WO2004092386A2

WO2004092386A2 - Inducing a t cell response with recombinant pestivirus replicons or recombinant pestivirus replicon-transfected dendritic cells

Info

Publication number: WO2004092386A2
Application number: PCT/US2004/011018
Authority: WO
Inventors: Barbara Rehermann; Vito Racanelli; Sven-Erik Behrens; Norbert Tautz
Original assignee: The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services; Justus-Liebig-Universität Giessen
Priority date: 2003-04-11
Filing date: 2004-04-10
Publication date: 2004-10-28
Also published as: WO2004092386A3

Abstract

The present disclosure relates to compounds and methods of generating T cell-mediated immunity, particularly T cell-mediated immunity to Hepatitis C Virus (HCV), Respiratory Syncytial Virus (RSV), Human Immunodeficiency Virus (HIV), Mycobacterium tuberculosis, Plasmodium falciparum, and tumors. The method includes (a) administering to the subject an amount of an antigen presenting cell (such as dendritic cell) sufficient to induce the response in the subject, wherein the antigen presenting cell expresses the antigen from apestivirus replicon or (b) directly administering the antigen expressing replicon in form of RNA or DNA.

Description

INDUCING A T CELL RESPONSE WITH RECOMBINANT PESTIVTRUS REPLICONS OR RECOMBINANT PESTIVTRUS REPLICON-TRANSFECTED DENDRITIC CELLS

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 60/462, 165, filed

April 11, 2003, and U.S. Provisional Application No. 60/463,097, filed April 14, 2003, each of which is incorporated in its entirety herein by reference.

FIELD OF THE DISCLOSURE The present disclosure relates to compounds and methods for generating T cell-mediated immunity, such as T cell-mediated immunity to Hepatitis C Virus O^CV), Human Immunodeficiency Virus (HIV), Respiratory Syncytial Virus (RSV), Mycobacterium tuberculosis, Plasmodium falciparum, or tumor antigens.

BACKGROUND

Effective vaccination requires the induction of the appropriate type of immunity. Most vaccines in current use work by stimulating the production of neutralizing antibodies. However, some pathogens, particularly Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV),Respiratory Syncytial Virus (RSV), Mycobacterium tuberculosis, and Plasmodium falciparum, and some tumors are more effectively dealt with by T cell-mediated immune responses. To date, there are no effective vaccines that induce strong T cell responses against these pathogens and tumors in humans. In particular, CD8+ T cell responses are difficult to induce, because CD8+ T cells do not directly respond to injected protein antigen, but require antigens to be generated and processed in antigen presenting cells. RNA replicons are positive-strand subgenomic viral RNAs that encode their own viral replicase, and can perform high-level cytoplasmic amplification. Replicons are capable of functioning autonomously without the support of a helper virus. Cytopathicreplicons eventually kill the transfected or infected cells, whereas noncytopathic replicons do not. Recombinant forms of RNA replicons contain the coding region of heterologous proteins (for example, antigens) and thus express the heterologous protein/antigen at high level in the cell. If the heterologous protein is an immunogenic antigen, replicon-based vectors can thus be used as vaccine delivery systems. Replicon- based expression vectors have been developed from representatives of most positive-strand RNA virus families, including alphaviruses, picornaviruses, and flaviviruses. However, the majority of the data on immunogenic properties of replicon vectors in laboratory animals has been accumulated using replicons of alphaviruses such as Sindbis virus, Semlicki Forest virus, and Venezuelan equine encephalitis.

Cross-priming is a specific capacity of dendritic cells that involves the acquisition of exogenous antigens from apoptotic or dead cells in the periphery and the migration to secondary lymphoid organs, where dendritic cells (DC) undergo apoptosis and are taken up by secondary antigen presenting cells (APC). These APC reprocess the antigen and present it to T cells (for review, see Zhou et al, "Current methods for loading dendritic cells with umor antigen for the induction of antitumor immunity," J. Immunother. 25(4):289-303, 2002). Cross-priming is particularly useful when immunity is based on T cell rather than on antibody responses (for example, for hepatitisC virus (HCV), respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), Mycobacterium tuberculosis and Plasmodium falciparum) and for immune responses against tumors.

There are no efficient vaccines currently available for such infections because of the difficulty of inducing strong T cell responses in non-human primates and animals using known methods, including methods involving Sindbis virus or Semlicki Forest virus replicons. Thus, methods are needed for inducing T cell-mediated immunity to tumors, and to pathogens such as Mycobacterium tuberculosis, Plasmodium falciparum RSV, HIV and HCV.

SUMMARY OF THE DISCLOSURE Disclosed herein is a method of vaccinating subjects against pathogens and tumors in which the immune response relies primarily on a T cell rather than an antibody-based immune response. The method involves administering to the subject an amount of an antigen-presenting cell (such as a dendritic cell) sufficient to induce an immune response in a subject, wherein the antigen presenting cell expresses the antigen from a pestivirus replicon. The replicon may be either cytopathic or noncytopathic. In some embodiments, the method further includes introducing into an antigen presenting cell (such as a dendritic cell) a self-replicating cytopathic or noncytopathic pestivirus replicon that expresses the antigen in the antigen-presenting cell. Alternatively, a DNA molecule that encodes the replicon can be introduced into the antigen presenting cell.

In certain examples, the replicon expresses an immunogenic antigen for example an antigen for which the immune system relies upon theT cell-mediated response to clear the antigen from the body. Examples of such antigens include a Hepatitis C virus (HCV) antigen, Respiratory Syncytial virus (RSV) antigen, Human Immunoώficiency virus (HIV) antigen, Mycobacterium tuberculosis antigen, Plasmodium falciparum antigen, and tumor antigen.

The dendritic cells that express the antigen may be introduced into the body of the subject, where they migrate to lymphoid tissue (such as lymph nodes or spleen), replicating the RNA to produce high cytoplasmic levels of the immunogenic antigen. When the dendritic cells die in the lymphatic tissue, the antigen efficiently induces Tcell immunity via cross-priming.

This method provides several advantages over known methods of inducing a Tcell response. First, pestivirus replicons replicate less efficiently than replicons from Semlicki Forest virus and Sindbis virus, which allows the transfected dendritic cells to survive longer. This increased survival time permits the dendritic cells to migrate to the lymph nodes and spleen, where the apoptotic dendritic cells are taken up by secondary antigen presenting cells which present the antigβi of interest to the T cells. This timing enhances the stimulation of a T cell-mediated immune response, such as a cytotoxic T cell response. Second, the pestivirus replicons are derived from viruses that are not capable of infecting humans, which represents an important safety concern. Third, recombinant pestivirus RNA replicons cannot integrate into the human cellular genome, and thus cannot activate potential oncogenes. In some examples, the dendritic cells are transfected in vitro before administering them to the subject to provide more efficient vaccination than the prior method of administering naked RNA or DNA directly to the subject. Either cytopathic or noncytopathic pestivirus replicons can be used. The cytopathic r.eplicon produces a higher level of antigen expression and induction of cell death fσ cross-priming. Conversely, the noncytopathic replicon ensures longer expression of antigen in living cells, which may be important to maintain immune responses.

The foregoing and other features and advantages will become more apparent from the following detailed description of a several embodiments.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows schematics of certain BVDV-based replicon embodiments. In particular,

FIG. 1A is a schematic diagram of the noncytopathic replicon, "BVDV Bi-ΔN^prononcp, GUS" (also called noncytopathic Repl-GUS) and the cytopathic replicon, "BVDV Bi-ΔN^procp, GUS" (also called cytopathic Repl-GUS). The relative positions of restriction sites used for the cloning procedures are indicated. FIG. IB shows schematic diagrams of the wild-type BVDV genome (RNA) and the noncytopathic and cytopathic BVDV replicons used to express the HCV protein NS3. The noncytopathic replicon was designated "BVDV Bi-noncp, HCV NS3" or "noncytopathic Repl-HCV '. The cytopathic replicon was designated "BVDV Bi-cp, HCV NS3" or "cytopathic Repl-HCV_NS3". The ΔN^pro-glucoronidase (GUS) sequence of the replicons shown in FIG.1A was replaced with the sequence encoding the complete W° protein and the HCV NS3 antigen to construct the noncytopathic and cytopathic BVDV HCV NS3 replicons. In FIG. IB, "A" refers to autoprotease sites, filled arrows refer to viral protease sites, and open arrows refer to cellular protease sites in the protein translated from the respective RNAs.

FIG. 2 is a composite of figures showing the in vitro expression and transfection of DC2.4 cells with cytopathic and noncytopathic Repl-HCV_NS3 RNA. Specifically FIG.2A is a digital image of an agarose gel electrophoresis showing molecular size markers (unmarked lane) and in vitro transcribed cytopathic (lane 1) and noncytopathic (lane2) Repl-HCV_NS3 RNA. FIG. 2B is a digital image showing the morphology of the dendritic cell line DC2.4. FIGS. 2C and D show a Western blot and flow cytometry histogram, respectively, each of whichdemonstrate the efficiency of DC2.4 transfection with cytopathic (lane 2 of Western blot and black histogram area) or noncytopathic (lane 3 of Western blot and gray histogram area) Repl-HCV_NS3 RNA. Lane 1 of the Western blot and unshaded histrogram area represent untransfected DC2.4. FIGS. 2E and F are digital micrographs of HCV_NS3^_specific indirect immunofluorescence (IF) microscopy of DC2.4 transfected with cytopathic Repl-HCV_NS3 RNA. FIG. 3 shows two sets of six graphs, which indicated the presence or absence of apoptosis over time in DC2.4 cells transfected with cytopathic (left row of graphs) and noncytopathic (right row of graphs) Repl-HCV_NS3 RNA. FIG. 3 A shows propidium iodide fluorescence intensity versus Annexin V FITC fluorescence intensity at 12, 24 and 48 hours after transfection of DC. FIG. 3B shows the results of TUNEL analysis of transfected DC cells at the indicated time points. The number of Annexin V-positive, propdium iodide-positive and TUNEL-positive cells (all indicators of apoptosis) increased over time in the DC cells transfected with cytopathic Repl-HCV_NS3 RNA, but not significantly in the cells transfected with noncytopathic Repl-HCV_NS3 RNA.

FIG. 4 is a schematic diagram of the strategy used for vaccination (by subcutaneous injection) of mice and the subsequent detection of primed CD8+ T cells in immunized (vaccinated) mice. FIG. 4A represents the transfection of the DC2.4 dendritic cells with cytopathic or noncytopathic Repl-HCV_NS3 RNA, respectively, by electroporation. FIG. 4B depicts the in vivo phenomena of direct priming and cross-priming of T cells in the immunized mouse. FIG. 4C depicts the immunological assays target cells to detect cross-primed T cells (white) and directly primed T cells (black). Note that all T cells that recognize peptide-loaded C1R-AAD target cells are cross-primed T cells. CIR-AAD are CIR cells expressing the AAD molecule. The AAD molecule consists of the αl and α2 chains of HLA-A2.1 and of the α3 chain of H-2d.

FIG. 5 is a series of graphs showing the frequency of in v/vo-primed HCV_Ns₃-specific CD8+ T cells as determined by ex vivo IFN-γ ELISpot assays. Assays were performed one week after a single immunization of mice with subcutaneously injected RNA-transfected DC or with intramuscularly injected plasmid DNA as indicated at the top of each column of graphs. HLA-A2-restricted CD8⁺ T cells were quantified with peptide-loaded CIR-AAD (filled squares) and H-2^b-restricted CD8⁺ T cells were quantified with peptide-loaded EL4 (H-2^b) cells (open circles). The top three rows of graphs show T cells tested against NS3 peptide pool 1 (top row), pool 2 (second row from top), and pool 3 (third row from top). The bottom row of graphs shows the sum of all peptide pools which equals the total NS3-specific response. As diagramed in FIG.4, HLA-A2-restricted CD8⁺ T cells are cross-primed, and H-2^b-restricted CD8⁺ T cells are either directly primed or cross-primed. Note that direct priming and cross-priming cannot be differentiated after immunization with naked plasmid DNA (right-most column). Each data point represents the HCV_NS3 peptide pool-specific response (mean ± SD) of 6-10 mice (p < 0.04 for DC/cytopathic Repl-HCV_NS3 RNA versus naked plasmid HCV_NS₃ DNA; p < 0.04 for DC/cytopathic Repl-HCV_NS3 RNA versus DC/noncytopathic Repl-HCV_NS3 RNA at 2.5 and 5 x 10⁵ T cells/well; p < 0.05 for (DC/noncytopathic Repl-HCV_NS3 RNA + cytopathic Repl-GUS RNA) versus DC/noncytopathic Rep HCV_NS3 RNA at 5 x 10⁵ T cells/well for HLA-A2-restricted responses only). FIG. 6 is a set of graphs showing the cytotoxic activity of HLA-A2-restricted and

H-2^b-restricted cytotoxic CD8⁺ T cells isolated from mice immunized with (A) cytopathic Repl-HCV_NS3 RNA-transfected DC2.4, (B) noncytopathic Repl-HCV_NS3 RNA-transfected DC2.4, (C) noncytopathic Repl-HCVrø RNA-transfected DC2.4, which were supertransfected with cytopathic Repl-GUS RNA, or (D) HCV_NS3 plasmid DNA. Cytotoxic activity of HLA-A2-restricted CD8⁺ T cells was measured against peptide-pulsed CIR-AAD targets (filled squares), and cytotoxic activity of H-2^b-restricted CD8⁺ T cells was measured against peptide-pulsed EL4 (H-2^b) targets (open circles). Three different NS3 peptide pools were used to pulse the targets. The result obtained using NS3 peptide pools 1, 2 and 3 are shown in the graphs of the top, middle and bottom row, respectively, in each column. Each data point represents the HCV_NS3 peptide pool-specific response of 6-10 mice (mean ± SD) (p < 0.05 for DC/cytopathic Repl-HCV_NS3 RNA versus naked plasmid HCV_NS3 DNA; p < 0.05 for DC/cytopathic Repl-HCV_NS3 RNA vs DC/noncytopathic Repl-HCV_NS3 RNA at 30:1, 60:1 and 120:1 E:T for HLA-A2-restricted responses only; p < 0.04 for (DC/noncytopathic Repl-HCV_NS3 RNA + cytopathic Repl-GUS RNA) versus DC/noncytopathic Repl-HCV_NS3 RNA at 60:1 and 120:1 E:T for HLA-A2-restricted responses only). Note that induction of cell death (induced in noncytopathic Repl-HCV_NS3 RNA transfected DC2.4 by supertransfection with a cytopathic replicon encoding the irrelevant antigen GUS instead of HCV_NS₃) enhanced the cross-primed, HLA-A2 restricted T cell response (compare column C to column B). FIG. 7 is a composite of two digital immunofluorescence micrograph images and a series of

FACS analyses. FIG. 7A shows that, 12 hours after injection, few CFSE-labeled DC2.4 transfected with Repl-HCV_NS3 RNA (arrow) are detectable as intact cells in the lymph node cell fraction of immunized mice. FIG.7B shows two CD1 lc-expressing host dendritic cells from the same immunized mice. The bright particles (arrows) in the cytoplasm of the host dendritic cells are cellular fragments of the injected DC2.4 "programmed" to undergo apoptosis 24-48 hours after transfection with cytopathic Repl-HCV_NS3 RNA. The FACS analyses of FIG. 7C show the phenotypes of CFSE-positive cells detected in low-density lymph node and spleen cell populations ofnonvaccinated (negative control mice, left column) and experimental mice, that had been vaccinated with CFSE-labeled DC2.4 transfected with noncytopathic or cytopathic RepkHCV_NS3 RNA, respectively (right 3 columns). In the vaccinated mice, the CFSE sgnal is exclusively found in HLA-A2 positive cells, indicating that these were not the injected Repl-HCV_NS3 RNA-transfected DC2.4 but host cells that had captured cellular fragments of the injected, apoptotic Repl-HCV_NS3 RNA-transfected DC2.4. The transfer of cell-associated HCV NS3 antigen to host dendritic cells results in CD8+ T cell cross-priming. FIG. 8 shows two graphs indicating the proliferative response of T cells isolated from draining lymph nodes (top) and spleen (bottom) of mice immunized as iπlicated across the top of the figure. Stimulation index (mean ± SD) of lymph node and spleen cells of groups of 610 immunized HLA-A2-transgenic mice are shown in each group.

FIG. 9A shows vaccinia virus (VV) titers (top row) and HCV- and VV-specific CD8⁺ T cell responses (bottom row) of groups of 5 HL A-A2-transgenic mice immunized with RNA-transfected DC or plasmid DNA as indicated at the top of each column and then challenged with recombinant HCV_NS3-encoding VV. The dotted line in the top row of graphs indicates the detection limit of the assay. The graphs in FIG. 9B show the frequency of HLA-A2-restricted IFN-γ-producing CD8⁺ T cells in HLA-A2-transgenic (left) and wildtype (right) mice immunized with cytopathic ReptHCV_NS3 RNA-transfected DC2.4.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS The nucleic acid and protein sequences listed in the accompanying sequence listing is shown using standard letter abbreviations for nucleotide bases, and triple letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing: SEQ ID NO: 1 shows the amino acid sequence of the cytopatiic pestivirus replicons

(BVDV Bi-cp).

SEQ ED NO: 2 shows the amino acid sequence of the noncytopathic pestivirus replicons (BVDV Bi-noncp).

SEQ ID NO: 3 shows the amino acid sequence for an HCV antigen, HCV NS3. SEQ ID NO: 4 shows the DNA sequence for an HCV antigen, HCV NS3.

SEQ ID NO: 5 shows an HSV NS3 sense primer. SEQ ID NO: 6 shows an HSV NS3 antisense primer.

SEQ ID NO: 7 shows the nucleic acid sequence for the full length cytopathicpestivirus replicon/HCV antigen construct, BVDV Bi-cp, HCV NS3 (also called cytopathic Repl-HCV_NS3). SEQ ID NO: 8 shows the nucleic acid sequence for the full length noncytopathic pestivirus replicon/HCV antigen construct, BVDV Bi-noncp, HCV NS3 (also called noncytopathic Repl-HCV_NS3).

SEQ ID NO: 9 shows the nucleic acid sequence for the full length cytopathicpestivirus replicon/GUS construct, BVDV Bi-ΔN^procp, GUS (also called cytopathic Repl-GUS). SEQ ID NO: 10 shows the nucleic acid sequence for the full length noncytopathicpestivirus replicon/GUS construct, BVDV Bi-ΔN^prononcp, GUS (also called noncytopathic Repl-GUS).

SEQ ID NO: 11 shows the nucleic acid sequence of the pestivirus replicon "BVDV DI9C". SEQ TD NO: 12 shows the amino acid sequence of theHLA-A2 restricted minimal optimal epitope HCV NS3ι₀₇3-i08i- SEQ ID NO: 13 shows the amino acid sequence of theHLA-A2 restricted minimal optimal epitope HCV NS3 ιos4-ιo92-

SEQ ID NO: 14 shows the amino acid sequence of theHLA-A2 restricted minimal optimal epitope HCV NS31169-1177.

SEQ ID NO: IS shows the amino acid sequence of theHLA-A2 restricted minimal optimal epitope HCV NS3ι ₀6-i4i5.

SEQ ID NO: 16 shows the amino acid sequence of the HLA- A2 restricted minimal optimal epitope HCV NS3ι₅₈5-i593-

SEQ ID NO: 17 shows the amino acid sequence of a vaccinia virus epitope. DETAILED DESCRIPTION

Abbreviations 143TK a human osteosarcoma cell line

A adenine

AA amino acid

AAD a hybrid MHC class I molecule consisting of theαl+ 2 domains of HLA-

A2.1 and the α3 domain of H-2D^d

APC antigen-presenting cell

BDV border diseases virus

BSA bovine serum albumin

BVDV bovine viral diarrhea virus

CIR-AAD transfectants of the HLA-A,B-negative human B lymphoblastoid cell line

C1R with AAD

C cytosine

CP cytopathic

CSFV classical swine fever virus

DC dendritic cell

DC2.4 an immature murine dendritic cell line

DMEM Dulbecco's Modified Eagle Medium

DMSO dimethyl sulfoxide

ECL enhanced chemiluminescence

EDTA ethylenediaminetetraacetic acid

EL4 murine thymoma cell line EL4

FACS fluorescence-activated cell sorting

FBS fetal bovine serum

FITC fluorescein isothiocyanate

G guanine

GUS glucoronidase

HCV Hepatitis C Virus

HCV NS3 amino acids 1192-1457 of HCV- 1

HIV Human Immunodeficiency Virus

HLA human major histocompatibility complex

IgG immunoglobulin

IIF indirect immunofluorescence microscopy

IRES internal ribozymal entry site

NBT/BCIP nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate

NCP noncytopathic

NS2 non-structural protein 2

NS3 non-structural protein 3

NSS non-structural protein 5

PBS phosphate buffered saline

PFA paraformaldehyde

PFU plaque-forming units

RSV Respiratory Syncytial Virus

SDS sodium dodecyl sulfate

SDS-PAGE SDS-polyacrylamide gel electrophoresis

SFC spot forming cells

T thymine

U uracil

V volts

Vol volume

Wt weight II. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin,Ge«es V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182- 9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments, the following explanations of specific terms are provided: Adjuvant: an adjuvant is any substance that enhances the immune response to an antigen with which it is mixed. Adjuvants enhance the immunogenicity of the antigen by helping to retain the antigen in the body and to promote its uptake by antigen-presenting cells. Adjuvants may include bacteria or bacterial components. Adjuvants may include but are not limited to aluminum hydroxide, CpG-containing nucleotide sequences, ISCOMS (immune stimulatory complexes, which are small micelles of detergent which contain the antigen, fuse with host cells and antigenpresenting cells and allow the antigen to enter the cytosol of the host cells and antigen presenting cells). Adjuvants may be administered with the immunogenic compositions disclosed in the specification.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. An"antigen" includes one or more antigenic epitopes, including, for example, a single epitope or a combination of epitopes. Epitopes constituting a combination of epitopes may be derived from a single compound, composition, or substance or from different compounds, compositions, or substances. The individual epitopes of a combination epitope may be directly linked one to the other (using, for example, recombinant techniques commonly known in the art) or individual epitopes of a combination epitope may be linked together via linkers (such as, relatively short, non-antigenic peptide sequence) that separate the individual epitopes. Antigens can also include fragments of known antigens that retain the ability to stimulate the production of antibodies or a T cell response in an animal. For example, an immunogenic composition of the type described herein may include a peptide of at least about 5, 10, 15 or 20 amino acid residues. Smaller immunogens may require the presence of a"carrier" polypeptide, for example as a fusion protein, aggregate, conjugate or mixture linked (chemically or otherwise) to the immunogen.

Antigen presenting cell (APC): A class of cells capable of presenting one or more antigens in the form of an antigen-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against theantigen or antigens being presented. An APC cell carries on its surface antigen bound to MCH Class I or Class II molecules, and presents the antigen in this "context" to T cells. Examples of antigen presenting cells include, but are not limited to macrophages, dendritic cells (DC), follicular dendritic cells (FDC), and Langerhans cells. Macrophages are large white blood cells that ingest antigens and other foreign substances. Each macrophage contains packets of chemicals and enzymes that digest the ingsted antigen or microbe. Dendritic cells are the principle APC involved in primary immune responses. Their major function is to obtain antigen in tissues, migrate to lymphoid organs and activate T cells. Langerhans cells are dendritic cells specific to the skin.

Autologous cell: The term "autologous cell" describes a cell, for example a cell that is used for vaccination, that is derived from the subject. The "autologous cell" therefore displays a histocompatibility complex (MHC), that is identical to that of the subject from which it is derived Bicistronic: A form of genomic organization enabling translation of two open reading frames from the same RNA molecule For example, a pestivirus replicon RNA sequence that contains two open reading frames is bicistronic.

BDV (Border Disease Virus): Border Disease Virus, along with CSFV and BVDV belongs to the genus of animal pathogens Pestivirus, family Flaviviridae. BDV is distributed worldwide and is the causative agent of a congenital disease of sheep. Its genome is 12,333 nucleotides long and contains one long open reading frame encoding 3,895 amino acids (Bechere? al, J. Virol. 1998; 72: 5165-5173).

BVDV: bovine viral diarrhea virus; a member of the Pestivirus genus of animal pathogens, family Flaviviridae. BVDV includes two different biotypes of viruses. Noncytopathic viruses express predominantly the nonstructural protein NS2-3, whereas cytopathic viruses express the nonstructural protein NS3. cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and transcriptional regulatory sequences. cDNA can also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells. Chemokine: A chemokine is a small chemoat ractant protein that stimulates the migration and activation of cells, especially phagocytic cells and lymphocytes. Chemokines have acentral role in inflammatory responses.

Cross-priming: Cross-priming is a specific capacity of antigen presenting cells (APC) that involves the acquisition of exogenous antigens from apoptotic or dead cells in the periphery and the migration to secondary lymphoid organs, where APC, for example dendritic cells (DC), undergo apoptosis and are taken up by secondary APC. These APC reprocess the antigen and present it to T cells. For vaccination purposes, part of the pathway can be bypassed by directly introducing(for example by transfection or injection) an antigen to an antigen presenting cell (APC), for example a dendritic cell (DC), and allowing the cell to migrate to the spleen or lymph node. Secondary APCs phagocytose dying primary APC and present the antigen to T cells (for review, see Zhou et al, "Current methods for loading dendritic cells with tumor antigen for the induction of antitumor immunity," J. Immunother. 25(4):289-303, 2002). Cross-priming is particularly useful when immunity is based on T cell rather than on antibody responses (for example, for hepatitis C virus (HCV), -respiratory syncytial virus (RSV), and human immunodeficiency virus (HIV), Plasmodium falciparum, and Mycobacterium tuberculosis) and for immune responses against tumors. CSFV (classical swine fever virus, formerly termed"hog cholera virus"): CSFV, along with

BDV and BVDV, belongs to the genus Pestivirus, family Flaviviridae. CSFV is the causative agent of classical swine fever. It is an enveloped virus with a 12.5 kb single-stranded RNA genome of positive polarity that encodes a 4,000 amino acid polyprotein (Risatti et al, Journal of Clinical Microbiology 2003; 41: 500-505). Cytokine: A cytokine is a protein made by cells that affect the behavior of other cells.

Cytokines act on specific cytokine receptors on the cells that they affect.

Cytopathic: Damaging to cells, causing them to exhibit signs of disease or die. The cytopathic or noncytopathic phenotype of the pestivirus replicon is determined by the second open reading frame. For the cytopathic replicon, it encodes an ubiquitin gene (ubi) and the pestiviral nonstructural (NS3 to NS5) proteins. For the noncytopathic replicon, it encodes the 3' -terminal of the p7 coding unit which comprises the cleavage site for the generation ofthe correct N-terminus of NS2. This sequence is followed by the sequences ofthe BVDV nonstructural proteins NS2-NS5.

The expression of NS3 alone results in a cytopathic phenotype, whereas the expression of NS2/NS3 as an uncleaved protein is associated with a noncytopathic phenotype. Dendritic cells: A diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues. Dendritic cells constitute the most potent APCs in an organism. A subset of dendritic cells are derived from bone marrow progenitor cells, circulate in small numbers in the peripheral blood and appear either as immature Langerhans' cells or terminally differentiated mature cells. Dendritic cells do not have the CD14 antigen maiker associated with monocytes.

Dendritic cells recognize and act against invading antigens ofthe lymphoid and hematopoietic systems and skin, and function as the principle APC involved in primary immune responses. Their major function is to obtain antigen in tissues, migrate to lymphoid organs and activate T cells, thereby stimulating cellular immunity. Dendritic cells are also known as interdigitating, reticular, and veiled cells. Dendritic cell lines include, but are not limited to DC2.4, NemodDC, Dl, and XS52. For vaccination of chimpanzees and humans, autologous, primary dendritic cells are isolated from peripheral blood monocytes precursors (Reddye al, Blood, 90: 3640-3646, 1997; Dhodapkar et al, J. Clin. Invest., 104: 173-180, 1990).

DNA (deoxyribonucleic acid): A long chain polymer that comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA)). The repeating units in DNA polymers are four different nucleotides, each of which comprises one ofthe fiur bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA or RNA molecule is intended to include the reverse complement of that molecule. Except where single-strandedness is required by the text herein, DNA or RNA molecules, though written to depict only a single strand, encompass both strands ofthe molecule. Thus, a reference to the nucleic acid molecule that encodes a specific protein, or a fragment thereof, encompasses both the sense strand and its reverse complement. Thus, for instance, it is appropriate to generate probes or primers from the reverse complement sequence of the disclosed nucleic acid molecules. Effective amount: An amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, application or dosages. The effective amount can be an amount that is effective alone, or i combination with other agents (such as otter anti-infective or anti-neoplastic chemotherapeutic agents).

Encode: A polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translaed to produce the mRNA for and/or the polypeptide or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic; that is, elicit a specific immune response. An antibody binds a particular antigenic epitope. Exemplary HCV epitopes are HCV core, HCV El, HCV E2, HCV p7, HCV NS2, HCV NS3, HCV NS4 and HCV NS5 sequences. Particular HIV epitopes are located within, but are not limited to the antigens HIV Nef, HIV gag-p24, HIV reverse transcriptase, HIV P17 gag. Particular RSV epitopes are located in, but not limited to RSV G, RSV F, RSV N, RSV M2. Particular tumor antigens include Her-2/neu and α-fetoprotein, human epithelial cell mucin, the Ha-ras oncogene product, p53, carcino-embryonic antigen, the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-AlMart-1, gpIOO, EBV-LNT 1, EBV-LNT 2, HPV-F4, HPV-F 6, HPV-F 7, prostatic serum antigen, C017-1A, GA733, gp72, p53, the ras oncogene product, BPV E7 or a melanoma ganglioside, or variants or fragments thereof that retain the desired antigenic activity. Particular Mycobacterium tuberculosis antigens include ESAT-6, MPT63, MPT64, MPT83, Antigen 85B, Antigen 85A, PstS-1, PstS-2, PstS-3, MTB41, hsp60 (reviewed in Anderson, Trends in Immunology, 22: 160-168, 2002). Particular Plasmodium falciparum antigens include circumsporozoite protein (CSP), thrombospondin-related adhesive protien (TRAP), sporozoite and liver-stage antigen (SALSA), sporozoite threonine- and asparagine- rich protein (STARP), merozoite surface protein (MSP)-1, -2, -3, -4, -5, erythrocyte-binding antigen (EBA)-175, apical membrane antigen (AMA)-1, rhoptry-associated protein (RAP)-l and -2, acidic-basic repeat antigen (ABRA), ring erythrocyte surface antigen (RESA), serine-rich protein (SERP), erythrocyte membrane protein (EMP)-1, -2 and -3, Glutamate-rich protein (GLURP), Glycosilphopatidylinositol (GPI), Ps25, Ps28, Ps48/45 and Ps230 (reviewed in Carvalho et al, Scand. J. Immunol, 56: 327-343, 2002).

Expression: A process by which mRNA is translated into peptides, polypeptides or proteins. Expression can also include transcription of polynucleotides from DNA into mRNA, or translation directly from RNA, for example an RNA replicon.

Fibroblast: A cell of connective tissue that is mesodermally derived, and that secretes fibrillar procollagen, fibronectin and collagenase. The extracellular matrix secreted by fibroblasts is rich in collagen and other extracellular matrix macromolecules. Fibroblasts migrate and proliferate readily in wounded tissue and in tissue culture. Fusion protein: A polypeptide formed by the joining of two or more polypeptides through a peptide bond formed by the amino terminus of one polypeptide and the carboxyl terminus ofthe other polypeptide. A fusion protein is typically expressed as a single polypeptide from a nucleic acid sequence encoding the single contiguous fusion protein. However, a fusion protein can also be formed by the chemical coupling ofthe constituent polypeptides. Genotype and subtype: The genotype is the genetic constitution of a cell, an individual or an organism. The term "genotype" can pertain to all genes or to a specific gene. Genotype is sometimes used as one ofthe characteristics to classify viruses.

Viruses are generally classified, at least, by family, genus, and species. Families are typically based on genome type, virion structure, and replication cycles that distinguish members of one family from other families. Within families, genera arecommonly established based on shared characteristics that distinguish one group of viruses from another. The criteriafor establishing genera can vary from family to family. In some diverse families, subfamilies have also been established. The species is the least rigorous taxonomic unit in viral taxonomy. Species are often defined by place of isolation, disease caused, and host range and more recently by immunological andnucleic acid sequence characterization. Species may be subdivided into subtypes (or subspecies, variants, or strains). Alternatively, in some genera, a species may be synonymous with asubtype (variants or strains).

HCV exists in 6 different genotypes (1 to 6) and more than 50 different subtypes (for example, la, lb, 2a, 2b...; B kh et al, Semin. Liver Dis., 15: 41-63, 1995). Different isolates of HCV ofthe same subtype can differ by 5% to 15%, subtypes by 10% to 30% and genotypes by as much as 30% to 50% in nucleotide sequence. The different HCV genotypes have marked geographic variation in their relative frequencies. In addition, HCV develops multiple quasispecies with further sequence variation in any given patient, because of its high replication rate and lack of proofreading capacity ofthe viral polymerase (Hoofhagle, Hepatology, 36: S21-S29, 2002). A subtype of HIV is made up of a group of related HIV isolates classified- according to their degree of genetic similarity (such as, the percentage of identity within their envelope genes). There are at least 3 groups of HIV-1 isolates, called M, N. and O. Isolate M (major strains) consists of at least 10 subtypes (or, clades), A through J. Group O (outer strains) may consist of a similar number of subtypes (or, clades).

Hepatitis C virus QHCV): Hepatitis C is a viral infection ofthe liver which had been referred to as parenterally transmitted "non A, non B hepatitis" until identification of the causative agent, the Hepatitis C virus (HCV), in 1989. HCV is a major cause of acute hepatitis and chronic liver disease, including cirrhosis and liver cancer. Globally, an estimated 170 million persons are chronically infected with HCV, and 3 to 4 million persons are newly infected each year. HCV is spread primarily by direct contact with human blood.

Hepatitis C virus (HCV) is one ofthe five viruses (Hepatitis A, B, C, D, and E), which together account for the vast majority of cases of viral hepatitis. A vaccine is not available. It is an enveloped RNA virus in the flaviviridae family that appears to have a narrow host range. Humans and chimpanzees are the only known species susceptible to infection, with both species developing similar disease.

Heterologous nucleic acid: Exogenous or non-native DNA, for example from a different genetic source or different species.

Human Immunodeficiency Virus (HIV): Acquired immunodeficiency syndrome (AIDS) is a disease characterized by a progressive loss of function ofthe immune system. As a result, those afflicted with the syndrome are susceptible to a variety of opportunistic infections. The etiologic agent of AIDS is a cytopathic retrovirus designated the Human Immunodeficiency virus (HIV). Hybridization: Oligonucleotides and their analogs hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, a nucleic acid molecule consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as "base pairing." More specifically, A will hydrogen bond to T or U, and G will bond to C. "Complementary" refers to the base pairing that occurs between to distinct nucleic acid sequences or two distinct regions ofthe same nucleic acid sequence.

Immune response: A response of a cell ofthe immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). An immune response can be a humoral (antibody or Bcell response) or cellular (cell mediated or T cell response). In one embodiment ofthe disclosed method, an immune response is a T cell response, such as a CD4+ T cell response or a CD8+ T cell response.

Introduce into a cell: A nucleotide is introduced into a cell in a variety of ways, for example by chemical transfection, transduction, injection, or electroporation. Introduction of an RNA molecule into a cell includes introduction into the cell of either an RNA molecule or of a DNA molecule that encodes the RNA.

Isolated: A biological component (such as a nucleic acid molecule, protein or organelle) that has been substantially completely separated or purified away from other biological components in the cell ofthe organism in which the component na^turally occurs, for example, other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

Major histocompatibility complex (MHC): The minor and major histocompatibility complexes are a cluster of genes that encode membrane glycoproteins. The MHC class I molecules present peptides generated in the cytosol to CD8+ T cells, and the MHC class II molecules present peptides degraded in intracellular vesicles to CD4+ T cells. The MHC also encodes proteins involved in antigen processing and other aspects of host defense. The MHC is the most polymorphic gene cluster in the human genome.

Monocistronic: A form of genomic organization resulting in transcription of an mRNA that contains the coding sequence for a single polyprotein. A "polyprotein" is a polypeptide that contains multiple individual protein sequences embedded within it and which must be proteolytically cleaved to yield the individual proteins.

Mycobacterium tuberculosis: Mycobacterium tuberculosis is the pathogen eliciting tuberculosis, a major global health problem causing more than 2 million deaths each year. The current vaccine, Mycobacterium bovis bacilli Calmette-Guerin (BCG) was developed at the start of the 20^th century, but has proven inefficient in several recent field trials, and multi-drug resistant mycobacteria have emerged (reviewed in Andersen, Trends in Immunology, 22: 160-168, 2001).

Nucleotide: This term includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine, or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide. Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurringnucleotides.

Oligonucleotide: A plurality of nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid (PNA) molecules. Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNAor RNA) that is at least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long, or from about 6 to about 50 bases, for example about 10-25 bases, such as 12, 15 or 20 bases. Open reading frame: A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide.

Operably linked: A coding sequence is linked to a regulatory sequence in a manner that allows expression ofthe coding sequence. Known regulatory sequences may be used for direct expression ofthe desired protein in an appropriate host cell.

Pestivirus: Pestiviruses belong to the Flaviviridae family of viruses. The pestivirus genus includes, but is not limited to bovine viral diarrhea virus 03VDV), classical swine fever virus (CSFV, also called hog cholera virus) and border disease virus (BDV) of sheep (Moennige al, Adv. Vir. Res., 41: 53-98, 1992). Pestivirus infections of domesticated livestock (cattle, pigs and sheep) cause significant economic losses worldwide. BVDV causes mucosal disease in cattle and is of significant economic importance to the livestock industry (Meyers and

in Virus Research, 47: 53-118, 1996; Moennig, et al, Adv. Vir. Res., 41: 53-98, 1992).

Pestivirus subtypes include Bovine viral diarrhea virus genotype 2 (BVDV-2), Pestivirus type 1, which includes Bovine viral diarrhea virus isolates and strains, Pestivirus type 1 isolates, and Pestivirus type 1 strain R2727, Pestivirus type 2, which includes Classical swine fever virus and Hog cholera virus, Pestivirus type 3, and unclassifiedpestiviruses.

Pestiviruses can be differentiated into cytopathic and noncytopathic strains according to the effect of an infection on cells in tissue cultures. In contrast to infection with noncytopathic strains, infection with cytopathic pestiviruses leads to the lysis ofthe cellular host. Cytopathic BVDV strains apparently develop from noncytopathic BVDV strains by rearrangement of the viral genome due to RNA recombination, for example, deletions, duplications of certain parts ofthe viral genome, and ' insertions of parts of cellular mRNAs (reviewed in Meyers and T iel,Adv. Virus Res. 47: 53-118, 1995; Thiel et al, Pestiviruses, In: Virology, 3rd edition, ed. by B. N. Fields, Philadelphia, PA: Lippincott-Raven, p. 1059-1074,1996). Pestiviruses are also discussed in detail in EP01149901. Pestivirus replicon: A pestivirus subgenomic RNA that encodes its own viral replicase, and can perform cytoplasmic amplification. A specific, non-limiting example of a pestivirus replicon is an sg BVDV replicon that encodes from the viral proteins only the first three amino acids ofthe autoprotease N(pro), in addition to nonstructural (NS) proteins NS3 to NS5B, and that replicates. Pestivirus replicons also include, but are not limited to four infectious BVDV cDNA clones, BVDV Bi-ΔN^procp (cytopathic), BVDV Bi-ΔN^procp (noncytopathic), BVDV Bi-cp (cytopathic), and BVDV Bi-noncp (noncytopathic), which are bicistronic replicons expressing proteins NS2-3 to NS5B. (Tautz et al, J. Virol, 73(11): 9422-9432, 1999). These replicons express, in addition to the viral proteins, the reporter gene encoding beta-glucuronidase.

Introduction of a heterologous coding region (encoding, for example, an antigen) into the pestivirus replicon produces a self-replicating RNA that expresses the heterologous protein at a high level in transfected or infected cells. If such a replicon is cytopathic, it eventually kills thetransfected or infected cells. Pestivirus replicon-based vectors can be used to express a heterologous protein at high levels, and thus serve as excellent vaccine delivery systems. Variations (such as substitutions, deletions or insertions) can be made in the sequence of the pestivirus replicon while retaining its ability to provide its desired function. In addition to BVDV-based pestivirus replicons, CSFV- and BDV-based replicons also are of use.

Because the genomic organization of many pestiviruses, for instance BDV and CSFV, is identical to that of BVDV, pestivirus replicons are derived from these viruses in the same manner used for the BVDV replicons described herein. In addition, the pestivirus replicons ofthe present disclosure can be altered by one or more nucleotides without changing the essential function ofthe replicons. For example, one or more nucleotides can be added, deleted, or changed within the 3' untranslated region of each replicons described herein. This region includes approxύnately the last 100 nucleotides of each pestivirus replicons sequence.

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly admuiEtered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulaticns suitable for phannaceutical delivery ofthe fusion proteins herein disclosed.

In general, the nature ofthe carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fuids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can containminor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Plasmodium falciparum: Plasmodium falciparum is the most prevalent Plasmodium species that causes human malaria. A vaccine that would protect from this pathogen is not available yet, and more than 750,000 deaths occur each year due to malaria (reviewed in Carvalhoe? al, Scand. J. Immunol, 56: 327-343, 2002). Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The term polypeptide or protein as used herein encompasses any amino acid sequence and includes modified sequences such as glycoproteins. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

The term polypeptide fragment refers to a portion of a polypeptide that exhibits at least one useful epitope. The phrase "functional fragments of a polypeptide" refers to all fragments of a polypeptide that retain an activity, or a measurable portion of an activity, ofthe polypeptide from which the fragment is derived. Fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptile capable of participating in the characteristic induction or programming of phenotypic changes within a cell. An epitope is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen. Thus, smaller peptides containing the biological activity of insulin, or conservative variants ofthe insulin, are thus included as being of use.

The term soluble refers to a form of a polypeptide that is not inserted into a cell membrane.

Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Variations in the cDNA or RNA sequence that result in amino acid changes, whether conservative or not, are usually minimized in order to preserve the functional and immunologic identity ofthe encoded protein. The immunologic identity ofthe protein may be assssed by determining whether it is recognized by an antibody; a variant that is recognized by such an antibody is immunologically conserved. Any cDNA or RNA sequence variant will preferably introduce no more than twenty, and preferably fewer than ten am υ acid substitutions into the encoded polypeptide. Variant amino acid sequences may, for example, be 80%, 90%, or even 95% or 98% identical to the native amino acid sequence. Programs and algorithms for determining percentage identity can be found at theNCBI website.

Protein: A biological molecule expressed by a gene and comprised of amino acids. Purified: In a more pure form than is found in nature. The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell.

The term "substantially purified" refers to a molecule (for example, a nucleic acid, polypeptide, oligonucleotide, etc.) that is substantially free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. In one embodiment, the molecule is a polypeptide that is at least 50% free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. In another embodiment, the polypeptide is at least at least 80% free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. In yet other embodiments, the polypeptide is at least 90% or at least 95% free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. RNA replicons are positive-strand RNA viral particles that encode their own viral replicase, and can perform high level cytoplasmic amplification. Pestivirus replicons are subgenomic viral RNAs, which replicate autonomously in a broad spectrum of transfected host cells. They lack the genes ofthe virus structural proteins, which can be substitited by heterologous genes for foreign protein (for example antigen) expression. Replicon-based vectors can be used to express a heterologous protein at high levels, and thus serve as excellent vaccine delivery systems.

Respiratory Syncytial Virus (RSV) is the most common respiratory virus in infants and young children. It infects virtually all infants by the age of two years. In most infants, the virus causes symptoms resembling those ofthe common cold. In infants born prematurely and/or with chronic lung disease, RSV can cause a severe or even life-threatening disease. Each year, RSV disease results in over 125,000 hospitalizations, and about 2% of these infants die. Exemplary RSV antigens include, but are not limited to RSV F and RSV G, and are dεcussed in detail in WO9940937.

Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms ofthe similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or orthologs of the pestivirus replicon construct protein, and the corresponding cDNA sequence, will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or cDNAs are derived from species that are more closely related (for example, human and chimpanzee sequences), compared to species more distantly related (for example, human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, J. Mol. Biol, 147(1): 195-197, 1981; Needleman and Wunsch, J. Mol. Biol, 48: 443-453, 1970; Pearson and

Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444-2448, 1988; Higgins and Sharp, Gene, Ti: 237-244, 1988; Higgins and Sharp, CABIOS, 5: 151-153, 1989; Corpet et al, Nuc. Acids Res., 16 : 10881-10890, 1988; Huang et al, Computer Appls. in the Biosciences, 8: 155-165, 1992; and Pearson et al, Meth. Mol. Bio., 24: 307-331, 1994. Furthermore, Altschul et al. (J. Mol. Biol, 215: 403-410, 1990) present a detailed consideration of sequence alignment methods and homology calculations. Default parameters may be used for alignment. In particular, preferred programs are BLASTIN and BLASTP, using the following default parameters: Genetic code=s1andard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR.

For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment is performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to a reference sequence will show increasing percentage identities when assessed by this method, such as at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J. Mol. Biol, 215: 403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. The Search Tool can be accessed at the NCBI website, together with a description of how to determine sequence identity using this program. BLAST searching permits the determination ofthe sequence identity between a given sequence, for example a nucleotide sequence and a reference sequence. Nucleotide sequences with even greater similarity to a reference sequence will show increasing percentage identities when assessed by this method, such as at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity

An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence- dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5° C to 20° C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence remains hybridized to a perfectly matched probe or complementary strand. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, CSHL, New York, and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes Part I, Chapter 2, Elsevier, New York. Nucleic acid molecules that hybridize under stringent conditions to a pestivirus replicon encoding sequence will typically hybridize to a probe based on either an entire human pestivirus replicon encoding sequence or selected portions ofthe gene under wash conditions of 2x SSC at 50 C.

Nucleic acid sequences that do not show a high degree of identity can nevertheless encode similar amino acid sequences, due to the degeneracy ofthe genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.

Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals. T cell: A white blood cell involved in the immune response. T cells include, but are not limited to, CD4+ T cells and CD8+ T cells. A CD4+ T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8+ T cells carry the "cluster of differentiation 8" (CD8) marker. In one embodiment, CD8+ T cells are cytotoxic T lymphocytes. In another embodiment, CD8+- T cells are IFN-gamma-producing T cells.

T cell response: A response of a T cell to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, a T cell response is a CD4+ T cell response or a CD8+ T cell response.

Transfected: A process by which a nucleic acid molecule is introduced into cell, for instance by molecular biology techniques, resulting in a transfected cell. As used herein, the term transfection encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transfection with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

Tumor: A neoplasm. The methods disclosed in this specification are suitable for treating a variety of tumors. These tumors include both solid and hematological (or liquid)tumors. Examples of hematological tumors include, but are not limited to: leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, mαiocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkiris disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenshom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, include, but are not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancey hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

Tumor antigens that can be expressed from the replicon to induce theT cell response include, but are not limited to human epithelial cell mucin (Mue- 1 ; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), the Ha-ras oncogene product, p53, carcino-embryonic antigen, the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase,^' gp75, Melan-AlMart-1, gpIOO, IHER2/neu, EBV-LNT 1 & 2, HPV-F4, 6, 7, prostatic serum antigen, alpha-fetoprotein, C017-1A, GA733, gp72, p53, the ras oncogene product, BPV E7 and melanoma gangliosides. Tumor antigens are discussed in greater detail in WO02061113, WO03025002, WO03024994, WO03024304, WO03024304, and WO03024302.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transfected host cell. Recombinant nucleic acid vectors are vectors having recombinant nucleic acid sequences. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art.

Virus: A microscopic infectious organism that reproduces inside living cells. An enveloped virus consists essentially of a core of a single nucleic acid surrounded by a protein coat, and has the ability to replicate only inside a living cell. "Viral replication" is the production of additional virus by the occurrence of at least one viral life cycle. A virus may subvert the host cells' normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so. Unless otherwise explained, 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 disαbsure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. "Comprises" means "includes." Hence "comprising A or B" means includes A or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing ofthe present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

III. Detailed Description A. Overview of several embodiments

Disclosed herein are pestivirus replicons having a nucleic acid sequence that includes a pestivirus nucleic acid sequence and a heterologous antigen-encoding sequence. The antigen-encoding sequence ofthe pestivirus replicon is inserted at a position in the pestivirus nucleic acid sequence that inhibits formation of infectious replicon particles by disrupting the expression of structural proteins required for formation of infectious replicons, including, for example, one or more ofthe C, E ^s, El, or E2 structural proteins. In some embodiments, the antigen-encoding sequence partially or completely replaces one or more or all ofthe C, E^ras, El, or E2 subunits ofthe pestivirus nucleic acid sequence. In more specific embodiments, the antigen-encoding sequence completely replaces the C, E^ms, El, and E2 subunits ofthe pestivirus nucleic acid sequence.

The pestivirus of some replicon embodiments is the bovine viral diarrhea virus (BVDV), the classical swine fever virus (CSFV) or the border disease virus (BDV). In a specific example, the pestivirus replicon is based on BVDV.

Some ofthe disclosed replicons are monocistronic, while others are bicistronic. Examples of bicistronic replicons can encode an N^pr0-antigen fusion protein in one open reading frame, and encode a polyprotein comprising NS2/NS3, NS4A, NS4B, NS5A, andNS5B in a second open reading frame. In these examples, NS2/N3 can be uncleaved NS2-NS3 polypeptide or NS3 polypeptide. In certain examples, pestivirus replicons include the following elements:

5'-IRES-Npro-Antigen-IRES-p7-(NS2-NS3)-NS4A-NS4B-NS5A-NS5B-3', wherein "Antigen" is a sequence encoding a heterologous antigen. More particular examples ofthe replicons are encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 8, such as the nucleic acid sequence set forth as SEQ ID NO: 8. In other examples, pestivirus replicons include the following elements:

5'-IRES-Npro-Antigen-IRES-ubi-NS3-NS4A-NS4B-NS5A-NS5B-3', wherein "Antigen" is a sequence encoding a heterologous antigen. More particular examples of replicons having this structure are encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 7, such as the nucleic acid sequence set forth as SEQ ID NO: 7. The antigen encoding-sequence of certain ofthe disclosed replicons encodes an antigen, which can be one or more epitopes and/or may be an antigenof a pathogen or tumor. In some cases, a pathogen antigen may derive from a virus (such as Hepatitis C virus, Human Immunodeficiency Virus, Respiratory Syncytial Virus), a bacteria, Mycobacterium tuberculosis, or Plasmodium falciparum. With regard to those replicon embodiments wherein the antigen is a Hepatitis C virus (HCV) antigen, the antigen can for example be HCV core, HCV El, HCV E2, HCV p7, HCV NS2, HCV NS3, HCV NS4, or HCV NS5. In some particular examples, an HCV antigen can have at least 90%, at least 95%, at least 98% sequence identity with, or even be, SEQ ID NO: 3. In addition, an HCV antigen can be a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: 3. In various applicable embodiments, respiratory syncytial virus antigens include RSV F, RSV

N, RSV M2 or RSV G. Human immunodeficiency virus antigen include pi 8, p24, p33, p39, p55, gp36, gp41, or gpl20. Mycobacterium tuberculosis antigens include ESAT-6, MPT63, MPT64, MPT83, Antigen 85B, Antigen 85A, PstS-1, PstS-2 or PstS-3, MTB41, or hsp60. Plasmodium falciparum antigens include circumsporozoite protein (CSP), thrombospondin-related adhesive protien (TRAP), sporozoite and liver-stage antigen (SALSA), sporozoite threonine- and asparagine- rich protein (STARP), merozoite surface protein (MSP)-l, -2, -3, -4, -5, erythrocyte-binding antigen (EBA)-175, apical membrane antigen (AMA 1, rhoptry-associated protein (RAP)-l and -2, acidic- basic repeat antigen (ABRA), ring erythrocyte surface antigen (RESA), serine-rich protein (SERP), erythrocyte membrane protein (EMP)-l, -2 and -3, Glutamate-rich protein (GLURP), Glycosilphopatidylinositol (GPI), Ps25, Ps28, Ps48/45 or Ps230. Tumor antigen include Her-2/neu, α-fetoprotein, human epithelial cell mucin, the Ha-ras oncogene product, p53, carcino-embryonic antigen, the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-AlMart- 1 , gpIOO, EBV-LNT 1 , EBV-LNT 2, HPV-F4, HPV-F 6, HPV-F 7, prostatic serum antigen, C017-1A, GA733, gp72, p53, the ras oncogene product, BPV E7 ormelanoma ganglioside.

This specification further discloses a method of producing aT cell response in a subject against a pathogen that is more effectively cleared by a T cell response rather than a Bcell (antibody- based) immune response. The method involves expressing an antigen encoded by a recombinant pestivirus replicon in an APC such as a dendritic cell. The pestivirus replicon can be any of those described herein. Such expression may be achieved, for example, by introducing a selfreplicating cytopathic or noncytopathic pestivirus replicon ex vivo or in vivo into the APC. The replicon expresses an immunogenic antigen, for example, a hepatitis C virus (HCV), respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), Mycobacterium tubeculosis, Plasmodium falciparum, or tumor antigen.

In vivo introduction ofthe replicon can occur in several ways. Replicon RNA or cDNA copies that encode the replicon RNA, can be injected directly into skin, muscle, lymph node or specific target organs such as the liver. Replication and expression ofthe heterologous antigen starts after the replicon or replicon-encoding cDNA is taken up by host cells, some of which will be antigen-presenting cells. Ex vivo introduction ofthe replicon into the APCs can occur in several ways. For example, (a) primary dendritic cells can be isolated from the blood of patients, transfected with the RNA replicon and injected (see Example 2), or (b) a cell line, such as the dendritic cell line DC2.4 for mice or an autologous fibroblast cell line or another autologous cell line for humans or chimpanzees, can be transfected with the RNA replicon and injected into a subject (see Example 1). Once in the body, the APCs migrate to the lymph tissue, replicating the RNA to produce high cytoplasmic levels ofthe immunogenic antigen. When the APC becomes apoptotic and dies in the lymph tissue (such as a lymph node or spleen), the antigen induces T cell immunity viacross-priming.

This method provides several advantages over previously known methods. Pestivirus has been found to replicate less efficiently than Semlicki Forest virus and Sindbis virus, which allows dendritic cells carrying the pestivirus replicon to survive longer. This increased survival time is believed to permit more time for the dendritic cells to migrate to the lymph nodes and spleen, where the cross-priming event occurs. It has been found that the pestivirus replicons are therefore superior cross-priming vectors.

An additional advantage is that the pestivirus replicons provide an extra margin of safety over other viral-based vaccines in that the pestivirus replicons are derived from viruses that are not capable of infecting humans, and therefore do not cause human diseases. Moreover, the viral RNA cannot integrate into the genome ofthe subject. Finally, in those embodiments ofthe method in which the replicons are introduced into APCs (such as dendritic cells) in vitro before administering them to the subject provides, more efficient immunization occurs thanwith the prior method of administering naked RNA or DNA directly to the subject. Without being bound by theory, this is believed to be because naked RNA and DNA will be randomly taken up by any host cell, the majority of them not being dendritic cells. Therefore, these transfected host cells need to undergo apoptosis in the periphery before they and the antigens they express will be taken up by dendritic cells, which then transport the antigens to lymph nodes and spleens and stimulate T cells.

In one specifically disclosed embodiment, the method of inducing aT cell response to an antigen in a subject is performed by administering to the subject an amount of an APC sufficient to induce an immune response in a subject, wherein the APC expresses the antigenfrom a cytopathic or noncytopathic pestivirus replicon. In certain embodiments, the pestivirus replicon is introduced into the APC prior to administering the antigen presenting cell to the subject. A dendritic cell is a particular example ofthe APC that is suitable for use in this method.

In particular examples, the replicon used as a starting material comprises SEQ ID NOs: 1 or 2, or comprises a sequence having at least 90%, 95%), or 98% sequence identity to SEQ ID NCs: 1 or 2, or encodes a sequence at least 90%, 95% or 98% identical to SEQ ID NOs: 1 or 2, or comprises a sequence that is a conservative variant of SEQ ID NOs: 1 or 2 (having, for example, not more than 1, 2, 5 or 10 conservative amino acid substitutions).

The replicon can be used to encode an antigen against which aT cell-mediated immune response is desired. For example, the antigen may be a tumor antigen or a pathogen antigen, such as a viral pathogen antigen, for example an antigen from Hepatitis C virus, a Human Immunodeficiency Virus, or a Respiratory Syncytial Virus. In some embodiments the repliconexpresses multiple antigens, such as multiple antigens from the same virus or tumor, or multiple antigens from a variety of different viruses or tumors. HCV, HIV, RSV, Mycobacterium tuberculosis, and Plasmodium falciparum antigens are particularly suitable antigens for use in the method, because the body does not usually produce neutralizing antibodies against them that are sufficient to clear the infection from the body. Instead, a T cell-mediated response is substantially relied upon to overcome the infection and to induce immunity against subsequent infections.

When the virus is Hepatitis C virus, examples ofthe antigen are one or more of HCV core, HCV El and E2, HCV p7, HCV NS2, HCV NS3, HCV NS4, HCV NS5, or variants or fragments thereof, for example, an antigen having at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the HCV genotype and/or to the HCV quasispecies sequence found in a given patient. In some embodiments, the sequence ofthe HCV subtype infecting a particular subject is determined, permitting the creation of a customizedpestivirus replicon construct that is specific to the particular HCV quasispecies. In alternative examples, the viral pathogen is a Respiratory Syncytial Virus, and the antigen is RSV F, RSV N, RSV M2, or RSV G, or a fragment or conservative variant thereof that retains the desired antigenic activity. In yet other examples, the viral pathogen is a Human Immunodeficiency Virus, and the antigen includes HIVpl8, p24, p33, p39, p55, gp36, gp41, or gpl20, or fragments or variants that retain the desired immunogenic activity.

In yet other examples the antigen is a tumor antigen, such as Her-2/neu, α-fetoprotein, human epithelial cell mucin, the Ha-ras oncogene product, p53, carcino-embryonic antigen, the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75,

Melan-AlMart- 1, gpIOO, EBV-LNT 1, EBV-LNT 2, HPV-F4, HPV-F 6, HPV-F 7, prostatic serum antigen, CO 17-1 A, GA733, gp72, p53, the ras oncogene product, BPV E7 or a melanoma ganglioside, or variants or fragments thereof that retain tiie desired antigenic activity.

In yet other examples, the antigen is a Mycobacterium tuberculosis antigen, for example ESAT-6, MPT63, MPT64, MPT83, Antigen 85B, Antigen 85A, PstS-1, PstS-2, PstS-3, MTB41, or hsp60 (reviewed in Anderson, Trends in Immunology 2002; 22: 160-168), or Plasmodium falciparum antigen, for instance circumsporozoite protein (CSP), thrombospondin-related adhesive protien (TRAP), sporozoite and liver-stage antigen (SALSA), sporozoite threonine- and asparagine-rich protein (STARP), merozoite surface protein (MSP)-l, -2, -3, -4, -5, erythrocyte-binding antigen (EBA)-175, apical membrane antigen (AMA)-1, rhoptry-associated protein (RAP)-l and -2, acidic-basic repeat antigen (ABRA), ring erythrocyte surface antigen (RESA), serine-rich protein (SERP), erythrocyte membrane protein (EMP 1, -2 and -3, Glutamate-rich protein (GLURP), Glycosilphopatidylinositol (GPI), Ps25, Ps28, Ps48/45 and Ps230 (reviewed in Carvalho et al, Scand. J. Immunol, 56: 327-343, 2002). In more specific examples, the pestivirus replicon encodes a Hepatitis C antigen. A specific example would be a pestivirus replicon that encodes SEQ ID NO: 3, or encodes a sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO:3, or a conservative variant of SEQ ID NO: 3, which retain immunogenecity ofthe antigen. Hence any variant ofthe antigen can be used, as long as it still provokes a desired immune response in the subject to whom it is administered. The replicon can be provided in the form of an isolated nucleic acid sequence, such as that set forth in SEQ ID NO: 8, including the corresponding RNA sequence.

In yet another example a dendritic cell is provided, into which thepestivirus replicon has been introduced, for example by transfection. The replicon may be a cytopathic replicon, such as that shown in SEQ ID NO: 1, or a replicon having at least 90%, 95%, 98% or 100% sequence identity to SEQ ID NO: 1, and which retains the ability to stimulate the desired immune response. Alternatively, the replicon may be a noncytopathic replicon, such as that shown in SEQ ID NO: 3, or a replicon having at least 90%, 95%, 98% or 100% sequence identity to SEQ ID NO: 3, and which retains the ability to stimulate the desired immune response. The pestivirus replicon expresses the antigen, such as an antigen or a tumor or pathogen, such as a viralantigen from HCV, HIV, or RSV, or an antigen from Mycobacterium tuberculosis, or Plasmodium falciparum. Also disclosed herein are compositions for inducing an immune response, wherein the composition comprises the replicon, or the dendritic cell into whidi the replicon has been introduced, and a pharmaceutically acceptable carrier.

B. Pestiviruses

Bovine viral diarrhea virus (BVDV type I and II), Classical swine fever virus (CSFV) and Border Disease virus of sheep (BDV) are the members ofthe Pestivirus genus of widespread animal pathogens. Along with the genera Flavivirus and Hepacivirus (hepatitisC viruses, HCVs), the pestiviruses compose the family Flaviviridae. The pestiviral genome is a positive-strand, single-stranded RNA, which has a length ofabout 12-16 kilobases. It consists of a long open reading frame (ORF) that is flanked by non-translated regions (NTRs) at the 5' and 3' ends. Following entry and uncoating, the viral RNA acts directly as a messenger in the host cells cytoplasm and replicates in the same compartment via a' negative-strand RNA intermediate and without a DNA stage. Translation initiates through a complex type IV IRES element in the 5 NTR and yields a polyprotein NH₂-N^pro, C, E^ms, El, E2, p7, NS2-NS3, NS4A, NS4B, NS5A, NS5B-COOH that is co- and post-translationally processed into the structural (C, E¹™, El, E2, p7) and non-structural (N^pro, NS2-NS5B) proteins. N^pro is an autoprotease that releases itself from the polyprotein precursor. The core (C) and Envelope proteins (Erns, El, E2) as well as p7 are destined to form the virus particle. A protease complex consisting of NS3 and NS4A generates the proteins NS3 to NS5B, all which were shown to be essentially involved in viral replication. Apart from the NS3/NS4A protease.the virus encodes two further enzymes that are crucial for the replication process, namely an RNA helicase harbored also by NS3 and the RNA-dependent RNA polymerase (RdRp), which is associated to the NS5B protein (for a review see Lindenbach and Rice, In: Fields Virology, ed. by Knipe et al., Philadelphia, PA: Lippincott Williams & Wilkins, pp. 991-1041, 2001; Behrens et al, J. Virol, 72: 2364-2372, 1998; Grassmann et al, J Virol, 73: 9196-9205, 1999; Grassmann et al, J. Virol, 75: 7791-7802, 2001).

The pestivirus genus includes, but is not limited to bovine viral diarrhea virus (BVDV type I and II), classical swine fever virus (CSFV, also called hog cholera virus) and border disease virus (BDV) of sheep (Moennig et al, Adv. Vir. Res., 41 : 53-98, 1992). Pestivirus infections of domesticated livestock (cattle, pigs and sheep) cause significant economic losses worldwide. BVDV causes mucosal disease in cattle and is of significant economic importance to the livestock industry (Meyers and Thiel, Advances in Virus Research, 47: 53-118, 1996; Moennig, et al, Adv. Vir. Res., 41: 53-98, 1992).

Bovine viral diarrhea virus As discussed above, bovine viral diarrhea viruses (BVDV) are members ofthe Pestivirus genus in the family Flaviviridae. BVDV are small, enveloped viruses having a single-stranded positive-sense RNA genome. The BVDV genome is approximately 12.3 kb in length with a 5'-nontranslated region O^TR), a single large open reading frame (ORF), and a 3' -NTR lacking a poly(A)tail. The 5'-NTR contains an internal ribosome entry site that initiates translation of BVDV mRNA in a cap-independent manner. The secondary structure ofthe 5 -NTR is involved in the regulation of both translation and genome replication. The ORF is translated into a single pol rotein of approximately 4000 amino acids that is co- and post-translationally cleaved into 11 or 12 mature proteins by viral and host proteases. The order of proteins in the polyprotein is Npro (a nonstructural autoprotease unique to pestiviruses), the capsid protein (C), the envelope glycoproteins (Erns, El, and E2), and the nonstructural proteins (p7, NS2/NS3, NS4A, NS4B, NS5A, and NS5B). NS3 (NS2/NS3) has helicase, serine protease, and NTPase activities, and NS5A has RNA-dependent RNA-polymerase activity. The NS2/NS3 complex is not cleaved in BVDV isolates that are noncytopathic in cell culture. In contrast, both NS2/NS3 and a discrete NS3 are observed in cytopathic BVDV isolates. The processing of NS2/NS3 appears to develop from RNA recombination events during the genomic replication of a noncytopathic virus. As a result, the genome of cytopathic isolates may contain genomic duplications, deletions, rearrangements, and/or insertions of cellular mRNA.

C. Pestivirus replicons

A pestivirus replicon is a pestivirus subgenomic RNA that encodes its own viral replicase and can perform cytoplasmic amplification. A specific, non-limiting example of a pestivirus replicon is an sg BVDV replicon that encodes from the viral proteins only the first three aminoacids ofthe autoprotease N^pro, in addition to nonstructural (NS) proteins NS3 to NS5B, and that replicates (for example, BVDV DI9c and derivatives thereof as described byBehrens et al, J. Virol, 72: 2364 2372, 1998). From the infectious BVDV cDNA construct BVDV CP7 (Meyers et al, J. Virol, 70: 8606-8613, 1996), cytopathic and noncytopathic replicons have been developed (Tautz et al, J. Virol, 73(11):9422-9432, 1999). These replicons may, but need not, be mono- or bi-cistronic. In some embodiments, these replicons express, in addition to the viral proteins, reporter genes such as that encoding beta-glucuronidase or selective markers such as theNEO or Hyg genes (see below). As previously discussed, a pestivirus replicon may contain a heterologous RNA sequence encoding, for example, an antigen against which an immune response is desired. In some embodiments, the antigen-encoding sequence is positioned within the pestivirus nucleic acid (RNA) sequence so as to functionally disrupt the coding sequence of one or more ofthe structural proteins, including for example, C, Erns, El, E2, and/or p7. Functional disruption ofthe structural protein(s), as used herein, means that expression of these proteins is altered such that thepestivirus replicon is substantially unable to form virus particles. Thus, functional disruption ofthe structural proteins will substantially inhibit infectivity ofthe pestivirus replicon. However, because the absence ofthe structural proteins does not appreciably affect replication processes, the pestivirus replicon will maintain its ability to self replicate in the absence ofthe structural proteins. In particular embodiments, an antigen-encoding sequence is inserted into the replicon sequence encoding the structural proteins, thereby functionally disrupting the structural proteins. In more specific embodiments, an antigen-encoding sequence totally or partially replaces replicon sequences that would have otherwise encoded the structural proteins, C, Erns, El, E2, and p7. It is further recognized that all but the N-terminal three amino acids of N¹"⁰ may also be deleted without affecting a pestivirus replicon' s ability to self-replicate. Thus, portions of an Npro sequence may also be deleted or interrupted by an antigen-encoding sequence in some embodiments ofthe pestivirus replicons described herein.

In use, a replicon is introduced into a host cell, where gene expression and hence protein production take place. Because the vector is capable of self-replication, multiple copies ofthe replicon will also be generated. This leads to an exponential increase in the number of replicons in the host cell as well as an exponential increase in the amount of protein that is produced. Introduction of a heterologous coding region (encoding for example, an antigen) into the pestivirus replicon produces a self-replicating RNA that expresses the heterologous protein at a high level in transfected or infected cells. If such a replicon is cytopathic, it eventually kills the transfected or hfected cells. If such a replicon is noncytopathic, it remains for a certain time persistently in the cell. Without being bound by theory, replication of a pestivirus genome is dependent on the proteins encoded by the nonstructural region ofthe genome. Preferably, any modification made to the nonstructural region should not interfere with the functional activity ofthe genes within the nontruc ural region ofthe genome.

Pestivirus replicon-based vectors can be used to express a heterologous protein at high levels, and thus serve as excellent vaccine delivery systems. Variations (such as substitutions, deletions or insertions) can be made in the sequence of the pestivirus replicon while retaining its ability to provide its desired function. In addition to BVDV-based pestivirus replicons, other Pestiviruses, for example CSFV- and BDV-based replicons, also are of use.

In some embodiments, the pestivirus replicon design for transfection into eukaryotic cells includes sequences to promote expression of the heterologous gene of interest, including appropriate transcription initiation, termination, and enhancer sequences; as well as sequences that enhance translation efficiency, such as the Kozak consensus sequence; and an internal ribosomal entry site (IRES) of picornaviruses. Therefore, while the nucleotide sequence may be placed under the control of pestivirus regulatory machinery in the replicon, it may alternatively be controlled by one or more alternate regulatory elements capable of promoting expression Such elements will be well known to those of ordinary skill in the field.

It will be appreciated that the nucleotide sequence inserted into the replicon may encode part or all of any natural or recombinant protein except for the structural protein sequence into which or in place of which the nucleotide sequence is inserted. For example, the nucleotide sequence may encode a single polypeptide sequence or a plurality of sequences linked together in such a way that each of the sequences retains its identity when expressed as an amino acid sequence. Where the nucleotide sequence encodes a plurality of peptides, the peptides are linked together in such a way that each retains its identity when expressed. Such polypeptides may be produced as a fiision protein or engineered in such a manner to result in separate polypeptide or peptide sequences.

Where the vector is used to deliver nucleotide sequences to a host cell to enable host cell expression of immunogenic polypeptides, the nucleotide sequence may encodeone or more immunogenic polypeptides in association with a range of epitopes which contribute toT cell activity. In such circumstances the heterologous nucleotide sequence preferably encodes epitopes capable of eliciting either a helper T cell response or a cytotoxic T cell (CTL) response or both. The replicon described herein may also be engineered to express multiple nucleotide sequences allowing co-expression of several proteins such as a plurality of antigens In some embodiments, the replicon further expresses cytokines or other immunomodulators to enhance the generation of an immune response.

By way of example, the nucleotide sequence may include the nucleic acid sequence of one or more ofthe following: Hepatitis C virus antigenHCV core, HCV El, HCV E2, HCV p7, HCV NS2, HCV NS3, HCV NS4, HCV NS5, Her-2/neu, α-fetoprotein, human epithelial cell mucin, the Ha-ras oncogene product, p53, carcino-embryonic antigen, the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-AlMart- 1, gpIOO, EBV-LNT 1, EBV-LNT 2, HPV-F4, HPV-F 6, HPV-F 7, prostatic serum antigen, C017-1A, GA733, gp72, p53, the ras oncogene product, BPV E7, amelanoma ganglioside, RSV F, RSV N, RSV M2, RSVG, pl8, p24, p33, p39, p55, gp36, gp41, gpl20, ESAT-6, MPT63, MPT64, MPT83, Antigen 85B, Antigen 85A, PstS-1, PstS-2 or PstS-3, MTB41, hsp60, circumsporozoite protein (CSP), thrombospondin-related adhesive protein (TRAP), sporozoite and liver-stage antigen (SALSA), sporozoite threonine- and asparagine-rich protein (STARP), merozoite surface protein (MSP 1, -2, -3, -4, -5, erythrocyte- binding antigen (EBA)-175, apical membrane antigen (AMA-1, rhoptry-associated protein (RAP)-l and -2, acidic-basic repeat antigen (ABRA), ring erythrocyte surface antigen (RESA), serine-rich protein (SERP), erythrocyte membrane protein (EMP)-1, -2 and -3, Glutamate-rich protein (GLURP), Glycosilphopatidylinositol (GPI), Ps25, Ps28, Ps48/45 or Ps230. In particular embodiments, the nucleotide sequence encodes a sequence having at least 70%, 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 3, a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: 3, or the amino acid sequence set forth as SEQ ID NO: 3. The present disclosure also provides stable cell lines capable of persistently producing replicon RNAs. To prepare such cell lines, the described vectors are constructed in selectable form by inserting a selectable marker gene, for example genes mediating resistance to G418 (NEO) or hygromycin (HYG) in place of a structural gene, or in another location. Useful host cell lines include any eukaryotic cell lines that can be immortalized, for example, are viable for multiple passages, (for example, greater than 50 generations), without significant reduction in growth rate or protein production. Useful cell lines also are easy to transfect, are capable of stably maintaining foreign RNA with an unarranged sequence, and have the necessary cellular components for efficient transcription, translation, post-translation modification, and secretion ofthe protein. Particularly useful cell lines include those having simple media component requirements, and which can be adapted for suspension culturing. In some embodiments, useful cell lines are mammalian cell lines that can be adapted to growth in low serum or serum-free medium. Representative host cell lines include BHK (baby hamster kidney), VERO, C6-36@ COS. CHO (Chinese hamster ovary), myeloma, HeLa, fibroblast, embryonic and various tissue cells, for example, kidney, liver, lung and the like. In some embodiments, a cell line is selected from BHK21 (hamster), SK6 (swine), VERO (monkey), L292 (mouse), HeLa (human), HEK (human), 2ffGH cells, HepG2, and Huh-7 (human). Useful cells can be obtained from the American Type Culture Collection (ATCC), Manassas, VA.

With respect to the transfection process used in the practice ofthe disclosure, all means for introducing nucleic acids into a cell are contemplated including, without limitation, CaPQ co-precipitation, electroporation, DEAE-dextran mediated uptake, protoplast fusion, microinjection, and lipofusion.

The present disclosure also provides virus like particles containing pestivirus replicons and a method for producing such particles. It will be appreciated by those skilled in the art that virus like particles that contain pestivirus derived replicons can be used to deliver any nucleotide sequence to a cell. Further, the replicons maybe of either DNA or RNA in structure. One particular use for such particles is to deliver nucleotide sequences coding for polypeptides that stimulate an immune response. Such particles may be employed as a therapeutic or in circumstances where the nucleotide sequence encodes peptides that are capable of eliciting a protective immune response so that they may be used as a vaccine. Another particular use is for transfecting an antigen presenting cell, such as a dendritic cell (DC). Such transfected DC are used to induce cross-priming, as described herein.

Because the genomic organization of most Pestiviruses, for example BDV and CSFV, is identical to that of BVDV, pestivirus replicons are derived from other Pestiviruses in the same manner used for the BVDV replicons described herein. In addition, the pestivirus replicons ofthe present disclosure can be altered by one or more nucleotides without changing the essential function ofthe replicons. For example, one or more nucleotides can be added, deleted, or changed within the 5 ' or 3 ' untranslated region of each replicon described herein. Nucleotides can be also exchanged in the ORF region of each replicon described herein.

There are numerous examples of replicons that can be used. Examples of pestivirus replicons lacking at least part ofthe coding sequence ofthe El or C protein are provided in WO 2004/016794. Other examples of useful non-infective (or reduced infectivity) pestivirus replicons are described in U.S. Pat. App. Nos.20020106641 and 20020086033.

Replicons obtained or derived from any Pestivirus species or subtype are contemplated. Pestivirus subtypes include Bovine viral diarrhea virus genotype 2 (BVDV-2), Pestivirus type 1 , which includes Bovine viral diarrhea virus isolates and strains, Pestivirus typel isolates, and

Pestivirus type 1 strain R2727, Pestivirus type 2, which includes Classical swine fever virus and Hog cholera virus, Pestivirus type 3, and unclassified pestiviruses. Bovine viral diarrhea virus genotype 2 (BVDV-2) includes but is not limited to Bovine viral diarrhea virus-2 isolate 230/98-K1 (Gi-4), Bovine viral diarrhea virus-2 isolate 230/98-K2 (Gi-5), Bovine viral diarrhea virus-2 isolate 230/98-K3 (Gi-6), Bovine viral diarrhea virus-2 isolate Giessen- 3, and Bovine viral diarrhea virus-2 isolate SCP. Pestivirus type 1 includes, but is not limited to Bovine viral diarrhea virus isolates and strains, for example Bovine viral diarrhea virus (isolate NADL), Bovine viral diarrhea virus (strain SD-1), Bovine viral diarrhea virus 1 77, Bovine viral diarrhea virus 104/98, Bovine viral diarrhea virus 1041/01, Bovine viral diarrhea virus 107/01, Bovine viral diarrhea virus 10846/91, Bovine viral diarrhea virus 1103/88, Bovine viral diarrhea virus 11207/98, Bovine viral diarrhea virus 1248/01, Bovine viral diarrhea virus 125 85, Bovine viral diarrhea virus 128/88, Bovine viral diarrhea virus 133/02, Bovine viral diarrhea virus 1372/01, Bovine viral diarrhea virus 16484/93, Bovine viral diarrhea virus 1891/99, Bovine viral diarrhea virus 1946/01, Bovine viral diarrhea virus 2032/01, Bovine viral diarrhea virus 2204/82, Bovine viral diarrhea virus 22146/81, Bovine viral diarrhea virus 2218/01, Bovine viral diarrhea virus 228/02, Bovine viral diarrhea virus 2318/01, Bovine viral diarrhea virus 2343/01, Bovine viral diarrhea virus 2430 95, Bovine viral diarrhea virus 25284, Bovine viral diarrhea virus 2555/01, Bovine viral diarrhea virus 2586X 99, Bovine viral diarrhea virus 2703D 99, Bovine viral diarrhea virus 2708/01, Bovine viral diarrhea virus 2750A 99, Bovine viral diarrhea virus 2900/83, Bovine viral diarrhea virus 3114 93, Bovine viral diarrhea virus 3251/01, Bovine viral diarrhea virus 3310/01, Bovine viral diarrhea virus 3336/00, Bovine viral diarrhea virus 3340/01, Bovine viral diarrhea virus 3417/00, Bovine viral diarrhea virus 3425/01,

Bovine viral diarrhea virus 3478/00, Bovine viral diarrhea virus 3499/00, Bovine viral diarrhea virus 3596/86, Bovine viral diarrhea virus 368/02, Bovine viral diarrhea virus 371 89, Bovine viral diarrhea virus 383 76, Bovine viral diarrhea virus 4050/00, Bovine viral diarrhea virus 4071/00, Bovine viral diarrhea virus 4092/00, Bovine viral diarrhea virus 4163/00, Bovine viral diarrhea virus 4283/00, Bovine viral diarrhea virus 4325/01, Bovine viral diarrhea virus 438/02, Bovine viral diarrhea virus 4382/01, Bovine viral diarrhea virus 4629/01, Bovine viral diarrhea virus 4771 94, Bovine viral diarrhea virus 4796 94, Bovine viral diarrhea virus 4898 94, Bovine viral diarrhea virus 4998/89, Bovine viral diarrhea virus 5284/00, Bovine viral diarrhea virus 551/02, Bovine viral diarrhea virus 5551/84, Bovine viral diarrhea virus 561/01, Bovine viral diarrhea virus 720/02, Bovine viral diarrhea virus 80/1 cp 82, Bovine viral diarrhea virus 80/1 ncp 82, Bovine viral diarrhea virus 8087 99, Bovine viral diarrhea virus 819 85, Bovine viral diarrhea virus 832/01, Bovine viral diarrhea virus 9466/91, Bovine viral diarrhea virus 985 84, Bovine viral diarrhea virus B551 98, Bovine viral diarrhea virus bo2340/01, Bovine viral diarrhea virus H686 98, Bovine viral diarrhea virus H851 98, Bovine viral diarrhea virus K869 98, Bovine viral diarrhea virus L1000 98, Bovine viral diarrhea virus L322 98, Bovine viral diarrhea virus Lamspringe/735, Bovine viral diarrhea virus Lamspringe/738, Bovine viral diarrhea virus Ln 68, Bovine viral diarrhea virus strain 104/98, Bovine viral diarrhea virus strain 10846/91, Bovine viral diarrhea virus strain 1103/88, Bovine viral diarrhea virus strain 11158/98, Bovine viral diarrhea virus strain 11202/98, Bovine viral diarrhea virus strain 11203/98, Bovine viral diarrhea virus strain 11205/98, Bovine viral diarrhea virus strain 11207/98, Bovine viral diarrhea virus strain 11253/98, Bovine viral diarrhea virus strain 11255/98, Bovine viral diarrhea virus strain 11336/98, Bovine viral diarrhea virus strain 11337/98, Bovine viral diarrhea virus strain 11357/98, Bovine viral diarrhea virus strain 11358/98, Bovine viral diarrhea virus strain 11359/98, Bovine viral diarrhea virus strain 11360/98, Bovine viral diarrhea virus strain 11361/98, Bovine viral diarrhea virus strain 11362/98, Bovine viral diarrhea virus strain 128/88, Bovine viral diarrhea virus strain 151/95, Bovine viral diarrhea virus strain 152/95, Bovine viral diarrhea virus strain 16484/93, Bovine viral diarrhea virus strain 17004/85, Bovine viral diarrhea virus strain 1891/99, Bovine viral diarrhea virus strain 2037/93, Bovine viral diarrhea virus strain 2204/82, Bovine viral diarrhea virus strain 22146/81, Bovine viral diarrhea virus strain 223/00, Bovine viral diarrhea virus strain 2324/94,

Bovine viral diarrhea virus strain 2336/85, Bovine viral diarrhea virus strain 2543/87, Bovine viral diarrhea virus strain 2583/86, Bovine viral diarrhea virus strain 2823/87, Bovine viral diarrhea virus strain 2900/83, Bovine viral diarrhea virus strain 3142, Bovine viral diarrhea virus strain 3185/83, Bovine viral diarrhea virus strain 3187/83, Bovine viral diarrhea virus strain 3206/83, Bovine viral diarrhea virus strain 3208/83, Bovine viral diarrhea virus strain 321/80, Bovine viral diarrhea virus strain 3596/86, Bovine viral diarrhea virus strain 3833/84, Bovine viral diarrhea virus strain 3887, Bovine viral diarrhea virus strain 4315/84, Bovine viral diarrhea virus strain 4979, Bovine viral diarrhea virus strain 4998/89, Bovine viral diarrhea virus strain 5.19006, Bovineviral diarrhea virus strain 5.19516, Bovine viral diarrhea virus strain 5059/89, Bovine viral diarrhea virus strain 5190/89, Bovine viral diarrhea virus strain 5551/84, Bovine viral diarrhea virus strain 5586/84, Bovine viral diarrhea virus strain 5769/84, Bovine viral diarrhea virus strain 5862/94, Bovine viral diarrhea virus strain 60.875, Bovine viral diarrhea virus strain 638/87, Bovine viral diarrhea virus strain 6384, Bovine viral diarrhea virus strain 6533/90, Bovine viral diarrhea virus strain 710/80, Bovine viral diarrhea virus strain 712/80, Bovine viral diarrhea virus strain 715/80, Bovine viral diarrhea virus strain 7331/92, Bovine viral diarrhea virus strain 7417/90, Bovine viral diarrhea virus strain 763+, Bovine viral diarrhea virus strain 763-, Bovine viral diarrhea virus strain 7923, Bovine viral diarrhea virus strain 799+, Bovine viral diarrhea virus strain 799-, Bovine viral diarrhea virus strain 829+, Bovine viral diarrhea virus strain 829-, Bovine viral diarrhea virus strain 839+, Bovine viral diarrhea virus strain 839-, Bovine viral diarrhea virus strain 9368/92, Bovine viral diarrhea virus strain 9466/91, Bovine viral diarrhea virus strain 97/123, Bovine viral diarrhea virus strain 97/360, Bovine viral diarrhea virus strain 97/730, Bovine viral diarrhea virus strain Bega, Bovine viral diarrhea virus strain Bovax20, Bovine viral diarrhea virus strain Braidwood, Bovine viral diarrhea virus strain Changchun 184, Bovine viral diarrhea virus strain Cumulus, Bovine viral diarrhea virus strainD, Bovine viral diarrhea virus strain H, Bovine viral diarrhea virus strain L1305, Bovine viral diarrhea virus strain L4262, Bovine viral diarrhea virus strain Lamspringe/735, Bovine viral diarrhea virus strain Lamspringe/738, Bovine viral diarrhea virus strain Oregon C24V, Bovine viral diarrhea virus strain Rit 4350, Bovine viral diarrhea virus strain Trangie Y546, Bovine viral diarrhea virus strain V071094, Bovine viral diarrhea virus strain VI 10794, Bovine viral diarrhea virus strain VI 11295, Bovine viral diarrhea virus strain V130995b, Bovine viral diarrhea virus sfrain V190695, Bovine viral diarrhea virus strain VEDEVAC, Bovine viral diarrhea virus sfrain Yak, Bovine viral diarrhea virus strain ZM-95, Bovine viral diarrhea virus type 2, and Bovine viral diarrhea virus- 1.

Pestivirus type 1 further includes Pestivirus type 1 isolates, for example Pestivirus isolate 17P, Pestivirus isolate 1R, Pestivirus isolate 1R93, Pestivirus isolate 25H, Pestivirus isolate 2B,

Pestivirus isolate 318, Pestivirus isolate 34B, Pestivirus isolate 354, Pestivirus isolate 3P, Pestivirus isolate 4H, Pestivirus isolate 65.2, Pestivirus isolate 66.1, Pestivirus isolate 66.3, Pestivirus isolate 66.5, Pestivirus isolate 66.6, Pestivirus isolate 68.88, Pestivirus isolate 76865, Pestivirus isolate 86713, Pestivirus isolate S21, and Pestivirus isolate TFB. Pestivirus type 1 also includes Pestivirus type 1 sfrain R2727.

Pestivirus type 2 includes, but is not limited to Classical swine fever virus, for example Classical swine fever virus 39, Classical swine fever virus isolate Schweinfurt, Classical Swine Fever virus strain 5440/99, and Classical swine fever virus strain Eystrup, in addition to Hog cholera virus, for example Hog cholera virus 'Switzerland 1/93', Hog cholera virus 'Switzerland 2/93', Hog cholera virus 'Switzerland 3/93/1', Hog cholera virus 'Switzerland 3/93/2', Hog cholera virus 'Switzerland 4/93', Hog cholera virus (strain Alfort), Hog cholera virus (strain Brescia), Hog cholera virus sfrain 'ATCC VR-531', Hog cholera virus strain 'Chinese vaccine, Wuhan', Hog cholera virus strain 'Jen Sal', Hog cholera virus strain 'Russian LK vaccine', Hog cholera virus strain 'Switz. IV/93', Hog cholera virus strain 'VRI 4425', Hog cholera virus strain Alfort/M, Hog cholera virus strain Cellpest, Hog cholera virus sfrain Duvaxin, Hog cholera virus strain EVIlOO, Hog cholera virus strain GPB,

Hog cholera virus strain Kanagawa, Hog cholera virus strain Norden, Hog cholera virus strain Osaka, Hog cholera virus strain Painswhin, Hog cholera virus strain Pestipan, Hog cholera virus strain Porcivac, Hog cholera virus strain PS Porco, Hog cholera virus strain Riems, Hog cholera virus sfrain Rovac, Hog cholera virus strain Steiermark, Hog cholera virus strain Tipest, Hog cholera virus strain TVM-1, Hog cholera virus strain VRI4061, Hog cholera virus sfrain Zoelen.

Pestivirus type 3 includes, but is not limited to Border disease virus sfrain 135661, Border disease virus strain 137/4, Border disease virus sfrain 170337, Border disease virus strain 8320-22NZ, Border disease virus strain 8320-31 NZ, Border disease virus strain A1263/2, Border disease virus strain A 1870, Border disease virus strain A841/1, Border disease virus sfrain BD31, Border disease virus strain Cumnock, Border disease virus strain D1586/2, Border disease virus strain Frijters, Border disease virus sfrain G1305, Border disease virus strain G2048, Border disease virus sfrain JH2816, Border disease virus strain K1729/3, Border disease virus strain L83-84, Border disease virus strain L991, Border disease virus strain Moredun, Border disease virus strain Moredun cp, Border disease virus strain Moredun ncp, Border disease virus strain Q1488/1, Border disease virus strain Q1488/6, Border disease virus strain Q1673/2, Border disease virus strain T1789/1, Border disease virus sfrain T1802/1, Border disease virus strain V-TOB, Border disease virus strain V1414, Border disease virus strain V2377/12, Border disease virus strain V2536/2, Border disease virus sfrain V3196/1, and Border disease virus sfrain X818. Unclassified Pestivirus subtypes include, but are not limited to, Border disease virus strain 2112/99, Border disease virus strain 79248/01, Border disease virus strain 80582/01, Border disease virus sfrain 87/6, Border disease virus strain 87877/01, Border disease virus strain 90/8320/31, Border disease virus sfrain 91/5809, Border disease virus strain A1263/1, Border disease virus strain CB2, Border disease virus strain CB5, Border disease virus sfrain Idaho207, Border disease virus strain Idaho209, Border disease virus strain Idaho211, Border disease virus strain R1292/01, Bovine viral diarrhea virus-1 strain R2727, Ovine pestivirus, Pestivirus Giraffe- 1, Pestivirus giraffe-1 H138, Pestivirus isolate 97-360, Pestivirus isolate Hay 87/2210, Pestivirus Reindeer- 1, Pestivirus reindeer- 1 V60-Krefeld, Porcine pestivirus, Pronghorn antelope pestivirus, Pestivirus sp., Pestivirus sp. Bison- 1, and Pestivirus sp. Reindeer- 1.

D. Cross-priming

Cross-priming is a specific capacity of antigen presenting cells (APC) that involves the acquisition of exogenous antigens from apoptotic or dead cells in the periphery and the migration to secondary lymphoid organs, where APC undergo apoptosis and are taken up by secondary APC.

These secondary APC reprocess the antigen and present it to T cells. For vaccination purposes, part ofthe pathway can be bypassed by directly introducing (for example by transfection or injection) an antigen to an antigen presenting cell (APC), for example a dendritic cell (DC), and allowing the cell to migrate to the spleen or lymph node. Secondary APCs phagocytose dyingprimary APC and present the antigen to T cells (for review, see Zhou et al, J. Immunother., 25(4): 289-303, 2002). Cross-priming is particularly useful when immuiity is based on T cell rather than on antibody responses (for example, for hepatitis C virus (HCV), respiratory syncytial virus (RSV), and human immunodeficiency virus (HIV), plasmodium falciparum and mycobacterium tuberculosis) and for immune responses against tumors.

E. Pharmaceutical Compositions

The pestivirus replicons and dendritic cells or other autologous cells fransfected with pestivirus replicons described herein may be formulated in a variety of ways depending on the desired route of administration to the subject. Therefore, the disclosure includes within its scope pharmaceutical compositions comprising at least one pestivirus replicon or dendritic cell transfected with a pestivirus replicon formulated for use inhuman or veterinary medicine.

Pharmaceutical compositions that include at least one pestivirus replicon or dendritic cell or other autologous cells transfected with apestivirus replicon as described herein as an active ingredient may be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen. The pharmaceutically acceptable carriers and excipients useful in this disclosure are conventional. For instance, parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients that can be included are, for instance, proteins, such as human serum albumh or plasma preparations. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Optionally, the pharmaceutical composition includes one or more adjuvants and/or cytokines and/or chemokines and or nucleotide sequences encoding cytokines and chemokines. Without being bound by theory, an adjuvant enhances the immunogenicity by helping to retain the antigen in the body and to promote its uptake by antigen-presenting cells. Adjuvants may include bacteria or bacterial components, and may include but are not limited to aluminum hydroxide, CpGcontaining nucleotide sequences, ISCOMS (immune stimulatory complexes, which are small micelles of detergent which contain the antigen, fuse with host cells and antigenpresenting cells and allow the antigen to enter the cytosol ofthe host cells and antigen presenting cells).

The pharmaceutical compositions that include thepestivirus replicon or dendritic cell or other autologous cell transfected with thepestivirus replicon, in some embodiments, will be formulated in unit dosage form, suitable for individual administration of precise dosages. The amount of active compound(s) administered will be dependent on the subject being treated, the severity ofthe affliction, and the manner of administration, and is best left to the judgment ofthe prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity ofthe active component(s) in amounts effective to achieve the desired effect in the subject being treated. In some embodiments, thepestivirus replicon or pestivirus replicon-encoding cDNA is administered directly to a subject.

F. Therapeutic Uses

A method is also disclosed for inducing a Tcell response in a subject. The method includes transfecting a dendritic cell or other autologous cell with apestivirus replicon (or a DNA molecule encoding the replicon) and administering the dendritic cell to ths subject, thereby inducing a T cell response in the subject. In order to induce the T cell response, a therapeutically effective amount of the pestivirus replicon DNA or RNA, or dendritic cells, or other autologous cells (for example fibroblasts) fransfected with the pestivirus replicon DNA or RNA (see above) is administered to the subject. In one embodiment, the adminisfration of thepestivirus replicon or dendritic cells transfected with a pestivirus replicon is systemic. Intravenous, infra-arterial, subcutaneous, infra-peritoneal, intralymphatic and infra-muscular administration is contemplated.

Effective doses of pestivirus replicon RNA or pestivirus replicon encoding cDNA, or dendritic cells transfected with a pestivirus replicon RNA or pestivirus replicon encoding cDNA can be readily determined by those who are skilled in the art and will depend, of course, upon the exact condition being treated or prevented, and by the particular therapy being employed. The cells can be transplanted to a desired location, or can be administered intravenously. Other agents, such as immunostimulants or immune modulators can be administered in conjunction with replicons or immune cells.

The composition can be administered to persons at risk of developing the disease being treated, inhibited or prevented, to provoke a protective T cell-mediated immune response. For example, the composition would be administered to a person at risk ofdeveloping HIV or HCV infection, such as persons engaging in high risk sexual behaviors or intravenous drug use. The composition could also be administered to subjects who are at risk ofdeveloping RSV infection (such as young children), tuberculosis, malaria or tumors (such as someone with a genetic or environmental risk for a particular neoplasm). Alternatively, the composition is administered to someone who already has the infection or tumor, for the purpose of stimulating aT cell-mediated immune response that will improve or cure the condition in the subject.

F. Kits

Further embodiments ofthe disclosure include kits useful for introducing thepestivirus replicon RNA or pestivirus replicon encoding cDNA into APCs, for example for transfecting dendritic cells with a pestivirus replicon. For example, a kit useful for transfecting dendritic cells with the pestivirus replicon would include an appropriate amount ofpestivirus replicon, as well as, optionally, any reagents useful for carrying out the frarsfection. Other embodiments further include instructions for using the kit, and/or frozen aliquots of dendritic cells. Thus, in one embodiment, a kit is provided including a container ofpestivirus replicon (a sufficient amount for either a single use ormultiple uses), and instructions for introducing the pestivirus replicon into APCs, such as dendritic cells, for example by transfection. The instructions can be in written form, or can be provided in an electronic format, such as on a diskette or a CD ROM. Instructions can also be provided in the form of a video cassette. Further embodiments ofthe disclosure include kits useful for inducing a Tcell response in a subject. For example, a kit useful for inducing a T cell response in a subject would inclide an appropriate amount of dendritic cells transfected with apestivirus replicon, as well as, optionally, any instructions for using the kit.

The subject matter ofthe present disclosure is further illustrated by the following non- limiting Examples.

EXAMPLES Example 1 CROSS-PRIMING OF HEPATITIS C VIRUS SPECIFIC CD8+ T CELLS IN MICE BY IMMUNIZATION WITH SELF-REPLICATING RNA TRANSFECTED DENDRITIC CELLS

At present, a protective HCV vaccine is not available and induction of strong Tcell responses by immunization is difficult to achieve. The methods disclosed herein are designed to achieve more effective T cell responses by cross-priming with dendritic cells (DCs) containing self replicating RNA to induce strong, HCV-specific cellular immune responses.

Specifically, an autonomous subgenomic pestivirus RNA replicon was used to amplify HCV NS3 in DCs. One characteristic of this vector is its ability to replicate in fransfected cells, which in turn leads to enhanced levels of production, processing and presentation of encoded antigens.

Moreover, the availability of cytopathic and noncytopathic forms ofthe same replicon enabled the demonstration ofthe immunologic implications of DC apoptosis. Murine DC2.4 cells were transfected with cytopathic and noncytopathic HCV NS3 replicons, respectively. HCV NS3 expression was detectable in more than 95% ofthe fransfected cells by immunofluorescence. The time kinetics of apoptosis induction was monitored by FACS using annexin V and propidium iodide staining. In contrast to the noncytopathic replicons which did not kill the dendritic cells, the cytopathic replicon led to the apoptosis ofthe DCs twelve hours after transfection. The cytopathic replicon produces a higher level of antigen expression and the induction of cell death for cross- priming. The noncytopathic replicon ensures longer expression of antigen in living cells, which, without being bound by theory, may be important to maintain immune responses.

DC2.4 cells transfected with the cytopathic replicon were then used to immunize HLA-A2+ transgenic C57BL/6 mice subcutaneously. The magnitude and quality ofthe HCVNS3 specific CD4+ and CD8+ T cell response were characterized as regards to proliferation, Interferon-γ (IFN-γ) production, and cytotoxicity. IFN-γ producing, proliferating CD4+ T cells and IFN-γ producing cytotoxic CD8+ T cells were induced by a single subcutaneous vaccination. Cross-presentation was confirmed when T cells, primed by injection of H2b+ DCs into the H-2b+ HLA-A2+ mice, were purified and tested against HLA-A2+ antigen-presenting cells in vitro. Upon challenge ofthe mice with recombinant HCV-NS3 -expressing vaccinia virus, vaccinia titers were 4 log 10 lower in vaccinated mice than in non- vaccinated mice, demonstrating the vivo function ofthe vaccine- induced T cells.

H2-b is the MHC allele expressed by the dendritic cells. Both the dendritic cell line and the mice are H-2b+, therefore the injected dendritic cells can directly prime T cells. Because only the mice and not the injected dendritic cells are HLA-A2+, HLA-A2 resfricted T cell responses cannot be induced by direct priming but must be induced by cross-priming. Thus, cross-priming of T cells with DCs that are transfected with self replicating RNA provides a powerful vaccination approach for inducing a Hepatitis C Virus-specific T cell response.

A. Materials and Methods

Mice C57BL/6 (H-2b haplotype) mice were purchased from the Jackson Laboratory. AAD transgenic mice expressing a hybrid class I molecule consisting of theαl and α2 domains from HLA-A2.1 and the α3 domain from H-2D^don a C57BL/6 background were provided by Dr. E. Engelhard (University of Virginia, Charlottesville, VA). All mice were bred and maintained in a pathogen-free environment at the National Institutes of Health (NIH).

Cell lines and media DC2.4 cells, which were previously characterized as an immature murine DC line (H2^b haplotype) were provided by Dr. K.L. Rock (Department of Pathology, University of Massachusetts Medical School, Worchester, MA) and were grown at 37°C and 5% CQj in RPMI 1640 (Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L- glutamine, 100 μM nonessential amino acids , 50 μM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin. CIR-AAD cells were maintained in the same medium with 400 μg/ml G418 (Sigma-Aldrich, St. Louis, MO). CIR-AAD are transfectants ofthe HLA-A,B-negative human B lymphoblastoid cell line C1R with AAD (a hybrid MHC class I molecule consisting of he αl+ α2 domains of HLA-A2.1 and the 3 domain of H-2D^d) and were obtained from Dr. J. Berzofsky, National Cancer Institute, National Institutes of Health. 143TK- cells, a human osteosarcoma cell line (ATCC, Rockville, MD), were grown in DMEM (Gibco) supplemented with 10% heat-inactivated

FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. EL4 murine thymoma cells (C57BL/6 origin, H-2^b haplotype; ATCC) were maintained in the same medium with 50 μM 2 mercaptoethanol.

Synthetic peptides and proteins

Recombinant HCV NS3 protein (amino acid residues 1192-1457 of HCV-1, genotype la, accession number M62321) was provided by Dr. Michael Houghton (Chiron Corporation, Emeryville, CA). Pentadecamer peptides, overlapping by 10 amino acids each, were synthesized at Mimotopes (Clayton, Australia) to span the complete amino acid sequence ofthe HCV NS3 protein. Peptides were resuspended at 20 mg/ml in dimethyl sulfoxide (DMSO) and further diluted with PBS into three pools designated as pool I (corresponding to aa residues 1021-1240 of HCV NS3), pool II (corresponding to aa residues 1231-1443 of HCV NS3), and pool III (corresponding to aa residues 1434-1660 of HCV NS3).

Recombinant Vaccinia Virus for re-challenge experiments

The NS3-encoding recombinant Vaccinia virus rVV-NS3-4B used for challenge experiments has previously been described (Wedemeyeret al, Gastroenterology, 121: 1158-1166, 2001; Bartenschlager et al, J. Virol, 68: 5045, 1994), and was derived from the WR strain and expressed amino acid 1007-1890 ofthe polyprotein.

Construction of recombinant plasmids encoding self replicating HCV NS3 RNA

The original constructs used for the generation of cytopathic andnoncytopathic BVDV replicons are described by Tautz et al. (J. Virol, 12>: 9422-9432, 1999). For the noncytopathic constructs, "BVDV Bi-ΔN^prononcp, GUS", the heterologous gene is GUS (β-glucoronidase). For the cytopathic constructs, "BVDV Bi-ΔN^procp, GUS", the heterologous gene is also GUS (glucoronidase). The relative positions of restriction sites used for the cloning procedures explained below are indicated (see, also FIG. 1 A). Both cDNAs were first modified by introduction (Quickchange procedure) of an additional

Srfl site at the immediate 3'-end ofthe RNA coding region. This was done by exchanging G at pos. 12290 by C (numbers refer to the original BVDV CP7 construct of Meyers et al, J. Virol, 70: 8606-8613, 1996). Linearization with Srfl allowed the generation of BVDV RNAs containing the authentic 3 '-terminus by run-off transcription. This step was necessary, because Srfl restriction sites are rare in foreign genes in confrast to Smal restriction sites, which was originally used to linearize the BVDV cDNAs.

The construct "BVDV Bi-ΔN^procp, GUS, Δla" was generated via deletion (Quickchange procedure) ofthe hairpin la motif at the immediate 5'-terminus ofthe 5'UTR of "BVDV Bi-ΔN^procp, GUS". This mutation (deletion of nucleotides 1-32 of BVDV CP7) was previously described to inhibit the replication ofBVDV RNA (Yu et al, J. Virol., 74: 5825-5835, 2000). The resulting viral transcript was used as a negative control.

An Fspl site was introduced into the coding region ofthe BVDV CP7 cDNA (Meyers et al, J. Virol, 70: 8606-8613, 1996) by changing tec at pos. 896-898 (5'-portion ofthe core-coding region) into gca (Quickchange). The modified N^pro/core region was amplified by PCR using appropriate oligonucleotide primers and introduced into "BVDV Bi-ΔN^procp, GUS" thus replacing the ΔN^pro region and the 5'-terminal portion ofthe GUS-gene. The modified plasmid was termed "BVDV Bi- cp, N^pr core Fsp".

The PCR product was cloned into the Fspl site and Clal (position 11083 ofBVDV CP7in the NS5B coding region) sites ofBVDV Bi-cp, N^pr7core Fsp". The resulting plasmid was cut with Nhel (upstream ofthe SP6 promoter) and Sail (3'-end ofthe inserted novel gene-product) and the fragment consisting ofthe BVDV5'UTR and the N^pro coding region fused in frame to the gene of interest cloned into "BVDV Bi-ΔN^prononcp, GUS" and "BVDV Bi-ΔN^procp, GUS", which were previously digested with Nhel and Xhol. Xhol, which is compatible to the ligation with a Sail site, cuts upstream ofthe EMCV IRES sequence. The resulting plasmids were designated as'ΕVDV Bi- noncp, HCV NS3" (also called, noncytopathic Repl-HCV_NS3) and "BVDV Bi-cp, HCV NS3" (also called, cytopathic Repl-HCV_NS3) (see, the middle and bottom constructs of FIG. IB).

For the introduction of other foreign genes or antigenic determinants of HCV, HIV, RSV, and bacteria, as well as tumor antigens (max. length 3 kb), a similar strategy is used,for example, cloning PCR products via Fspl and Clal into "Bi-cp, N ^r core Fsp ", followed by completion ofthe constructs via Nhel and Sall/Xho ligation as described. When using alternative pestivirus replicon vectors, a similar strategy can be adapted as well. In vitro RNA preparation

Srfl (Stratagene, La Jolla, CA) was used to linearize the DNA templates ofthe bicistronic cytopathic BVDV HCV NS3 replicon and the bicistronic noncytopathic BVDV HCV NS3 replicon replicon. After purification with MiniElute Reaction Cleanup Kit (Qiagen Inc., Valencia, CA), the linearized plasmids were in vitro transcribed with SP6 RNA polymerase (Roche Diagnostics, Indianapolis, IN) in a standard reaction. After removal of DNA by digestion with RNase-free DNase I (Roche) and purification of transcribed RNA with the Rneasy Mini Kit (Qiagen), RNA concentration was determined at OD 260 mn. The integrity and quantity of RNA transcripts wee further checked by denaturing gel electrophoresis. RNA aliquots of 5 μg, suitable for transfection of dendritic cells were stored at -70°C.

RNA transfection of dendritic cells

Subconfluent monolayers of DC2.4 cells were harvested from the culture after a five-minute incubation with PBS / 0.02% EDTA. After washing twice in PBS, 5 x 10⁶ cells were pelletted and resuspended in Cytomix containing 1.25% DMSO. This suspension was mixed with 5 μg RNA, transferred to a 2 mm-gap cuvette, and elecfroshocked twice with a Gene Pulser apparatus (Bio-Rad, Hercules, CA) using a voltage pulse of 300 V in combination with a capacitance of 150 μF. After incubation on ice for ten minutes, the cells were seeded in fresh complete medium and incubated at 37°C and 5% C0₂. Transfection efficiency was evaluated by indirect immunofluorescence (UF) microscopy and fluorescence-activated cell sorting (FACS). In brief, transfected cells were either maintained as monolayers in chamber slides (Nunc Inc., Naperville, IL) or harvested into FACS tubes, fixed with 4% (wt/vol) paraformaldehyde, permeabilized with 0.1 % (wt/vol) saponin, and incubated with the murine monoclonal anti-HCV NS3 antibody (1B6) in PBS containing 3% (wt/vol) bovine serum albumin (BSA) and 0.1% (wt/vol) saponin. Bound primary antibody was stained with a fluorescein isothiocyanate (FITC)-conjugated goat F(ab')₂ fragment to mouse immunoglobulin G (IgG) (Caltag Laboratories, Burlingame, CA). Slides were mounted and examined with a Zeiss Axioskop2 microscope (Carl Zeiss Inc., Thornwood, NY) equipped with an epifluorescence attachment. Images were processed with Adobe Photoshop 3.0.5. (Adobe System, San Jose, CA). For flow cytometry, cell suspensions were fixed in 1% PFA in PBS and analyzed on a FACSCalibur flow cjtometer (Becton Dickinson, San Jose, CA) with CellQuest (Becton Dickinson) and Flow Jo (Flow Jo, San Carlos, California) software. At least 10,000 events were acquired using forward and side scatter gating to exclude cell debris.

HCV NS3 protein expression in DC2.4 cells was further confirmed by Western blot analysis. For this assay, transfected cells were lysed in a buffer containing 150 mM NaCl, 1 % Nonidet P40, 0.5% deoxicolate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-Cl, pH 8.0, 1 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml aprotin, 1 μg/ml, leupeptin, and 1 μg/ml pepstatin A. Immunoblotting was performed after SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according to a standard protocol. Briefly, proteins were electrotransferred onto Immohilon-P membranes (Millipore, Bedford, MA) and membranes were blocked with PBS containing 3% nonfat dry milk and 3% BSA for 2 h at 20°C. Blots were subsequently incubated for one hour with 1B6 anti HCV NS3 MoAb (1/500 dilution). Washing steps were performed with TBS containing 0.1% Tween 20. Horseradish peroxidase-labeled sheep anti-mouse Ig was used at a dilution of 1/1000 for detection of bound primary antibody by enhanced chemiluminescence (ECL; Amersham, Arlington Height, IL).

Cell death analysis To monitor replicon-induced apoptosis of transfected DC2.4 cells, annexin V/propidium iodide double staining followed by FACS analysis was performed. This assay allows the quantitation of different cell populations: living cells that were not labeled by ether annexin V or propidium iodide, necrotic cells that were stained by both agents, and apoptotic cells that were labeled only by annexin V. DC2.4 cells were harvested after different posttransfection periods, washed twice with PBS, and subjected to the assay according to the manufacturer's protocol (Annexin V-FITC Apoptosis Detection Kit I, BD PharMingen, San Diego, CA).

Adoptive immunization

DC2.4 were harvested 12 h after transfection with cytopathic replicon RNA, washed twice and resuspended in PBS. 5 x 10⁵ cells / 100 μl PBS were injected subcutaneously at the base of tail.

Cell and tissue preparations from immunized mice

Spleens and lymph nodes were collected from immunized and euthanized mice,perfused with 400 μg/ml of Liberase CI solution (Roche Diagnostics) in PBS , incubated for 30 min at 37°C, and forced through a cell strainer (Falcon, Franklin Lakes, NJ) to obtain single cell suspensions.

After lysis of red blood cells in ACK lysing buffer cells were washed and resuspended in RPMI 1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

T cells were isolated with anti-CD3 coated magnetic beads (MACS Column Purification System, Miltenyi Biotec (Auburn, CA)), according to the manufacturer's instructions. Recovered cells were washed in RPMI 1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine,

100 U/ml penicillin, and 100 μg/ml streptomycin, counted by trypan blue exclusion and resuspended at concentrations suitable for the various assays.

Ex vivo detection ofHCVNS3peptide-speciflc CD8⁺ T cells: IFN-j ELISPOT Assay

The frequency of peptide-specific, IFN-γ producing CD8⁺ T cells was determined in an ELISPOT assay as described previously (Wedemeyer et al, Gastroenterology 2001; 121: 1158-1166) MultiScreen-HA IP plates (Millipore, Bedford, MA) were coated overnight with 0.5 μg/ml anti mouse IFN-γ antibody (clone R4-6A2; Pharmingen, San Diego, CA) in PBS, blocked for one hour at 25°C with PBS / 1% bovine serum albumin (BSA) (Sigma) and washed three times with PBS. Serial dilutions (5 x 10⁵, 2.5 x 10⁵, 1.25 x 10⁵, and 0.625 x 10⁵) of freshly purified splenic T cells from immunized mice were plated in triplicate into wells containing 10^s irradiated (10,000 rad) EL4 or C IR-AAD cells with each HCV NS3 peptide pool, with a final concenfration of 1 μg/ml per peptide. Negative control wells did not contain antigens, positive control wells contained phytohemagglutinin (Murex Biotech Limited, Dartford, England). All conditions were set up in triplicates. After 30 hours of incubation at 37°C, 5% C0₂, the plates were washed with PBST (PBS containing 0.05% Tween 20) and incubated with 0.5 μg/ml biotinylated IFN-γ antibody (clone XMG1.2; Pharmingen) at 4°C overnight. After washing with PBST, alkaline phosphatase-conjugated streptavidin (1 :2,000)

(DAKO, Glostrup, Denmark) was added to each well. The plates were incubated at room temperature for two hours, washed with PBS, and developed with 100 μl NBT/BCIP (nitroblue tetrazolium-5- bromo-4-chloro-3-indolylphosphate) solution (Bio-Rad, Hercules, CA). The reaction was stopped after 10-20 minutes with distilled water. The resulting spots were counted on a KS ELISPOT Reader (Carl Zeiss Inc., New York, New York) based on size, shape, contrast and density to gate-out speckles and noise caused by spontaneous substrate precipitation and nonspecific antibody binding. The number of peptide-specific IFN-γ-positive spots was obtained by subtracting the mean number of spots in the negative control wells from the mean number of spots in the wells containing peptides.

Detection ofHCV-NS3-specific CDX T cells: Proliferation Assay

A total of 5 x 10⁵ total spleens or lymph node cells or 5 x 10⁵ isolated CD3+ cells were placed in triplicates into 96-well round-bottomed plates in RPMI 1640 (Gibco) supplemented with 10% FBS, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin. 10⁵ autologous irradiated (3,000 rad) splenocytes (as stimulators) were added to the wells containing CD3+ T cells. Cultures were stimulated with 1 μg/ml HCV NS3 protein, buffer control or 1 μg/ml PHA, respectively. After 48 hours of incubation at 37°C and 5% C0₂, 1 μCi tritiated thymidine (ICN, Costa Mesa, CA) was added per well. Cells were harvested 16 hours later with a Packard Filtermate 196 cell harvester and incorporated radioactivity was measured as counts per minute on a Packard beta counter. Results were expressed as stimulation index (^!H-thymidine incorporation in the presence of antigen / incorporation in the absence of antigen).

In vitro expansion and detection of HCV NS3-specific CTLs: ⁵ Cr release assay

A total of 5 x 10⁵ purified CD3+ spleen cells were cultured with lO⁵ irradiated (10,000 rad) EL4 (H2^b) or CIR-AAD (HLA-A2.1) cells in 96-well round-bottomed plates in RPMI 1640 (Gibco) supplemented with 10% FBS, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin with one ofthe HCV NS3 peptide pools at a final concentration of 1 μg/ml per peptide. On day two, 10% Rat-T-stim (Collaborative Biomedical Products, Bedford, MA) was added. On day 7, cytotoxicity was determined in a six hour⁵¹Cr-release assay. Target cells were either EL4 (H2^b) or CIR-AAD (HLA-A2.1) cells, respectively, that had been pulsed with an individual HCV NS3 peptide pool at 1 μg/ml per peptide overnight and then labeled with 100 μCi ⁵¹Cr for one hour at 37°C. After labeling with⁵¹Cr, target cells were washed and plated at a concentration of 3 x 10³ cells per well in a 96-well plate. Effector cells were added at effectoπtarget (E:T) ratios of 120:1, 60:1, 30:1, 15:1 in triplicate wells. After a six hour incubation, 40 μl of supernatant was collected and ⁵¹Cr release was determined in a beta counter (Packard). The percentage of killed cells was calculated by the formula: (experimental release- spontaneous release)/(maximum release- spontaneous release) x 100, in which spontaneous release represented target cells cultured in the absence of effector cells, and maximum release represented target cells lysed with 1% Triton X-100 (Sigma). Spontaneous release was <10% of maximum release in all experiments. A specific cytotoxic activity of >10% was considered positive.

Vaccinia virus challenge and plaque assay Two weeks after immunization with replicon-RNA fransfected or non-transfected DC2.4, respectively, mice were challenged intra-peritoneally with 10⁸ or 10⁷ plaque-forming units (pfus) rVV-NS3-4B. Five days later, mice were euthanized, and the ovaries, the main organs of VV replication, were harvested, homogenized and sonicated. VV titers were determined after plating tenfold dilutions ofthe homogenate on 143TK-cell monolayers (ATCC, Rockville, MD) in 6-well plates and after staining with 0.075 wt/vol% crystal vfalet 48 hours later.

Visualisation of in vivo phagocytosed dendritic cells

Twelve hours after transfection with the cytopathic BVDV HCV NS3 replicon, 5x 10⁵ CFSE-labeled DC2.4 cells were subcutaneously injected at the base ofthe tail of AAD mice. Unlabeled DC2.4 cells and DC2.4 transfected with a replication-incompetent construct were used as controls. Twelve hours after injection, three mice per group were sacrificed and, spleen and lymph node cell suspensions were analysed for the presence of CFSE^1" by immunofluorescence microscopy and FACS. Seven 7 μm cryosections of spleen tissue were fixed with acetone for ten minutes, and then incubated successively with antiHLA-A2.1, PE-conjugated goat F(ab')2 anti-mouse IgG (Immunotech), anti GDI lc, APC-conjugated anti-mouse F4/80 and anti-B220 antibody.

B. Genomic organization ofthe in vitro transcribed bicistronic cytopathic (cp) and noncytopathic (ncp) BVDV HCV NS3 replicon RNA constructs

As shown in FIG. 1, boxes indicate the protein-coding regions; horizontal lines represent the untranslated regions (UTR). The 5'-terminal open reading frame (ORF) consists

region which was fused in such a way to the coding region ofthe HCV NS3 protein that protein expression leads to the autoproteolytic generation ofthe authentic N-terminus ofthe HCV NS3 protein. The HCV NS3 coding region contains an artificial franslational stop codon. The second ORF is located downstream ofthe encephalomyocarditis virus (EMCV) IRES and determines the cytopathic or noncytopathic phenotype ofthe replicon. For the cytopathic replicon, it encodes an ubiquitin gene (ubi) and the pestiviral nonstructural NS3 to NS5B proteins. The ubiquitin gene was inserted to enable post translation the generation ofthe authentic Nterminus of the BVDV NS3 protein by ubiquitine carboxy-terminal hydrolase. For the noncytopathic replicon, it encodes the 3 '-terminal portion ofthe p7 coding unit. Post translation, this unit gives rise to the signal peptide necessary for generation ofthe correct N-terminus of NS2. The p7 coding unit is followed by the sequences ofthe BVDV nonstructural proteins NS2-NS5B. Importantly, the expression of NS3 alone results in a cytopathic phenotype, whereas the expression ofthe NS2/NS3 polypeptide as an uncleaved protein is associated with a noncytopathic phenotype.

FIG. 2A is a digital image of a denaturing agarose gel electrophoresis showing the linearized RNA ofthe cytopathic replicon in lane 1. FIG. 2B is a digital image showing the morphology ofthe dendritic cell line DC2.4. FIG. 2E is a digital image showing the transfection efficiency of RNA-elecfroporated DC2.4 as determined by immunofluorescence microscopy. Transfected DC2.4, which expressed the HCV NS3 protein, were stained with a primary antibody directed against the HCV NS3 protein and a FITC-labeled secondary antibody as described in Materials and Methods (above). FIG. 2F is a higher magnification view ofthe digital image shown inFIG. 2E. FIG. 2D is a 3D histogram showing the transfection efficiency of RNA-elecfroporated DC2.4 as determined by flow cytometry analysis. The unshaded histogram represents untransfected DC2.4, and the solid histogram represents DC2.4 transfected with the cytopathic replicon RNA (cytopathic Repl-HCV_HS₃)- Both cell populations were permeabilized and stained with the primary antibody directed against the HCV NS3 protein and a FITC-labeled secondary antibody as described in the Materials and Methods, above. FIG. 2C is a digital image of a Western blot analysis of DC2.4 cells fransfected with the cytopathic replicon RNA. Lane 1 shows total protein from an untransfected DC2.4 cell(negative control). Lane 2 shows cytopathic RepHdCV_NS3-transfected DC2.4 cell lysate. Lane 3 shows noncytopathic Repl-HCV_NS3-transfected DC2.4 cell lysate. The Western blot was probed with an antibody directed against the HCV NS3 protein. Each lysate was derived from a similar number of transfected cells. These results demonstrate that more than 95% ofthe dendritic cells are fransfected with the cytopathic replicon and express the HCV NS3.

C. Detection of apoptosis in DC2.4 transfected with the cytopathic and noncytopathic replicon RNA

To monitor replicon-induced apoptosis of DC2.4 cells fransfected with cytopathic and noncytopathic Repl-HCV_NS3 RNA, annexin V and propidium iodide double staining followed by FACS analysis was performed 12h, 24h and 48h after transfection, respectively (see, FIG. 3 A). This assay allows the quantitation of different cell populations. Living cells are not labeled by either annexin V or propidium iodide and are found the lower left quadrant of each graph. When cells undergo apoptosis, they are labeled only by annexin V and are therefore found in the lower right quadrant. Necrotic (dead) cells are stained by both agents and are found in the upper right quadrant of each graph. The percentage of apoptotic and necrotic cells increases with time if cells were fransfected with the noncytopathic replicon, but does not increase significantly if the eels were fransfected with the noncytopathic replicon. These results demonstrate that the cytopathic replicon induces cell death 24h to 48 hours after transfection. Thus, the cytopathic replicon is preferred for vaccination purposes because ofthe induction of cell death for cross-priming. The noncytopathic replicon ensures longer expression of antigen in living cells, which may be important to maintain immune responses.

D. Vaccination and detection of primed CD8+ T cells

FIG. 4 shows a schematic outline ofthe strategy used for vaccination of mice with transfected DC and the subsequent detection of direct primed and/or cross-primed CD8+ T cells in the immunized mice. FIG. 4A shows the transfection ofthe DC2.4 dendritic cells with cytopathic or noncytopathic replicon RNA, respectively, by electroporation. Twelve hours after elecfroporation, transfected DC2.4 were then injected subcutaneously into AAD mice as described in the Materials and Methods section. AAD mice express both murine H-2b and human HLA-A2.1 as major histocompatibility complexes, whereas the DC2.4 cell line expresses only murine H-2b.

FIG. 4B depicts the in vivo phenomenon of direct priming and cross-priming of T cells in the immunized mouse. Direct priming occurs when the injected DC24 cells present HCV NS3 peptides on their cell surface H-2b molecules and directly induce NS3-specific T cells in the mouse. These T cells are then H-2b resfricted. Cross-priming occurs when the injected DC2.4 cells undergo apoptosis and are taken up by the dendritic (or other antigen-presenting) cells ofthe immunized mouse. These dendritic (or other antigen-presenting) cells ofthe immunized mouse display both H2b and AAD on their cell surface and, after processing HCV NS3, they can present NS3 peptiώs on both H-2b and AAD to T cells. These T cells are cross-primed. Thus, antigen-specific H-2b restricted T cells can be induced by either direct priming or by cross-priming, whereas AAD-restricted T cells are induced exclusively by cross-priming.

FIG.4C depicts the immunological assays used to detect cross-priming and direct priming. CD3+ T cells are isolated from the immunized mouse and tested against C1RAAD cells (HLA-A2.1 positive) or against EL-4 cells (H-2b positive) that have been loaded with HCV NS3 peptides. If the T cells recognize NS3 peptides in the context of H-2b on EL-4 cells, they are either directly primed or cross-primed T cells. If the T cells recognize NS3 peptides in the context of HLA-A2.1 on CIR-AAD cells, they are cross-primed T cells. E. Qμantitation ofHCV-NS3 specific, IFN-γ-producing T cell and proliferative capacity of HCV-NS3 specific T cells isolated from mice immunized with the C2.4 transfected with cytopathic replicon RNA

The number of HCV-NS3 specific, IFNγ-producing T cells isolated from mice immunized with DC2.4 fransfected with cytopathic replicon RNA was determined by Elispot analysis as described in the Materials and Methods section, above. As shown in FIG.5, column A, T cells from mice immunized with cytopathic Repl-HCVNS3 RNA-transfected DC were tested against NS3 peptide pool 1, pool 2, and pool 3. The sum of all pools, which equaled the total NS3 -specific response, for mice immunized with cytopathic RepKHCVNS3 RNA-transfected DC is shown in lower bottom graph of FIG. 5, column A. In each graph, open circles indicate experiments performed with peptide-presenting EL-4 cells (to detect cross-priming plus direct priming) and filled squares indicate experiments performed with CIR-AAD cells (to detect cross-priming alone). Collectively, the results shown in FIG. 5, column A demonstrate that the cytopathic Repl-HCVNS3 RNA-transfected DC vaccine induces T cells not only by direct priming but to a significant extent by cross-priming.

F. Cytotoxic activity ofHCV-NS3 specific T cells isolated from mice immunized with the DC2.4 transfected with cytopathic or noncytopathic replicon RNA

The cytotoxic activity of HCV-NS3 specific T cells isolated from mice immunized with the DC2.4 transfected with cytopathic and noncytopathic replicon RNA is shown in FIG. 7, columns A and B, respectively. Cytotoxicity (% specific lysis of peptide-loaded target cells) of T cells from immunized mice is determined as described in the Materials and Methods section, above. T cells were tested against NS3 peptide pool 1 (top graph in each column), pool 2 (middle graph in each column) or pool 3 (bottom graph in each column)-loaded target cells, respectively. In each graph, open circles indicate experiments performed with peptide-presenting EL-4 cells as target cells (to detect cross-priming plus direct priming) and filled squares indicate experiments performed with

CIR-AAD cells as target cells (to detect cross-priming alone). Collectively, these results demonstrate that the cytopathic and noncytopathic replicon RNA vaccine induces T cells not only by direct priming but to a significant extent by cross-priming.

G. HCV-specific T cells induced by the replicon-DC-vaccine are functionally active in vivo and protect mice against challenge with recombinant vaccinia virus

To address the question whether the in v/tro-detected HCV-specific T cells are functionally active in vivo, mice, which had received a single vaccination of cytotoxic ReplHCV_NS3-transfected DC, were challenged with 10⁷ plaque forming units recombinant HCV NS3-expressing vaccinia virus. Five days after challenge, mice were killed and the vaccinia virus titer was determined in the ovaries, the organ where the virus preferentially replicates. As shown inthe top, leftmost graph of FIG. 9A, the vaccinia virus titer was below the detectable limit in four ofthe five mice tested These results show that a single vaccination is sufficient to induce HCV-specific T cells that are functionally active in vivo and protect the mice against challenge with a recombinant, HCV NS3-expressing vaccinia virus.

Example 2 VACCINATION OF CHIMPANZEES AND HUMANS WITH AUTOLOGOUS DENDRITIC CELLS TRANSFECTED WITH THE SELF-REPLICATING, CYTOPATHIC REPLICON

A. Generation of autologous dendritic cells

Instead of using the fransfected murine dendritic cell line DC2.4, chimpanzees and humans are vaccinated with autologous cells such as but not limited to autologous dendritic cells and fibroblasts that are transfected with the self replicating, cytopathic replicon. Autologous dendritic cells are isolated from peripheral blood monocytes precursors (Reddy et al, Blood; 1997; 90: 3640- 3646; Dhodapkar et al, J. Clin. Invest. 1999; 104: 173-180).

Specifically, peripheral blood mononuclear cells are isolated from blood via ficoll density gradient cenfrifugation, resuspended in RPMI 1640 cell culture medium supplemented with 5% autologous serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM Lglutamine at 8 x 10⁶ cells / ml and incubated in 6-well culture plates at 37°C, 5% C0₂. After 2 hours, nonadherent cells are removed by gentle pipetting and washing with PBS and adherent cells are cultured in RPMI 1640 cell culture medium supplemented with 1% autologous serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine for 7 days. A total of 800 U/ml GMP grade IL4 (Cell Genix, Freiburg, Germany) and 100 IU/ml clinical grade GMCSF (Immunex, Seattle, Washington, USA) is added on days 0, 2, 4 and 6 of culture. On day 7, cells are transferred to new plates and cultured in the presence of 50% (vol/vol) monocytes-conditioned medium (MCM) for 2 additional days (Dhodapkar et al, J. Clin. Invest. 1999; 104: 173-180). Sixteen hours prior to the end ofthe culture, dendritic cells are transfected with self-replicating, cytopathic replicon RNA in the same manner as described above for the murine cell line DC2.4

Autologous fibroblasts are isolated from 2 mm x 2 mm skin biopsies. Briefly, skin biopsies are cut in small pieces of less than 1 mm, placed in a 6-well plate under a cover glass in 1 ml RPMI 1640 cell culture medium containing 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM Lglutamine and 5% human AB-serum. 0.5 ml ofthe cell culture medium is exchanged every week. Growing fibroblast populations are expanded into cell culture flasks and aliquots are used for transfection.

B. Quality control

Phenotype and purity of dendritic cells is evaluated for expression ofthe maturationmarker CD83. Assays to exclude potential bacterial and fungal contaminationare performed prior to injection. C. DC injection

Dendritic cells are removed from the culture plates, washed in PBS and resuspended in 2 aliquots of 0.2 ml PBS containing 5% autologous serum at a concenfration of 10⁷/ml. DC are injected subcutaneously in 2 adjacent sites on the upper inner arm, approximately 4 inches from the axilla as previously described (Dhodapkar et al, J. Clin. Invest., 104: 173-180, 1999).

Subjects are evaluated 48 hours after injection for a local reaction and seven and thirty cays after injection for evidence of an immune response To analyze immune responses, 50 ml of peripheral blood is drawn, and lymphocytes are isolated via Ficoll gradients as previously described, (Takaki et al, Nature Medicine, 6: 578-582, 2000) and tested for HCV-specific effector functions as described in Example 1 (Ex vivo detection of HCV NS3 peptide-specific CD8+ T cells: IFN-γ Elispot assay // Detection of HCV NS3-specific CD4+ T cells: Proliferation assay II In vitro expansion and detection of HCV NS3-specific CTLs: 51Cr release assay) using autologous Epstein-Barr- Virus transformed B cells as target cells as previously described (Takaki et al, Nature Medicine, 6: 578-582, 2000).

Example 3 VACCINATION WITH CYTOPATHIC REPLICON RNA ENCODING TUMOR ANTIGENS

Another application ofthe cytopathic BVDV replicons is the induction of cellular immune responses against tumors. In this example, cytopathic BVDV replicons are constructed that encode the tumor antigens Her-2/neu (HER-2) and -fetoprotein (AFP), respectively.

Overexpression ofthe 185-kDa glycoprotein HER-2 is associated with malignant transformation of epithelial cells. Furthermore, HER-2 is overexpressed in breast cancers, ovarian cancer, gastric cancer and colorectal carcinomas. T cells have been shown to recognize sequences encoded by HER-2 and several rounds of DNA immunization with HER2 expressing plasmids have been shown to protect mice from rechallenge with tumors that express the same antigen

(J. Immunology, 170: 1202-1208, 2003). However, rejection of tumors that have been established prior to vaccination has not been shown previously, presumably because induction of a much stronger immune response is required and cannot be achieved with standard DNA immunization.

Therefore, tumors are established in mice by injecting the HER-2 fransfected mouse mammary tumor cell lines D2F2. The tumor itself does not induce tumor-specific T cell immunity (J. Immunology, 170: 1202-1208, 2003). Mice are vaccinated with autologous dendritic cells fransfected with the HER-2 expressing cytopathic BVDV replicon RNA to inhibit tumor growth and/or reduce tumor size.

AFP is a tissue-specific tumor-associated self antigen, which is expressed during fetal development ofthe liver and reexpressed at high levels in patients with hepatocellular carcinoma.

Hepatocellular carcinoma is the most common primary malignant tumor ofthe liver and ranks fifth in frequency (fifth in men and eighth in women) and fourth in annual mortality rate. An estimated 372,000 new cases of hepatocellular carcinoma are diagnosed each year, constituting 4.6% of all new human cancers (6.3% in men; 2.7% in women). The annual mortality rate from the tumor is virtually the same as its annual incidence. Because of its poor response to conservative treatment, low resectabilty rate when symptomatic, high recurrence rate after resection and liver transplantation, and grave prognosis, hepatocellular carcinoma is now regarded as one ofthe major malignant diseases (Kew, Toxicology, 27(181-182):35-38, 2002).

When mice bearing AFP-expressing murine hepatocellular carcinoma (for example, the cell line Hepa 1-6 (CRL-1830; ATCC)) were vaccinated with DNA expression vectors encoding AFP and cytokines, tumor-specific T cells could be induced and partial regression ofthe tumors was achieved (Grimm et al, Gastroenterology, 119: 1104-1112, 2000). However, complete tumor regression and cure was not achieved, apparently because the cellular immune response was not strong enough.

Thus, expressing the sequence of AFP from the cytopathic BVDV replicon and vaccinating mice with autologous dendritic cells that are fransfected with the replicon RNA produces a stronger immune response and a better outcome for the subject.

Example 4

KINETICS OF CD4⁺ AND CD8⁺ MEMORY T CELL RESPONSES DURING HEPATITIS C VπtUS RECHALLENGE OF RECOVERED CHIMPANZEES

This example (fromNascimbeni et al, J. Virol, 11: 4781-4793, 2003) demonstrates that strong memory T cell responses after recovery from HCV infection are associated with prrtective immunity upon reinfection. This protective immunity provides strong evidence that generating a vaccine that induces sfrong T cell responses, such as by inducing cross-priming with pestivirus replicons as described above, is beneficial in preventing HCV infection.

In addition, this example demonstrates how HCV-specific memory T cells react upon reexposure to the virus. Analysis ofthe peripheral blood and intrahepatic cellular immune response during HCV rechallenge is done with an animal model andthe only non-human animal susceptible to HCV infection is the chimpanzee. Three previously recovered chimpanzees were rechallenged with increasing doses of homologous HCV. All rechallenged animals cleared HCV below the detection level of real-time PCR more rapidly and displayed significantly less liver disease than during primary infection (Major et al, J. Virol, 76: 6586-6595, 2002). HCV-specific T cell responses in the blood and liver of these animals during rechallenge was analyzed. Although all rchallenged chimpanzees rapidly controlled HCV to levels below 400 copies/ml, distinct virological differences were detected at the RT-PCR level and correlated with the cellular immune response. Vigorous HCV-specific T cell responses were associated with rapid HCV control in chimpanzee Ch4X0186, whereas weak proliferation of HCV-specific T cells was associated with persistence of trace amounts of HCV in Chi 605 and loss of proliferative HCV-specific T cell responses was associated with HCV recrudescence in Chl552 (Nascimbeni et al, J. Virol, 11: 4781-4793, 2003).

Several conclusions can be drawn from these studies. First, protection correlates with rapid memory responses of HCV-specific T cells in all animals. Vigorous proliferation of IFNy-producing, circulating CD4⁺T cells is followed by an increased frequency and a phenotypic and functional change ofthe teframer⁺ CD8⁺ T cell population from CCR7 ^" central memory to CCRT, IFNγ-producing effector memory cells (Nascimbeni et al, J. Virol, 11: 4781-4793, 2003). At the same time, upregulation of IFNy-mRNA is detectable in the liver (Major et al, J. Virol, 76: 6586-6595, 2002).

Second, HCV-specific T cell responses need to be maintained for at least 4 months to prevent HCV recrudescence in the blood (Nascimbeni et al, J. Virol, 11: 4781-4793, 2003). Third, antibodies to HCV envelope glycoproteins may not be necessary for viral control, because they remain undetectable in at least three ofthe rechallenged animals (Nascimbeni et al, J. Virol, 77: 4781-4793, 2003). Finally, different levels of HCV-specific immunity may exist in animals even if they had recovered from infection with HCV of identical sequence because the animal with the most vigorous and multispecific cellular immune response was profected against the highest rechallenge dose and did most rapidly clear the rechallenge virus from the circulation and liver (Nascimbeni et al, J. Virol, 11: 4781-4793, 2003).

Example 5 DENDRITIC CELLS TRANSFECTED WITH CYTOPATHIC SELF-REPLICATING RNA INDUCE CROSS-PRIMING OF CD8+ T CELLS AND ANTIVIRAL IMMUNITY A potential shortcoming of non-live vaccines is their relative inefficiency in generating Tcell responses, thus limiting their application in infections requiring cellular immunity. This example expands upon Example 1, and describes in more detail the system introduced in Example 1. The system, which is useful to induce cellular immunity, uses a self-replicating cytopathic pestivirus RNA to enhance production and presentation of hepatitis C virus (HCV) antigens and to induce apoptosis in dendritic cells (DC) 24-48 hours after transfection. Replicon-transfected H-2b DCs used to immunize HLA-A2 transgenic mice induced protection upon challenge with a vaccinia virus expressing HCV antigens. Induction of cell death enhanced the immunogenicity of DGassociated antigen. Transfer of cellular material from vaccine DCs to endogenous antigen presenting cells wasshown in lymph nodes and spleen and cross-primed CD8+ T cells were characterized.

The NS3 protein of HCV was selected as a model antigen for several reasons. First, given the global disease burden and public health impact of HCV infection, a protective or therapeutic vaccine would be highly desirable (Davis et al, Liver Transpl, 9: 331-338, 2003). Second, HCV clearance and protective immunity are associated with strong, durable T cell responses against multiple HCV epitopes (Nascimbeni et al, J. Virol, 11: 4781-4793, 2003; Shoukry et al, J. Exp. Med., 197: 1645-1655,2003; Thimme et α/., J Exp. Med., 194: 1395-1406, 2001), whereas HCV-specific antibodies can disappear after recovery (Takaki et al, Nature Medicine, 6: 578-582, 2000) and do not to contribute to immune protection (Nascimbeni et al, J. Virol, 11: 4781-4793, 2003; Shoukry et al, J. Exp. Med., 197: 1645-1655,2003). Third, HCVNS3 harbors some ofthe most frequently targeted and highly conserved CD4+ (Diepolderet al, Lancet, 346: 1006-1007, 1995) and CD8+ T cell epitopes (Takaki et al, Nature Medicine, 6: 578-582, 2000). Fourth, HCV is a tissue-tropic, noncytopathic virus that does not infect professional antigen presenting cells (APCs) and rarely induces death of infected cells, thus reducing the main source of exogenous antigens for crosspresentation. Poor priming of cellular immune response is therefore regarded as an important reason for the high incidence of HCV persistence (Thimme et al, J. Exp. Med., 194: 1395-1406,

2001). Because of these observations, a method that forces exogenous antigens into the MHC classl pathway and promotes crosspresentation is believed to be particularly useful to generate effective T cell responses against HCV.

Using a murine H-2b DC line and HLA-A2 transgenic mice it is shown in this example that a single injection of DCs transfected with cytopathic replicon RNA induced efficient T cell responses that conferred antiviral immunity. Thus, this example further demonstrates the usefulness of self-replicating cytopathic pestivirus vectors as vaccines against noncytopathic pathogens, such as HCV.

A. Materials and Methods

Cell lines

The immature murine DC2.4 line (C57BL/6 origin, H-2^b haplotype) (Shen et al, J. Immunol, 158: 2723-2730, 1997), generously provided by Dr. K.L. Rock and the Dana Farber Cancer Institute (Boston, MA), was grown in complete RPMI 1640 medium (10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 μM nonessential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin) containing 50 μM 2-mercaptoethanol (Gibco BRL, Grand Island, NY). This DC line has the full functional capacity of primary DC, including the capacity to mature, to process antigens, to upregulate expression of MHC I and II molecules as well as CD80, CD86, CD40, CD54 and to migrate (Okada et al, Cancer Res., 61 : 7913-7919, 2001 ). The HLA-A, B-negative human B lymphoblastoid cell line C1R transfected with AAD (a hybrid MHC class I molecule consisting ofthe αl+ <x2 domains of HLA-A2.1 and the α3 domain of H-2D^d) (Newberg et al, J. Immunol, 156: 2473-80, 1996) was provided by Dr. J. Berzofsky (National Institutes of Health, Bethesda, MD) and propagated in complete medium with 400 μg/ml G418 (Sigma-Aldrich, St. Louis, MO) and without 2-mercaptoethanol. The murine H-2^b thymoma cell line EL4 and the human osteosarcoma cell line 143TK (ATCC, Rockville, MD) were grown in complete DMEM (Gibco BRL, Grand Island, NY) medium in the presence or absence of 50 μM 2-mercaptoethanol, respectively.

Recombinant proteins and synthetic peptides

Recombinant HCVNS₃ protein (aa 1192-1457 of HCV-1 genotype la; GenBank Accession No. M62321) was kindly provided by Dr. M. Houghton (Chiron Corporation, Emeryville, CA). One hundred twenty-six pentadecamer peptides (Mimotopes, Clayton, Australia), overlapping by 10 amino acids each and spanning the complete amino acid sequence ofthe HCV_NS3 protein, were divided into three pools designated pool 1 (aa 1021-1240), pool 2 (aa 1231-1443), and pool 3 (aa 1434-1660) at 24 μg/ml per peptide. The HLA-A2 restricted minimal optimal epitopes HCV NS3₁₀₇₃-₁₀₈₁ CVNGVCWTV (SEQ ID NO: 12), NS3i₀₈₄-io₉₂ GAGTRTIAS (SEQ ID NO: 13), NS3_n69.₁₁₇₇ LLCPAGHAV (SEQ ID NO: 14), NS3₁₄₀6-i4i5 KLVALGINAV (SEQ ID NO: 15), NS3i₅₈₅.₁₅₉₃ YLVAYQATV (SEQ ID NO: 16) (Major et al, Hepatitis C Viruses, In: Fields Virology, ed. by Knipe et al, Philadelphia, PA: Lippincott-Raven Publishers, pp. 1127-1161, 2001) and the vaccinia virus epitope SLSAYIIRV (SEQ ID NO: 17) (Drexler et al, Proc. Natl. Acad. Sci. USA, 100: 217-222, 2003) were synthesized at >80% purity at Research Genetics (Huntsville, AL) or at the Facility for Biotechnology Resources, Center for Biologies Evaluation and Research, Food and Drug Adminisfration (Bethesda, MD).

Construction of recombinant plasmids

The plasmid encoding cytopathic Repl-HCV_NS3 was generated by modifying the original, GUS-expressing cDNA construct Bi-ubi-NS3-NS5B (Tautz et al, J. Virol, 73:9422-9432, 1999). First, g at position 12290 (numbering refers to the sequence ofthe BVDV CP7 construct (Meyers et al, J. Virol, 70: 8606-8613, 1996)) was substituted (Quickchange procedure) bye to introduce an additional Srfl resfriction site at the immediate 3' end ofthe RNA coding region. This change allowed the generation ofthe authentic 3 'terminus in runoff viral RNA transcripts from the linearized cDNA construct. Second, tec at position 896-898 (BVDV CP7, (Meyers et al, J Virol, 70: 8606-8613, 1996)) was exchanged (Quickchange procedure) intogc to introduce an Fspl resfriction site at the 3' end ofthe BVDV N^pr0 coding region. The resulting plasmid was termed

Bi-ubi-NS3-NS5B (Fsp). Third, the HCV_NS3 gene was amplified by PCR from the HCV Conl cDNA isolate (Lohmann et al, Science, 285: 110-113,1999) using a sense primer

(5'-caagctgcgcacctattacggcc-3'; SEQ ID NO: 5) with an Fspl restriction site and an antisense primer (5'-gtacatcgatatcgtcgactacgtgacgacctcca-3'; SEQ ID NO 6) with an artificial stop-codon and additional Sail and Clal resfriction sites. The PCR product was sequenced and cloned via Fspl and Clal (position 11083 ofBVDV CP7 in the NS5B coding region) into Bi-ubi-NS3-NS5B (Fsp). The resulting plasmid was digested with Nhel (cutting upstream ofthe SP6 promoter) and Sail (cutting downstream ofthe inserted HCV_NS3 gene) to obtain a DNA fragment consisting ofthe BVDV 5 'UTR and theN^pro coding region fused in frame to the HCVNS₃ gene. This fragment was finally cloned into the original Bi-ubi-NS3-NS5B plasmid that had been digested with Nhel and Xhol (Xhol cuts immediately upstream ofthe EMCV IRES). The plasmid encoding noncytopathic Repϋ-HCV_NS3 RNA was generated from the previously described Bi-NS2-NS5B cDNA construct (Tautz et al, J. Virol, 73:9422-9432, 1999) using the same strategy. The plasmid pcDNA3/NS3 was generated by cloning the HCV_NS3 gene ofthe Conl cDNA into the BamHI and Xbal sites of pcDNA3 (Invifrogen, Carlsbad, CA).

For the introduction of other foreign genes or antigenic determinants of HCV, HIV, RSV, and bacteria, as well as tumor antigens (max. length 3 kb), a similar strategy is used, for example, cloning PCR products via Fspl and Clal into "Bi-cp, N^pr core Fsp", followed by completion ofthe constructs via Nhel and Sall/Xho ligation as described. When using alternative pestivirus replicon vectors, a similar strategy can be adapted as well.

In vitro transcription of RNA Srfl and Smal (Stratagene, La Jolla, CA) were used to linearize DNA templates of

Repl-HCV_NS3 and Repl-GUS constructs, respectively. After purification with MiniElute Reaction Cleanup Kit (Qiagen Inc., Valencia, CA), linearized plasmids were in vitro transcribed with SP6 RNA polymerase (Roche Diagnostics, Indianapolis, IN) in a standard reaction. After removal of DNA by digestion with RNase-free DNase I (Roche Diagnostics, Indianapolis, IN) and purification of synthesized RNA with the Rneasy Mini Kit (Qiagen Inc., Valencia, CA),RNA concentration and integrity were determined by UV spectrophotometry (OD 250 nm) and gel electrophoresis.

RNA transfection of DCs and detection ofHCVχs₃ expression

Five hundred thousand DC2.4 were pelleted, resuspended in Cytomix(van den Hoff et al, Nucleic Acids Res., 20: 2902, 1992) containing 1.25% DMSO, mixed with 5 μg RNA, fransferred to a 2 mm-gap cuvette, and elecfroshocked twice at a voltage of 300 V and a capacitance of 150 μF using a Gene Pulser apparatus (Bio-Rad, Hercules, CA). After a 10-min incubation on ice, cells were seeded in fresh complete medium and incubated at 37 °C and 5% C0₂.

Transfection efficiency was evaluated after 24 or 48 h of culture. For indirect IF microscopy and flow cytometry, transfected cells were fixed on chamber slides or in tubes, permeabilized, and incubated with 1B6 anti-HCV_NS , (Wolk et al, J. Virol, 74: 2293-2304, 2000) followed by fluorescein isothiocyanate (FITC)-conjugated F(ab')₂ anti-mouse IgG (Caltag Laboratories, Burlingame, CA). For Western Blot analysis, transfected and untransfected DC2.4 lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto Immobilon-P membranes (Millipore, Bedford, MA) and immunobloted using 1B6 anti-HCV_NS3, and horseradish peroxidase-conjugated anti-mouse Ig followed by enhanced chemilumirescence (ECL; Amersham, Arlington Height, IL).

Cell death analysis Replicon-induced apoptosis of transfected DC2.4 was demonstrated by annexin V and propidium iodide double staining (Annexin V-FITC Apoptosis Detection Kit I, PharMingen, San Diego, CA) and TUNEL assay (DeadEnd Fluorimetric TUNEL System, Promega, Madison, WI) followed by flow cytometry.

Mouse immunization

H-2^b C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) and AAD-fransgenic mice (HLA-A2 on H-2^b C57BL/6 background) (Newberg et al, J. Immunol, 156: 2473-2480, 1996), kindly provided by Dr. V. Engelhard (University of Virginia, Charlottesville, VA), were used under a protocol approved by the Animal Care and Use Committee ofthe National Institute of Diabetes and Digestive and Kidney Diseases. For DC immunization, DC2.4 cells were harvested 12 or 48 h after transfection with cytopathic or noncytopathic ReptHCV_NS3 RNA, washed twice and resuspended in PBS. For selected immunizations, 5 x 10⁵ transfected DC2.4 cells were labeled with 5 μM CFSE (Molecular Probes) at 37 °C for 10 min and washed once with PBS/0.1% FCS and twice with PBS. Five-hundred thousand fransfected DC2.4 cells in 100 μl PBS were injected subcutaneously (s.c.) (at the base ofthe tail) into 6- to 8-week-old female mice. For DNA immunization, 50 μg pcDNA/NS3 was injected into each regenerating tibialis anterior muscle 5 days after intramuscular (i.m.) injection of 10 μM cardiotoxin.

Primary cell preparations

Mice were sacrificed 7 days after immunization. Draining lymph nodes and spleens were isolated, injected with 400 μg/ml Liberase CI (Roche Diagnostics, Indianapolis, IN), incubated at 37 °C for 30 min and forced through a cell strainer (Falcon, Franklin Lakes, NJ) to obtain single cell suspensions. T cells were isolated with anti-CD90 (Thy 1.2) coated magnetic beads and MACS columns (Miltenyi Biotec, Auburn, CA). In other experiments, splenic low-density cells were isolated by density centrifugation over a 30% BSA (Sigma-Aldrich, St. Louis, MO) step gradient.

Visualisation of adoptively transferred DCs Draining lymph nodes and spleens were isolated and analyzed for the presence of CFSE 12h after immunization with CFSE-labeled DC2.4. For flow cytometry, low-density spleen cells and lymph node cells were incubated with anti-HLA-A2.1 (ATCC, Manassas, VA) and biotin-conjugated anti-mouse GDI lc followed by phycoeryfrin (PE conjugated anti-mouse F(ab')₂ IgG (Immunotech, Marseille, France), PerCP-conjugated Streptavidin (PharMingen, San Diego, CA) and allophycocyanin (APC)-conjugated anti-mouse F4/80 OPharMingen). Cells were analysed on a

FACSCalibur with CellQuest (Becton & Dickinson, San Jose, CA) and Flow Jo (Flow Jo, San Carlos, CA) software. For IF, lymph node cell cytospins and spleen cryosections were acetone-fixed, stained with PE-conjugated anti-mouse CD1 lc (PharMingen), counterstained with DAPI (Molecular Probes, Eugene, OR), mounted, and examined with a Zeiss Axioskop2 microscope (Carl Zeiss Inc., Thornwood, NY) equipped with an epifluorescence source.

ELISpot Assay

Ex vivo IFN-γ-ELISpot assays were performed as previously described (Wedemeyer et al, Gastroenterology, 121: 1158-1166, 2001) with the following modifications. Individual HCV_NS3 peptide pools (1 μg/ml per peptide) or HLA-A2-restricted HCV_NS3 and VV peptides (10 μg/ml) were used to stimulate for 30 h (a) serial dilutions of freshly purified splenic T cells in the presence of \ irradiated (10,000 rad) EL4 or CIR-AAD cells per well, or (b) 2.5 x 10⁵ unseparated splenocytes per well. All cultures were set up in triplicates with appropriate negative (no peptide) and positive (0.2 μg/ml phytohemagglutinin, Murex Biotech Limited, Dartford, England) controls. Spots were evaluated and counted with a KS ELISpot Reader (Carl Zeiss Inc., Thornwood, NY). The number of specific spots was obtained by subtracting the mean number of spots in negative control wells from the mean number of spots in experimental wells.

Proliferation Assay

Triplicate cultures of 5 x 10⁵ T cells were stimulated for 5 days with 10⁵ irradiated (10,000 rad) EL4 cells and with either 1 μg/ml HCV_NS3 protein or buffer control or 1 μg/ml PHA in complete RPMI 1640 medium. For the last 16 h, 1 μCi ³H-thymidine (ICN, Costa Mesa, CA) was added and the incorporated radioactivity was measured and expressed as stimulation index (cpm in presence of antigen / cpm in absence of antigen).

Chromium release assay

Five-hundred thousand purified splenic T cells were cultured with IG? irradiated (10,000 rad) EL4 or CIR-AAD cells in complete RPMI 1640 medium containing individual HCV_NS3 peptide pools (1 μg/ml per peptide). On day 2, 10% Rat-T-Stim (Collaborative Biomedical Products, Bedford, MA) was added. On day 7, a 6-h ^5ICr-release assay was performed using EL4 or CIR-AAD target cells that had been pulsed with individual HCV_NS3 peptide pools at 1 μg/ml per peptide overnight and then labeled with 100 μCi ^5ICr (Amersham) for 1 h at 37 °C. Triplicate cultures of 3,000 target cells were incubated with effector cells. Percent specific lysis was calculated as (experimental release- spontaneous release) x 100 / (maximum release- spontaneous release), in which spontaneous and maximum release reflected target cell lysis in the absence of effector cells and in the presence of 10% Triton X-100 (Sigma- Aldrich, St. Louis, MO), respectively.

Recombinant vaccinia virus (VV) challenge and plaque assay

Two weeks after immunization, mice were intraperitoneally challenged with 10⁷ plaque-forming units of HCV_NS3-encoding recombinant VV (Bartenschlager et al, J. Virol, 68: 5045-5055, 1994; Wedemeyer et al, Gastroenterology, 121: 1158-1166, 2001). Five days later, mice were sacrificed and VV titers were detennined by plating 10-fold dilutions of homogenized and sonicated ovaries on 143TK^" monolayers that were stained with 0.075 wt/vol% crystal violet 48 h later.

Statistical analysis

Ap value <0.05 (Mann- Whitney U test) was considered statistically significant.

B. Cytopathic and noncytopathic self-replicating RNAs encoding HCVNS3 can be originated from the bovine viral diarrhea virus genome To produce an antigen expression system, a self replicating positive-strand viral RNA (replicon) from the genome ofthe pestivirus bovine viral diarrhea virus(BVDV) was generated. This recombinant bicistronic replicon, termed Repl-HCV_NS3, contains a 5' terminal open reading frame (ORF) encoding a fusion protein ofthe pestiviral autoprotease N⁵™ (Wiskerchen et al, J. Virol, 65: 4508-4514, 1991) and the heterologous antigen HCV_NS3, and a 3' terminal ORF encoding the BVDV nonstructural proteins (see, FIG. IB). When transfected into host cells, Repl-HCV_NS3 RNA operates initially as mRNA. Translation ofthe ORFs is mediated by internal ribosomal entry sites (IRES) located in the BVDV 5' untranslated region and downstream ofthe HCV_NS3 coding region. Translation products are co- and posttranslationally processed by viral and cellular proteases into mature proteins. In particular, the authentic N-terminus of HCV_NS3 is generated by autoproteolytic cleavage of N^pro. The BVDV nonstructural proteins are active components ofthe viral replication complex, that multiplies the RNA copies in the cytoplasm via negative-strand intermediates (Behrens et al, J. Virol, 72: 2364-2372, 1998; Grassmann et al, J. Virol, 75: 7791-7802, 2001). It is believed that production of BVDV_NS3 as a single protein (cytopathic Repl-HCV_NS3) rather than as a part ofthe uncleaved BVDV_Ns₂-₃ polypeptide (noncytopathic Repl-HCV_NS3) is the molecular correlate for the cytopathogenicity observed for the replicon in cell culture as well as for BVDV in the natural infection (Thiel et al., Pestiviruses, In: Fields Virology, ed. by Knipe et al, Philadelphia, PA: Lippincott-Raven, pp. 1059-1074, 1996).

C. Cytopathic Repl-HCV_NS3 RNA induces high levels of HCV_NS3 expression in transfected

DCs

Induction of effective T cell responses depends on the amount of antigen delivered and the number and type of APCs that actively process and present the antigen. To provide cytoplasmic HCV_NS3 protein for antigen processing and presentation, in vitro transcribed cytopathic or noncytopathic Repl-HCV_NS3 RNA (FIG. 2A) was introduced into DC2.4 lines (Shen et al,

J. Immunol, 158: 2723-2730, 1997) (FIG. 2B) by electroporation. Efficiency of transfection, RNA replication and HCV_NS3 expression were monitored by flow cytometry, immunofluorescence (IF) microscopy and Western Blot (FIGS. 2C-F). After transfection with cytopathic Repl-HCV_Ns₃ RNA, almost the entire DC population (95-100%) expressed high levels of HCV_NS3 (FIGS. 2C-F) homogenously disfributed in the cytoplasm (FIGS. 2E-F). This level was similar to that of other bicistronic replicon constructs tested in different cell lines, i.e. in the range of approximately lOOng protein per 10⁷ cells (Tautz et al, J. Virol, 73: 9422-9432, 1999). Lower level of antigen expression was instead achieved after transfection with noncytopathic ReplHCVrø RNA (FIG. 2C-D). RNA replication was confirmed when DC2.4 transfected with RNAs encoding a defective BVDV replicase revealed no staining. This was consistent with previous reports that showed replicon-mediated protein synthesis only in the presence of RNA replication (Behrenset al, J. Virol, 72: 2364-2372, 1998). D. Cytopathic Repl-HCV_NS3 RNA induces apoptosis of transfected DCs

To demonstrate the immunologic effects of DC apoptosis and antigen reprocessing, the survival kinetics of DC2.4 were compared after fransfection with cytopathic or noncytopathic Repl-HCV_NS3 RNA. Major differences were revealed on examination ofthe cultures by light microscopy. Floating cells appeared in the DC2.4 cultures within 1 or 2 days after transfection with cytopathic Repl-HCVNS3 RNA and, after that, the cultures turned into single cell suspensions containing loose clumps of large, dendritic-shaped cells. In confrast, in the cultures transfected with noncytopathic Repl-HCV_NS3 RNA, cells remained as adherent and proliferative as non-transfected control cells, and cell detachment started only after prolonged culture. To show that cell death results from the cytopathic effect ofthe replicon, DC surface expression of phosphatidylserine and uptake of propidium iodide as well as DNA fragmentationwas determined. As depicted in FIG. 3, the percentage of Annexin V-positive (FIG. 3A) and TUNEL-positive (FIG. 3B) apoptotic cells as well as the percentage of propidium iodide-positive dead cells (FIG. 3A) increased over time after transfection with cytopathic ReptHCV_NS3 RNA, but not after fransfection with noncytopathic Repl-HCV_NS3 RNA. Induction of apoptosis and cell death was, therefore, dependent upon the cytopathic effect ofthe replicon and the time after transfection and not due to the electroporation itself.

E. DCs transfected with cytopathic Repl-HCV_NS3 RNA induce cross-priming of HCV_NS3-specific CD8+ T cells

To demonstrate the in v vo-priming capacity of DCs fransfected with cytopathic Repl-HCV_NS3 RNA, DC2.4 cells transfected with cytopathic or noncytopathic Repl-HCV_NS3 RNA were subcutaneously (s.c.) injected into HLA-A2-transgenic mice. The number of apoptotic and dead cells was negligible at the time of injection. By adoptively transferring H-2b cells into mice that express both murine H-2b and human HLA-A2 alleles, it was possible to specifically address the impact of cross-presentation of exogenous cell-associated antigens on in vivo priming of CD8+ T cells. When the injected DC2.4 directly prime HCVNS3-specific CD8+ T cells, this protocol results in the expansion of H-2b-restricted T cells. On the contrary, when host resident APCs prime HCVNS3 -specific CD8+ T cells (as a consequence of reprocessing and cross-presentation of antigens acquired from apoptotic or dead DC2.4), this protocol (see, for example, FIG.4) results in the expansion of H-2b-restricted and HLA-A2-restricted T cells. Thus, HLA-A2-restricted T cell responses can only be induced by cross-priming.

The frequency ofthe in vivo primed HCV_NS3-specific CD8+ T cells was determined one week after a single immunization in ex vivo IFN-γ ELISpot assays in which purified splenic T cells were stimulated with either CIR-AAD (HLA-A2) or EL4 (H-2b) cells in the presence of individual peptide pools covering the complete HCVNS3 sequence. The peptide pools did not crossreact, because H-2b-resfricted T cells from C57BL/6 mice immunized with cytopathic ReptHCVNS3 RNA-transfected DC2.4 did not recognize these peptides on HLA-A2-positive APCs (not shown). As shown in FIG. 5, column A, immunization with cytopathic ReptHCVNS3 RNA-transfected DC2.4 induced a substantial number of IFN-γ-producing T cells directed against all three HCVNS3 peptide pools. HLA-A2-resfricted T cell responses were as vigorous as H-2b-restricted T cell responses, thus indicating the relevance of cross-priming. A significantly lesser response was achieved with DC2.4 fransfected with noncytopathic Repl-HCV_NS3 RNA (p < 0.04) (FIG. 5, column B) and with a single intramuscular (i.m.) injection of plasmid DNA encoding HCV_NS3 (p < 0.04) (FIG. 5, column D).

To measure the cytotoxic activity of the in vivo primed HCVNS3 -specific CD8+ T cells, purified splenic T cells from immunized mice wererø vitro restimulated with either CIR-AAD (HLA-A2) or EL4 (H-2b) cells in the presence of individual HCV_NS3 peptide pools and, after 7 days, tested for lysis of peptide-pulsed CIR-AAD and EL4 target cells. As shown in FIG. 6, column A, CTL responses obtained by immunization with cytopathic ReplHCV_NS3 RNA-transfected DC2.4 were significantly stronger than those obtained by immunization with DC2.4 transfected with noncytopathic Repl-HCV_NS3 RNA (p < 0.05) (FIG. 6, column B) or with a single i.m. injection of plasmid DNA (p < 0.05) (FIG. 6, column D). CTL activities were detected against HLA-A2 and H-2b target cells pulsed with all 3 HCV_NS3 peptide pools, thus confirming the ELISpot results and the contribution of cross-priming to the in vivo induction of CD8+ T cell responses.

F. Cell death enhances immunogenicity of DC-associated HCV_NS3 antigen

The relative contribution of cell death to the immunogenicity ofthe DGassociated antigens is demonstrated in this subsection. Because the HCV_NS3 expression level differed between DC2.4 fransfected with cytopathic or noncytopathic Repl-HCV_NS3 RNA, the priming capacity of DC2.4 transfected with noncytopathic Repl-HCV_Ns₃ RNA was compared with the priming capacity of DC2.4 transfected with noncytopathic Repl-HCV_NS3 RNA and supertransfected 48 h later with a cytopathic replicon encoding the irrelevant antigen glucoronidase instead of HCV_NS3 (cytopathic Repl-GUS RNA). Although the level of HCV_NS3 expression was equivalent in both immunization regimens, the induced CD8+ T cell responses differed. Number and cytotoxic activity of CD8+ T cells were significantly enhanced in mice s.c. injected with supertransfected DCs (p < 0.04)(see, FIG. 5, column C and FIG. 6, column C). Notably, this increase was exclusively observed for the HLA A2-restricted T cell response reflecting cross-priming.

G. Resident host APCs internalize cellular fragments of injected DC2.4 transfected with cytopathic RepI-HCV_NS3 RNA

To study the fate of injected DCs and to visualize the transfer of cell-associated antigens that underlies cross-priming in vivo, cytopathic Repl-HCV_NS3 RNA-transfected DC2.4 were labeled with CFSE and s.c. injected into HLA-A2-transgenic mice. Twelve hours after injection, draining lymph nodes and spleen were isolated and analyzed for the presence of CFSE fluorescence. Although a distinct, CFSE-positive cell population was found in the lymph nodes, the green fluorescence was rarely homogenously disfributed in intact, live cells (FIG. 7A). Instead, it was almost exclusively detected in the form of cellular fragments in the cytoplasn (FIG. 7B) of HLA-A2-positive (FIG. 7C) cells, indicating that these were not the injected DC2.4, but host cells thathad captured cellular fragments ofthe injected, apoptotic DC2.4. Their CD1 lc negative and F4/80 positive phenotype (FIG. 7C) and their location in the marginal zone ofthe spleen indicate that the majority of these host cells were macrophages.

When the same experiments were performed with DC2.4 transfected with noncytopathic Repl-HCV_NS3 RNA, both size and phenotype ofthe CFSE-positive HLA-A2-positive host cell population differed. More CFSE-positive cells in the lymph nodes were HLA-A2-negative and the HLA-A2-positive cells displayed a CD1 lc+ rather than CD1 lc- phenotype.

H. DCs transfected with cytopathic Repl-HCV_NS3 RNA induce HCV_NS3-specific CD4+

T cell responses

Effective induction of CD4+ T cell responses is an important component of any vaccinaton strategy because maintenance of CD8+ T cell responses often requires CD4+ T cell help. In parallel with the CD8+ T cell analysis, the potential of DCs transfected with noncytopathic and/or cytopathic replicons to induce CD4+ T cell responses in vivo was compared in HLA-A2-transgenic mice. As demonstrated in FIG 8, T cells isolated from draining lymph nodes and spleen of mice immunized with cytopathic Repl-HCV_NS₃ RNA-transfected DC2.4 showed a vigorous proliferative response to recombinant HCV_NS3 protein which was significantly stronger than those induced by DC2.4 transfected with noncytopathic ReplΗCV_Ns₃ RNA (p < 0.002) and by conventional i.m. DNA immunization (p < 0.002).

I. DCs transfected with the cytopathic Repl-HCV_Ns3 RNA induce protective antiviral immunity Because HCV does not infect rodents, a previously described surrogate challenge model

(Wedemeyer et al, Gastroenterology, 121: 1158-1166, 2001) was used to demonstrate the protective capacity ofthe induced T cells in vivo. Three weeks after a single s.c. immunization with DC2.4 transfected with cytopathic or noncytopathic Repl-HCV_NS3 RNA, HLA-A2-fransgenic mice were intraperitoneally challenged with 10⁷ pfu recombinant vaccinia virus encoding HCV_NS3. In parallel, a control group of mice was immunized with 100 μg plasmid DNA encoding HCV_NS3 and challenged with the same type and dose of vaccinia virus. Five days after challenge, mice were sacrificed, vaccinia virus titers were determined, and cellular immune responses analyzed. As shown in FIG. 9A (top row), the group of mice immunized with DC2.4 fransfected with cytopathic ReplHCV_NS3 RNA displayed lower vaccinia virus titers than the group of mice immunized with DC2.4 transfected with noncytopathic Repl-HCV_NS₃ RNA or with naked plasmid DNA. The adjuvant effect of cell death was confirmed by the intermediate level of protection displayed by mice immunized with DC2.4 transfected with noncytopathic Repl-HCV_NS3 RNA and supertransfected with cytopathic Repl-GUS RNA. Protection correlated with the strength ofthe T cell response to pools of overlapping HCV_NS3 peptides (FIG. 9A; bottom row) and to the HLA-A2-restricted minimal optimal HCV epitopes (FIG. 9B) and not with the response to the vaccinia virus epitope (FIG. 9A; bottom row). Nonspecific responses did not significantly contribute to the antiviral protection as determined in a control vaccination experiment with DC2.4 fransfected with cytopathic ReplGUS RNA.

J. Advantages of self-replicating, cytopathic, antigen-expressing RNA replicon as demonstrated in this Example 5

Immunization with antigen-presenting cells, such as dendritic cells, with a self-replicating cytopathic RNA replicon, such as described herein, has several advantages. First, self-amplification ofthe fransfected RNA in DCs mimics viral infection and results in the synthesis of double stranded RNA intermediates which are known to induce DC maturation, MHC expression and cytokine production (Cella et al, J. Exp. Med., 189: 821-829, 1999). Second, self-replicating RNA allows high-level expression and endogenous processing ofthe encoded antigens in DC, thus overcoming one ofthe main limiting factors in the priming of Tcell responses. Both factors provide an advantage over the use ofnonreplicating RNA(Saeboe-Larssen et α/., J. Immunol. Methods, 259: 191-203, 2002) and peptide-loaded DC (Dhodapkar et al, J. Clin. Invest., 104: 173-180, 1999). Third, the cytopathic effect ofthe self-replicating RNA triggers time-delayed apoptosis ofthe vaccine DCs in vivo, allowing sufficient time for migration to secondary lymphoid organs.

The relatively high levels of antigen expression and cytopathic nature of the self-replicating RNA replicons described herein may be particularly useful to exploit the cross-presentation and cross-priming processes ofthe immune system. Under normal circumstances, when antigen levels are within the physiologic range and no significant tissue damage occurs, crosspresentation is thought to be less efficient than direct presentation, because it requires the additional step of antigen transfer from one cell to another (Zinkernagel, Eur. J. Immunol, 32: 2385-2392, 2002). Most model systems that describe cross-presentation therefore use high levels of antigen expression (Heath and Carbone, Ann. Rev. Immunol, 19: 47-64, 2001). This example demonstrates that induction of cell death (by the cytopathic Repl-GUS RNA) can help to overcome the limitations of low antigen le\els in noncytopathic Repl-HCV_NS3 RNA-transfected DCs in the priming of cellular immune responses.

The transfer of antigens from the injected cells to the APCs ofthe host may occur either in the periphery or in the secondary lymphoid organs. The first scenario occurs when antigen-expressing, nonmigratory cells are injected (Berd, Vaccine, 19: 2565-2570, 2001), or when naked DNA (Gurunathan et al, Curr. Opin. Immunol, 12: 442-447, 2000) or RNA (Ying et al, Nat. Med., 5: 823-827, 1999) is locally injected (for example, by intramuscular or subcutaneous injection) Efficient transfer of an antigen in the periphery may depend upon the presence ofadditional inflammatory signals, which attract and activate migratory DCs (Gurunathan et al, Curr. Opin. Immunol, 12: 442-447, 2000). In comparison, a migratory cell transfected with an antigen-expressing, cytopathic replicon actively migrates to the draining lymph nodes or spleen and undergoes apoptosis in these secondary lymphoid organs. In principle, any cell could be fransfected with a self-replicating, cytopathic, antigen-expressing replicon RNA; however, in some embodiments it is preferable to use migratory cells, such as DCs, which can transport the antigen directly to secondary lymphoid organs.

It will be apparent that the precise details ofthe methods described may be varied or modified without departing from the spirit ofthe described disclosure. We claim all such modifications and variations that fall within the scope and spirit of he claims below.

Claims

CLAIMSWe claim:

1. A pestivirus replicon, comprising a pestivirus nucleic acid sequence and a heterologous antigen-encoding sequence, wherein the antigen-encoding sequence is inserted at a position in the pestivirus nucleic acid sequence that inhibits formation of infectious replicon particles by disrupting the expression of structural proteins required for formation of infectious replicons.

2. The replicon of claim 1, wherein the pestivirus is bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV) or border disease virus (BDV).

3. The replicon of claim 2, wherein the pestivirus is BVDV.

4. The replicon of claim 1, wherein the antigen-encoding sequence disrupts the expression of at least one of the C, E^ms, E 1 , or E2 subunits of the pestivirus nucleic acid sequence.

5. The replicon of claim 4, wherein the antigen-encoding sequence partially or completely replaces the C, E^ms, El, or E2 subunits ofthe pestivirus nucleic acid sequence.

6. The replicon of claim 5, wherein the antigen-encoding sequence completely replaces the C, E^ms, El, and E2 subunits ofthe pestivirus nucleic acid sequence.

7. The replicon of claim 1, wherein the replicon is monocistronic orbicistronic.

8. The replicon of claim 7, wherein the replicon is bicistronic.

9. The replicon of claim 8, wherein one open reading frame ofthe bicistronic replicon comprises an N^pro-encoding sequence and the heterologous antigen-encoding sequence.

10. The replicon of claim 8, comprising an RNA sequence that encodes an N^-antigen fusion protein and a polyprotein comprising NS2/NS3, NS4A, NS4B, NS5A, and NS5B; wherein NS2/N3 comprises uncleaved NS2-NS3 polypeptide or NS3 polypeptide.

11. The replicon of claim 10, wherein NS2/N3 is uncleaved NS2-NS3 polypeptide.

12. The replicon of claim 10, wherein NS2/N3 isNS3 polypeptide.

13. The replicon of claim 11, wherein the RNA sequence comprises the following elements:

5'-IRES-Npro-Antigen-IRES-p7-(NS2-NS3)-NS4A-NS4B-NS5A-NS5B-3' wherein Antigen is the heterologous antigen-encoding sequence.

14. The replicon of claim 13, wherein the RNA sequence is encoded by a nucleic acid sequence comprising: a) a sequence having at least 90% sequence identity to SEQ ID NO: 8; or b) the nucleic acid sequence set forth as SEQ ID NO: 8.

15. The replicon of claim 12, wherein the RNA sequence comprises the following elements:

5'-IRES-Npro-Antigen-IRES-ubi-NS3-NS4A-NS4B-NS5A-NS5B-3' wherein Antigen is the heterologous antigen-encoding sequence

16. The replicon of claim 15, wherein the RNA sequence is encoded by a nucleic acid sequence comprising:: a) a sequence having at least 90% sequence identity to SEQ ID NO: 7; or b) the nucleic acid sequence set forth as SEQ ID NO: 7.

17. The replicon of claim 1, wherein the antigen-encoding sequence encodes an antigen of a pathogen or tumor.

18. The replicon of claim 17, wherein the antigen comprises one or more epitopes.

19. The replicon of claim 17, wherein the antigen is an antigen of a pathogen.

20. The replicon of claim 19, wherein the pathogen is a viral pathogen.

21. The replicon of claim 20, wherein the viral pathogen is a Hepatitis C virus, a

Human Immunodeficiency Virus, or a Respiratory Syncytial Virus.

22. The replicon of claim 21, wherein the viral pathogen is Hepatitis C virus.

23. The replicon of claim 22, wherein the antigen is HCV core, HCV E 1 , HCV E2,

HCV p7, HCV NS2, HCV NS3, HCV NS4, or HCV NS5.

24. The replicon of claim 23, wherein the antigen is HCV NS3.

25. The replicon of claim 24, wherein the antigen comprises: a) a sequence having at least 90% sequence identity to SEQ ID NO: 3 b) a sequence having at least 95% sequence identity to SEQ ID NO: 3 c) a sequence having at least 98% sequence identity to SEQ ID NO: 3 d) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: 3; or e) the amino acid sequence set forth as SEQ ID NO: 3.

26. The replicon of claim 20, wherein the viral pathogen is a Respiratory Syncytial

Virus.

27. The replicon of claim 26, wherein the antigen is RSV F, RSV N, RSV M2 or RSV

G.

28. The replicon of claim 20, wherein the viral pathogen is a Human Immunodeficiency

Virus.

29. The replicon of claim 28, wherein the antigen is pl8, p24, p33, p39, p55, gp36, gp41, or gpl20.

30. The replicon of claim 19, wherein the pathogen is a bacterial pathogen.

31. The replicon of claim 19, wherein the pathogen is Mycobacterium tuberculosis or Plasmodium falciparum.

32. The replicon of claim 31 , wherein the pathogen is Mycobacterium tuberculosis .

33. The replicon of claim 32, wherein the antigen is ESAT-6, MPT63, MPT64, MPT83, Antigen 85B, Antigen 85A, PstS-1, PstS-2 or PstS-3, MTB41, or hsp60.

34. The replicon of claim 31, wherein the pathogen is Plasmodium falciparum.

35. The replicon of claim 34, wherein the antigen is circumsporozoite protein (CSP), thrombospondin-related adhesive protien (TRAP), sporozoite and liver-stage antigen (SALSA), sporozoite threonine- and asparagine-rich protein (STARP), merozoite surface protein (MSP)-l, -2, 3, -4, -5, erythrocyte-binding antigen (EBA)-175, apical membrane antigen (AMA)-1, rhoptry- associated protein (RAP)-l and -2, acidic-basic repeat antigen (ABRA), ring erythrocyte surface antigen (RES A), serine-rich protein (SERP), erythrocyte membrane protein (EMP)-1, -2 and -3, Glutamate-rich protein (GLURP), Glycosilphopatidylinositol (GPI), Ps25, Ps28, Ps48/45 or Ps230.

36. The replicon of claim 17, wherein the antigen is a tumor antigen.

37. The replicon of claim 36, wherein the tumor antigen is Her-2/neu, α-fetoprotein, human epithelial cell mucin, the Ha-ras oncogene product, p53, carcino-embryonic antigen, the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-AlMart- 1, gpIOO, EBV-LNT 1, EBV-LNT 2, HPV-F4, HPV-F 6, HPV-F 7, prostatic serum antigen, CO 17- 1 A, GA733, gp72, p53, the ras oncogene product, BPV E7 or melanoma ganglioside.

38. A method of inducing aT cell response to an antigen in a subject, comprising: administering to a subject an amount of an antigen presenting cell sufficient to induce a T cell response in the subject; wherein the antigen presenting cell expresses an antigen from the pestivirus replicon of claim 1, thereby inducing a Tcell response to the antigen in the subject.

39. The method of claim 38, further comprising transfecting the antigen presenting cell with the pestivirus replicon prior to administering the antigen presenting cell to the subject.

40. The method of claim 38,wherein the antigen presenting cellis a dendritic cell or a fibroblast.

41. The method of claim 38,wherein the antigen presenting cell is a dendritic cell.

42. The method of claim 40,wherein the antigen presenting cell is an autologous cell.

43. The method of claim 38,wherein the replicon is a cytopathic replicon.

44. The method of claim 43, wherein the replicon encodes a sequence that comprises: a) a sequence having at least 90% sequence identity to SEQ ID NO: 1; b) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: l; or c) the amino acid sequence set forth as SEQ ID NO: 1.

45. The method of claim 38,wherein the replicon is a noncytopathic replicon.

46. The method of claim 45, wherein the replicon encodes a sequence that comprises: a) a sequence having at least 90% sequence identity to SEQ ID NO: 2; b) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: 2; or c) the amino acid sequence set forth as SEQ ID NO: 2.

I

47. The method of claim 38,wherein the antigen is an antigen of a pathogen σ tumor.

48. The method of claim 47, wherein the antigen is an antigen of a pathogen.

49. The method of claim 48, wherein the pathogen is a viral pathogen.

50. The method of claim 49, wherein the viral pathogen is a Hepatitis C virus, a Human Immunodeficiency Virus, or a Respiratory Syncytial Virus.

51. The method of claim 50, wherein the viral pathogen is Hepatitis C virus.

52. The method of claim 51 , wherein the antigen comprises HCV core, HCV E 1 , HCV E2, HCV p7, HCV NS2, HCV NS3, HCV NS4, or HCV NS5.

53. The method of claim 52, wherein the antigen comprises HCV NS3.

54. The method of claim 51 , wherein the antigen comprises a) a sequence having at least 90%) sequence identity to SEQ ID NO: 3 b) a sequence having at least 95%) sequence identity to SEQ ID NO: 3 c) a sequence having at least 98% sequence identity to SEQ ID NO: 3 d) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: 3; or e) the amino acid sequence set forth as SEQ ID NO: 3.

55. The method of claim 48, wherein the pathogen is bacterial pathogen.

56. The method of claim 48, wherein the pathogen is Mycobacterium tuberculosis or Plasmodium falciparum.

57. The method of claim 56, wherein the pathogen is Mycobacterium tuberculosis.

58. The method of claim 57, wherein the antigen is ESAT-6, MPT63, MPT64, MPT83, Antigen 85B, Antigen 85A, PstS-1, PstS-2 or PstS-3, MTB41, or hsp60.

59. The method of claim 56, wherein the pathogen is Plasmodium falciparum.

60. The method of claim 59, wherein the antigen is circumsporozoite protein (CSP), thrombospondin-related adhesive protien (TRAP), sporozoite and liver-stage antigen (SALSA), sporozoite threonine- and asparagine-rich protein (STARP), merozoite surface protein (MSP)-1, -2, - 3, -4, -5, erythrocyte-binding antigen (EBA)-175, apical membrane antigen (AMA)-1, rhoptry- associated protein (RAP)-l and -2, acidic-basic repeat antigen (ABRA), ring erythrocyte surface antigen (RES A), serine-rich protein (SERP), erythrocyte membrane protein (EMP>1, -2 and -3, Glutamate-rich protein (GLURP), Glycosilphopatidylinositol (GPI), Ps25, Ps28, Ps48/45 or Ps230.

61. The method of claim 47, wherein the antigen is a tumor antigen.

62. The method of claim 61, wherein the tumor antigen is Her-2/neu, α-fetoprotein, human epithelial cell mucin, the Ha-ras oncogene product, p53, carcino-embryonic antigen, the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-AlMart- 1, gpIOO, EBV-LNT 1, EBV-LNT 2, HPV-F4, HPV-F 6, HPV-F 7, prostatic serum antigen, CO 17- 1 A, GA733, gp72, p53, the ras oncogene product, BPV E7 or melanoma ganglioside.

63. The method of claim 49, wherein the viral pathogen is a Respiratory Syncytial Virus.

64. The method of claim 63, wherein the antigen is RSV F, RSV N, RSV M2 or

RSV G.

65. The method of claim 49, wherein the viral pathogen is a Human Immunodeficiency Virus.

66. The method of claim 65, wherein the antigen isplδ, p24, p33, p39, p55, gp36, gp41, or gpl20.

67. The method of claim 38,wherein the pestivirus replicon is derived from classical swine fever virus or border disease virus.

68. The method of claim 51, wherein the pestivirus nucleic acid sequence encodes a protein comprising: a) a sequence having at least 90% sequence identity to SEQ ID NO: 1; b) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: 1; or c) the amino acid sequence set forth as SEQ ID NO: 1; and wherein the antigen-encoding sequence encodes a Hepatitis C virus antigen comprising: i) a sequence having at least 90% sequence identity to SEQ ID NO: 3; ii) a sequence having at least 95% sequence identity to SEQ ID NO: 3; iii) a sequence having at least 98% sequence identity to SEQ ID NO: 3; iv) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: 3; or v) the amino acid sequence set forth as SEQ ID NO: 3.

69. An isolated nucleic acid molecule, wherein the nucleic acid molecule comprises: a) a sequence having at least 90% sequence identity to SEQ ID NO: 7; or b) the nucleic acid sequence set forth as SEQ ID NO: 7.

70. An isolated nucleic acid molecule, wherein the nucleic acid comprises: a) a sequence having at least 90% sequence identity to SEQ ID NO: 8; or b) the sequence of SEQ ID NO: 8.

71. A fransfected dendritic cell into which a pestivirus replicon lhat expresses an antigen has been introduced ex vivo.

72. The transfected dendritic cell of claim 71 , wherein the replicon is a cytopathic replicon.

73. The transfected dendritic cell of claim 72, wherein the replicon encodes a sequence comprising: a) a sequence having at least 90% sequence identity to SEQ ID NO: 1 ; b) a conservative variant of the amino acid sequence set forth as

SEQ ID NO: l; or c) the amino acid sequence set forth as SEQ ID NO: 1.

74. The transfected dendritic cell of claim 71, wherein the replicon is a noncytopathic replicon.

75. The fransfected dendritic cell of claim 74, wherein the replicon encodes a sequence that comprises: a) a sequence having at least 90% sequence identity to SEQ ID NO: 2; b) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: 2; or c) the amino acid sequence set forth as SEQ ID NO: 2.

76. A transfected dendritic cell into which thepestivirus replicon of claim 1 has been introduced ex vivo.

77. The transfected dendritic cell of claim 71 , wherein the antigen is an antigen of a pathogen or tumor.

78. The transfected dendritic cell of claim 77, wherein the antigen is a pathogen antigen.

79. The transfected dendritic cell of claim 78, wherein the pathogen is a viral pathogen.

80. The transfected dendritic cell of claim 79, wherein the viral pathogen is a Hepatitis C virus, a Human Immunodeficiency Virus, or a Respiratory Syncytial Virus.

81. The transfected dendritic cell of claim 71 , wherein the pathogen is a bacterial pathogen.

82. The transfected dendritic cell of claim 71 , wherein the pathogen is Mycobacterium tuberculosis or Plasmodium falciparum.

83. The transfected dendritic cell of claim 82, wherein the pathogen is Mycobacterium tuberculosis.

84. The transfected dendritic cell of claim 83, wherein the antigen is ESAT-6, MPT63, MPT64, MPT83, Antigen 85B, Antigen 85A, PstS-1, PstS-2 or PstS-3, MTB41, orhspόO.

85. The transfected dendritic cell of claim 82, wherein the pathogen is Plasmodium falciparum.

86. The transfected dendritic cell of claim 85, wherein the Plasmodium falciparum antigen is circumsporozoite protein (CSP), thrombospondin-related adhesive protien (TRAP), sporozoite and liver-stage antigen (SALSA), sporozoite threonine- and asparagine-rich protein (STARP), merozoite surface protein (MSP)-l, -2, -3, -4, -5, erythrocyte-binding antigen (EBA>175, apical membrane antigen (AMA)-1, rhoptry-associated protein (RAP)-l and -2, acidic-basic repeat antigen (ABRA), ring erythrocyte surface antigen (RESA), serine-rich protein (SERP), erythrocyte membrane protein (EMP)-1, -2 and -3, Glutamate-rich protein (GLURP), Glycosilphopatidylinositol (GPI), Ps25, Ps28, Ps48/45 or Ps230.

87. The transfected dendritic cell of claim 79, wherein the viral pathogen is Hepatitis C virus.

88. The transfected dendritic cell of claim 87, wherein the antigen is HCV core, HCV El, HCV E2, HCV p7, HCV NS2, HCV NS3, HCV NS4, or HCV NS5.

89. The transfected dendritic cell of claim 87, wherein the antigen comprises: a) a sequence having at least 90% sequence identity to SEQ ID NO: 3 ; b) a sequence having at least 95% sequence identity to SEQ ID NO: 3; c) a sequence having at least 98% sequence identity to SEQ ID NO: 3; d) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO:3; or e) the amino acid sequence set forth as SEQ ID NO: 3.

90. The transfected dendritic cell of claim 77, wherein the antigen is a tumor antigen.

91. The fransfected dendritic cell of claim 90, wherein the tumor antigen is Her-2/neu, α-fetoprotein, human epithelial cell mucin, the Ha-ras oncogene product, p53, carcino-embryonic antigen, the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-AlMart- 1, gpIOO, EBV-LNT 1, EBV-LNT 2, HPV-F4, HPV-F 6, HPV-F 1, prostatic serum antigen, C017-1A, GA733, gp72, p53, the ras oncogene product, BPV E7 or a melanoma ganglioside.

92. The transfected dendritic cell of claim 79, wherein the viral pathogen is a

Respiratory Syncytial Virus.

93. The transfected dendritic cell of claim 92, wherein the antigen is RSV F, RSV N, RSV M2, or RSV G.

94. The fransfected dendritic cell of claim 79, wherein the viral pathogen is a Human Immunodeficiency Virus.

95. The fransfected dendritic cell of claim 94, wherein the antigen is pl8, p24, p33, _P39, p55, gp36, gp41, or gpl20.

96. The fransfected dendritic cell of claim 87, wherein the antigen comprises: a) a sequence having at least 90% sequence identity to SEQ ID NO: 3 b) a sequence having at least 95% sequence identity to SEQ ID NO: 3 c) a sequence having at least 98% sequence identity to SEQ ID NO: 3 d) a conservative variant ofthe am o acid sequence set forth as SEQ ID NO: 3; or e) the amino acid sequence set forth as SEQ ID NO: 3; and wherein the pestivirus replicon comprises: i) a sequence having at least 90% sequence identity to SEQ ID NO: 1 ii) a conservative variant ofthe amino acid sequence set forth as

SEQ ID NO: l; or iii) the amino acid sequence set forth as SEQ ID NO: 1.

97. A composition for inducing an immune response, vJ erein the composition comprises a) a sequence having at least 90% sequence identity to SEQ ID NO: 1; b) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: l; or c) the amino acid sequence set forth as SEQ ID NO: 1.

98. A composition for inducing an immune response, wherein the composition comprises a) a sequence having at least 90% sequence identity to SEQ ID NO: 2; b) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: 2; or c) the amino acid sequence set forth as SEQ ID NO: 2.

99. A composition for inducing an immune response, wherein the composition comprises a pestivirus replicon, wherein the pestivirus replicon comprises: a) a nucleic acid sequence encoding a protein comprising: i) a sequence having at least 90% sequence identity to SEQ ID

NO: l; ii) a conservative variant ofthe amino acid sequence set forth as

SEQ ID NO: l; or iii) the amino acid sequence set forth as SEQ ID NO: 1, and b) a nucleic acid sequence encoding a Hepatitis C virus antigen comprising: i) a sequence having at least 70% sequence identity to SEQ ID

NO: 3; ii) a sequence having at least 80% sequence identity to SEQ ID

5 NO: 3; iii) a sequence having at least 90% sequence identity to SEQ ID

NO: 3; iv) a sequence having at least 95% sequence identity to SEQ ID

NO: 3; 10 v) a sequence having at least 98% sequence identity to SEQ ID

I NO: 3; vi) a conservative variant ofthe amino acid sequence set forth as

SEQ ID NO: 3; or vii) the amino acid sequence set forth as SEQ ID NO: 3; and 15 wherein the nucleic acid sequence encoding the Hepatitis C virus antigen is operably linked to the nucleic acid sequence encoding thepestivirus replicon.

100. A pharmaceutical composition, wherein the composition comprises a pharmaceutically acceptable carrier and 20 a) a sequence having at least 90% sequence identity to SEQ ID NO: 1 ; b) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: l; or c) the amino acid sequence set forth as SEQ ID NO: 1.

25 101. A pharmaceutical composition, wherein the composition comprises a pharmaceutically acceptable carrier and a) a sequence having at least 90%) sequence identity to SEQ ID NO: 2; b) a conservative variant ofthe amino acid sequence set forth as SEQ ID NO: 2; or

30 c) the amino acid sequence set forth as SEQ ID NO: 2.

102. A pharmaceutical composition, wherein the composition comprises a pharmaceutically acceptable carrier and a pestivirus replicon, wherein the pestivirus replicon comprises: a) a nucleic acid sequence encoding a protein comprising: i) a sequence having at least 90% sequence identity to SEQ ID

NO: l; ii) a conservative variant ofthe amino acid sequence set forth as

SEQ ID NO: l; or iii) the amino acid sequence set forth as SEQ ID NO: 1, and b) a nucleic acid sequence encoding a Hepatitis C virus antigen comprising: i) a sequence having at least 90% sequence identity to SEQ ID

NO: 3; ii) a sequence having at least 95% sequence identity to SEQ ID

NO: 3; iii) a sequence having at least 98% sequence identity to SEQ ID

NO: 3; iv) a conservative variant ofthe amino acid sequence set forth as

SEQ ID NO: 3; or v) the amino acid sequence set forth as SEQ ID NO: 3; and wherein the nucleic acid sequence encoding the Hepatitis C virus antigen is operably linked to the nucleic acid sequence encoding the pestivirus replicon.