids from SEQ ID NO:1.

Those skilled in the art will appreciate that other attenuating insertion sequences comprising a fragment of the sequence set forth above, or which incorporate conservative amino acid substitutions into the sequence set forth above, may be routinely identified by those of ordinary skill in the art (as described above). While not wishing to be bound by any theory, it appears that the insertion sequence of SEQ ID NO:1 is highly phosphorylated at serine residues, which confers an attenuated phenotype. Thus, other attenuating insertion sequences which serve as substrates for serine (or threonine) phosphorylation may be identified by conventional techniques known to those skilled in the art.

Alternatively, or additionally, the attenuating mutation comprises a Tyr->Ser substitution at amino acid 385 of the S.A.AR86 nsP3 (i.e., just prior to the insertion sequence above). This sequence is conserved in the non-virulent Sindbis-group viruses, but is deleted from S.A.AR86.

Other attenuating mutations for S.A.AR86 include attenuating mutations at those positions that diverge between S.A.AR86 and non-neurovirulent Sindbis group viruses, including attenuating mutations at nsP2 amino acid position 256 (preferably Arg -> Ala), 648 (preferably lie -> VaI) or 651 (preferably Lys -> GIu), attenuating mutations at nsP3 amino acid position 344 (preferably GIy -> GIu), 441 (preferably Asp -> GIy) or 445 (preferably Ne -> Met), attenuating mutations at E2 amino acid position 243 (preferably Ser -> Leu), attenuating mutations at 6K amino acid position 30 (preferably VaI -> lie), and attenuating mutations at E1 amino acid positions 112 (preferably VaI -> Ala) or 169 (preferably Leu -> Ser). As a further alternative, the alphavirus vector can be an alphavirus particle comprising an alphavirus capsid protein in which there is an attenuating mutation in the capsid protease that reduces, preferably ablates, the autoprotease activity of the capsid and results, therefore, in non-viable virus. Capsid mutations that reduce or ablate the autoprotease activity of the alphavirus capsid are known in the art, see e.g., WO 96/37616 to Johnston et al., the disclosure of which is incorporated herein in its entirety. In particular embodiments, the alphavirus comprises a VEE capsid protein in which the capsid protease is reduced or ablated, e.g., by introducing an amino acid substitution at VEE capsid position 152, 174, or 226. Alternatively, one or more of the homologous positions in other alphaviruses may be altered to reduce capsid protease activity.

If the alphavirus comprises a Sindbis-group virus (e.g., Sindbis, TR339, S.A.AR86, GirdwoodSA, Ockelbo) capsid protein, the attenuating mutation may be a mutation at capsid amino acid position 215 (e.g., a Ser->Ala) that reduces capsid autoprotease activity (see, Hahn et al., (1990) J. Virology 64:3069).

It is not necessary that the attenuating mutations eliminate all pathology or adverse effects associated with administration of the alphavirus vector, as long as there is some improvement or benefit (e.g., increased safety and/or reduced morbidity and/or reduced mortality) as a result of the attenuating mutation.

In particular embodiments, the attenuating mutation is an attenuating mutation in one or more of the cleavage domains between the alphavirus nonstructural (nsp) genes, e.g., the nsP1/nsP2 cleavage region, the nsP2/nsP3 cleavage region, and/or the nsP3/nsP4 cleavage region as described in PCT Application No. PCT/US01/27644 (the disclosure of which is incorporated herein in its entirety). An exemplary attenuating mutation is a mutation at S.A.AR86 nsP1 amino acid 538 (position P3), more preferably a substitution mutation at S.A.AR86 nsP1 amino acid 538, still more preferably a Thr->lle substitution at S.A.AR86 nsP1 amino acid 538.

In particular embodiments, the attenuating mutation reduces (e.g., by at least 25%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) the pathology induced by a vector derived from a wild-type virus, for example, neurovirulence of the alphavirus vector (e.g., as determined by intracerebral injection in weanling or adult mice).

Those skilled in the art may identify attenuating mutations other than those specifically disclosed herein using other methods known in the art, e.g., looking at neurovirulence in weanling or adult mice following intracerebral injection. Methods of identifying attenuating mutations in alphaviruses are described by Olmsted et al., (1984) Science 225:424 and Johnston and Smith, (1988) Virology 162:437; the disclosures of which are incorporated herein in their entireties.

To identify other attenuating mutations other than those specifically disclosed herein, amino acid substitutions may be based on any characteristic known in the art, including the relative similarity or differences of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

Mutations can be introduced into the alphavirus genome by any method known in the art. For example, mutations may be introduced into the alphavirus RNA by performing site-directed mutagenesis on a cDNA that encodes the RNA, in accordance with known procedures (see, Kunkel, Proc. Natl. Acad. Sci. USA 82, 488 (1985), the disclosure of which is incorporated herein by reference in its entirety). Alternatively, mutations may be introduced into the RNA by replacement of homologous restriction fragments in a cDNA that encodes the RNA in accordance with known procedures. V. Methods of Administering and Using DC-Based Vaccines.

The cells, compositions, pharmaceutical formulations, and methods of the invention can be used for a variety of purposes. For example, the present invention finds use in methods of producing antibodies in vivo for passive immunization techniques. In this embodiment, a DC cell according to the invention and expressing an antigen of interest is administered to a subject. The antibody can then be collected from the subject using routine methods known in the art. The invention further finds use in methods of producing antibodies against an antigen for any other purpose, e.g., for diagnostics or for use in histological techniques. The present invention can also be practiced to study the function of DC in the immune system and the process of DC maturation.

The cells, compositions and pharmaceutical formulations of the invention can further be used in therapeutic and/or prophylactic methods, for veterinary and/or medical purposes. Suitable subjects according to the present invention can be any animal subject (e.g., avians and mammalian subjects). Mammalian subjects include but are not limited to humans, non-human primates, dogs, cats, pigs, goats, sheep, cattle, horses, mice, rats and rabbits. Avian subjects include but are not limited to chickens, turkeys, ducks, geese, quail, and birds kept as pets (e.g., parakeets, parrots, macaws, cockatoos, and the like). In particular embodiments, the subject is an animal model of cancer, including tumor models. In other embodiments, the subject has cancer or is a subject believed at risk for cancer. In still other embodiments, the subject is infected with an infectious agent or is believed at risk for infection with an infectious agent. Optionally, the subject is a subject "in need of the cells, compositions, pharmaceutical formulations and methods of the invention (e.g., to protect against cancer or an infectious agent).

In particular embodiments, the invention provides a method of inducing an immune response against an infectious agent in a subject, the method comprising: administering a cell, composition or pharmaceutical formulation of the invention comprising a heterologous nucleic acid encoding an infectious disease antigen to the subject, optionally in an immunogenically effective amount. The invention also provides a method of treating a subject against an infectious disease (therapeutically or prophylactically), the method comprising: administering a cell, composition or pharmaceutical formulation of the invention comprising a heterologous nucleic acid encoding an infectious disease antigen to the subject, optionally in a treatment effective amount.

As used herein, a "treatment effective amount" is an amount that is sufficient to treat (as defined herein) the subject.

Also provided is a method of inducing an anti-cancer immune response (including an anti-tumor immune response) in a subject, the method comprising: administering a cell, composition, or pharmaceutical formulation of the invention comprising a heterologous nucleic acid encoding a cancer antigen to the subject, optionally in an immunogenically effective amount. In particular embodiments, the method is practiced to induce an immune response against a cancer cell, comprising administering a cell, composition, or pharmaceutical formulation of the invention comprising a heterologous nucleic acid encoding a cancer cell antigen (e.g., displayed on the surface of the cancer cell) to the subject, optionally in an immunogenically effective amount.

The invention further provides a method of treating cancer (therapeutic or prophylactic), the method comprising: administering a cell, composition, or pharmaceutical formulation of the invention comprising a heterologous nucleic acid encoding a cancer antigen to a subject, optionally in a treatment effective amount. In particular embodiments, the heterologous nucleic acid encodes a cancer cell antigen (e.g., displayed on the surface of the cancer cell), optionally in a treatment effective amount. In a representative embodiment, the invention provides a method of inducing an immune response against an infectious agent or cancer in a subject, the method comprising: introducing an alphavirus (e.g., VEE) particle, or a nucleic acid encoding an alphavirus particle into a DC (e.g., a human DC such as an immature human DC), wherein the alphavirus particle comprises a heterologous nucleic acid sequence encoding an infectious disease antigen or a cancer antigen, respectively; and administering the modified DC to a subject, optionally in an immunogenically effective amount. As an alternative method, an alphavirus vector RNA or nucleic acid encoding an alphavirus particle or alphavirus vector RNA is introduced into the DC. In particular embodiments, the cancer antigen is a cancer cell antigen (e.g., displayed on the surface of the cancer cell).

In other embodiments, the invention provides a method of treating an infectious disease or cancer (therapeutic or prophylactic), the method comprising: introducing an alphavirus (e.g., VEE) particle or a nucleic acid encoding an alphavirus particle into a DC (e.g., a human DC such as an immature human DC), wherein the alphavirus particle comprises a heterologous nucleic acid sequence encoding an infectious disease antigen or a cancer antigen, respectively; and administering the modified DC to a subject, optionally in a treatment effective amount. As an alternative method, an alphavirus vector RNA or nucleic acid encoding an alphavirus particle or alphavirus vector RNA is introduced into the DC. In particular embodiments, the cancer antigen is a cancer cell antigen (e.g., displayed on the surface of the cancer cell). The invention can be practiced to treat subjects with existing cancers

(e.g., tumors) or to prevent or delay cancers from occurring. Further, the inventive methods can be used to treat both a primary tumor and to prevent metastasis. Alternatively, the inventive methods can be advantageously employed to reduce or prevent growth of metastatic nodules (e.g., following surgical removal of a primary tumor). The methods of the invention can also be prophylactic, e.g., to treat a subject believed at risk for cancer. In particular embodiments, individuals with specific cancers are administered an autologous vaccine generated by isolating DC from peripheral blood monocytes. Alphavirus vectors encoding cancer antigens such as HER-2/neu (or a modified form thereof that induces an immune response in a subject) can be introduced into a DC for administration to a subject to treat cancer.

The term "cancer" has its understood meaning in the art, for example, an uncontrolled growth of tissue that has the potential to spread to distant sites of the body (i.e., metastasize). Exemplary cancers include, but are not limited to, B cell lymphoma, T cell lymphoma, myeloma, leukemia, hematopoietic neoplasias, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, uterine cancer, adenocarcinoma, breast cancer, pancreatic cancer, colon cancer, lung cancer, renal cancer, bladder cancer, liver cancer, prostate cancer, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcimona, gastric cancer, brain cancer, angiosarcoma, hemangiosarcoma, head and neck cancer, thyroid carcinoma, soft tissue sarcoma, bone sarcoma, testicular cancer, uterine cancer, cervical cancer, gastrointestinal cancer, and any other cancer now known or later identified (see, e.g., Rosenberg (1996) Ann. Rev. Med. 47:481-491 , the entire contents of which are incorporated by reference herein). The term "cancer" further includes tumor-forming cancers. The term "tumor" is also understood in the art, for example, as an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. Preferably, the inventive methods disclosed herein are used to prevent and treat malignant tumors.

As another aspect, the invention provides for the use of the DC, compositions and pharmaceutical formulations of the invention for producing an immune response against an infectious agent or cancer. Also encompassed by the invention is the use of a cell or composition of the invention for the preparation of medicament to protect against an infectious agent or cancer.

An "immunogenically effective amount" is an amount of the DC, composition or pharmaceutical formulation of the invention that is sufficient to evoke an active immune response (cellular and/or humoral). Optionally, an immunogenically effective amount is sufficient to produce a protective immune response. The degree of protection conferred need not be complete or permanent.

An "active immune response" or "active immunity" is characterized by "participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both." Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to immunogens by infection or by vaccination. Active immunity can be contrasted with passive immunity, which is acquired through the "transfer of preformed substances (antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a non-immune host." Id.

A "protective" immune response or "protective" immunity as used herein indicates that the immune response confers some benefit to the subject in that it prevents or reduces the incidence and/or severity of disease. Alternatively, a protective immune response or protective immunity may be useful in the therapeutic treatment of disease.

Suitable dosages of the DC, compositions and pharmaceutical formulations of the invention will vary depending upon the condition, age and species of the subject, the nature of the immunogen, the nature of the alphavirus vector, the level of immunogenicity and enhancement desired, and like factors, and can be readily determined by those skilled in the art. In representative embodiments, a subject is administered 10⁵ to 10⁹, and more preferably 10⁶ to 10⁸ DC. Single or multiple (Ae., booster) dosages of the DC, compositions and pharmaceutical formulations of the invention can be administered, and, further, administration can be to multiple sites. The cells, compositions and pharmaceutical formulations can be administered by any method known in the art including but not limited to intraperitoneal, intravenous, intraarterial, intramuscular, intraventricular, intrathecal, transdermal, intradermal, subcutaneous, topical administration, and foot pad administration. In representative embodiments, administration is into or near a tumor. For example, in the treatment of cancer, administration can be in or near a lymph node (e.g., in or near the lymph node where tumor lymph is draining). To illustrate, for breast tumors, administration can be in or near an axillary lymph gland, and for cancers in the pelvic regions (e.g., uterine, ovarian, prostate, colon), administration can be in or near an inguinal lymph gland. In particular embodiments, administration in or near a lymph gland is by subcutaneous or intradermal route of administration.

Vl. Pharmaceutical Formulations.

The invention further provides pharmaceutical formulations comprising the DC of the invention in a pharmaceutically acceptable excipient. Formulation of pharmaceutical compositions is well known in the pharmaceutical arts (see, e.g., Remington's Pharmaceutical Sciences, (15th Edition, Mack Publishing Company, Easton, Pa. (1975)).

By "pharmaceutically acceptable" it is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects such as toxicity.

The DC of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9^th Ed. 1995). In the manufacture of a pharmaceutical formulation according to the invention, the DC is typically admixed with, inter alia, an acceptable carrier. The carrier can be a solid or a liquid, or both, and is preferably formulated as a unit-dose formulation. The formulations of the invention can optionally comprise additional medicinal agents, pharmaceutical agents, carriers, buffers, adjuvants, dispersing agents, diluents, and the like.

The formulations of the invention include those suitable for intraperitoneal, intravenous, intraarterial, intramuscular, intraventricular, intrathecal, transdermal, intradermal, subcutaneous, topical administration, and foot pad administration as well administration in or near a tumor or in or near a lymph node. The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular DC and antigen that is being delivered.

For injection, the carrier will typically be a liquid, such as sterile pyrogen- free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.). For other methods of administration, the carrier can be either solid or liquid.

Formulations of the present invention suitable for parenteral administration can comprise sterile aqueous and non-aqueous injection solutions of the DC, which preparations are generally isotonic with the blood of the intended recipient. These preparations can contain anti-oxidants, buffers, bacteriostats and solutes, which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The formulations can be presented in unit\dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for- injection immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising a DC of the invention, in a unit dosage form in a sealed container. The DC is provided in the form of a lyophilizate that is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject.

Formulations suitable for topical application to the skin can take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers that can be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

Formulations suitable for transdermal administration can be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of the DC. Suitable formulations comprise citrate or bis\tris buffer (pH 6) or ethanol/water.

Alternatively, one can administer the DC in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention. Example 1

Materials and Methods Antibodies and Reagents

Phycoerythrin (PE)-conjugated monoclonal antibodies specific for human CD8 (SK2), CD11c (B-LY6), CD14 (M5E2), CD40 (5C3), CD80 (L307.4), CD83 (HB15e) and HLA-DR (G46-6) were purchased from BD Pharmingen (San Diego, CA). Anti-human CD86-PE (HA5.2B7) was purchased from Beckman Coulter (San Diego, CA). Mouse anti-influenza A matrix protein (FMP) monoclonal antibody was purchased from Serotec (Raleigh, NC). All isotype control antibodies were purchased from BD Pharmingen. Recombinant human GM-CSF, IL-4, IL-2, IL-7 and TNF-σ were purchased from Peprotech (Rocky Hill, NJ). Human AB serum (HABS) was purchased from Gemini Bioproducts (Woodland, CA).

Generation of Human Monocyte-Derived DCs

Peripheral blood was obtained from volunteer donors by venipuncture and diluted 1 :2 with phosphate buffered saline (PBS). Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over lymphocyte separation medium (ICN Biomedicals, Aurora, OH), washed twice with PBS, and resuspended in serum-free AIM-V media (Invitrogen, Carlsbad, CA). Monocytes were enriched by culturing 10⁷ PBMCs/well in 6-well tissue culture plates for 2 hours. Nonadherent PBMCs were removed and cryopreserved in 90% fetal bovine serum/10% DMSO. In experiments evaluating cytokine secretion, highly purified monocytes (>90% CD14⁺) were obtained by immunodepletion of non- monocytic cells using the Monocyte Isolation Kit Il (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. Monocytes isolated by either method were cultured at 37°C/5% CO₂ in complete AIM-V/10% HABS supplemented with GM-CSF (800U/ml) and IL-4 (500U/ml). Fresh cytokine was added on days 3 and 6 of culture. The cells were harvested on day 6 as immature DCs, or further matured for 24-48 hours with lipopolysaccharide (LPS) (0.1 - 1μg/ml) or for 48 hours with recombinant human TNF-σ (20ng/ml) added daily. All of the clinical reagents were generated under protocols approved by the Committee for the Protection of the Rights of Human Subjects at the University of North Carolina School of Medicine.

Generation of Recombinant VRPs The production of VRPs that encode the green fluorescent protein (GFP-

VRP) has been previously described (MacDonald et al., (2000) J. Virol. 74:914). The absence of propagating recombinant virus was confirmed by passage in BHK cells. VRPs were concentrated from supernatants by centrifugation through a 20% sucrose cushion and resuspended in PBS. Titration of GFP- VRPs was determined by infecting BHK monolayers with 10-fold dilutions of VRPs for 16-18 hours at 37°C/5% CO₂. The infected cells were fixed with 4% paraformaldehyde and GFP-expressing cells were directly visualized by fluorescent microscopy. VRPs that encode influenza matrix protein (FMP) (FMP-VRP) were generated by directionally cloning the FMP cDNA (kindly provided by P. Palese, Mt. Sinai School of Medicine, New York, NY) immediately downstream of the 26S mRNA promoter of the pVR21 replicon plasmid; proper orientation was confirmed by DNA sequencing. The FMP replicon plasmid was used to generate FMP-VRPs. For titration, BHK monolayers were infected with 10-fold dilutions of FMP-VRPs for 16-18 hours at 37°C/5% CO₂. Infected cells were fixed with ice-cold methanol and sequentially stained with mouse anti-FMP monoclonal antibody, biotinylated anti-mouse IgG, and FITC-conjugated streptavidin. FITC-positive cells were directly enumerated by fluorescent microscopy.

Infection of Human DCs with VRPs

Immature or mature DCs were resuspended in serum-free AIM-V at 0.5- 1.0 x 10⁶ cells/ml and seeded at 1-2 x 10⁵ DCs per well in 24-well ultra low attachment plates (Corning Inc., Corning, NY). For infectivity experiments, 1-2 x 10⁵ DCs were infected with VRPs at different multiplicities of infection (MOIs) over specific time intervals as indicated in the figure legends. Infections were performed in serum-free conditions at 37°C/5% CO₂. After 1-2 hours, DCs were washed with AIM-V/10% HABS, resuspended in media supplemented with GM- CSF (800U/ml) and IL-4 (500U/ml), and cultured in 24-well ultra low attachment plates at 37°C/5% CO₂.

Flow Cytometry Analysis For quantification of VRP transduction efficiency, GFP-VRP- or mock- infected DCs were harvested at 6, 12 or 24 hpi and washed once with cold FACS buffer (PBS/0.5% human serum albumin). DCs were fixed with PBS/1 % formaldehyde before FACS analysis. In some experiments, DC viability was determined using the Fixation and Dead Cell Discrimination Kit (Miltenyi Biotec) according to the manufacturer's instructions. For phenotypic analysis, 5 x 10⁴ DCs were incubated with 200μg/ml mouse IgG (Sigma) at 4°C for 20 minutes. Following blocking, the DCs were stained with 2//I of PE-conjugated specific or isotype control antibodies for 30 minutes at 4°C, washed once with FACS buffer, and fixed with PBS/1 % formaldehyde. FACS data was acquired using a FACScan flow cytometer (BD Biosciences, San Jose, CA), and analyzed using FlowJo software (Tree Star, Ashland, OR).

Cytokine Assays

For evaluation of cytokine secretion by DCs, immature DCs were either mock-infected or infected with GFP-VRPs (MOI = 20) for 2 hours at 37°C/5% CO₂. Fully mature DCs were generated by treatment for either 24 hours with LPS (100 ng/ml) or 48 hours with TNF-σ (20 ng/ml). Mock-infected immature DCs, VRP-infected immature DCs, or fully mature DCs were washed and seeded into 96-well flat bottom tissue culture plates at 10⁵ DCs/well. Supematants were harvested at 12, 24, 36 or 48 hours post-treatment and stored at -80°C. Quantification of IL-6, IL-8, IL-10, IL-12p70, and TNF-α in the supematants was performed using the cytometric bead array (CBA) according to the manufacturer's instructions (BD Pharmingen, Franklin Lakes NJ). Measurement of IFN-α was determined by ELISA (Biosource International, Camarillo CA) according to the manufacturer's instructions. Allogeneic Mixed Leukocyte Reactions (MLR)

Mock- or GFP-VRP-infected (MOI = 10) DC cultures were harvested after 1 hour of infection, washed with media, and resuspended in AIM-V/10% HABS. Decreasing numbers of DCs were added in triplicate to 1 x 10⁵ nonadherent allogeneic PBMCs per well in 96-well round bottom plates and T cell proliferation assays were performed as previously described (Wysocki et al., (2004) J. Immunol. 173:845).

In Vitro Expansion of FMP-Specific T Cells Immature DCs from HLA-A*0201 -positive donors were infected for 2 hours with either GFP-VRPs or FMP-VRPs (MOI = 10) and washed with AIM- V/10% HABS media. DCs (0.2-2 x 10⁵) were added to 2 x 10⁶ autologous nonadherent PBMCs per well in 24-well tissue culture plates. For comparative stimulation of T cells with peptide-pulsed DCs, TNF-α-matured or LPS-matured DCs from the same donors were incubated with 10 μg/ml of FMP peptide in AIM- V/10% HABS for 2 hours. FMP peptide-pulsed DCs were washed with media and added to autologous nonadherent PBMCs as described above. PBMCs were incubated for 7 days in AIMV/10% HABS supplemented with IL-2 (20U/ml) and IL-7 (10ng/ml). Fresh cytokine was added on days 3 and 6 of culture, and cell density was maintained at < 2 x 10⁶ cells/ml during the entire assay. On day 7, the responders were harvested and evaluated for either antigen-specific expansion by tetramer staining, or for specific lysis of peptide-pulsed T2 cells by a conventional ⁵¹Cr release assay (Serody et al., (1997) Cancer Research 57:1547). Percent specific lysis was determined using the following formula:

Percent specific lysis = 100 * [(sample cpm - spontaneous cpm) / (total cpm - spontaneous cpm)]

For tetramer staining, 1 x 10⁶ responders were stained for 30 minutes with 20 μ\ of anti-human CD8-FITC (Pharmingen) and 10 μ\ of either PE- conjugated H LA-A*0201 /Influenza M1 peptide tetramer or HLA- A2*0201 /Negative tetramer (Beckman Coulter). Cells were washed with PBS, fixed with PBS/0.5% formaldehyde, and analyzed by FACS within 6 hours.

Statistical Analysis Statistical differences were calculated using a Student's t-test when sample data distribution was parametric. Sample data that exhibited nonparametric distribution were evaluated using a Mann-Whitney rank sum test. Differences in costimulatory molecule expression between mock- and VRP- infected DCs from several donors were analyzed using a Wilcoxon signed rank test. P values < 0.05 were considered significant. All statistical analyses were performed with SigmaStat 3.0 software (Port Richmond, CA).

Example 2

Results VRPs Can Efficiently Transduce Human Immature DCs

It has previously demonstrated that VRPs can infect mouse DCs in vivo following foot pad injection (MacDonald et al, (2000) J. Virol. 74:914). To determine if human DCs could be infected with VRPs in vitro, immature monocyte-derived DCs were generated from normal donors. By day 6 of culture, DCs exhibited a typical immature phenotype (CDHc⁺, HLA-DR⁺, CD86⁺, CD14^', CD40^', CD80^") when evaluated by flow cytometry (data not shown). Immature day 6 DCs were infected with GFP-VRPs at an MOI of 10. GFP expression in DCs was first detectable at around 4 hpi, and reached a maximum between 6-12 hpi (Figure 1, panel A). To quantify VRP transduction efficiency, mock- or GFP-VRP-infected

(MOI = 10) immature DCs were harvested at 6, 12 or 24 hpi and analyzed for GFP expression by flow cytometry. As shown in Figure 1, panel B, VRPs could infect human immature DCs at an MOI of 10, with a mean of 10.8% expressing GFP by 6 hpi. The mean percentage of GFP-positive immature DCs peaked at 18.4% at 12 hpi and then decreased to 15.5% by 24 hpi. To determine if the maturation status of the DCs affected the transduction efficiency, immature DCs were stimulated with LPS for two days, resulting in marked up-regulation of CD40, CD80, CD83 and CD86 (data not shown). LPS-matured DCs were minimally transduced by GFP-VRPs (Figure 1. panel B). DCs matured with TNF-α for two days were also less susceptible to VRP infection (mean 6.2% GFP-positive at 12 hpi), although not to the same degree as found using LPS- matured DCs. Thus, VRP transduction efficiency was inversely related to the degree of DC maturation.

In order to verify that the GFP-positive cells exhibited a DC phenotype, two-color FACS analysis was performed on infected DC cultures. GFP-VRP- infected DCs were harvested at 24 hpi and stained with PE-conjugated antibodies specific for CD11c, HLA-DR and CD14. GFP-positive cells expressed high levels of CD11c and HLA-DR, and did not express the monocyte-marker CD14 (Figure 1, panel C). To demonstrate that VRPs specifically infected immature DCs, the ability of VRPs to transduce peripheral blood T cells, B cells and monocytes was determined. Infection of CD3⁺ T cells and CD19⁺ B cells with GFP-VRPs (MOI = 10) was not observed, with only minimal (-2%) transduction of CD14⁺ monocytes (data not shown). Thus, VRPs specifically infected immature DCs.

Approaches were next evaluated that could enhance the efficiency of VRP transduction of immature DCs. Increasing the MOI improved the transduction of immature DCs by GFP-VRPs. At an MOI of 100, approximately 50% of DCs expressed GFP (Figure 2, panel A). The percent of GFP-positive DCs began to plateau between an MOI of 50 and 100, suggesting that transduction efficiency was near maximal. In an effort to maximize transduction efficiency at a lower MOI, the duration of infection and the DC concentration during infection was increased. By doubling both the time of infection and the DC concentration during infection at an MOI of 20, the transduction efficiency increased from a mean of 22.5% to 37.0% (n =3, p = 0.002) (Figure 2, panel B). Thus, immature DCs can be efficiently transduced with relatively small quantities of VRPs.

The percentage of GFP-expressing DCs began to decline between 12 and 24 hpi (Figure 1, panel B), suggesting that VRP infection may be cytopathic to human DCs. Alphaviruses and alphaviral vectors induce apoptosis in cultured cells (Li et al., (2004) Int. Rev. Immunol. 23:7), although their ability to similarly induce cell death in human DCs is unknown. The viability of VRP-infected (GFP- positive) DCs to uninfected (GFP-negative) DCs in the culture by exclusion of a vital dye was therefore compared. VRP-infected DCs exhibited >90% viability between 6-12 hpi, and remained -75% viable at 24 hpi (Figure 3). However, by 48 hpi only 26% of the DCs remained viable compared to 66% of the uninfected DCs. This loss in viability was associated with increased expression of annexin- V by VRP-infected DCs (data not shown), suggesting that VRP-induced apoptosis was likely responsible for the death of human DCs. In summary, the viability of VRP-transduced DCs remained high for 24 hours following infection, but steadily decreased between 24-72 hpi.

VRP Infection Induces DC Maturation and Secretion of Proinflammatory Cytokines

The studies described above indicated that immature DCs could be easily transduced with VRPs. However, immature DCs are poor stimulators of antigen- specific T cells and have been shown to induce tolerance (Hawiger et al., (2001 ) J. Exp. Med. 194:769; Dhodapkar et al., (2001 ) J. Exp. Med. 193:233). Thus, it was determined if VRP-infection induced maturation of immature DCs by evaluating expression of costimulatory and maturation surface markers (Figure 4). At 12 hpi, the expression of various costimulatory/matu ration markers in VRP-infected DC cultures was similar to DCs that were mock-infected or treated with TNF-σ. In contrast, DCs treated with a strong maturation stimulus (100 ng/ml of LPS) had upregulated CD80 and CD86 expression at this time. By 24 hpi, however, the expression of CD40, CD80 and CD86 was significantly elevated in VRP-infected DC cultures when compared to mock-infected or TNF- σ-treated DCs. CD86 expression in VRP-infected DC cultures at 24 hpi was comparable to that seen with LPS treatment, while LPS induced higher levels of CD80 and CD83. Interestingly, VRP-infection induced higher levels of CD40 expression when compared to LPS treatment, a trend that was consistent in four different experiments. It was next determined if VRP infection induced maturation of both infected and uninfected bystander DCs by analyzing costimulatory/maturation marker expression on GFP-positive and -negative DCs in the culture (Table 1). The expression of costimulatory/maturation molecules was increased on both GFP-positive and GFP-negative DCs, although the latter exhibited the highest expression levels at 24 hpi. These observations indicate that VRP infection resulted in phenotypic maturation of both infected and uninfected immature DCs within the same culture.

Secretion of proinflammatory cytokines by GFP-VRP-infected DC cultures was next evaluated (Figure 5). Because DC cultures generated from adherent PBMCs contained a small but significant population of contaminating lymphocytes (15-45%), highly purified DCs were generated from monocytes that had been isolated by negative selection using immunomagnetic beads. DC cultures generated by this method were >95% CD11 C⁺, and were similar to adherent monocyte-derived DCs in both surface marker phenotype and susceptibility to VRP infection (data not shown). These DCs were mock- or VRP-infected and supematants were collected and assayed for proinflammatory cytokines at various time points post-infection. In contrast to mock-infected DCs, VRP-infected immature DCs secreted significant amounts of TNF-σ, IL-6 and IFN-σ at 24 to 48 hours following infection. Low but statistically significant levels of IL-12p70 were detected at later time points (36-48 hpi). IL-10 was also barely detectable in the supematants from VRP-infected DCs, but the levels were not significantly higher than in mock-infected DC supematants. By comparison, DCs that had been matured by either 24 hours with LPS or 48 hours with TNF-α did not secrete significant amounts of TNF-α, IL-6 and IFN-σ. LPS- and TNF-α- matured DCs secreted IL-8 (Figure 5) and displayed increased costimulatory molecule expression (Figure 4), demonstrating that these cells had been activated. Additionally, incubating mature DCs with an MHC class l-restricted peptide (FMP peptide 58-66) did not affect cytokine secretion (data not shown). In summary, VRP infection of immature human DCs induced maturation and proinflammatory cytokine secretion. Table I.1

¹ Median fluorescence intensity (MFI) of costimulatory/maturation markers following VRP infection of DCs. DC cultures were mock-infected or infected with GFP-VRP (MOI = 10-20) and evaluated 24 hours later for the expression of costimulatory/maturation markers with PE-conjugated antibodies. Numbers represent the median PE fluorescence from eight donors (minimum - maximum). In VRP-infected cultures, the MFI for infected and uninfected DCs was determined by gating on GFP-positive and GFP-negative DCs, respectively. ^ap < 0.05 compared to mock-infected DCs (Wilcoxon sign rank test); ^Dp < 0.01 compared to mock-infected DCs.

VRP-lnfected Human DCs Can Stimulate Allogeneic and Antigen-Specific T Cells

To initially evaluate the functionality of VRP-infected DC cultures, standard allospecific T cell stimulation assay were performed. DCs infected with GFP-VRP stimulated substantial proliferation of allogeneic T cells, indicating that VRP infection did not have a detrimental effect on DC function (data not shown). More importantly, it was determined if VRP-transduced DCs could stimulate expansion of autologous T cells specific for a VRP-encoded antigen. For this set of experiments, recombinant VRPs expressing FMP were utilized. When autologous PBMCs were stimulated with an irrelevant VRP expressing GFP, there was no significant increase in the percentage of FMP-specific CD8⁺ T cells (Figure 6, panels A-B). However, stimulation of PBMCs with FMP-VRP- transduced DCs led to a significant increase in the percentage of FMP-specific CD8⁺ T cells (Figure 6, panels A-B). VRP-transduced DCs were highly efficient at expanding FMP-specific CD8⁺ T cells at even low DC numbers (Figure 6, panel C). Furthermore, the expanded FMP-specific CD8⁺ T cells were functional as they could lyse T2 cells pulsed with the FMP peptide (Figure 6, panel D). When FMP-VRP-transduced DCs were compared with TNF-σ-matured DCs pulsed with FMP peptide, it was found that FMP-VRPs were significantly more effective at inducing expansion of FMP-specific CD8⁺ T cells compared to FMP- pulsed DCs (Figure 6, panels A-B). However, peptide-pulsed DCs matured with a more potent stimulus (100 ng/ml of LPS) induced comparable expansion of FMP-specific CD8⁺ T cells when compared to FMP-VRP-infected DCs (42% and 38% tetramer-positive cells, respectively, respondeπstimulator ratio = 10:1 ). VRP-infected DC can thus process and present vector-encoded antigens to reactive T cells, resulting in significant T cell expansion and acquisition of effector function.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

We claim:

1. An isolated human dendritic cell, wherein said human dendritic cell comprises a recombinant Venezuelan Equine Encephalitis virus (VEE) vector RNA that comprises a heterologous nucleic acid sequence encoding an antigen.

2. The human dendritic cell of claim 1 , wherein said human dendritic cell is an immature human dendritic cell.

3. The human dendritic cell of claim 1 , wherein said human dendritic cell is derived from a monocyte.

4. The human dendritic cell of claim 1 , wherein said human dendritic cell is isolated from skin.

5. The human dendritic cell of claim 4, wherein said human dendritic cell is a Langerhans cell.

6. The human dendritic cell of claim 1 , wherein said human dendritic cell is derived from a CD34⁺ bone marrow progenitor cell.

7. The human dendritic cell of claim 1 , wherein said human dendritic cell is isolated from spleen.

8. The human dendritic cell of claim 1 , wherein said recombinant VEE vector RNA is a VEE replicon vector RNA.

9. The human dendritic cell of claim 1 , wherein said heterologous nucleic acid sequence encodes an infectious disease antigen.

10. The human dendritic cell of claim 9, wherein said antigen is a human immunodeficiency virus antigen, a simian immunodeficiency virus antigen, a human papilloma virus antigen or an influenza virus antigen.

11. The human dendritic cell of claim 1 , wherein said antigen is a cancer antigen.

12. The human dendritic cell of claim 11 , wherein said cancer antigen is a cancer cell antigen.

13. The human dendritic cell of claim 12, wherein said cancer cell antigen is a Her2/Neu antigen.

14. A pharmaceutical formulation comprising the human dendritic cell of any of claims 1-13 in a pharmaceutically acceptable carrier.

15. A method of inducing an immune response against an infectious agent in a subject, the method comprising: administering the human dendritic cell of claim 9 or the pharmaceutical formulation of claim 14 when dependent on claim 9 in an immunogenically effective amount to the subject.

16. A method of treating an infectious disease in a subject, the method comprising: administering the human dendritic cell of claim 9 or the pharmaceutical formulation of claim 14 when dependent on claim 9 in a treatment effective amount to the subject.

17. A method of inducing an anti-cancer immune response in a subject, the method comprising: administering the human dendritic cell of claim 11 or the pharmaceutical formulation of claim 14 when dependent on claim 11 in an immunogenically effective amount to the subject.

18. A method of treating cancer in a subject, the method comprising: administering the human dendritic cell of claim 11 or the pharmaceutical formulation of claim 14 when dependent on claim 11 in a treatment effective amount to the subject.

19. The method of any of claims 15-18, wherein the subject is a human subject.

20. The method of any of claims 15-18, wherein the human dendritic cell or pharmaceutical formulation is administered intradermally.

21. The method of any of claims 15-18, wherein the dendritic cell or pharmaceutical formulation is administered subcutaneously.

22. The method of any of claims 15-18, wherein the dendritic cell or pharmaceutical formulation is administered near a lymph node.

23. A method of inducing an immune response against an infectious agent in a subject, the method comprising: introducing a Venezuelan Equine Encephalitis (VEE) particle into an immature human dendritic cell, wherein the VEE particle comprises a heterologous nucleic acid sequence encoding an infectious disease antigen; and administering the modified human dendritic cell in an immunogenically effective amount to the subject.

24. A method of treating an infectious disease in a subject, the method comprising: introducing a Venezuelan Equine Encephalitis (VEE) particle into an immature human dendritic cell, wherein the VEE particle comprises a heterologous nucleic acid sequence encoding an infectious disease antigen; and administering the modified human dendritic cell in a treatment effective amount to the subject.

25. A method of inducing an anti-cancer immune response in a subject, the method comprising: introducing a Venezuelan Equine Encephalitis (VEE) particle into an immature human dendritic cell, wherein the VEE particle comprises a heterologous nucleic acid sequence encoding a cancer antigen; and administering the modified human dendritic cell in an immunogenically effective amount to the subject.

26. A method of treating cancer in a subject, the method comprising: introducing a Venezuelan Equine Encephalitis (VEE) particle into an immature human dendritic cell, wherein the VEE particle comprises a heterologous nucleic acid sequence encoding a cancer antigen; and administering the modified human dendritic cell in a treatment effective amount to the subject.