Scholm, Jeffrey (Potomac, MD, US)
Hodge, James (Gaithersburg, MD, US)
Panicali, Dennis (Acton, MA, US)

Plaque It!

10/406317

01/29/2004

04/04/2003

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435/456

435/320.100, 424/93.600, 536/23.100, 424/93.400, 536/23.700, 424/184.100, 536/23.720

(IPC1-7): C12N015/70; C12N015/86; C07H021/04; A61K039/38; A01N063/00; C12N015/74; C12N015/63; C12N015/09; C07H021/02

HELLER EHRMAN WHITE & MCAULIFFE LLP (1666 K STREET,NW, WASHINGTON, DC, 20006, US)

We claim:

1. A recombinant vector comprising foreign nucleic acid sequences encoding multiple costimulatory molecules or functional portions thereof.

2. The recombinant vector according to claim 1 wherein the nucleic acid sequences encode at least three costimulatory molecules.

3. The recombinant vector according to claim 1 wherein the nucleic acid sequence encoding each costimulatory molecule is derived from a mammalian source.

4. The recombinant vector according to claim 1 wherein the multiple costimulatory molecules are selected from the group consisting of B7-1, B7-2, ICAM-1, LFA-3, 4-1BBL, CD59, CD40, CD70, OX-40L, VCAM-1 and mammalian homologs thereof.

5. The recombinant vector according to claim 5 wherein the multiple costimulatory molecules are B7-1, ICAM-1 and LFA-3.

6. The recombinant vector according to claim 1 further comprising a foreign nucleic acid sequence encoding at least one cytokine, chemokine, Flt-3L, or combination thereof.

7. The recombinant vector according to claim 1 further comprising a multiplicity of promoters.

8. The recombinant vector according to claim 7 wherein the promoters are derived from a eukaryotic source, prokaryotic source, or viral source.

9. The recombinant vector according to claim 7 wherein the promoters are selected from the group consisting of an SV40 early promoter, RSV promoter, adenovirus major late promoter, human CMV immediate early I promoter, poxvirus promoter, 30K promoter, I3 promoter, sE/L promoter, 7.5K promoter, 40K promoter, and C1 promoter.

10. The recombinant vector according to claim 1 wherein the recombinant vector is selected from the group consisting of a bacteria, virus, and nucleic acid-based vector.

11. The recombinant vector according to claim 1 wherein the recombinant vector is selected from the group consisting of poxvirus, adenovirus, Herpes virus, alphavirus, retrovirus, picornavirus, and iridovirus.

12. The recombinant vector according to claim 11 wherein the recombinant vector is a recombinant poxvirus.

13. The recombinant vector according to claim 12 wherein the recombinant poxvirus is a replicating virus or a non-replicating virus.

14. The recombinant vector according to claim 12 wherein the recombinant poxvirus is orthopox, avipox, capripox or suipox.

15. The recombinant vector according to claim 14 wherein the avipox is fowlpox, canary pox or derivatives thereof.

16. The recombinant vector according to claim 14 wherein the orthopox is vaccinia, vaccinia-Copenhagen strain, vaccinia-Wyeth strain, NYVAC, vaccinia-MVA strain, raccoon pox or rabbit pox.

17. The recombinant vector according to claim 1 further comprising a foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof.

18. The recombinant vector according to claim 17 wherein the target antigen has an amino acid sequence selected from the group consisting of SEQ ID NO: 2 through SEQ ID NO: 40.

19. The recombinant vector according to claim 17 wherein the target antigen is selected from the group consisting of a tumor specific antigen, tumor associated antigen, tissue-specific antigen, bacterial antigen, viral antigen, yeast antigen, fungal antigen, protozoan antigen, and parasite antigen and mitogen.

20. The recombinant vector according to claim 18 wherein the bacterial antigen is derived from a bacterium selected from the group consisting of Chiamydia, Mycobacteria. Legionella, Meningiococcus, Group A Streptococcus, Hemophilus influenzae, Salmonella, and Listeria.

21. The recombinant vector according to claim 18 wherein the viral antigen is derived from a virus selected from the group consisting of Lentivirus, Herpes virus, Hepatitis virus, Orthomyxovirus and Papillomavirus.

22. The recombinant vector according to claim 21 wherein the Lentivirus is HIV-1 or HIV-2.

23. The recombinant vector according to claim 21 wherein the Herpes virus is HSV or CMV.

24. The recombinant vector according to claim 21 wherein the Hepatitis virus is Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D or Hepatitis E.

25. The recombinant vector according to claim 21 wherein the orthomyxovirus is influenza virus.

26. The recombinant vector according to claim 18 wherein the tumor associated antigen, tumor specific antigen or tissue-specific antigen is selected from the group consisting of CEA, MART-1, MAGE-1, MAGE-3, GP-100, MUC-1, MUC-2, pointed mutated ras oncogene, normal or point mutated p53, overexpressed p53, CA-125, PSA, C-erb/B2, BRCA I, BRCA II, PSMA, tyrosinase, TRP-1, TRP-2, NY-ESO-1, TAG72, KSA, HER-2/neu, bcr-abl, pax3-fkhr, ews-fli-1, modified TAAs, splice variants of TAAs, functional epitopes and epitope agonists thereof.

27. The recombinant vector according to claim 26 wherein the antigen is CEA (6D) having an amino acid aspartic acid at amino acid position 576.

28. The recombinant vector according to claim 26 wherein the antigen is PSA and PSMA.

29. The recombinant vector according to claim 26 wherein the antigen is MUC-1 encoded by a truncated MUC-1 gene consisting of a signal sequence, ten copies of a tandem repeat sequence, and a 3′ coding sequence.

30. The recombinant vector according to claim 18 wherein the yeast or fungal antigen is derived from a yeast or fungus selected from the group consisting of Aspergillus, Nocardia, Histoplasmosis, Candida, and Cryptosporidia.

31. The recombinant vector according to claim 18 wherein the parasitic antigen is derived from a Plasmodium species, Toxoplasma gondii, Pneumocystis carinii, Trypasosoma species, or Leishmania species.

32. The recombinant vector according to claim 1 wherein the vector further comprises a selectable marker.

33. The recombinant vector according to claim 32 wherein the selectable marker is selected from the group consisting of lacZ gene, thymidine kinase, gpt, GUS and a vaccinia K1L host range gene.

34. A pharmaceutical composition comprising at least one recombinant vector according to any of claims 1-33 and a pharmaceutically acceptable carrier.

35. A pharmaceutical composition comprising at least one recombinant vector according to any of claims 1-16, a second recombinant vector comprising at least one nucleic acid sequence encoding at least one target antigen or immunological epitope thereof and a pharmaceutically acceptable carrier.

36. A pharmaceutical composition according to claim 34 or 35 further comprising a cytokine, chemokine or Flt-3L.

37. A host cell infected, transfected or induced with the recombinant vector according to any of claims 1-33.

38. The host cell infected, transfected or induced with the recombinant vector according to claims 1-16 and infected, transfected or induced with a second recombinant vector comprising at least one foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof.

39. The host cell according to claim 38 wherein the host cell is an antigen presenting cell or precursor thereof, a premalignant cell, a hyperplastic cell or tumor cell.

40. The host cell according to claim 39 wherein the antigen presenting cell is a dendritic cell or precursor thereof, a monocyte, macrophage, B-cell, fibroblast or muscle cell.

41. The host cell according to claim 39 wherein the antigen presenting cell is derived from bone marrow, spleen, skin, peripheral blood, tumor, lymph node, or muscle.

42. The host cell according to claim 38 wherein the host cell is an antigen presenting cell or precursor thereof, a premalignant cell, a hyperplastic cell or tumor cell.

43. The host cell according to claim 42 wherein the antigen presenting cell is a dendritic cell, precursor or derivative thereof, a monocyte, macrophage, B-cell, fibroblast or muscle cell.

44. The host cell according to claim 43 wherein the derivative is a TNF α-treated dendritic cell, a CD40-treated dendritic cell, or a subpopulation of adherent cells.

45. A dendritic cell or precursor thereof comprising a foreign nucleic acid sequence encoding multiple costimulatory molecules.

46. A tumor cell or precursor thereof comprising a foreign nucleic acid sequence encoding multiple costimulatory molecules.

47. The cell according to claims 45 or 46 wherein the cell comprises a foreign nucleic acid sequence encoding at least three costimulatory molecules.

48. The cell according to claims 45 or 46 wherein the multiple costimulatory molecules are selected from the group consisting of B7-1, B7-2, ICAM-1, LFA-3, 4-1BBL, CD59, CD40, CD70, OX-40L, VCAM-1, mammalian homologs thereof and combinations thereof.

49. The cells according to claims 45 or 46 wherein the multiple costimulatory molecules are at least B7-1, ICAM-1 and LFA-3.

50. The cells according to claims 45 or 46 further comprising a foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof.

51. The cells according to claim 50 wherein the foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof is provided by a recombinant vector, RNA or DNA from a tumor cell lysate, or by fusion with a tumor cell comprising said sequence.

52. The cells according to claim 50 wherein the target antigen or immunological epitope thereof is selected from the group consisting of a tumor specific antigen, tumor associated antigen, tissue-specific antigen, bacterial antigen, viral antigen, yeast antigen, fungal antigen, protozoan antigen, parasite antigen and mitogen.

53. A pharmaceutical composition comprising the cells according to claims 45-52 and optionally an exogenous source of target antigen or immunological epitope thereof.

54. A recombinant poxvirus having integrated into a viral genome foreign DNA encoding multiple costimulatory molecules produced by a process comprising: allowing a plasmid vector comprising the foreign DNA encoding multiple costimulatory molecules to undergo recombination with a parental poxvirus genome to produce a recombinant poxvirus having inserted into its genome the foreign DNA.

55. A recombinant poxvirus having integrated into a viral genome foreign DNA encoding LFA-3, ICAM-1 and at least one B7 molecule produced by a process comprising: allowing a plasmid vector comprising the foreign DNA encoding LFA-3, ICAM-1 and at least one B7 molecule to undergo recombination with a parental poxvirus genome to produce a recombinant poxvirus having inserted into its genome the foreign DNA.

56. The recombinant poxvirus according to claim 54 or 55, wherein the genome further comprises a multiplicity of poxvirus promoters capable of controlling expression of the foreign DNA.

57. The recombinant poxvirus according to claims 54 or 55 further comprising a foreign gene encoding at least one target antigen or immunological epitope thereof.

58. A pharmaceutical composition comprising a recombinant poxvirus according to any of claims 54-57 and a pharmaceutically acceptable carrier.

59. A pharmaceutical composition comprising a recombinant poxvirus according to any of claims 54-57 further comprising a second recombinant poxvirus comprising at least one foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof.

60. A host cell infected with the recombinant poxvirus according to claims 54-57.

61. The host cell according to claim 60, wherein the cell is a progenitor antigen presenting cell, an antigen presenting cell or an engineered antigen presenting cell.

62. The host cell according to claim 61, wherein the cell is a progenitor dendritic cell, dendritic cell, monocyte, macrophage, B-cell, fibroblast or muscle cell.

63. The host cell according to claim 60, wherein the cell is a hyperplastic cell, premalignant cell or a tumor cell.

64. A plasmid vector comprising nucleic acid sequences encoding multiple costimulatory molecules or functional portions thereof.

65. The plasmid vector according to claim 64 further comprising a gene encoding a selectable marker.

66. The plasmid vector according to claim 64 wherein the costimulatory molecules are human derived, non-human primate derived or murine derived.

67. The plasmid vector according to claim 64 further comprising a foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof.

68. The plasmid vector according to claim 67 wherein the target antigen is selected from the group consisting of a tumor specific antigen, tumor associated antigen, tissue specific antigen, bacterial antigen, viral antigen, yeast antigen, fungal antigen, protozoan antigen, parasite antigen, and mitogen.

69. The plasmid vector according to claim 68 wherein the target antigen is carcinoembryonic antigen (CEA) or immunological epitope thereof.

70. A plasmid vector for recombination with a poxvirus designed to produce a recombinant poxvirus capable of expressing foreign nucleic acid sequences encoding three costimulatory molecules, LFA-3, ICAM-1 and at least one B7 molecule which comprises (a) a multiplicity of poxviral promoters, (b) the foreign nucleic acid sequences encoding LFA-3, ICAM-1 and at least one B7 molecule, (c) DNA sequences flanking the construct of elements (a) and (b), the flanking sequences of both the 5′ and 3′ ends being homologous to a region of a parental poxvirus genome where elements (a) and (b) are to be inserted.

71. The plasmid vector according to claim 64 designated pT5064 deposited with the ATCC under Accession No. 203482

72. The plasmid vector according to claim 67, designated pT5049 deposited with the ATCC under Accession No. 203481.

73. A kit for use in making a recombinant poxvirus comprising a plasmid vector according to any of claims 64-72 and optionally a parental poxvirus.

74. A method of making a recombinant poxvirus comprising allowing the plasmid vector according to claim 64-72 to undergo recombination with a parental poxvirus genome to produce a recombinant poxvirus having inserted into its genome the foreign DNA and a multiplicity of poxvirus promoters capable of controlling the expression of the foreign DNA.

75. A method of enhancing an immune response in an individual comprising administration of a recombinant vector according to claims 1-33 in an amount sufficient to enhance the immune response.

76. The method according to claim 75 wherein a route of administration is intravenous, subcutaneous, intralymphatic, intratumoral, intradermal, intramuscular, intraperitoneal, intrarectal, intravaginal, intranasal, oral, via bladder instillation, or via scarification.

77. The method according to claim 75 wherein the enhanced immune response is a cell mediated or humoral response.

78. The method according to claim 75, wherein the enhancement is of CD4+ T cell proliferation, CD8+ T cell proliferation, or combination thereof.

79. The method according to claim 75, wherein the enhancement is of CD4+ T cell function, CD8+ T cell function or combination thereof.

80. The method according to claim 75, wherein the enhancement is in IL-2 production, IFN-γ production or combination thereof.

81. The method according to claim 75, wherein the enhancement is of antigen presenting cell proliferation, function or combination thereof.

82. A method of enhancing an antigen-specific T-cell response in an individual to a target antigen or immunological epitope thereof comprising administering a recombinant poxvirus comprising a foreign nucleic acid sequence encoding at least one B7 molecule, a foreign nucleic acid sequence encoding ICAM-1, and a nucleic acid sequence encoding LFA-3, and optionally a nucleic acid sequence encoding a target antigen or immunological epitope thereof, each nucleic acid sequence expressed in an infected cell in the individual in an amount effective to enhance at least one T-cell response, wherein the enhancement is greater than the enhancement obtained using a single costimulatory molecule or two costimulatory molecules.

83. The method according to claim 82, wherein the enhancement is of CD4+ T cell proliferation, CD8+ T cell proliferation, or combination thereof.

84. The method according to claim 82, wherein the enhancement is in IL-2 production, IFN-γ production or combination thereof.

85. The method according to claim 82, wherein the enhancement is of antigen-specific cytotoxicity.

86. The method according to claim 82 wherein the infected cell is an antigen presenting cell.

87. The method according to claim 86, wherein the cell is a dendritic cell, precursor thereof, monocyte, macrophage, B-cell fibroblast or muscle cell.

88. The method according to claim 82, wherein the infected cell is a tumor cell or precursor thereof.

89. A method of treatment or prevention of disease in an individual comprising: (a) activating a T lymphocyte by exposing the T lymphocyte in vitro to a cell according to claim 37 alone or in combination with a target antigen or immunological epitope thereof; (b) administering the activated T lymphocyte to an individual alone, or in combination with the target antigen in an amount sufficient to enhance an immune response.

90. The method according to claim 89 wherein the T lymphocytes are autologous with the individual.

91. The method according to claim 89, further comprising the administration of a cytokine, chemokine, flt-31 or combination thereof.

92. The method according to claim 89 wherein the immune response is against the target antigen selected from the group consisting of a tumor specific antigen, tumor associated antigen, tissue-specific antigen, bacterial antigen, viral antigen, yeast antigen, fungal antigen, protozoan antigen, and parasite antigen.

93. The method according to claim 89 wherein the immune response prevents or treats a disease caused by a cell or organism selected from the group consisting of viruses, bacteria, protozoans, parasites, premalignant cells and tumor cells.

94. A method of enhancing an immune response in an individual comprising administration of a cell according to any of claims 45-52 in an amount effective to enhance an immune response.

95. A method of enhancing an immune response in an individual comprising administration of a tumor cell, or precursors thereof according to claim 46 in an amount effective to enhance an immune response.

96. The method according to claims 94 or 95 wherein the cells are autologous, syngeneic or allogeneic with the individual.

97. The method according to claims 94 or 95 wherein the cells have been pulsed with a target antigen or epitope thereof.

98. The method according to claims 94 or 95 further comprising the administration of a target cell, target antigen or immunological epitope thereof.

99. The method according to claim 94 further comprising the administration of activated, target antigen specific lymphocytes.

100. A method for making a progenitor dendritic cell or dendritic cell that overexpresses multiple costimulatory molecules, said method comprising: (a) providing the cell with a recombinant vector comprising foreign genes encoding multiple costimulatory molecules for a period of time sufficient to cause overexpression of the multiple costimulatory molecules by the cells.

101. The method according to claim 100 wherein the cells are isolated from bone marrow or peripheral blood mononuclear cells.

102. The method according to claim 100 wherein the recombinant vector further comprises a foreign gene encoding at least one target antigen or immunological epitope thereof.

103. The method according to claim 100 further comprising (b) providing the cell with a second recombinant vector comprising a foreign gene encoding at least one target antigen or immunological epitope thereof.

104. An in vitro method of assessing efficacy of a vaccine against a target antigen comprising: (a) obtaining lymphocytes from an individual previously vaccinated with a target antigen or epitope thereof, (b) determining the number and function of target-antigen specific lymphocytes in the presence of antigen presenting cells according to claim 39, an increase in number, function or combination thereof of target-antigen specific lymphocytes being indicative of efficacy of the vaccine.

105. A method of screening for novel immunogenic peptides from a multiplicity of peptides comprising: (a) pulsing antigen presenting cells infected with a recombinant vector encoding multiple costimulatory molecules with a multiplicity of peptides to form peptide-pulsed antigen presenting cells; (b) measuring lymphoid immunoreactivity in the presence of the peptide-pulsed antigen presenting cells, wherein enhanced immunoreactivity is indicative of an immunogenic peptide on the peptide-pulsed antigen presenting cell.

106. The method according to claim 105, wherein the source of the multiplicity of peptides is a combinatorial peptide library.

FIELD OF THE INVENTION

[0001] The present invention relates to a recombinant vector comprising foreign genes encoding multiple costimulatory molecules and optionally a foreign gene encoding a target antigen. The invention further relates to a recombinant virus comprising foreign genes encoding at least three costimulatory molecules and optionally a foreign gene encoding at least one target antigen or immunological epitope thereof. More specifically, the present invention relates to a recombinant poxvirus comprising foreign genes encoding at least the costimulatory molecules: one molecule from the B7 family, LFA-3 and ICAM-1 and optionally a foreign gene encoding at least one target antigen or immunological epitope thereof and uses thereof as immunogens and vaccines. The invention further relates to antigen presenting cells transfected, infected or transduced by a recombinant vector comprising foreign genes encoding multiple costimulatory molecules and optionally a foreign gene encoding at least one target antigen or immunological epitope thereof.

BACKGROUND OF THE INVENTION

[0002] The extent of the primary response of T cells, which involves their activation, expansion, and differentiation, is paramount to a successful immune response to an antigen. The initiation of an immune response requires at least two signals for the activation of naive T cells by antigen presenting cells (APC) (1-5). The first signal is antigen specific, delivered through the T-cell receptor via the peptide/major histocompatibility complex, and causes the T cell to enter the cell cycle. The second, or “costimulatory,” signal is required for cytokine production and proliferation. At least three distinct molecules normally found on the surface of professional APC have been proposed as capable of providing the second signal critical for T-cell activation: B7.1 (CD80), Intercellular adhesion molecule-1 (ICAM-1; CD54), and Leukocyte function-associated antigen-3 (LFA-3; human CD58; murine CD48) (2, 6, 7). The T-cell ligands for these costimulatory molecules are distinct. B7-1 interacts with the CD28 and CTLA4 molecules, ICAM-1 interacts with the CD11a/CD18 (LFA-1/2 integrin) complex, and LFA-3 interacts with the CD2 (LFA-2) molecules. It is not known whether these costimulatory molecules perform equivalent functions or carry out specialized functions at specific stages of an induced immune response (2). These molecules have been individually shown to costimulate T-cell proliferation in vitro (6). However, because they may be expressed simultaneously on APC, it has been difficult to examine relative potencies of individual costimulatory molecules during the induction of T-cell proliferation (2).

[0003] As it has been proposed that both antigen and costimulatory molecules must be expressed in proximity to each other to properly co-engage the T cell and costimulatory receptors (8, 9), the admixture of several recombinant viruses could be utilized to explore the potential cooperation of costimulatory molecules. The disadvantage of this approach, however, is that the admixture of three or more viruses has a statistically diminished probability of co-infecting the same cell, thereby making a multi-gene construct much more desirable for use with multiple costimulatory molecule genes.

[0004] WO 91/02805, published Mar. 7, 1991, discloses a recombinant retrovirus vector construct which directs the expression of a target antigen, an MHC protein and other proteins involved in immune interactions which are missing or under-represented in a target cell.

[0005] Akagi, et al. 1997, J. Immunotherapy Vol. 20 (1):38-47 disclose an admixture of a recombinant vaccinia virus containing a modified MUC1 gene (rV-MUC1), and a recombinant vaccinia virus containing the gene for the murine costimulatory molecule B7 (rV-B7).

[0006] Cavallo, P. et al. 1995, Eur. J. Immunol., 25:1154-1162 disclose that transfection of B7-1 cDNA into three ICAM-1+ tumor cell lines is sufficient to induce rejection in syngeneic mice.

[0007] Chen, L. et al. 1994, J. Exp. Med., 179:523-532 disclose a recombinant retrovirus vector containing cDNA for murine B7 and the use of the vector in transducing various tumors.

[0008] Damle, N. K. et al 1992, J. Immunol Vol 148 (No. 7): 1985-1992 disclose the use of an antigen presenting cell (APC)-independent in vitro culture system consisting of immobilized combinations of monoclonal antibodies directed at the TCR/CD3 complex and soluble Ig chimeras (RG) of four distinct APC-associated costimulatory molecules to compare the abilities of these molecules to costimulate T cell proliferation.

[0009] Dubey, C. et al 1995, J Immunol 155: 45-57 disclose a study of the relative contribution of ICAM-1: LFA-1 and B7: CD28/CTLA-4 costimulatory pathways in naïve T cell activation, using either anti-CD28 antibody or fibroblast cell lines transfected with I-Ek, which express either no costimulatory molecules, ICAM-1 alone, B7-1 alone, or ICAM-1 and B7-1 together.

[0010] Fenton, R. G. et al, 1998 Vol. 21, No. 2, pp 95-108, disclose transfection of the costimulatory molecule B7-1 gene into three HLA-A2-expressing human melanoma cell lines, and their capacity to stimulate primary human T cells. The three melanoma lines also expressed detectable levels of the costimulatory molecules ICAM-1 (CD54) and LFA-3 (CD58).

[0011] Gjorloff Wingren, A. et al 1995, Critical Reviews in Immunol 15 (3 & 4): 235-253 disclose that with co-transfection of HLA-DR, B7 and LFA-3 into CHO cells, these molecules cooperate in activation of both naïve and memory T cells and allow responses at picomolar concentrations of the antigen, staphylococcal enterotoxin B (SEB).

[0012] Goldbach-Mansky, R. et al 1992, International Immunol. 4(No. 12): 1351-1360 disclose that CD4+ T cells respond to staphylococcal enterotoxin B (SEB) in the presence of the LFA-3, ICAM-1 and B7 positive erythroleukemic cell line K562, murine L cells, and human B7 transfected L cells.

[0013] Hodge, J. W. et al 1994, Cancer Research 54:5552-5555 disclose the construction and characterization of recombinant vaccinia viruses containing the murine B7.1 and B7.2 genes.

[0014] Hodge, J. W. et al 1995, Cancer Research 55: 3598-3603 Cancer Research 55:3598-3603 disclose an admixture of recombinant vaccinia murine B7.1 (rV-B7) plus recombinant vaccinia expressing the human carcinoembryonic antigen gene (rV-CEA) and the use of this admixture for anti-tumor activity.

[0015] Parra, et al 1993, Scand J. Immunol 38: 508-514, Parra, E. et al 1994, J. Immunol 153: 2479-2487, and Parra, et al. 1997, J. Immunol., 458:637-642 disclose CHO cells transfected with the human HLA-DR4 molecule (CHO-DR4); HLA-DR4 and B7 (CHO-DR4/B7), HLA-DR4 and LFA-3 (CHO-DR4/LFA3); HLA-DR4 and ICAM-1 (CHO-DR4/ICAM-1); or DR4, B7 and LFA-3 (CHO-DR4/B7/LFA-3) genes.

[0016] Thomas, R. et al. 1993 J. Immunol. 151:6840-6852 disclose that freshly obtained dendritic cells (DC) express similar densities of HLA-DR and the accessory molecules LFA-3, ICAM-1 and B7 as monocytes.

[0017] Uzendoski, K et al. May 1997, Human Gene Therapy 8:851-860 disclose the construction, characterization and immunological consequences of a recombinant vaccinia virus expressing the murine costimulatory molecule, ICAM-1.

[0018] WO 96/10419, published Apr. 11, 1996, of PCT/US95/12624 discloses subject matter relating to a single recombinant viral vector which has incorporated one or more genes or portion thereof encoding an immunostimulatory molecule and one or more genes or portion thereof encoding an antigen of a disease state.

[0019] Robinson et al U.S. Pat. No. 5,738,852 discloses a retroviral vector containing a polynucleotide sequence encoding a target antigen of an infectious agent and a polynucleotide sequence encoding a B7 costimulatory molecule.

[0020] The present invention is a vector containing foreign DNA encoding at least three costimulatory molecules, alone or in combination with foreign DNA encoding at least one target antigen or immunological epitope thereof which allows functional expression of each foreign DNA in an infected host cell.

SUMMARY OF THE INVENTION

[0021] The present invention provides a recombinant vector comprising foreign or exogenous genes or portions thereof encoding multiple costimulatory molecules. Genes or functional portions thereof encoding costimulatory molecules having utility in the present invention include but are not limited to a B7 family member, ICAM-1, LFA-3, 4-1BBL, CD59, CD40, CD70, VCAM-1, OX-40L, functional portions and homologs thereof. The vector of the invention may further provide a foreign gene encoding at least one target antigen or immunological epitope thereof in combination with the foreign genes encoding multiple costimulatory molecules. The foreign gene encoding at least one target antigen or immunological epitope thereof may be derived from cells, tissues or organisms such as viruses, bacteria, protozoans, parasites, yeast, tumor cells, preneoplastic cells, hyperplastic cells, tissue specific cells, or synthetic antigens. The vector may further provide a foreign gene encoding at least one or a combination of cytokines, chemokines and flt-3L.

[0022] The recombinant vector for use in the present invention group consisting of bacterial vectors, virus vectors, nucleic acid based vectors and the like. The recombinant virus vectors include but are not limited to poxvirus, adenovirus, herpes virus, alphavirus, retrovirus, picornavirus, iridovirus and the like. The poxvirus include but are not limited to the orthopox, avipox, suipox and capripox.

[0023] The present invention provides a recombinant virus comprising foreign genes or portions thereof encoding multiple costimulatory molecules for providing an enhanced immune response to a target cell, target antigen or immunological epitope thereof which is greater than a response provided by a recombinant virus comprising a foreign gene or genes encoding single or double costimulatory molecules. The recombinant virus of the invention may further provide a foreign gene encoding at least one target antigen or immunological epitope thereof in combination with the foreign genes encoding multiple costimulatory molecules. The recombinant virus may further provide a foreign gene encoding other classes of immunostimulatory molecules such as cytokines including but not limited to IL-2, IL-12, GM-CSF and the like, chemokines such as MIP1, MIP2, RANTES and the like, and Flt-3L which stimulates DC proliferation.

[0024] The present invention further provides a recombinant poxvirus comprising foreign genes or portions thereof encoding multiple costimulatory molecules for providing an enhanced immune response to a target cell, target antigen or immunological epitope thereof which is greater than a response provided by a recombinant poxvirus comprising a foreign gene or genes encoding single or double costimulatory molecules. The recombinant poxvirus of the invention may further provide a foreign gene encoding at least one target antigen or immunological epitope thereof in combination with the foreign genes encoding multiple costimulatory molecules.

[0025] The present invention also provides a recombinant poxvirus comprising a nucleic acid sequence encoding and expressing multiple costimulatory molecules, said nucleic acid sequence comprising a nucleic acid sequence encoding at least one molecule from the B7 family of costimulatory molecules, a nucleic acid sequence encoding an ICAM-1 costimulatory molecules, and a nucleic acid sequence encoding an LFA-3 costimulatory molecule. The recombinant virus further provides a multiplicity of poxvirus promoters which regulate expression of each foreign gene.

[0026] The present invention provides a recombinant virus produced by allowing a plasmid vector comprising foreign DNA encoding multiple costimulatory molecules to undergo recombination with a parental virus genome to produce a recombinant virus having inserted into its genome the foreign DNA. The recombinant virus produced by recombination may further contain a foreign gene encoding at least one target antigen or immunological epitope thereof provided by the plasmid vector.

[0027] The present invention also provides a recombinant poxvirus produced by allowing a plasmid vector comprising foreign DNA encoding the costimulatory molecule, LFA-3, ICAM-1 and at least one molecule from the B7 family to undergo recombination with a parental poxvirus genome to produce a recombinant poxvirus having inserted into its genome the foreign DNA and a multiplicity of poxvirus promoters capable of controlling the expression of the foreign DNA. The recombinant poxvirus produced by recombination may further contain a foreign gene encoding at least one target antigen or immunological epitope thereof provided by the plasmid vector.

[0028] An object of the invention is to provide an immunogen for enhancement of immune responses against target cells, target antigens or immunological epitopes thereof comprising a recombinant vector having foreign nucleic acid sequences encoding multiple costimulatory molecules. The vector may further comprise a foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof.

[0029] Another object of the invention is to provide an immunogen for enhancement of immune responses against target cells, target antigens or immunological epitopes thereof comprising a recombinant virus vector having foreign nucleic acid sequences encoding three or more costimulatory molecules. The recombinant virus vector may further comprise a foreign nucleic acid sequence encoding at least one or more target antigens or immunological epitopes thereof.

[0030] Yet another object of the invention is to provide an immunogen for enhancement of immune responses against target cells, target antigens or immunological epitopes thereof comprising a recombinant poxvirus vector comprising a foreign nucleic acid sequence encoding the costimulatory molecules LFA-3, ICAM-1 and at least one molecule from the B7 family and a foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof.

[0031] The vector of the present invention provides a vaccine for eliciting and enhancing immune responses against target cells, target antigens or epitopes thereof for protection and/or treatment of disease states. The vector vaccine comprises foreign nucleic acid sequences encoding multiple costimulatory molecules. The vector vaccine may also comprise foreign nucleic acid sequences encoding one or more target antigens or immunological epitopes thereof for producing a monovalent or polyvalent vaccine against a disease.

[0032] The present invention provides pharmaceutical compositions comprising a vector having foreign nucleic acid sequences encoding multiple costimulatory molecules and a pharmaceutically acceptable carrier. The vector may further comprise a foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof. The vector may additionally comprise a nucleic sequence encoding a cytokine, chemokine, flt-3L, or combination thereof.

[0033] The present invention provides a pharmaceutical composition comprising a recombinant virus vector which comprises foreign or exogenous genes or functional portions thereof encoding three or more costimulatory molecules, a foreign gene encoding at least one target antigen or immunological epitope thereof, and a pharmaceutically acceptable carrier.

[0034] The present invention also provides pharmaceutical compositions comprising a recombinant poxvirus comprising foreign genes or portions thereof encoding multiple costimulatory molecules and a pharmaceutically acceptable carrier. The recombinant poxvirus may further comprise a foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof.

[0035] Another aspect of the invention is a pharmaceutical composition comprising a recombinant poxvirus comprising foreign genes or portions thereof encoding three or more costimulatory molecules, and may further comprise a foreign gene or portion thereof encoding at least one target antigen or immunological epitope thereof, and a pharmaceutically acceptable carrier or immunological epitope thereof.

[0036] The present invention also provides a pharmaceutical composition comprising a first vector comprising foreign genes or functional portions thereof encoding multiple costimulatory molecules and a second vector comprising foreign genes encoding at least one target antigen or immunological epitope thereof and a pharmaceutically acceptable carrier.

[0037] The present invention provides host cells infected, transfected or transduced with a first vector comprising foreign genes encoding multiple costimulatory molecules causing expression of the multiple costimulatory molecules in the host cells. The first vector or a second vector may further provide a foreign gene encoding at least one target antigen or immunological epitope thereof to the host cell.

[0038] The present invention provides antigen-presenting cells (APCs) or tumor cells infected, transfected or transduced with a first vector comprising foreign or exogenously provided genes encoding multiple costimulatory molecules causing expression or overexpression of the multiple costimulatory molecules. The first vector or a second vector may further provide a foreign gene encoding at least one target antigen or immunological epitope thereof to the host cell.

[0039] The present invention further provides host cells infected with a recombinant poxvirus causing expression of the multiple costimulatory molecules, and optionally causing expression of a target antigen or immunological epitope thereof.

[0040] Another aspect of the invention is a dendritic cell (DC) and precursor thereof infected, transfected or genetically engineered to overexpress genes encoding multiple exogenous costimulatory molecules. The DCs and precursors thereof may further be engineered to express foreign genes encoding at least one target antigen or immunological epitope thereof.

[0041] Yet another aspect of the invention is a DC and precursors thereof genetically engineered to overexpress genes encoding at least three exogenous costimulatory molecules. The DCs and precursor thereof may further be engineered to express foreign genes encoding at least one target antigen or immunological epitope thereof.

[0042] The present invention further provides a DC and precursors thereof genetically engineered to overexpress genes encoding at least one B7 molecule, ICAM-1 and LFA-3. The DCs and precursor thereof may further be engineered to express foreign genes encoding at least one target antigen or immunological epitope thereof.

[0043] The present invention provides methods and a plasmid vector for recombination with a parental virus designed to produce a recombinant virus capable of expressing foreign nucleic acid sequences encoding multiple costimulatory molecules comprising (a) a multiplicity of viral promoters, (b) the foreign nucleic acid sequences encoding the multiple costimulatory molecules, (c) DNA sequences flanking the constructs of elements (a) and (b), the flanking sequences at both the 5′ and 3′ ends being homologous to a region of a parental virus genome where elements (a) and (b) are to be inserted. The plasmid vector may further provide a foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof. The plasmid vector may also provide a gene encoding a selectable marker.

[0044] The present invention also provides methods and a plasmid vector for recombination with a parental poxvirus designed to produce a recombinant poxvirus capable of expressing foreign nucleic acid sequences encoding the costimulatory molecules LFA-3, ICAM-1 and at least one B7 molecule which comprises (a) a multiplicity of poxviral promoters, (b) the foreign nucleic acid sequences encoding the LFA-3, ICAM-1 and at least one B7 molecule, (c) DNA sequences flanking the construct of elements (a) and (b), the flanking sequences at both 5′ and 3′ ends being homologous to a region of a parental poxvirus genome where elements (a) and (b) are to be inserted. The plasmid vector may further provide a foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof. The plasmid vector may also provide a gene encoding a selectable marker.

[0045] One aspect of the invention is a method of enhancing immunological responses in a mammal to at least one target cell, target antigen or immunological epitope thereof comprising administration of a first vector comprising foreign nucleic acid sequences encoding multiple costimulatory molecules, each costimulatory molecule expressed in a cell in the mammal in an amount effective to enhance at least one immunological response in the mammal. Genes or functional portions thereof encoding costimulatory molecules having utility in the present invention include but are not limited to a B7 family member, ICAM-1, LFA-3, 4-1 BBL, CD59, CD40, CD70, VCAM-1, OX-40L and homologs and portions thereof. A foreign nucleic acid sequence encoding at least one target antigen or immunological epitope thereof may further be provided in the method by the first vector or by a second vector.

[0046] In addition to genes or portion thereof encoding multiple costimulatory molecules, a foreign or exogenous nucleic acid sequence or functional portions thereof encoding at least one or a combination of other classes of immunostimulatory molecules may also be provided by the first vector, by the second vector, or by a third vector. Other classes of immunostimulatory molecules includes cytokines such as IL-2, IL-12, GM-CSF and the like, chemokines such as MIP1, MIP2, RANTES and the like and Flt-3L.

[0047] An aspect of the invention is a method of enhancing an antigen-specific T cell immune response in a mammal to a target cell, target antigen or immunological epitope thereof comprising administration of a foreign recombinant poxvirus comprising nucleic acid sequences encoding multiple costimulatory molecules LFA-3, ICAM-1 and at least one B7 molecule, each costimulatory molecule expressed in a cell in the mammal in an amount effective to enhance at least one T-cell immune response in which the enhancement is greater than the additive sum of enhancement provided by administration of single or double costimulatory molecules.

[0048] In another method of enhancing immunological responses, APCs or tumor cells expressing foreign or exogenously provided genes encoding multiple costimulatory molecules are provided to a mammal in an effective amount to enhance immunological responses. The APC or tumor cell may further express foreign genes encoding at least one target antigen or immunological epitope thereof for enhancement of immune responses. A target antigen or immunological epitope thereof may be administered to the mammal prior to, concurrently with or subsequent to the administration of the APC or tumor cell. In addition, or alternatively, APCs or tumor cells are pulsed with at least one target antigen or immunological epitope thereof prior to administration to the mammal.

[0049] The present invention provides methods of enhancing humoral responses in a mammal to a target cell, target antigen or immunological epitope thereof comprising administration of a recombinant vector comprising foreign nucleic acid sequences encoding multiple costimulatory molecules to a mammal in an amount effective to enhance an humoral response. The vector may further comprise nucleic acid sequences encoding at least one target antigen or immunological epitope thereof. The invention further provides an isolated antibody or functional portion thereof against a target cell, target antigen or immunological epitope thereof produced by the method.

[0050] The present invention also provides antibody specific for a target antigen or immunological epitope thereof produced in response to administration of a recombinant poxvirus comprising foreign genes encoding B7, ICAM-1 and LFA-3 and genes encoding one or more target antigens or epitopes thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the detailed description of the invention.

[0052] FIG. 1. Genomic structure of plasmid pT5032 comprising nucleic acid sequences encoding murine LFA-3, ICAM-1 and B7.1, flanked by portions of the Hind III M region of the vaccinia genome.

[0053] FIG. 2. Genomic structure of plasmid pT5047 comprising nucleic acid sequences encoding murine LFA-3, ICAM-1, B7.1, and the lacZ gene, flanked by portions of the Hind III J region of the vaccinia genome.

[0054] FIG. 3. Genomic structure of plasmid pT5031 comprising nucleic acid sequences encoding murine LFA-3, ICAM-1 and B7.1 and a nucleic acid sequence encoding CEA, flanked by portions of the Hind III M region of the vaccinia genome.

[0055] FIGS. 4A through 4C. Genomic structure of recombinant vaccinia viruses expressing three murine costimulatory molecules with (FIG. 4C) or without (FIGS. 4A and B) a tumor-associated antigen. FIG. 4A shows the genomic structure of recombinant vaccinia, vT171. FIG. 4B shows the genomic structure of recombinant vaccinia vT199. FIG. 4C shows the genomic structure of recombinant vaccinia vT172. Hind III M and Hind III J are the sites of insertion in the poxvirus genomes of the foreign genes. Promoters 30K, I3, sE/L, 7.5K, 40K and C1 are poxviral promoters. Bam HI and Hind III restriction sites in the inserted sequences are shown, with the distance of each site (in kilobase pairs) from the 5′ end of the insertion (0) listed above each site in parentheses (not drawn to scale).

[0056] FIG. 5. Genomic structure of plasmid pT8001 comprising nucleic acid sequences encoding murine B7.1, LFA-3, ICAM-1 and the lacZ gene, flanked by portions of the BamHI J region of the fowlpox genome.

[0057] FIG. 6. Genomic structure of plasmid pT5049 comprising a nucleic acid sequence encoding the tumor associated antigen, CEA, and murine B7.1, LFA-3, and ICAM-1, in combination with the lacZ gene, flanked by portions of the BamHI J region of the fowlpox genome.

[0058] FIGS. 7A through 7D. Genomic structure of recombinant fowlpox viruses expressing three murine costimulatory molecules with (FIGS. 7B, 7C and 7D) or without (FIG. 7A) a tumor-associated antigen (TAA). FIG. 7A shows the genomic structure of recombinant fowlpox vT222. FIG. 7B shows the genomic structure of recombinant fowlpox vT194. FIG. 7C shows the genomic structure of recombinant fowlpox expressing MUC-1, B7.1, ICAM-1 and LFA-3. FIG. 7D shows the genomic structure of recombinant fowlpox expressing a tumor-associated antigen, B7.1, ICAM-1 and LFA-3. BamHI J is the site of insertion in the fowlpox virus genome of the foreign genes. sE/L, I3, 7.5K, C1, 40K and 30 K are poxviral promoters. P1-P5 denote five different poxvirus promoters. BamHI and HindIII restriction sites in the inserted sequences are shown, with the distance of each site (in kilobase pairs) from the 5′ end of the insertion (0) listed above each site in parentheses (not drawn to scale).

[0059] FIG. 8. Genomic structure of plasmid pT5064 comprising nucleic acid sequences encoding human LFA-3, human ICAM-1, human B7.1 and the lacZ gene, flanked by portions of the HindIII J region of the vaccinia genome.

[0060] FIGS. 9A through 9C Genomic structure of recombinant poxvirus expressing three human costimulatory molecules LFA-3, ICAM-1 and B7.1 along with the lacZ gene with (FIGS. 9B, C) or without (FIG. 9A) a tumor associated antigen, HindIII J is the site of insertion in the vaccinia virus genome of the foreign genes. BamHI J is the site of insertion in the fowlpox virus genome. 30K, I3, sE/L, 40K and C1 are poxviral promoters. BglII and HindIII restriction sites in the inserted sequences are shown, with the distance of each site (in kilobase pairs) from the 5′ end of the insertion (0) listed above each site in parentheses (not drawn to scale).

[0061] FIG. 10. Genomic structure of plasmid pT8016 comprising nucleic acid sequences encoding CEA (6D) and human LFA-3, ICAM-1, B7.1, and the E. coli lacZ gene, flanked by portions of the HindIII J region of the vaccinia genome.

[0062] FIG. 11. Genomic structure of recombinant vaccinia virus vT238 expressing CEA (6D) and three human costimulatory molecules. HindIII J is the site of insertion in the poxvirus genome of the foreign genes. 40K, 30K, I3, sE/L, and C1 are poxviral promoters.

[0063] FIG. 12. Genomic structure of plasmid pT8019 comprising nucleic acid sequences encoding murine LFA-3, ICAM-1, B7.1, and the E. coli lacZ gene, flanked by portions of the BamHI J region of the fowlpox genome.

[0064] FIGS. 13A and 13B. Genomic structure of recombinant fowlpox viruses expressing murine or human costimulatory molecules. FIG. 13A shows the genomic structure of recombinant fowlpox vT251. FIG. 13B shows the genomic structure of recombinant fowlpox vT232. BamHI J is the site of insertion in the poxvirus genome of the foreign genes. 30K, I3, sE/L and C1 are poxviral promoters.

[0065] FIG. 14. Genomic structure of plasmid pT5072 comprising nucleic acid sequences encoding human LFA-3, ICAM-1, B7.1, and the E. coli lacZ gene, flanked by portions of the BamHI J region of the fowlpox genome.

[0066] FIG. 15. Genomic structure of plasmid pT8020 comprising nucleic acid sequences encoding MUC-1, murine LFA-3, ICAM-1, B7.1, and the E. coli lacZ gene, flanked by portions of the BamHI J region of the fowlpox genome.

[0067] FIGS. 16A through 16D. Genomic structure of recombinant fowlpox viruses expressing murine or human costimulatory molecules with at least one tumor-associated antigen. FIG. 16A shows the genomic structure of recombinant fowlpox vT250. FIG. 16B shows the genomic structure of recombinant fowlpox vT242. FIG. 16C shows the genomic structure of recombinant fowlpox vT236. FIG. 16D shows the genomic structure of recombinant fowlpox vT257. BamHI J is the site of insertion in the poxvirus genome of the foreign genes. 40K, 7.5K, 30K, I3, sE/L, and C1 are poxviral promoters.

[0068] FIG. 17. Genomic structure of plasmid pT2186 comprising nucleic acid sequences encoding MUC-1, human LFA-3, ICAM-1, B7.1, and the E. coli lacZ gene, flanked by portions of the BamHI J region of the fowlpox genome.

[0069] FIG. 18. Genomic structure of plasmid pT2187 comprising nucleic acid sequences encoding CEA (6D), human LFA-3, ICAM-1, B7.1, and the E. coli lacZ gene, flanked by portions of the BamHI J region of the fowlpox genome.

[0070] FIG. 19. Genomic structure of plasmid pT5080 comprising nucleic acid sequences encoding PSA, PSMA, human LFA-3, ICAM-1, B7.1, and the E. coli lacZ gene, flanked by portions of the BamHI J region of the fowlpox genome.

[0071] FIG. 20. Genomic structure of plasmid pT5085 comprising nucleic acid sequences encoding murine LFA-3, ICAM-1, B7.1, and the E. coli lacZ gene, flanked by portions of the deletion III region of the MVA genome.

[0072] FIGS. 21A and 21B. Genomic structure of recombinant MVA viruses expressing murine or human costimulatory molecules with or without tumor-associated antigens. FIG. 21A shows the genomic structure of recombinant MVA vT264. FIG. 21B shows the genomic structure of recombinant MVA vT260. Deletion III is the site of insertion in the poxvirus genome of the foreign genes. 40K, 7.5K, 30K, I3, sE/L, and C1 are poxviral promoters.

[0073] FIG. 22. Genomic structure of plasmid pT5084 comprising nucleic acid sequences encoding PSA, PSMA, human LFA-3, ICAM-1, B7.1, and the E. coli lacZ gene, flanked by portions of the deletion III region of the MVA genome.

[0074] FIG. 23. Costimulatory molecule surface expression following infection with recombinant viruses. MC38 tumor cells were infected for 5 hours at 5 MOI (multiplicity of infection; pfu/cell) with the indicated virus. After infection, cells were immunostained with FITC-labeled monoclonal antibodies (MAb) specific for the costimulatory molecule. Shaded areas are fluorescence intensity of the specific MAb while unshaded areas are the fluorescence intensity of the appropriate isotype control antibody (see Materials and Methods).

[0075] FIGS. 24A and 24B. Effect of multiple costimulatory molecules on T-cell proliferation. Naive murine T cells, in the presence of varying concentrations of Con A to provide the first signal, were co-cultured with MC38 stimulator cells infected with either recombinant vaccinia (FIG. 24A) or recombinant fowlpox (FIG. 24B) vectors. Recombinant vectors were wild-type (i.e., V-Wyeth or WT-FP [open squares]), rV-LFA-3 (closed triangle), rV-ICAM-1 or rF-ICAM-1 (closed circles), rV-B7-1 or rF-B7-1 (closed diamonds), and rV-B7-1/ICAM-1/LFA-3 or rF-CEA/B7-1/ICAM-1/LFA-3 (closed squares). Uninfected MC38 cells are open circles. Proliferation assay is as described in Materials and Methods.

[0076] FIGS. 25A through 25D. Specificity of costimulation delivered via recombinant vaccinia viruses. T cells, in the presence of Con A, were co-cultured with MC38 stimulator cells infected with V-Wyeth (FIG. 25A), rV-B7-1 (FIG. 25B), rV-ICAM-1 (FIG. 25C), and rV-LFA-3 (FIG. 25D), as denoted by open circles. Infected stimulator cells in the presence of costimulatory molecule-specific MAb are denoted by closed circles, and isotype control antibody is denoted by closed triangles.

[0077] FIG. 26. Relative capacity of B7-1, ICAM-1, LFA-3 and the coexpression of all three costimulatory molecules to deliver the second signal for T-cell proliferation. In the presence of Con A (2.5 μg/ml), 100,000 T cells were co-cultured with 10,000 MC38 cells. The stimulator MC38 cells expressing one or all of the costimulatory molecules were added to the wells in various ratios in combination with V-Wyeth-infected stimulator cells to a total of 104 MC38 cells/well. MC38 cells were infected with V-Wyeth (open square), rV-LFA-3 (closed triangles), rV-ICAM-1 (closed circles), rV-B7-1 (closed diamonds), or rV-B7-1-ICAM-1-LFA-3 (closed squares). Cells were co-cultured for 48 hours. During the final 18 hours, 3H-Thymidi ne was added to measure T-cell proliferation. Inset panel depicts proliferation values obtained from a culture in which 3% of the MC38 stimulator cells were infected with the vectors shown. Thus, in this experiment, the final ratio of stimulator cells to T cells was 0.003. Note the relatively poor effect of rVB7.1/ICAM under these conditions as compared to rV-B7/ICAM/LFA-3.

[0078] FIGS. 27A through 27D. Effect of costimulation on specific T-cell populations. Murine CD4+ (FIG. 27A) or CD8+ T cells (FIG. 27B) were co-cultured with uninfected MC38 cells (open circle), or cells infected with V-Wyeth (open squares), rV-LFA-3 (closed triangles), rV-ICAM-1 (closed circles), rV-B7-1 (closed diamonds) or rV-B7-1/ICAM-1/LFA-3 (closed squares) at a 10:1 ratio for 48 hours in the presence of various concentrations of Con A. During the final 18 hours, 3H-Thymidi ne was added to measure T-cell proliferation. FIGS. 27C and 27D show the proliferative responses of purified CD4+ and CD8+ cells, respectively, when co-cultured in the presence of vector-infected MC38 stimulator cells at a low Con A concentration (0.625 μg/ml).

[0079] FIGS. 28A through 28D. Effect of costimulation on cytokine production. Murine CD4+ (FIGS. 28A and 28C) or CD8+ (FIGS. 28B and 28D) T cells were purified as described in Materials and Methods and co-cultured with the indicated MC38 vector-infected stimulator cells for 24 hours in the presence of 2.5 μg/ml Con A. Supematant fluids were analyzed for production of IL-2 (FIGS. 28A and 28B) and IFN-γ (FIGS. 28C and 28D) by capture ELISA.

[0080] FIGS. 29A through 29C. Effect of costimulation on cytokine RNA expression. FIG. 29A: murine CD4+ or CD8+ T cells were co-cultured with MC38 stimulator cells infected with V-Wyeth (lane A), rV-B7-1(lane B), rV-ICAM-1 (lane C), rV-LFA-3 (lane D) or rV-B7-1/ICAM-1/LFA-3 (lane E) at a T-cell to stimulator cell ratio of 10:1 for 24 hours in the presence of 2.5 μg/ml Con A. Following culture, T-cell RNA was analyzed by multiprobe RNAse protection assay. The quantitative representation of results from the autoradiograph is normalized for expression of the housekeeping gene L32 in FIG. 29B (CD4+ cells) and FIG. 29C (CD8+ cells). Order of histogram bars (from left to right) is MC38/V-Wyeth, MC38/B7-1, MC38/ICAM-1, MC38/LFA-3, and MC38/B7-1/ICAM-1/LFA-3.

[0081] FIG. 30. C57BL/6 mice (5/group) were administered HBSS (closed squares) or vaccinated with 107 pfu rV-CEA (closed triangles) or rV-CEA/TRICOM (closed circle). One hundred days later, mice were inoculated with 1×106MC38 carcinoma cells expressing CEA and survival was monitored. All mice other than the rV-CEA/TRICOM group developed tumors and were sacrificed when tumors exceeded 20 mm in length or width, or when the mice were moribund. FIG. 30: In a second experiment, C57BL/6 mice (5/group) were vaccinated with 107 pfu rV-CEA, rV-CEA/B7.1, rV-CEA/TRICOM or HBSS buffer. Lymphoproliferative responses from pooled splenic T cells were analyzed 22 days following vaccination. Values represent the stimulation index of the mean cpm of triplicate sames vs. media. Standard deviation never exceeded 10%. Antigens used were Con A (5 μg/ml), CEA (100 μg/ml) and ovalbumin (100 μg/ml).

[0082] FIG. 31 shows a schematic of an in vitro costimulation assay of dendritic cells.

[0083] FIGS. 32A and 32B show the proliferative response of naive CD4+ (FIG. 32A) or naive CD8+ (FIG. 32B) T cells stimulated with progenitor DCs infected with rV-B7/ICAM-1/LFA-3 or DCs (noninfected, ie, CD34+ cells treated with GM-CSF+IL-4 for 6 days) in the presence of Con A.

[0084] FIGS. 33A and 33B show the proliferative response of naïve CD4+ (FIG. 33A) or naïve CD8+ (FIG. 33B) T cells stimulated with progenitor DCs infected with rV-B7/ICAM-1/LFA-3 or DCs infected with rV-B7/ICAM-1/LFA-3 or V-Wyeth (control).

[0085] FIG. 34 shows the mixed lymphocyte reaction (MLR) of Balb/C splenocytes vs. irradiated C57bl/6 dendritic cells infected with 25 MOI of V-Wyeth or rV-TRICOM. 3H-thymidi ne pulsed on day 3, harvest on day 4, □DC (uninfected), ▪ DC (V-Wyeth infected), □DC (rV-TRICOM infected).

[0086] FIG. 35 shows the proliferative response of responder T cells (CAP-M8 T-cell line specific for CEA peptide 8) at various APC ratios harvested on day 5 after stimulation with peptide-pulsed DCs infected with rV-TRICOM and rested 2 days with 10 μ/ml IL2 (no APC or peptide). Peptide 8-(EAQNTTYL) in assay at 1 μg/ml final concentration. 3H-thymidi ne added on day 2, T cells harvested on day 3. 0=DC(v-Wyeth)−pep and Δ=DC (rV-TRICOM)−pep results are at baseline.

[0087] FIGS. 36A and 36B. Efficiency of poxviral infection of murine dendritic cells (DC). DC were infected with 25 MOI rV-TRICOM or 50 MOI rF CEA/TRICOM for 5 h. DC infected with TRICOM vectors exhibit enhanced capacity to stimulate naïve T-cells. All DC populations were co-cultured for 48 h with T-cells at a ratio of 10:1 in the presence of different concentrations of Con A to provide signal-1. 3H-thymidi ne was added during the final 18 h. FIG. 36A: Uninfected DC (closed squares), mock-infected DC (closed diamonds), or DC infected with V-WT (closed inverse triangles), rV-B7.1 (open triangles) or rV-TRICOM (open circles). FIG. 36B: DC (closed squares), mock-infected DC (closed diamonds), or DC infected with WT-FP (closed inverse triangles), rF-B7.1 (open triangles) or rF-TRICOM (open circles).

[0088] FIGS. 37A through 37F. Enhanced allostimulatory activity by DC infected with vaccinia (FIGS. 37A, C, E) or fowlpox (FIGS. 37B, D, F) vectors. Uninfected DC (closed squares); mock-infected DC (closed diamonds); or DC infected with wild-type poxviral vectors (V-WT or F-WT, closed inverse triangles), rV-B7.2 or rF-B7.1 (open triangles), or rV-TRICOM or rF-TRICOM (open circles) were co-cultured with allogeneic (FIGS. 37A-D) or syngeneic T cells (FIGS. 37E-F) for 5 days. 3H-thymidi ne was added during the final 18 h.

[0089] FIGS. 38A through 38F. Effect of vaccinia infection of DC on peptide-specific T-cell proliferation. Uninfected DC (closed squares), or DC infected with V-WT (closed inverse triangles), rV-B7.1 (open triangles) or rV-TRICOM (open circles) were co-cultured with OVA peptide-specific T cells (FIGS. 38A, C, E) or CAP-M8 peptide-specific T cells (FIGS. 38B, D, F). Experimental conditions included a fixed effector:stimulator cell ration of 10:1 in the presence of various concentrations of the appropriate peptides (FIGS. 38A-D), negative control peptides (open squares, either VSVN (FIG. 38A), or FLU-NP (FIG. 38B), or a fixed peptide concentration of 1 μM in the presence of various effector:stimulator cell ratios (FIGS. 38E and F).

[0090] FIGS. 39A and 39B. Effect of rV-TRICOM infection with DC matured with TNF-α or CD40. DC (closed squares), or DC cultured with either 100 ng/ml TNF-α (open triangles), or 5 μg/ml CD40 mAb (open circles) for the final 24 h of culture were used to stimulate CAP-M8-specific effector T cells (FIG. 39A). The proliferation of CAP-M8 T cells in response to these DC populations after infection with 25 MOI rV-TRICOM (FIG. 39B). For all panels, the T-cell:DC ratio was 10:1, while the CAP-M8 peptide concentration was 1 μg/ml. Closed circles denote proliferation of CAP-M8 T cells stimulated with all DC populations in the presence of 1 μg/ml VSVN peptide.

[0091] FIGS. 40A through 40H: Effect of vaccinia infection of DC on induction of CTL activity. DC (FIG. 40B), or DC infected with V-WT (FIG. 40C), or rV-TRICOM (FIG. 40D) were pulsed with 10 μM OVA peptide for 2 h. DC populations were administered intravenously to mice (1×105 cells/mouse). Control mice were immunized subcutaneously with 100 μg OVA peptide in Ribi/Detox adjuvant (FIG. 40A). Fourteen days later spleens were harvested, restimulated for 6 days with the corresponding peptide, and assessed for lytic ability against EL-4 cells pulsed with either OVA (closed squares) or VSVN peptides (open squares). Inset numbers depict CTL activity as expressed in lytic units. Also shown is the effect of vaccinia infection of DC on induction of CTL activity. DC (FIG. 40F), or DC infected with V-WT (FIG. 40G), or rV-TRICOM (FIG. 40H) were pulsed with 10 μM CAP-M8 peptide for 2 h. DC populations were administered intravenously to mice (1×105 cells/mouse). Control mice were immunized subcutaneously with 100 μg CAP-M8 peptide in Ribi/Detox adjuvant (FIG. 40E). Fourteen days later spleens were harvested, restimulated for 6 days with the corresponding peptide, and assessed for lytic ability against EL-4 cells pulsed with either CAP-M8 (closed squares) or FLU-NP peptides (open squares). Inset numbers depict CTL activity as expressed in lytic units.

[0092] FIGS. 41A through 41C: Effect of multiple immunizations with vaccinia-infected DC on induction of CTL activity. DC (closed squares), or DC infected with V-WT (closed inverse triangles) or rV-TRICOM (open circles) were pulsed with 10 μM CAP-M8 peptide for 2 h. DC populations were administered intravenously to mice (1×105 cells/mouse) 1, 2 or 3 times at 7 day intervals. Control mice were immunized subcutaneously with 100 μg CAP-M8 peptide in Ribi/Detxo adjuvant (crosses). Fourteen days after the final immunization, spleens were harvested, restimulated for 6 days with CAP-M8, and assessed for lytic ability against EL-4 cells pulsed with CAP-M8 or control peptide VSVN (not shown).

[0093] FIGS. 42A and 42B. Effect of vaccinia and fowlpox TRICOM-infected splenocytes on T cell proliferation. Naïve murine T cells were co-cultured with autologous splenocytes infected with either recombinant vaccinia or fowlpox vectors. Co-culture was performed in varying concentrations of Con-A as Signal-1. Recombinant vectors were wild type (i.e. V-WT, FP-WT, open diamond), rV-B7-1 or rF-B7-1, (open circles) or rV-TRICOM or rF-TRICOM (closed squares). Uninfected splenocytes are shown as open triangles.

[0094] FIGS. 43A through 43D. Effect of TRICOM vector infected splenocytes on allogeneic T cells. Naïve Balb/C T cells were co-cultured with C57B1/6 splenocytes infected with recombinant vaccinia (FIGS. 43A and C) or fowlpox (FIGS. 43B and D) vectors for either 2 days (FIGS. 43A and B) or 5 days (FIGS. 43C and 43D). Recombinant vectors were V-WT or FP-WT, open diamonds, rV-B7-1 or rF-B7-1 (open circles), or rV-TRICOM or rF-TRICOM (closed squares). Uninfected splenocytes are indicated as open triangles. Proliferation induced by DC is indicated as closed squares.

[0095] FIGS. 44A through 44F. Effect of rV-TRICOM-infected splenocytes on specific T cell populations. Naïve murine T cells were fractionated with CD3+, CD4+, and CD8+ subpopulations. T cells were co-cultured with either uninfected autologous BMDC or splenocytes infected with recombinant vaccinia vectors. Varying Con-A concentrations (FIGS. 44A-C) or varying number of stimulator cells (FIGS. 44D-F) provided the first signal. T cell proliferation in response to mature BMDC is indicated by open squares, and to uninfected splenocytes by open triangles. Recombinant vectors were wild-type (V-WT, open diamonds) or rV-TRICOM (closed squares).

[0096] FIGS. 45A through 45F. Effect of rV-TRICOM-infected bone marrow cells on specific T cell populations. Naïve murine T cells were fractionated into CD3+, CD4+, and CD8+ subpopulations. T cells were co-cultured with either uninfected autologous BMDC or splenocytes infected with recombinant vaccinia vectors. Varying Con-A concentrations (FIGS. 45A-C) or varying number of stimulator cells (FIGS. 45D-F) provided the first signal. T cell proliferation in response to mature BMDC is indicated by open squares, and to uninfected splenocytes by open triangles. Recombinant vectors were wild-type (V-WT, open diamonds) or rV-TRICOM (closed squares).

[0097] FIGS. 46A through 46D. Effect or rV-TRICOM-infected splenocytes or bone marrow (BM) cells on peptide-specific memory CD8+ T cells. CAP-M8-specific T cells were co-cultured with autologous splenocytes (FIGS. 46A and B) or bone marrow cells (FIGS. 46C and D) infected with recombinant vaccinia vectors. The analysis was carried out using two sets of conditions: a) a 10:1 fixed ratio of responder:stimulator cells that were cultured in the presence of several concentrations of CAP-M8 peptide (FIGS. 46A and 46C), or b) a fixed concentration of peptide (1 uM) at various responder:stimulator ratios (FIGS. 46B and 46D). Recombinant vectors were wild type (open diamonds), and rV-TRICOM (closed squares). Uninfected splenocytes are shown as open triangles. BM are shown as open squares.

[0098] FIG. 47. Shows production of IFN-γ by human T cells isolated from peripheral blood mononuclear cells (PBMC) using rF-TRICOM-infected human dendritic cells pulsed with CEA peptides, CAP-1 or CAP1, 6D.

[0099] FIG. 48. Shows production of IFN-γ by human T cells using rF-TRICOM-infected human dendritic cells pulsed with PSA peptide, PSA-3.

[0100] FIG. 49. Shows production of IFN-γ by human T cells isolated from PBMC using rF-TRICOM-infected human dendritic cells pulsed with Flu peptide 58-66.

[0101] FIG. 50. Shows production of IFN-γ by human T cells isolated from PBMC using rF-TRICOM- or rF-B7.1-infected human dendritic cells pulsed with Flu peptide 58-66 at various effector:APC ratios.

[0102] FIG. 51. Shows production of IFN-γ by human T cells from donor 868 using rF-TRICOM-infected human dendritic cells pulsed with HPV peptide (11-20) after one or two in vitro stimulation (IVS).

[0103] FIG. 52. Shows production of IFN-γ by human T cell line using rF-TRICOM- or rF-B7.1-infected human dendritic cells pulsed with HPV peptide (11-20).

[0104] FIG. 53. Shows production of IFN-γ by a human T cell line using rF-TRICOM- or rF-B7.1-infected human dendritic cells pulsed with various concentrations of HPV peptide (11-20).

[0105] FIG. 54. Shows production of IFN-γ by human T cells using rF-TRICOM or rF-B7.1-infected human dendritic cells pulsed with HPV E7 peptide 11-20 at various effector:APC ratios.

DETAILED DESCRIPTION OF THE INVENTION

[0106] The present invention is a recombinant vector comprising foreign genes encoding multiple costimulatory molecules, in combination, or the functionally active portions of each costimulatory molecule. Multiple costimulatory molecules as used herein are at least three or more costimulatory molecules. As used herein a functionally active portion is that portion of the molecule responsible for binding to its respective ligand, triggering an appropriate costimulatory signal for immune-cell activation. One method of determining functional activity is to access the induction of naïve T-cell proliferation by delivering the costimulatory molecule to a target cell in vitro as described herein. A functional portion of a costimulatory molecule stimulates at least 20% increase in T cell proliferation.

[0107] The term foreign gene or foreign nucleic acid sequence or functional portion thereof as used herein is a gene, nucleic acid sequence or functional portion thereof that is exogenously provided by a recombinant vector to a host cell or organism. The exogenous gene or portion thereof which is provided to the host cell or host organism may be one which is not endogenously present in the host cell or organism or may be endogenously present and functional or non-functional. In the case in which a functional endogenous gene is present in the host cell or organism, the foreign or exogenously provided gene or functional portion thereof results in overexpression of the gene product.

[0108] The recombinant vectors of the present invention have utility in providing enhanced immunological response to cells of the immune system including but not limited to T lymphocytes, B lymphocytes, NK cells, antigen-presenting cells (APCs) and the like. The enhancement of the immunological response using the recombinant vectors expressing multiple costimulatory molecules is synergistic as compared to the use of a single costimulatory molecule or the use of two costimulatory molecules in enhancing immunological responses. The immunological response may be a cellular and/or humoral immune response and may be directed to a specific target antigen or epitope thereof or may be a generalized immune enhancing or upregulating effect as demonstrated by increased cytokine release, increase proliferation by immune cells, increased mitogen responsiveness and the like. The enhancement in an immune response preferably includes hyperstimulation or high intensity T cell stimulation (HITS) as a result of stimulation using the recombinant vectors of the present invention or cells transfected, transduced or induced by the recombinant vector of the present invention.

[0109] The foreign genes encoding the costimulatory molecules may be obtained from a variety of sources. The selection of the source of foreign genes encoding the costimulatory molecules may depend on the species to be immunized or treated using the recombinant vector.

[0110] The foreign genes encoding the costimulatory molecules may be murine-derived, human-derived, simian-derived, other mammalian homologs and may be chemically synthesized based on mammalian genes. The foreign genes encoding the costimulatory molecules may also be avian-derived or chemically synthesized based on avian costimulatory molecule genes. The recombinant vectors of the present invention are useful as immunogens and as vaccines in stimulating an enhancement of immunological responses to target cells, target antigens and immunological epitopes thereof. Such level of enhancement of a immune response using the present recombinant vectors comprising genes encoding multiple costimulatory molecules has not been obtainable using a single or double costimulatory molecule.

[0111] Genes or functional portions thereof encoding costimulatory molecules having utility in the present invention include but are not limited to B7.1, B7.2, ICAM-1, LFA-3, 4-1BBL, CD59, CD40, CD70, VCAM-1, OX-40L, mammalian homologs and the like. The recombinant vector of the present invention comprises genes encoding at least three costimulatory molecules for synergistic enhancement of immune responses which is not obtainable by the use of a single or a double costimulatory molecule. Genes encoding various combinations of costimulatory molecules are an ambit of the invention for use in the recombinant vector and may include such combinations as B7.1, B7.2, ICAM-1, LFA-3; B7.1, B7.2, ICAM-1, LFA-3; B7.1, B7.2, ICAM-1, 4-1BBL; B7.1, B7.2, ICAM-1, LFA-3, 4-1BBL; CD59, VCAM-1; and B7.1, B7.2; CD59, CD40, 4-1 BBL, CD70 and VCAM-1, B7.1, B7.2; OX-40L, 4-1BBL; and the like depending on the desired immune response and the disease or condition to be treated. Based on the dramatic synergistic immune responses achieved using a recombinant vector encoding three costimulatory molecules as compared to the use of a recombinant vector encoding one or two costimulatory molecules, a recombinant vector encoding four, five or more costimulatory molecules will result in a synergistic immune response or immune response equal to/or greater than that using a recombinant vector encoding three costimulatory molecules.

[0112] B7 represents a family of costimulatory molecules which are members of the Ig gene superfamily. The members include murine B7.1 (CD80) and B7.2 (CD86). B7.1 and B7.2 are the natural ligands of CD28/CTLA-4 (CD152). The gene sequence of murine B7.1 is disclosed in Freeman et al (J. Immunol. 143:2714-2722, 1989) and in GENBANK under Accession No. X60958. The gene sequence of murine B7.2 is disclosed in Azuma et al (Nature 366:76-79, 1993) and in GENBANK under Accession No. L25606 and MUSB72X.

[0113] The human homologs of the murine B7 costimulatory molecules and functional portions thereof are an ambit of the present invention and have particular utility in recombinant vectors for human clinical use. The human homolog of the murine B7 costimulatory molecules include CD80, the homolog of murine B7.1, and CD86, the homolog of B7.2. The gene sequence of human B7.1 (CD80) is disclosed in GENBANK under Accession No. M27533, and the gene sequence of human B7.2 (CD86) is disclosed under Accession No. U04343 and AF099105. A license may be required to practice this invention.

[0114] For use in the present invention, a recombinant vector may contain a foreign nucleic acid sequence encoding at least one molecule from the B7 costimulatory molecule family, or a combination of B7 costimulatory molecules or functional portions thereof in addition to other costimulatory molecules. The combination of B7 costimulatory molecules includes but is not limited to two or more B7.1 molecules, two or more B7.2 molecules, B7.1 and B7.2 and the like. In one embodiment the recombinant vector contains a foreign nucleic acid sequence encoding the B7.1 molecule in combination with foreign nucleic acid sequences encoding LFA-3 and ICAM-1.

[0115] Intercellular adhesion molecule-1 (murine ICAM-1, CD54) and the human homolog, CD54, also acts as a costimulatory molecule. Its ligand is leukocyte function-associated antigen-1 (LFA-1, CD11a/CD 18) which is expressed on the surface of lymphocytes and granulocytes. The gene for murine ICAM-1 is disclosed in GenBank under Accession No. X52264 and the gene for the human ICAM-1 homolog, (CD54), is disclosed in Accession No. J03132. In one embodiment, the recombinant vector of the present invention contains a foreign nucleic acid sequence encoding at least one murine ICAM-1 molecule, human homolog, other mammalian homolog or functional portion thereof in addition to foreign nucleic acid sequences encoding two or more additional costimulatory molecules.

[0116] The costimulatory molecule leukocyte function antigen 3, murine LFA-3 (CD48), and its human homolog LFA-3 (CD58), a glycosyl-phosphatidylinositol-linked glycoprotein, is a member of the CD2 family within the immunoglobulin gene superfamily. The natural ligand of LFA-3 is CD2 (LFA-2) which is expressed on thymocytes, T cells, B cells and NK cells. The gene for murine LFA-3 is disclosed in GenBank under Accession No. X53526 and the gene for the human homolog is disclosed in Accession No. Y00636.

[0117] The T cell antigen 4-1BBL is a costimulatory molecule that relays costimulatory signals in antigen-stimulated primary T cell cultures and in lectin-driven activation of thymocytes (Hurtado, J. C. et al J. Immunol. 158(6):2600-2609, 1997). 4-1BBL belongs to the tumor necrosis factor receptor superfamily, a group of cysteine-rich cell surface molecules (Vinay, D. S. et al, Seminars in Immunolozy, 1998, Vol.10, pp. 481-489). The gene for the murine 4-1BBL is disclosed in GenBank under Accession No. U02567. The gene for the human homolog, hu4-1BBL is disclosed in GenBank under Accession No. U03397.

[0118] OX-40L is a type II membrane protein with limited homology to TNF and is stimulatory to OX-40+ T cells in vitro. The murine and human OX-40L cDNAs have 68% homology at the nucleotide level and 46% at the amino acid level. Human OX-40L stimulates human T cells exclusively, while murine OX-40L stimulates both human and mouse T cells. APC express OX-40L and can transmit the OX-40L: OX40R signal during presentation of antigen to CD4+ T cells. OX-40L signaling is important for differentiation of human dendritic cells and leads to increased production of IL-12, TNF-α, IL-1B, and IL-6. (Weinberg, A. D. et al 1998 Seminars in Immunology, Vol.10:471-480). OX-40L is a potent costimulatory molecule for sustaining primary CD4+ T cell responses, used in combination with B7-1 (Gramaglia, I. et al 1998 J. Immunology, Vol. 161:6510-7.

[0119] Vectors having utility in the present invention are capable of causing expression of at least three or more foreign genes, preferably five or more foreign genes. Vectors having utility in the present invention include any vector capable of causing functional expression of at least three foreign costimulatory molecules gene products in a host cell. In addition to the genes encoding at least three costimulatory molecules, the vector is also capable of causing the expression of at least one foreign gene encoding at least one target antigen or immunological epitope thereof as well as a selectable marker.

[0120] Vectors of the present invention include but are not limited to bacterial vectors such as Salmonella, viral vectors, nucleic acid based vectors and the like. Viral vectors include but are not limited to poxvirus, Herpes virus, adenovirus, alphavirus, retrovirus, picornavirus, iridovirus, and the like. Poxviruses having utility in the present invention include replicating and non-replicating vectors. Such poxviruses include but are not limited to orthopox such as vaccinia, raccoon pox, rabbit pox and the like, avipox, suipox, capripox and the like. Poxviruses may be selected from the group consisting of vaccinia-Copenhagen, vaccinia-Wyeth strain, vaccinia-MVA strain, NYVAC, fowlpox, TROVAC, canarypox, ALVAC, swinepox, and the like. In one embodiment, the recombinant vector is a vaccinia virus. In another embodiment, the recombinant vector is fowlpox.

[0121] A preferred vector of the present invention is a recombinant virus, preferably a poxvirus. The recombinant poxviruses having utility in the present invention have a number of attributes, including (i) efficient delivery of genes to multiple cell types, including APC and tumor cells; (ii) high levels of protein expression; (iii) optimal presentation of antigens to the immune system; (iv) the ability to elicit cell-mediated immune responses as well as antibody responses; (v) transient, rather than permanent, genetic modification of cells, and (vi) the ability to use combinations of poxviruses from different genera, as they are not immunologically cross-reactive. Parental poxviruses useful in constructing the recombinant poxvirus of the present invention include but are not limited to orthopox virus such as replicating vaccinia virus (Perkus et al Science 229:981-984, 1985; Kaufman et al Int. J. Cancer 48:900-907, 1991, Moss Science 252:1662, 1991), highly attenuated vaccinia viruses such as MVA, modified vaccinia Ankara (Sutter and Moss, Proc. Nat'l Acad. Sci. U.S.A. 89:10847-10851; Sutter et al Virology 1994), vaccinia-Copenhagen and NYVAC: avipoxviruses (15) such as fowlpox virus (15), canary poxviruses, such as ALVAC and the like (Baxby and Paoletti, Vaccine 10:8-9, 1992; Rinns, M. M. et al (Eds) Recombinant Poxviruses CRC Press, Inc, Boca Raton 1992; Paoletti, E. Proc. Nat'l Acad. Sci. USA 93:11349-11353, 1996), and suipoxvirus, capripoxvirus and the like.

[0122] In one embodiment, the parental poxvirus is a vaccinia virus. In a particular embodiment, the vaccinia virus is a Wyeth strain or derivative thereof. A derivative of the Wyeth strain includes but is not limited to vTBC33 which lacks a functional K1L gene and the like. In yet another embodiment, the virus is Dry-Vax available as a smallpox vaccine from the Centers for Disease Control, Atlanta, Ga. In another embodiment, the parental poxvirus is a strain of fowlpox, for example POXVAC-TC (Schering-Plough Corporation), and the like.

[0123] The recombinant vector of the present invention is able to infect, transfect or transduce host cells in a host. The host includes but is not limited to mammals, birds, fish and the like. The host cells are any cell amenable to infection, transfection or transduction by the recombinant vector and capable of expressing the foreign genes from the recombinant vector at functional levels. The host cells include but are not limited to professional APC and antigen presenting precursor cells such as monocytes, macrophages, DC, Langerhans cells and the like. The recombinant vector of the present invention may also infect tumor cells or other cell types such as fibroblasts or muscle cells. Infection of the host cells allows expression of each foreign, exogenous costimulatory molecule and expression of the foreign nucleic acid sequence encoding target antigen(s) if present in the recombinant vector. The host cells express, or are engineered to express, the appropriate MHC (HLA) Class I or II molecules for appropriate antigenic presentation to CD4+ and/or CD8+ T cells. As such virtually any mammalian cell may be engineered to become an appropriate antigen presenting cell expressing multiple costimulatory molecules.

[0124] The recombinant vector of the present invention comprises at least one expression control element operably linked to the nucleic acid sequence. The expression control elements are inserted in the vector to control and regulate the expression of the nucleic acid sequence (Ausubel et al, 1987, in “Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y.). Expression control elements are known in the art and include promoters. Promoters useful in the present invention are poxviral promoters as are known in the art which include but are not limited to 30K, I3, sE/L, 7.5K, 40K, C1 and the like. The nucleic acid sequence of the 30K promoter is disclosed in GenBank Accession No. M35027 at base numbers 28,012 through 28,423 (antisense). The nucleic acid sequence of I3 is disclosed in GenBank Accession No. J03399 at base numbers 1100 through 1301 (antisense). The nucleic acid sequence of the 7.5K promoter is disclosed in GenBank Accession No. M35027 at base numbers 186550 through 186680. The nucleic acid sequence of the 40K promoter is disclosed in GenBank Accession No. M13209 at base numbers 9700 through 9858 (antisense). The nucleic acid sequence of the C1 promoter is disclosed in GenBank Accession No. M59027 at base numbers 1 through 242 and in U.S. Pat. No. 5,093,258. The sequence of the sE/L promoter is disclosed in Reference 16. Other poxvirus promoters may be used, such as, those described by Davison and Moss (J. Mol. Biol. 210:749-769, (1989). Any of these promoters can be synthesized by using standard methods in the art. The selection of an appropriate promoter is based on its timing and level of expression. Early or early/late promoters are preferred. In a preferred embodiment, the promoter or combination of promoters utilized allow for optimal expression of each costimulatory molecule in an infected host to provide a synergistic immune response. In a preferred embodiment, each foreign gene encoding a costimulatory molecule is controlled by a separate and distinct promoter.

[0125] In the case of nucleic acid-based vectors, the constructs may be either nucleic acid (DNA or RNA) or associated with/or encapsulated in a lipid carrier. Optionally, the lipid carrier molecule and/or construct may provide targeting and/or expression in a particular target cell type or types. Naked DNA vectors may be prepared by methods described in U.S. Pat. No. 5,827,703. For the transcriptional initiation region, or promoter element, any region may be used with the proviso that it provides the desired level of transcription of the DNA sequence of interest. The transcriptional initiation region may be native to or homologous to the host cell and/or to the DNA to be transcribed, or foreign or heterologous to the host cell and/or the DNA sequence to be transcribed. Efficient promoter elements for transcription initiation of naked DNA include but are not limited to the SV40 (simian virus 40) early promoter, the RSV (Rous sarcoma virus) promoter, the adenovirus major late promoter, the human CMV (cytomegalovirus) immediate early I promoter, and the like. Nucleic acid-based vectors may be delivered to a host using a syringe, a catheter, or a needle-free injection device such as a gene gun.

[0126] In an embodiment of the invention, a recombinant vector is provided comprising a foreign nucleic acid sequence encoding a first costimulatory molecule or functional portion thereof under control of a first promoter, a foreign nucleic acid sequence encoding a second costimulatory molecule or functional portion thereof under control of a second promoter, and a foreign nucleic acid sequence encoding a third costimulatory molecule or functional portion thereof under control of a third promoter. The recombinant vector may further provide a foreign nucleic acid sequence encoding a target antigen or immunological portion thereof under control of a fourth promoter.

[0127] In one embodiment of the present invention, a recombinant poxvirus is provided comprising a nucleic acid sequence encoding LFA-3 or functional portion thereof under control of a 30K poxviral promoter, a nucleic acid sequence encoding ICAM-1 or portion thereof under control of an I3 poxviral promoter, and a nucleic acid sequence encoding B7.1 or portion thereof under control of an sE/L poxviral promoter. One example of such a recombinant poxvirus construct is vaccinia vT171 as depicted in FIG. 11A. The recombinant poxvirus may further provide a nucleic acid sequence encoding a tumor associated antigen or immunological portion thereof. One embodiment of the invention is recombinant vaccinia vT172 as depicted in FIG. 4C.

[0128] In another embodiment of the present invention, a recombinant poxvirus is provided comprising a nucleic acid sequence encoding B7.1 under control of a sE/L poxviral promoter, a nucleic acid sequence encoding LFA-3 or portion thereof under control of the I3 poxviral promoter, and a nucleic acid sequence encoding ICAM-1 or portion thereof under control of the 7.5K poxvirus promoter. Optionally the construct further comprises a nucleic acid sequence encoding at least one target antigen or immunological epitope thereof and/or a nucleic acid sequence encoding a selectable marker. One embodiment of such a recombinant poxvirus construct is vaccinia vT199 as depicted in FIG. 4B containing a lacZ gene as the selectable marker.

[0129] In an embodiment of the invention a recombinant fowlpox virus comprises a nucleic acid sequence encoding B7.1 or portion thereof under control of the sE/L poxviral promoter, a nucleic acid sequence encoding LFA-3 or portion thereof under control of the I3 poxviral promoter, and a nucleic acid sequence encoding ICAM-1 or portion thereof under control of the 7.5K poxviral promoter. An example of this embodiment is fowlpox vT222 as depicted in FIG. 4A. A recombinant fowlpox virus may further comprise a nucleic acid sequence encoding a target antigen, CEA, under control of the 40K poxviral promoter and a nucleic acid sequence encoding the selectable marker, lacZ under control of the C1 poxviral promoter. An example of this embodiment is fowlpox vT194 as depicted in FIG. 4B.

[0130] In another embodiment, a recombinant fowlpox virus comprises a nucleic acid sequence encoding the tumor-associated antigen MUC-1 or portion thereof under the control of the 40K promoter, a nucleic acid sequence encoding LFA-3 or portion thereof under the control of the 30K promoter, a nucleic acid sequence encoding ICAM-1 or portion thereof under the control of the I3 promoter, and a nucleic acid sequence encoding B7.1 or portion thereof under the control of the sE/L promoter, as depicted in FIG. 14C. The recombinant fowlpox virus may comprise a nucleic acid sequence encoding any tumor-associated antigen or portion thereof and nucleic acid sequences encoding LFA-3, ICAM-1 and B7.1, under the control of a multiplicity of promoters, as depicted in FIG. 4D.

[0131] Another embodiment of the present invention is a recombinant vector comprising nucleic acid sequences encoding the human homologs of the costimulatory molecules LFA-3, B7 and ICAM-1. The recombinant vector may further provide the appropriate promoters to allow expression of each sequence in an infected host cell. One embodiment of the recombinant vector is vT224 depicted in FIG. 9.

[0132] The present invention provides plas