WO2002074920A2 - A replication-defective alphavirus vaccine linking antigen with an immunogenicity-potentiating polypeptide and a method of delivery the same - Google Patents

A replication-defective alphavirus vaccine linking antigen with an immunogenicity-potentiating polypeptide and a method of delivery the same Download PDF

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WO2002074920A2
WO2002074920A2 PCT/US2002/008033 US0208033W WO02074920A2 WO 2002074920 A2 WO2002074920 A2 WO 2002074920A2 US 0208033 W US0208033 W US 0208033W WO 02074920 A2 WO02074920 A2 WO 02074920A2
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nucleic acid
cell
cells
antigen
polypeptide
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WO2002074920A3 (en
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Tzyy-Choou Wu
Chien-Fu Hung
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Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention in the fields of molecular biology, immunology and medicine relates to a PCL-generated replication-defective alphavirus replicons as vectors for chimeric nucleic acid vaccines encoding fusion proteins. These vectors are used as vaccines to enhance immune responses, primarily cytotoxic T lymphocyte (CTL) responses to specific antigens such as tumor or viral antigens.
  • CTL cytotoxic T lymphocyte
  • the fusion protein comprises an antigenic polypeptide fused to an immunogenicity-potentiating polypeptide that promotes intercellular transport ofthe antigen, processing via the MHC class I pathway, stimulation of dendritic cell development or function, and the like. Description of the Background Art Naked DNA vaccines have emerged as attractive approaches for vaccine development
  • the "gene gun” strategy provides efficient delivery of DNA into epidermal bone marrow-derived APCs termed Langerhans cells, which move to draining lymph nodes where they enter the lymphatic system.
  • the present inventors and their colleagues have successfully used this system of DNA delivery to test various intracellular targeting strategies (Chen et al., 2000, Cancer Res. 60:1035-1042; Ji et al., 1999, Human Gene Therapy 10:2727-2740); co-pending, commonly assigned U.S. patent applications USSN 09/421,608; 09/501,097, 09/693,450??? and 60/281,003???).
  • RNA replicon vaccines may be derived from alphavirus vectors, such as Sindbis virus (Hariharan, JM et al., 1998, J. Virol. 72:950-958), Semliki Forest virus (Berglund, P et al, 1997, AIDS Res. Hum. Retrovir. 73:1487- 1495; Berglund, P. et al, 1998, Nat. Biotech.
  • Alphavirus vectors such as Sindbis virus (Hariharan et al, 1998; Xiong et al, 1989. Science 243:1188-1191) and Semliki Forest virus (Berglund et al, 1997, AIDS Res Hum Retroviruses 13: 1487-1495; Daemen et al, 2000, Gene Ther. 7: 1859-1866), have become an important strategy for the development of vaccines and gene therapy applications because of their high levels of R ⁇ A replication and gene expression in cells, their ability to infect a variety of diverse cell types, and the relative ease of manipulating cD ⁇ A clones for transcription of vectors and infectious viral R ⁇ A (for review, see (Dubensky et al, In: Gene Therapy: Therapeutic Mechanisms and Strategies.
  • alphavirus-based expression vectors The general strategy for construction of alphavirus-based expression vectors has been to substitute viral structural protein genes with a heterologous gene, while preserving transcriptional control via the highly active subgenomic RNA promoter (Frolov et al, supra; Huang, supra; Xiong et al, supra). These vectors are self-replicating in cells and may be administered as either RNA, DNA, or infectious propagation-incompetent alphavirus particles. Since alphavirus vectors eventually trigger apoptosis of transfected cells (Ying et al, 1999, Nat Med. 5:823-827), they do not raise the concern associated with DNA integration into the host genome. This is particularly important for vaccine development approaches targeting proteins that are potentially oncogenic, such as the HPN E6 and E7 proteins.
  • infectious alphavirus replicon particles provide a highly efficient method of introducing heterologous genes into target cells and the opportunity to generate vaccines at a large scale.
  • significant concerns have been raised about potential contamination with replication-competent virus.
  • an alphavirus replicon packaging cell line (PCL) was developed (Polo, JM et al, 1999, Proc ⁇ atl Acad Sci U S A.
  • HSP-based protein vaccines involved fusing antigens to HSPs (Suzue (1996) J. Immunol. 156:873-879) wherein, HSP70 fusion protein elicited humoral and cellular immune responses to an HIV-l protein. While these investigations have made HSPs more attractive for use in immunotherapy, there have been no reports or suggestions of making HSP- linked molecular vaccine using replication defective alphavirus vectors as described herein.
  • the centrosome also called the microtubule organizing center (MTOC)
  • MTOC microtubule organizing center
  • MTOC microtubule organizing center
  • proteins notably ⁇ -tubulin and ⁇ -tubulin, are localized and concentrated at the centrosome.
  • the centrosome has been implicated as an important intracellular compartment for proteasomal degradation of certain antigens (Anton, supra).
  • Calreticulin an abundant 46 kilodalton (kDa) protein located in the lumen ofthe cell's endoplasmic reticulum (ER), displays lectin activity and participates in the folding and assembly of nascent glycoproteins. See, e.g.,, Nash (1994) Mol Cell. Biochem. 235:71-78; Hebert (1997) J Cell Biol. 139:613-623; Nassilakos (1998) Biochemistry 37:3480-3490; Spiro
  • CRT associates with peptides transported into the ER by transporters that are associated with antigen processing, such as TAP-1 and TAP-2 (Spee).
  • CRT also forms complexes with peptides in vitro. Upon adminsitration to mice, these complexes, elicited peptide-specific CD8+ T cell responses (Basu (1999) J. Exp. Med. 59:797-802; ⁇ air (1999) J. Immunol 162:6426-6432). CRT purified from murine tumors elicited immunity specific for the tumor from which the CRT was taken, but not for an antigenically distinct tumor (Basu, supra).
  • DCs mouse dendritic cells
  • MHC class I molecules By pulsing mouse dendritic cells (DCs) in vitro with a CRT-peptide complex, the peptide was re-presented by MHC class I molecules on the DCs to stimulate a peptide-specific CTL response( ⁇ air, supra).
  • CRT also has anti-angiogenic effects.
  • CRT and a fragment comprising amino acid residues 1-180, which has been called "vasostatin,” are endothelial cell inhibitors that can suppress tumor growth (Pike (1999) Blood. 94:2461-2468).
  • Tumor growth and metastasis depend on the existence of an adequate blood supply. As tumors grow larger, adequate blood supply to the tumor tissue is often ensured by new vessel formation, a process termed angiogenesis. (Folkman (1982) Ann. NY Acad. Sci. 401:212-27; Hanahan (1996) Cell 86:353- 364).
  • Therapeutic agents that target and damage tumor vasculature can prevent or delay tumor growth and even promote regression or dormancy.
  • DNA vaccines are their potency, since they do not have the intrinsic ability to amplify and spread in vivo as some replicating viral vaccine vectors do.
  • the present inventors conceived a strategy that facilitates the spread of antigen may significantly enhance the potency of naked DNA vaccines.
  • VP22 a herpes simplex virus (HSN-1) protein has demonstrated the remarkable property of intercellular transport and is capable of distributing protein to many surrounding cells(4) (U.S. Patent 6,017,735, O'Hare & Elliott, 25 Jan 2000).
  • NP22 has been linked to p53 (Phelan, A.
  • Marek's disease virus type 1 (MDN-1) UL49 shares homology with HSN-1 NP22 (Koptidesova et al, 1995, Arch Virol. 140:355-362) and has been shown to be capable of intercellular transport after exogenous application (Dorange et al, 2000, J Gen Virol. 81 Pt 9:2219-2230).
  • Flt3-ligand (Maraskovsky, E et al, JExp Med 184: 1953-62, 1996, Shurin, MR et al, Cell Lmmunol 179: 174-84, 1997). FL has emerged as an important molecule in the development of tumor vaccines that augment numbers and action of DCs in vivo.
  • Flt3 a murine tyrosine kinase receptor, first described in 1991 (Rosnet, O et al, Oncogene.
  • the predominant form of FL is synthesized as a transmembrane protein from which the soluble form is believed to be generated by proteolytic cleavage.
  • the soluble form of FL (the extracellular domain or "ECD") is functionally similar to intact FL (Lyman, SD et al, Cell. 75: 1157-67, 1993). These proteins function by binding to and activating unique tyrosine kinase receptors. Expression ofthe Flt3 receptor is primarily restricted, among hematopoietic cells, to the most primitive progenitor cells, including DC precursors.
  • the soluble ECD of FL induced strong anti-tumor effects against several murine model tumors including f ⁇ brosarcoma (Lynch, DH et al, Nat Med.
  • the potency of naked DNA molecular vaccines is limited by their inability to amplify and spread in vivo.
  • Inclusion of nucleic acid sequences that encode polypeptides that modify the way the antigen encoded by molecular vaccine is "received” or “handled” by the immune system serve as a basis for enhancing vaccine potency.
  • Polypeptides that have such modes of action are termed herein "mimunogenicity-potentiating (or -promoting) polypeptide" or "IPP" to reflect this general property, even though these IPP's may act by any of a number of cellular and molecular mechanisms that may or may not share common steps.
  • IPP's may be produced as fusion or chimeric polypeptides with the antigen, or may be expressed from the same nucleic acid vector but produced as distinct expression products.
  • the present invention provide a recombinant, replication-defective alphavirus-based replicon particles that encode a fusion of a polypeptide antigen of choice with an intercellular transport protein that, when expressed in a transfected cell, is capable of distributing the antigen to many surrounding cells.
  • This has been accomplished by the use of a stable packaging cell line (PCL), which is capable of generating alphavirus replicon particles without contamination from replication-competent virus.
  • PCL stable packaging cell line
  • This invention has been exemplified using the HSV-1 VP22 protein linked to a model tumor antigen, human papillomavirus type 16 (HPV-16) E7 oncoprotein and included in a nucleic acid which is a Sindbis virus (SIN)-based replicon particle encoding the VP22-E7 fusion and using a PCL termed SLN-PCL.
  • SIN Sindbis virus
  • the linkage of NP22 to E7 in these SP replicon particles resulted in a significant increase in the number of E7-specific CD8 + T cell precursors and a strong antitumor effect against E7-expressing tumors in vaccinated C57BL/6 mice relative to wild-type E7 SIN replicon particles.
  • NP22/E7-containing naked D ⁇ A, naked R ⁇ A replicons, or R ⁇ A replicon particle vaccines indicated that SI ⁇ rep5-VP22/E7 replicon particles generated the most potent therapeutic antitumor effect.
  • SI ⁇ rep5-VP22/E7 replicon particles generated the most potent therapeutic antitumor effect.
  • Cheng, WF et al, 2002, Hum Gene Ther, 2002, Mar;73: 553-568 a publication by the present inventors and colleagues after the priority date of this application.
  • the present strategy used in the context of SIN replicon particles produce with a PCL facilitates the generation of a highly effective vaccines for widespread immunization.
  • the present invention is directed to a nucleic acid molecule encoding a fusion polypeptide useful as a vaccine composition, which molecule comprises: (a) a first nucleic acid sequence encoding a first polypeptide that comprises at least one immunogenicity-potentiating polypeptide;
  • the first polypeptide is preferably one that acts by promoting: (a) processing of the linked antigenic polypeptide via the MHC class I pathway or targeting of a cellular compartment that increases the processing;
  • the first polypeptide is:
  • a viral intercellular spreading protein selected from the group of herpes simplex virus- 1 VP22 protein, Marek's disease virus NP22 protein or a functional homologue or derivative thereof;
  • an endoplasmic reticulum chaperone polypeptide selected from the group of calreticulin, ER60, GRP94, gp96, or a functional homologue or derivative thereof
  • cytoplasmic translocation polypeptide domains of a pathogen toxin selected from the group of domain II of Pseudomonas exotoxin ETA ( ⁇ TAdll) or a functional homologue or derivative thereof;
  • a polypeptide that targets the centrosome compartment of a cell selected from ⁇ -tubulin or a functional homologue or derivative thereof;
  • a polypeptide that stimulates dendritic cell processors or activates dendritic cell activity selected from the group of GM-CSF, Flt3 -ligand extracellular domain, or a functional homologue or derivative thereof
  • the first polypeptide above is selected from the group consisting of Mycobacterium tuberculosis HSP70, the HSP70 C-terminal domain, HSN-1 NP22, MDN NP22, calreticulin, Pseudomonas ETAdll, GM-CSF, Flt-3 ligand extracellular domain or ⁇ -tubulin.
  • the first polypeptide is a transport polypeptide comprising SEQ ID ⁇ O:5 or 7 or an active fragment thereof.
  • the antigenic polypeptide preferably comprises an epitope that binds to, and is presented on the cell surface by, an MHC class I protein.
  • the epitope is between about 8 and about 11 amino acid residues in length.
  • the antigen is preferably one which is present on, or cross-reactive with an epitope of, a pathogenic organism, cell, or virus.
  • a preferred virus is a human papilloma virus.
  • a preferred antigen is the E7 polypeptide of HPV-16 or an antigenic fragment thereof.
  • the pathogenic organism may be a bacterium.
  • the pathogenic cell is preferably a tumor cell.
  • the antigen is a tumor-specific or tumor-associated antigen, for example, a peptide ofthe HER-2/neu protein.
  • the above nucleic acid molecule may be operatively linked to a promoter.
  • the promoter is preferably one which is expressed in an APC, preferably a DC.
  • the above nucleic acid molecule is preferably an RNA replicon wherein the alphavirus is Sindbis virus, Semliki forest virus or Venezuelan equine encephalitis virus, most preferably Sindbis virus.
  • the nucleic acid molecule may have the sequence ofthe SINrep5 molecule
  • the packaging cell line is preferably one in which genes encoding capsid and envelope glycoproteins ofthe alphavirus are separated in distinct cassettes to minimize formation of replication competent virus during replicon production.
  • a most preferred packaging cell line is 987dlsplit #24.
  • an expression vector comprising any ofthe nucleic acid molecules described above, operatively linked to (a) a promoter; and (b) optionally, additional regulatory sequences that regulate expression ofthe nucleic acid in a eukaryotic cell.
  • the present invention is also directed to a cell which has been modified to comprise the nucleic acid or expression vector as above
  • the cell expresses the nucleic acid molecule.
  • Preferred cells as above are APCs, for example, a dendritic cell, a keratinocyte, a macrophage, a monocyte, a B lymphocyte, a microglial cell, an astrocyte, or an activated endothelial cell.
  • the present invention is directed to a pharmaceutical composition capable of inducing or enhancing an antigen-specific immune response, comprising:
  • composition selected from the group consisting of:
  • a method of inducing or enhancing an antigen specific immune response in a subject comprising administering to the subject an effective amount ofthe above pharmaceutical composition, thereby inducing or enhancing the response.
  • the response is preferably one which is mediated at least in part by CD8 cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • the response may be mediated at least in part by antibodies.
  • the present invention includes a method of inducing or enhancing an antigen specific immune response in cells or in a subject comprising administering to the cells or to the subject an effective amount ofthe pharmaceutical composition as above, thereby inducing or enhancing the response.
  • the composition may be is administered ex vivo to the cells.
  • These cells may comprise APCs, such as DCs.
  • the APCs are human APCs.
  • These APCs are preferably isolated from a living subject.
  • This method may further comprising a step of administering the ex vzv ⁇ -freated cells to a histocompatible subject.
  • the cells are human cells and the subject is a human.
  • the administering is preferably by a intramuscular, intradermal, or subcutaneous route.
  • the administering may be intratumoral or peritumoral.
  • the present invention provides a method of increasing the numbers or lytic activity of CD8 + CTLs specific for a selected antigen in a subject, comprising administering to the subject an effective amount of a composition selected from the group consisting of:
  • the antigen comprises an epitope that binds to, and is presented on the cell surface by, MHC class I proteins, thereby increasing the numbers or activity ofthe CTLs.
  • Also provided is a method of inhibiting growth or preventing re-growth of a tumor in a subject comprising administering to the subject, preferably intratumorally or peritumorally, an effective amount of a composition selected from the group consisting of:
  • the antigen comprises one or more tumor-associated or tumor-specific epitopes present on the tumor in the subject thereby inhibiting the growth or preventing the re-growth.
  • FIG. 1 A) BHK21 cells infected with SINrep5-E7 replicon particles and stained for E7 at 48 hours after infection. Note: E7 protein was predominantly located in the nucleus.
  • Fig. IB BHK21 cells infected with SINrep5-E7 replicon particles and stained for E7 at 72 hours after infections. Note: E7 protein remained in the nucleus 72 hours after infection.
  • Fig. IC BHK21 cells infected with SINre ⁇ 5-VP22/E7 replicon particles and stained for E7 at 48 hours after infection. Note: VP22/E7 protein was mostly located in the cytoplasm.
  • Fig. ID BHK21 cells infected with SLNrep5-VP22/E7 replicon particles and stained for E7 at 72 hours after infections. Note: intercellular spreading of VP22/E7 protein to many neighboring cells became apparent 72 hours after infection.
  • FIG. 2A Intracytoplasmic cytokine staining followed by flow cytometry analysis to demonstrate that SEMrep5-VP22/E7 replicon particles can enhance E7-specific CD8 + but not CD4 + T cell immunologic responses.
  • Vaccination of mice and preparation of splenocytes is described in Example I.
  • Fig. 2A Representative figure of flow cytometric analysis demonstrating E7-specific CD8 + T cell precursors in splenocytes from vaccinated mice. The number of IFN- ⁇ -secreting CD8 T cell precursors is shown in the upper right corner.
  • Fig. 2B Histogram to show E7-specific IFN- ⁇ -secreting CD8 + T cell precursors in vaccinated mice.
  • the number of IFN- ⁇ -producing E7-specific CD8 + T cells was determined using flow cytometry in the presence (solid columns) or absence (open columns) of MHC class I restricted E7 peptide (aa 49-57). Data are expressed as mean number of CD8 + ,LFN- ⁇ + cells/3xl0 splenocytes; bars, SE. Fig. 2C: Histogram to show E7-specific IFN- ⁇ -secreting CD4 + T cell precursors. The number of LFN- ⁇ -producing E7-specific CD4 + T cells was determined using flow cytometry in the presence (solid columns) or absence (open columns) of MHC class LI restricted E7 peptide (aa 30-67).
  • FIG. 3A Mice were immunized with 5x10 IU/mouse of SLNrep5-VP22 E7 replicon particles via intramuscular, intraperitoneal, or subcutaneous injection.
  • FIG. 3B Mice were immunized intramuscularly with different dosages of SINrep5-VP22/E7 replicon particles as described in Example I. With increasing dosages of SINrep5-VP22/E7 particles, the number of E7-specific CD8 + T cell precursors increased gradually, reaching a plateau at the dose of 5x10 6 IU/mouse. The number of IFN- ⁇ -producing E7-specif ⁇ c CD8 + T cells was determined using flow cytometry in the presence (solid columns) or absence (open columns) of MHC class I restricted E7 peptide (aa 49-57).
  • FIG. 5A In vivo tumor treatment experiments to demonstrate the antitumor effect generated by SLNrep5 replicon particles against TC-1 tumors. Mice were challenged and treated as described in the Materials and Methods.
  • Fig. 5A Treatment of pulmonary nodules with SINrep5-VP22/E7 replicons relative to other SINrep5 constructs. Mice treated with SINrep5-VP22/E7 replicon particles displayed a significantly lower mean number of pulmonary nodules 3 days after tumor challenge (0.7+0.3) than mice treated with the other SINrep5 replicon particle vaccines.
  • Fig. 5A Treatment of pulmonary nodules with SINrep5-VP22/E7 replicons relative to other SINrep5 constructs. Mice treated with SINrep5-VP22/E7 replicon particles displayed a significantly lower mean number of pulmonary nodules 3 days after tumor challenge (0.7+0.3) than mice treated with the other SINrep5 re
  • mice treated with STNrep5-VP22/E7 replicon particles exhibited a significantly lower mean number of pulmonary nodules three days (0.7+0.3), seven days (0.5+0.3), or fourteen days (25.0+4.0) after tumor challenge compared to SLNrep5 control (no insert) (one-way ANOVA, P ⁇ 0.05).
  • Figure 6A and 6B In vivo tumor treatment experiment to compare the antitumor effect in mice treated with VP22/E7 naked DNA, naked SLNrep5-VP22/E7 RNA replicons, or SINrep5-VP22/E7 RNA replicon particles. Mice were challenged with TC-1 and treated with VP22/E7 naked DNA, naked SINrep5-VP22/E7 RNA replicons, or SINrep5-VP22/E7 RNA replicon particles as described in Example I.
  • Fig 6A Treatment of pulmonary tumor nodules with various VP22/E7-containing vaccines.
  • mice treated with SINrep5-VP22/E7 replicon particles displayed a significantly lower mean lung weight after tumor challenge than mice treated with VP22/E7 DNA and naked SPNrep5-VP22/E7 RNA replicon vaccines.
  • Fig IB Representative gross pictures of pulmonary metastatic nodules in mice treated with the different VP22/E7-containing vaccines.
  • FIG. 7 In vivo antibody depletion experiments to determine the effect of lymphocyte subsets on the potency of SINrep5-VP22/E7 replicon particles as a vaccine. Mice were immunized intramuscularly with 5x 10 6 IU/mouse of SINrep5-VP22/E7 replicon particles. CD4, CD8 and NK1.1 depletions were initiated one week after vaccination. Two weeks after vaccination, mice were challenged with 10 4 TC-1 cells/mouse subcutaneously. All na ⁇ ve mice and all mice depleted of CD8 + T cells grew tumors within 14 days after tumor challenge.
  • FIG. 8A-8D TUNEL assay of apoptotic cells in the skeletal muscle of vaccinated mice. These photomicrographs show mus-cle tissue at the injection sites from (A) control mice immunized with normal saline, (B) mice immunized with VP22-E7 DNA, (C) mice immunized with VP22-E7 RNA, and (D) mice immunized with SINre ⁇ 5-VP22/E7 replicon particles.
  • Vaccination with SLNrep5-VP22/E7 replicon particles induced a greater degree of apoptosis in muscle tissue compared with the other groups.
  • FIG. 9 Activity of E7-specific CTL.
  • BHK21 cells were first infected with various SLNrep5 replicon particles. Infected BHK21 cells were co-incubated with bone marrow-derived DCs. DCs were used as target cells and an E7-specific CD8 + T cell line served as effector cells.
  • CTL assays with various E:T ratios were performed.
  • the SINrep5-VP22/E7 replicon particle vaccine generated greater cytotoxicity (measured at E:T ratios of 9 and 27 (p ⁇ 0.01)) compared to BHK21 cells infected with SINrep5-E7 replicon particles.
  • the CTL assays shown here are from one representative experiment of two performed. DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the invention provides compositions and methods for enhancing the immune responses, particularly cytotoxic T cell immune responses, induced by ex vivo or in vivo administration of nucleic acid vaccines that encode chimeric polypeptides.
  • the preferred chimeric or fusion polypeptide comprises (1) at least one first polypeptide or peptide that, upon introduction to cells ofthe host immune system, in vitro or in vivo, promotes or potentiates immunogenicity ofthe second polypeptide or peptide (the antigen). For this reason, the first polypeptide has been termed an "immunogenicity-potentiating (or promoting) polypeptide, abbreviated "IPP". These are described in more detail below.
  • the nucleic acid vaccine further comprises (2) at least one second polypeptide or peptide that is an antigenic polypeptide or peptide in the host against which it is desired to induce an immune response.
  • the chimeric or fusion polypeptides are "indirectly" administered by administration of a nucleic acid vector that encodes the chimeric molecule; the nucleic acid construct, and thus the fusion protein, is expressed in vivo.
  • this nucleic acid construct of vector is in the form of replication-defective alphaviruses generated from stable alphavirus packaging cell lines ("PCL").
  • the IPP is a polypeptide that acts by promoting (1) processing via the MHC class I pathway.
  • MHCi-PP polypeptide or peptide that promotes processing via the MHC class I pathway.
  • MHCi-PP polypeptide that promotes processing via the MHC class I pathway.
  • Hsp70 a polypeptide or peptide that promotes processing via the MHC class I pathway.
  • a preferred fragment is a C-terminal domain ("CD") of Hsp70, which is designated "Hsp70cD"-
  • Hsp70cD spans from about residue 312 to the C terminus of Hsp70 (SEQ ID NO:9).
  • a preferred shorter polypeptide spans from about residue 517 to the C-terminus of SEQ ID NO:9. Shorter peptides from that sequence that have the ability to promote protein processing via the MHC-1 class I pathway are also included, and may be defined by routine experimentation.
  • MHQ-PP Another category of MHQ-PP is an ER chaperone polypeptide such as calreticulin, ER60, GRP94 or gp96, well-characterized ER chaperone polypeptides that representatives ofthe HSP90 family of stress-induced proteins (Argon (1999) Semin. Cell Dev.. Biol. 10:495-505; Sastry (1999) J Biol. Chem. 274:12023-12035; Nicchitta (1998) Curr. Opin. Immunol. 10:103-109; U.S. Patent 5,981,706))
  • cytoplasmic translocation polypeptide domains of pathogen toxins such as domain II of Pseudomonas exotoxin ETA (ETAd ⁇ ) or of similar toxins from Diptheria, Clostridium, Botulinum, Bacillus, Yersinia, Vibrio cholerae, or B or detella pertussis; or active fragments or domains of any ofthe foregoing polypeptides.
  • pathogen toxins such as domain II of Pseudomonas exotoxin ETA (ETAd ⁇ ) or of similar toxins from Diptheria, Clostridium, Botulinum, Bacillus, Yersinia, Vibrio cholerae, or B or detella pertussis; or active fragments or domains of any ofthe foregoing polypeptides.
  • Polypeptides that route a linked protein to the cell centrosome compartment promote processing for antigen presentation.
  • linkage of ⁇ -tubulin to an antigen (E7 protein) efficiently re-routed E7 into the centrosome compartment, making ⁇ -tubulin a useful IPP according to this invention.
  • DC-PP a polypeptide or peptide that promotes development or activity of APCs or targeting of APCs, , preferably DCs, is termed a "DC-PP".
  • IP Ps immunostimulatory cytokines that target APCs, primarily eferably DCs, such as granulocyte macrophage colony stimulating factor (GM-CSF), or active fragments or domains thereof.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • DNA encoding the cytokine GM-CSF gene to DNA encoding an antigen e.g., an HTV or hepatitis C antigen
  • an antigen e.g., an HTV or hepatitis C antigen
  • the chimeric GM- CSF/antigen is believed to act as an immunostimulatory signal to DCs, inducing their differentiation from an immature form (Banchereau, J et al, Nature 392: 245-52, 1998). Since DCs and their precursors express high levels of GM-CSF receptors, the chimeric GM- CSF/antigen should target and concentrate the linked antigen to the DCs and further improve the vaccine's potency.
  • the Flt-3 ligand (FL) stimulates growth of DC precursors.
  • the constructs ofthe present invention include FL, preferably its ECD. FL also targets a linked antigen to DCs thereby promoting antigen presentation.
  • the APCs targeted by the compositions ofthe present invention jnclude DCs keratinocytes, astrocytes, monocytes, macrophages, B lymphocytes, a microglial cell, or activated endothelial cells, and the like, although DC are a preferred target.
  • VP22 a herpes simplex virus type 1 (HSV-1) protein
  • HSV-1 herpes simplex virus type 1
  • MDV avian Marek's Disease Virus
  • both polypeptides enhanced DNA vaccine potency when linked to a "model” antigen, E7 9, as disclosed by the present inventors in WO 02/09645. It is important to note that not all molecules with "trafficking properties" have this action of enhancing vaccine potency.
  • the present inventors found, when analyzing naked DNA vaccines comprising E7 DNA fused to DNA encoding sequences derived from proteins with trafficking properties such as HIV TAT protein, the membrane-translocating sequence and the third helix ofthe Antennapedia homeodomain did not generate CD8+ T cell- responses of similar potency as those induced by VP22/E7. Therefore, they concluded that VP22 and homologues thereof have a unique property or properties that distinguish them from these other constructs.
  • the order in which the two (or more) component polypeptides ofthe present fusion protein of this invention are arranged, and therefore, the order ofthe encoding nucleic acid fragments in the nucleic acid vector, can be altered without affecting immunogenicity ofthe fusion polypeptides proteins and the utility ofthe composition.
  • the Hsp70- encoding (or FL -encoding) DNA sequences may be located 5' or 3' to the target antigen- encoding sequences, h one embodiment, these polypeptide-encoding nucleic acid domains are in-frame so that the DNA construct encodes a recombinant fusion polypeptide in which the antigen is located N- terminal to the Hsp70 or FL derived polypeptide.
  • the vaccines ofthe present invention include, the antigenic epitope itself and an IPP, such as an MHQ-PP like Hsp70 or its active domain (CD), or a DC-PP such as FL, or a intercellular spreading protein such as VP22, as summarized above and describe in more detail below.
  • an IPP such as an MHQ-PP like Hsp70 or its active domain (CD), or a DC-PP such as FL, or a intercellular spreading protein such as VP22, as summarized above and describe in more detail below.
  • the vaccine construct ofthe present invention optionally, may also include more than one ofthe foregoing IPPs.
  • Another useful polypeptide for the present constructs is a costimulatory signal, such as a B7 family protein, including B7-DC (see commonly assigned U.S. patent application Serial No. 09/794,210), B7.1, B7.2, soluble CD40, etc.).
  • B7-DC see commonly assigned U.S. patent application Serial No. 09/794,210
  • HPV-16 E7 was used as a model antigen for vaccine development because human papillomaviruses (HPVs), particularly HPV-16, are associated with most human cervical cancers.
  • HPVs human papillomaviruses
  • HPV-16 human papillomaviruses
  • the oncogenic HPV protein E7 is important in the induction and maintenance of cellular transformation and co-expressed in most HPV-containing cervical cancers and their precursor lesions. Therefore, cancer vaccines, such as the compositions ofthe invention, that target E7 can be used to control of HPV-associated neoplasms (Wu (1994) Curr. Opin. Immunol. 6:746-754).
  • antigen or "immunogen” as used herein refers to a compound or composition comprising a peptide, polypeptide or protein which is “antigenic” or “immunogenic” when administered (or expressed in vivo by an administered nucleic acid, e.g., a DNA vaccine) in an appropriate amount (an “immunogenically effective amount"), i.e., capable of inducing, eliciting, augmenting or boosting a cellular and/or humoral immune response either alone or in combination or linked or fused to another substance (which can be administered at once or over several intervals).
  • An immunogenic composition can comprise an antigenic peptide of at least about 5 amino acids, a peptide of 10 amino acids in length, a polypepide fragment of 15 amino acids in length, 20 amino acids in length or longer.
  • Smaller immunogens may require presence of a "carrier" polypeptide e.g., as a fusion protein, aggregate, conjugate or mixture, preferablyl linked (chemically or otherwise) to the immunogen.
  • the immunogen can be recombinantly expressed from a vaccine vector, which can be naked DNA comprising the immunogen' s coding sequence operably linked to a promoter, e.g., an expression cassette as described herein.
  • the immunogen includes one or more antigenic determinants or epitopes which may vary in size from about 3 to about 15 amino acids.
  • epitope refers to an antigenic determinant or antigenic site that interacts with an antibody or a T cell receptor (TCR), e.g., the MHC class I-binding peptide compositions (or expressed products ofthe nucleic acid compositionsof the invention) used in the methods ofthe invention.
  • TCR T cell receptor
  • An "antigen” is a molecule or chemical structure that either induces an immune response or is specifically recognized or bound by the product or mediator of an immune response, such as an antibody or a CTL.
  • TCRs bind to peptide epitopes which are physically associated with a third molecule, a major histocompatibility complex (MHC) class I or class U protein.
  • MHC major histocompatibility complex
  • recombinant refers to (1) a nucleic acid or polynucleotide synthesized or otherwise manipulated in vitro, (2) methods of using recombinant DNA technology to produce gene products in cells or other biological systems, or (3) a polypeptide encoded by a recombinant nucleic acid.
  • the FL-encoding nucleic acid or polypeptide, the nucleic acid encoding an MHC class I-binding peptide epitope (antigen) or the peptide itself can be rececombinant.
  • Recombinant means includes ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into a single unit in the form of an expression cassette or vector for expression ofthe coding sequences in the vectors resulting in production ofthe encoded polypeptide.
  • the present inventors investigated the novel use of VP22 proteins linked to a model antigen (HPV-16 E7) in the context of a PCL-generated replication-defective Sinbis virus replicon vaccine and found that it led to the spread of linked antigen to surrounding cells and enhanced antigen-specific immune responses and antitumor effects.
  • HPV-16 E7 a model antigen linked to a model antigen linked to a PCL-generated replication-defective Sinbis virus replicon vaccine
  • HPV-E7 protein The "wild-type” amino acid sequence of HPV-E7 protein is provided below:
  • Production of various vectors may result in loss of certain residues ofthe antigen without affecting the immunogenicity ofthe vaccine and the specificity ofthe immune response.
  • the present inventors have described elsewhere a pcDNA3 naked DNA vector wherein only 96 ofthe 98 residues of E7 are present and the C-terminal two residues of wild-type E7, Lys and Pro are absent from this contstruct.
  • This is an example of a deletion variant.
  • Such deletion variants e.g., terminal truncation of two or a small number of amino acids
  • of other antigenic polypeptides are examples ofthe embodiments intended within the scope ofthe fusion polypeptides of this invention.
  • Such a modified HPV-E7 (nucleic acid sequence is SEQ ID NO:2; amino acid sequence is SEQ ID NO:3) is shown below: 1/1 31/11 atg cat gga gat aca ect aca ttg cat gaa tat atg tta gat ttg caa cca gag aca act
  • a preferred intercellular spreading protein is preferably a viral spreading protein, most preferably a herpesvirus VP22 protein.
  • a viral spreading protein most preferably a herpesvirus VP22 protein.
  • HVP22 herpes simplex virus- 1
  • MDV-VP22 or MVP-22 herpes simplex virus
  • homologues of VP22 from other members ofthe herpesviridae or polypeptides from nonviral sources that are considered to be homologous and share the functional characteristic of promoting intercellular spreading of a polypeptide or peptide that is fused or chemically conjugated thereto.
  • DNA encoding HVP22 has the sequence SEQ ID NO:4 which is shown as nucleotides 1- 903 below.
  • the VP22 amino acid sequence (1-301) is SEQ ID NO:5. 1/1 31/11
  • GAG glu DNA encoding MVP22 is SEQ ID NO:6 shown below: atg ggg gat tct gaa agg egg aaa teg gaa egg cgt cgt tec ctt gga 48 tat ccc tct gca tat gat gac gtc teg att ect get cgc aga cca tea 96 aca cgt act cag cga aat tta aac cag gat gat ttg tea aaa cat gga 144 cca ttt ace gac cat cca aca caa aaa cat aaa teg gcg aaa gcc gta 192 teg gaa gac gtt teg tct ace ace egg ggt ggc ttt aca aac aaa
  • the amino acid sequence ofthe MDV PV22, SEQ ID NO:7, is shown below:
  • nucleic acid is SEQ LD NO:8; amino acids are SEQ ID NO:9)
  • the unmodified GENBANK nucleotide sequence encoding HSP70, SEQ LD NO: 12 is atggctcg tgcggtcggg atcgacctcg ggaccaccaa ctccgtcgtc tcggttctgg aaggtggcga cccggtcgtc gtcgccaact ccgagggctc caggaccacc ccgtcaattg tcgcgttcggccagcc cgccaagaac caggcagtga ccaacgtcga tcgcaccgtg cgctcggtca agcgacacat gggcagcgac tggtccatag agattgacgg caagaaatac acc
  • the cDNA sequence of Mouse GM-CSF (SEQ LD NO: 13) is as follows: gagctcagca agcgctctcc cccaattccc ttagccaaag tggacgccac cgaacagaca 61 gacctaggct aagaggtttg atgtctctgg ctacccgact ttgaaaattt tccgcaaagg 121 aaggcctttt gactacaatg gcccacgaga gaaaggctaa ggtcctgagg aggatgtggc 181 tgcagaattt acttttcctg ggcattgtgg tctacagcct ctcagcaccc acccgctcac 241 ccatcactgt cacccggct tggaagcatg tagaggcca
  • amino acid sequence of mouse GM-CSF (161 residues) (SEQ ID NO: 14) is :
  • a cDNA sequence encoding Pseudomonas exotoxin A (ETA) (SEQ ID NO: 15) is ctgcagctgg tcaggccgtt tccgcaacgc ttgaagtcct ggccgatata ccggcagggc 61 cagccatcgt tcgacgaata aagccacctc agccatgatg ccctttccat ccccagcgga 121 accccgacat ggacgccaaa gccctgctcc tcggcagcct ct ctggccg 181 ccgacgcggc gacgctcgac aatgctctct ct cgg cggtg 181 ccgacggc gacgctcgac aatgct
  • the encoded amino acid sequence of ETA (SEQ ID NO: 16) is
  • nucleic acid is SEQ ID NO: 17; amino acids are SEQ LD NO: 18)
  • a fusion polypeptide FL-E7 is shown below: (nucleic acid is SEQ ID NO:19; amino acids are SEQ ID NO:20). The N-terminal sequence is FL, followed by E7 (underscored, nucleic acids capitalized)
  • the antigen ofthe present invention against which immunity is desired and which may be as short as an MHC class I-binding peptide epitope is derived from a pathogen, e.g., it comprises a peptide expressed by a pathogen.
  • the pathogen can be a virus, such as, e.g. , a papilloma virus, a herpesvirus, a retrovirus (e.g. , an immunodeficiency virus, such as HIV-1), an adenovirus, and the like.
  • the papilloma virus can be a human papilloma virus; for example, the antigen (e.g., the Class I-binding peptide) can be derived from an HPV- 16 E7 polypeptide.
  • the HPV-16 E7 polypeptide is substantially non- onco genie, i.e., it does not bind retinoblastoma polypeptide (pRB) or binds pRB with such low affinity that the HP V- 16 E7 polypeptide is effectively non-oncogenic when expressed or delivered in vivo.
  • the pathogen is a bacteria, such ⁇ SB or detella pertussis; Ehrlichia chaffeensis; Staphylococcus aureus; Toxoplasma gondii; Legionella pneumophila; Brucella suis; Salmonella enterica; Mycobacterium avium; Mycobacterium tuberculosis; Listeria monocytogenes; Chlamydia trachomatis; Chlamydia pneumoniae; Rickettsia rickettsii; or, a fungi, such as, e.g., Paracoccidioides brasiliensis; or other pathogen, e.g., Plasmodium falciparum.
  • a bacteria such as ⁇ SB or detella pertussis; Ehrlichia chaffeensis; Staphylococcus aureus; Toxoplasma gondii; Legionella pneumophila; Brucella suis; Salmonella enterica; Mycobacterium avi
  • the MHC class I-binding peptide epitope is derived from a tumor cell.
  • the tumor cell-derived peptide epitope can comprise a tumor associated antigen, e.g., a tumor specific antigen, such as, e.g., a HER-2/neu antigen.
  • the methods ofthe invention provide for the administration of nucleic acid vectors encoding a fusion protein between an antigen, preferably a MHC Class I epitope binding polypeptide or peptide, used to an IPP, as has been described above.
  • Recombinant IPP- containing fusion proteins can be synthesized in vitro or in vivo.
  • Nucleic acids encoding these compositions can be prepared in in the form of "naked DNA” or they can be incorporated in plasmids, vectors, recombinant viruses (e.g., "replicons") and the like.
  • the present invention is directed ot one class of vectors, replication defective alphavirus vectors, prefereably Sinbis virus, for in vivo or ex vivo administration.
  • Nucleic acids and vectors ofthe invention can be made and expressed in vitro or in vivo, a variety of means of making and expressing these genes and vectors can be used.
  • desired expression can be obtained by modulating the activity ofthe nucleic acids (e.g., promoters) within vectors used to practice the invention.
  • nucleic acid is synonymous with "polynucleotide” and is intended to include a gene, a cDNA molecule, an mRNA molecule, as well as a fragment of any of these such as an oligonucleotide, and further, equivalents thereof (explained more fully below). Sizes of nucleic acids are stated either as kilobases (kb) or base pairs (bp).
  • Protein size is stated as molecular mass in kilodaltons (kDa) or as length (number of amino acid residues). Protein size is estimated from PAGE, from sequencing, from presumptive amino acid sequences based on the coding nucleic acid sequence or from published amino acid sequences.
  • cDNA molecules encoding the amino acid sequence corresponding to the fusion polypeptide ofthe present invention or fragments or derivatives thereof can be synthesized by the polymerase chain reaction (PCR) (see, for example, U.S. 4,683,202) using primers derived the sequence ofthe protein disclosed herein. These cDNA sequences can then be assembled into a eukaryotic or prokaryotic expression vector and the resulting vector can be used to direct the synthesis ofthe fusion polypeptide or its fragment or derivative by appropriate host cells, for example COS or CHO cells.
  • This invention includes isolated nucleic acids having a nucleotide sequence encoding the novel fusion polypeptides that comprise a spreading protein and an antigen, fragments thereof or equivalents thereof.
  • nucleic acid as used herein is intended to include such fragments or equivalents.
  • the nucleic acid sequences of this invention can be DNA or RNA.
  • a cDNA nucleotide sequence the fusion polypeptide can be obtained by isolating total mRNA from an appropriate cell line. Double stranded cDNA is prepared from total mRNA.
  • cDNA can be inserted into a suitable plasmid, bacteriophage or viral vector using any one of a number of known techniques.
  • the term "equivalent" is intended to include sequences encoding structurally homologous and/or a functionally equivalent proteins.
  • a natural polymorphism of a sequence encoding an LPP such as viral VP22 spreading protein or CRT, or the like, (especially at the third base of a codon) may be manifest as "silent" mutations which do not change the amino acid sequence.
  • an LPP such as viral VP22 spreading protein or CRT, or the like
  • isoforms or family members are defined as proteins that share function amino acid sequence similarity to the reference protein.
  • a fragment ofthe nucleic acid sequence is defined as a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length IPP, antigenic polypeptide or the fusion thereof.
  • This invention includes such nucleic acid fragments that encode polypeptides which retain (1) the ability ofthe fusion polypeptide to induce increases in frequency or reactivity of T cells, preferably CD8+ T cells, that are specific for the antigen portion ofthe fusion polypeptide.
  • a nucleic acid fragment as intended herein encodes a VP22 or HSP70 or
  • CRT or FL or other type of IPP that retains the ability to improve the immunogenicity of an antigen when administered as a fusion polypeptide with an antigenic polypeptide or peptide.
  • the nucleic acid sequence encoding a fragment of an IPP polypeptide comprises nucleotides from the sequence encoding the mature protein (i.e., excluding signal peptide sequences).
  • Nucleic acid sequences of this invention may also include linker sequences, natural or modified restriction endonuclease sites and other sequences that are useful for manipulations related to cloning, expression or purification of encoded protein or fragments. These and other modifications of nucleic acid sequences are described herein or are well-known in the art.
  • the techniques for assembling and expressing DNA coding sequences for ffPs such as spreading proteins, proteins or ER-such as VP22 and antigenic polypeptides such as synthesis of oligonucleotides, PCR, transforming cells, constructing vectors, expression systems, and the like are well-established in the art. Those of ordinary skill are familiar with the standard resource materials for specific conditions and procedures. EXPRESSION VECTORS AND HOST CELLS
  • This invention includes an expression vector comprising a nucleic acid sequence encoding a spreading protein/antigen fusion polypeptide operably linked to at least one regulatory sequence.
  • "Operably linked” means that the coding sequence is linked to a regulatory sequence in a manner that allows expression ofthe coding sequence.
  • Known regulatory sequences are selected to direct expression ofthe desired protein in an appropriate host cell. Accordingly, the term “regulatory sequence” includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in, for example, Goeddel, Gene
  • an expression vector of this invention depends on considerations such as the host cell to be transfected and/or the type of protein to be expressed.
  • the present expression vectors comprise the full range of nucleic acid molecules encoding the various embodiments ofthe fusion polypeptide and its functional derivatives (defined herein) including polypeptide fragments, variants, etc.
  • Such expression vectors are used to transfect host cells for expression ofthe DNA and production ofthe encoded proteins which include fusion proteins or peptides. It will be understood that a genetically modified cell expressing the fusion polypeptide may transiently express the exogenous DNA for a time sufficient for the cell to be useful for its stated purpose.
  • the present in invention provides methods for producing the fusion polypeptides, fragments and derivatives.
  • a host cell transfected with a nucleic acid vector that encodes the fusion polypeptide is cultured under appropriate conditions to allow expression of the polypeptide.
  • Host cells may also be transfected with one or more expression vectors that singly or in combination comprise DNA encoding at least a portion ofthe fusion polypeptide and DNA encoding at least a portion of a second protein, so that the host cells produce yet further fusion polypeptides that include both the portions.
  • a culture typically includes host cells, appropriate growth media and other byproducts. Suitable culture media are well known in the art.
  • the fusion polypeptide can be isolated from medium or cell lysates using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, fractionation column chromatography (e.g.
  • the recombinant polypeptides ofthe invention can be utilized in pharmaceutical compositions as described in more detail herein.
  • Prokaryotic or eukaryotic host cells transformed or transfected to express the fusion polypeptide or a homologue or functional derivative thereof are within the scope ofthe invention.
  • the fusion polypeptide may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO) or human cells.
  • Other suitable host cells may be found in Goeddel, (1990) supra or are otherwise known to those skilled in the art.
  • Expression in eukaryotic cells leads to partial or complete glycosylation and/or formation of relevant inter- or infra-chain disulfide bonds ofthe recombinant protein.
  • Vector systems for the expression of heterologous genes have been developed from full- length cDNA clones of three members ofthe alphavirus genus, Sindbis virus ("SIN”), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE) (Xiong et al, 1989; Huang, HV et al, 1989, Virus Genes 3:85-91; Liljestrom, P et al, , 1991, BioTechnology 9:1356-1361; Bredenbeek, PJ et ⁇ /., 1993, J. Virol. 67:6439-6446; Zhou, X et al, 1994, Vaccine 12:1510- 1514; Davis et al, 1996, J.
  • Sindbis virus Sindbis virus
  • SFV Semliki Forest virus
  • VEE Venezuelan equine encephalitis virus
  • the viral structural protein genes are deleted and replaced by a heterologous gene under the control ofthe 26S promoter.
  • the structural genes are provided in trans from a helper construct( s) consisting of a partial clone ofthe viral genome which is missing all or part ofthe genes for the nonstructural proteins, including the putative cis-acting RNA packaging signals (Weiss, B et a/., 1989, J Virol. ⁇ 3:5310-5318; Weiss, B. et al, 1994, Nucleic Acids Res. 22:780-786).
  • Both the replicon and the helper RNAs retain the cis-acting terminal sequences required for genome replication and the promoter for transcription ofthe subgenomic mRNA.
  • the replicon RNA supplies the nonstructural proteins in trans for the expression ofthe helper RNA.
  • only the replicon RNA retains the packaging signal(s), and it is packaged into replicon particles by the viral structural proteins provided in trans by the helper. Infection of cells by these replicon particles results in amplification of replicon RNA and expression ofthe heterologous gene, but there is no further spread to other cells.
  • Alphavirus replicon vectors have been used to vaccinate against microbial pathogens (Zhou, X et al, 1995, Proc. Natl. Acad. Sci. USA 92:3009-3013; Mossman, SP et al, 1996, J. Virol. 70:1953-1960) and offer several potential advantages as delivery systems.
  • replicons typically express heterologous genes to high levels, permittin a relatively low dose of replicon particles to produce a large dose of immunogen in vivo. Because they lack a complete complement of viral genes, after replication and expression ofthe heterologous gene in the cells initially infected, no additional infectious particles would be produced to spread to other tissues.
  • VEE glycoproteins will preferentially target heterologous gene expression to lymphoid tissues (Davis, NL et al, 1996, J. Virol.
  • VEE-based replicon vector either (1) included previously defined attenuating mutations in the replicon and/or its helper so that any viable recombinant virus is could not initiate a virulent infection; or (2) used a bipartite helper to supply structural proteins for packaging ofthe replicon into particles, thus requiring at least two recombination events for the generation of viable virus.
  • VEE replicon particle vaccine prepared in this manner, Pushko and colleagues induced potent protective immunity to a heterologous mucosal pathogen in naive animals and have achieved an equally high level, protective response in animals previously immunized with VEE replicon particle-expressing genes from another pathogen.
  • DHRNAs Defective-helper RNAs
  • DHRNAs Defective-helper RNAs
  • Packaging of SLN replicons is achieved by efficient cotransfection of BHK cells with both RNAs by electroporation (Liljestrom et al, 1991, supra) (See also, Frolov et al, 1996, supra; Fig. 3).
  • Replicase/franscriptase functions supplied by the vector RNA lead not only to its own amplification but also act in trans to allow replication and transcription ofthe helper RNA. This results in synthesis of structural proteins that can package the replicon with >10 infectious particles per ml (5xl0 9 infectious particles per electroporation) being produced after only 16-24 h. Such stocks can be used without further phenotypic selection to infect cells for expression studies or high-level protein production. According to Frolov et al, supra, it should be possible to package replicons containing at least 5 kb of heterologous sequence. A spectrum of DHRNAs have been characterized that differ in their ability to be packaged.
  • DHRNAs that allow packaging ofthe replicon as well as themselves are useful under conditions where extensive amplification by passaging is advantageous.
  • Other DHRNAs allow efficient packaging of replicons but are packaged very poorly themselves (Frolov et al, supra; Liljestrom et al, supra; Bredenbeek et al, supra; Geigenmuller-Gnirke et al, supra). These latter helpers are useful when expression of viral structural proteins and virus spread are not desired.
  • DHRNAs One approach to minimize the possibility of recombination between replicon and helper RNAs to produce wild-type virus is to use two DHRNAs, one that expresses the capsid protein and a second that expresses the viral glycoproteins (Frolov et al, supra).
  • the capsid protein expressed independently, accumulates at high levels, but to achieve similar levels of viral glycoprotein expression retention ofthe 5' terminus ofthe capsid protein mRNA, which acts as a translational enhancer, is required.
  • capsid protein genes that preserve both the 5' terminus (the enhancer region) and the 3' half (the sequences that code for the autoprotease activity) but eliminate the region that binds RNA, produce high levels of glycoprotein expression from a second DHRNA.
  • Capsid protein genes from heterologous alphaviruses can also be used to enhance translation ofthe glycoproteins and should further reduce the probability of wild-type virus generation via recombination.
  • RNA replicons In addition to packaging of alphavirus RNA replicons by cotransfection with DHRNAs, continuous packaging cell lines have been developed that express a DHRNA under the control of a nuclear promoter. Such cells may be useful for rescuing transfected RNA replicons, titering packaged replicons, and production of large quantities of packaged replicon stocks by low- multiplicity passage.
  • Variants ofthe prototype alphavirus, SLN, with differential abilities to infect human dendritic cells were described by Gardner JP et al. , 2000, J Virol 74:11849- 11857.
  • the genetic determinant for human DC tropism maps to a single amino acid substitution at residue 160 ofthe envelope glycoprotein E2.
  • Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant confers a similar ability to efficiently infect immature human DC, whhich are induced to undergo rapid activation and maturation.
  • the SIN replicon particles infected skin- resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of MHC and costimulatory molecules.
  • the present invention provides a dual approach to enhancing the potency of nucleic acid vaccines.
  • the present nucleic acid constructs are designed to target MHC class I processing pathways, to target DCs, to stimulate DC maturation, activation, etc., as described.
  • the vector system provides a potential of using a directed approach to generate ialphavirus vaccine vectors that target and activate APCs.
  • alphavirus replicons as expression vectors are noted above are well-known in the art, as are corresponding packaging cells that permit their production in relatively high quantities.
  • a proteolytic cleavage site may be introduced at the junction ofthe reporter group and the target protein to enable separation ofthe target protein from the reporter group subsequent to purification ofthe fusion protein.
  • Proteolytic enzymes for such cleavage and their recognition sequences include Factor Xa, thrombin and enterokinase.
  • One embodiment of this invention is a transfected cell which expresses novel fusion polypeptide.
  • DNA sequences which are used for the present constructs are available from a number of sources.
  • Backbone vectors and control systems are generally found on available "host" vectors which are used for the bulk ofthe sequences in construction.
  • initial construction maybe, and usually is, a matter of retrieving the appropriate sequences from cDNA or genomic DNA libraries.
  • sequence is disclosed it is possible to synthesize the entire gene sequence in vitro starting from the individual nucleotide derivatives.
  • genes of sizeable length e.g., 500-1000 bp may be prepared by synthesizing individual overlapping complementary oligonucleotides and filling in single stranded nonoverlapping portions using DNA polymerase in the presence ofthe deoxyribonucleoti.de triphosphates.
  • This approach has been used successfully in the construction of several genes of known sequence. See, for example, Edge, M. D., Nature (1981) 292:756; Nambair, K. P., et al, Science (1984) 223:1299; and Jay, E., JBiol Chem (1984) 259:6311.
  • Synthetic oligonucleotides are prepared by either the phosphotriester method as described by references cited above or the phosphoramidite method as described by Beaucage, S. L., and Caruthers, M. H., TetLett (1981) 22:1859; and Matteucci, M. D., and Caruthers, M. H., J Am Chem Soc (1981) 103:3185 and can be prepared using commercially available automated oligonucleotide synthesizers.
  • kinase treatment of single strands prior to annealing or for labeling is achieved using an excess, e.g., about 10 units of polynucleotide kinase to 1 nmole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl 2 , 5 mM dithiothreitol, 1-2 mM ATP, 1.7 pmoles ⁇ - 32 P-ATP (2.9 mCi/mmole), 0.1 mM spermidine, 0.1 mM EDTA.
  • an excess e.g., about 10 units of polynucleotide kinase to 1 nmole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl 2 , 5 mM dithiothreitol, 1-2 mM ATP, 1.7 pmoles ⁇ - 32 P-ATP (2.9 mCi/mmole), 0.1 mM sperm
  • the components ofthe desired vectors can be excised and ligated using standard restriction and ligation procedures.
  • Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog, hi general, about 1 mg of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ml of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to insure complete digestion ofthe DNA substrate, hicubation times of about one hour to two hours at about 37°C. are workable, although variations can be tolerated.
  • protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol.
  • size separation ofthe cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymology (1980) 65:499-560.
  • Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleoti.de triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20° to 25° C. in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 0.1-1.0 mM dNTPs.
  • the Klenow fragment fills in at 5' single-stranded overhangs but chews back protruding 3 ' single strands, even though the four dNTPs are present.
  • selective repair can be performed by supplying only one ofthe, or selected, dNTPs within the limitations dictated by the nature ofthe overhang. After treatment with Klenow, the mixture is extracted with phenol/chloroform and ethanol precipitated. Treatment under appropriate conditions with SI nuclease or BAL-31 results in hydrolysis of any single-stranded portion.
  • Ligations are typically performed in 15-50 ml volumes under the following standard conditions and temperatures: for example, 20 mM Tris-HCl pH 7.5, lOmM MgCl 2 , 10 mM DTT, 33 ⁇ g/ml BSA, 10-50mM NaCl, and either 40 ⁇ M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for "sticky end” ligation) or ImM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for "blunt end” ligation).
  • Intermolecular "sticky end” ligations are usually performed at 33-100 ⁇ g/ml total DNA concentrations (5-100 nM total end concentration). Intermolecular blunt end ligations are performed at 1 mM total ends concentration.
  • vector construction employing "vector fragments” the fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase (CIAP) in order to remove the 5' phosphate and prevent self-ligation. Digestions are conducted at pH 8 in approximately 10 mM Tris-HCl, 1 mM EDTA using BAP or CIAP at about 1 unit/mg vector at 60° for about one hour. The preparation is extracted with phenol/chloroform and ethanol precipitated. Alternatively, re-ligation can be prevented in vectors which have been double digested by additional restriction enzyme and separation ofthe unwanted fragments.
  • Any of a number of methods are used to introduce mutations into the coding sequence to generate the variants ofthe invention. These mutations include simple deletions or insertions, systematic deletions, insertions or substitutions of clusters of bases or substitutions of single bases.
  • modifications ofthe IPP or the antigenic polypeptide DNA sequence are created by site-directed mutagenesis, a well-known technique for which protocols and reagents are commercially available (Zoller, MJ et al, Nucleic Acids Res (1982) 10:6487-6500 and Adelman, JP et al, DNA (1983) 2:183-193)).
  • Correct ligations for plasmid construction are confirmed, for example, by first transforming E. coli strain MCI 061 (Casadaban, M., et al, J Mol Biol (1980) 138: 179-207) or other suitable host with the ligation mixture.
  • transformants are selected based on the presence ofthe ampicillin-, tetracycline- or other antibiotic resistance gene (or other selectable marker) depending on the mode of plasmid construction. Plasmids are then prepared from the transformants with optional chloramphenicol amplification optionally following chloramphenicol amplification ((Clewell,
  • vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dexfran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. supra and other standard texts.
  • a proteolytic cleavage site is introduced at the junction ofthe reporter group and the target protein to enable separation ofthe target protein from the reporter group subsequent to purification ofthe fusion protein.
  • Proteolytic enzymes for such cleavage and their recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-fransferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • hiducible non-fusion expression vectors include pTrc (Amann et al, (1988) 7ene 69: 301-315) and pET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). While target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc, expression of target genes inserted into pET lid relies on transcription from the T7 gnlO-lacO fusion promoter mediated by coexpressed viral RNA polymerase (T7gnl). Th is viral polymerase is supplied by host strains BL21 (DE3) or HMS 174(DE3) from a resident ⁇ prophage harboring a T7gnl under the transcriptional control ofthe lacUV 5 promoter.
  • a promoter region of a DNA or RNA molecule binds RNA polymerase and promotes the transcription of an "operably linked" nucleic acid sequence.
  • a "promoter sequence” is the nucleotide sequence ofthe promoter which is found on that strand ofthe DNA or RNA which is transcribed by the RNA polymerase.
  • Two sequences of a nucleic acid molecule, such as a promoter and a coding sequence are "operably linked” when they are linked to each other in a manner which permits both sequences to be transcribed onto the same RNA transcript or permits an RNA transcript begun in one sequence to be extended into the second sequence.
  • two sequences such as a promoter sequence and a coding sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript ofthe operably linked coding sequence.
  • a promoter sequence and a coding sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript ofthe operably linked coding sequence.
  • two sequences In order to be "operably linked" it is not necessary that two sequences be immediately adjacent to one another in the linear sequence.
  • the preferred promoter sequences ofthe present invention must be operable in mammalian cells and may be either eukaryotic or viral promoters. Although preferred promoters are described in the Examples, other useful promoters and regulatory elements are discussed below. Suitable promoters may be inducible, repressible or constitutive. An example of a constitutive promoter is the viral promoter MS V-LTR, which is efficient and active in a variety of cell types, and, in contrast to most other promoters, has the same enhancing activity in arrested and growing cells. Other preferred viral promoters include that present in the CMV-LTR (from cytomegalovirus) (Bashart, M.
  • TK promoter of Herpes virus McKnight, S., Cell 31:355-365 (1982)
  • SV40 early promoter Boist, C, et al, Nature 290:304-310 (1981)
  • yeast gal4 gene promoter Johnston, S.A., et al, Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P.A., et al, Proc. Natl. Acad. Sci. (USA) 5 :5951-5955 (1984)).
  • the promoter region may further include an octamer region which may also function as a tissue specific enhancer, by interacting with certain proteins found in the specific tissue.
  • the enhancer domain ofthe DNA construct ofthe present invention is one which is specific for the target cells to be transfected, or is highly activated by cellular factors of such target cells.
  • vectors examples include vectors or retrovirus
  • retroviral enhancers e.g., viral LTR
  • the enhancer is preferably placed upstream from the promoter with which it interacts to stimulate gene expression.
  • the endogenous viral LTR may be rendered enhancer-less and substituted with other desired enhancer sequences which confer tissue specificity or other desirable properties such as transcriptional efficiency.
  • the nucleic acid sequences ofthe invention can also be chemically synthesized using standard techniques.
  • Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated with commercially available DNA synthesizers (See, e.g., Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
  • the present invention includes a molecular vaccine encoding a "functional derivative" of an IPP such as the intercellular spreading protein VP22.
  • a functional derivative is an amino acid substitution variant, a "fragment,” or a “chemical derivative” of VP22, which terms are defined below.
  • a functional derivative retains measurable VP22-like activity, preferably that of promoting intercellular spreading and immunogenicity of one or more antigenic epitopes fused thereto, which permits its utility in accordance with the present invention.
  • “Functional derivatives” encompass “variants” and “fragments” regardless of whether the terms are used in the conjunctive or the alternative herein.
  • a functional homologue must possess the above biochemical and biological activity, hi view of this functional characterization, use of homologous VP22 proteins including proteins not yet discovered, fall within the scope ofthe invention if these proteins have sequence similarity and the recited biochemical and biological activity.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • Cys residues are aligned.
  • the length of a sequence being compared is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% ofthe length ofthe reference sequence (e.g., VP22, SEQ LD NO:5 ).
  • the amino acid residues (or nucleotides) at corresponding amino acid positions (or nucleotide) positions are then compared. When a position in the first sequence is occupied by the same amino acid residue (or nucleotide) as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").
  • the percent identity between the two sequences is a function ofthe number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment ofthe two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol Biol. - 5:444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences ofthe present invention can further be used as a "query sequence" to perform a search against public databases, for example, to identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J Mol. Biol. 275:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters ofthe respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • a homologue of HVP22 described above is characterized as having (a) functional activity of native HVP22 and (b) sequence similarity to a native VP22 protein (such as SEQ ID NO:5) when determined above, of at least about 20% (at the amino acid level), preferably at least about 40%, more preferably at least about 70%, even more preferably at least about 90%.
  • the fusion protein's biochemical and biological activity can be tested readily using art-recognized methods such as those described herein, for example, a T cell proliferation, cytokine secretion or a cytolytic assay, or an in vivo assay of tumor protection or therapy.
  • a biological assay ofthe stimulation of antigen-specific T cell reactivity will indicate whether the homologue has the requisite activity to qualify as a "functional" homologue.
  • a “variant” of a HVP22 refers to a molecule substantially identical to either the full protein or to a fragment thereof in which one or more amino acid residues have been replaced (substitution variant) or which has one or several residues deleted (deletion variant) or added (addition variant).
  • a “fragment” of HVP22 refers to any subset ofthe molecule, that is, a shorter polypeptide ofthe full-length protein.
  • a number of processes can be used to generate fragments, mutants and variants ofthe isolated DNA sequence. Small subregions or fragments ofthe nucleic acid encoding the spreading protein, for example 1-30 bases in length, can be prepared by standard, chemical synthesis. Antisense oligonucleotides and primers for use in the generation of larger synthetic fragment.
  • a preferred group of variants are those in which at least one amino acid residue and preferably, only one, has been substituted by different residue.
  • the types of substitutions that may be made in the protein molecule may be based on analysis ofthe frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al. (supra) and Figure 3-9 of Creighton (supra). Based on such an analysis, conservative substitutions are defined herein as exchanges within one ofthe following five groups:
  • substitutions are (i) substitution of Gly and/or Pro by another amino acid or deletion or insertion of Gly or Pro; (ii) substitution of a hydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobic residue, e.g.,, Leu, Ile, Phe, Val or Ala; (iii) substitution of a Cys residue for (or by) any other residue; (iv) substitution of a residue having an electropositive side chain, e.g.,, Lys, Arg or His, for (or by) a residue having an electronegative charge, e.g.,, Glu or Asp; or (v) substitution of a residue having a bulky side chain, e.g., Phe, for (or by) a residue not having such a side chain, e.g., Gly.
  • a hydrophilic residue e.g., Ser or Thr
  • a hydrophobic residue e.g., Leu, Ile,
  • deletions, insertions and substitutions according to the present invention are those that do not produce radical changes in the characteristics ofthe LPP, e.g., HVP22, in terms of its intercellular spreading activity and its ability to stimulate antigen specific T cell reactivity to an antigenic epitope or epitopes that are fused to the spreading protein.
  • the effect can be evaluated by routine screening assays such as those described here, without requiring undue experimentation.
  • the preferred longer chain variants are typically made by site-specific mutagenesis of the nucleic acid encoding the IPP, expression ofthe variant nucleic acid in cell culture, and, optionally, purification ofthe polypeptide from the cell culture, for example, by immunoaffinity chromatography using specific antibody immobilized to a column (to absorb the variant by binding to at least one epitope).
  • “Chemical derivatives” ofthe IPP e.g., HVP22, or a fusion polypeptide thereof, contain additional chemical moieties not normally a part ofthe protein. Covalent modifications ofthe polypeptide are included within the scope of this invention. Such derivatized moieties may improve the solubility, absorption, biological half life, and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington 's Pharmaceutical Sciences, 16 th ed., Mack Publishing Co., Easton, PA (1980).
  • Such modifications may be introduced into the molecule by reacting targeted amino acid residues ofthe polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • Another modification is cyclization ofthe protein. Examples of chemical derivatives ofthe polypeptide follow. Lysinyl and amino terminal residues are derivatized with succinic or other carboxylic acid anhydrides. Derivatization with a cyclic carboxylic anhydride has the effect of reversing the charge ofthe lysinyl residues.
  • Suitable reagents for derivatizing amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonia.
  • the present invention includes longer polypeptides in which a basic peptidic sequence obtained from the sequence of either the IPP, such as HVP22, or the antigenic polypeptide or peptide unit, is repeated from about two to about 100 times, with or without intervening spacers or linkers. It is understood that such multimers may be built from any ofthe peptide variants defined herein. Moreover, a peptide multimer may comprise different combinations of peptide monomers and the disclosed substitution variants thereof. Such oligomeric or multimeric peptides can be made by chemical synthesis or by recombinant DNA techniques as discussed herein. When produced chemically, the oligomers preferably have from 2-8 repeats ofthe basic peptide sequence.
  • the multimers When produced recombinantly, the multimers may have as many repeats as the expression system permits, for example from two to about 100 repeats.
  • tandem multimers preferably dimers and trimers, ofthe fusion polypeptide, the chains bonded by interchain disulfide bonds or other "artificial" covalent bonds between the chains such that the chains are "side-by-side” rather than "end to end.”
  • a vaccine composition comprising the nucleic acid encoding the fusion polypeptide, or a cell expressing this nucleic acid is administered to a mammalian subject, preferably a human.
  • the vaccine composition is administered in a pharmaceutically acceptable carrier in a biologically effective or a therapeutically effective amount .
  • the composition may be given alone or in combination with another protein or peptide such as an immunostimulatory molecule.
  • Treatment may include administration of an adjuvant, used in its broadest sense to include any nonspecific immune stimulating compound such as an interferon.
  • Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylehe oleyl ether and n- hexadecyl polyethylene ether.
  • a therapeutically effective amount is a dosage that, when given for an effective period of time, achieves the desired immunological or clinical effect.
  • a therapeutically active amount of a nucleic acid encoding the fusion polypeptide may vary according to factors such as the disease state, age, sex, and weight ofthe individual, and the ability ofthe peptide to elicit a desired response in the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies ofthe therapeutic situation.
  • a therapeutically effective amounts ofthe protein, in cell associated form may be stated in terms ofthe protein or cell equivalents.
  • an effective amount is between about 1 nanogram and about 10 milligram per kilogram of body weight ofthe recipient, more preferably between about 0.1 ⁇ g and 1 ⁇ g/kg.
  • Dosage forms suitable for internal administration preferably contain (for the latter dose range) from about 0.01 ⁇ g to 100 ⁇ g of active ingredient (nucleic acid or polypeptide) per unit.
  • the active ingredient may vary from 0.5 to 95% by weight based on the total weight ofthe composition.
  • an effective dose of cells expressing the nucleic acid is between about 10 and 10 cells.
  • the active compound maybe administered in a convenient manner, e.g., injection or infusion by a convenient and effective route.
  • Preferred routes include subcutaneous, intradermal, intravenous and intramuscular routes.
  • Other possible routes include oral administration, infrathecal, inhalation, fransdermal application, or rectal administration.
  • direct intratumoral or peritumoral injection is also intended.
  • the active compound may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
  • a material to prevent its inactivation.
  • an enzyme inhibitors of nucleases or proteases e.g., pancreatic trypsin inhibitor, diisopropylfluorophosphate and trasylol
  • liposomes including water-in-oil-in- water emulsions as well as conventional liposomes (Strejan et al, (1984) J. Neuroimmunol 7:27).
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • Preferred pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • Isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride may be included in the phannaceutical composition.
  • the composition should be sterile and should be fluid. It should be stable under the conditions of manufacture and storage and must include preservatives that prevent contamination with microorganisms such as bacteria and fungi.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants.
  • Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • compositions are preferably formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for a mammalian subject; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • aerosolized solutions are used.
  • the active protein may be in combination with a solid or liquid inert carrier material. This may also be packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant.
  • the aerosol preparations can contain solvents, buffers, surfactants, and antioxidants in addition to the protein ofthe invention.
  • compositions according to the present invention are liposomes, pharmaceutical compositions in which the active protein is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers.
  • the active protein is preferably present in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non- homogeneous system generally known as a liposomic suspension.
  • the hydrophobic layer, or lipidic layer generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • a major utility for the present invention is the use ofthe present nucleic acid compositions in therapeutic vaccine for cancer and for major chronic viral infections that cause morbidity and mortality worldwide. Such vaccines are designed to eliminate infected cells - this requires T cell responses as antibodies are often ineffective.
  • the vaccines ofthe present invention include, the antigenic epitope itself and an IPP such as an intercellular spreading protein like HVP22. h addition to the specific antigens and first LPP polypeptide in the present vectors as employed in the Examples, the present invention is intended to encompass
  • additional vectors such as naked DNA, naked RNA, self replicating RNA replicons and viruses including vaccinia, adenoviruses, adeno-associated virus (AAV), lentiviruses and RNA alphaviruses;
  • an additional IPP such as HSP70, calreticulin, the extracellular domain of FL, , domain II of Pseudomonas exotoxin ETA; and/or
  • a costimulatory signal such as a B7 family protein, including B7-DC (see commonly assigned U.S. patent application Serial No. 09/794,210 which is incorporated by reference in its entirety), B7.1, B7.2, soluble CD40, etc.).
  • Preferred antigens are preferably epitopes of pathogenic microorganisms against which the host is defended by effector T cells responses, including cytotoxic T lymphocyte (CTL) and delayed type hypersensitivity. These typically include viruses, infracellular parasites such as malaria, and bacteria that grow mfracellularly such as mycobacteria and listeria.
  • CTL cytotoxic T lymphocyte
  • the types of antigens included in the vaccine compositions of this invention are any of those associated with such pathogens (in addition, of course, to tumor-specific antigens). It is noteworthy that some viral antigens are also tumor antigens in the case where the virus is a causative factor in cancer.
  • Hepatitis B virus(HBV) (Beasley, R.P. et al, Lancet 2, 1129- 1133 (1981) has been implicated as etiologic agent of hepatomas.
  • 80-90% of cervical cancers express the E6 and E7 antigens (exemplified herein) from one of four "high risk" human papiUomavirus types: HPV-16, HPV-18, HPV-31 and HPV-45 (Gissmann, L. et al, Ciba Found Symp. 120, 190-207 (1986); Beaudenon, S., et al. Nature 321, 246-249 (1986).
  • HPV E6 and E7 antigens are the most promising targets for virus associated cancers in immunocompetent individuals because of their ubiquitous expression in cervical cancer, hi addition to their importance as targets for therapeutic cancer vaccines, virus associated tumor antigens are also ideal candidates for prophylactic vaccines. Indeed, introduction of prophylactic HBV vaccines in Asia have decreased the incidence of hepatoma (Chang, M.H., et al. New Engl J. Med. 336, 1855-1859 (1997), representing a great impact on cancer prevention.
  • HBN human papiUomavirus
  • HCV hepatitis B virus
  • HCV hepatitis C Virus
  • HAV human immunodeficiency virus
  • EBV Epstein Barr Virus
  • HSV herpes simplex virus
  • the present invention is also intended for use in treating animal diseases in the veterinary medicine context.
  • herpesvirus infections including equine herpesviruses, bovine herpesviruses, Marek's disease virus in chickens and other fowl; animal retroviral diseases; pseudorabies and rabies and the like.
  • DNA delivery for example to effect what is generally known as “gene therapy” involves introduction of a "foreign” DNA into a cell and ultimately, into a live animal.
  • gene therapy involves introduction of a "foreign” DNA into a cell and ultimately, into a live animal.
  • Several general strategies for gene therapy have been studied and have been reviewed extensively (Yang, N-S., Crit. Rev. Biotechnol. 12:335-356 (1992); Anderson, W.F., Science 255:808-813 (1992); Miller, A.S., Nature 357:455-460 (1992); Crystal, R.G., Amer. J Med. 92(suppl 6A):44S>-52S> (1992); Zwiebel, J.A. et al, Ann. NY. Acad. Sci.
  • nucleic acid transfer into primary cells in culture followed by autologous transplantation ofthe ex vivo transformed cells into the host, either systemically or into a particular organ or tissue.
  • nucleic acid therapy would be accomplished by direct transfer of a the functionally active vectors into mammalian somatic tissue or organ in vivo.
  • Nucleic acid or replicon transfer can be achieved using a number of approaches described below.
  • retroviral vectors are effective for in vivo and in situ infection of blood vessel tissues;
  • retroviral vectors are effective for in vivo and in situ infection of blood vessel tissues;
  • portal vein injection and direct injection of retrovirus preparations into liver effected gene transfer and expression in vivo Horzaglou, M. et al, J. Biol. Chem.
  • Refroviral-mediated human therapy utilizes amphofrophic, replication-deficient retrovirus systems (Temin, H.M., Human Gene Therapy 7:111 (1990); Temin et ⁇ /., U.S. Patent 4,980,289; Temin et al, U.S. Patent 4,650,764; Temin et al, U.S. Patent No. 5,124,263; Wills, J.W. U.S. Patent 5,175,099; Miller, A.D., U.S. Patent No. 4,861,719).
  • Retro virus-mediated gene delivery generally requires target cell proliferation for gene transfer (Miller, D.G. et al, Mol. Cell. Biol. 10:4239 (1990). This condition is met by certain of the preferred target cells into which the present DNA molecules are to be introduced, i.e., actively growing tumor cells.
  • Gene therapy of cystic fibrosis using transfection by plasmids using any of a number of methods and by retroviral vectors has been described by Collins et al, U.S. Patent 5,240,846.
  • vectors that may be used in conjunction with the present vectors to include DNA packaged into retrovirus vectors using packaging cell lines that produce replication-defective retroviruses, as is well-known in the art (see, for example, Cone, R.D. et al, Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Mann, R.F. et al, Cell 33:153-159 (1983); Miller, A.D. et al, Molec. Cell. Biol. 5:431-437 (1985),; Sorge, J., et al, Molec. Cell. Biol. 4:1730-1737 (1984); Hock, R.A. et al, Nature 320:257 (1986); Miller, A.D. et al, Molec. Cell Biol. 6:2895-2902 (1986).
  • Newer packaging cell lines which are efficient an safe for gene fransfer have also been described (Bank et al, U.S. 5,278,056.
  • This approach can be utilized in a site specific manner to deliver the retroviral vector to the tissue or organ of choice.
  • a catheter delivery system can be used (Nabel, EG et al, Science 244:1342 (1989)).
  • Such methods using either a retroviral vector or a liposome vector, are particularly useful to deliver the nucleic acid to be expressed to a blood vessel wall, or into the blood circulation of a tumor.
  • Other virus vectors may also be used, including recombinant adenoviruses (Horowitz, M.S., In: Virology, Fields, BN et al, eds, Raven Press, New York, 1990, p.
  • adenovirus vectors for human gene therapy include the fact that recombination is rare, no human malignancies are known to be associated with such viruses, the adenovirus genome is double stranded DNA which can be manipulated to accept foreign genes of up to 7.5 kb in size, and live adenovirus is a safe human vaccine organisms.
  • Adeno- associated virus is also useful for human therapy (Samulski, R.J. et al, EMBO J. 10:3941 (1991) according to the present invention.
  • Vaccinia virus which can be rendered non-replicating (U.S. Patents 5,225,336; 5,204,243; 5,155,020; 4,769,330; Sutter, G et al, Proc. Natl. Acad. Sci. USA (1992) 59:10847-10851; Fuerst, T.R. et al, Proc. Natl Acad. Sci. USA (1989) 55:2549-2553; Falkner F.G. et al; Nucl. Acids Res (1987) 75:7192; Chakrabarti, S et al, Molec. Cell. Biol. (1985) 5:3403-3409).
  • Carrier mediated gene transfer has also been described (Wu, CH. et al, J. Biol. Chem. 264:16985 (1989); Wu, G.Y. et al, J. Biol. Chem. 263:14621 (1988); Soriano, P. et al, Proc. Natl. Acad. Sci. USA 50:7128 (1983); Wang, C-Y. et al, Proc. Natl. Acad. Sci. USA 84:7851 (1982); Wilson, J.M. et al, J. Biol. Chem. 267:963 (1992)).
  • Preferred carriers are targeted liposomes ( ⁇ icolau, C. et al, Proc. Natl. Acad. Sci.
  • D ⁇ A used for transfection or microinjection may be prepared using methods well-known in the art, for example using the Quiagen procedure (Quiagen), followed by D ⁇ A purification using known methods, such as the methods exemplified herein.
  • the packaging cell line (987dlsplit #24) has been described previously (Polo et al, supra) and was grown in DMEM supplemented with 10% FBS, antibiotics and G418.
  • the production and maintenance of TC-1 cells has been described previously (Lin, KY et al, 1996, Cancer
  • TC-1 cells were harvested by trypsinization, washed twice with IX Hanks buffered salt solution (HBSS), and finally resuspended in IX HBSS to the designated concenfration for injection.
  • Baby hamster kidney (BHK21) cells were obtained from the ATCC (RockviUe, MD) and grown in Glasgow MEM supplemented with 5% FBS, 10%) tryptose phosphate broth, 2 mM glutamine, and antibiotics.
  • Sindbis virus RNA replicon vector SINrep5
  • SINrep5 The Sindbis virus RNA replicon vector, SINrep5
  • Dr. Charles Rice was provided by Dr. Charles Rice at the Washington University School of Medicine, St. Louis.
  • SEQ ID NO:23 The sequence of SLNrep5 self replicating replicon (SEQ ID NO:23) is shown below and includes cloning sites
  • SLNrep5-VP22, SLNrep5-E7, and SLNrep5-VP22/E7 constructs have been described previously by the present inventors (Wu, TC et al, co-pending International patent application published as WOOl/29233 26 April 2001; see also Cheng, WF et al, 2001, J Virol. 75:2368-2376).
  • the pcDNA3 expression vector and pcDNA3-E7 have been described (Chen, CH et al, 1999, Gene Ther 6:1972-81; Ji, H. et al, 1999, Human Gene Therapy 10:2727-2740).
  • pcDNA3 has been used successfully in DNA vaccine induced immune responses and antitumor effects (Chen, CH et al, 2000, Cancer Res 60:1035-42; co-pending, commonly assigned patent pubications or applications WOOl/29233, USSN 09/421,608, filed 20 October 1999, USSN 09/501,097, filed 09 February 2000, which are inco ⁇ orated by reference).
  • VP22 was subcloned from pVP22/myc-His (Invitrogen, Carlsbad, CA) into the unique EcoRV and BamHI cloning sites ofthe pcDNA3.1(-) expression vector (Invitrogen, Carlsbad, CA) downstream ofthe CMV promoter.
  • pcDNA3-E7 The generation of pcDNA3-E7 has been described previously (Chen et al, supra).
  • VP22 was subcloned from pcDNA3-VP22 into the unique EcoRV and BamHI cloning sites ofthe pcDNA3-E7.
  • PCR was used to amplify the E7 fragment with pcDNA3-E7 and a set of primers: 5' -ggggaattcatggagatacaccta-3' (SEQ ID NO: 24) and 5' -ggtggatccttgagaacagatgg-3' ' . (SEQ ID NO:25).
  • the amplified product was further cloned into the EcoRI/BamHI sites of pcDNA3.
  • pcDNA3-VP22(l-267)/E7 a DNA fragment encoding VP22(l-267) was first amplified using PCR with pcDNA3-VP22 and a set of primers: 5 ' -gggtctagaatgacctctcgccgctccgt-3 ' ( SEQ ID NO : 26 ) and 5 ' -ggggaattcgtcctgcaccacgtctggat-3 ' ( SEQ ID NO : 27 ) .
  • the amplified product was cloned into the Xbal/EcoRI cloning sites of pcDNA3-E7(E/B).
  • DNA fragment encoding GFP was first amplified using PCR with pEGFPNl DNA (Clontech, Palo Alto, CA) and a set of primers:
  • VP22 was subcloned from pcDNA3-VP22 into the unique EcoRV and BamHI cloning sites ofthe pcDNA3-GFP.
  • GFP was isolated from pcDNA3-GFP and cloned into BamHI/Hindm sites of pcDNA3-E7(E+B).
  • VP22/E7/GFP was amplified by and a set of primers:
  • VP22(l-267) was isolated from pcDNA3-VP22( 1-267) and cloned into
  • pSCl 1-E7 The generation of pSCl 1-E7 has been described previously (Wu et al, 1995, . Proc. Natl. Acad. Sci. 92: 11671-11675).
  • VP22 was isolated from pcDNA3-
  • VP22 was isolated from ⁇ cDNA3-VP22 by Notl/Pmel and cloned into Notl/Saml sites of pSCll vector.
  • oligomers were annealed and cloned into the Xbal/EcoRI sites of pcDNA3-E7(E/B).
  • pcDNA3-E7/MTS the following complementary oligomers encoding
  • AAVLLPVLLAAP (SEQ ID NO:12) (Rojas, M et al, 1998, Nat Biotechnol 16:370-5) were synthesized: 5' -gatccgcagccgttcttctccctgttcttcttgccgcacccta-3' (SEQ ID NO:35) and 5' -agcttagggtgcggcaagaagaacagggagaagaacggctgcg-3' (SEQ ID NO: 36).
  • the oligomers were annealed and cloned into the BamHI/Hindi ⁇ sites of pcDNA3-E7(E/B).
  • the oligomers were annealed and cloned into the Xbal/EcoRI sites of pcDNA3-E7(E/B). The accuracy of all the DNA constructs was confirmed by sequencing.
  • RNA transcripts from SLNrep5-VP22, SLNrep5-E7, SP rep5-VP22/E7 and SINrep5 was performed using a protocol described previously (WO 02/09645 07-Feb-02; Cheng et al, supra). Briefly, Spel was used to linearize DNA templates. RNA replicons were transcribed in vitro and capped using SP6 RNA polymerase and capping analog from an in vitro transcription kit (Life Technologies, RockviUe, MD) according to the vendor's manual. After synthesis, DNA was removed by digestion with DNase I. Synthesized RNA was then purified by precipitation. RNA concenfration was determined by optical density measured at 260 nm.
  • RNA transcripts were further checked using denaturing gel electrophoresis.
  • the purified RNA was divided into aliquots to be used for vaccination in animals and for transfection of BHK21 cells.
  • the protein expression ofthe transcripts was characterized by transfection ofthe RNA into BHK21 cells using the Cell-Porator Electroporation System (Life Technologies, RockviUe, MD) according to the vendor's manual, followed by Western blot analysis.
  • SLNrep5 replicon particles were made using a protocol described protocol by Polo et al. supra. Briefly, 4 ⁇ g of mRNA synthesized in vitro was electroporated into 10 7 cells ofthe PCL. The PCL cells were incubated in 23 ml DMEM supplemented with 10% FBS, antibiotics and G418 at 5% CO 2 , 37°C. After 72 hr, culture supernatants were collected. The titer of SLNrep5 replicon particles in clarified PCL culture supernatants was determined by infection of na ⁇ ve BHK-21 monolayers, followed by indirect E7 immunofluorescence staining (Wu et al, 1995, . Proc. Natl. Acad. Sci. 92:11671-11675) with serial dilution and quantitation ofthe total number of green stained cells per well at each dilution. Vector titer is designated as infectious units (IU)/ml, and represents the population of functional particles.
  • BHK21 cells were cultured in 2-well culture chamber slides (Nalge Nunc Int., Naperville, EL) until they reached 50% confluency.
  • the BHK21 cells were infected with a serial dilution of replicon particles. After 48 and 72 hours of infection, the cells were fixed in 10%> formalin for 20 min. Diluted anti-E7 Ab (1 : 200 dilution, Zymed, San Francisco, CA) was added into the chamber and incubated for 30 min.
  • mice were vaccinated with 5x10 6 IU/mouse of SINrep5-VP22/E7 via different routes of administration (intramuscular, intraperitoneal, subcutaneous), hi another experiment, another group of mice (5 per group) was vaccinated intramuscularly with different titers of SrNrep5 replicon particles (5xl0 7 , 5xl0 6 , 5xl0 5 IU 5xl0 4 , and 5xl0 3 IU/mouse). Na ⁇ ve mice served as negative controls.
  • Splenocytes from vaccinated mice were collected seven days after vaccination and incubated either with the E7 peptide (aa 49-57, RAJHYNTVTF ) containing the MHC class I epitope (Feltkamp et al, 1993, Eur J Immunol. 23:2242-2249) (to detect E7-specific CD8 + cytotoxic T cell precursors) or with the E7 peptide (aa 30-67,
  • Cells were subjected to infracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacturer's instructions (PharMingen, San Diego, CA). Analysis was done on a Becton Dickinson FACScan with CELLQuest software (Becton Dickinson hnmunocytometry System, Mountain View, CA).
  • Anti-HPV 16 E7 antibodies in the sera from vaccinated mice (5 per group) were determined by a direct ELISA as described previously (Wu et al, supra). The ELISA plate was read with a standard ELISA reader at 450 nm. The quantity of E7 protein in cell lysates from BHK21 cells infected with SIN5rep-E7 or
  • SINrep5-NP22/E7 replicon particles was determined by an indirect ELISA method as described previously (Cheng et al, supra). Briefly, lxlO 7 BHK21 cells were infected with 5xl0 7 IU of SI ⁇ rep5, SLNrep5-E7, SINrep5-NP22 or SL ⁇ rep5-VP22/E7 particles. The transfected BHK21 cells were collected 40-44 hrs after infection. The quantity of E7 protein in cell lysates from transfected BHK21 cells was determined by ELISA with a standard ELISA reader at 450 nm.
  • mice (5 per group) were immunized intramuscularly with 5xl0 6 IU/mouse of SLNrep5-E7, SINrep5-VP22, SINrep5-VP22/E7 or control SLNrep5 replicon particles.
  • mice were subcutaneously challenged with 1 x 10 4 TC-1 cells/mouse in the right leg. Mice were monitored for evidence of tumor growth by palpation and inspection twice a week until they were sacrificed at day 60.
  • C57BL/6 mice (5 each group) were challenged with 10 4 cells/mouse TC-1 tumor cells i.v. in the tail vein on day 0.
  • mice were treated intramuscularly with 5xl0 6 IU/mouse of SLNrep5-E7, SINrep5-VP22, SINrep5-VP22/E7 or SINrep5 control replicon particles. Mice were sacrificed on day 21 after tumor challenge. The number of pulmonary tumor nodules on the surface of lungs in each mouse was determined by experimenters blinded to the sample identity. For the head-to-head comparison of various VP22/E7-containing vaccines, we performed another in vivo tumor treatment experiment. C57BL/6 mice (5 each group) were intravenously challenged with 10 4 cells/mouse TC-1 tumor cells in the tail vein on day 0.
  • mice were treated intramuscularly with optimized vaccine doses determined from previous studies: 2 ⁇ g/mouse SLNrep5-VP22/E7 DNA (Hung et al, 2001, supra), 1 ⁇ g/mouse SLNrep5-VP22/E7 RNA (Cheng et al, 2001a), or 5xl0 6 IU/mouse of SINrep5-VP22/E7 replicon particles. Na ⁇ ve mice were used as a negative control. Mice were sacrificed on day 28 after tumor challenge and mean lung weight was measured by experimenters blinded to the sample identity. In Vivo Antibody Depletion Experiments
  • mice were vaccinated intramuscularly with 5x10 6 IU/mouse of SLNrep5-VP22/E7 replicon particles. Depletions were started on day 7 after immunization and mice were challenged with 1 x 10 cells/mouse TC-1 tumor cells on day 14 after immunization.
  • MAb GK1.5 Dialynas et al, 1983, J. Immunol. J. Immunol. 737:2445:2445
  • MAb 2.43 Sa ⁇ niento et al, 1980, J. Immunol.
  • mice were immunized with 5x10 IU/mouse of SLNrep5-VP22/E7 replicon particles intramuscularly in the right leg. Normal saline without replicon particles was injected intramuscularly into the left leg as a control. Mice were sacrificed 7 days after intramuscular injection. For the detection of apoptotic cells, a modified TUNEL method was used as described previously (Cheng et al, supra). Apoptotic index is used as a measure ofthe extent of apoptosis in the stained slides following inspection under a light microscope. Apoptotic index is defined as the percentage of apoptotic cells and apoptotic bodies per 100 cells (Lipponen et al, 1994, J Pathol.
  • the infected BHK21 cells were collected 40-44 hrs later.
  • the levels of E7 protein expression for all replicon-infected BHK21 cells were similar, as determined by ELISA.
  • 3x10 5 of infected BHK21 cells were then co-incubated with lxlO 5 of bone marrow-derived DCs at 37°C for 48 hr. These prepared DCs were then used as target cells and an E7-specific CD8 T cell line (Wang et al, 2000, supra) served as effector cells.
  • CTL assays were performed with effector cells and target cells (lxlO 4 per well) mixed together at various ratios (1:1, 3:1, 9:1, and 27:1) in a final volume of 200 ⁇ l. After a 5 hr incubation at 37°C, 50 ⁇ l ofthe cultured media was collected to assess the amount of LDH using the CytoTox assay kit (Promega, Madison, WI). The percentage of lysis was calculated from the formula:
  • % Lysis [(A-B)/(C-D)] x 100 where A is the experimental-effector signal value, B is the effector spontaneous background signal value, C is maximum signal value from target cells, D is the target spontaneous background signal value.
  • DCs co-incubated with uninfected BHK21 cells, infected BHK21 cells alone, untreated DCs alone, and CD8 + T cells alone were included as negative controls. Fluorescence Microscopy for In vitro Distribution of VP22/E7
  • 293 D K b cells (provided by Dr. JC Yang, National Cancer Institute, NTH; Bloom, MB et al, 1997, JExp Med 185:453-459) were utilized for an in vitro assay of GFP expression.
  • 20 ⁇ g of VP22, E7/GFP, VP22(l-267)/E7/GFP or VP22/E7/GFP DNA were transfected into 5xl0 6 293 D K cells using lipofectamine 2000 (Life Technologies, RockviUe, MD).
  • Transfected cells were fixed with 4 % paraformaldehyde in IX PBS, permeabilized with IX PBS containing 0.05%) saponin and 1% BSA, then incubated with 0.5 ⁇ g/ml of primary anti-camexin antibody (Stressgen Biotechnologies, Victoria, BC). Samples were acquired with the Noran Oz confocal laser scanning microscope system using Invertension® software (v. 6.5). Slides were imaged with an Olympus IX-50 inverted microscope (lOOx magnification). Immunohistochemical Staining for In Vivo Distribution of VP22/E7
  • mice were sacrificed 3 days after vaccination with pcDNA3-VP22/GFP or pcDNA3-. Skin was biopsied, fixed, paraffin-embedded, and cut into 5 ⁇ m sections. After deparaffinization and hydration, slides were incubated with rabbit anti-GFP polyclonal antibody (1:200 dilution; Molecular Probes, Eugene, OR) followed by biotinylated goat anti-rabbit IgG (1 :200 dilution) and avidin-biotin complex (1:100 dilution; Vector, Burlingame, CA). The slides were developed by adding DAB substrate solution (DAKO, Ca ⁇ enteria, CA) and counterstained with Mayer's hematoxylin. Stained slides were dehydrated, mounted and observed by light microscopy.
  • DAKO DAB substrate solution
  • E7 protein was determined using lysates from cells infected with 5x10 7 IU of
  • E7 and VP22/E7 protein expression were comparable between cells infected with SINrep5-E7 and SINrep5-VP22/E7 replicon particles 48 hours after infection.
  • the subcellular localization of E7 and VP22/E7 proteins in the infected BHK21 cells was also evaluated by immunofluorescence 48 hours after infection.
  • E7 protein was mainly located in the nucleus ( Figure 1 A), while chimeric VP22/E7 protein was located in the cytoplasm
  • mice vaccinated with SINrep5-VP22/E7 replicon particles were vaccinated with SINrep5-VP22/E7 replicon particles.
  • infracellular cytokine staining was performed as described above.
  • mice vaccinated with SINrep5-VP22/E7 replicon particles generated the greatest number of E7-specific CD8 + T cell precursors compared to the other vaccination groups (p ⁇ .001).
  • Anti-E7 antibody titers generated by SUMrep5-VP22/E7 replicon particles were not increased compared to the other groups. These results were consistent with the observed lack of E7-specific CD4 + T helper cell enhancement. Fusion of E7 to VP22 was required for enhanced CD8 + T cell activity, since VP22 mixed with E7 (VP22 + E7) did not cause such enhancement of CD8 + T cell activity. Furthermore, E7 linked to an irrelevant protein such as green fluorescent protein (GFP) did not enhance E7- specific CD8 + T cell activity.
  • GFP green fluorescent protein
  • mice were injected i.m., l.p, and s.c, with 5x10 IU/mouse of SLNrep5-VP22/E7 replicon particles.
  • Intramuscular injection generated more E7-specif ⁇ c CD8 + T cells than the other two routes ( Figure 3A).
  • Figure 3A The correlation between replicon particle dosages and responses were also evaluated.
  • Different doses of SINrep5-VP22/E7 replicon particles were injected i.m. into mice. With increasing doses of replicon particles, the number of E7-specific
  • CD8 T cell precursors progressively increased until reaching a plateau at the dose of 5x10 IU/mouse ( Figure 3B).
  • mice freated with SLNrep5-VP22/E7 replicon particles three days after tumor challenge exhibited a significantly lower mean number of pulmonary nodules (0.7+ 0.3) than mice vaccinated with wild-type E7 replicon particles (72.5+ 8.5) or VP22 replicon particles (79.0+17.0).
  • mice freated with SINrep5-VP22/E7 replicon particles displayed a significantly fewer pulmonary nodules on day 3 (0.7+0.3), day 7 (0.5+0.3) and day 14 (25.0+4.0) after tumor challenge compared to mice freated with the SINrep5 control particles (no insert)(one-way ANOVA, p ⁇ 0.05).
  • Treatment with SPNrep5-VP22/E7 replicon particles reduced grossly visible tumors even if administered as late as 14 days after tumor challenge, hi general, grossly visible lung nodules could be detected 14 days after i.v. tumor (TC-1) cell injection.
  • Sindbis Virus Replicon Particles are the Most Effective Delivery Vector for VP22/E7 Vaccines used to Controlling Established Pulmonary Tumors
  • FIG. 6A demonstrated that mice treated with SLNrep5-VP22/E7 replicon particles exhibited a significantly lower mean lung weight (208+13 mg) than did mice vaccinated with VP22/E7 naked DNA (256 ⁇ 33 mg) or naked SINrep5-VP22/E7 RNA replicons (521+53 mg), or na ⁇ ve mice (644+44 mg).
  • Figure 6B displays representative gross pictures of pulmonary metastatic nodules derived from mice treated with different VP22/E7-containing vaccines. These results indicated that treatment of mice with SLNrep5-VP22/E7 replicon particles generated the greatest therapeutic antitumor effect among the VP22/E7 chimeric vaccines.
  • CD8 + T cells, CD4 + T cells and NK cells are Essential for the Anti-tumor Effect Generated by SINrep5-VP22/E7 Replicon Particles
  • mice were immunized ⁇ .m. with 5x10 IU each of SLNrep5-VP22/E7 replicon particles and were sacrificed 7 days later. Tissue sections of muscle were stained using the TUNEL method described above. Cells undergoing apoptosis exhibited brown staining of nuclei. As shown in Figure 8, vaccination with SIN replicon vaccines led to a significantly higher number of apoptotic cells in muscle tissue as compared to tisse from mice given normal saline.
  • the apoptotic index was 74.5 ⁇ 4.5 for the SINrep5-VP22/E7-treated group and 30.5+2.5 for the saline control (p ⁇ 0.01). These results indicated that muscle cells infected with SIN replicon particles underwent apoptosis.
  • BHK21 cells were first infected with various antigen-containing and control SIN replicon particles. These infected BHK21 cells were then incubated with bone marrow-derived DCs and used as target cells. Cytotoxic effector cells were T cells of an E7- specific CD8 + T cell line.
  • PCL for vaccine development.
  • the use of a stable PCL allowed the production of high titers of SLN replicon particles free of replication-competent virus, representing an important advance in the preparation of vaccines for mass immunization.
  • SIN infection in humans typically has limited climcal manifestations, fever, skin rash, and arthritic joint pain have been reported in people infected with certain Sindbis virus strains (Strauss & Strauss, supra).
  • the separation of structural protein cassettes in alphavirus PCLs significantly decreases the possibility of producing replication-competent virus (Polo et al, supra) and therefore decreases the likelihood of such undesired clinical effects.
  • replication-defective viral vectors whicha re relatively safe, one is their intrinsic inability to spread in vivo as effectively as do replication-competent viruses .
  • an intercellular spreading protein exemplified as HSV-1 VP22
  • HSV-1 VP22 fused to antigen in the context of SLN replicon vectors, facilitated the spread of antigen to surrounding cells in vivo resulting in a significantly enhanced E7- specific CD8 + T cell response and consequent antitumor effects.
  • the strategy of using a an intercellular spreading protein fused to an antigen and producing the vectors containing the nucleic acid expressing this fusion polypeptide by employing a stable PCL represents a unique and novel approach for generating a safe, potent vaccine in high quantities.
  • This strategy provides several advantages over other vaccine approaches. Compared to naked nucleic acid vaccines, SLN replicon particles are capable of infecting/fransfecting a higher proportion of "target" cells.
  • the linkage of the intercellular spreading protein, e.g., HSV VP22, further enhances vaccine potency.
  • Another advantage is that SIN replicon RNA does not integrate into the host genome, which is a potential concern with naked DNA vaccines or DNA-based viral vectors.
  • composition comprising SLN replicon vectors that is generated from a stable PCL such as that exemplifed here is free of replication-competent virus particles without sacrificing the efficiency of gene delivery. This feature maximizes vaccine potency while minimizing the risk associated with replication-competent viral vectors.
  • stable PCLs are also versatile, allowing for the packaging of different alphavirus-derived replicon vectors, in the present example, either Sindbis or Semliki Forest virus derived replicon vectors.
  • mice with SLNrep5-VP22/E7 replicon particles led to a more potent antitumor effect than did treatment with VP22/E7 naked DNA or naked SINrep5- VP22/E7 RNA replicon vaccines.
  • mice vaccinated with VP22/E7 DNA (as reported elsewhere), a higher frequency of antigen-specific CD8+ T cell precursors were detected (576/3xl0 5 splenocytes) (Hung et al, 2001, supra) compared to the present examples of mice vaccinated with SINrep5-VP22/E7 replicon particles (219/3xl0 5 splenocytes, Figure 2B) at one week after the final vaccination.
  • SINrep5-VP22/E7 replicon particles (219/3xl0 5 splenocytes, Figure 2B
  • Sindbis virus replicon particle vaccine resulted in peak numbers of antigen-specific CD8+ T cells earlier than vaccination with a DNA vaccine (3 days vs. 11 days.
  • a DNA vaccine 3 days vs. 11 days.
  • the present inventors tested the strategy of combining an intercellular spreading protein, such as HSV-1 VP22, with antigen while comparing different delivery vectors: naked DNA (pcDNA3) and naked SIN RNA (SINrep5).
  • pcDNA3 naked DNA
  • SIrep5 naked SIN RNA
  • CD8 + T cells were important components ofthe responses to all ofthe vectors tested, while CD4 + T cells were only essential for the antitumor effect generated by the VP22/E7 SLN particle-based vaccine. This conclusion is based on the observation that depleting CD4 + T cells did not diminish antitumor effects ofthe naked DNA vaccine ⁇ pcDNA3-VP22/E7 (Hung et al, 2001, supra) or the naked SIN replicon RNA vaccine - SIN replicon RNA-VP22/E7 (Cheng et al, 2001, J. Virol, supra).
  • CD4 T cells appeared to be needed for an optimal antitumor effect in response to the VP22/E7 SLN particle-based vaccine described herein, this vaccine did not actively induce E7-specific CD4+ T cells. This suggested that these CD4 T cells were contributing to an antitumor effect via a non-antigen-specific mechanism. Indeed, NK cells were needed for the present antitumor effect but were not as important in response to the VP22/E7 SLN particle-based vaccine or the naked DNA vaccine. Thus, different types of vaccines encoding the same protein construct may activate different subsets of effector cells in the vaccinated host and activate different immune or nonimmune antitumor mechanisms.
  • the enhanced E7-specific CD8 + T cell responses induced by the present VP22/E7 SIN replicon particle vaccine compared to a "control" E7 SIN replicon particle vaccine are believed to result, at least in part, from a process whereby infectected apoptotic cells are endocytosed and processed by APCs for MHC class I antigen presentation to CD8 + T cells (Albert, ML et al, 1998, JExp Med. 188:1359-1368.; Albert, ML et al, 1998, Nature. 392:86-89).
  • apoptotic cells may release chimeric VP22/E7 proteins that are taken up and processed by other APCs via a MHC class I-restricted pathway (Huang et al, supra).
  • MHC class I-restricted pathway Human et al, supra.

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Abstract

Superior molecular vaccines comprise nucleic acids in the form of PCL-generated replication-defective alphavirus replicons, preferably Sindbis virus, that encode a fusion polypeptide that includes an antigenic peptide or polypeptide against which an immune response is desired. Fused to the antigenic peptide is at least a second polypeptide that is an immunogenicity-potentiating olypeptide acting by any of a number of mechanisms to promote immunogenicity of the antigen. Examples include intercellular spreading proteins, in particular a herpes virus protein VP22 or a homologue or functional derivative thereof. Other examples are proteins that stimulate MHC class I processing of the antigen, target the antigen to APCs promote development and growth of immature DCs or stimulate DC antigen presenting activity. The nucleic acid can encode any antigenic epitope of interest, preferably an epitope that is processed and presented by MHC class I proteins. Antigens of pathogenic organisms and cells such as tumor cells are preferred. Vaccines comprising HPV-16 E7 oncoprotein are exemplified. Also disclosed are methods of using the vaccines to induce heightened T cell mediated immunity, in particular by cytotoxic T lymphocytes, leading to protection from or treatment of a tumor.

Description

MOLECULAR VACCINE LINKING ANTIGEN WITH AN IMMUNOGENICITY- POTENTIATING POLYPEPTIDE DELIVERED AS REPLICATION DEFECTIVE ALPHAVIRUS REPLICONS FROM STABLE PACKAGING CELLS
BACKGROUND OF THE INVENTION Field of the Invention
The present invention in the fields of molecular biology, immunology and medicine relates to a PCL-generated replication-defective alphavirus replicons as vectors for chimeric nucleic acid vaccines encoding fusion proteins. These vectors are used as vaccines to enhance immune responses, primarily cytotoxic T lymphocyte (CTL) responses to specific antigens such as tumor or viral antigens. The fusion protein comprises an antigenic polypeptide fused to an immunogenicity-potentiating polypeptide that promotes intercellular transport ofthe antigen, processing via the MHC class I pathway, stimulation of dendritic cell development or function, and the like. Description of the Background Art Naked DNA vaccines have emerged as attractive approaches for vaccine development
(Hoffman, SL et al., 1995, Ann N Y Acad Sci 772: 88-94; Donnelly, JJ et al., 1997 ', Annu Rev Immunol 15:617-648; Gurunathan, S et al, 2000, Annu Rev Immunol 18:927-74). Infradermal administration of DNA vaccines via gene gun represents a convenient way of delivering DNA vaccines into professional antigen presenting cells (APCs) in vivo. Professional APCs are a superior candidate for mediating presentation of an antigen encoded by such a DNA vaccine to T lymphocytes of the immune system. The "gene gun" strategy provides efficient delivery of DNA into epidermal bone marrow-derived APCs termed Langerhans cells, which move to draining lymph nodes where they enter the lymphatic system. The present inventors and their colleagues have successfully used this system of DNA delivery to test various intracellular targeting strategies (Chen et al., 2000, Cancer Res. 60:1035-1042; Ji et al., 1999, Human Gene Therapy 10:2727-2740); co-pending, commonly assigned U.S. patent applications USSN 09/421,608; 09/501,097, 09/693,450??? and 60/281,003???).
Recently, self-replicating RNA vaccines (RNA replicons) have also emerged as an important strategy to enhance the potency of nucleic acid vaccines for cancer immunotherapy (for review, see Leitner, W et al, 1999, Vaccine 18:765-777. RNA replicon vaccines may be derived from alphavirus vectors, such as Sindbis virus (Hariharan, JM et al., 1998, J. Virol. 72:950-958), Semliki Forest virus (Berglund, P et al, 1997, AIDS Res. Hum. Retrovir. 73:1487- 1495; Berglund, P. et al, 1998, Nat. Biotech. 16:562-565) or Venezuelan equine encephalitis virus (Pushko, P et al, 1997, Virology 239:389^101) vectors. These vaccines are self-replicating and self-limiting and may be administered as either RΝA or DΝA, which is then transcribed into RΝA replicons in transfected cells or in vivo (Berglund et al, supra; Leitner, WW et al, 2000, Cancer Res. (50:51-55). Self-replicating RΝA eventually causes lysis of transfected cells (Ying, H et al, 1999, Nat. Med. 5:823-827). These vectors do not raise the concern about integration into the host genome associated with naked DΝA vectors. This is particularly important for development of vaccines that target potentially oncogenic proteins such as the human papillomavirus (HPV) E6 and E7 proteins. One limitation on the potency of RΝA replicon vaccines is their inability to spread in vivo. The present inventors conceived a strategy that facilitates the spread of antigen to enhance significantly the potency of RΝA replicon vaccines.
Alphavirus vectors, such as Sindbis virus (Hariharan et al, 1998; Xiong et al, 1989. Science 243:1188-1191) and Semliki Forest virus (Berglund et al, 1997, AIDS Res Hum Retroviruses 13: 1487-1495; Daemen et al, 2000, Gene Ther. 7: 1859-1866), have become an important strategy for the development of vaccines and gene therapy applications because of their high levels of RΝA replication and gene expression in cells, their ability to infect a variety of diverse cell types, and the relative ease of manipulating cDΝA clones for transcription of vectors and infectious viral RΝA (for review, see (Dubensky et al, In: Gene Therapy: Therapeutic Mechanisms and Strategies. Templeton, ΝS et al, eds, pp.109-129, Marcel Dekker hie: New York, 2000.; Frolov et al, 1996, Proc Natl Acad Sci U S A. 93: 11371-11377; Garoff & Li, 1998, Curr Opin Biotechnol. 9, 464-469; Huang, HV, 1996, Curr Opin Biotechnol. 7, 531- 535; Schlesinger & Dubensky, 1999, Curr Opin Biotechnol. i 0:434-439 ; Strauss & Strauss, 1994, Microbiol Rev. 55:491-562)). The general strategy for construction of alphavirus-based expression vectors has been to substitute viral structural protein genes with a heterologous gene, while preserving transcriptional control via the highly active subgenomic RNA promoter (Frolov et al, supra; Huang, supra; Xiong et al, supra). These vectors are self-replicating in cells and may be administered as either RNA, DNA, or infectious propagation-incompetent alphavirus particles. Since alphavirus vectors eventually trigger apoptosis of transfected cells (Ying et al, 1999, Nat Med. 5:823-827), they do not raise the concern associated with DNA integration into the host genome. This is particularly important for vaccine development approaches targeting proteins that are potentially oncogenic, such as the HPN E6 and E7 proteins.
Unlike DΝA- or RΝA-based nucleic acid vaccines, infectious alphavirus replicon particles provide a highly efficient method of introducing heterologous genes into target cells and the opportunity to generate vaccines at a large scale. However, significant concerns have been raised about potential contamination with replication-competent virus. Recently, an alphavirus replicon packaging cell line (PCL) was developed (Polo, JM et al, 1999, Proc Νatl Acad Sci U S A. 9(5:4598-4603) to produce alphavirus replicon particle stocks, including Sindbis virus and Semliki Forest virus-derived vectors, free of detectable contaminating replication-competent virus, hi this PCL (987dlsplit #24), genes encoding the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in undetectable levels of contaminating replication- competent virus while maintaining relatively high levels of viral particle production (approximately 107 infectious units/ml). The availability of such a PCL allows for large-scale vector production that may be useful in vaccine applications. HSP70: A Protein that Promotes Antigen Processing via the MHC Class I Pathway
The present inventors and their colleagues (Chen (2000) Cane Resh (50:1035-1042) demonstrated that linkage of human papillomavirus type 16 (HPN- 16) E7 antigen to Mycobacterium tuberculosis heat shock protein 70 (HSP70) led to enhancement of DΝA vaccine potency. Other studies have demonstrated that immunization with heat shock protein (HSP) complexes isolated from tumor or virus-infected cells are able to induce potent anti-tumor
(Janetzki (1998) J. Immunother. 21:269-276) or antiviral immunity (Heikema (1997) Immunol. Lett. 57:69-74). Some HSP-based protein vaccines involved fusing antigens to HSPs (Suzue (1996) J. Immunol. 156:873-879) wherein, HSP70 fusion protein elicited humoral and cellular immune responses to an HIV-l protein. While these investigations have made HSPs more attractive for use in immunotherapy, there have been no reports or suggestions of making HSP- linked molecular vaccine using replication defective alphavirus vectors as described herein.
The centrosome, also called the microtubule organizing center (MTOC), is a perinuclear organelle which contains a high density of proteasomes (Anton, LC et al, 1999, J Cell Biol. 146: 113-24; Wigley, WC, 1999, J Cell Biol. i ¥5:481-90; Fabunmi, RP et al, J Biol Chem. 275:409-13, 2000). Several proteins, notably γ-tubulin and β-tubulin, are localized and concentrated at the centrosome. The centrosome has been implicated as an important intracellular compartment for proteasomal degradation of certain antigens (Anton, supra).
Endoplasmic Reticulum Chaperone Polypeptides Calreticulin (CRT), an abundant 46 kilodalton (kDa) protein located in the lumen ofthe cell's endoplasmic reticulum (ER), displays lectin activity and participates in the folding and assembly of nascent glycoproteins. See, e.g.,, Nash (1994) Mol Cell. Biochem. 235:71-78; Hebert (1997) J Cell Biol. 139:613-623; Nassilakos (1998) Biochemistry 37:3480-3490; Spiro
(1996) J. Biol. Chem. 271:11588-11594. CRT associates with peptides transported into the ER by transporters that are associated with antigen processing, such as TAP-1 and TAP-2 (Spee
(1997) Eur. J. Immunol. 27:2441-2449). CRT also forms complexes with peptides in vitro. Upon adminsitration to mice, these complexes, elicited peptide-specific CD8+ T cell responses (Basu (1999) J. Exp. Med. 59:797-802; Νair (1999) J. Immunol 162:6426-6432). CRT purified from murine tumors elicited immunity specific for the tumor from which the CRT was taken, but not for an antigenically distinct tumor (Basu, supra). By pulsing mouse dendritic cells (DCs) in vitro with a CRT-peptide complex, the peptide was re-presented by MHC class I molecules on the DCs to stimulate a peptide-specific CTL response(Νair, supra).
CRT also has anti-angiogenic effects. CRT and a fragment comprising amino acid residues 1-180, which has been called "vasostatin," are endothelial cell inhibitors that can suppress tumor growth (Pike (1999) Blood. 94:2461-2468). Tumor growth and metastasis depend on the existence of an adequate blood supply. As tumors grow larger, adequate blood supply to the tumor tissue is often ensured by new vessel formation, a process termed angiogenesis. (Folkman (1982) Ann. NY Acad. Sci. 401:212-27; Hanahan (1996) Cell 86:353- 364). Therapeutic agents that target and damage tumor vasculature can prevent or delay tumor growth and even promote regression or dormancy.
Viral Polypeptides that Promote Intercellular Transport and Spread
One limitation of DNA vaccines is their potency, since they do not have the intrinsic ability to amplify and spread in vivo as some replicating viral vaccine vectors do. The present inventors conceived a strategy that facilitates the spread of antigen may significantly enhance the potency of naked DNA vaccines. VP22, a herpes simplex virus (HSN-1) protein has demonstrated the remarkable property of intercellular transport and is capable of distributing protein to many surrounding cells(4) (U.S. Patent 6,017,735, O'Hare & Elliott, 25 Jan 2000). For example, NP22 has been linked to p53 (Phelan, A. et al, 1998, Nat Biotechnol 16:440-3) or thymidine kinase (Dilber, MS et al, 1999, Gene Ther 6:12-21), facilitating the spread of linked proteins to surrounding cells in vitro and the treatment of model tumors. Marek's disease virus type 1 (MDN-1) UL49 shares homology with HSN-1 NP22 (Koptidesova et al, 1995, Arch Virol. 140:355-362) and has been shown to be capable of intercellular transport after exogenous application (Dorange et al, 2000, J Gen Virol. 81 Pt 9:2219-2230). Polypeptide Stimulators of Growth, Differentiation or Activation of Antigen Presenting Cells A molecule that stimulates growth of DC precursors and can help in generating large numbers of DCs in vivo is Flt3-ligand ("FL") (Maraskovsky, E et al, JExp Med 184: 1953-62, 1996, Shurin, MR et al, Cell Lmmunol 179: 174-84, 1997). FL has emerged as an important molecule in the development of tumor vaccines that augment numbers and action of DCs in vivo. Flt3, a murine tyrosine kinase receptor, first described in 1991 (Rosnet, O et al, Oncogene. 6: 1641-50, 1991), was found to be a member ofthe type HI receptor kinase family which includes - kit and c-frns (for review, see (Lyman, SD Curr Opin Hematol 5:192-6, 1998). hi hematopoietic tissues, the Flt3 expression is restricted to the CD34+ progenitor population. Flt3 has been used to identify and subsequently clone the corresponding ligand, Flt3 -ligand or "FL" (Lyman, SD et al, Cell. 75: 1157-67, 1993; Hannum, C et al, Nature. 368: 643-8, 1994).
The predominant form of FL is synthesized as a transmembrane protein from which the soluble form is believed to be generated by proteolytic cleavage. The soluble form of FL (the extracellular domain or "ECD") is functionally similar to intact FL (Lyman, SD et al, Cell. 75: 1157-67, 1993). These proteins function by binding to and activating unique tyrosine kinase receptors. Expression ofthe Flt3 receptor is primarily restricted, among hematopoietic cells, to the most primitive progenitor cells, including DC precursors. The soluble ECD of FL induced strong anti-tumor effects against several murine model tumors including fϊbrosarcoma (Lynch, DH et al, Nat Med. 3: 625-31, 1997), breast cancer (Chen, K et al Cancer Res. 57: 3511-6, 1997; Braun, SE et al, Hum Gene Ther. 10: 2141-51, 1999), liver cancer (Peron, JM et al, J Immunol 161: 6164-70, 1998), lung cancer (Chakravarty, PK et al, Cancer Res. 59: 6028-32, 1999), melanoma and lymphoma (Esche, C et al, Cancer Res. 58: 380-3, 1998).
SUMMARY OF THE INVENTION
The potency of naked DNA molecular vaccines is limited by their inability to amplify and spread in vivo. Inclusion of nucleic acid sequences that encode polypeptides that modify the way the antigen encoded by molecular vaccine is "received" or "handled" by the immune system serve as a basis for enhancing vaccine potency. Polypeptides that have such modes of action are termed herein "mimunogenicity-potentiating (or -promoting) polypeptide" or "IPP" to reflect this general property, even though these IPP's may act by any of a number of cellular and molecular mechanisms that may or may not share common steps. IPP's may be produced as fusion or chimeric polypeptides with the antigen, or may be expressed from the same nucleic acid vector but produced as distinct expression products.
The present invention provide a recombinant, replication-defective alphavirus-based replicon particles that encode a fusion of a polypeptide antigen of choice with an intercellular transport protein that, when expressed in a transfected cell, is capable of distributing the antigen to many surrounding cells. This has been accomplished by the use of a stable packaging cell line (PCL), which is capable of generating alphavirus replicon particles without contamination from replication-competent virus.
This invention has been exemplified using the HSV-1 VP22 protein linked to a model tumor antigen, human papillomavirus type 16 (HPV-16) E7 oncoprotein and included in a nucleic acid which is a Sindbis virus (SIN)-based replicon particle encoding the VP22-E7 fusion and using a PCL termed SLN-PCL. The linkage of NP22 to E7 in these SP replicon particles resulted in a significant increase in the number of E7-specific CD8+ T cell precursors and a strong antitumor effect against E7-expressing tumors in vaccinated C57BL/6 mice relative to wild-type E7 SIN replicon particles. Furthermore, a head-to-head comparison of
NP22/E7-containing naked DΝA, naked RΝA replicons, or RΝA replicon particle vaccines indicated that SIΝrep5-VP22/E7 replicon particles generated the most potent therapeutic antitumor effect. For additional disclosure, see also, Cheng, WF et al, 2002, Hum Gene Ther, 2002, Mar;73: 553-568, a publication by the present inventors and colleagues after the priority date of this application. Thus, the present strategy used in the context of SIN replicon particles produce with a PCL facilitates the generation of a highly effective vaccines for widespread immunization.
The present invention is directed to a nucleic acid molecule encoding a fusion polypeptide useful as a vaccine composition, which molecule comprises: (a) a first nucleic acid sequence encoding a first polypeptide that comprises at least one immunogenicity-potentiating polypeptide;
(b) optionally, fused in frame with the first nucleic acid sequence, a linker nucleic acid sequence encoding a linker peptide; and
(c) a second nucleic acid sequence that is linked in frame to the first nucleic acid sequence or to the linker nucleic acid sequence and that encodes an antigenic polypeptide or peptide, which nucleic acid is in the form of a replication-defective alphavirus replicon particle prepared using a packaging cell line. h the above nucleic acid molecule, the first polypeptide is preferably one that acts by promoting: (a) processing of the linked antigenic polypeptide via the MHC class I pathway or targeting of a cellular compartment that increases the processing;
(b) development, accumulation or activity of antigen presenting cells or targeting of antigen to compartments ofthe antigen presenting cells leading to enhanced antigen presentation;
(c) intercellular transport and spreading ofthe antigen; or (d) any combination of (a)-(c).
Preferably the first polypeptide is:
(a) a mycobacterial HSP70 polypeptide, the C-terminal domain thereof, or a functional homologue or derivative ofthe polypeptide or domain;
(b) a viral intercellular spreading protein selected from the group of herpes simplex virus- 1 VP22 protein, Marek's disease virus NP22 protein or a functional homologue or derivative thereof;
(c) an endoplasmic reticulum chaperone polypeptide selected from the group of calreticulin, ER60, GRP94, gp96, or a functional homologue or derivative thereof
(d) a cytoplasmic translocation polypeptide domains of a pathogen toxin selected from the group of domain II of Pseudomonas exotoxin ETA (ΕTAdll) or a functional homologue or derivative thereof; (e) a polypeptide that targets the centrosome compartment of a cell selected from γ-tubulin or a functional homologue or derivative thereof; or
(f) a polypeptide that stimulates dendritic cell processors or activates dendritic cell activity selected from the group of GM-CSF, Flt3 -ligand extracellular domain, or a functional homologue or derivative thereof
More preferably, the first polypeptide above is selected from the group consisting of Mycobacterium tuberculosis HSP70, the HSP70 C-terminal domain, HSN-1 NP22, MDN NP22, calreticulin, Pseudomonas ETAdll, GM-CSF, Flt-3 ligand extracellular domain or γ-tubulin. a preferred embodiment, the first polypeptide is a transport polypeptide comprising SEQ ID ΝO:5 or 7 or an active fragment thereof. h the above nucleic acid molecule, the antigenic polypeptide preferably comprises an epitope that binds to, and is presented on the cell surface by, an MHC class I protein. Preferably, the epitope is between about 8 and about 11 amino acid residues in length.
In the above nucleic acid molecule, the antigen is preferably one which is present on, or cross-reactive with an epitope of, a pathogenic organism, cell, or virus. A preferred virus is a human papilloma virus. A preferred antigen is the E7 polypeptide of HPV-16 or an antigenic fragment thereof. h the above nucleic acid molecule, the pathogenic organism may be a bacterium.
Further, the pathogenic cell is preferably a tumor cell. In that case, the antigen is a tumor-specific or tumor-associated antigen, for example, a peptide ofthe HER-2/neu protein.
The above nucleic acid molecule may be operatively linked to a promoter. The promoter is preferably one which is expressed in an APC, preferably a DC.
The above nucleic acid molecule is preferably an RNA replicon wherein the alphavirus is Sindbis virus, Semliki forest virus or Venezuelan equine encephalitis virus, most preferably Sindbis virus. The nucleic acid molecule may have the sequence ofthe SINrep5 molecule
In the above nucleic acid molecule, the packaging cell line is preferably one in which genes encoding capsid and envelope glycoproteins ofthe alphavirus are separated in distinct cassettes to minimize formation of replication competent virus during replicon production. A most preferred packaging cell line is 987dlsplit #24. Also provided herein is an expression vector comprising any ofthe nucleic acid molecules described above, operatively linked to (a) a promoter; and (b) optionally, additional regulatory sequences that regulate expression ofthe nucleic acid in a eukaryotic cell.
The present invention is also directed to a cell which has been modified to comprise the nucleic acid or expression vector as above Preferably, the cell expresses the nucleic acid molecule. Preferred cells as above are APCs, for example, a dendritic cell, a keratinocyte, a macrophage, a monocyte, a B lymphocyte, a microglial cell, an astrocyte, or an activated endothelial cell.
In another embodiment, the present invention is directed to a pharmaceutical composition capable of inducing or enhancing an antigen-specific immune response, comprising:
(a) pharmaceutically and immunologically acceptable excipient in combination with;
(b) a composition selected from the group consisting of:
(i) the above nucleic acid molecule or expression vector;
(ii) the above cell; and (iii) any combination of (i) and (ii).
Also provided is a method of inducing or enhancing an antigen specific immune response in a subject comprising administering to the subject an effective amount ofthe above pharmaceutical composition, thereby inducing or enhancing the response. The response is preferably one which is mediated at least in part by CD8 cytotoxic T lymphocytes (CTL). Alternatively, or additionally, the response may be mediated at least in part by antibodies.
The present invention includes a method of inducing or enhancing an antigen specific immune response in cells or in a subject comprising administering to the cells or to the subject an effective amount ofthe pharmaceutical composition as above, thereby inducing or enhancing the response. In the foregoing method, the composition may be is administered ex vivo to the cells.
These cells may comprise APCs, such as DCs. Preferably, the APCs are human APCs. These APCs are preferably isolated from a living subject. This method may further comprising a step of administering the ex vzvø-freated cells to a histocompatible subject. Preferably, the cells are human cells and the subject is a human. In all the foregoing method o in vivo treatment, the administering is preferably by a intramuscular, intradermal, or subcutaneous route. Alternatively, when treating a tumor, the administering may be intratumoral or peritumoral.
The present invention provides a method of increasing the numbers or lytic activity of CD8+ CTLs specific for a selected antigen in a subject, comprising administering to the subject an effective amount of a composition selected from the group consisting of:
(a) the nucleic acid molecule or expression vector as above;
(b) the cell as above, and
(c) any combination of (a) and (b), wherein (i) the nucleic acid molecule, the expression vector or the cell comprises the antigen,
(ii) the antigen comprises an epitope that binds to, and is presented on the cell surface by, MHC class I proteins, thereby increasing the numbers or activity ofthe CTLs.
Also provided is a method of inhibiting growth or preventing re-growth of a tumor in a subject, comprising administering to the subject, preferably intratumorally or peritumorally, an effective amount of a composition selected from the group consisting of:
(a) the above nucleic acid molecule or expression vector;
(b) the above cells; and
(c) any combination of (a) and (b), wherein (i) the nucleic acid molecule, the expression vector or the cell comprises the antigen,
(ii) the antigen comprises one or more tumor-associated or tumor-specific epitopes present on the tumor in the subject thereby inhibiting the growth or preventing the re-growth.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A, IB, IC and ID. hnmunofluorescence staining to demonstrate the expression and distribution of E7 and chimeric VP22/E7 protein. BHK 21 cells were infected with SINrep5-E7 (Fig. 1 A and B) or SINrep5-VP22/E7 (Fig. IC and D) replicon particles. Infected cells were fixed in 10% formalin and stained for HPV-16 E7 protein at 48 hrs (Fig. 1A and C) or 72 hrs (Fig. IB and D) after infection. See Example I. Slides were mounted and observed immediately under a fluorescence microscope. (Fig. 1 A) BHK21 cells infected with SINrep5-E7 replicon particles and stained for E7 at 48 hours after infection. Note: E7 protein was predominantly located in the nucleus. Fig. IB: BHK21 cells infected with SINrep5-E7 replicon particles and stained for E7 at 72 hours after infections. Note: E7 protein remained in the nucleus 72 hours after infection. Fig. IC: BHK21 cells infected with SINreρ5-VP22/E7 replicon particles and stained for E7 at 48 hours after infection. Note: VP22/E7 protein was mostly located in the cytoplasm. Fig. ID: BHK21 cells infected with SLNrep5-VP22/E7 replicon particles and stained for E7 at 72 hours after infections. Note: intercellular spreading of VP22/E7 protein to many neighboring cells became apparent 72 hours after infection.
Figure 2A. 2B and 2C. Intracytoplasmic cytokine staining followed by flow cytometry analysis to demonstrate that SEMrep5-VP22/E7 replicon particles can enhance E7-specific CD8+ but not CD4+ T cell immunologic responses. Vaccination of mice and preparation of splenocytes is described in Example I. Fig. 2A: Representative figure of flow cytometric analysis demonstrating E7-specific CD8+ T cell precursors in splenocytes from vaccinated mice. The number of IFN-γ-secreting CD8 T cell precursors is shown in the upper right corner. Fig. 2B: Histogram to show E7-specific IFN-γ-secreting CD8+ T cell precursors in vaccinated mice. The number of IFN-γ -producing E7-specific CD8+ T cells was determined using flow cytometry in the presence (solid columns) or absence (open columns) of MHC class I restricted E7 peptide (aa 49-57). Data are expressed as mean number of CD8+,LFN-γ+ cells/3xl0 splenocytes; bars, SE. Fig. 2C: Histogram to show E7-specific IFN-γ-secreting CD4+ T cell precursors. The number of LFN-γ -producing E7-specific CD4+ T cells was determined using flow cytometry in the presence (solid columns) or absence (open columns) of MHC class LI restricted E7 peptide (aa 30-67). Data are expressed as mean number of CD4+, IFN-γ+ cells/3xl0 splenocytes; bars, SE. The intracellular cytokine staining results are from one representative experiment of two performed. Figures 3A and 3B. Intracytoplasmic cytokine staining followed by flow cytometry analysis to demonstrate the generation of E7-specific CD8+ T cell precursors using different routes of administration and dosages of the SINrep5-VP22/E7 particle vaccine. Fig. 3A: Mice were immunized with 5x10 IU/mouse of SLNrep5-VP22 E7 replicon particles via intramuscular, intraperitoneal, or subcutaneous injection. Note: The intramuscular route generated the highest number of E7-specific CD8+ T cell precursors. Fig. 3B: Mice were immunized intramuscularly with different dosages of SINrep5-VP22/E7 replicon particles as described in Example I. With increasing dosages of SINrep5-VP22/E7 particles, the number of E7-specific CD8+ T cell precursors increased gradually, reaching a plateau at the dose of 5x106 IU/mouse. The number of IFN-γ-producing E7-specifϊc CD8+ T cells was determined using flow cytometry in the presence (solid columns) or absence (open columns) of MHC class I restricted E7 peptide (aa 49-57). Data are expressed as mean number of IFN-γ-secreting CD8+ T cells/3xl05 splenocytes; bars, SE. The results shown here are from one representative experiment of two performed. Figure 4. In vivo tumor protection experiments to demonstrate the antitumor effect generated by SINrep5 replicon particles against TC-1 tumors. Mice were immunized with various SLNrep5 replicon particles as described in Example I. One week after vaccination, mice were challenged with 104 TC-1 cells/mouse subcutaneously and monitored for evidence of tumor growth by palpation and inspection twice a week. 100% of mice receiving the SINrep5- VP22/E7 replicon particles remained tumor-free 60 days after TC-1 challenge. All ofthe mice in the other vaccination groups exhibited tumor growth within 20 days after tumor challenge. The data shown here are from one representative experiment of two performed.
Figure 5A and 5B. In vivo tumor treatment experiments to demonstrate the antitumor effect generated by SLNrep5 replicon particles against TC-1 tumors. Mice were challenged and treated as described in the Materials and Methods. Fig. 5A: Treatment of pulmonary nodules with SINrep5-VP22/E7 replicons relative to other SINrep5 constructs. Mice treated with SINrep5-VP22/E7 replicon particles displayed a significantly lower mean number of pulmonary nodules 3 days after tumor challenge (0.7+0.3) than mice treated with the other SINrep5 replicon particle vaccines. Fig. 5B: Treatment of pulmonary nodules with SLNrep5-VP22 E7 three, seven, and fourteen days after tumor challenge. Mice treated with STNrep5-VP22/E7 replicon particles exhibited a significantly lower mean number of pulmonary nodules three days (0.7+0.3), seven days (0.5+0.3), or fourteen days (25.0+4.0) after tumor challenge compared to SLNrep5 control (no insert) (one-way ANOVA, P<0.05).
Figure 6A and 6B. In vivo tumor treatment experiment to compare the antitumor effect in mice treated with VP22/E7 naked DNA, naked SLNrep5-VP22/E7 RNA replicons, or SINrep5-VP22/E7 RNA replicon particles. Mice were challenged with TC-1 and treated with VP22/E7 naked DNA, naked SINrep5-VP22/E7 RNA replicons, or SINrep5-VP22/E7 RNA replicon particles as described in Example I. Fig 6A: Treatment of pulmonary tumor nodules with various VP22/E7-containing vaccines. Mice treated with SINrep5-VP22/E7 replicon particles displayed a significantly lower mean lung weight after tumor challenge than mice treated with VP22/E7 DNA and naked SPNrep5-VP22/E7 RNA replicon vaccines. Fig IB: Representative gross pictures of pulmonary metastatic nodules in mice treated with the different VP22/E7-containing vaccines.
Figure 7. In vivo antibody depletion experiments to determine the effect of lymphocyte subsets on the potency of SINrep5-VP22/E7 replicon particles as a vaccine. Mice were immunized intramuscularly with 5x 106 IU/mouse of SINrep5-VP22/E7 replicon particles. CD4, CD8 and NK1.1 depletions were initiated one week after vaccination. Two weeks after vaccination, mice were challenged with 104 TC-1 cells/mouse subcutaneously. All naϊve mice and all mice depleted of CD8+ T cells grew tumors within 14 days after tumor challenge. 80% of mice depleted of CD4+ T cells and 60% of mice depleted of NK1.1 cells developed tumors within 60 days after tumor challenge. Note: these results suggested that CD8+ T cells, CD4+ T cells and NK cells are all important for the anti-tumor immunity generated by the SINrepS- VP22/E7 replicon particles. Data from the antibody depletion experiment shown here are from one representative experiment of two performed.
FIG. 8A-8D. TUNEL assay of apoptotic cells in the skeletal muscle of vaccinated mice. These photomicrographs show mus-cle tissue at the injection sites from (A) control mice immunized with normal saline, (B) mice immunized with VP22-E7 DNA, (C) mice immunized with VP22-E7 RNA, and (D) mice immunized with SINreρ5-VP22/E7 replicon particles.
Vaccination with SLNrep5-VP22/E7 replicon particles induced a greater degree of apoptosis in muscle tissue compared with the other groups.
Figure 9. Activity of E7-specific CTL. BHK21 cells were first infected with various SLNrep5 replicon particles. Infected BHK21 cells were co-incubated with bone marrow-derived DCs. DCs were used as target cells and an E7-specific CD8+ T cell line served as effector cells. CTL assays with various E:T ratios were performed. The SINrep5-VP22/E7 replicon particle vaccine generated greater cytotoxicity (measured at E:T ratios of 9 and 27 (p<0.01)) compared to BHK21 cells infected with SINrep5-E7 replicon particles. The CTL assays shown here are from one representative experiment of two performed. DESCRIPTION OF THE PREFERRED EMBODIMENTS
All references cited above and below are incorporated by reference in their entirety herein, whether specifically incorporated or not.
The invention provides compositions and methods for enhancing the immune responses, particularly cytotoxic T cell immune responses, induced by ex vivo or in vivo administration of nucleic acid vaccines that encode chimeric polypeptides. The preferred chimeric or fusion polypeptide comprises (1) at least one first polypeptide or peptide that, upon introduction to cells ofthe host immune system, in vitro or in vivo, promotes or potentiates immunogenicity ofthe second polypeptide or peptide (the antigen). For this reason, the first polypeptide has been termed an "immunogenicity-potentiating (or promoting) polypeptide, abbreviated "IPP". These are described in more detail below. The nucleic acid vaccine further comprises (2) at least one second polypeptide or peptide that is an antigenic polypeptide or peptide in the host against which it is desired to induce an immune response.
In a preferred embodiment, the chimeric or fusion polypeptides are "indirectly" administered by administration of a nucleic acid vector that encodes the chimeric molecule; the nucleic acid construct, and thus the fusion protein, is expressed in vivo. In the present invention, this nucleic acid construct of vector is in the form of replication-defective alphaviruses generated from stable alphavirus packaging cell lines ("PCL").
The IPP is a polypeptide that acts by promoting (1) processing via the MHC class I pathway.
(2) development or activity of APCs or targeting of DCs for antigen presentation
(3) intercellular transport and spreading thereby conferring such properties on a linked antigenic polypeptide or peptide.
It is important to note that these categories are artificial and are being made to assist in classifying the polypeptides that are disclosed herein. A number ofthe polypeptides that are ascribed ton one category, act in ways that would place them in another of these categories.
IPP's that Promote Processing via the MHC Class I Pathway.
For convenience, a polypeptide or peptide that promotes processing via the MHC class I pathway is abbreviated herein as "MHCi-PP". One exemplary MHCi -PP described herein is Hsp70. However, it is understood that any protein, or functional fragment or variant thereof, that has this activity can be used in the invention. A preferred fragment is a C-terminal domain ("CD") of Hsp70, which is designated "Hsp70cD"- One Hsp70cD spans from about residue 312 to the C terminus of Hsp70 (SEQ ID NO:9). A preferred shorter polypeptide spans from about residue 517 to the C-terminus of SEQ ID NO:9. Shorter peptides from that sequence that have the ability to promote protein processing via the MHC-1 class I pathway are also included, and may be defined by routine experimentation.
Another category of MHQ-PP is an ER chaperone polypeptide such as calreticulin, ER60, GRP94 or gp96, well-characterized ER chaperone polypeptides that representatives ofthe HSP90 family of stress-induced proteins (Argon (1999) Semin. Cell Dev.. Biol. 10:495-505; Sastry (1999) J Biol. Chem. 274:12023-12035; Nicchitta (1998) Curr. Opin. Immunol. 10:103-109; U.S. Patent 5,981,706))
Another group of proteins that act as MHQ-PP are cytoplasmic translocation polypeptide domains of pathogen toxins, such as domain II of Pseudomonas exotoxin ETA (ETAdπ) or of similar toxins from Diptheria, Clostridium, Botulinum, Bacillus, Yersinia, Vibrio cholerae, or B or detella pertussis; or active fragments or domains of any ofthe foregoing polypeptides.
Polypeptides that route a linked protein to the cell centrosome compartment promote processing for antigen presentation. Thus, linkage of γ-tubulin to an antigen (E7 protein) efficiently re-routed E7 into the centrosome compartment, making γ-tubulin a useful IPP according to this invention.
IPP's that Promote Development or Activity of APCs or Targeting of DC
For convenience, a polypeptide or peptide that promotes development or activity of APCs or targeting of APCs, , preferably DCs, is termed a "DC-PP". One class of such IP Ps are immunostimulatory cytokines that target APCs, primarily eferably DCs, such as granulocyte macrophage colony stimulating factor (GM-CSF), or active fragments or domains thereof. DNA encoding the cytokine GM-CSF gene to DNA encoding an antigen (e.g., an HTV or hepatitis C antigen) enhanced the potency of DNA vaccines (Lee, AH et al, Vaccine 17: 473-9, 1999; Lee, SW et al, J Virol. 72: 8430-6, 1998). The chimeric GM- CSF/antigen is believed to act as an immunostimulatory signal to DCs, inducing their differentiation from an immature form (Banchereau, J et al, Nature 392: 245-52, 1998). Since DCs and their precursors express high levels of GM-CSF receptors, the chimeric GM- CSF/antigen should target and concentrate the linked antigen to the DCs and further improve the vaccine's potency. The Flt-3 ligand (FL) stimulates growth of DC precursors. Thus, the constructs ofthe present invention include FL, preferably its ECD. FL also targets a linked antigen to DCs thereby promoting antigen presentation.
The APCs targeted by the compositions ofthe present invention jnclude DCs keratinocytes, astrocytes, monocytes, macrophages, B lymphocytes, a microglial cell, or activated endothelial cells, and the like, although DC are a preferred target..
IPPs that Promote Intercellular Transport and Spread
Examples of such proteins are VP22, a herpes simplex virus type 1 (HSV-1) protein and its "homologues" in other herpes viruses, such as the avian Marek's Disease Virus (MDV) have the property of intercellular transport that provide an approach for enhancing vaccine potency. In commonly assigned patent application WO 02/09645 , published 07-FEB-02 (Wu, TC et al. , filed Ol-AUG-01) incorporated by reference herein, and in several publications with their colleagues (Hung, CF et al, 2002, J Virol 76:2676-2682; Hung, CF et al, 2001, J Immunol; 166:5733-5744; Cheng, WF et al, 2001, J Virol. 75:2368-2376 ), the present inventors disclosed novel fusions of VP22 and its homologues with a model antigen, human papillomavirus type 16 (HPV-16) E7, in a DNA vaccine which generated enhanced spreading and MHC class I presentation of antigen. These properties led to a dramatic increase in the number of E7-specific CD8 T cell precursors in vaccinated mice (at least 50-fold) and converted a less effective DNA vaccine into one with significant potency against E7-expressing tumors, comparison, a non-spreading mutant, VP22(1 -267), failed to enhance vaccine potency. Thus the potency of DNA vaccines was dramatically improved through enhanced intercellular spreading and MHC class I presentation ofthe antigen.
Despite the limited identity between the amino acid sequence of MDV- 1 UL49 (VP22) and that of HSV-1 VP22 ~ approximately 20% — both polypeptides enhanced DNA vaccine potency when linked to a "model" antigen, E7 9, as disclosed by the present inventors in WO 02/09645. It is important to note that not all molecules with "trafficking properties" have this action of enhancing vaccine potency. The present inventors found, when analyzing naked DNA vaccines comprising E7 DNA fused to DNA encoding sequences derived from proteins with trafficking properties such as HIV TAT protein, the membrane-translocating sequence and the third helix ofthe Antennapedia homeodomain did not generate CD8+ T cell- responses of similar potency as those induced by VP22/E7. Therefore, they concluded that VP22 and homologues thereof have a unique property or properties that distinguish them from these other constructs.
The order in which the two (or more) component polypeptides ofthe present fusion protein of this invention are arranged, and therefore, the order ofthe encoding nucleic acid fragments in the nucleic acid vector, can be altered without affecting immunogenicity ofthe fusion polypeptides proteins and the utility ofthe composition. For example, the Hsp70- encoding (or FL -encoding) DNA sequences may be located 5' or 3' to the target antigen- encoding sequences, h one embodiment, these polypeptide-encoding nucleic acid domains are in-frame so that the DNA construct encodes a recombinant fusion polypeptide in which the antigen is located N- terminal to the Hsp70 or FL derived polypeptide.
The vaccines ofthe present invention include, the antigenic epitope itself and an IPP, such as an MHQ-PP like Hsp70 or its active domain (CD), or a DC-PP such as FL, or a intercellular spreading protein such as VP22, as summarized above and describe in more detail below. hi fact, the vaccine construct ofthe present invention optionally, may also include more than one ofthe foregoing IPPs. Another useful polypeptide for the present constructs is a costimulatory signal, such as a B7 family protein, including B7-DC (see commonly assigned U.S. patent application Serial No. 09/794,210), B7.1, B7.2, soluble CD40, etc.). For description of some ofthe foregoing, see, for example, commonly owned
International patent publications WO 01/29233 (26-APR-Ol), WO 02/09645 (07-FEB-02) and WO 02/12281 (14-FEB-02).
The Examples described herein demonstrate that the methods ofthe invention can enhance a cellular immune response, particularly, tumor-destructive CTL reactivity, induced by a DNA vaccine encoding an epitope of a human pathogen. Human HPV-16 E7 was used as a model antigen for vaccine development because human papillomaviruses (HPVs), particularly HPV-16, are associated with most human cervical cancers. The oncogenic HPV protein E7 is important in the induction and maintenance of cellular transformation and co-expressed in most HPV-containing cervical cancers and their precursor lesions. Therefore, cancer vaccines, such as the compositions ofthe invention, that target E7 can be used to control of HPV-associated neoplasms (Wu (1994) Curr. Opin. Immunol. 6:746-754).
Unless defined otherwise, all known technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art of this invention. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
The term "antigen" or "immunogen" as used herein refers to a compound or composition comprising a peptide, polypeptide or protein which is "antigenic" or "immunogenic" when administered (or expressed in vivo by an administered nucleic acid, e.g., a DNA vaccine) in an appropriate amount (an "immunogenically effective amount"), i.e., capable of inducing, eliciting, augmenting or boosting a cellular and/or humoral immune response either alone or in combination or linked or fused to another substance (which can be administered at once or over several intervals). An immunogenic composition can comprise an antigenic peptide of at least about 5 amino acids, a peptide of 10 amino acids in length, a polypepide fragment of 15 amino acids in length, 20 amino acids in length or longer. Smaller immunogens may require presence of a "carrier" polypeptide e.g., as a fusion protein, aggregate, conjugate or mixture, preferablyl linked (chemically or otherwise) to the immunogen. The immunogen can be recombinantly expressed from a vaccine vector, which can be naked DNA comprising the immunogen' s coding sequence operably linked to a promoter, e.g., an expression cassette as described herein. The immunogen includes one or more antigenic determinants or epitopes which may vary in size from about 3 to about 15 amino acids.
The term "epitope" as used herein refers to an antigenic determinant or antigenic site that interacts with an antibody or a T cell receptor (TCR), e.g., the MHC class I-binding peptide compositions (or expressed products ofthe nucleic acid compositionsof the invention) used in the methods ofthe invention. An "antigen" is a molecule or chemical structure that either induces an immune response or is specifically recognized or bound by the product or mediator of an immune response, such as an antibody or a CTL. The specific conformational or stereochemical "domain" to which an antibody or a TCR bind is an "antigenic determinant" or "epitope." TCRs bind to peptide epitopes which are physically associated with a third molecule, a major histocompatibility complex (MHC) class I or class U protein.
The term "recombinant" refers to (1) a nucleic acid or polynucleotide synthesized or otherwise manipulated in vitro, (2) methods of using recombinant DNA technology to produce gene products in cells or other biological systems, or (3) a polypeptide encoded by a recombinant nucleic acid. For example, the FL-encoding nucleic acid or polypeptide, the nucleic acid encoding an MHC class I-binding peptide epitope (antigen) or the peptide itself can be rececombinant. "Recombinant means" includes ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into a single unit in the form of an expression cassette or vector for expression ofthe coding sequences in the vectors resulting in production ofthe encoded polypeptide.
Specifically, the present inventors investigated the novel use of VP22 proteins linked to a model antigen (HPV-16 E7) in the context of a PCL-generated replication-defective Sinbis virus replicon vaccine and found that it led to the spread of linked antigen to surrounding cells and enhanced antigen-specific immune responses and antitumor effects.
The following sections provide various nucleic acid and amino acid sequences of a model antigenic (HPV16-E7) protein and various ofthe IPP's as listed above.
The "wild-type" amino acid sequence of HPV-E7 protein is provided below:
MHGDTPTLHEYMLDLQPETTD YCYEQ NDSSEEEDEIDGPAGQAEPDRAHYMIVTFCCKCDST LRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP
(SEQ ID NO:l), GENBANK AccessionNo. AAD33353.
Production of various vectors may result in loss of certain residues ofthe antigen without affecting the immunogenicity ofthe vaccine and the specificity ofthe immune response. For example, the present inventors have described elsewhere a pcDNA3 naked DNA vector wherein only 96 ofthe 98 residues of E7 are present and the C-terminal two residues of wild-type E7, Lys and Pro are absent from this contstruct. This is an example of a deletion variant. Such deletion variants (e.g., terminal truncation of two or a small number of amino acids) of other antigenic polypeptides are examples ofthe embodiments intended within the scope ofthe fusion polypeptides of this invention. Such a modified HPV-E7 (nucleic acid sequence is SEQ ID NO:2; amino acid sequence is SEQ ID NO:3) is shown below: 1/1 31/11 atg cat gga gat aca ect aca ttg cat gaa tat atg tta gat ttg caa cca gag aca act
Met hi s gly asp thr pro thr l eu hi s gl u tyr met l eu asp leu gi n pro glu thr thr
61/21 91/31 gat etc tac tgt tat gag caa tta aat gac age tea gag gag gag gat gaa ata gat gqt asp l eu tyr cys tyr gl u gi n l eu asn asp ser ser gl u gl u gl u asp gl u i l e asp gly
121/41 151/51 cca get gqa caa gca gaa ccg gac aga gcc cat tac aat att gta ace ttt tgt tgc aag pro al a gly gi n al a gl u pro asp arg al a hi s tyr asn i l e val thr phe cys cys lys 181/61 211/71 tgt gac tct acg ctt egg ttg tgc gta caa age aca cac gta gac att cgt act ttg gaa cys asp ser thr l eu arg l eu cys val gin ser thr hi s val asp i l e arg thr l eu gl u
241/81 271/91 gac ctg tta atg ggc aca eta gqa att gtq tgc ccc ate tgt tct cag gat aag ctt asp leu l eu met gly thr leu gly i le val cys pro i l e cys ser gi n asp lys leu
After cloning, the last 9 nucleotides (3 amino acids were modified, as shown above underscored and/or bold. The original GENBANK sequence shown above has a Lys-Pro after the Gin in position 96 (encoded by aaa/cca/t aa, rather than Asp-Lys-Leu (encoded by gat/aag/ctt).
A preferred intercellular spreading protein is preferably a viral spreading protein, most preferably a herpesvirus VP22 protein. Exemplified herein are fusion constructs that comprise herpes simplex virus- 1 (HSV-1) VP22 (abbreviated HVP22). Also shown below is its homologue from Marek's disease virus (MDV) termed MDV-VP22 or MVP-22). Also included in the invention are homologues of VP22 from other members ofthe herpesviridae or polypeptides from nonviral sources that are considered to be homologous and share the functional characteristic of promoting intercellular spreading of a polypeptide or peptide that is fused or chemically conjugated thereto.
DNA encoding HVP22 has the sequence SEQ ID NO:4 which is shown as nucleotides 1- 903 below. The VP22 amino acid sequence (1-301) is SEQ ID NO:5. 1/1 31/11
ATG ACC TCT CGC CGC TCC GTG AAG TCG GGT CCG CGG GAG GTT CCG CGC GAT GAG TAC GAG
Met thr ser arg arg ser val lys ser gly pro arg glu val pro arg asp glu tyr glu 61/21 91/31 GAT CTG TAC TAC ACC CCG TCT TCA GGT ATG GCG AGT CCC GAT AGT CCG CCT GAC ACC TCC asp leu tyr tyr thr pro ser ser gly met ala ser pro asp ser pro pro asp thr ser
121/41 151/51
CGC CGT GGC GCC CTA CAG ACA CGC TCG CGC CAG AGG GGC GAG GTC CGT TTC GTC CAG TAC arg arg gly ala leu gin thr arg ser arg gin arg gly glu val arg phe val gin tyr 181/61 211/71
GAC GAG TCG GAT TAT GCC CTC TAC GGG GGC TCG TCT TCC GAA GAC GAC GAA CAC CCG GAG asp glu ser asp tyr ala leu tyr gly gly ser ser ser glu asp asp glu his pro glu
241/81 271/91
GTC CCC CGG ACG CGG CGT CCC GTT TCC GGG GCG GTT TTG TCC GGC CCG GGG CCT GCG CGG val pro arg thr arg arg pro val ser gly ala val leu ser gly pro gly pro ala arg 301/101 331/111
GCG CCT CCG CCA CCC GCT GGG TCC GGA GGG GCC GGA CGC ACA CCC ACC ACC GCC CCC CGG ala pro pro pro pro ala gly ser gly gly ala gly arg thr pro thr thr ala pro arg 361/121 391/131 GCC CCC CGA ACC CAG CGG GTG GCG TCT AAG GCC CCC GCG GCC CCG GCG GCG GAG ACC ACC ala pro arg thr gin arg val ala ser lys ala pro ala ala pro ala ala glu thr thr
421/141 , 451/151
CGC GGC AGG AAA TCG GCC CAG CCA GAA TCC GCC GCA CTC CCA GAC GCC CCC GCG TCG ACG arg gly arg lys ser al a gi n pro gl u ser al a al a l eu pro asp al a pro al a ser thr 481/161 511/171
GCG CCA ACC CGA TCC AAG ACA CCC GCG CAG GGG CTG GCC AGA AAG CTG CAC TTT AGC ACC ala pro thr arg ser lys thr pro ala gin gly leu ala arg lys leu his phe ser thr 541/181 571/191 GCC CCC CCA AAC CCC GAC GCG CCA TGG ACC CCC CGG GTG GCC GGC TTT AAC AAG CGC GTC ala pro pro asn pro asp ala pro trp thr pro arg val ala gly phe asn lys arg val
601/201 631/211
TTC TGC GCC GCG GTC GGG CGC CTG GCG GCC ATG CAT GCC CGG ATG GCG GCT GTC CAG CTC phe cys al a ala val gly arg l eu al a al a met hi s al a arg met ala al a val gi n l eu 661/221 691/231
TGG GAC ATG TCG CGT CCG CGC ACA GAC GAA GAC CTC AAC GAA CTC CTT GGC ATC ACC ACC trp asp met ser arg pro arg thr asp glu asp leu asn glu leu leu gly ile thr thr
721/241 751/251
ATC CGC GTG ACG GTC TGC GAG GGC AAA AAC CTG CTT CAG CGC GCC AAC GAG TTG GTG AAT ile arg val thr val cys glu gly lys asn leu leu gin arg ala asn glu leu val asn 781/261 811/271
CCA GAC GTG GTG CAG GAC GTC GAC GCG GCC ACG GCG ACT CGA GGG CGT TCT GCG GCG TCG pro asp val val gin asp val asp ala ala thr ala thr arg gly arg ser ala ala ser 841/281 871/291 CGC CCC ACC GAG CGA CCT CGA GCC CCA GCC CGC TCC GCT TCT CGC CCC AGA CGG CCC GTC arg pro thr glu arg pro arg ala pro ala arg ser ala ser arg pro arg arg pro val
901/301
GAG glu DNA encoding MVP22 is SEQ ID NO:6 shown below: atg ggg gat tct gaa agg egg aaa teg gaa egg cgt cgt tec ctt gga 48 tat ccc tct gca tat gat gac gtc teg att ect get cgc aga cca tea 96 aca cgt act cag cga aat tta aac cag gat gat ttg tea aaa cat gga 144 cca ttt ace gac cat cca aca caa aaa cat aaa teg gcg aaa gcc gta 192 teg gaa gac gtt teg tct ace ace egg ggt ggc ttt aca aac aaa ccc 240 cgt ace aag ccc ggg gtc aga get gta caa agt aat aaa ttc get ttc 288 agt acg get ect tea tea gca tct age act tgg aga tea aat aca gtg 336 gca ttt aat cag cgt atg ttt tgc gga gcg gtt gca act gtg get caa 384 tat cac gca tac caa ggc gcg etc ' gcc ctt tgg cgt caa gat ect ccg 432 cga aca aat gaa gaa tta gat gca ttt ctt tec aga get gtc att aaa 480 att ace att eaa gag ggt cca aat ttg atg ggg gaa gcc gaa ace tgt 528 gcc cgc aaa eta ttg gaa gag tct gga tta tec cag ggg aac gag aac 576 gta aag tec aaa tct gaa cgt aca ace aaa tct gaa cgt aca aga cgc 624 ggc ggt gaa att gaa ate aaa teg cca gat ccg gga tct cat cgt aca 672 cat aac ect cgc act ccc gca act teg cgt cgc cat cat tea tec gcc 720 cgc gga tat cgt age agt gat age gaa taa 747
The amino acid sequence ofthe MDV PV22, SEQ ID NO:7, is shown below:
Met Gly Asp Ser Glu Arg Arg Lys Ser Glu Arg Arg Arg Ser Leu Gly 16
Tyr Pro Ser Ala Tyr Asp Asp Val Ser Ile Pro Ala Arg Arg Pro Ser 32 Thr Arg Thr Gin Arg Asn Leu Asn Gin Asp Asp Leu Ser Lys His Gly 48
Pro Phe Thr Asp His Pro Thr Gin Lys His Lys Ser Ala Lys Ala Val 64
Ser Glu Asp Val Ser Ser Thr Thr Arg Gly Gly Phe Thr Asn Lys Pro 80
Arg Thr Lys Pro Gly Val Arg Ala Val Gin Ser Asn Lys Phe Ala Phe 96
Ser Thr Ala Pro Ser Ser Ala Ser Ser Thr Trp Arg Ser Asn Thr Val 112 Ala Phe Asn Gin Arg Met Phe Cys Gly Ala Val Ala Thr Val Ala Gin 128
Tyr His Ala Tyr Gin Gly Ala Leu Ala Leu Trp Arg Gin Asp Pro Pro 144
Arg Thr Asn Glu Glu Leu Asp Ala Phe Leu Ser Arg Ala Val Ile Lys 160
Ile Thr Ile Gin Glu Gly Pro Asn Leu Met Gly Glu Ala Glu Thr Cys 176
Ala Arg Lys Leu Leu Glu Glu Ser Gly Leu Ser Gin Gly Asn Glu Asn 192 Val Lys Ser Lys Ser Glu Arg Thr Thr Lys Ser Glu Arg Thr Arg Arg 208
Gly Gly Glu lie Glu lie Lys Ser Pro Asp Pro Gly Ser His Arg Thr 224
His Asn Pro Arg Thr Pro Ala Thr Ser Arg Arg His His Ser Ser Ala 240
Arg Gly Tyr Arg Ser Ser Asp Ser Glu - 249 The sequences of Hsp70 from tuberculosis is shown below
(nucleic acid is SEQ LD NO:8; amino acids are SEQ ID NO:9)
1/1 31/11 atg get cgt gcg gtc ggg ate gac etc ggg ace ace aac tec gtc gtc teg gtt etg gaa
Met ala arg ala val gly ile asp leu gly thr thr asn ser val val ser val leu glu
61/21 91/31 ggt ggc gac ccg gtc gtc gtc gcc aac tec gag ggc tec agg ace ace ccg tea att gtc gly gly asp pro val val val ala asn ser glu gly ser arg thr thr pro ser ile val
121/41 151/51 gcg ttc gcc cgc aac ggt gag gtg etg gtc ggc cag ccc gcc aag aac cag gca gtg ace ala phe ala arg asn gly glu val leu val gly gin pro ala lys asn gin ala val thr
181/61 211/71 aac gtc gat cgc ace gtg cgc teg gtc aag cga cac atg ggc age gac tgg tec ata gag asn val asp arg thr val arg ser val lys arg his met gly ser asp trp ser ile glu 241/81 271/91 att gac ggc aag aaa tac ace gcg ccg gag ate age gcc cgc att etg atg aag etg aag ile asp gly lys lys tyr thr ala pro glu ile ser ala arg ile leu met lys leu lys
301/101 331/111 cgc gac gcc gag gcc tac etc g t gag gac att ace gac gcg gtt ate acg acg ccc gcc arg asp ala glu ala tyr leu gly glu asp ile thr asp ala val ile thr thr pro ala
361/121 391/131 tac ttc aat gac gcc cag cgt cag gcc ace aag gac gcc ggc cag ate gcc ggc etc aac tyr phe asn asp ala g n arg gin ala thr lys asp ala gly gin ile ala gly leu asn
421/141 451/151 gtg etg egg ate gtc aac gag ccg ace gcg gcc gcg etg gcc tac ggc etc gac aag ggc val leu arg ile val asn glu pro thr ala ala ala leu ala tyr gly leu asp lys gly
481/161 511/171 gag aag gag cag cga ate etg gtc ttc gac ttg ggt ggt ggc act ttc gac gtt tec etg glu lys glu gin arg ile leu val phe asp leu gly gly gly thr phe asp val ser leu 541/181 571/191 etg gag ate ggc gag ggt gtg gtt gag gtc cgt gcc act teg ggt gac aac cac etc ggc leu glu ile gly glu gly val val glu val arg ala thr ser gly asp asn his leu gly
601/201 631/211 ggc gac gac tgg gac cag egg gtc gtc gat tgg etg gtg gac aag ttc aag ggc ace age gly asp asp trp asp gin arg val val asp trp leu val asp lys phe lys gly thr ser
661/221 691/231 ggc ate gat etg ace aag gac aag atg gcg atg cag egg etg egg gaa gcc gcc gag aag gly ile asp leu thr lys asp lys met ala met gin arg leu arg glu ala ala glu lys
721/241 751/251 gca aag ate gag etg agt teg agt cag tec ace teg ate aac etg ccc tac ate ace gtc ala lys ile glu leu ser ser ser gin ser thr ser ile asn leu pro tyr ile thr val
781/261 811/271 gac gcc gac aag aac ccg ttg ttc tta gac gag cag etg ace cgc gcg gag ttc caa egg asp ala asp lys asn pro leu phe leu asp glu gin leu thr arg ala glu phe gin arg 841/281 871/291 ate act cag gac etg etg gac cgc act cgc aag ccg ttc cag teg gtg ate get gac ace ile thr gin asp leu leu asp arg thr arg lys pro phe gin ser val ile ala asp thr
901/301 931/311 ggc att teg gtg teg gag ate gat cac gtt gtg etc gtg ggt ggt teg ace egg atg ccc gly ile ser val ser glu ile asp his val val leu val gly gly ser thr arg met pro
961/321 991/331 gcg gtg ace gat etg gtc aag gaa etc ace ggc ggc aag gaa ccc aac aag ggc gtc aac ala val thr asp leu val lys glu leu thr gly gly lys glu pro asn lys gly val asn
1021/341 1051/351 ccc gat gag gtt gtc gcg gtg gga gcc get etg cag gcc ggc gtc etc aag ggc gag gtg pro asp glu val val ala val gly ala ala leu gin ala gly val leu lys gly glu val
1081/361 1111/371 aaa gac gtt etg etg ctt gat gtt ace ccg etg age etg ggt ate gag ace aag ggc ggg lys asp val leu leu leu asp val thr pro leu ser leu gly ile glu thr lys gly gly 1141/381 1171/391 gtg atg ace agg etc ate gag cgc aac ace acg ate ccc ace aag egg teg gag act ttc val met thr arg leu ile glu arg asn thr thr ile pro thr lys arg ser glu thr phe
1201/401 1231/411 ace ace gcc gac gac aac caa ccg teg gtg cag ate cag gtc tat cag ggg gag cgt gag thr thr ala asp asp asn gin pro ser val gin ile gin val tyr gin gly glu arg glu
1261/421 1291/431 ate gcc gcg cac aac aag ttg etc ggg tec ttc gag etg ace ggc ate ccg ccg gcg ccg ile ala ala his asn lys leu leu g _lyy sseerr pphhee gglluu lleeuu tthhrr ggϊlvy ile pro pro ala pro 1321/441 1351/451 gg ggg att ccg cag ate gag gtc act ttc gac ate gac gcc aac ggc att gtg cac gtc arg gly ile pro gin ile glu val thr phe asp ile asp ala asn gly ile val his val 1381/461 1411/471 ace gcc aag gac aag ggc ace ggc aag gag aac acg ate cga ate cag gaa ggc teg ggc thr ala lys asp lys gly thr gly lys glu asn thr ile arg ile gin glu gly ser giy
1441/481 1471/491 etg tec aag gaa gac att gac cgc atg ate aag gac gcc gaa gcg cac gcc gag gag gat leu ser lys glu asp ile asp arg met ile lys asp ala glu ala his ala glu glu asp
1501/501 1531/511 cgc aag cgt cgc gag gag gcc gat gtt cgt aat caa gcc gag aca ttg gtc tac cag acg arg lys arg arg glu glu ala asp val arg asn gin ala glu thr eu val tyr gin thr
1561/521 1591/531 gag aag ttc gtc aaa gaa cag cgt g g gcc gag ggt ggt teg aag gta ect gaa gac acg glu lys phe val llyyss g glluu g gilnή aarrgg g glluu aallaa g glluu g glTyy g glTyy sseerr llyyss vvaall p prroo g glluu aasspp tthhrr 540
1621/541 1651/551 etg aac aag gtt gat gcc gcg gtg gcg gaa gcg aag gcg gca ctt ggc gga teg gat att leu asn lys val aasspp aallaa aallaa vvaall aallaa g qlluu aallaa llyyss aallaa aallaa lleeuu g qllyy g qllyy sseerr aasspp iillee 560
1681/561 1711/571 teg gcc ate aag teg gcg atg gag aag etg ggc cag gag teg cag etg ggg caa gcg ser al a i l e lys sseerr aallaa mmeett g qlluϋ llyyss lleeuϋ g qllyy g qilnή g qlluύ sseerr g qilnή aallaa lleeuύ g qllyy g qilnn aallaa 580
1741/581 1771/591 ate tac gaa gca get cag get gcg tea cag gcc act ggc get gcc cac ccc ggc tcq qct ile tyr glu ala ala gin ala ala ser gin ala thr gly ala ala his pro gly ser ala
1801/601 gat qaA AGC asp gl u ser
GENBANK Z95324 AL123456. This protein is encoded by nucleotides 10633-12510 of
Mycobacterium tuberculosis genome). As a result of cloning, this was modified from the original GENBANK sequence which had at its 3' end: ggc gag ccg ggc ggt gcc cac ccc ggc teg get gat gac gtt gtg gac gcg gag gtg gtc gac gac ggc egg gag gcc aag (SEQ ID NO:10) which was replaced in the cloned version described above by teg get gat gaa agc (SEQ ID NO: 11) which is bold and underlined above.
The unmodified GENBANK nucleotide sequence encoding HSP70, SEQ LD NO: 12 (Accession numbers Z95324 and AL123456, is atggctcg tgcggtcggg atcgacctcg ggaccaccaa ctccgtcgtc tcggttctgg aaggtggcga cccggtcgtc gtcgccaact ccgagggctc caggaccacc ccgtcaattg tcgcgttcgc ccgcaacggt gaggtgctgg tcggccagcc cgccaagaac caggcagtga ccaacgtcga tcgcaccgtg cgctcggtca agcgacacat gggcagcgac tggtccatag agattgacgg caagaaatac accgcgccgg agatcagcgc ccgcattctg atgaagctga agcgcgacgc cgaggcctac ctcggtgagg acattaccga cgcggttatc acgacgcccg cctacttcaa tgacgcccag cgtcaggcca ccaaggacgc cggccagatc gccggcctca acgtgctgcg gatcgtcaac gagccgaccg cggccgcgct ggcctacggc ctcgacaagg gcgagaagga gcagcgaatc ctggtcttcg acttgggtgg tggcactttc gacgtttccc tgctggagat cggcgagggt gtggttgagg tccgtgccac ttcgggtgac aaccacctcg gcggcgacga ctgggaccag cgggtcgtcg attggctggt ggacaagttc aagggcacca gcggcatcga tctgaccaag gacaagatgg cgatgcagcg gctgcgggaa gccgccgaga aggcaaagat cgagctgagt tcgagtcagt ccacctcgat caacctgccc tacatcaccg tcgacgccga caagaacccg ttgttcttag acgagcagct gacccgcgcg gagttccaac ggatcactca ggacctgctg gaccgcactc gcaagccgtt ccagtcggtg atcgctgaca ccggcatttc ggtgtcggag atcgatcacg ttgtgctcgt gggtggttcg acccggatgc ccgcggtgac cgatctggtc aaggaactca ccggcggcaa ggaacccaac aagggcgtca accccgatga ggttgtcgcg gtgggagccg ctctgcaggc cggcgtcctc aagggcgagg tgaaagacgt tctgctgctt gatgttaccc cgctgagcct gggtatcgag accaagggcg gggtgatgac caggctcatc* gagcgcaaca ccacgatccc caccaagcgg tcggagactt tcaccaccgc cgacgacaac caaccgtcgg tgcagatcca ggtctatcag ggggagcgtg agatcgccgc gcacaacaag ttgctcgggt ccttcgagct gaccggcatc ccgccggcgc cgcgggggat tccgcagatc gaggtcactt tcgacatcga cgccaacggc attgtgcacg tcaccgccaa ggacaagggc accggcaagg agaacacgat ccgaatccag gaaggctcgg gcctgtccaa ggaagacatt gaccgcatga tcaaggacgc cgaagcgcac gccgaggagg atcgcaagcg tcgcgaggag gccgatgttc gtaatcaagc cgagacattg gtctaccaga cggagaagtt cgtcaaagaa cagcgtgagg ccgagggtgg ttcgaaggta cctgaagaca cgctgaacaa ggttgatgcc gcggtggcgg aagcgaaggc ggcacttggc ggatcggata tttcggccat caagtcggcg atggagaagc tgggccagga gtcgcaggct ctggggcaag cgatctacga agcagctcag gctgcgtcac aggccactgg cgctgcccac cccggcggcg agccgggcgg tgcccacccc ggctcggctg atgacgttgt ggacgcggag gtggtcgacg acggccggga ggccaagtga
The cDNA sequence of Mouse GM-CSF (SEQ LD NO: 13) is as follows: gagctcagca agcgctctcc cccaattccc ttagccaaag tggacgccac cgaacagaca 61 gacctaggct aagaggtttg atgtctctgg ctacccgact ttgaaaattt tccgcaaagg 121 aaggcctttt gactacaatg gcccacgaga gaaaggctaa ggtcctgagg aggatgtggc 181 tgcagaattt acttttcctg ggcattgtgg tctacagcct ctcagcaccc acccgctcac 241 ccatcactgt cacccggcct tggaagcatg tagaggccat caaagaagcc ctgaacctcc 301 tggatgacat gcctgtcaca ttgaatgaag aggtagaagt cgtctctaac gagttctcct 361 tcaagaagct aacatgtgtg cagacccgcc tgaagatatt cgagcagggt ctacggggca 421 atttcaccaa actcaagggc gccttgaaca tgacagccag ctactaccag acatactgcc 481 ccccaactcc ggaaacggac tgtgaaacac aagttaccac ctatgcggat ttcatagaca 541 gccttaaaac ctttctgact gatatcccct ttgaatgcaa aaaaccagtc caaaaatgrag 601 gaagcccagg ccagctctga atccagcttc tcagactgct gcttttgtgc ctgcgtaatg 661 agccaggaac tcggaatttc tgccttaaag ggaccaagag atgtggcaca gccacagttg, 721 gagggcagta tagccctctg aaaacgctga ctcagcttgg acagcggcaa gacaaacgag 781 agatattttc tactgatagg gaccattata tttatttata tatttatatt ttttaaatat 841 ttatttattt atttatttaa ttttgcaact ctatttattg agaatgtctt accagataat 901 aaattattaa aacttt
GENBANK Accession No. X02333; start sites and termination codon are italicized and bolded. For additional mRNA for murine GM-CSF, See GENBANK # X05906
The amino acid sequence of mouse GM-CSF (161 residues) (SEQ ID NO: 14) is :
MWLQNLLFLG IWYSLSAPT RSPITVTRPW KHVEAIKEAL NLLDDMPVTL NEEVEWSNE FSFKKLTCVQ TRLKIFEQGL RGNFTKLKGA LN TASYYQT YCPPTPETDC ETQVTTYADF
IDSLKTFLTD IPFECKKPVQ K GENBANK Accession No. X02333.
A cDNA sequence encoding Pseudomonas exotoxin A (ETA) (SEQ ID NO: 15) is ctgcagctgg tcaggccgtt tccgcaacgc ttgaagtcct ggccgatata ccggcagggc 61 cagccatcgt tcgacgaata aagccacctc agccatgatg ccctttccat ccccagcgga 121 accccgacat ggacgccaaa gccctgctcc tcggcagcct ctgcctggcc gccccattcg 181 ccgacgcggc gacgctcgac aatgctctct ccgcctgcct cgccgcccgg ctcggtgcac 241 cgcacacggc ggagggccag ttgcacctgc cactcaccct tgaggcccgg cgctccaccg 301 gcgaatgcgg ctgtacctcg gcgctggtgc gatatcggct gctggccagg ggcgccagcg 361 ccgacagcct cgtgcttcaa gagggctgct cgatagtcgc caggacacgc cgcgcacgct 421 gaccctggcg gcggacgccg gcttggcgag cggccgcgaa ctggtcgtca ccctgggttg 481 tcaggcgcct gactgacagg ccgggctgcc accaccaggc cgagatggac gccctgcatg 541 tatcctccga tcggcaagcc tcccgttcgc acattcacca ctctgcaatc cagttcataa 601 atcccataaa agccctcttc cgctccccgc cagcctcccc gcatcccgca ccctagacgc 661 cccgccgctc tccgccggct cgcccgacaa gaaaaaccaa ccgctcgatc agcctcatcc 721 ttcacccatc acaggagcca tcgcgatgca cctgataccc cattggatcc ccctggtcgc 781 cagcctcggc ctgctcgccg gcggctcgtc cgcgtccgcc gccgaggaag ccttcgacct 841 ctggaacgaa tgcgccaaag cctgcgtgct cgacctcaag gacggcgtgc gttccagccg 901 catgagcgtc gacccggcca tcgccgacac caacggccag ggcgtgctgc actactccat 961 ggtcctggag ggcggcaacg acgcgctcaa gctggccatc gacaacgccc tcagcatcac 1021 cagcgacggc ctgaccatcc gcctcgaagg cggcgtcgag ccgaacaagc cggtgcgcta 1081 cagctacacg cgccaggcgc gcggcagttg gtcgctgaac tggctggtac cgatcggcca 1141 cgagaagccc tcgaacatca aggtgttcat ccacgaactg aacgccggca accagctcag 1201 ccacatgtcg ccgatctaca ccatcgagat gggcgacgag ttgctggcga agctggcgcg 1261 cgatgccacc ttcttcgtca gggcgcacga gagcaacgag atgcagccga cgctcgccat 1321 cagccatgcc ggggtcagcg tggtcatggc ccagacccag ccgcgccggg aaaagcgctg 1381 gagcgaatgg gccagcggca aggtgttgtg cctgctcgac ccgctggacg gggtctacaa 1441 ctacctcgcc cagcaacgct gcaacctcga cgatacctgg gaaggcaaga tctaccgggt 1501 gctcgccggc aacccggcga agcatgacct ggacatcaaa cccacggtca tcagtcatcg 1561 cctgcacttt cccgagggcg gcagcctggc cgcgctgacc gcgcaccagg cttgccacct 1621 gccgctggag actttcaccc gtcatcgcca gccgcgcggc tgggaacaac tggagcagtg 1681 cggctatccg gtgcagcggc tggtcgccct ctacctggcg gcgcggctgt cgtggaacca 1741 ggtcgaccag gtgatccgca acgccctggc cagccccggc agcggcggcg acctgggcga 1801 agcgatccgc gagcagccgg agcaggcccg tctggccctg accctggccg ccgccgagag 1861 cgagcgcttc gtccggcagg gcaccggcaa cgacgaggcc ggcgcggcca acgccgacgt 1921 ggtgagcctg acctgcccgg tcgccgccgg tgaatgcgcg ggcccggcgg acagcggcga 1981 cgccctgctg gagcgcaact atcccactgg cgcggagttc ctcggcgacg gcggcgacgt 2041 cagcttcagc acccgcggca cgcagaactg gacggtggag cggctgctcc aggcgcaccg 2101 ccaactggag gagcgcggct atgtgttcgt cggctaccac ggcaccttcc tcgaagcggc 2161 gcaaagcatc gtcttcggcg gggtgcgcgc gcgcagccag gacctcgacg cgatctggcg 2221 cggtttctat atcgccggcg atccggcgct ggcctacggc tacgcccagg accaggaacc 2281 cgacgcacgc ggccggatcc gcaacggtgc cctgctgcgg gtctatgtgc cgcgctcgag 2341 cctgccgggc ttctaccgca ccagcctgac cctggccgcg ccggaggcgg cgggcgaggt 2401 cgaacggctg atcggccatc cgctgccgct gcgcctggac gccatcaccg gccccgagga 2461 ggaaggcggg cgcctggaga ccattctcgg ctggccgctg gccgagcgca ccgtggtgat 2521 tccctcggcg atccccaccg acccgcgcaa cgtcggcggc gacctcgacc cgtccagcat 2581 ccccgacaag gaacaggcga tcagcgccct gccggactac gccagccagc ccggcaaacc 2641 gccgcgcgag gacctgaagt aactgccgcg accggccggc tcccttcgca ggagccggcc 2701 ttctcggggc ctggccatac atcaggtttt cctgatgcca gcccaatcga atatgaattc 2761
GENBANK Accession No. K01397 M23348.
The encoded amino acid sequence of ETA (SEQ ID NO: 16) is
MHLIPHWIPL VASLGLLAGG SSASAAEΞAF DLWNECAKAC VLDLKDGVRS SRMSVDPAIA DTNGQGVLHY SMVLEGGNDA LKLAIDNALS ITSDGLT RL EGGVEPNKPV RYSYTRQARG SWSLNWLVPI GHEKPSNIKV FIHELNAGNQ LSHMSPIYTI EMGDELLAKL ARDATFFVRA HESNEMQPTL AISHAGVSW MAQTQPRREK RWSEWASGKV LCLLDPLDGV YNYLAQQRCN LDDTWEGKIY RVLAGNPAKH DLDIKPTVIS HRLHFPEGGS LAALTAHQAC HLPLETFTRH RQPRG EQLE QCGYPVQRLV ALYLAARLSW NQVDQVIRNA LASPGSGGDL GEAIREQPEQ ARLALTLAAA ESERFVRQGT GNDEAGAANA D SLTCPVA AGECAGPADS GDALLERNYP TGAEFLGDGG DVSFSTRGTQ NWTVERLLQA HRQLEERGYV FVGYHGTFLE AAQSIVFGGV RARSQDLDAI WRGFYIAGDP ALAYGYAQDQ EPDARGRIRN GALLRVYVPR SSLPGFYRTS LTLAAPEAAG EVERLIGHPL PLRLDAITGP EEEGGRLETI LGWPLAERTV VIPSAIPTDP RNVGGDLDPS SIPDKEQAIS ALPDYASQPG KPPREDLK
GENBANK Accession No. K01397 M23348; residues 1-25 = signal peptide; start of mature peptide is the underscored "A"; the ETA translocation domain, which is a useful IPP according to this invention, spans residues 247-417 (underscored, bolded) of SEQ ID NO_, above. The sequence ofthe Flt3 Ligand (FL) extracellular domain is shown below :
(nucleic acid is SEQ ID NO: 17; amino acids are SEQ LD NO: 18)
1/1 31/11 atg aca gtg etg gcg cca gcc tgg agc cca aat tec tec etg ttg etg etg ttg etg etg
Met thr val leu ala pro ala trp ser pro asn ser ser leu leu leu leu leu leu leu 61/21 91/31 etg agt ect tgc etg egg ggg aca ect gac tgt tac ttc agc cac agt ccc ate tec tec leu ser pro cys leu arg gly thr pro asp cys tyr phe ser his ser pro ile ser ser
121/41 151/51 aac ttc aaa gtg aag ttt aga gag ttg act gac cac etg ctt aaa gat tac cca gtc act asn phe lys val lys phe arg glu leu thr asp his leu leu lys asp tyr pro val thr
181/61 211/71 gtg gcc gtc aat ctt cag gac gag aag cac tgc aag gcc ttg tgg agc etc ttc eta gcc val ala val asn leu gin asp glu lys his cys lys ala leu trp ser leu phe leu ala 241/81 271/91 cag cgc tgg ata gag caa etg aag act gtg gca ggg tct aag atg caa acg ctt etg gag gin arg trp ile glu gin leu lys thr val ala gly ser lys met gin thr leu leu glu
301/101 331/111 gac gtc aac ace gag ata cat ttt gtc ace tea tgt ace ttc cag ccc eta cca gaa tgt asp val asn thr glu ile his phe val thr ser cys thr phe gin pro leu pro glu cys
361/121 391/131 etg cga ttc gtc cag ace aac ate tec cac etc etg aag gac ace tgc aca cag etg ctt leu arg phe val gin thr asn ile ser his leu leu lys asp thr cys thr gin leu leu
421/141 451/151 get etg aag ccc tgt ate ggg aag gcc tgc cag aat ttc tct egg tgc etg gag gtg cag ala leu lys pro cys ile gly lys ala cys gin asn phe ser arg cys leu glu val gin
481/161 511/171 tgc cag ccg gac tec tec ace etg etg ccc cca agg agt ccc ata gcc eta gaa gcc acg cys gin pro asp ser ser thr leu leu pro pro arg ser pro ile ala leu glu ala thr 541/181 gag etc cca gag ect egg ccc agg cag glu leu pro glu pro arg pro arg gin
A fusion polypeptide FL-E7 is shown below: (nucleic acid is SEQ ID NO:19; amino acids are SEQ ID NO:20). The N-terminal sequence is FL, followed by E7 (underscored, nucleic acids capitalized)
1/1 31/11 atg aca gtg etg gcg cca gcc tgg agc cca aat tec tec etg ttg etg etg ttg etg etg
Met thr val leu ala pro ala trp ser pro asn ser ser leu leu leu leu leu leu leu 61/21 91/31 etg agt ect tgc etg egg ggg aca ect gac tgt tac ttc agc cac agt ccc ate tec tec leu ser pro cys leu arg gly thr pro asp cys tyr phe ser his ser pro ile ser ser
121/41 151/51 aac ttc aaa gtg aag ttt aga gag ttg act gac cac etg ctt aaa gat tac cca gtc act asn phe lys val lys phe arg glu leu thr asp his leu leu lys asp tyr pro val thr
181/61 211/71 gtg gcc gtc aat ctt cag gac gag aag cac tgc aag gcc ttg tgg agc etc ttc eta gcc val ala val asn leu gin asp glu lys his cys lys ala leu trp ser leu phe leu ala
241/81 271/91 cag cgc tgg ata gag caa etg aag act gtg gca ggg tct aag atg caa acg ctt etg gag gin arg trp ile glu gin leu lys thr val ala gly ser lys met gin thr leu leu glu
301/101 331/111 gac gtc aac ace gag ata cat ttt gtc ace tea tgt ace ttc cag ccc eta cca gaa tgt asp val asn thr glu ile his phe val thr ser cys thr phe gin pro leu pro glu cys 361/121 391/131 etg cga ttc gtc cag ace aac ate tec cac etc etg aag gac ace tgc aca cag etg ctt leu arg phe val gin thr asn ile ser his leu leu lys asp thr cys thr gin leu leu
421/141 451/151 get etg aag ccc tgt ate ggg aag gcc tgc cag aat ttc tct egg tgc etg gag gtg cag ala leu lys pro cys ile gly lys ala cys gin asn phe ser arg cys leu glu val gin
481/161 511/171 tgc cag ccg gac tec tec ace etg etg ccc cca agg agt ccc ata gcc eta gaa gcc acg cys gin pro asp ser ser thr leu leu pro pro arg ser pro ile ala leu glu ala thr
541/181 571/191 gag etc cca gag ect egg ccc agg cag gaa ttc ATG CAT GGA GAT ACA CCT ACA TTG CAT glu leu pro glu pro arg pro arg gin glu phe met his qly asp thr pro thr leu his
601/201 631/211
GAA TAT ATG TTA GAT TTG CAA CCA GAG ACA ACT GAT CTC TAC TGT TAT GAG CAA TTA AAT glu tyr met leu asp leu gin pro glu thr thr asp leu tyr cvs tyr glu gin leu asn 661/221 691/231
GAC AGC TCA GAG GAG GAG GAT GAA ATA GAT GGT CCA GCT GGA CAA GCA GAA CCG GAC AGA asp ser ser gl u gl u gl u asp gl u i l e asp qlv pro al a qly gi n al a gl u pro asp arg 721/241 751/251
GCC CAT TAC AAT ATT GTA ACC TTT TGT TGC AAG TGT GAC TCT ACG CTT CGG TTG TGC GTA aallaa hhiiss tvr asn ile val thr phe cvs cvs lys cvs asp ser thr leu arq leu cvs val 7 78811//226611 811/271 CCAAAA AAGGCC ACA CAC GTA GAC ATT CGT ACT TTG GAA GAC CTG TTA ATG GGC ACA CTA GGA ATT ggiinn sseerr thr his val asp ile arg thr leu qlu asp leu leu met qly thr leu qly ile
841/281
GTG TGC CCC ATC TGT TCT CAA GGA TCC val cvs pro ile cys ser gin gly ser
The sequences of CRT, including human CRT, are well known in the art (McCauliffe (1990) J. Clin. Invest. 86:332-335; Burns (1994) Nature 367:476-480; Coppolino (1998) Int. J. Biochem. Cell Biol. 30:553-558). The nucleic acid sequence appears as GenBank Accession No. NM 004343 and is SEQ ID NO:21.
1 gtccgtactg cagagccgct gccggagggt cgttttaaag ggccgcgttg ccgccccctc
61 ggcccgccat gctgctatcc gtgccgctgc tgctcggcct cctcggcctg gccgtcgccg
121 agcccgccgt ctacttcaag gagcagtttc tggacggaga cgggtggact tcccgctgga
181 tcgaatccaa acacaagtca gattttggca aattcgttct cagttccggc aagttctacg
241 gtgacgagga gaaagataaa ggtttgcaga caagccagga tgcacgcttt tatgctctgt
301 cggccagttt cgagcctttc agcaacaaag gccagacgct ggtggtgcag ttcacggtga
361 aacatgagca gaacatcgac tgtgggggcg gctatgtgaa gctgtttcct aatagtttgg
421 accagacaga catgcacgga gactcagaat acaacatcat gtttggtccc gacatctgtg
481 gccctggcac caagaaggtt catgtcatct tcaactacaa gggcaagaac gtgctgatca
541 acaaggacat ccgttgcaag gatgatgagt ttacacacct gtacacactg attgtgcggc
601 cagacaacac ctatgaggtg aagattgaca acagccaggt ggagtccggc tccttggaag
661 acgattggga cttcctgcca cccaagaaga taaaggatcc tgatgcttca aaaccggaag
721 actgggatga gcgggccaag atcgatgatc ccacagactc caagcctgag gactgggaca
781 agcccgagca tatccctgac cctgatgcta agaagcccga ggactgggat gaagagatgg
841 acggagagtg ggaaccccca gtgattcaga accctgagta caagggtgag tggaagcccc
901 ggcagatcga caacccagat tacaagggca cttggatcca cccagaaatt gacaaccccg
961 agtattctcc cgatcccagt atctatgcct atgataactt tggcgtgctg ggcctggacc
1021 tctggcaggt caagtctggc accatctttg acaacttcct catcaccaac gatgaggcat
1081 acgctgagga gtttggcaac gagacgtggg gcgtaacaaa ggcagcagag aaacaaatga
1141 aggacaaaca ggacgaggag cagaggctta aggaggagga agaagacaag aaacgcaaag
1201 aggaggagga ggcagaggac aaggaggatg atgaggacaa agatgaggat gaggaggatg
1261 aggaggacaa ggaggaagat gaggaggaag atgtccccgg ccaggccaag gacgagctgt
1321 agagaggcct gcctccaggg ctggactgag gcctgagcgc tcctgccgca gagcttgccg
1381 cgccaaataa tgtctctgtg agactcgaga actttcattt ttttccaggc tggttcggat
1441 ttggggtgga ttttggtttt gttcccctcc tccactctcc cccaccccct ccccgccctt
1501 tttttttttt tttttaaact ggtattttat cctttgattc tccttcagcc ctcacccctg
1561 gttctcatct ttcttgatca acatcttttc ttgcctctgt gccccttctc tcatctctta
1621 gctcccctcc aacctggggg gcagtggtgt ggagaagcca caggcctgag atttcatctg
1681 ctctccttcc tggagcccag aggagggcag cagaaggggg tggtgtctcc aaccccccag
1741 cactgaggaa gaacggggct cttctcattt cacccctccc tttctcccct gcccccagga
1801 ctgggccact tctgggtggg gcagtgggtc ccagattggc tcacactgag aatgtaagaa
1861 ctacaaacaa aatttctatt aaattaaatt ttgtgtctc 1899
Human CRT protein (GenBank Accession No. NM 004343), (SEQ LD NO:22) is shown below:
1 MLLSVPLLLG LLGLAVAEPA VYFKEQFLDG DGWTSR IES KH SDPGKFV LSSGKFYGDE 61 EKDKGLQTSQ DARFYALSAS FEPFSN GQT LWQFTVKHE QNIDCGGGYV KLFPNSLDQT
121 DMHGDSEYNI MFGPDICGPG TKKVHVIFNY KGKNVLINKD IRC DDΞFTH LYTLIVRPDN
181 TYEVKIDNSQ VESGSLEDD DFLPP KIKD PDASKPED D ERAKIDDPTD SKPED DKPΞ
241 HIPDPDAKKP EDWDEEMDGE EPPVIQNPE YKGEWKPRQI DNPDYKGT I HPEIDNPEYS
301 PDPSIYAYDN FGVLGLDLWQ VKSGTIFDNF LITNDEAYAE EFGNETWGVT KAAEKQMKDK 361 QDEEQRLKEE EEDKKRKEEE EAEDKEDDED KDEDΞEDEED KEEDEEΞDVP GQAKDEL 417 ANTIGENS -
In one embodiment, the antigen ofthe present invention against which immunity is desired and which may be as short as an MHC class I-binding peptide epitope, is derived from a pathogen, e.g., it comprises a peptide expressed by a pathogen. The pathogen can be a virus, such as, e.g. , a papilloma virus, a herpesvirus, a retrovirus (e.g. , an immunodeficiency virus, such as HIV-1), an adenovirus, and the like. The papilloma virus can be a human papilloma virus; for example, the antigen (e.g., the Class I-binding peptide) can be derived from an HPV- 16 E7 polypeptide. In one embodiment, the HPV-16 E7 polypeptide is substantially non- onco genie, i.e., it does not bind retinoblastoma polypeptide (pRB) or binds pRB with such low affinity that the HP V- 16 E7 polypeptide is effectively non-oncogenic when expressed or delivered in vivo. h alternative embodiments, the pathogen is a bacteria, such ΆSB or detella pertussis; Ehrlichia chaffeensis; Staphylococcus aureus; Toxoplasma gondii; Legionella pneumophila; Brucella suis; Salmonella enterica; Mycobacterium avium; Mycobacterium tuberculosis; Listeria monocytogenes; Chlamydia trachomatis; Chlamydia pneumoniae; Rickettsia rickettsii; or, a fungi, such as, e.g., Paracoccidioides brasiliensis; or other pathogen, e.g., Plasmodium falciparum.
In another embodiment, the MHC class I-binding peptide epitope is derived from a tumor cell. The tumor cell-derived peptide epitope can comprise a tumor associated antigen, e.g., a tumor specific antigen, such as, e.g., a HER-2/neu antigen.
Generating and Manipulating of Nucleic Acids
The methods ofthe invention provide for the administration of nucleic acid vectors encoding a fusion protein between an antigen, preferably a MHC Class I epitope binding polypeptide or peptide, used to an IPP, as has been described above. Recombinant IPP- containing fusion proteins can be synthesized in vitro or in vivo.
Nucleic acids encoding these compositions can be prepared in in the form of "naked DNA" or they can be incorporated in plasmids, vectors, recombinant viruses (e.g., "replicons") and the like. The present invention is directed ot one class of vectors, replication defective alphavirus vectors, prefereably Sinbis virus, for in vivo or ex vivo administration. Nucleic acids and vectors ofthe invention can be made and expressed in vitro or in vivo, a variety of means of making and expressing these genes and vectors can be used. One of skill will recognize that desired expression can be obtained by modulating the activity ofthe nucleic acids (e.g., promoters) within vectors used to practice the invention. Any ofthe known methods described for increasing or decreasing expression or activity, or tissue specificity, of genes can be used for this invention. The invention can be practiced in conjunction with any method or protocol known in the art, which are well described in the scientific and patent literature. GENERAL RECOMBINANT DNA METHODS
Basic texts disclosing general methods of molecular biology, all of which are incorporated by reference, include: Sambrook, J et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989; Ausubel, FM et al Current Protocols in Molecular Biology, Vol. 2, Wiley-Interscience, New York, (current edition); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); Glover, DM, ed, DNA Cloning: A Practical Approach, vol. I & II, IRL Press, 1985; Albers, B. et al, Molecular Biology ofthe Cell, 2nd Ed., Garland Publishing, Inc., New York, NY (1989); Watson, JD et al, Recombinant DNA, 2nd Ed., Scientific American Books, New York, 1992; and Old, RW et al, Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2 Ed., University of California Press, Berkeley, CA (1981).
Unless otherwise indicated, a particular nucleic acid sequence is intended to encompasses conservative substitution variants thereof (e.g., degenerate codon substitutions) and a complementary sequence. The term "nucleic acid" is synonymous with "polynucleotide" and is intended to include a gene, a cDNA molecule, an mRNA molecule, as well as a fragment of any of these such as an oligonucleotide, and further, equivalents thereof (explained more fully below). Sizes of nucleic acids are stated either as kilobases (kb) or base pairs (bp). These are estimates derived from agarose or polyacrylamide gel electrophoresis (PAGE), from nucleic acid sequences which are determined by the user or published. Protein size is stated as molecular mass in kilodaltons (kDa) or as length (number of amino acid residues). Protein size is estimated from PAGE, from sequencing, from presumptive amino acid sequences based on the coding nucleic acid sequence or from published amino acid sequences.
Specifically, cDNA molecules encoding the amino acid sequence corresponding to the fusion polypeptide ofthe present invention or fragments or derivatives thereof can be synthesized by the polymerase chain reaction (PCR) (see, for example, U.S. 4,683,202) using primers derived the sequence ofthe protein disclosed herein. These cDNA sequences can then be assembled into a eukaryotic or prokaryotic expression vector and the resulting vector can be used to direct the synthesis ofthe fusion polypeptide or its fragment or derivative by appropriate host cells, for example COS or CHO cells. This invention includes isolated nucleic acids having a nucleotide sequence encoding the novel fusion polypeptides that comprise a spreading protein and an antigen, fragments thereof or equivalents thereof. The term nucleic acid as used herein is intended to include such fragments or equivalents. The nucleic acid sequences of this invention can be DNA or RNA. A cDNA nucleotide sequence the fusion polypeptide can be obtained by isolating total mRNA from an appropriate cell line. Double stranded cDNA is prepared from total mRNA. cDNA can be inserted into a suitable plasmid, bacteriophage or viral vector using any one of a number of known techniques. In reference to a nucleotide sequence, the term "equivalent" is intended to include sequences encoding structurally homologous and/or a functionally equivalent proteins. For example, a natural polymorphism of a sequence encoding an LPP such as viral VP22 spreading protein or CRT, or the like, (especially at the third base of a codon) may be manifest as "silent" mutations which do not change the amino acid sequence. Furthermore, there may be one or more naturally occurring isoforms or related, immunologically cross-reactive family members of the an LPP such as VP22. Such isoforms or family members are defined as proteins that share function amino acid sequence similarity to the reference protein.
Fragment of Nucleic Acid
A fragment ofthe nucleic acid sequence is defined as a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length IPP, antigenic polypeptide or the fusion thereof. This invention includes such nucleic acid fragments that encode polypeptides which retain (1) the ability ofthe fusion polypeptide to induce increases in frequency or reactivity of T cells, preferably CD8+ T cells, that are specific for the antigen portion ofthe fusion polypeptide. For example, a nucleic acid fragment as intended herein encodes a VP22 or HSP70 or
CRT or FL or other type of IPP that retains the ability to improve the immunogenicity of an antigen when administered as a fusion polypeptide with an antigenic polypeptide or peptide.
Generally, the nucleic acid sequence encoding a fragment of an IPP polypeptide comprises nucleotides from the sequence encoding the mature protein (i.e., excluding signal peptide sequences).
Nucleic acid sequences of this invention may also include linker sequences, natural or modified restriction endonuclease sites and other sequences that are useful for manipulations related to cloning, expression or purification of encoded protein or fragments. These and other modifications of nucleic acid sequences are described herein or are well-known in the art. The techniques for assembling and expressing DNA coding sequences for ffPs such as spreading proteins, proteins or ER-such as VP22 and antigenic polypeptides such as synthesis of oligonucleotides, PCR, transforming cells, constructing vectors, expression systems, and the like are well-established in the art. Those of ordinary skill are familiar with the standard resource materials for specific conditions and procedures. EXPRESSION VECTORS AND HOST CELLS
This invention includes an expression vector comprising a nucleic acid sequence encoding a spreading protein/antigen fusion polypeptide operably linked to at least one regulatory sequence. "Operably linked" means that the coding sequence is linked to a regulatory sequence in a manner that allows expression ofthe coding sequence. Known regulatory sequences are selected to direct expression ofthe desired protein in an appropriate host cell. Accordingly, the term "regulatory sequence" includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in, for example, Goeddel, Gene
Expression Technology. Methods in Enzymology, vol. 185, Academic Press, San Diego, Calif. (1990)).
Those skilled in the art appreciate that the particular design of an expression vector of this invention depends on considerations such as the host cell to be transfected and/or the type of protein to be expressed.
The present expression vectors comprise the full range of nucleic acid molecules encoding the various embodiments ofthe fusion polypeptide and its functional derivatives (defined herein) including polypeptide fragments, variants, etc.
Such expression vectors are used to transfect host cells for expression ofthe DNA and production ofthe encoded proteins which include fusion proteins or peptides. It will be understood that a genetically modified cell expressing the fusion polypeptide may transiently express the exogenous DNA for a time sufficient for the cell to be useful for its stated purpose.
The present in invention provides methods for producing the fusion polypeptides, fragments and derivatives. For example, a host cell transfected with a nucleic acid vector that encodes the fusion polypeptide is cultured under appropriate conditions to allow expression of the polypeptide.
Host cells may also be transfected with one or more expression vectors that singly or in combination comprise DNA encoding at least a portion ofthe fusion polypeptide and DNA encoding at least a portion of a second protein, so that the host cells produce yet further fusion polypeptides that include both the portions. A culture typically includes host cells, appropriate growth media and other byproducts. Suitable culture media are well known in the art. The fusion polypeptide can be isolated from medium or cell lysates using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, fractionation column chromatography (e.g. ion exchange, gel filtration, affinity chromatography, etc.) and or electrophoresis (see generally, "Enzyme Purification and Related Techniques", Meth Enzymol 22: 233-577 (1971)). Once purified, partially or to homogeneity, the recombinant polypeptides ofthe invention can be utilized in pharmaceutical compositions as described in more detail herein.
Prokaryotic or eukaryotic host cells transformed or transfected to express the fusion polypeptide or a homologue or functional derivative thereof are within the scope ofthe invention. For example, the fusion polypeptide may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO) or human cells. Other suitable host cells may be found in Goeddel, (1990) supra or are otherwise known to those skilled in the art. Expression in eukaryotic cells leads to partial or complete glycosylation and/or formation of relevant inter- or infra-chain disulfide bonds ofthe recombinant protein. PCL-generated replication-defective Alpha virus replicons
Vector systems for the expression of heterologous genes have been developed from full- length cDNA clones of three members ofthe alphavirus genus, Sindbis virus ("SIN"), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE) (Xiong et al, 1989; Huang, HV et al, 1989, Virus Genes 3:85-91; Liljestrom, P et al, , 1991, BioTechnology 9:1356-1361; Bredenbeek, PJ et α/., 1993, J. Virol. 67:6439-6446; Zhou, X et al, 1994, Vaccine 12:1510- 1514; Davis et al, 1996, J. Virol. 70:3781-3787; Dryga, SA et al, 1996, (Russian). Vopr. Virusol 3:100-104; reviewed in Frolov et al, supra. The systems are of two general types: "double promoter' ' vectors and ' 'replicon' ' vectors.
In alphavirus replicon vectors, the viral structural protein genes are deleted and replaced by a heterologous gene under the control ofthe 26S promoter. The structural genes are provided in trans from a helper construct( s) consisting of a partial clone ofthe viral genome which is missing all or part ofthe genes for the nonstructural proteins, including the putative cis-acting RNA packaging signals (Weiss, B et a/., 1989, J Virol. ^3:5310-5318; Weiss, B. et al, 1994, Nucleic Acids Res. 22:780-786). Both the replicon and the helper RNAs retain the cis-acting terminal sequences required for genome replication and the promoter for transcription ofthe subgenomic mRNA. The replicon RNA supplies the nonstructural proteins in trans for the expression ofthe helper RNA. However, only the replicon RNA retains the packaging signal(s), and it is packaged into replicon particles by the viral structural proteins provided in trans by the helper. Infection of cells by these replicon particles results in amplification of replicon RNA and expression ofthe heterologous gene, but there is no further spread to other cells.
Alphavirus replicon vectors have been used to vaccinate against microbial pathogens (Zhou, X et al, 1995, Proc. Natl. Acad. Sci. USA 92:3009-3013; Mossman, SP et al, 1996, J. Virol. 70:1953-1960) and offer several potential advantages as delivery systems. As noted above, replicons typically express heterologous genes to high levels, permittin a relatively low dose of replicon particles to produce a large dose of immunogen in vivo. Because they lack a complete complement of viral genes, after replication and expression ofthe heterologous gene in the cells initially infected, no additional infectious particles would be produced to spread to other tissues. This property contributes significantly to the inherent safety of alphavirus replicon vectors. hi addition, the self-limiting nature ofthe replicon particle infection and the lack of structural protein expression should minimize the induction of an immune response to the vector, allowing the sequential use of these vectors for immunization ofthe same individual with immunogens of different pathogens. As described in Pushko et al, 1997, VEE was the only alphavirus for which a live, attenuated vaccine strain (TC-83) has been developed for veterinary and human use (Jahrling, m et al, 1984, J Clin. Microbiol 19:429^131; Kinney, R et al, 1993, J. Virol. 67:1269-1277). Newer live, attenuated VEE vaccines with improvements over TC-83, were been developed from a full-length cDNA clone (Davis, NL et al, 1989, Virology 171:189-204), by the introduction of multiple attenuating mutations into the structural genes. Thus, vectors derived from such vaccine strains are inherently safer than those derived from wild-type virus. Several of these strains, including V3014, replicate in lymphoid tissue without causing disease (Pushko et al, supra), h the context of a vector, the VEE glycoproteins will preferentially target heterologous gene expression to lymphoid tissues (Davis, NL et al, 1996, J. Virol. 70:3781- 3787; Caley, U et al, 1997, J. Virol. 77:3031-3038), which would be expected to increase immuno genicity ofthe heterologous gene product. Two disclosed strategies for improving the safety and efficacy of a VEE-based replicon vector (Pushko et al, supra) either (1) included previously defined attenuating mutations in the replicon and/or its helper so that any viable recombinant virus is could not initiate a virulent infection; or (2) used a bipartite helper to supply structural proteins for packaging ofthe replicon into particles, thus requiring at least two recombination events for the generation of viable virus. This strategy greatly reduces the probability that a viable recombinant VEE virus would be generated during packaging. Using a VEE replicon particle vaccine prepared in this manner, Pushko and colleagues induced potent protective immunity to a heterologous mucosal pathogen in naive animals and have achieved an equally high level, protective response in animals previously immunized with VEE replicon particle-expressing genes from another pathogen.
The utility ofthe alphavirus replicon expression systems has been markedly improved by development of a series of defective helper RNAs that allow efficient packaging of RNA replicons (Liljestrom et al, supra; Bredenbeek et al, supra). Defective-helper RNAs (DHRNAs) are designed to contain the cis-acting sequences required for replication as well as the subgenomic RNA promoter driving expression ofthe structural protein genes. Packaging of SLN replicons is achieved by efficient cotransfection of BHK cells with both RNAs by electroporation (Liljestrom et al, 1991, supra) (See also, Frolov et al, 1996, supra; Fig. 3). Replicase/franscriptase functions supplied by the vector RNA lead not only to its own amplification but also act in trans to allow replication and transcription ofthe helper RNA. This results in synthesis of structural proteins that can package the replicon with >10 infectious particles per ml (5xl09 infectious particles per electroporation) being produced after only 16-24 h. Such stocks can be used without further phenotypic selection to infect cells for expression studies or high-level protein production. According to Frolov et al, supra, it should be possible to package replicons containing at least 5 kb of heterologous sequence. A spectrum of DHRNAs have been characterized that differ in their ability to be packaged. Some DHRNAs that allow packaging ofthe replicon as well as themselves are useful under conditions where extensive amplification by passaging is advantageous. Other DHRNAs allow efficient packaging of replicons but are packaged very poorly themselves (Frolov et al, supra; Liljestrom et al, supra; Bredenbeek et al, supra; Geigenmuller-Gnirke et al, supra). These latter helpers are useful when expression of viral structural proteins and virus spread are not desired. One approach to minimize the possibility of recombination between replicon and helper RNAs to produce wild-type virus is to use two DHRNAs, one that expresses the capsid protein and a second that expresses the viral glycoproteins (Frolov et al, supra). The capsid protein, expressed independently, accumulates at high levels, but to achieve similar levels of viral glycoprotein expression retention ofthe 5' terminus ofthe capsid protein mRNA, which acts as a translational enhancer, is required. Deletions in the capsid protein gene that preserve both the 5' terminus (the enhancer region) and the 3' half (the sequences that code for the autoprotease activity) but eliminate the region that binds RNA, produce high levels of glycoprotein expression from a second DHRNA. Capsid protein genes from heterologous alphaviruses can also be used to enhance translation ofthe glycoproteins and should further reduce the probability of wild-type virus generation via recombination.
In addition to packaging of alphavirus RNA replicons by cotransfection with DHRNAs, continuous packaging cell lines have been developed that express a DHRNA under the control of a nuclear promoter. Such cells may be useful for rescuing transfected RNA replicons, titering packaged replicons, and production of large quantities of packaged replicon stocks by low- multiplicity passage.
Variants ofthe prototype alphavirus, SLN, with differential abilities to infect human dendritic cells were described by Gardner JP et al. , 2000, J Virol 74:11849- 11857. The genetic determinant for human DC tropism maps to a single amino acid substitution at residue 160 ofthe envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant confers a similar ability to efficiently infect immature human DC, whhich are induced to undergo rapid activation and maturation. The SIN replicon particles infected skin- resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of MHC and costimulatory molecules. SIN replicon particles encoding HIV 1 p55(Gag) elicited potent specific T cell responses in vitro and in vivo , demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Human and mouse DC were differentially infected by selected SLN variants, suggesting differences in receptor expression between human and murine DC.
Thus, the present invention provides a dual approach to enhancing the potency of nucleic acid vaccines. On the one hand, the present nucleic acid constructs are designed to target MHC class I processing pathways, to target DCs, to stimulate DC maturation, activation, etc., as described. However, independently, the vector system provides a potential of using a directed approach to generate ialphavirus vaccine vectors that target and activate APCs.
Although preferred vectors and packaging cells are described in the Examples, other examples of alphavirus replicons as expression vectors are noted above are well-known in the art, as are corresponding packaging cells that permit their production in relatively high quantities.
For certain fusion expression vectors, a proteolytic cleavage site may be introduced at the junction ofthe reporter group and the target protein to enable separation ofthe target protein from the reporter group subsequent to purification ofthe fusion protein. Proteolytic enzymes for such cleavage and their recognition sequences include Factor Xa, thrombin and enterokinase. One embodiment of this invention is a transfected cell which expresses novel fusion polypeptide. Vector Construction
In construction of suitable vectors containing the desired coding and confrol sequences in the process of arriving at the PCL-generated replication-defective Sinbis virus replicons of the present invention, standard ligation and restriction techniques which are well understood in the art are employed. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and re-ligated in the form desired.
The DNA sequences which are used for the present constructs are available from a number of sources. Backbone vectors and control systems are generally found on available "host" vectors which are used for the bulk ofthe sequences in construction. For the pertinent coding sequence, initial construction maybe, and usually is, a matter of retrieving the appropriate sequences from cDNA or genomic DNA libraries. However, once the sequence is disclosed it is possible to synthesize the entire gene sequence in vitro starting from the individual nucleotide derivatives. The entire gene sequence for genes of sizeable length, e.g., 500-1000 bp may be prepared by synthesizing individual overlapping complementary oligonucleotides and filling in single stranded nonoverlapping portions using DNA polymerase in the presence ofthe deoxyribonucleoti.de triphosphates. This approach has been used successfully in the construction of several genes of known sequence. See, for example, Edge, M. D., Nature (1981) 292:756; Nambair, K. P., et al, Science (1984) 223:1299; and Jay, E., JBiol Chem (1984) 259:6311. Synthetic oligonucleotides are prepared by either the phosphotriester method as described by references cited above or the phosphoramidite method as described by Beaucage, S. L., and Caruthers, M. H., TetLett (1981) 22:1859; and Matteucci, M. D., and Caruthers, M. H., J Am Chem Soc (1981) 103:3185 and can be prepared using commercially available automated oligonucleotide synthesizers. Kinase treatment of single strands prior to annealing or for labeling is achieved using an excess, e.g., about 10 units of polynucleotide kinase to 1 nmole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol, 1-2 mM ATP, 1.7 pmoles γ-32P-ATP (2.9 mCi/mmole), 0.1 mM spermidine, 0.1 mM EDTA.
Once the components ofthe desired vectors are thus available, they can be excised and ligated using standard restriction and ligation procedures. Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog, hi general, about 1 mg of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ml of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to insure complete digestion ofthe DNA substrate, hicubation times of about one hour to two hours at about 37°C. are workable, although variations can be tolerated. After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol. If desired, size separation ofthe cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymology (1980) 65:499-560.
Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleoti.de triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20° to 25° C. in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl2, 6 mM DTT and 0.1-1.0 mM dNTPs. The Klenow fragment fills in at 5' single-stranded overhangs but chews back protruding 3 ' single strands, even though the four dNTPs are present. If desired, selective repair can be performed by supplying only one ofthe, or selected, dNTPs within the limitations dictated by the nature ofthe overhang. After treatment with Klenow, the mixture is extracted with phenol/chloroform and ethanol precipitated. Treatment under appropriate conditions with SI nuclease or BAL-31 results in hydrolysis of any single-stranded portion.
Ligations are typically performed in 15-50 ml volumes under the following standard conditions and temperatures: for example, 20 mM Tris-HCl pH 7.5, lOmM MgCl2 , 10 mM DTT, 33 μg/ml BSA, 10-50mM NaCl, and either 40 μM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for "sticky end" ligation) or ImM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for "blunt end" ligation). Intermolecular "sticky end" ligations are usually performed at 33-100 μg/ml total DNA concentrations (5-100 nM total end concentration). Intermolecular blunt end ligations are performed at 1 mM total ends concentration. In vector construction employing "vector fragments", the fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase (CIAP) in order to remove the 5' phosphate and prevent self-ligation. Digestions are conducted at pH 8 in approximately 10 mM Tris-HCl, 1 mM EDTA using BAP or CIAP at about 1 unit/mg vector at 60° for about one hour. The preparation is extracted with phenol/chloroform and ethanol precipitated. Alternatively, re-ligation can be prevented in vectors which have been double digested by additional restriction enzyme and separation ofthe unwanted fragments.
Any of a number of methods are used to introduce mutations into the coding sequence to generate the variants ofthe invention. These mutations include simple deletions or insertions, systematic deletions, insertions or substitutions of clusters of bases or substitutions of single bases.
For example, modifications ofthe IPP or the antigenic polypeptide DNA sequence are created by site-directed mutagenesis, a well-known technique for which protocols and reagents are commercially available (Zoller, MJ et al, Nucleic Acids Res (1982) 10:6487-6500 and Adelman, JP et al, DNA (1983) 2:183-193)). Correct ligations for plasmid construction are confirmed, for example, by first transforming E. coli strain MCI 061 (Casadaban, M., et al, J Mol Biol (1980) 138: 179-207) or other suitable host with the ligation mixture. Using conventional methods, transformants are selected based on the presence ofthe ampicillin-, tetracycline- or other antibiotic resistance gene (or other selectable marker) depending on the mode of plasmid construction. Plasmids are then prepared from the transformants with optional chloramphenicol amplification optionally following chloramphenicol amplification ((Clewell,
DB et al. , Proc Natl Acad Sci USA (1969) 62:1159; Clewell, D. B., JBacteriol (1972) 110:667). Several mini DNA preps are commonly used. See, e.g. „ Holmes, DS, et al. , Anal Biochem (1981) 114:193-197; Birnboim, HC et al. , Nucleic Acids Res (1979) 7:1513-1523. The isolated DNA is analyzed by restriction and/or sequenced by the dideoxy nucleotide method of S anger (Proc Natl Acad Sci USA (1977) 74:5463) as further described by Messing, et al, Nucleic Acids Res (1981) 9:309, or by the method of Maxam et al Methods in Enzymology (1980) 65:499. During the process of preparing the nucleic acids ofthe present invention, vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dexfran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. supra and other standard texts.
Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction ofthe reporter group and the target protein to enable separation ofthe target protein from the reporter group subsequent to purification ofthe fusion protein. Proteolytic enzymes for such cleavage and their recognition sequences include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-fransferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein. hiducible non-fusion expression vectors include pTrc (Amann et al, (1988) 7ene 69: 301-315) and pET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). While target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc, expression of target genes inserted into pET lid relies on transcription from the T7 gnlO-lacO fusion promoter mediated by coexpressed viral RNA polymerase (T7gnl). Th is viral polymerase is supplied by host strains BL21 (DE3) or HMS 174(DE3) from a resident λ prophage harboring a T7gnl under the transcriptional control ofthe lacUV 5 promoter.
Promoters and Enhancers
A promoter region of a DNA or RNA molecule binds RNA polymerase and promotes the transcription of an "operably linked" nucleic acid sequence. As used herein, a "promoter sequence" is the nucleotide sequence ofthe promoter which is found on that strand ofthe DNA or RNA which is transcribed by the RNA polymerase. Two sequences of a nucleic acid molecule, such as a promoter and a coding sequence, are "operably linked" when they are linked to each other in a manner which permits both sequences to be transcribed onto the same RNA transcript or permits an RNA transcript begun in one sequence to be extended into the second sequence. Thus, two sequences, such as a promoter sequence and a coding sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript ofthe operably linked coding sequence. In order to be "operably linked" it is not necessary that two sequences be immediately adjacent to one another in the linear sequence.
The preferred promoter sequences ofthe present invention must be operable in mammalian cells and may be either eukaryotic or viral promoters. Although preferred promoters are described in the Examples, other useful promoters and regulatory elements are discussed below. Suitable promoters may be inducible, repressible or constitutive. An example of a constitutive promoter is the viral promoter MS V-LTR, which is efficient and active in a variety of cell types, and, in contrast to most other promoters, has the same enhancing activity in arrested and growing cells. Other preferred viral promoters include that present in the CMV-LTR (from cytomegalovirus) (Bashart, M. et al, Cell 41:521 (1985)) or in the RSV-LTR (from Rous sarcoma virus) (Gorman, CM., Proc. Natl. Acad. Sci. USA 79:6777 (1982). Also useful are the promoter ofthe mouse metallothionein I gene (Hamer, D., et al, J. Mol Appl Gen. :273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C, et al, Nature 290:304-310 (1981)); and the yeast gal4 gene promoter (Johnston, S.A., et al, Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P.A., et al, Proc. Natl. Acad. Sci. (USA) 5 :5951-5955 (1984)). Other illustrative descriptions of transcriptional factor association with promoter regions and the separate activation and DNA binding of transcription factors include: Keegan et al, Nature (1986) 237:699; Fields et al, Nature (1989) 340:245; Jones, Cell (1990) 6~7:9; Lewin, Cell (1990) 61:1161; Ptashne et al, Nature (1990) 346:329; Adams et al, Cell (1993) 72:306. The relevant disclosure of all of these above-listed references is hereby incorporated by reference.
The promoter region may further include an octamer region which may also function as a tissue specific enhancer, by interacting with certain proteins found in the specific tissue. The enhancer domain ofthe DNA construct ofthe present invention is one which is specific for the target cells to be transfected, or is highly activated by cellular factors of such target cells.
Examples of vectors (plasmid or retrovirus) are disclosed in (Roy-Burman et al, U.S. Patent No. 5,112,767). For a general discussion of enhancers and their actions in transcription, see, Lewin, B.M., Genes IV, Oxford University Press, Oxford, (1990), pp. 552-576. Particularly useful are retroviral enhancers (e.g., viral LTR). The enhancer is preferably placed upstream from the promoter with which it interacts to stimulate gene expression. For use with retroviral vectors, the endogenous viral LTR may be rendered enhancer-less and substituted with other desired enhancer sequences which confer tissue specificity or other desirable properties such as transcriptional efficiency.
The nucleic acid sequences ofthe invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated with commercially available DNA synthesizers (See, e.g., Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
PROTEINS AND POLYPEPTIDES
While the present disclosure exemplifies the use of a particular class of JPP, an intercellular spreading protein, particularly, the full length VP22 protein of HSV-1, it is to be understood that homologues a useful IPP, such as VP22 from other viruses or from non- viral origin, and mutants thereof that possess the characteristics disclosed herein are intended within the scope of this invention.
Thus, the present invention includes a molecular vaccine encoding a "functional derivative" of an IPP such as the intercellular spreading protein VP22. Described in terms of VP22 merely for the sake of simplicity and clarity, but not limitation, such a functional derivative is is an amino acid substitution variant, a "fragment," or a "chemical derivative" of VP22, which terms are defined below. A functional derivative retains measurable VP22-like activity, preferably that of promoting intercellular spreading and immunogenicity of one or more antigenic epitopes fused thereto, which permits its utility in accordance with the present invention. "Functional derivatives" encompass "variants" and "fragments" regardless of whether the terms are used in the conjunctive or the alternative herein. A functional homologue must possess the above biochemical and biological activity, hi view of this functional characterization, use of homologous VP22 proteins including proteins not yet discovered, fall within the scope ofthe invention if these proteins have sequence similarity and the recited biochemical and biological activity.
To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred method of alignment, Cys residues are aligned.
In a preferred embodiment, the length of a sequence being compared is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% ofthe length ofthe reference sequence (e.g., VP22, SEQ LD NO:5 ). The amino acid residues (or nucleotides) at corresponding amino acid positions (or nucleotide) positions are then compared. When a position in the first sequence is occupied by the same amino acid residue (or nucleotide) as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function ofthe number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment ofthe two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol Biol. - 5:444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. hi yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. hi another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences ofthe present invention can further be used as a "query sequence" to perform a search against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J Mol. Biol. 275:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to HVP22 nucleic acid molecules. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to HVP22 protein molecules. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters ofthe respective programs (e.g.,, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Thus, a homologue of HVP22 described above is characterized as having (a) functional activity of native HVP22 and (b) sequence similarity to a native VP22 protein (such as SEQ ID NO:5) when determined above, of at least about 20% (at the amino acid level), preferably at least about 40%, more preferably at least about 70%, even more preferably at least about 90%.
It is within the skill in the art to obtain and express such a protein using DNA probes based on the disclosed sequences of VP22. Then, the fusion protein's biochemical and biological activity can be tested readily using art-recognized methods such as those described herein, for example, a T cell proliferation, cytokine secretion or a cytolytic assay, or an in vivo assay of tumor protection or therapy. A biological assay ofthe stimulation of antigen-specific T cell reactivity will indicate whether the homologue has the requisite activity to qualify as a "functional" homologue.
A "variant" of a HVP22 refers to a molecule substantially identical to either the full protein or to a fragment thereof in which one or more amino acid residues have been replaced (substitution variant) or which has one or several residues deleted (deletion variant) or added (addition variant). A "fragment" of HVP22 refers to any subset ofthe molecule, that is, a shorter polypeptide ofthe full-length protein. A number of processes can be used to generate fragments, mutants and variants ofthe isolated DNA sequence. Small subregions or fragments ofthe nucleic acid encoding the spreading protein, for example 1-30 bases in length, can be prepared by standard, chemical synthesis. Antisense oligonucleotides and primers for use in the generation of larger synthetic fragment.
A preferred group of variants are those in which at least one amino acid residue and preferably, only one, has been substituted by different residue. For a detailed description of protein chemistry and structure, see Schulz, GE et al, Principles of Protein Structure, Springer- Verlag, New York, 1978, and Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. The types of substitutions that may be made in the protein molecule may be based on analysis ofthe frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al. (supra) and Figure 3-9 of Creighton (supra). Based on such an analysis, conservative substitutions are defined herein as exchanges within one ofthe following five groups:
Figure imgf000046_0001
The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking a side chain and thus imparts flexibility to the chain. Pro, because of its unusual geometry, tightly constrains the chain. Cys can participate in disulfide bond formation, which is important in protein folding.
More substantial changes in biochemical, functional (or immunological) properties are made by selecting substitutions that are less conservative, such as between, rather than within, the above five groups. Such changes will differ more significantly in their effect on maintaining (a) the structure ofthe peptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk ofthe side chain. Examples of such substitutions are (i) substitution of Gly and/or Pro by another amino acid or deletion or insertion of Gly or Pro; (ii) substitution of a hydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobic residue, e.g.,, Leu, Ile, Phe, Val or Ala; (iii) substitution of a Cys residue for (or by) any other residue; (iv) substitution of a residue having an electropositive side chain, e.g.,, Lys, Arg or His, for (or by) a residue having an electronegative charge, e.g.,, Glu or Asp; or (v) substitution of a residue having a bulky side chain, e.g., Phe, for (or by) a residue not having such a side chain, e.g., Gly.
Most acceptable deletions, insertions and substitutions according to the present invention are those that do not produce radical changes in the characteristics ofthe LPP, e.g., HVP22, in terms of its intercellular spreading activity and its ability to stimulate antigen specific T cell reactivity to an antigenic epitope or epitopes that are fused to the spreading protein. However, when it is difficult to predict the exact effect ofthe substitution, deletion or insertion in advance of doing so, one skilled in the art will appreciate that the effect can be evaluated by routine screening assays such as those described here, without requiring undue experimentation. Whereas shorter chain variants can be made by chemical synthesis, for the present invention, the preferred longer chain variants are typically made by site-specific mutagenesis of the nucleic acid encoding the IPP, expression ofthe variant nucleic acid in cell culture, and, optionally, purification ofthe polypeptide from the cell culture, for example, by immunoaffinity chromatography using specific antibody immobilized to a column (to absorb the variant by binding to at least one epitope). Chemical Derivatives
"Chemical derivatives" ofthe IPP, e.g., HVP22, or a fusion polypeptide thereof, contain additional chemical moieties not normally a part ofthe protein. Covalent modifications ofthe polypeptide are included within the scope of this invention. Such derivatized moieties may improve the solubility, absorption, biological half life, and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington 's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980).
Such modifications may be introduced into the molecule by reacting targeted amino acid residues ofthe polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Another modification is cyclization ofthe protein. Examples of chemical derivatives ofthe polypeptide follow. Lysinyl and amino terminal residues are derivatized with succinic or other carboxylic acid anhydrides. Derivatization with a cyclic carboxylic anhydride has the effect of reversing the charge ofthe lysinyl residues. Other suitable reagents for derivatizing amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
Carboxyl side groups, aspartyl or glutamyl, may be selectively modified by reaction with carbodiimides (R-N=C=N-R') such as l-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodumide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodumide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonia.
Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation ofthe amino group of lysine (Creighton, supra, pp. 79-86 ), acetylation ofthe N-terminal amine, and amidation ofthe C- terminal carboxyl groups. Also included are peptides wherein one or more D-amino acids are substituted for one or more L-amino acids.
Multimeric Peptides
The present invention includes longer polypeptides in which a basic peptidic sequence obtained from the sequence of either the IPP, such as HVP22, or the antigenic polypeptide or peptide unit, is repeated from about two to about 100 times, with or without intervening spacers or linkers. It is understood that such multimers may be built from any ofthe peptide variants defined herein. Moreover, a peptide multimer may comprise different combinations of peptide monomers and the disclosed substitution variants thereof. Such oligomeric or multimeric peptides can be made by chemical synthesis or by recombinant DNA techniques as discussed herein. When produced chemically, the oligomers preferably have from 2-8 repeats ofthe basic peptide sequence. When produced recombinantly, the multimers may have as many repeats as the expression system permits, for example from two to about 100 repeats. In tandem multimers, preferably dimers and trimers, ofthe fusion polypeptide, the chains bonded by interchain disulfide bonds or other "artificial" covalent bonds between the chains such that the chains are "side-by-side" rather than "end to end."
THERAPEUTIC COMPOSITIONS AND THEIR ADMINISTRATION A vaccine composition comprising the nucleic acid encoding the fusion polypeptide, or a cell expressing this nucleic acid is administered to a mammalian subject, preferably a human. The vaccine composition is administered in a pharmaceutically acceptable carrier in a biologically effective or a therapeutically effective amount . The composition may be given alone or in combination with another protein or peptide such as an immunostimulatory molecule. Treatment may include administration of an adjuvant, used in its broadest sense to include any nonspecific immune stimulating compound such as an interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylehe oleyl ether and n- hexadecyl polyethylene ether.
A therapeutically effective amount is a dosage that, when given for an effective period of time, achieves the desired immunological or clinical effect.
A therapeutically active amount of a nucleic acid encoding the fusion polypeptide may vary according to factors such as the disease state, age, sex, and weight ofthe individual, and the ability ofthe peptide to elicit a desired response in the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies ofthe therapeutic situation. A therapeutically effective amounts ofthe protein, in cell associated form may be stated in terms ofthe protein or cell equivalents.
Thus an effective amount is between about 1 nanogram and about 10 milligram per kilogram of body weight ofthe recipient, more preferably between about 0.1 μg and 1 μg/kg. Dosage forms suitable for internal administration preferably contain (for the latter dose range) from about 0.01 μg to 100 μg of active ingredient (nucleic acid or polypeptide) per unit. The active ingredient may vary from 0.5 to 95% by weight based on the total weight ofthe composition. Alternatively, an effective dose of cells expressing the nucleic acid is between about 10 and 10 cells. Those skilled in the art of immunotherapy will be able to adjust these doses without undue experimentation. The active compound maybe administered in a convenient manner, e.g., injection or infusion by a convenient and effective route. Preferred routes include subcutaneous, intradermal, intravenous and intramuscular routes. Other possible routes include oral administration, infrathecal, inhalation, fransdermal application, or rectal administration. For the treatment of tumors which have not been completely resected, direct intratumoral or peritumoral injection is also intended.
Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. Thus it may be necessary to coat the composition with, or co-administer the composition with, a material to prevent its inactivation. For example, an enzyme inhibitors of nucleases or proteases (e.g., pancreatic trypsin inhibitor, diisopropylfluorophosphate and trasylol).or in an appropriate carrier such as liposomes (including water-in-oil-in- water emulsions as well as conventional liposomes (Strejan et al, (1984) J. Neuroimmunol 7:27). As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incoφorated into the compositions.
Preferred pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride may be included in the phannaceutical composition. In all cases, the composition should be sterile and should be fluid. It should be stable under the conditions of manufacture and storage and must include preservatives that prevent contamination with microorganisms such as bacteria and fungi. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants.
Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
Parenteral compositions are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for a mammalian subject; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
For lung instillation, aerosolized solutions are used. In a sprayable aerosol preparations, the active protein may be in combination with a solid or liquid inert carrier material. This may also be packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant. The aerosol preparations can contain solvents, buffers, surfactants, and antioxidants in addition to the protein ofthe invention.
Other pharmaceutically acceptable carriers for the nucleic acid vaccine compositions according to the present invention are liposomes, pharmaceutical compositions in which the active protein is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The active protein is preferably present in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non- homogeneous system generally known as a liposomic suspension. The hydrophobic layer, or lipidic layer, generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid, and/or other materials of a hydrophobic nature.
ANTIGENS ASSOCIATED WITH PATHOGENS
A major utility for the present invention is the use ofthe present nucleic acid compositions in therapeutic vaccine for cancer and for major chronic viral infections that cause morbidity and mortality worldwide. Such vaccines are designed to eliminate infected cells - this requires T cell responses as antibodies are often ineffective. The vaccines ofthe present invention, include, the antigenic epitope itself and an IPP such as an intercellular spreading protein like HVP22. h addition to the specific antigens and first LPP polypeptide in the present vectors as employed in the Examples, the present invention is intended to encompass
(a) use of additional vectors such as naked DNA, naked RNA, self replicating RNA replicons and viruses including vaccinia, adenoviruses, adeno-associated virus (AAV), lentiviruses and RNA alphaviruses;
(b) an additional IPP such as HSP70, calreticulin, the extracellular domain of FL, , domain II of Pseudomonas exotoxin ETA; and/or
(c) a costimulatory signal, such as a B7 family protein, including B7-DC (see commonly assigned U.S. patent application Serial No. 09/794,210 which is incorporated by reference in its entirety), B7.1, B7.2, soluble CD40, etc.).
Preferred antigens are preferably epitopes of pathogenic microorganisms against which the host is defended by effector T cells responses, including cytotoxic T lymphocyte (CTL) and delayed type hypersensitivity. These typically include viruses, infracellular parasites such as malaria, and bacteria that grow mfracellularly such as mycobacteria and listeria. Thus, the types of antigens included in the vaccine compositions of this invention are any of those associated with such pathogens (in addition, of course, to tumor-specific antigens). It is noteworthy that some viral antigens are also tumor antigens in the case where the virus is a causative factor in cancer.
In fact, the two most common cancers worldwide, hepatoma and cervical cancer, are associated with viral infection. Hepatitis B virus(HBV) (Beasley, R.P. et al, Lancet 2, 1129- 1133 (1981) has been implicated as etiologic agent of hepatomas. 80-90% of cervical cancers express the E6 and E7 antigens (exemplified herein) from one of four "high risk" human papiUomavirus types: HPV-16, HPV-18, HPV-31 and HPV-45 (Gissmann, L. et al, Ciba Found Symp. 120, 190-207 (1986); Beaudenon, S., et al. Nature 321, 246-249 (1986). The HPV E6 and E7 antigens are the most promising targets for virus associated cancers in immunocompetent individuals because of their ubiquitous expression in cervical cancer, hi addition to their importance as targets for therapeutic cancer vaccines, virus associated tumor antigens are also ideal candidates for prophylactic vaccines. Indeed, introduction of prophylactic HBV vaccines in Asia have decreased the incidence of hepatoma (Chang, M.H., et al. New Engl J. Med. 336, 1855-1859 (1997), representing a great impact on cancer prevention.
Among the most important viruses in chronic human viral infections are human papiUomavirus (HPN) hepatitis B virus (HBN), hepatitis C Virus (HCV), human immunodeficiency virus (HIV), Epstein Barr Virus (EBV) and herpes simplex virus (HSV). In addition to its applicability to human cancer and infectious diseases,, the present invention is also intended for use in treating animal diseases in the veterinary medicine context. Thus, the approaches described herein may be readily applied by one skilled in the art to treatment of veterinary herpesvirus infections including equine herpesviruses, bovine herpesviruses, Marek's disease virus in chickens and other fowl; animal retroviral diseases; pseudorabies and rabies and the like.
The following references set forth principles and current information in the field of basic, medical and veterinary virology and are incorporated by reference: Fields Virology, Fields, BΝ et al, eds., Lippincott Williams & Wilkins, ΝY, 1996; Principles of Virology: Molecular Biology, Pathogenesis, and Control, Flint, S.J. et al, eds., Amer Society for Microbiology, Washington, 1999; Principles and Practice of Clinical Virology, 4th Edition, Zuckerman. A.J. et al, eds, John Wiley & Sons, ΝY, 1999; The Hepatitis C Viruses, by Hagedorn, CH et al, eds., Springer Verlag, 1999; Hepatitis B Virus: Molecular Mechanisms in Disease and Novel Strategies for Therapy, Koshy, R. et al, eds,, World Scientific Pub Co, 1998; Veterinary
Virology, Murphy, F.A. et al, eds., Academic Press, ΝY, 1999; Avian Viruses: Function and Corøtro/ Ritchie, B.W., Iowa State University Press, Ames , 2000; Virus Taxonomy: Classification and Nomenclature of Viruses: Seventh Report ofthe International Committee on Taxonomy of Viruses, by M. H. V. Van Regenmortel, MHV et al, eds., Academic Press; ΝY, 2000. DELIVERY OF VACCINE NUCLEIC ACID TO CELLS AND ANIMALS
The Examples below describe certain preferred approaches to delivery ofthe vaccines of the present invention.
DNA delivery, for example to effect what is generally known as "gene therapy" involves introduction of a "foreign" DNA into a cell and ultimately, into a live animal. Several general strategies for gene therapy have been studied and have been reviewed extensively (Yang, N-S., Crit. Rev. Biotechnol. 12:335-356 (1992); Anderson, W.F., Science 255:808-813 (1992); Miller, A.S., Nature 357:455-460 (1992); Crystal, R.G., Amer. J Med. 92(suppl 6A):44S>-52S> (1992); Zwiebel, J.A. et al, Ann. NY. Acad. Sci. 575:394-404 (1991); McLachlin, J.R. et al, Prog. Nucl. Acid Res. Molec. Biol. 35:91-135 (1990); Kohn, D.B. et al, Cancer Invest. 7:179-192 (1989), which references are herein incorporated by reference in their entirety).
One approach comprises nucleic acid transfer into primary cells in culture followed by autologous transplantation ofthe ex vivo transformed cells into the host, either systemically or into a particular organ or tissue. For accomplishing the objectives ofthe present invention, nucleic acid therapy would be accomplished by direct transfer of a the functionally active vectors into mammalian somatic tissue or organ in vivo. Nucleic acid or replicon transfer can be achieved using a number of approaches described below. These systems can be tested for successful expression in vitro by use of a selectable marker (e.g., G418 resistance) to select transfected clones expressing the DNA, followed by detection ofthe presence ofthe antigen-containing expression product (after treatment with the inducer in the case of an inducible system) using an antibody to the product in an appropriate immunoassay. Efficiency ofthe procedure, including DNA uptake, integration and stability of integrated DNA, can be improved using known methods, and co-transfection using high molecular weight mammalian DNA as a "carrier". Examples of successful "gene transfer" reported in the art include: (a) direct injection of plasmid DNA into mouse muscle tissues, which led to expression of marker genes for an indefinite period of time (Wolff, J.A. et al, Science 247:1465 (1990); Acsadi, G. et al, The New Biologist 3:71 (1991)); (b) retroviral vectors are effective for in vivo and in situ infection of blood vessel tissues; (c) portal vein injection and direct injection of retrovirus preparations into liver effected gene transfer and expression in vivo (Horzaglou, M. et al, J. Biol. Chem.
265:17285 (1990); Koleko, M. et al, Human Gene Therapy 2:27 (1991); Ferry, N. et al, Proc. Natl Acad. Sci. USA 88:8387 (1991)); (d) intratracheal infusion of recombinant adenovirus into lung tissues was effective for in vivo fransfer and prolonged expression of foreign genes in lung respiratory epithelium (Rosenfeld, M.A. et al, Science 252:431 (1991); (e) Herpes simplex virus vectors achieved in vivo gene transfer into brain tissue (Ahmad, F. et al, eds, Miami Short Reports - Advances in Gene Technology: The Molecular Biology of Human Genetic Disease, Vol 1, Boehringer Manneheiml Biochemicals, USA, 1991).
Refroviral-mediated human therapy utilizes amphofrophic, replication-deficient retrovirus systems (Temin, H.M., Human Gene Therapy 7:111 (1990); Temin et α/., U.S. Patent 4,980,289; Temin et al, U.S. Patent 4,650,764; Temin et al, U.S. Patent No. 5,124,263; Wills, J.W. U.S. Patent 5,175,099; Miller, A.D., U.S. Patent No. 4,861,719). Such vectors have been used to introduce functional DNA into human cells or tissues, for example, the adenosine deaminase gene into lymphocytes, the NPT-IJ gene and the gene for tumor necrosis factor into tumor infiltrating lymphocytes. Retro virus-mediated gene delivery generally requires target cell proliferation for gene transfer (Miller, D.G. et al, Mol. Cell. Biol. 10:4239 (1990). This condition is met by certain of the preferred target cells into which the present DNA molecules are to be introduced, i.e., actively growing tumor cells. Gene therapy of cystic fibrosis using transfection by plasmids using any of a number of methods and by retroviral vectors has been described by Collins et al, U.S. Patent 5,240,846.
Other vectors that may be used in conjunction with the present vectors to include DNA packaged into retrovirus vectors using packaging cell lines that produce replication-defective retroviruses, as is well-known in the art (see, for example, Cone, R.D. et al, Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Mann, R.F. et al, Cell 33:153-159 (1983); Miller, A.D. et al, Molec. Cell. Biol. 5:431-437 (1985),; Sorge, J., et al, Molec. Cell. Biol. 4:1730-1737 (1984); Hock, R.A. et al, Nature 320:257 (1986); Miller, A.D. et al, Molec. Cell Biol. 6:2895-2902 (1986). Newer packaging cell lines which are efficient an safe for gene fransfer have also been described (Bank et al, U.S. 5,278,056.
This approach can be utilized in a site specific manner to deliver the retroviral vector to the tissue or organ of choice. Thus, for example, a catheter delivery system can be used (Nabel, EG et al, Science 244:1342 (1989)). Such methods, using either a retroviral vector or a liposome vector, are particularly useful to deliver the nucleic acid to be expressed to a blood vessel wall, or into the blood circulation of a tumor. Other virus vectors may also be used, including recombinant adenoviruses (Horowitz, M.S., In: Virology, Fields, BN et al, eds, Raven Press, New York, 1990, p. 1679; Berkner, K.L., Biotechniques 6:616 9191988), Strauss, S.E., In: The Adenoviruses, Ginsberg, HS, ed., Plenum Press, New York, 1984, chapter 11), herpes simplex virus (HSV) for neuron-specific delivery and persistence. Advantages of adenovirus vectors for human gene therapy include the fact that recombination is rare, no human malignancies are known to be associated with such viruses, the adenovirus genome is double stranded DNA which can be manipulated to accept foreign genes of up to 7.5 kb in size, and live adenovirus is a safe human vaccine organisms. Adeno- associated virus is also useful for human therapy (Samulski, R.J. et al, EMBO J. 10:3941 (1991) according to the present invention.
Vaccinia virus, which can be rendered non-replicating (U.S. Patents 5,225,336; 5,204,243; 5,155,020; 4,769,330; Sutter, G et al, Proc. Natl. Acad. Sci. USA (1992) 59:10847-10851; Fuerst, T.R. et al, Proc. Natl Acad. Sci. USA (1989) 55:2549-2553; Falkner F.G. et al; Nucl. Acids Res (1987) 75:7192; Chakrabarti, S et al, Molec. Cell. Biol. (1985) 5:3403-3409). Descriptions of recombinant vaccinia viruses and other viruses containing heterologous DNA and their uses in immunization and DNA therapy are reviewed in: Moss, B., Curr. Opin. Genet. Dev. (1993) 3:86-90; Moss, B. Biotechnology (1992) 20: 345-362; Moss, B., Curr Top Microbiol Immunol (1992) 755:25-38; Moss, B., Science (1991) 252:1662-1667; Piccini, A et al, Adv. Virus Res. (1988) 34:43-64; Moss, B. et al, Gene AmplifAnal (1983) 3:201-213. hi addition engineered bacteria may be used as additional vectors. A number of bacterial strains including Salmonella, BCG and Listeria monocytogenes(LM) (Hoiseth & Stocker, Nature 291, 238-239 (1981); Pokier, TP et al. J. Exp. Med. 168, 25-32 (1988); (Sadoff, J.C., et al, Science 240, 336-338 (1988); Stover, C.K., et al, Nature 351, 456-460 (1991); Aldovini, A. et al„ Nature 351, 479-482 (1991); Schafer, R., et al, J. Immunol. 149, 53-59 (1992);
Ikonomidis, G. et al, J. Exp. Med. 180, 2209-2218 (1994)). These organisms display two promising characteristics for use as vaccine vectors: (1) enteric routes of infection, providing the possibility of oral vaccine delivery; and (2) infection of monocytes/macrophages thereby targeting antigens to professional APCs. In addition to the forms of nucleic acid transfer in vivo described above, physical means well-known in the art can be used for direct fransfer of DNA, including administration of plasmid DNA (Wolff et al, 1990, supra) and particle-bombardment mediated gene transfer (Yang, N.-S., et al, Proc. Natl. Acad. Sci. USA 57:9568 (1990); Williams, R.S. et al, Proc. Natl. Acad. Sci. USA 88:2726 (1991); Zelenin, AN. et al, FEBSLett. 280:94 (1991); Zelenin, AN. et al, FEBSLett. 244:65 (1989); Johnston, S.A. et al, In Vitro Cell. Dev. Biol. 27:11 (1991)). Furthermore, electroporation, a well-known means to transfer genes into cell in vitro, can be used to transfer DΝA molecules according to the present invention to tissues in vivo (Titomirov, AN. et al, Biochim. Biophys. Ada 1088:131 ((1991)).
"Carrier mediated gene transfer" has also been described (Wu, CH. et al, J. Biol. Chem. 264:16985 (1989); Wu, G.Y. et al, J. Biol. Chem. 263:14621 (1988); Soriano, P. et al, Proc. Natl. Acad. Sci. USA 50:7128 (1983); Wang, C-Y. et al, Proc. Natl. Acad. Sci. USA 84:7851 (1982); Wilson, J.M. et al, J. Biol. Chem. 267:963 (1992)). Preferred carriers are targeted liposomes (Νicolau, C. et al, Proc. Natl. Acad. Sci. USA 80:1068 (1983); Soriano et al, supra) such as immunoliposomes, which can incoφorate acylated mAbs into the lipid bilayer (Wang et al, supra). Polycations such as asialoglycoprotein/polylysine maybe used, where the conjugate includes a molecule which recognizes the target tissue (e.g., asialoorosomucoid for liver) and a DΝA binding compound to bind to the DΝA to be transfected. Polylysine is an example of a DΝA binding molecule which binds DΝA without damaging it. This conjugate is then complexed with plasmid DΝA according to the present invention for fransfer.
DΝA used for transfection or microinjection may be prepared using methods well-known in the art, for example using the Quiagen procedure (Quiagen), followed by DΝA purification using known methods, such as the methods exemplified herein.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.
EXAMPLE I
Materials and Methods Cell Lines
The packaging cell line (987dlsplit #24) has been described previously (Polo et al, supra) and was grown in DMEM supplemented with 10% FBS, antibiotics and G418. The production and maintenance of TC-1 cells has been described previously (Lin, KY et al, 1996, Cancer
Research. 56:21-26). On the day of tumor challenge, TC-1 cells were harvested by trypsinization, washed twice with IX Hanks buffered salt solution (HBSS), and finally resuspended in IX HBSS to the designated concenfration for injection. Baby hamster kidney (BHK21) cells were obtained from the ATCC (RockviUe, MD) and grown in Glasgow MEM supplemented with 5% FBS, 10%) tryptose phosphate broth, 2 mM glutamine, and antibiotics. Plasmid DNA Constructs
The Sindbis virus RNA replicon vector, SINrep5, previously described (Bredenbeek, PJ et al, supra) was provided by Dr. Charles Rice at the Washington University School of Medicine, St. Louis.
The sequence of SLNrep5 self replicating replicon (SEQ ID NO:23) is shown below and includes cloning sites
ATTGACGGCG TAGTACACAC TATTGAATCA AACAGCCGAC CAATTGCACT ACCATCACAA TGGAGAAGCC AGTAGTAAAC GTAGACGTAG ACCCCCAGAG TCCGTTTGTC GTGCAACTGC AAAAAAGCTT CCCGCAATTT GAGGTAGTAG CACAGCAGGT CACTCCAAAT GACCATGCTA ATGCCAGAGC ATTTTCGCAT CTGGCCAGTA AACTAATCGA GCTGGAGGTT CCTACCACAG CGACGATCTT GGACATAGGC AGCGCACCGG CTCGTAGAAT GTTTTCCGAG CACCAGTATC ATTGTGTCTG CCCCATGCGT AGTCCAGAAG ACCCGGACCG CATGATGAAA TACGCCAGTA AACTGGCGGA AAAAGCGTGC AAGATTACAA ACAAGAACTT GCATGAGAAG ATTAAGGATC TCCGGACCGT ACTTGATACG CCGGATGCTG AAACACCATC GCTCTGCTTT CACAACGATG TTACCTGCAA CATGCGTGCC GAATATTCCG TCATGCAGGA CGTGTATATC AACGCTCCCG GAACTATCTA TCATCAGGCT ATGAAAGGCG TGCGGACCCT GTACTGGATT GGCTTCGACA CCACCCAGTT CATGTTCTCG GCTATGGCAG GTTCGTACCC TGCGTACAAC ACCAACTGGG CCGACGAGAA AGTCCTTGAA GCGCGTAACA TCGGACTTTG CAGCACAAAG CTGAGTGAAG GTAGGACAGG AAAATTGTCG ATAATGAGGA AGAAGGAGTT GAAGCCCGGG TCGCGGGTTT ATTTCTCCGT AGGATCGACA CTTTATCCAG AACACAGAGC CAGCTTGCAG AGCTGGCATC TTCCATCGGT GTTCCACTTG AATGGAAAGC AGTCGTACAC TTGCCGCTGT GATACAGTGG TGAGTTGCGA AGGCTACGTA GTGAAGAAAA TCACCATCAG TCCCGGGATC ACGGGAGAAA CCGTGGGATA CGCGGTTACA CACAATAGCG AGGGCTTCTT GCTATGCAAA GTTACTGACA CAGTAAAAGG AGAACGGGTA TCGTTCCCTG TGTGCACGTA CATCCCGGCC ACCATATGCG ATCAGATGAC TGGTATAATG GCCACGGATA TATCACCTGA CGATGCACAA AAACTTCTGG TTGGGCTCAA CCAGCGAATT GTCATTAACG GTAGGACTAA CAGGAACACC AACACCATGC AAAATTACCT TCTGCCGATC ATAGCACAAG GGTTCAGCAA ATGGGCTAAG GAGCGCAAGG ATGATCTTGA TAACGAGAAA ATGCTGGGTA CTAGAGAACG CAAGCTTACG TATGGCTGCT TGTGGGCGTT TCGCACTAAG AAAGTACATT CGTTTTATCG CCCACCTGGA ACGCAGACCT GCGTAAAAGT CCCAGCCTCT TTTAGCGCTT TTCCCATGTC GTCCGTATGG ACGACCTCTT TGCCCATGTC GCTGAGGCAG AAATTGAAAC TGGCATTGCA ACCAAAGAAG GAGGAAAAAC TGCTGCAGGT CTCGGAGGAA TTAGTCATGG AGGCCAAGGC TGCTTTTGAG GATGCTCAGG AGGAAGCCAG AGCGGAGAAG CTCCGAGAAG CACTTCCACC ATTAGTGGCA GACAAAGGCA TCGAGGCAGC CGCAGAAGTT GTCTGCGAAG TGGAGGGGCT CCAGGCGGAC ATCGGAGCAG CATTAGTTGA AACCCCGCGC GGTCACGTAA GGATAATACC TCAAGCAAAT GACCGTATGA TCGGACAGTA TATCGTTGTC TCGCCAAACT CTGTGCTGAA GAATGCCAAA CTCGCACCAG CGCACCCGCT AGCAGATCAG GTTAAGATCA TAACACACTC CGGAAGATCA GGAAGGTACG CGGTCGAACC ATACGACGCT AAAGTACTGA TGCCAGCAGG AGGTGCCGTA CCATGGCCAG AATTCCTAGC ACTGAGTGAG AGCGCCACGT TAGTGTACAA CGAAAGAGAG TTTGTGAACC GCAAACTATA CCACATTGCC ATGCATGGCC CCGCCAAGAA TACAGAAGAG GAGCAGTACA AGGTTACAAA GGCAGAGCTT GCAGAAACAG AGTACGTGTT TGACGTGGAC AAGAAGCGTT GCGTTAAGAA GGAAGAAGCC TCAGGTCTGG TCCTCTCGGG AGAACTGACC AACCCTCCCT ATCATGAGCT AGCTCTGGAG GGACTGAAGA CCCGACCTGC GGTCCCGTAC AAGGTCGAAA CAATAGGAGT GATAGGCACA CCGGGGTCGG GCAAGTCAGC TATTATCAAG TCAACTGTCA CGGCACGAGA TCTTGTTACC AGCGGAAAGA AAGAAAATTG TCGCGAAATT GAGGCCGACG TGCTAAGACT GAGGGGTATG CAGATTACGT CGAAGACAGT AGATTCGGTT ATGCTCAACG GATGCCACAA AGCCGTAGAA GTGCTGTACG TTGACGAAGC GTTCGCGTGC CACGCAGGAG CACTACTTGC CTTGATTGCT ATCGTCAGGC CCCGCAAGAA GGTAGTACTA TGCGGAGACC CCATGCAATG CGGATTCTTC AACATGATGC AACTAAAGGT ACATTTCAAT CACCCTGAAA AAGACATATG CACCAAGACA TTCTACAAGT ATATCTCCCG GCGTTGCACA CAGCCAGTTA CAGCTATTGT ATCGACACTG CATTACGATG GAAAGATGAA AACCACGAAC CCGTGCAAGA AGAACATTGA AATCGATATT ACAGGGGCCA CAAAGCCGAA GCCAGGGGAT ATCATCCTGA CATGTTTCCG CGGGTGGGTT AAGCAATTGC AAATCGACTA TCCCGGACAT GAAGTAATGA CAGCCGCGGC CTCACAAGGG CTAACCAGAA AAGGAGTGTA TGCCGTCCGG CAAAAAGTCA ATGAAAACCC ACTGTACGCG ATCACATCAG AGCATGTGAA CGTGTTGCTC ACCCGCACTG AGGACAGGCT AGTGTGGAAA ACCTTGCAGG GCGACCCATG GATTAAGCAG CCCACTAACA TACCTAAAGG AAACTTTCAG GCTACTATAG AGGACTGGGA AGCTGAACAC AAGGGAATAA TTGCTGCAAT AAACAGCCCC ACTCCCCGTG CCAATCCGTT CAGCTGCAAG ACCAACGTTT GCTGGGCGAA AGCATTGGAA CCGATACTAG CCACGGCCGG TATCGTACTT ACCGGTTGCC AGTGGAGCGA ACTGTTCCCA CAGTTTGCGG ATGACAAACC ACATTCGGCC ATTTACGCCT TAGACGTAAT TTGCATTAAG I I I I I CGGCA TGGACTTGAC AAGCGGACTG TTTTCTAAAC AGAGCATCCC ACTAACGTAC CATCCCGCCG ATTCAGCGAG GCCGGTAGCT CATTGGGACA ACAGCCCAGG AACCCGCAAG TATGGGTACG ATCACGCCAT TGCCGCCGAA CTCTCCCGTA GATTTCCGGT GTTCCAGCTA GCTGGGAAGG GCACACAACT TGATTTGCAG ACGGGGAGAA CCAGAGTTAT CTCTGCACAG CATAACCTGG TCCCGGTGAA CCGCAATCTT CCTCACGCCT TAGTCCCCGA GTACAAGGAG AAGCAACCCG GCCCGGTCAA AAAATTCTTG AACCAGTTCA AACACCACTC AGTACTTGTG GTATCAGAGG AAAAAATTGA AGCTCCCCGT AAGAGAATCG AATGGATCGC CCCGATTGGC ATAGCCGGTG CAGATAAGAA CTACAACCTG GCTTTCGGGT TTCCGCCGCA GGCACGGTAC GACCTGGTGT TCATCAACAT TGGAACTAAA TACAGAAACC ACCACTTTCA GCAGTGCGAA GACCATGCGG CGACCTTAAA AACCCTTTCG CGTTCGGCCC TGAATTGCCT TAACCCAGGA GGCACCCTCG TGGTGAAGTC CTATGGCTAC GCCGACCGCA ACAGTGAGGA CGTAGTCACC GCTCTTGCCA GAAAGTTTGT CAGGGTGTCT GCAGCGAGAC CAGATTGTGT CTCAAGCAAT ACAGAAATGT ACCTGATTTT CCGACAACTA GACAACAGCC GTACACGGCA ATTCACCCCG CACCATCTGA ATTGCGTGAT TTCGTCCGTG TATGAGGGTA CAAGAGATGG AGTTGGAGCC GCGCCGTCAT ACCGCACCAA AAGGGAGAAT ATTGCTGACT GTCAAGAGGA AGCAGTTGTC AACGCAGCCA ATCCGCTGGG TAGACCAGGC GAAGGAGTCT GCCGTGCCAT CTATAAACGT TGGCCGACCA GTTTTACCGA TTCAGCCACG GAGACAGGCA CCGCAAGAAT GACTGTGTGC CTAGGAAAGA AAGTGATCCA CGCGGTCGGC CCTGATTTCC GGAAGCACCC AGAAGCAGAA GCCTTGAAAT TGCTACAAAA CGCCTACCAT GCAGTGGCAG ACTTAGTAAA TGAACATAAC ATCAAGTCTG TCGCCATTCC ACTGCTATCT ACAGGCATTT ACGCAGCCGG AAAAGACCGC CTTGAAGTAT CACTTAACTG CTTGACAACC GCGCTAGACA GAACTGACGC GGACGTAACC ATCTATTGCC TGGATAAGAA GTGGAAGGAA AGAATCGACG CGGCACTCCA ACTTAAGGAG TCTGTAACAG AGCTGAAGGA TGAAGATATG GAGATCGACG ATGAGTTAGT ATGGATTCAT CCAGACAGTT GCTTGAAGGG AAGAAAGGGA TTCAGTACTA CAAAAGGAAA ATTGTATTCG TACTTCGAAG GCACCAAATT CCATCAAGCA GCAAAAGACA TGGCGGAGAT AAAGGTCCTG TTCCCTAATG ACCAGGAAAG TAATGAACAA CTGTGTGCCT ACATATTGGG TGAGACCATG GAAGCAATCC GCGAAAAGTG CCCGGTCGAC CATAACCCGT CGTCTAGCCC GCCCAAAACG TTGCCGTGCC TTTGCATGTA TGCCATGACG CCAGAAAGGG TCCACAGACT TAGAAGCAAT AACGTCAAAG AAGTTACAGT ATGCTCCTCC ACCCCCCTTC CTAAGCACAA AATTAAGAAT GTTCAGAAGG TTCAGTGCAC GAAAGTAGTC CTGTTTAATC CGCACACTCC CGCATTCGTT CCCGCCCGTA AGTACATAGA AGTGCCAGAA CAGCCTACCG CTCCTCCTGC ACAGGCCGAG GAGGCCCCCG AAGTTGTAGC GACACCGTCA CCATCTACAG CTGATAACAC CTCGCTTGAT GTCACAGACA TCTCACTGGA TATGGATGAC AGTAGCGAAG GCTCACTTTT TTCGAGCTTT AGCGGATCGG ACAACTCTAT TACTAGTATG GACAGTTGGT CGTCAGGACC TAGTTCACTA GAGATAGTAG ACCGAAGGCA GGTGGTGGTG GCTGACGTTC ATGCCGTCCA AGAGCCTGCC CCTATTCCAC CGCCAAGGCT AAAGAAGATG GCCCGCCTGG CAGCGGCAAG AAAAGAGCCC ACTCCACCGG CAAGCAATAG CTCTGAGTCC CTCCACCTCT CTTTTGGTGG GGTATCCATG TCCCTCGGAT CAATTTTCGA CGGAGAGACG GCCCGCCAGG CAGCGGTACA ACCCCTGGCA ACAGGCCCCA CGGATGTGCC TATGTCTTTC GGATCGTTTT CCGACGGAGA GATTGATGAG CTGAGCCGCA GAGTAACTGA GTCCGAACCC GTCCTGTTTG GATCATTTGA ACCGGGCGAA GTGAACTCAA TTATATCGTC CCGATCAGCC GTATCTTTTC CACTACGCAA GCAGAGACGT AGACGCAGGA GCAGGAGGAC TGAATACTGA CTAACCGGGG TAGGTGGGTA CATATTTTCG ACGGACACAG GCCCTGGGCA CTTGCAAAAG AAGTCCGTTC TGCAGAACCA GCTTACAGAA CCGACCTTGG AGCGCAATGT CCTGGAAAGA ATTCATGCCC CGGTGCTCGA CACGTCGAAA GAGGAACAAC TCAAACTCAG GTACCAGATG ATGCCCACCG AAGCCAACAA AAGTAGGTAC CAGTCTCGTA AAGTAGAAAA TCAGAAAGCC ATAACCACTG AGCGACTACT GTCAGGACTA CGACTGTATA ACTCTGCCAC AGATCAGCCA GAATGCTATA AGATCACCTA TCCGAAACCA TTGTACTCCA GTAGCGTACC GGCGAACTAC TCCGATCCAC AGTTCGCTGT AGCTGTCTGT AACAACTATC TGCATGAGAA CTATCCGACA GTAGCATCTT ATCAGATTAC TGACGAGTAC GATGCTTACT TGGATATGGT AGACGGGACA GTCGCCTGCC TGGATACTGC AACCTTCTGC CCCGCTAAGC TTAGAAGTTA CCCGAAAAAA CATGAGTATA GAGCCCCGAA TATCCGCAGT GCGGTTCCAT CAGCGATGCA GAACACGCTA CAAAATGTGC TCATTGCCGC AACTAAAAGA AATTGCAACG TCACGCAGAT GCGTGAACTG CCAACACTGG ACTCAGCGAC ATTCAATGTC GAATGCTTTC GAAAATATGC ATGTAATGAC GAGTATTGGG AGGAGTTCGC TCGGAAGCCA ATTAGGATTA CCACTGAGTT TGTCACCGCA TATGTAGCTA GACTGAAAGG CCCTAAGGCC GCCGCACTAT TTGCAAAGAC GTATAATTTG GTCCCATTGC AAGAAGTGCC TATGGATAGA TTCGTCATGG ACATGAAAAG AGACGTGAAA GTTACACCAG GCACGAAACA CACAGAAGAA AGACCGAAAG TACAAGTGAT ACAAGCCGCA GAACCCCTGG CGACTGCTTA CTTATGCGGG ATTCACCGGG AATTAGTGCG TAGGCTTACG GCCGTCTTGC TTCCAAACAT TCACACGCTT TTTGACATGT CGGCGGAGGA TTTTGATGCA ATCATAGCAG AACACTTCAA GCAAGGCGAC CCGGTACTGG AGACGGATAT CGCATCATTC GACAAAAGCC AAGACGACGC TATGGCGTTA ACCGGTCTGA TGATCTTGGA GGACCTGGGT GTGGATCAAC CACTACTCGA CTTGATCGAG TGCGCCTTTG GAGAAATATC ATCCACCCAT CTACCTACGG GTACTCGTTT TAAATTCGGG GCGATGATGA AATCCGGAAT GTTCCTCACA C I I I I I GTCA ACACAGTTTT GAATGTCGTT ATCGCCAGCA GAGTACTAGA AGAGCGGCTT AAAACGTCCA GATGTGCAGC GTTCATTGGC GACGACAACA TCATACATGG AGTAGTATCT GACAAAGAAA TGGCTGAGAG GTGCGCCACC TGGCTCAACA TGGAGGTTAA GATCATCGAC GCAGTCATCG GTGAGAGACC ACCTTACTTC TGCGGCGGAT TTATCTTGCA AGATTCGGTT ACTTCCACAG CGTGCCGCGT GGCGGATCCC CTGAAAAGGC TGTTTAAGTT GGGTAAACCG CTCCCAGCCG ACGACGAGCA AGACGAAGAC AGAAGACGCG CTCTGCTAGA TGAAACAAAG GCGTGGTTTA GAGTAGGTAT AACAGGCACT TTAGCAGTGG CCGTGACGAC CCGGTATGAG GTAGACAATA TTACACCTGT CCTACTGGCA TTGAGAACTT TTGCCCAGAG CAAAAGAGCA TTCCAAGCCA TCAGAGGGGA AATAAAGCAT CTCTACGGTG GTCCTAAATA GTCAGCATAG TACATTTCAT CTGACTAATA CTACAACACC ACCACCTCTA GACGCGTAGA TCTCACGTGA GCATGCAGGC CTTGGGCCCA ATGATCCGAC CAGCAAAACT CGATGTACTT CCGAGGAACT GATGTGCATA ATGCATCAGG CTGGTACATT AGATCCCCGC TTACCGCGGG CAATATAGCA ACACTAAAAA CTCGATGTAC TTCCGAGGAA GCGCAGTGCA TAATGCTGCG CAGTGTTGCC ACATAACCAC TATATTAACC ATTTATCTAG CGGACGCCAA AAACTCAATG TATTTCTGAG GAAGCGTGGT GCATAATGCC ACGCAGCGTC TGCATAACTT TTATTATTTC TTTTATTAAT CAACAAAATT TTG I I I I I AA CATTTCAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAGGGAATT CCTCGATTAA TTAAGCGGCC GCTCGAGGGG AATTAATTCT TGAAGACGAA AGGGCCAGGT GGCACTTTTC GGGGAAATGT GCGCGGAACC CCTATTTGTT TATTTTTCTA AATACATTCA AATATGTATC CGCTCATGAG ACAATAACCC TGATAAATGC TTCAATAATA TTGAAAAAGG AAGAGTATGA GTATTCAACA TTTCCGTGTC GCCCTTATTC CCI I I I I I GC GGCATTTTGC CTTCCTGTTT TTGCTCACCC AGAAACGCTG GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCACGAG TGGGTTACAT CGAACTGGAT CTCAACAGCG GTAAGATCCT TGAGAGTTTT CGCCCCGAAG AACGTTTTCC AATGATGAGC ACTTTTAAAG TTCTGCTATG TGGCGCGGTA TTATCCCGTG TTGACGCCGG GCAAGAGCAA CTCGGTCGCC GCATACACTA TTCTCAGAAT GACTTGGTTG AGTACTCACC AGTCACAGAA AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA GTGCTGCCAT AACCATGAGT GATAACACTG CGGCCAACTT ACTTCTGACA ACGATCGGAG GACCGAAGGA GCTAACCGCT TTTTTGCACA ACATGGGGGA TCATGTAACT CGCCTTGATC GTTGGGAACC GGAGCTGAAT GAAGCCATAC CAAACGACGA GCGTGACACC ACGATGCCTG TAGCAATGGC AACAACGTTG CGCAAACTAT TAACTGGCGA ACTACTTACT CTAGCTTCCC GGCAACAATT AATAGACTGG ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG CCCTTCCGGC TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA CGGGGAGTCA GGCAACTATG GATGAACGAA ATAGACAGAT CGCTGAGATA GGTGCCTCAC TGATTAAGCA TTGGTAACTG TCAGACCAAG TTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCI I I I I CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC AGCGTGAGCA TTGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC GAGCTCGTAT GGACATATTG TCGTTAGAAC GCGGCTACAA TTAATACATA ACCTTATGTA TCATACACAT ACGATTTAGG GGACACTATA G
The generation of SLNrep5-VP22, SLNrep5-E7, and SLNrep5-VP22/E7 constructs have been described previously by the present inventors (Wu, TC et al, co-pending International patent application published as WOOl/29233 26 April 2001; see also Cheng, WF et al, 2001, J Virol. 75:2368-2376). The pcDNA3 expression vector and pcDNA3-E7 have been described (Chen, CH et al, 1999, Gene Ther 6:1972-81; Ji, H. et al, 1999, Human Gene Therapy 10:2727-2740). pcDNA3 has been used successfully in DNA vaccine induced immune responses and antitumor effects (Chen, CH et al, 2000, Cancer Res 60:1035-42; co-pending, commonly assigned patent pubications or applications WOOl/29233, USSN 09/421,608, filed 20 October 1999, USSN 09/501,097, filed 09 February 2000, which are incoφorated by reference). For the generation of pcDNA3-VP22, VP22 was subcloned from pVP22/myc-His (Invitrogen, Carlsbad, CA) into the unique EcoRV and BamHI cloning sites ofthe pcDNA3.1(-) expression vector (Invitrogen, Carlsbad, CA) downstream ofthe CMV promoter. The generation of pcDNA3-E7 has been described previously (Chen et al, supra). For the generation of pcDNA3- VP22/E7, VP22 was subcloned from pcDNA3-VP22 into the unique EcoRV and BamHI cloning sites ofthe pcDNA3-E7. For the generation of pcDNA3-E7(E/B), which contains E7 with EcoRI and BamHI restriction sites on the flanking ends of E7, PCR was used to amplify the E7 fragment with pcDNA3-E7 and a set of primers: 5' -ggggaattcatggagatacaccta-3' (SEQ ID NO: 24) and 5' -ggtggatccttgagaacagatgg-3'' . (SEQ ID NO:25).
The amplified product was further cloned into the EcoRI/BamHI sites of pcDNA3. For the generation of pcDNA3-VP22(l-267)/E7, a DNA fragment encoding VP22(l-267) was first amplified using PCR with pcDNA3-VP22 and a set of primers: 5 ' -gggtctagaatgacctctcgccgctccgt-3 ' ( SEQ ID NO : 26 ) and 5 ' -ggggaattcgtcctgcaccacgtctggat-3 ' ( SEQ ID NO : 27 ) . The amplified product was cloned into the Xbal/EcoRI cloning sites of pcDNA3-E7(E/B).
For the generation of pcDNA3-GFP, DNA fragment encoding GFP was first amplified using PCR with pEGFPNl DNA (Clontech, Palo Alto, CA) and a set of primers:
5' -atcggatccatggtgagcaagggcgaggag-3' (SEQ ID NO:28)and 5' -gggaagctttacttgtacagctcgtccatg-3'' . (SEQ ID NO:29)
The amplified product was cloned into the BamHI Hindlll cloning sites of pcDNA3. For the generation of pcDNA3-VP22/GFP, VP22 was subcloned from pcDNA3-VP22 into the unique EcoRV and BamHI cloning sites ofthe pcDNA3-GFP. For construction of pcDNA3- E7/GFP, GFP was isolated from pcDNA3-GFP and cloned into BamHI/Hindm sites of pcDNA3-E7(E+B). For construction of VP22/E7/GFP, VP22 was amplified by and a set of primers:
5' -gggtctagaatgacctctcgccgctccgt-3' (SEQ ID NO: 30) and 5' -ggggaattcctcgacgggccgtctggggc-3' (SEQ ID NO: 31) and cloned into Xbal/EcoRI sites of pcDNA3-E7/GFP. For construction of pcDNA3-VP22(l-
267VE7/GFP, VP22(l-267) was isolated from pcDNA3-VP22( 1-267) and cloned into
Xbal/EcoRI sites of pcDNA3-E7/GFP.
The generation of pSCl 1-E7 has been described previously (Wu et al, 1995, . Proc. Natl. Acad. Sci. 92: 11671-11675). For cloning pSCl 1-VP22/E7, VP22 was isolated from pcDNA3-
VP22/E7 by Notl/Pmel and coned into Notl Saml sites of pSCl 1 vector. To generate pSCl 1-
VP22, VP22was isolated from ρcDNA3-VP22 by Notl/Pmel and cloned into Notl/Saml sites of pSCll vector.
For the generation of pcDNA3-TAT/E7, the following complementary oligomers encoding MRKKRRQRRR (SEQ ID NO:32) (Green, M et al, 1988, Cell 55:1179-88;
Schwarze, SR et al, 1999, Science 285:1569-72) were synthesized:
5' -ctagaatgtacggccgcaagaaacgccgccagcgccgccgcg-3' (SEQ ID NO:33) and 5' -aattcgcggcggcgctggcggcgtttcttgcggccgtacatt-3' (SEQ ID NO:34).
The oligomers were annealed and cloned into the Xbal/EcoRI sites of pcDNA3-E7(E/B). For the generation of pcDNA3-E7/MTS, the following complementary oligomers encoding
AAVLLPVLLAAP (SEQ ID NO:12) (Rojas, M et al, 1998, Nat Biotechnol 16:370-5) were synthesized: 5' -gatccgcagccgttcttctccctgttcttcttgccgcacccta-3' (SEQ ID NO:35) and 5' -agcttagggtgcggcaagaagaacagggagaagaacggctgcg-3' (SEQ ID NO: 36).
The oligomers were annealed and cloned into the BamHI/Hindiπ sites of pcDNA3-E7(E/B).
For the generation of pcDNA3-AH/E7, the following complementary oligomers encoding MRQIKIWFQNRRMKWKK (SEQ ID NO:15) (Derossi, D et al, 1994, JBiol Chem 269:10444- 10450) were synthesized:
5' -ctagaatgcgccaaatcaaaatctggttccagaatcgacgaatgaagtggaaaaaag-3' (SEQ ID NO:37) and
5' -aattcttttttccacttcattcgtcgattctggaaccagattttgatttggcgcatt-3' (SEQ ID NO:38).
The oligomers were annealed and cloned into the Xbal/EcoRI sites of pcDNA3-E7(E/B). The accuracy of all the DNA constructs was confirmed by sequencing.
In Vitro RNA Preparation
The generation of RNA transcripts from SLNrep5-VP22, SLNrep5-E7, SP rep5-VP22/E7 and SINrep5 was performed using a protocol described previously (WO 02/09645 07-Feb-02; Cheng et al, supra). Briefly, Spel was used to linearize DNA templates. RNA replicons were transcribed in vitro and capped using SP6 RNA polymerase and capping analog from an in vitro transcription kit (Life Technologies, RockviUe, MD) according to the vendor's manual. After synthesis, DNA was removed by digestion with DNase I. Synthesized RNA was then purified by precipitation. RNA concenfration was determined by optical density measured at 260 nm. The integrity and quantity of RNA transcripts were further checked using denaturing gel electrophoresis. The purified RNA was divided into aliquots to be used for vaccination in animals and for transfection of BHK21 cells. The protein expression ofthe transcripts was characterized by transfection ofthe RNA into BHK21 cells using the Cell-Porator Electroporation System (Life Technologies, RockviUe, MD) according to the vendor's manual, followed by Western blot analysis. Generation of SINrep5 Replicon Particles and Determination of the Vector Titer
SLNrep5 replicon particles were made using a protocol described protocol by Polo et al. supra. Briefly, 4 μg of mRNA synthesized in vitro was electroporated into 107 cells ofthe PCL. The PCL cells were incubated in 23 ml DMEM supplemented with 10% FBS, antibiotics and G418 at 5% CO2, 37°C. After 72 hr, culture supernatants were collected. The titer of SLNrep5 replicon particles in clarified PCL culture supernatants was determined by infection of naϊve BHK-21 monolayers, followed by indirect E7 immunofluorescence staining (Wu et al, 1995, . Proc. Natl. Acad. Sci. 92:11671-11675) with serial dilution and quantitation ofthe total number of green stained cells per well at each dilution. Vector titer is designated as infectious units (IU)/ml, and represents the population of functional particles.
Immunofluorescence Staining for E7 and VP22/E7 Expression
Immunofluorescence staining was performed as described by (Wu et al, supra). Briefly, BHK21 cells were cultured in 2-well culture chamber slides (Nalge Nunc Int., Naperville, EL) until they reached 50% confluency. The BHK21 cells were infected with a serial dilution of replicon particles. After 48 and 72 hours of infection, the cells were fixed in 10%> formalin for 20 min. Diluted anti-E7 Ab (1 : 200 dilution, Zymed, San Francisco, CA) was added into the chamber and incubated for 30 min. Diluted FITC goat anti-mouse IgG (10 μg/ml, Jackson hnmunoReseach Laboratories, West Grove, PA), was added and incubated for 30 min. The slides were mounted and observed immediately under a fluorescence microscope. Mice
6- to 8-week-old female C57BL/6 mice from the National Cancer Institute (Frederick, MD) were purchased and kept in the oncology animal facility ofthe Johns Hopkins Hospital (Baltimore, MD). All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals. Intracytoplasmic Cytokine Staining and Flow Cytometry Analysis
Intracytoplasmic cytokine staining and flow cytometry analysis were performed as described previously (Cheng et al, 2001b, Human Gene Therapy. 72:235-252). hi brief, mice (5 per group) were vaccinated with 5x106 IU/mouse of SINrep5-VP22/E7 via different routes of administration (intramuscular, intraperitoneal, subcutaneous), hi another experiment, another group of mice (5 per group) was vaccinated intramuscularly with different titers of SrNrep5 replicon particles (5xl07, 5xl06, 5xl05IU 5xl04, and 5xl03 IU/mouse). Naϊve mice served as negative controls. Splenocytes from vaccinated mice were collected seven days after vaccination and incubated either with the E7 peptide (aa 49-57, RAJHYNTVTF ) containing the MHC class I epitope (Feltkamp et al, 1993, Eur J Immunol. 23:2242-2249) (to detect E7-specific CD8+ cytotoxic T cell precursors) or with the E7 peptide (aa 30-67,
DSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRL; SEQ LD NO:39) that includes the MHC class E epitope (Tindle et al, 1991, Proc Natl Acad Sci USA 55:5887-5891) (to detect E7- specific CD4+ T helper cell precursors) for 20 hours. Cells were subjected to infracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacturer's instructions (PharMingen, San Diego, CA). Analysis was done on a Becton Dickinson FACScan with CELLQuest software (Becton Dickinson hnmunocytometry System, Mountain View, CA). ELISA
Anti-HPV 16 E7 antibodies in the sera from vaccinated mice (5 per group) were determined by a direct ELISA as described previously (Wu et al, supra). The ELISA plate was read with a standard ELISA reader at 450 nm. The quantity of E7 protein in cell lysates from BHK21 cells infected with SIN5rep-E7 or
SINrep5-NP22/E7 replicon particles was determined by an indirect ELISA method as described previously (Cheng et al, supra). Briefly, lxlO7 BHK21 cells were infected with 5xl07 IU of SIΝrep5, SLNrep5-E7, SINrep5-NP22 or SLΝrep5-VP22/E7 particles. The transfected BHK21 cells were collected 40-44 hrs after infection. The quantity of E7 protein in cell lysates from transfected BHK21 cells was determined by ELISA with a standard ELISA reader at 450 nm.
The quantity of E7 protein of cell lysates was then calculated and determined by comparing with the standardized E7 protein. In Vivo Tumor Protection
For the tumor protection experiment, mice (5 per group) were immunized intramuscularly with 5xl06 IU/mouse of SLNrep5-E7, SINrep5-VP22, SINrep5-VP22/E7 or control SLNrep5 replicon particles. One week after vaccination, mice were subcutaneously challenged with 1 x 104 TC-1 cells/mouse in the right leg. Mice were monitored for evidence of tumor growth by palpation and inspection twice a week until they were sacrificed at day 60. In Vivo Tumor Therapy C57BL/6 mice (5 each group) were challenged with 104 cells/mouse TC-1 tumor cells i.v. in the tail vein on day 0. Three, seven or fourteen days after tumor challenge, mice were treated intramuscularly with 5xl06 IU/mouse of SLNrep5-E7, SINrep5-VP22, SINrep5-VP22/E7 or SINrep5 control replicon particles. Mice were sacrificed on day 21 after tumor challenge. The number of pulmonary tumor nodules on the surface of lungs in each mouse was determined by experimenters blinded to the sample identity. For the head-to-head comparison of various VP22/E7-containing vaccines, we performed another in vivo tumor treatment experiment. C57BL/6 mice (5 each group) were intravenously challenged with 104 cells/mouse TC-1 tumor cells in the tail vein on day 0. 7 days after tumor challenge, mice were treated intramuscularly with optimized vaccine doses determined from previous studies: 2 μg/mouse SLNrep5-VP22/E7 DNA (Hung et al, 2001, supra), 1 μg/mouse SLNrep5-VP22/E7 RNA (Cheng et al, 2001a), or 5xl06 IU/mouse of SINrep5-VP22/E7 replicon particles. Naϊve mice were used as a negative control. Mice were sacrificed on day 28 after tumor challenge and mean lung weight was measured by experimenters blinded to the sample identity. In Vivo Antibody Depletion Experiments
The procedure for in vivo antibody depletion has been described previously. Briefly, mice were vaccinated intramuscularly with 5x106 IU/mouse of SLNrep5-VP22/E7 replicon particles. Depletions were started on day 7 after immunization and mice were challenged with 1 x 10 cells/mouse TC-1 tumor cells on day 14 after immunization. MAb GK1.5 (Dialynas et al, 1983, J. Immunol. J. Immunol. 737:2445:2445) was used for CD4 depletion, MAb 2.43 (Saπniento et al, 1980, J. Immunol. 725:2665) was used for CD8 depletion, and MAb PK136 (Koo et al, 1986, J. Immunol. 737:3742) was used for NK1.1 depletion. Flow cytometry analysis revealed that >99%> ofthe appropriate lymphocyte subsets were depleted while maintaining normal levels of other subsets. Depletion was terminated on day 40 after tumor challenge. In Vivo Assay for Apoptotic Cells in the Tissue of Vaccinated Mice
Mice were immunized with 5x10 IU/mouse of SLNrep5-VP22/E7 replicon particles intramuscularly in the right leg. Normal saline without replicon particles was injected intramuscularly into the left leg as a control. Mice were sacrificed 7 days after intramuscular injection. For the detection of apoptotic cells, a modified TUNEL method was used as described previously (Cheng et al, supra). Apoptotic index is used as a measure ofthe extent of apoptosis in the stained slides following inspection under a light microscope. Apoptotic index is defined as the percentage of apoptotic cells and apoptotic bodies per 100 cells (Lipponen et al, 1994, J Pathol. 173:333-339). CTL Assay Using DCs Co-Incubated with SIN Replicon-Infected BHK21 Cells DCs were generated by culturing bone marrow cells in the presence of GM-CSF as described previously (Cheng et al, Human Gene Ther., 2001). CTL assays, using DCs co- incubated with transfected cells as targets, were performed using a previously described protocol (Cheng et al, supra). Briefly, 107 BHK21 cells were infected with 5xl07 IU of SINrep5, SLNrep5-E7, SINrep5-NP22 or SLΝrep5-NP22/E7 replicon particles. The infected BHK21 cells were collected 40-44 hrs later. The levels of E7 protein expression for all replicon-infected BHK21 cells were similar, as determined by ELISA. 3x105 of infected BHK21 cells were then co-incubated with lxlO5 of bone marrow-derived DCs at 37°C for 48 hr. These prepared DCs were then used as target cells and an E7-specific CD8 T cell line (Wang et al, 2000, supra) served as effector cells. CTL assays were performed with effector cells and target cells (lxlO4 per well) mixed together at various ratios (1:1, 3:1, 9:1, and 27:1) in a final volume of 200 μl. After a 5 hr incubation at 37°C, 50 μl ofthe cultured media was collected to assess the amount of LDH using the CytoTox assay kit (Promega, Madison, WI). The percentage of lysis was calculated from the formula:
% Lysis = [(A-B)/(C-D)] x 100 where A is the experimental-effector signal value, B is the effector spontaneous background signal value, C is maximum signal value from target cells, D is the target spontaneous background signal value. DCs co-incubated with uninfected BHK21 cells, infected BHK21 cells alone, untreated DCs alone, and CD8+ T cells alone were included as negative controls. Fluorescence Microscopy for In vitro Distribution of VP22/E7
293 D Kb cells (provided by Dr. JC Yang, National Cancer Institute, NTH; Bloom, MB et al, 1997, JExp Med 185:453-459) were utilized for an in vitro assay of GFP expression. 20μg of VP22, E7/GFP, VP22(l-267)/E7/GFP or VP22/E7/GFP DNA were transfected into 5xl06 293 D K cells using lipofectamine 2000 (Life Technologies, RockviUe, MD). Transfected cells were fixed with 4 % paraformaldehyde in IX PBS, permeabilized with IX PBS containing 0.05%) saponin and 1% BSA, then incubated with 0.5 μg/ml of primary anti-camexin antibody (Stressgen Biotechnologies, Victoria, BC). Samples were acquired with the Noran Oz confocal laser scanning microscope system using Invertension® software (v. 6.5). Slides were imaged with an Olympus IX-50 inverted microscope (lOOx magnification). Immunohistochemical Staining for In Vivo Distribution of VP22/E7
Mice were sacrificed 3 days after vaccination with pcDNA3-VP22/GFP or pcDNA3-. Skin was biopsied, fixed, paraffin-embedded, and cut into 5 μm sections. After deparaffinization and hydration, slides were incubated with rabbit anti-GFP polyclonal antibody (1:200 dilution; Molecular Probes, Eugene, OR) followed by biotinylated goat anti-rabbit IgG (1 :200 dilution) and avidin-biotin complex (1:100 dilution; Vector, Burlingame, CA). The slides were developed by adding DAB substrate solution (DAKO, Caφenteria, CA) and counterstained with Mayer's hematoxylin. Stained slides were dehydrated, mounted and observed by light microscopy.
EXAMPLE II
Expression and Distribution of E7 and Chimeric VP22/E7 Protein in Cells Infected with SINrep5-E7 or SINrep5-VP22/E7 Replicon Particles
To demonstrate the expression of E7 protein in cells infected with E7-encoding SINrep5 replicon particles, indirect ELISA and immunofluorescence staining were performed. The quantity of E7 protein was determined using lysates from cells infected with 5x107 IU of
SLNrep5, SLNrep5-E7, SLNrep5-VP22 or SINrep5-VP22/E7 replicon particles.
The level of E7 and VP22/E7 protein expression was comparable between cells infected with SINrep5-E7 and SINrep5-VP22/E7 replicon particles 48 hours after infection. The subcellular localization of E7 and VP22/E7 proteins in the infected BHK21 cells was also evaluated by immunofluorescence 48 hours after infection. E7 protein was mainly located in the nucleus (Figure 1 A), while chimeric VP22/E7 protein was located in the cytoplasm
(Figure IB). 72 hours after infection, E7 protein remained localized predominantly in the nucleus ofthe initially infected BHK21 cells (Figure IC). Meanwhile, chimeric VP22/E7 protein was transferred to cells neighboring the initially infected BHK21 cells 72 hours after infection (Figure ID).
The results indicated that the linkage of VP22 to antigen in SINrep5 replicon particles led to the intercellular spread of linked antigen, which was observable at a later stage of infection (72 hours after infection).
EXAMPLE III
SINrep5-VP22/E7 Replicon Particles Significantly Enhance E7-Specific CD8+ T Cell Responses but not CD4+ Cell-Mediated Immune Responses
To determine the quantity of E7-specific CD8+ T cell precursors in mice vaccinated with SINrep5-VP22/E7 replicon particles, infracellular cytokine staining was performed as described above. As shown in Figure 2A, mice vaccinated with SINrep5-VP22/E7 replicon particles generated the greatest number of E7-specific CD8+ T cell precursors compared to the other vaccination groups (pθ.001). SUSfrep5-VP22/E7 replicon particles generated a significant 18- fold increase in the number of E7-specific CD8 T cell precursors compared to that generated by wild-type E7 expressing particles (219+12.7 versus 13.5+2.1 per 3xl05 splenocytes, p<0.001) (Figure 2B). hi comparison, the number of E7-specific CD4+ T cell precursors generated by the different
SLN replicon particles were not significantly different (Figure 2C).
Anti-E7 antibody titers generated by SUMrep5-VP22/E7 replicon particles were not increased compared to the other groups. These results were consistent with the observed lack of E7-specific CD4+ T helper cell enhancement. Fusion of E7 to VP22 was required for enhanced CD8+ T cell activity, since VP22 mixed with E7 (VP22 + E7) did not cause such enhancement of CD8+ T cell activity. Furthermore, E7 linked to an irrelevant protein such as green fluorescent protein (GFP) did not enhance E7- specific CD8+ T cell activity.
EXAMPLE IV Different Routes and Doses of SINrep5-VP22/E7 Replicon Particles
Influence the Antigen-Specific CD8+ T Cell-mediated Response
The relationship between different routes of vaccine administration and the resultig immune responses were evaluated. Mice were injected i.m., l.p, and s.c, with 5x10 IU/mouse of SLNrep5-VP22/E7 replicon particles. Intramuscular injection generated more E7-specifϊc CD8+ T cells than the other two routes (Figure 3A). The correlation between replicon particle dosages and responses were also evaluated. Different doses of SINrep5-VP22/E7 replicon particles were injected i.m. into mice. With increasing doses of replicon particles, the number of E7-specific
4- • f_
CD8 T cell precursors progressively increased until reaching a plateau at the dose of 5x10 IU/mouse (Figure 3B). These results suggested that i.m. delivery of SLNrep5-VP22/E7 replicon particles at a dose of 5x106 IU/mouse was optimal for generating an E7-specific CD8+ T cell immune response.
EXAMPLE V
Antitumor Effects of SINrep5-VP22 E7 Replicon Particle Vaccine: Tumor Protection
To determine whether vaccination with the various SLN replicon particles protected mice against E7-expressing tumors, in vivo tumor protection experiments were performed as described in Example I. Figure 4 shows that all mice receiving SINrep5-VP22/E7 particles remained tumor-free 60 days after TC-1 challenge. In contrast, all ofthe unvaccinated mice and mice receiving SINrep5 with no insert, VP22, wild-type E7, or VP22 + E7 developed visible tumors within 20 days after tumor challenge. These results also indicated that fusion of E7 to NP22 was required for antitumor immunity since NP22 mixed with E7 (NP22 + E7) did not improve the antitumor effect.
EXAMPLE VI
Anitutmor Effects of SIΝrep5-VP22/E7 Replicon Particles: Eradication of Established Lung Tumors in the Lungs
To determine the therapeutic potential ofthe SLNrep5-VP22/E7 vaccine, C57BL/6 mice were challenged i.v. with 104 TC-1 tumor cells per mouse in the tail vein to establish TC-1 tumors in the lungs (Ji et al, 1998, hit J Cancer. 78:41-45). This model permits a more quantitative assessment of antitumor effects than the subcutaneous model. Figure 5A demonstrated that mice freated with SLNrep5-VP22/E7 replicon particles three days after tumor challenge exhibited a significantly lower mean number of pulmonary nodules (0.7+ 0.3) than mice vaccinated with wild-type E7 replicon particles (72.5+ 8.5) or VP22 replicon particles (79.0+17.0). As shown in Figure 5B, mice freated with SINrep5-VP22/E7 replicon particles displayed a significantly fewer pulmonary nodules on day 3 (0.7+0.3), day 7 (0.5+0.3) and day 14 (25.0+4.0) after tumor challenge compared to mice freated with the SINrep5 control particles (no insert)(one-way ANOVA, p < 0.05). Treatment with SPNrep5-VP22/E7 replicon particles reduced grossly visible tumors even if administered as late as 14 days after tumor challenge, hi general, grossly visible lung nodules could be detected 14 days after i.v. tumor (TC-1) cell injection. The results indicated that SINrep5-VP22/E7 particles can confrol established life- threatening tumor growth, even up to 14 days after the tumor has been implanted. EXAMPLE VII
Sindbis Virus Replicon Particles Are the Most Effective Delivery Vector for VP22/E7 Vaccines used to Controlling Established Pulmonary Tumors
Previously, the prseent inventors and their colleagues developed a DNA vaccine (Hung et al., 2001, supra) and naked RNA replicon vaccine (Cheng et al., 2001a, supra ; Wu et al, WO02/09645) encoding VP22/E7 and found that they were effective for treatment of TC- 1 tumor cells. To compare the relative efficacy of VP22/E7 naked DNA, naked SP reρ5-VP22/E7 RNA replicons, and SUSfrep5-VP22/E7 RNA replicon particles, C57BL/6 mice were challenged with 104 TC-1 tumor cells per mouse i.v. via the tail vein to establish pulmonary TC-1 tumors, followed by treatment with optimized doses ofthe various VP22/E7-containing vaccines 7 days later. Figure 6A demonstrated that mice treated with SLNrep5-VP22/E7 replicon particles exhibited a significantly lower mean lung weight (208+13 mg) than did mice vaccinated with VP22/E7 naked DNA (256±33 mg) or naked SINrep5-VP22/E7 RNA replicons (521+53 mg), or naϊve mice (644+44 mg). Figure 6B displays representative gross pictures of pulmonary metastatic nodules derived from mice treated with different VP22/E7-containing vaccines. These results indicated that treatment of mice with SLNrep5-VP22/E7 replicon particles generated the greatest therapeutic antitumor effect among the VP22/E7 chimeric vaccines.
EXAMPLE VIII
CD8+ T cells, CD4+ T cells and NK cells are Essential for the Anti-tumor Effect Generated by SINrep5-VP22/E7 Replicon Particles
To determine which subset(s) of lymphocytes that are important for the rejection of E7+ tumor cells, in vivo antibody depletion experiments were done. The completeness of depletion was assessed on the day of tumor injection and weekly thereafter by flow cytometric analysis of spleen cells. Typically, the appropriate subset was depeleted by > 99% while other lymphocytes populations remained at normal levels. As shown in Figure 7, tumors grew in all naϊve mice and all mice depleted of CD8+ T cells within 14 days after implantation. 80%> of mice depleted of CD4+ T cells and 60% of mice depleted of NKl.l cells developed tumors within 60 days of implantation. These results suggested that CD8+ T cells, CD4+ T cells and NK cells all participate in the anti-tumor immunity induced by the SLNrep5-VP22/E7 replicon particles.
EXAMPLE IX Apoptotic in Cells Infected with SIN Replicon Particles in Vaccinated Mice
To evaluate whether SLN replicon particles induce apoptosis in vivo, mice were immunized ι.m. with 5x10 IU each of SLNrep5-VP22/E7 replicon particles and were sacrificed 7 days later. Tissue sections of muscle were stained using the TUNEL method described above. Cells undergoing apoptosis exhibited brown staining of nuclei. As shown in Figure 8, vaccination with SIN replicon vaccines led to a significantly higher number of apoptotic cells in muscle tissue as compared to tisse from mice given normal saline. The apoptotic index was 74.5±4.5 for the SINrep5-VP22/E7-treated group and 30.5+2.5 for the saline control (p<0.01). These results indicated that muscle cells infected with SIN replicon particles underwent apoptosis.
EXAMPLE X
Enhanced Presentation of E7 Through the MHC Class I Pathway in Dendritic Cells Pulsed With Lysate of Cells Infected by SINrep5-VP22/E7 Replicon Particles
Ofthe various treatments, vaccination with SINrep5-VP22/E7 replicon particles resulted in the most potent immune response measured as the number of E7-specific CD8+ T cell precursors (Figure 3A). One mechanism for enhanced E7-specific CD8+ T cell responses in vivo is presentation of E7 through the MHC class I pathway by APCs that have taken up apoptotic cells in whichthe various antigen constructs were expressed. This phenomenon is known as "cross-priming".
To determine whether SLNrep5-VP22/E7 particles induce a cell-mediated immune response via cross-priming, BHK21 cells were first infected with various antigen-containing and control SIN replicon particles. These infected BHK21 cells were then incubated with bone marrow-derived DCs and used as target cells. Cytotoxic effector cells were T cells of an E7- specific CD8+ T cell line. As shown in Figure 9, the E7-specific CTL lysed DCs that had been "pulsed" with BHK21 cells infected with SINrep5-VP22/E7 more effectively than they lysed DCs pulsed with BHK21 cells that had been infected with other replicon particles (at E:T ratios of 9 and 27 (pO.01). Thus, DCs "pulsed" with SINrep5-VP22/E7 infected cells present E7 antigen through the MHC class I pathway more efficiently than do DCs pulsed with SINrep5-E7 infected cells.
DISCUSSION OF EXAMPLES I-X As described above, the present inventors generated SLN replicon vectors from a stable
PCL for vaccine development. The use of a stable PCL allowed the production of high titers of SLN replicon particles free of replication-competent virus, representing an important advance in the preparation of vaccines for mass immunization. Although SIN infection in humans typically has limited climcal manifestations, fever, skin rash, and arthritic joint pain have been reported in people infected with certain Sindbis virus strains (Strauss & Strauss, supra). The separation of structural protein cassettes in alphavirus PCLs significantly decreases the possibility of producing replication-competent virus (Polo et al, supra) and therefore decreases the likelihood of such undesired clinical effects.
An important consideration for vaccine development is the titer of vector stocks produced from this or other stable PCL. As shown above, the present inventors successfully produced SIN replicon particle stocks with titers up to 5x107 IU/ml, which was slightly higher than that described by Polo et al, supra. Wild-type Sindbis virus infection is known to generate titers of greater than 109 PFU/ml. Research has focused on improving transport between the nucleus and cytoplasm as well as stabilizing primary alphavirus RNA transcripts (Polo et al, supra ), two approaches that would contribute to higher titers ofthe replicon vectors approaching the level of wild-type alphavirus. Indeed, a panel of recently constructed SLN replicon PCL consistently produced particle titers of >08 IU/ml (??PFU) (C. Greer, B. Belli, and J. Polo unpublished data).
One limitation of such replication-defective viral vectors, whicha re relatively safe, one is their intrinsic inability to spread in vivo as effectively as do replication-competent viruses . As described herein, the present invention's inclusion of an intercellular spreading protein, exemplified as HSV-1 VP22, fused to antigen in the context of SLN replicon vectors, facilitated the spread of antigen to surrounding cells in vivo resulting in a significantly enhanced E7- specific CD8+ T cell response and consequent antitumor effects. Thus, the strategy of using a an intercellular spreading protein fused to an antigen and producing the vectors containing the nucleic acid expressing this fusion polypeptide by employing a stable PCL represents a unique and novel approach for generating a safe, potent vaccine in high quantities. This strategy provides several advantages over other vaccine approaches. Compared to naked nucleic acid vaccines, SLN replicon particles are capable of infecting/fransfecting a higher proportion of "target" cells. The linkage of the intercellular spreading protein, e.g., HSV VP22, further enhances vaccine potency. Another advantage is that SIN replicon RNA does not integrate into the host genome, which is a potential concern with naked DNA vaccines or DNA-based viral vectors. The composition comprising SLN replicon vectors that is generated from a stable PCL such as that exemplifed here is free of replication-competent virus particles without sacrificing the efficiency of gene delivery. This feature maximizes vaccine potency while minimizing the risk associated with replication-competent viral vectors. Finally, stable PCLs are also versatile, allowing for the packaging of different alphavirus-derived replicon vectors, in the present example, either Sindbis or Semliki Forest virus derived replicon vectors. As disclosed above, treatment of mice with SLNrep5-VP22/E7 replicon particles led to a more potent antitumor effect than did treatment with VP22/E7 naked DNA or naked SINrep5- VP22/E7 RNA replicon vaccines. It is noteworthy that that in mice vaccinated with VP22/E7 DNA (as reported elsewhere), a higher frequency of antigen-specific CD8+ T cell precursors were detected (576/3xl05 splenocytes) (Hung et al, 2001, supra) compared to the present examples of mice vaccinated with SINrep5-VP22/E7 replicon particles (219/3xl05 splenocytes, Figure 2B) at one week after the final vaccination. One explanation for this difference is a difference in the kinetics of generation of antigen-specific T cells by the various types of vaccines. Thus, vaccination with a Sindbis virus replicon particle vaccine resulted in peak numbers of antigen-specific CD8+ T cells earlier than vaccination with a DNA vaccine (3 days vs. 11 days. Thus, the rapid expansion of antigen-specific T cells after vaccination with Sindbis virus replicon particles is expected to exert more effective responses against rapidly growing tumors than would the relatively slower expansion of antigen-specific T cells following DNA vaccination. The present inventors tested the strategy of combining an intercellular spreading protein, such as HSV-1 VP22, with antigen while comparing different delivery vectors: naked DNA (pcDNA3) and naked SIN RNA (SINrep5). Each vaccine enhanced E7-specific CD8+ T cell- mediated immune responses and antitumor effects, although the effector cell involvement was different. CD8+ T cells were important components ofthe responses to all ofthe vectors tested, while CD4+ T cells were only essential for the antitumor effect generated by the VP22/E7 SLN particle-based vaccine. This conclusion is based on the observation that depleting CD4+ T cells did not diminish antitumor effects ofthe naked DNA vaccine ~ pcDNA3-VP22/E7 (Hung et al, 2001, supra) or the naked SIN replicon RNA vaccine - SIN replicon RNA-VP22/E7 (Cheng et al, 2001, J. Virol, supra). Although CD4 T cells appeared to be needed for an optimal antitumor effect in response to the VP22/E7 SLN particle-based vaccine described herein, this vaccine did not actively induce E7-specific CD4+ T cells. This suggested that these CD4 T cells were contributing to an antitumor effect via a non-antigen-specific mechanism. Indeed, NK cells were needed for the present antitumor effect but were not as important in response to the VP22/E7 SLN particle-based vaccine or the naked DNA vaccine. Thus, different types of vaccines encoding the same protein construct may activate different subsets of effector cells in the vaccinated host and activate different immune or nonimmune antitumor mechanisms.
The enhanced E7-specific CD8+ T cell responses induced by the present VP22/E7 SIN replicon particle vaccine compared to a "control" E7 SIN replicon particle vaccine are believed to result, at least in part, from a process whereby infectected apoptotic cells are endocytosed and processed by APCs for MHC class I antigen presentation to CD8+ T cells (Albert, ML et al, 1998, JExp Med. 188:1359-1368.; Albert, ML et al, 1998, Nature. 392:86-89). Alternatively, apoptotic cells may release chimeric VP22/E7 proteins that are taken up and processed by other APCs via a MHC class I-restricted pathway (Huang et al, supra). Because different types of SINrep5 replicon particles induced similar degrees of apoptosis, the distinct enhancement in E7- specific CD8+ T cell activity was inteφreted by the presen inventors as most likely due to the linkage of VP22 with E7. It is unlikely that the observed enhancement occurs as a result of improved direct MHC class I presentation of E7 to CTLs by cells expressing VP22/E7 because the replicon-infected cells eventually undergo apoptosis.
Recently, Gardner et al. (2000, J Virol. 74: 11849-11857) reported that SLN replicon particles encoding a modified E2 glycoprotein successfully delivered genes of interest into DCs to create a DC -based vaccine that could induce potent immune responses in vaccinated mice. DCs are the most potent APC and play a major role in the activation of both memory and naϊve T cells. Therefore, as conceived herein, the employment of SLN replicon particles for preparing effective DC compositions is a significant extension ofthe heretofore dislcosed uses of these recombinant alphaviral vectors. Thus, it is expected that the combination of intercellular spreading protein fusion strategy with the convenience of generating SIN replicon particles from stable PCLs will improve the efficiency of antigen delivery into DCs and permit development of improved DC -based immunotherapeutic vaccines. h summary, these results revealed that the combined usage ofthe viral spreading protein strategy along with an efficient method of producing safe SLN replicon particle preparations free of replication-competent virus was effective in generating potent antigen-specific immune responses and a strong antitumor effect. Furthermore, the availability of this stable alphavirus PCL makes it possible to generate a large quantity ofthe replicon particle-based vaccine for mass immunization. These strategies may also be applied to other cancer systems and infectious

Claims

WHAT IS CLAIMED IS
1. A nucleic acid molecule encoding a fusion polypeptide useful as a vaccine composition, which molecule comprises:
(a) a first nucleic acid sequence encoding a first polypeptide that comprises at least one im unogenicity-potentiating polypeptide;
(b) optionally, fused in frame with the first nucleic acid sequence, a linker nucleic acid sequence encoding a linker peptide; and
(c) a second nucleic acid sequence that is linked in frame to said first nucleic acid sequence or to said linker nucleic acid sequence and that encodes an antigenic polyp eptide or peptide, which nucleic acid is in the form of a replication-defective alphavirus replicon particle prepared using a packaging cell line.
2. The nucleic acid molecule of claim 1 wherein the first polypeptide is one that acts by promoting: (a) processing ofthe linked antigenic polypeptide via the MHC class I pathway or targeting of a cellular compartment that increases said processing;
(b) development, accumulation or activity of antigen presenting cells or targeting of antigen to compartments of said antigen presenting cells leading to enhanced antigen presentation; (c) intercellular transport and spreading ofthe antigen; or
(d) any combination of (a)-(c).
3. The nucleic acid molecule of claim 1 or 2 wherein the first polypeptide is: (a) a mycobacterial HSP70 polypeptide, the C-terminal domain thereof, or a functional homologue or derivative of said polypeptide or domain; (b) a viral intercellular spreading protein selected from the group of heφes simplex virus- 1 VP22 protein, Marek's disease virus VP22 protein or a functional homologue or derivative thereof;
(c) an endoplasmic reticulum chaperone polypeptide selected from the group of caheticulin, ER60, GRP94, gp96, or a functional homologue or derivative thereof (d) a cytoplasmic translocation polypeptide domains of a pathogen toxin selected from the group of domain II of Pseudomonas exotoxin ETA (ETAdH) or a functional homologue or derivative thereof;
(e) a polypeptide that targets the centrosome compartment of a cell selected from γ- tubulin or a functional homologue or derivative thereof; or
(f) a polypeptide that stimulates dendritic cell processors or activates dendritic cell activity selected from the group of GM-CSF, Flt3-ligand extracellular domain, or a functional homologue or derivative thereof
4. nucleic acid molecule of any of claims 1-3 wherein the first polypeptide is selected from the group consisting of Mycobacterium tuberculosis HSP70, the HSP70 C- tenninal domain, HSV-1 VP22, MDV VP22, caheticulin, Pseudomonas ETAdE, GM-CSF, Flt-3 ligand extracellular domain or γ-tubulin.
5. The nucleic acid molecule of claim wherein the first polypeptide is a transport polypeptide comprising SEQ ED NO: 5 or 7 or an active fraagment thereof.
6. The nucleic acid molecule of any of claims 1 -4 wherein the antigenic polypeptide comprises an epitope that binds to, and is presented on the cell surface by, an MHC class I protein.
7. The nucleic acid molecule of claim 5, wherein said epitope is between about 8 and about 11 amino acid residues in length.
8. The nucleic acid molecule of any of claims 1-8 wherein the antigen is one which is present on, or cross-reactive with an epitope of, a pathogenic organism, cell, or virus.
9. The nucleic acid molecule of claim 8, wherein the virus is a human papilloma virus.
10. The nucleic acid molecule of claim 9, wherein the antigen is the E7 polypeptide of HPV-16 or an antigenic fragment thereof.
11. The nucleic acid molecule of claim 8, wherein the pathogenic organism is a bacterium.
12. The nucleic acid molecule of claim 8, wherein the pathogenic cell is a tumor cell.
13. The nucleic acid molecule of claim 12, wherein the antigen is a tumor-specific or tumor-associated antigen.
14. The nucleic acid molecule of claim 13, wherein the antigen comprises a peptide ofthe HER-2/neu protein.
15. The nucleic acid molecule of any of claims 1-14 operatively linked to a promoter.
16. The nucleic acid molecule of claim 15 , wherein the promoter is one which is expressed in an antigen presenting cell (APC).
17. The nucleic acid molecule of claim 16, wherein the APC is a dendritic cell.
18. The nucleic acid molecule of any of claims 1-17 wherein the alphavirus is Sindbis virus, Semliki forest virus or Venezuelan equine encephalitis virus.
19. The nucleic acid molecule of claim 18 wherein the alphavirus is Sindbis virus.
20. The nucleic acid molecule of claim 19 wherein the Sindbis virus replicon is SINrep5.
21. The nucleic acid molecule of any of claims 1 -20 wherein the packaging cell line is one in which genes encoding capsid and envelope glycoproteins of said alphavirus are separated in distinct cassettes to minimize formation of replication competent virus during replicon production.
22. The nucleic acid molecule of any of claims 18-21 wherein the packaging cell line is 987dlsplit #24.
23. An expression vector comprising the nucleic acid molecule of any of claims 1 -22 operatively linked to
(a) a promoter; and
(b) optionally, additional regulatory sequences that regulate expression of said nucleic acid in a eukaryotic cell.
24. A cell which has been modified to comprise the nucleic acid or expression vector of any of claims 1-23.
25. The cell of claim 31 which expresses said nucleic acid molecule.
26. The cell of claim 24 or 25 which is an APC.
27. The cell of claim 26, wherein the APC is a dendritic cell, a keratinocyte, a macrophage, a monocyte, a B lymphocyte, a microglial cell, an astrocyte, or an activated endothelial cell.
28. A pharmaceutical composition capable of inducing or enhancing an antigen- specific immune response, comprising:
(a) pharmaceutically and immunologically acceptable excipient in combination with;
(b) a composition selected from the group consisting of:
(i) the nucleic acid molecule or expression vector of any of claims 1 -23 ; (ii) the cell of any of claims 24-27
(iii) any combination of (i) and (ii).
29. A method of inducing or enhancing an antigen specific immune response in a subject comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim 28, thereby inducing or enhancing said response.
30. The method of claim 28 or 29, wherein the response is mediated at least in part by
CD8+ cytotoxic T lymphocytes (CTL).
31. The method of claim 28 or 29, wherein the response is mediated at least in part by antibodies.
32. A method of inducing or enhancing an antigen specific immune response in cells or in a subject comprising administering to said cells or to said subject an effective amount of the pharmaceutical composition of claim 28, thereby inducing or enhancing said response.
33. The method of claim 32, wherein the composition is administered ex vivo to said cells.
34. The method of claim 32 wherein said cells comprise APCs.
35. The method of claim 54, wherein said APCs are dendritic cells.
36. The method of claim 34 or 35, wherein the APCs are human APCs.
37. The method of any of claims 34-36, wherein the APCs are isolated from a living subject.
38. The method of any of claims 32-37, further comprising a step of administering the ex vzvo-treated cells to a histocompatible subject.
39. The method of any of claims 29-38 wherein said cells are human cells and said subject is a human.
40. The method of any of claims 29-32, 38 and 39 wherein said administering is by a intramuscular, intradermal, or subcutaneous route.
41. The method of any of claims 29-32 and 38-40 wherein the administering is infratumoral or peritumoral.
42. A method of increasing the numbers or lytic activity of CD8+ CTLs specific for a selected antigen in a subject, comprising administering to said subject an effective amount of a composition selected from the group consisting of:
(a) the nucleic acid molecule or expression vector of any of claims 1 -23 ; (b) the cell of any of claims 24-27
(c) any combination of (a) and (b). wherein
(i) said nucleic acid molecule, said expression vector or said cell comprises said antigen, (i) said antigen comprises an epitope that binds to, and is presented on the cell surface by, MHC class I proteins, thereby increasing the numbers or activity of said CTLs.
43. A method of inhibiting growth or preventing re-growth of a tumor in a subj ect, comprising administering to said subject an effective amount of a composition selected from the group consisting of:
(a) the nucleic acid molecule or expression vector of any of claims 1-23; (b) the cell of any of claims 24-27; and
(c) any combination of (a) and (b). wherein
(i) said nucleic acid molecule, said expression vector or said cell comprises said antigen, (ii) said antigen comprises one or more tumor-associated or tumor-specific epitopes present on said tumor in said subject thereby inhibiting said growth or preventing said re-growth.
44. The method of claim 43, wherein said administering is intratumoral or periturnoral.
PCT/US2002/008033 2001-03-16 2002-03-18 A replication-defective alphavirus vaccine linking antigen with an immunogenicity-potentiating polypeptide and a method of delivery the same WO2002074920A2 (en)

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