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| PCT Patent | WO 03/048366 |
| Sung , et al. | June 12, 2003 |
SIVmac239 Immunogenic Plasmids and AIDS DNA Vaccine Containing the Same
The present invention relates to immunogenic plasmids showing excellent expression efficiency of immunogens and immune efficacy in the SIVmac239/rhesus monkey model and AIDS human patients. Also, the present invention relates to DNA vaccines for prophylaxis or treatment of AIDS containing the above immunogenic plasmids.
What is claimed is:
1. A vector pGX 10.
2. A plasmid pGXIO-SIV/GE.
3. A plasmid comprising the vector pGXIO and the SIVmac239 pol gene encoding reverse transcriptase and integrase and a DNA sequence encoding a signal peptide of secretory protein fused to the 3' end of the SIVmac239 pol gene which are operably linked to the vector.
4. The plasmid of claim 3, in which said pol gene is mutated so that the enzymatic activity of integrase can be inhibited.
5. The plasmid of claim 4, in which the mutation inhibiting the enzymatic activity of integrase comprises the deletion of nucleotides 5130-5132 site and the substitution of nucleotides 5133-5135 site for a serine codon.
6. The plasmid of claim 3, in which the DNA sequence encoding a signal peptide of secretory protein comprises the DNA sequence encoding a signal peptide of glycoprotein.
7. The plasmid of claim 6, in which the DNA sequence encoding a signal peptide of glycoprotein comprises one derived from herpes simplex virus.
8. The plasmid of claim 3 which is pGXlO-SIV/dpol.
9. A plasmid comprising the SIVmac239 vif gene and a DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 nef gene fused to the 3' and 5' ends of the SIVmac239 vif gene, respectively.
10. The plasmid of claim 9, in which the DNA sequence encoding a signal peptide of secretory protein fused to the 3' end of the SIVmac239 vif gene comprises the DNA sequence encoding a signal peptide of glycoprotein.
11. The plasmid of claim 10, in which the DNA sequence encoding a signal peptide of glycoprotein comprises one derived from herpes simplex virus.
12. The plasmid of claim 9, in which the mutation of the SIVmac239 nef gene fused to the 5'end of the SIVmac239 vif gene comprises the deletion of codons for Argl37 and Argl38.
13. The plasmid of claim 12, in which the mutation of the SIVmac239 nef gene fused to the 5'end of the SIVmac239 vif gene comprises the deletion of codons for Argl37 and Argl38.
14. The plasmid of claim 9 which is pGXIO-SIV/VN.
15. A plasmid comprising any one of genes having from exon 1 to the full length of the SIVmac239 tat gene, and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively.
16. The plasmid of claim 15, in which the signal sequence DNA of secretory protein fused to the 3' end of any one of genes having from exon 1 to a full length of the SIVmac239 tat gene comprises the DNA sequence encoding a signal peptide of glycoprotein.
17. The plasmid of claim 16, in which the DNA sequence encoding a 4 signal peptide of glycoprotein comprises one derived from herpes simplex virus.
18. The plasmid of claim 15, in which the SIVmac239 tat gene is exon 1 of the SIVmac239 tat gene.
19. The plasmid of claim 15, which is pGXIO-SIV/TV.
20. A plasmid comprising (i) the SIVmac239 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 nef gene fused to the 3' and 5' ends of the SIVmac239 vif gene, respectively, and (ii) any one of genes having from exon 1 to a full length of the SIVmac239 tat gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 vpx gene fused to the 3' and 5' ends of the SIVmac239 tat gene, respectively.
21. The plasmid of claim 20, in which the signal sequence DNA of secretory protein fused to the 3'end of the SIVmac239 vif gene comprises the DNA sequence encoding a signal peptide of glycoprotein.
22. The plasmid of claim 21, in which the DNA sequence encoding a signal peptide of glycoprotein comprises one derived from herpes simplex virus.
23. The plasmid of claim 20, in which the mutation of the SIVmac239 nef gene fused to the 5'end of the SIVmac239 vif gene comprises the deletion of codons for Argl37 and Argl38.
24. The plasmid of claim 23, in which the mutation of the SIVmac239 nef gene fused to the 5'end of the SIVmac239 vif gene comprises the deletion of codons for Argl37 and Argl38.
25. The plasmid of claim 20, in which the signal sequence DNA of secretory protein fused to the 3'end of any one of genes having from exon 1 to a full length of the SIVmac239 tat gene comprises the DNA sequence encoding a signal peptide of glycoprotein.
26. The plasmid of claim 25, in which the DNA sequence encoding a signal peptide of glycoprotein comprises one derived from herpes simplex virus.
27. The plasmid of claim 25, in which the SIVmac239 tat gene is exon 1 of the SIVmac239 tat gene.
28. The plasmid of claim 25, which is pGXIO-SIV/VNTV.
29. The plasmid of claim 25, which is pGX10-SIV/TVVN.
30. A plasmid pGXlO-HIV/GE.
31. A plasmid comprising the vector pGXIO, and the HIV-1 pol gene encoding reverse transcriptase and integrase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene which are operably linked to the vector.
32. The plasmid of claim 31, in which said pol gene is mutated so that the enzymatic activity of integrase can be inhibited.
33. The plasmid of claim 32, in which the mutation inhibiting the enzymatic activity of integrase comprises the deletion of nucleotides 5130-5132 and the substitution of nucleotides 5133-5135 for a serine codon.
34. The plasmid of claim 31, in which the signal sequence DNA of secretory protein comprises the DNA sequence encoding a signal peptide of glycoprotein.
35. The plasmid of claim 34, in which the DNA sequence encoding a signal peptide of glycoprotein comprises one derived from herpes simplex virus.
36. The plasmid of claim 31 which is pGXlO-HIV/dpol.
37. A plasmid comprising the HIV-1 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the HIV-1 nef gene fused to the 3' and 5'ends of the HIV-1 vif gene, respectively.
38. The plasmid of claim 37, in which the signal sequence DNA of secretory protein fused to the 3'end of the HIV-1 vif gene comprises the DNA sequence encoding a signal peptide of glycoprotein.
39. The plasmid of claim 38, in which the DNA sequence encoding a signal peptide of glycoprotein comprises one derived from herpes simplex virus.
40. The plasmid of claim 37, in which the mutation of the HIV-1 nef gene fused to the 5' end of the HIV-1 vif gene comprises the deletion of codons for Argl37 and Arg 138.
41. The plasmid of claim 40, in which the mutation of the HIV-1 nef gene fused to the 5' end of the HIV-1 vif gene comprises the deletion of codons for Argl37 and Argl38.
42. The plasmid of claim 37, which is pGXIO-HIV/VN.
43. A plasmid comprising any one of genes having from exon 1 to a full length of HIV-1 tat gene and the DNA sequence encoding a signal peptide of secretory protein and HIV-1 vpx gene fused to the 3' and 5' ends of the HIV-1 tat gene, respectively.
44. The plasmid of claim 43, in which the signal sequence DNA of secretory protein fused to the 3' end of any one of genes having from exon 1 to a full length of HIV-1 tat gene comprises the DNA sequence encoding a signal peptide of glycoprotein.
45. The plasmid of claim 44, in which the DNA sequence encoding a signal peptide of glycoprotein comprises one derived from herpes simplex virus.
46. The plasmid of claim 43, in which the HIV-1 tat gene is exon 1 of the HIV-1 tat gene.
47. The plasmid of claim 43, which is pGXlO-HIV/TV.
48. A plasmid comprising (i) HIV-1 vif gene and the DNA sequence encoding a signal peptide of secretory protein and HIV-1 nef gene fused to the 3'and 5'ends of the HIV-1 vif gene, respectively, and (ii) any one of genes having from exon 1 to a full length of HIV-1 tat gene and the DNA sequence encoding a signal peptide of secretory protein and HIV-1 vpx gene fused to the 3'and 5'ends of the HIV-1 tat gene, respectively.
49. The plasmid of claim 48, in which the signal sequence DNA of secretory protein fused to the 3'end of the HIV-1 vif gene comprises the DNA sequence encoding a signal peptide of glycoprotein.
50. The plasmid of claim 49, in which the DNA sequence encoding a signal peptide of glycoprotein comprises one derived from herpes simplex virus.
51. The plasmid of claim 48, in which the mutation of the HIV-1 nef gene fused to the 5'end of the HIV-1 vif gene comprises the deletion of codons for Argl37 and Argl38.
52. The plasmid of claim 51, in which the mutation of the HIV-1 nef gene fused to the 5'end of the HIV-1 vif gene comprises the deletion of codons for Argl37 and Argl38.
53. The plasmid of claim 48, in which the signal sequence DNA of secretory protein fused to the 3'end of any one of genes having from exon 1 to a full length of HIV-1 tat gene comprises the DNA sequence encoding a signal peptide of glycoprotein.
54. The plasmid of claim 53, in which the DNA sequence encoding a signal peptide of glycoprotein comprises one derived from herpes simplex virus.
55. The plasmid of claim 48, in which the HIV-1 tat gene is exon 1 of the tat gene.
56. The plasmid of claim 48, which is pGXIO-HIV/VNTV.
57. The plasmid of claim 48, which is pGXIO-HIV/TVVN.
58. A plasmid pGX10-hIL-12m.
59. A DNA vaccine composition for prophylaxis or treatment of AIDS comprising (i) plasmid pGXIO-SIV/GE and (ii) plasmid comprising the vector pGXIO and the SIVmac239 pol gene encoding reverse transcriptase and integrase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene which are operably linked to the vector.
60. The DNA vaccine composition of claim 59, which further comprises plasmid pGX10-hIL-12m.
61. A DNA vaccine composition for prophylaxis or treatment of AIDS comprising (i) plasmid comprising the SIVmac239 gag, dpol, env and rev genes, (ii) plasmid comprising the SIVmac239 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene, and (iii) plasmid comprising the SIVmac239 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 nef gene fused to the 3'and 5'ends of the vif gene, respectively, or plasmid comprising any one of genes having from exon 1 to a full length of the SIVmac239 tat gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively.
62. The DNA vaccine composition of claim 61, in which the plasmid comprising the SIVmac239 gag, dpol, env and rev genes is pTV-SIV/GE or pGXIO- SIV/GE.
63. The DNA vaccine composition of claim 61, in which the pol gene in the plasmid comprising the SIVmac239 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the pol gene is mutated so that the enzymatic activity of integrase can be inhibited.
64. The DNA vaccine composition of claim 63, in which the mutation inhibiting the enzymatic activity of integrase comprises the deletion of nucleotides 5130-5132 and the substitution of nucleotides 5133-5135 for a serine codon.
65. The DNA vaccine composition of claim 61, in which the plasmid comprising the SIVmac239 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene is pTV-SIV/dpol or pGXIO-SIV/dpol.
66. The DNA vaccine composition of claim 61, which further comprises plasmid pGX10-hIL-12m.
67. A DNA vaccine composition for prophylaxis or treatment of AIDS comprising (i) plasmid comprising the SIVmac239 gag, dpol, env and rev genes, (ii) plasmid comprising the SIVmac239 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene, (iii) plasmid comprising the SIVmac239 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 nef gene fused to the 3'and 5'ends of the vif gene, respectively, and (iv) plasmid comprising any one of genes having from exon 1 to a full length of the SIVmac239 tat gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively.
68. The DNA vaccine composition of claim 67, in which the plasmid comprising the SIVmac239 gag, dpol, env and rev genes is pTV-SIV/GE or pGXIO- SIV/GE.
69. The DNA vaccine composition of claim 67, in which the pol gene in the plasmid comprising the SIVmac239 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the pol gene is mutated so that the enzymatic activity of integrase can be inhibited.
70. The DNA vaccine composition of claim 69, in which the mutation inhibiting the enzymatic activity of integrase comprises the deletion of nucleotides 5130-5132 site and the substitution of nucleotides 5133-5135 site for a serine codon.
71. The DNA vaccine composition of claim 67, in which the plasmid comprising the SIVmac239 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene is pTV-SIV/dpol or pGXIO-SIV/dpol.
72. The DNA vaccine composition of claim 67, which further comprises plasmid pGX1O-hIL-12m.
73. A DNA vaccine composition for prophylaxis or treatment of AIDS comprising (i) plasmid comprising the SIVmac239 gag, dpol, env and rev genes, (ii) plasmid comprising the SIVmac239 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene, and (iii) plasmid comprising (a) SIVmac239 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 nef gene fused to the 3'and 5'ends of the SIVmac239 vif gene, respectively, and (b) any one of genes having from exon 1 to a full length of the SIVmac239 tat gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 vpx gene fused to the 5'end of the SIVmac239 tat gene, respectively.
74. The DNA vaccine composition of claim 73, in which the plasmid comprising the SIVmac239 gag, dpol, env and rev genes is pTV-SIV/GE or pGXIO- SIV/GE.
75. The DNA vaccine composition of claim 73, in which the pol gene in plasmid comprising the SIVmac239 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene is mutated so that the enzymatic activity of integrase can be inhibited.
76. The DNA vaccine composition of claim 75, in which the mutation inhibiting the enzymatic activity of integrase comprises the deletion of nucleotides 5130-5132 site and the substitution of nucleotides 5133-5135 site for a serine codon.
77. The DNA vaccine composition of claim 73, in which the plasmid comprising the SIVmac239 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene is pTV-SIV/dpol or pGXIO-SIV/dpol.
78. The DNA vaccine composition of claim 73, which further comprises plasmid pGX1O-hIL-12m.
79. A DNA vaccine composition for prophylaxis or treatment of AIDS comprising (i) plasmid pGXIO-HIV/GE and (ii) plasmid comprising the vector pGXIO and the HIV-1 pol gene encoding reverse transcriptase and integrase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV- 1 pol gene which are operably linked to the vector.
80. The DNA vaccine composition of claim 79, which further comprises plasmid pGX10-hIL-12m.
81. A DNA vaccine composition for prophylaxis or treatment of AIDS comprising (i) plasmid comprising HIV-1 gag, dpol, env and rev genes, (ii) plasmid comprising the HIV-1 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene, (iii) plasmid comprising HIV-1 vif gene and the DNA sequence encoding a signal peptide of secretory protein and HIV-1 nef gene fused to the 3'and 5'ends of the vif gene, respectively, or plasmid comprising any one of genes having from exon 1 to a full length of the HIV-1 tat gene and the DNA sequence encoding a signal peptide of secretory protein and HIV-1 vpx gene fused to the 3'and 5'ends of the HIV-1 tat gene, respectively.
82. The DNA vaccine composition of claim 81, in which the plasmid comprising the HIV-1 gag, dpol, env and rev genes is pTV-HIV/GE or pGXIO- HIV/GE.
83. The DNA vaccine composition of claim 81, in which the pol gene in plasmid comprising the HIV-1 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3' end of the pol gene is mutated so that the enzymatic activity of integrase can be inhibited.
84. The DNA vaccine composition of claim 83, in which the mutation inhibiting the enzymatic activity of integrase comprises the deletion of nucleotides 5130-5132 site and the substitution of nucleotides 5133-5135 site for a serine codon.
85. The DNA vaccine composition of claim 81, in which the plasmid comprising the HIV-1 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene is pTV-HIV/dpol or pGXIO-HIV/dpol.
86. The DNA vaccine composition of claim 81, which further comprises plasmid pGX10-hIL-12m.
87. A DNA vaccine composition for prophylaxis or treatment of AIDS comprising (i) plasmid comprising the HIV-1 gag, dpol, env and rev genes, (ii) plasmid comprising HIV-1 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV- 1 pol gene, and (iii) plasmid comprising (a) HIV-1 vif gene and the DNA sequence encoding a signal peptide of secretory protein and HIV-1 nef gene fused to the 3'and 5'ends of the vif gene, respectively, and (b) any one of genes having from exon 1 to a full length of HIV-1 tat gene and the DNA sequence encoding a signal peptide of secretory protein and HIV-1 vpx gene fused to the 5'end of the HIV-1 tat gene, respectively.
88. The DNA vaccine composition of claim 87, in which the plasmid comprising HIV-1 gag, dpol, env and rev genes is pTV-HIV/GE or pGXlO-HIV/GE.
89. The DNA vaccine composition of claim 87, in which the pol gene in plasmid comprising HIV-1 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene is mutated so that the enzymatic activity of integrase can be inhibited.
90. The DNA vaccine composition of claim 89, in which the mutation inhibiting the enzymatic activity of integrase comprises the deletion of nucleotides 5130-5132 site and the substitution of nucleotides 5133-5135 site for a serine codon.
91. A DNA vaccine composition for prophylaxis or treatment of AIDS comprising (i) plasmid comprising HIV-1 gag, dpol, env and rev genes, (ii) plasmid comprising the HIV-1 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene, (iii) plasmid comprising HIV-1 vif gene and the DNA sequence encoding a signal peptide of secretory protein and HIV-1 nef gene fused to the 3'and 5'ends of the vif gene, respectively, and (iv) plasmid comprising any one of genes having from exon 1 to a full length of the HIV-1 tat gene and the DNA sequence encoding a signal peptide of secretory protein and HIV-1 vpx gene fused to the 3'and 5'ends of the HIV-1 tat gene, respectively.
92. The DNA vaccine composition of claim 91, in which the plasmid comprising the HIV-1 gag, dpol, env and rev genes is pTV-HIV/GE or pGXIO- EV/GE.
93. The DNA vaccine composition of claim 91, in which the pol gene in plasmid comprising the HIV-1 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3' end of the pol gene is mutated so that the enzymatic activity of integrase can be inhibited.
94. The DNA vaccine composition of claim 93, in which the mutation inhibiting the enzymatic activity of integrase comprises the deletion of nucleotides 5130-5132 site and the substitution of nucleotides 5133-5135 site for a serine codon.
95. The DNA vaccine composition of claim 91, in which the plasmid comprising the HIV-1 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene is pTV-HIV/dpol or pGXlO-HIV/dpol.
96. The DNA vaccine composition of claim 91, which further comprises plasmid pGX1O-hIL-12m.
97. A DNA vaccine composition for prophylaxis or treatment of AIDS comprising (i) plasmid comprising the HIV-1 gag, dpol, env and rev genes, (ii) plasmid comprising. HIV-1 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV- 1 pol gene, and (iii) plasmid comprising (a) HIV-1 vif gene and the DNA sequence encoding a signal peptide of secretory protein and HIV-1 nef gene fused to the 3'and 5'ends of the vif gene, respectively, and (b) any one of genes having from exon 1 to a full length of HIV-1 tat gene and the DNA sequence encoding a signal peptide of secretory protein and HIV-1 vpx gene fused to the 5'end of the HIV-1 tat gene, respectively.
98. The DNA vaccine composition of claim 97, in which the plasmid comprising HIV-1 gag, dpol, env and rev genes is pTV-HIV/GE or pGXIO-HIV/GE.
99. The DNA vaccine composition of claim 97, in which the pol gene in plasmid comprising HIV-1 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene is mutated so that the enzymatic activity of integrase can be inhibited.
100. The DNA vaccine composition of claim 99, in which the mutation inhibiting the enzymatic activity of integrase comprises the deletion of nucleotides 5130-5132 site and the substitution of nucleotides 5133-5135 site for a serine codon.
101. The DNA vaccine composition of claim 97, in which the plasmid comprising the HIV-1 pol gene encoding reverse transcriptase and invertase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene is pTV-HIV/dpol or pGXIO-HIV/dpol.
102. The DNA vaccine composition of claim 97, which further comprises plasmid pGX10-hIL-12m.
TECHNICAL FIELD
The present invention relates to an AIDS DNA vaccine. More particularly, the present invention relates to SIVmac239 and HIV immunogenic plasmids and AIDS DNA vaccines containing the plasmids.
BACKGROUND ART
DNA vaccination is the most recently developed vaccination method. Ertl et al. disclosed a method for administering a live or dead vaccine in a strategy for common immunization (Ertl et al. , J. Immunol. 156, 3579-3582 (1996) ). Hassett et al. warned that a live vaccine may be dangerous in case of a human or animal patient with lowered immunity and or pregnant patient, since the antigen in the vaccine can revert to or mutate into a pathogenic form, though it can generally elicit an effective immune response in a vaccinated body (Hassett et al. , Trends in Microbiol. 8,307-312 (1996) ).
Recently, Tang et al. found that an expression plasmid encoding an antigenic protein that is directly injected into mouse can induce an antibody response (Tang, D. C. , et al., Nature (Lond. ) 356,152-154 (1992) ). This suggested that injection of naked DNA may express an antigen type capable of inducing an immune response. Also, there has been a report that intramuscular injection of plasmid DNA encoding nucleoprotein of influenza may protect a mouse infected with a live influenza virus, thereby opening a new era for development of vaccines (Ulmer, J. B. , et al., Science 259,1745-1749 (1993); and Fynan, E. F. , et al., Proc. Nat. Acad. Sci. USA 90,11, 478-482 (1993)).
Also, it has been shown that immunization with plasmid DNA may activate both humoral immunity and cellular immunity, including production of antigen-specific CD8+ cytotoxic T cells as well as CD4+ T helper cell (Donnellym, J. J. , et al. , Ann Rev. Immunol. 15,617-648 (1997) ). In addition, Feigner et al. disclosed a method for delivering an isolated polynucleotide such as DNA or RNA, in which the polynucleotide is intramuscularly administered to mammals so that muscle cells absorb the polynucleotide, the method having therapeutic effects in the mammals (US PAT. No. 5, 589,466).
In general, it is thought that the protection level achieved by a DNA vaccine is lower than those observed in cases naturally recovered from infection with virus (Manickan et al., Critical Review Immunol. 17, 139-154 (1997) ) but is similar to levels induced by conventional protein-specific antigen vaccines and dead or attenuated virus vaccines. However, a DNA vaccine comprising naked plasmid DNA has some merits, as compared to immunization methods depending upon injection of purified or recombinant protein, or attenuated live or recombinant virus, as follows. (1) Genes encoding a particular tumor antigen and/or immunomodulatory cytokine can be introduced at the same time, since one or more genes can be easily introduced at the same time. (2) Only a desired gene can be transcribed without immunological interference from virus protein in vivo and in vitro. (3) There is no risk of recombination, which can occur when using replication-defective viral vectors. (4) It is substantially impossible for foreign DNA to be inserted into the host genome due to the transient nature of gene transfer. (5) DNA vectors which will be used in this method can be readily prepared. (6) It is possible to simultaneously induce humoral and cellular immune responses against a variety of antigens and to control characteristics of immune responses by simultaneously delivering genes encoding immunoregulatory cytokines and costimulatory molecules.
AIDS (Acquired Immune Deficiency Syndrom) is a disease which has been continuously studied since the first diagnosis in 1981. Upon infection with HIV, the number of CD4+ T cells is dramatically reduced, causing serious damage to the immune system. As a result, complex opportunistic infections or neoplasias arise due to the compromised immune system. So far, in order to treat AIDS, numerous types of vaccines has been developed, including for example, inactivated whole virus vaccines, live recombinant virus vaccines, attenuated virus vaccines, specific antigen subunit vaccines, synthetic peptide vaccines and anti-idiotype antibodies, etc. For example, an inactivated whole virus vaccine is disclosed in US PAT. No. 5,698, 432 to John Sidney, an attenuated virus vaccine is disclosed in US PAT. No. 6,004, 799 to Luciw et al. , specific antigen subunit vaccines are disclosed in US PAT. No. 6,331, 404 to Bermann et al. , US PAT No. 6,083, 504 to Cotropia, and a report by Dolin et al. (Ann Intern. Med. , 114,119-127 (1991) ), synthetic peptide vaccines are disclosed in US PAT. No. 6,139, 843 to Rubinstein et al. and US PAT. No. 5,817, 318 to Sia et al. However, the methods using these vaccines have problems, for example, evasion of immune response in vivo, such as HIV escaping immune recognition through its mutation, and risk of infection due to recovery of pathogenicity of a virus vector which has been administered as an attenuated vaccine. Furthermore, the methods failed to show desired prophylactic or therapeutic effects versus AIDS.
To the contrary, it has been found that plasmid DNA, when administered to primates, induced humoral immune response and cell-mediated immune respone, and effectively induced Thl (T helper-1) bias immune response and CTL response, which are known to be important for protection against viruses such as HIV-1, especially in small animals, monkeys, chimpanzees and humans. There are many DNA vaccines against AIDS. For example, EP 0276591 disclosed a vaccine consisting of a viral vector and recombinant DNA coding for the p25 protein of the AIDS virus. The viral vector is characterized by comprising a part of the genome of a vector virus; the complete gag gene or one of its fragments, especially a gene coding for the p25 protein or a gene coding for the pl8 protein of the HIV virus responsible for AIDS ; and elements ensuring the expression of these proteins in cells during culturing.
EP 0572737 disclosed a substantially pure HIV antigen comprising a Gag-Env fusion protein consisting of a Gag polypeptide fused at its C-end to an Env peptide.
FR 2596771 disclosed a viral vector characterized by comprising : a portion of the genome of a virus, a gene encoding one of the glycoproteins (gp) of the envelope of the virus responsible for AIDS ; and elements ensuring the expression of this glycoprotein in cells.
WO 99277958 disclosed an AIDS vaccine based on HIV-1 Tat as immunogen, in which HIV-1 Tat is inoculated either as DNA and/or recombinant protein or as peptides; alone or in combination with other genes or viral gene products (Nef, Rev, Gag) or parts thereof ; or in combination with various immunomodulant cytokines (IL- 12, IL-15) or with the gene coding for an immunomodulant cytokine or part thereof.
According to this patent, Tat, Nef, Rev, Gag and the immunomodulant cytokines are administered both as a mixture of recombinant proteins, peptides or fusion proteins (Tat/Nef, Tat/Rev, Tat/Gag, Tat/IL-12, Tat/IL-15), or as plasmid DNA.
Also, Roger Miller and Nava Sarver reported that when a Rhesus macaques monkey was inoculated with vif gene-deficient SIVmac239 wherein and the deficient virus was attenuated expression type, the monkeys formed a low level of antibody against SIVmac239 (Roger Miller and Nava Sarver, HIV Vif as a Therapeutic Target, DAIDS, NIAID, Sept. 18,2000). However, it was described that the monkeys immunized with vif-deficient SIV cannot be protected against infection of wild-type SIV H. Zhang, et al. disclosed that a mutated Gag could induce increased cytotoxicity in cells, as compared to a wild-type Gag (H. Zhang and L. Qiao, 184 HIV Gag DNA Vaccine, AIDS VACCINE, Foundation for AIDS Vaccine Research and Development, (2001) ). However, such result was obtained in immunized mice with a DNA vaccine coding the mutated Gag.
As described in AIDS WEEKLY Plus, Monday, (AW) Conference Coverage (Retrovirus), March 24,1997, Britta Wahren et al. showed that a plasmid containing attenuated HIV-1 nef, rev and tat genes, a gene encoding p24 structural protein and a gene encoding gap 160 envelope precursor glycoproteins can induce cellular and humoral anti-HIV immune response. However, this study was carried using mice as an experimental animal.
It was also described in AIDS WEEKLY Plus, Monday, (AW) Conference Coverage (NCVDG), June 23,1997 that Velpandi Ayyavoo and his colleagues showed that a naked DNA vaccine containing the attenuated HIV-1 genes, vif, nef, vpr and vpu could induce cellular and humoral anti-HIV immune response.
Amara et al. reported a study in which primates were immunized with a DNA vaccine containing many HIV and SIV genes (HIV-1 envelope, tat and rev genes and SIV gag, pol, vif, vpx and vpr genes) and a MVA (modified Vaccinia Ankara) booster vaccine containing HIV-and SIV-derived genes (Amara R. R., et al., Science 292,69- 74, (2001). According to their report, it was demonstrated that the strong vaccine- induced immune response in rhesus macaques monkey/SHIV model can control viral replication and diseases progression.
However, in spite of various studies in the prior arts, there has been no descriptions or suggestions of a DNA vaccine capable of successfully preventing or treating AIDS. Therefore, there are still demands for a novel immunogenic plasmid with improved expression efficiency and immunogenicity, and an effective and safe DNA vaccine.
DISCLOSURE OF THE INVENTION
Now, the inventors have developed immunogenic plasmids showing excellent expression efficiency and immunogenicity in SIVmac239/rhesus macaques monkey and AIDS human patients and DNA vaccines for prophylaxis or therapy of AIDS containing the same. We have developed two types of vaccine plasmids for AIDS in the previous study (Korean Patent Application Laid-Open No. 2001-0054338 and its corresponding US Patent Publication No. 2001004531). The first immunogenic plasmid comprises the vector pTV2 which was developed as a basic vector for a DNA vaccine, and the gag, dpol (corresponding to protease) and env genes derived from the virus SIVmac239 and the regulatory gene rev derived from the virus SIVmac239, but does not comprise the regulatory genes tat and nef ; and the second immunogenic plasmid comprises the vector pTV2 which was developed as a basic vector for a DNA vaccine, and the SIVmac239 pol gene encoding reverse transcriptase (RT) and integrase and a DNA sequence encoding a signal peptide of glycoprotein D (gD) of HSV (Herpes Simplex Virus) fused to the pol gene. These first and second immunogenic plasmids were developed to make up for defects of conventionally developed AIDS DNA vaccines in which the regulatory genes nef and tat, known to inhibit or disturb immune response ex vivo, were used, and the pol gene including many CTL epitopes which are known to be important for immunogenicity was not effectively used. According to the results of experiments, these immunogenic plasmids were shown to have excellent expression efficiencies and immune efficacies to some degrees in the SIVmac239/rhesus monkey model.
However, the inventors have now developed a novel ADIS DNA vaccine showing improved effects by augmenting our previous invention. One feature of the present invention is the vector pGXIO developed as a basic vector for a DNA vaccine.
The pGXIO is confirmed to have a higher level of expression upon cell infection ex vivo, and to induce more excellent immune response in vivo (upon immunization of mouse), as compared to the vector pTV2. Also, in the present invention, in order to minimize the down-regulation of immune responses by the regulatory genes nef and tat, which are excluded in the previous invention and increase the expression efficiency of a gene to be introduced, portions of the nef and tat genes are used, not their full-length sequence, and the vector is designed so that the nef and tat genes are expressed through fusion with another regulatory gene, thereby achieving codon optimization. As a result, we have succeeded in developing immunogenic plasmids showing significantly increased expression efficiency of immunogens and immune efficacy as compared to the previous invention, and effective and safe DNA vaccines containing the same.
In accordance with one aspect, the present invention provides the vector pGXIO as a basic vector for producing immunogenic plasmids which are used in the AIDS DNA vaccine according to the present invention.
In accordance with another aspect, the present invention provides the immunogenic plasmid pGXIO-SIV/GE, which is used in the AIDS DNA vaccine composition according to the present invention, characterized by comprising the SIVmac239 gag, dpol and env genes and rev regulatory gene.
In accordance with another aspect, the present invention provides an immunogenic plasmid, which is used in the AIDS DNA vaccine composition according to the present invention and characterized by comprising the vector pGXIO and the SIVmac239 pol gene encoding reverse transcriptase (RT) and integrase (INT) and a DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene which are operably linked to the vector.
In accordance with another aspect, the present invention provides an immunogenic plasmid, which is used in the AIDS DNA vaccine according to the present invention and characterized by comprising the SIVmac239 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 nef gene fused to the 3'and 5'ends of the SIVmac239 vif gene, respectively.
In accordance with another aspect, the present invention provides an immunogenic plasmid, which is used in the AIDS DNA vaccine according to the present invention and characterized by comprising any one of genes having from exon 1 to the full length of the SIVmac239 tat gene, and a signal sequence of secretory protein and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively.
In accordance with another aspect, the present invention provides an immunogenic plasmid, which is used in the AIDS DNA vaccine according to the present invention and characterized by comprising (i) the SIVmac239 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 nef gene fused to the 3'and 5'ends of the SIVmac239 vif gene, respectively, and (ii) any one of genes having from exon 1 to a full length of the SIVmac239 tat gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively.
In accordance with another aspect, the present invention provides the immunogenic plasmid pGX10-HIV/GE, which is used in the AIDS DNA vaccine (composition) according to the present invention, characterized by comprising the HIV- 1 gag, protease and env genes and rev regulatory gene.
In accordance with another aspect, the present invention provides an immunogenic plasmid, which is used in the AIDS DNA vaccine (composition) according to the present invention and characterized by comprising the vector pGXIO and the HIV-1 pol gene encoding reverse transcriptase (RT) and integrase (INT) and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene which are operably linked to the vector.
In accordance with another aspect, the present invention provides an immunogenic plasmid, which is used in the AIDS DNA vaccine according to the present invention and characterized by comprising the HIV-1 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the HIV-1 nef gene fused to the 3'and 5'ends of the HIV-1 vif gene, respectively.
In accordance with another aspect, the present invention provides an immunogenic plasmid, which is used in the AIDS DNA vaccine according to the present invention and characterized by comprising any one of genes having from exon 1 to the full length of the HIV-1 tat gene, and the DNA sequence encoding a signal peptide of secretory protein and the HIV-1 vpx gene fused to the 3'and 5'ends of the HIV-1 tat gene, respectively.
In accordance with another aspect, the present invention provides an immunogenic plasmid, which is used in the AIDS DNA vaccine according to the present invention and characterized by comprising (i) the HIV-1 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the HIV-1 nef gene fused to the 3'and 5'ends of the HIV-1 vif gene, respectively, and (ii) any one of genes having from exon 1 to a full length of the HIV-1 tat gene and the DNA sequence encoding a signal peptide of secretory protein and the HIV-1 vpx gene fused to the 3' and 5'ends of the HIV-1 tat gene, respectively.
In accordance with another aspect, the present invention provides the adjuvant plasmid pGX10-hIL-12m, which can be used in the AIDS DNA vaccine according to the present invention.
In accordance with another aspect, the present invention provides a DNA vaccine composition for prophylaxis or treatment of AIDS, characterized by comprising (i) plasmid pGXIO-SIV/GE and (ii) plasmid comprising the vector pGXIO and the SIVmac239 pol gene encoding reverse transcriptase and integrase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene which are operably linked to the vector pGX10.
In accordance with another aspect, the present invention provides a DNA vaccine composition for prophylaxis or treatment of AIDS, characterized by comprising (i) plasmid comprising the SIVmac239 gag, dpol, env and rev genes, (ii) plasmid comprising the SIVmac239 pol gene encoding reverse transcriptase and integrase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene, (iii) plasmid comprising the SIVmac239 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 nef gene fused to the 3'and 5'ends of the vif gene, respectively, and/or (iv) plasmid comprising any one of genes having from exon 1 to a full length of the SIVmac239 tat gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively.
In accordance with another aspect, the present invention provides a DNA vaccine composition for prophylaxis or treatment of AIDS, characterized by comprising (i) plasmid comprising the SIVmac239 gag, dpol, env and rev genes, (ii) plasmid comprising the SIVmac239 pol gene encoding reverse transcriptase and integrase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene, and (iii) plasmid comprising (a) SIVmac239 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 nef gene fused to the 3'and 5'ends of the SIVmac239 vif gene, respectively, and (b) any one of genes having from exon 1 to a full length of the SIVmac239 tat gene and the DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 vpx gene fused to the 5'end of the SIVmac239 tat gene, respectively.
In accordance with another aspect, the present invention provides a DNA vaccine composition for prophylaxis or treatment of AIDS, characterized by comprising (i) plasmid pGX10-HIV/GE and (ii) plasmid comprising the vector pGXIO and the HIV-1 pol gene encoding reverse transcriptase and integrase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV- 1 pol gene which are operably linked to the vector pGX10.
In accordance with another aspect, the present invention provides a DNA vaccine composition for prophylaxis or treatment of AIDS, characterized by comprising (i) plasmid comprising the HIV-1 gag, dpol, env and rev genes, (ii) plasmid comprising the HIV-1 pol gene encoding reverse transcriptase and integrase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene, (iii) plasmid comprising the HIV-1 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the HIV-1 nef gene fused to the 3' and 5'ends of the vif gene, respectively, and/or (iv) plasmid comprising any one of genes having from exon 1 to a full length of the HIV-1 tat gene and the DNA sequence encoding a signal peptide of secretory protein and the HIV-1 vpx gene fused to the 3' and 5'ends of the HIV-1 tat gene, respectively.
In accordance with another aspect, the present invention provides a DNA vaccine composition for prophylaxis or treatment of AIDS, characterized by comprising (i) plasmid comprising the HIV-1 gag, dpol, env and rev genes, (ii) plasmid comprising the HIV-1 pol gene encoding reverse transcriptase and integrase and the DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene, and (iii) plasmid comprising (a) HIV-1 vif gene and the DNA sequence encoding a signal peptide of secretory protein and the HIV-1 nef gene fused to the 3'and 5'ends of the HIV-1 vif gene, respectively, and (b) any one of genes having from exon 1 to a full length of the HIV-1 tat gene and the DNA sequence encoding a signal peptide of secretory protein and the HIV-1 vpx gene fused to the 5' end of the HIV-1 tat gene, respectively.
BRIEF DESCRIPTION OF THE DRAWINGS
The above objects, and other features and advantages of the present invention will become more apparent after a reading of the following detailed description when taken in conjunction with the drawings, in which:
Fig. 1 is a gene map of the vector pGXIO used to. express the SIVmac239 immunogenic gene according to the present invention, wherein SV40 pA refers to SV40 polyA ;
Fig. 2 is a gene map of the vector pGXIO (3.6 kb) used to express the SIVmac239 immunogenic gene according to the present invention;
Fig. 3 is a restriction map of the vector pGXIO (3.6kb) comprising 3641 nucleotides, which is used to express the SIVmac239 immunogenic gene according to the present invention, wherein the CMV promoter corresponds to nucleotides 3619- 3641 and nucleotides 1 to 661 ; the TPL corresponds to nucleotides 666 to 1101 ; the SV40 late polyA sequence corresponds to nucleotides 1236 to 1457; the SV40 enhancer corresponds to nucleotides 1469 to 1713; the kanamycin resistance ORF corresponds to nucleotides 1727 to 2521; and the ColEl origin corresponds to nucleotides 2907 to 3580, and the specific restriction sites are underlined;
Fig. 4 is a restriction map of the SIVmac239 clone;
Fig. 5 is a construction map of pGXIO-SIV/GE according to the present invention, which is used as the SIVmac239 immunogenic plasmid;
Fig. 6 is a construction map of pGXIO-SIV/dpol according to the present invention, which is used as the SIVmac239 immunogenic plasmid;
Fig. 7 is a construction map of pGXIO-SIV/VN according to the present invention, which is used as the SIVmac239 immunogenic plasmid ;
Fig. 8 is a construction map of pGXIO-SIV/TV according to the present invention, which is used as the SIVmac239 immunogenic plasmid;
Fig. 9 is a construction map of pGXIO-SIV/VNTV according to the present invention, which is used as the SIVmac239 immunogenic plasmid ;
Fig. 10 is a construction map of pGXIO-SIV/TVVN according to the present invention, which is used as the SIVmac239 immunogenic plasmid;
Fig. 11 is a construction map of pGXIO-HIV/GE according to the present invention, which is used as the HIV-1 immunogenic plasmid ;
Fig. 12 is a construction map of pGX10-HIV/dpol according to the present invention, which is used as the HIV-1 immunogenic plasmid;
Fig. 13 is a construction map of pGXIO-HIV/VN according to the present invention, which is used as the HIV-1 immunogenic plasmid;
Fig. 14 is a construction map of pGXIO-HIV/TV according to the present invention, which is used as the HIV-1 immunogenic plasmid;
Fig. 15 is a construction map of pGXIO-HIV/VNTV according to the present invention, which is used as the HIV-1 immunogenic plasmid;
Fig. 16 is a construction map of pGXIO-HIV/TVVN according to the present invention, which is used as the HIV-1 immunogenic plasmid;
Fig. 17 is a construction map of the adjuvant plasmid pGX1O-hp35/IRES/hp40 (pGX10-hIL-12m) according to the present invention, which is used as an adjuvant for the AIDS DNA vaccine of the present invention;
Fig. 18 is a graph showing expression levels of the human growth hormone expressed by the vector pGXIO, which is used to express the SIVmac239 immunogenic gene according to the present invention, and by the conventional vectors pTV2 and pGXI which are used as controls;
Fig. 19 is a construction map of the plasmid pTV2/hGH, which is used as a control for comparison of levels of the human growth hormone expressed by the vector pGXIO, which is used to express the SIVmac239 immunogenic gene according to the present invention;
Fig. 20 is a construction map of the plasmid pGXI/hGH, which is used as a control for comparison of levels of the human growth hormone expressed by the vector pGXIO, which is used to express the SIVmac239 immunogenic gene according to the present invention;
Fig. 21 is a restriction map of the adjuvant plasmid pAGGSIL-4, which is used as an adjuvant for the AIDS DNA vaccine according to the present invention;
Fig. 22 is a restriction map of the adjuvant plasmid pCAGGSIL-12, which is used as an adjuvant for the AIDS DNA vaccine according to the present invention;
Fig. 23 is a construction map of the plasmid pGXO/hGH, which is used as a control for comparison of abilities to induce immune response, as evaluated by expression of the human growth hormone, with the vector pGXIO, which is used to express the SIVmac239 immunogenic gene according to the present invention;
Fig. 24 is a construction map of the plasmid pGXIO/hGH, which is used as a control for comparison of abilities to induce immune response, through expression of the human growth hormone, with the vector pGXIO, which is used to express the SIVmac239 immunogenic gene according to the present invention;
Fig. 25 is a graph showing the abilities to induce immune response, through expression of human growth hormone, of the vector pGX10, which is used to express the SIVmac239 immunogenic gene according to the present invention, and the plasmid pTV2/hGH and pGXO/hGH, which are used as controls ;
Fig. 26 is a experimental protocol for evaluating the vaccine efficiencies of the immunogenic plasmids according to the present invention;
Fig. 27 is a graph showing the vacine efficiencies of the immunogenic plasmids according to the present invention, evaluated by counting the number of peripheral blood mononuclear cells (PBMC) in blood of Rhesus monkeys infected with SIVmac239. The results are expressed as the number of infectious PBMC per one million PBMC in blood of the treated monkey at various points of time after infection of the monkeys with SIVmac239. The X axis represents time (weeks) elapsing after infection with SIVmac239, the Y axis represents the number of infectious PBMC, and the 4 to 5 digit numbers represent the assigned numbers of respective monkeys;
Fig. 28 is a graph showing the vaccine efficiencies of the immunogenic plasmids according to the present invention, evaluated by counting copies of SIV RNA in blood plasma of Rhesus monkeys infected with SIVmac239. The results are expressed as the titers of SIV RNA detected in Iml of the blood plasma of the treated monkeys at various points of time after infection of the monkeys with SIVmac239. The X axis represents time (weeks) elapsing after infection with SIVmac239, the Y axis represents the number of SIVmac239 RNA molecules per lml of the blood plasma, and the 4 to 5 digit numbers represent the assigned numbers of respective monkeys;
Fig. 29 is a graph showing the vaccine efficiencies of the immunogenic plasmids according to the present invention, evaluated by measuring the number of absolute CD4+ cells in blood of Rhesus monkeys infected with SIVmac239. The results are expressed as percentages of the absolute number of CD4+ cells per unit volume of blood, relative to the absolute number of CD4+ cells per unit volume of blood before infection in the treated monkeys at various points of time after infection of the monkeys with SIVmac239. The X axis represents time (week) elapsing after infection with SIVmac239, the Y axis represents the percentages of the number of CD4+ cells in 1 je of blood relative to the CD4+ cells in 1, ue of blood before infection in the treated monkeys at various points of time, and the 4 to 5 digit numbers represent the assigned numbers of respective monkeys;
Fig. 30 is a graph showing the vaccine efficiencies of the immunogenic plasmids according to the present invention, evaluated by measuring gag-specific T-cell response induced by SIV DNA immunization. The results are obtained by measuring the T-cell immune response induced by immunization in monkeys undergoing respective treatments just before infection with SIVmac239. The X axis represents the assigned numbers of respective monkeys, and the Y axis represents the number of cells secreting IFN-in response to stimulation by the gag peptide per one million PBMC;
Fig. 31 is a view showing the results of a Western blot analysis in which the plasmids pGXIO-SIV/GE and pGXIO-SIV/dpol according to the present invention, and the immunogenic plasmid pTV2-SIV/GE as a control, were transfected into HeLa cells and examined for the expression of antigen proteins by immunoblotting;
Fig. 32 is a view showing the results of a Western blot analysis in which the plasmids pGXIO-SIV/GE and pGXIO-SIV/dpol according to the present invention, and the immunogenic plasmid pTV2-SIV/GE and pTV2-SIV/dpol as controls, were transfected into HeLa cells and examined for the expression of antigen proteins by immunoblotting;
Fig. 33 is a view showing the results of a Western blot analysis in which the plasmids pGXIO-SIV/VN and pGXIO-SIV/VNTV according to the present invention were transfected into HeLa cells and examined for the expression of adjuvant antigen proteins (Vif-Nef) by immunoblotting; and,
Fig. 34 is a view showing the results of a Western blot analysis in which the plasmid pGXIO-SIV/TV according to the present invention was. transfected into HeLa cells and examined for the expression of adjuvant antigen protein (Tat-Vpx) by immunoblotting.
BEST MODE FOR CARRYING OUT THE INVENTION
As described above, there are many reports describing protective effects of vaccines against AIDS virus in monkeys. In order to determine the efficacy of a vaccine substance in a primate model, various species of monkeys are infected with various viruses and examined for protective effects of the vaccine. Here, the model can be diverse according to the species of the used non-human primates (chimpanzee, monkey) and the types of infecting virus (HIV-1, HIV-2, SHIV, SIVmne, SIVmac).
Some models readily induce protection, while some models using a certain combination of monkeys and virus do not induce AIDS. Thus, these models can be classified into various types according to (1) the levels of virulence and severity of disease conditions, and (2) levels of induced protection, and efficacy of vaccines in the above various models are now carefully studied.
A representative model known to induce AIDS in monkey is a model in which a monkey is infected with SHIV89.6P. However, this model (1) uses virus which is produced by artificial recombination; (2) induces abnormally rapid decline in CD4 levels, thereby leading to death ; (3) shows conditions by infection itself as well as symptoms of an immune deficiency disease condition ; and (4) readily induces protection, as compared to the SIVmac239/monkey, as observed from many cases achieving successful protection using attenuated virus, DNA and recombinant virus vector, though it has been very recently developed, unlike SIVmac239. However, when using an attenuated virus as a vaccine, there are safety problems, since the attenuated virus can transform into a pathogenic virus. As another example, it has been attempted to induce protection through immunization with DNA and infection of blood with the virus SIVmac251. However, this attempt failed to induce protection, which caused reduction of CD4 levels in monkey, leading to death.
The present invention uses a model in which a DNA vaccine is administered to Rhesus macaques, which is then blood-infected with SIVmac239 to determine whether the vaccine can protect the monkey against the virus. The virus SIVmac239 is obtained by subjecting the virus SIVmac251 twice to in vivo passage, and hence has very similar base sequence, virulence and pathology to SIVmac251. There is no DNA vaccine which shows protective effects against blood infection with the virus SIVmac239. This model is characterized in that it can induce AIDS and also has an infection route, immunological indices after infection (CD4 number, CD29+CD4+T cell), and time to reach the maximum virus titer, which are all similar to the HIV-1 infection process in human beings. For this reason, the model used in the present invention is used in studies to determine the natures of protective immunization against HIV-1 in human beings, i. e. to determine which immune response should be induced to protect against the infection by HIV-1. Defects of this model are rapid development of AIDS and inevitable death. However, the rapid development can be an advantage of the model in that results can be obtained in a short time. Another feature of this model is that it does not readily induce protection, as seen from the fact that only an immunization method using a vaccine of attenuated virus has succeeded in protection.
A DNA vaccine has been developed, which can successfully protect against the blood infection with the virus SIVmac239. In vaccine fields, "successful protection" means that, upon infection with virus after administration of a vaccine, one of the following conditions is observed: (1) no proliferation of the virus is observed and the virus is removed (sterilizing immunity has been induced); (2) proliferation of the virus is observed in the early infection stage but removed later (without development of any disease); (3) proliferation of the virus is suppressed for a long period of time and no disease is developed (no infection); and (4) the patient slowly develops a disease condition while maintaining the virus titer at a low level, thereby preventing infection.
It is believed that the reason that the DNA vaccine of the present invention can succeed in protection against blood infection of the virus SIVmac239 is by virtue of development of an excellent vaccine vector, codon optimization and effective use of adjuvant (regulatory) gene. The vector pGXIO which is developed as one aspect of the present invention is formed by augmenting the vector pTV2, which has been previously developed and filed by the present inventors, and the vector pTX, which has been disclosed by Lee A. H. et al. (Vaccine 1999,17 : 473-9). This vector has been proven to have a high level of expression in vitro (10 times higher than the vector pTV2) and also to show excellent immune response in vivo (inducing 10 times more antibody response than the vector pTX). As for the immunogenic plasmid which is used in the DNA vaccine according to the present invention, the gene pol and adjuvant (regulatory) gene are fused with the DNA sequence encoding a signal peptide of secretory protein for codon optimization. The signal sequence of the secretory protein, for example, a signal sequence of gD (glycoprotein D) of Herpes Simplex virus has been shown to increase an expression level of a gene which has been fused thereto and immune response, particularly cell immune response, in vivo (Lee et al. , J. Virol 72 (10), 8430-36 (1998); in a DNA immunization using HCV structural gene, an increase of CTL responses were observed, as compared to immunization of ST DNA) Thus, in addition to the genes gag, pol and env, which have been conventionally used, adjuvant genes vif, nef, tat and vpx are used in the immunization. The gene vpr is not used as an adjuvant gene, since it is known to have an immune inhibitory effect (Ayyavoo Y, et al. , Int Immunol. , 14 (1), 13-22 (2002) ). Meanwhile, the genes nef and tat have not been used, due to their immune disturbance effects. Therefore, in the present invention, the genes nef and tat are fused to other adjuvant genes vif and vpx, respectively to produce expression vectors, so that their immune disturbance activities can be reduced. Also, where appropriate, not the full-length nef and tat genes, but a portion of them is used.
DEFINITION OF TERMINOLOGY
All the technically and scientifically related terminologies, which are included in the specification but are not defined, have meanings commonly accepted in the pertinent field of the present invention. Some of them are herein after defined in order to make clear their meanings, as follows: The term "vector" as used herein refers to a DNA molecule which acts as a carrier which can safely deliver a foreign gene into a host cell. Also, in order to be a useful vector, it should be replicable and should have a device by which it can be introduced into the host cell, and there should be provided means of detecting its presence. Here, examples of the foreign gene include structural and adjuvant genes of SIVmac239 and HIV The term "plasmid" as used herein generally refers to a circular DNA molecule in which a foreign gene is operably linked to a vector so as to be expressed in a host cell. However, a plasmid can be a vector in that it is used to carry a foreign gene by treatment of certain restriction enzymes. Therefore, in this application, the terms plasmid and vector are interchangeably used, but may be distinguished in their meanings by those having ordinary knowledge in the genetic engineering field without clarification of their meanings.
The term "immunogenic plasmid" as used herein refers to a circular DNA molecule which includs a gene encoding an antigen and induces antigen-specific humoral and cell-mediated immune responses.
The term "adjuvant plasmid" as used herein refers to a circular DNA molecule which expresses an immunoregulatory molecule to promote antigen-specific humoral and cell-mediated immune responses induced by an immunogenic plasmid.
The term "structural gene" as used herein refers to gag, env and pol genes coding for structural proteins of SIVmac239 and HIV-1. The gag gene produces proteins having molecular weights of 55,000 (p55) daltons, 24,000 (p24) daltons, 17,000 (pl7) daltons and 15,000 (pl5) daltons. The p55 antigen is a precursor which is formed in the early stage of infection and then divided into different core proteins.
The gag protein exists in an inner nucleocapsid of virus. The pl7 protein forms the matrix between the core and envelope and is buried in an inner part of the envelope.
The p24 and pi 5 proteins form the core coats enclosing the nucleic acids. The env gene produces glycoproteins having molecular weights of 160,000 (gp160) daltons, 120,000 (gel20) daltons and 41,000 (gp41) daltons. The gpl60 is a precursor of the glycoproteins gpl20 and gp41 and is not a constitutional component of mature virus.
The gp41 protein exists between the inner membrane and outer membrane and therefore, is also called a transmembrane protein. The gp120 protein forms 72 nobs over the envelope. The gp41 protein is involved in binding to CD4 molecule of a host cell, together with the gp120. The pol gene produces p66, p51 (reverse transcriptase), and p31 (integrase or endonuclease) proteins. The polymerase component plays a role in reverse-transcription of RNA to DNA and integration of DNA into cellular DNA, and functions to cleave a precursor into smaller active materials. The polymerase antigen exists within the core, in connection with nucleic acids. The gpl60 and p55 proteins are precursors, which are secreted to blood during replication of the virus to produce antibodies, whereby they can be used to detect antibodies against these precursors by a serological method. In the present invention, the first immunogenic plasmid construct includes the nucleotide sequence corresponding to the protease coding part (not the full-length sequence) of the structural gene pol, which is expressed herein as "dpol". Also, the second immunogenic plasmid construct includes only the nucleotide sequence encoding reverse-transcriptase and integrase (not the full-length sequence) of the structural gene pol, which is expressed herein as"RT-INT". The RT- INT coding part can be optionally mutated.
The term "regulatory gene" or "adjuvant gene" as used herein refers to the nef, vpr, vpu, tat, rev and vif genes, which encode regulatory proteins of SIVmac239 and HIV-1. Products of these regulatory genes function to modify expression of viral proteins and replication of virus, and regulate infectivity of the virus. Although exact roles of these genes are not yet known, it has been found that tat (pu4) has transcription activity, rev (pi 9/20) regulates expression of virus mRNA, nef (p27) has various functions such as inhibition of CD4 receptor and regulation of T cell activity, vif (p23) increses infectivity of the virus, vpr (p15) supports replication of virus, vpu (p16) is involved in release of virus and vpx (pal5) affects infectivity of the virus. However, in the present invention, vpr among the foregoing regulatory genes, is not used.
The term "operably linked" as used herein means that the respective components of a plasmid or vector are arranged so as to exert their own functions.
Therefore, a control sequence operably linked to a coding sequence can affect expression of the coding sequence. The control sequence does not need to lie adjacent to the coding sequence as long as the control sequence can act to regulate the expression of the coding sequence. For example, when an intervening sequence is disposed between a promoter sequence and a coding sequence, the promoter sequence can be said to be"operably linked"to the coding sequence.
Vector pGX10. The present inventors developed a basic vector for an immunogenic plasmid to be contained in an AIDS DNA vaccine, and designated the pGXIO. As shown in Fig. 2 and Fig. 3, the vector pGXIO of the invention is a novel vector of 3.6 kb, characterized by comprising SV40 ori, simian virus 40 replication origin, cytomegalovirus (CMV) early promoter/enhancer sequence, adenovirus tripartite leader sequence (TPL), multi-cloning site (MCS), simian virus 40 polyadenylation sequence (SV40PA), simian virus 40 enhancer sequence (SV40Eh), ColEl Ori and a kanamycin resistance gene. This vector can proliferate in E. coli, and has a plurality of particular restriction sites.
The vector pGXIO was prepared from a known vector, that is pTX, which had been previously disclosed by the present inventors (Lee A. H. , et al., Vaccine 17: 473-9 (1999) ), as described and illustrated in Example 1 and Fig. 1, and can be prepared using pTV2 vector, which has been used as a DNA vaccine vector in studies on small animals (Lee, et al. , J Virol. 72, 8430 (1998) ; and Cho, et al. , Vaccine 17,1136 (1999) ), as a starting vector according to a known method. The vector pGXIO was deposited with Korean Collection of Type Cultures (KCTC), one of international depository authorities, on March, 2002, as Accession No. KCTC 10212BP, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. It is apparent to those skilled in the art that types of the promoter and types and lengths of the glycoprotein signal sequence can be changed in various ways depending on the purpose for practicing the present invention. For example, the promoters which can be used include viral promoters such as RSV promoter, cellular promoters such as EF1, MCK (muscle specific promoter), LCK (T cell specific promoter). For the glycoprotein, VZV (varicella zoster virus) gB, HCMV (human cytomegalovirus) gH, gL, GO, VSV (vesicular stomatitis virus) G protein, rotavirus outer capsid glycoprotein), VP7 can be substituted.
The vector pGXIO is an improvement of the vector pTX and pTV2, which are already known, as described above, and shows a high level of expression in vitro (10 times higher than pTV2, Fig. 18) and moreover, generates a superior immune response, even in vivo. It was observed that the vector pGXIO induces immune responses at a level 10 times stronger than pTX, and pGXIO-HIV/RT (reverse transcriptase) produces anti-RT antibodies in an amount 10 times greater than pTXIO-HIV/RT (data not shown). The known vector pTV2 is disclosed in Korean Patent Application Laid- open No. 2001-0054338 (July 2, 2001) and its corresponding US Patent Publication No. 2001004531 (June 21,2001).
SIV immunogenic plasmids. According to the present invention, the following four types of basic immunogenic plasmids were constructed for use as AIDS DNA vaccines to be examined for their efficacy in rhesus macaques monkeys: 1) An immunogenic plasmid (hereinafter referred to as "first SIV immunogenic plasmid) comprising: the vector pGXIO, and (i) the SIVmac239 gag gene encoding matrix protein (MA), capsid protein (CA) and nucleocapsid protein (NC); (ii) the SIVmac239 dpol sequence in the pol gene, encoding protease; (iii) the SIVmac239 env gene encoding envelope protein; and (iv) the SIVmac239 regulatory gene rev, encoding the protein Rev, operably linked thereto; 2) An immunogenic plasmid (hereinafter referred to as "second SIV immunogenic plasmid) comprising: the vector pGXIO, and the SIVmac239 pol gene encoding reverse transcriptase (RT) and integrase (INT) and a DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene, operably linked thereto; 3) An immunogenic plasmid (hereinafter referred to as "third SIV immunogenic plasmid) comprising: the SIVmac239 vif gene, and a DNA sequence encoding signal peptide of secretory protein and the SIVmac239 nef gene, fused to the 3'and 5'ends of the SIVmac239 vif gene, respectively; and 4) An immunogenic plasmid (hereinafter referred to as "fourth SIV immunogenic plasmid) comprising: a DNA sequence comprising any one of genes having from exon 1 to a full-length sequence of the SIVmac239 tat gene, and a DNA sequence encoding signal peptide of secretory protein and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively.
Now, the above four types of immunogenic plasmids will be described in detail.
(1) First SIV immunogenic plasmid. This novel immunogenic plasmid of 8.7 kb is formed by introducing the gag, dpol (protease) and env genes, and the SIVmac239 rev regulatory gene to be operably linked to the MCS (multi-cloning site) of the vector pGXIO according to the present invention, which is used in the DNA vaccine against the SIVmac239/rhesus macaques monkey according to the present invention. The detailed procedure for constructing this plasmid is described in Example 2 and illustrated Fig. 5. This plasmid is designated pGXlO-SIV/GE and was deposited with Korean Collection of Type Cultures (KCTC), one of international depository authorities, on March, 2002, as Accession No. KCTC 10215BP, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
The plasmid pGXIO-SIV/GE shows a superior expression efficiency, as compared to the immunogenic plasmid pTV-SIV/GE of 10.0 kb disclosed in pending Korean Patent Application Laid-open No. 2001-0054338 and its corresponding US Patent Publication No. 2001004531, which were filed by the present inventors. This can be confirmed by Fig. 31 showing a photograph of Western blotting analyses of pGXIO-SIV/GE and pTV-SIV/GE.
2) Second SIV immunogenic plasmid. This novel immunogenic plasmid is formed by introducing the SIVmac239 pol gene encoding reverse transcriptase and integrase and the DNA sequence encoding signal peptide of secretory protein fused to the 3'end of the SIVmac239 pol gene into the MCS (multi-cloning site) to be operably linked to the vector pGXIO according to the present invention, which is used in the DNA vaccine against the SIVmac239/rhesus macaques monkey according to the present invention, together with the first SIV immunogenic plasmid.
In a preferred embodiment of the present invention, the pol gene may be mutated so that its integrase activity is suppressed. When using such mutated gene in a DNA vaccine, possibility of production of a virus which can proliferate in the subject inoculated with the DNA vaccine is further lowered, thereby leading to improvement in safety. For example, nucleotides 5130-5135 site in the integrase region is known to be very important for the enzyme activity of integrase (codon for Asp 116, Fields Virology, Third edition p1893, Lippincott-Raven Co. , 1996). Therefore, it is possible to inhibit the activity of integrase by modifying nucleotides 5130-5135 site so as to prevent proliferation of the virus in host cells. Particularly, nucleotides 5130-5132 site of integrase is deleted and/or nucleotides 5133-5135 site is substituted with codon for serine. Consequently, the gene was mutated to express Ser117, instead of Asn117.
In our own experiments, it was confirmed that such mutated SIVmac239 virus did not proliferate in host cells. The above-described position numbers of the base sequence followed the SIVmac239 clone of GeneBank Accession Number M33262 (Fig. 4).
In addition, it is possible to inhibit the activity of integrase by modifying coding sequences in which Asp116, Asp64 and Glu152 are conserved. These amino acids are known to stabilize transition state by coupling with a divalent metal ion such as Mg2-at the active site of the integrase. For HIV-1 integrase, Hisl2, Hixl6, Cys40 and Cys43 are important amino acids to form DNA binding structure (metal- finger) (Field Virology supra, pl893).
To the 3'end of the SIVmac239 pol gene ecoding reverse transcriptase and integrase (expressed as RT-INT in Fig. 6), a DNA sequence encoding signal peptide of secretory protein is fused. As a result of such fusion, transcription of transcriptase and integrase is directly controlled by CMV promoter, thereby increasing expression levels of the enzymes. Preferably, a signal peptide of glycoprotein is used as a signal sequence of secretory protein. Examples of the glycoprotein include herpes simplex virus glycoprotein gD, varicella zoster virus (VZV) gB, human cytomegalovirus (HCMV) gH, gL, gO, vesicular stomatitis virus (VSV) G protein, rotavirus outer capsid glycoprotein, VP7 and the like.
In a preferred embodiment, a second immunogenic plasmid of 6.3 kb is formed by inserting the SIVmac239 pol gene encoding reverse transcriptase and integrase in which the nucleotides 5130-5132 site is deleted and the nucleotides 5133-5135 site is substituted with serine codon, and a DNA sequence encoding a signal peptide of glycoprotein D (gD) derived from herpes simplex virus (HSV) which is fused to the 3' end of the SIVmac239 pol gene, into the MCS (multi-cloning site) to be operably linked to the vector pGXIO according to the present invention. The pol gene comes under direct transcriptional control of CMV promoter due to the DNA signal sequence encoding 33 N-terminal amino acids of HSV gD, which is fused to the 3'end of the pol gene, thereby increasing expression strength of reverse transcriptase and integrase.
This plasmid is used as a second SIV immunogenic plasmid of 8.7 kb in the DNA vaccine against the SIVmac239/rhesus macaques monkey according to the present invention. The detailed procedure for constructing this plasmid is described in Example 3 and illustrated Fig. 6. This plasmid is designated pGX-SIV/dpol and was also deposited with Korean Collection of Type Cultures (KCTC), one of international depository authorities, on March, 2002, as Accession No. KCTC 10214BP, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
Meanwhile, it should be understood that various changes and modifications of methods for mutagenesis, types of promoter, and types and lengths of the glycoprotein signal sequence, other than those described above, can be made according to the purpose for practicing the present invention, and such changes and modifications will be apparent to those skilled in the art.
The plasmid pGXlO-SIV/dpol shows a superior expression efficiency (data not shown), as compared to the immunogenic plasmid pTV-SIV/dpol of 10.0 kb disclosed in Korean Patent Application Laid-open No. 2001-0054338 and its corresponding US Patent Publication No. 2001004531, which were filed by the present inventors.
(3) Third SIV immunogenic plasmid This novel plasmid is constructed by inserting the SIVmac239 vif gene, and a DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 nef gene fused to the 3'and 5'ends of the SIVmac239 vif gene, respectively, to a vector, in which the genes and signal sequence are operably linked to the vector. The vector which can be used includes any mammalian cell expression vectors, preferably, a DNA vaccine vector optimized to induce immune response upon expression in muscle cells, DC (dendritic cells), and T cells. More preferably, the vector is a vector including CMV promoter and optionally TPL sequence SV40 pA. Concrete examples of vectors which can be used in the present invention include, but are not limited to, pGXIO and pTV2; pVXI, pRC/CMV, pREP4 and pREP10 (though including RSV promoter), pcDNAl, pcDNAI. 1, pcDNA3, pcDNA3/CAT, pRC/CMV and pRC/CMV2 supplied by Invitrogen Corp.; pCMV-script supplied by Stratagene; and pSI, pCI and pCI-neo supplied by Promega. The most preferred is pGXIO.
The third immunogenic plasmid thus constructed is delivered along with the first SIV immunogenic plasmid pGXIO-SIV/GE and the second SIV immunogenic plasmid (for example, pGXIO-SIV/dpol) to enhance the protection induced by the first and second immunogenic plasmids.
In addition, the third immunogenic plasmid is delivered along with the first SIV immunogenic plasmid pGXIO-SIV/GE and the second SIV immunogenic plasmid (for example, pGXIO-SIV/pol) and the fourth SIV immunogenic plasmid (for example, pGXIO-SIV/TV) to enhance the protection induced by the first and second immunogenic plasmids.
In a preferred embodiment of the present invention, the SIVmac239 vif regulatory gene and the SIVmac239 nef regulatory gene, which is fused to the 5'end of the SIVmac239 vif regulatory gene, may be modified to remove immunosuppressive effects. The modification can be effected by various methods. For example, Serl 14- Leul50 in the vif gene can be modified (Fields Virology Third edition, pl901, Lippincott-Raven Col, 1996) and Argl37, Argl38 and Gly2 (involved in myristylation) in the nef gene can be modified.
To the 3'end of the SIVmac239 vif gene, a DNA sequence encoding a signal peptide of secretory protein is fused. As a result of such fusion, the transcription of the vif gene is directly controlled by CMV promoter, thereby increasing expression levels of the Vif and Nef proteins. Preferably, the DNA sequence encoding a signal peptide of glycoprotein is used as the DNA sequence encoding a signal peptide of secretory protein. Examples of the glycoprotein include herpes simplex virus glycoprotein gD, varicella zoster virus (VZV) gB, human cytomegalovirus (HCMV) gH, gL, gO; vesicular stomatitis virus (VSV) G protein, rotavirus outer capsid glycoprotein, VP7 and the like.
In one embodiment, another immunogenic plasmid of 5.1 kb is constructed by inserting the SIVmac239 vif gene, and a DNA sequence encoding a signal peptide of HSV gD and the modified SIVmac239 nef gene fused to the 3'and 5'ends of the SIVmac239 vif gene, respectively, to the MCS (multi-cloning site) of the vector pGXIO according to the present invention, in which the genes and the signal sequence are operably linked to the vector. This plasmid is used as a third or fourth SIV immunogenic plasmid in the DNA vaccine against the SIVmac239/rhesus macaques monkey according to the present invention. The detailed procedure for constructing this plasmid is described in Example 4 and illustrated Fig. 7. This plasmid is designated pGX-SIV/VN and was also deposited with Korean Collection of Type Cultures (KCTC), one of international depository authorities, on March, 2002, as Accession No. KCTC 10213BP, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
To be concrete, the third SIV immunogenic plasmid pGXIO-SIV/VN comprises a gene (VN) formed by binding the SIVmac239 vif and nef. The nef gene is modified by the deletion of codons for Argl37 and Argl38 which are known to play an important role in the downregulation activity of CD4 (J. Biol. Chem. 270 ; 15307, 1995) so as to prevent the immunosuppressive effects of the Nef protein. Thus, this plasmid was devised to increase expression of fused Vif-Nef by fusing the signal sequence encoding 33 N-terminal amino acids of HSV gD to the 3'end of the VN gene so that the VN gene comes under direct transcription control of CMV promoter.
Meanwhile, it should be understood that various changes and modifications of methods for mutagenesis, types of promoter, and types and lengths of the glycoprotein signal sequence, other than those described above, can be made according to the purpose for practicing the present invention, and such changes and modifications will be apparent to those skilled in the art.
(4) Forth SIV immunogenic plasmid. This novel plasmid is constructed by inserting a DNA sequence comprising any one of genes having from exon 1 to a full-length sequence of the SIVmac239 tat gene, and a DNA sequence encoding signal peptide of secretory protein and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively, into a vector, in which the genes and signal sequence are operably linked to the vector. The vector which can be used includes any mammalian cell expression vectors, preferably, a DNA vaccine vector optimized to induce immune response upon expression in muscle cells, DC, and T cells. More preferably, the vector is a vector including CMV promoter and optionally TPL sequence SV40 pA. Concrete examples of vectors which can be used in the present invention include, but are not limited to, pGXIO and pTV2; pVXI, pRC/CMV, pREP4 and pREP10 (though including RSV promoter), pcDNAI, pcDNAl. 1, pcDNA3, pcDNA3/CAT, pRC/CMV and pRC/CMV2 supplied by Invitrogen Corp.; pCMV-script supplied by Stratagene; and pSI, pCI and pCI-neo supplied by Promega. The most preferred is pGXIO.
The fourth plasmid thus constructed is delivered along with the first SIV immunogenic plasmid pGXIO-SIV/GE and the second SIV immunogenic plasmid (for example, PGX10-SIV/dpol) to enhance the protection induced by the first and second immunogenic plasmids.
Additionally, the fourth SIV immunogeinc plasmid is delivered along with the first SIV immunogenic plasmid pGXlO-SIV/GE, the second SIV immunogenic plasmid (for example, pGXIO-SIV/dpol) and the third SIV immunogenic plasmid (for example, pGXIO-SIV/VN) to enhance the protection induced by the first and second immunogenic plasmids.
The tat gene is preferably used in a modified form since it can be bring about suppression of immune responses in a immunized host. A region in the tat gene which can be modified comprises the entire gene except exon 1. The exon 1 of the tat gene expresses the enzyme activity (immune disturbance) of the Tat but the effect is less than that of exon 1+exon 2. The immune epitope included in exon 2 of the tat gene cannot be used. Therefore, it is the most preferable to use only the exon 1 site of the tat gene.
To the 3'end of the SIVmac239 tat regulatory gene, a DNA sequence encoding a signal peptide of secretory protein is fused. As a result of such fusion, the transcription of the tat gene is directly controlled by the CMV promoter, thereby increasing expression levels of the Tat and Vpx proteins. Preferably, the DNA sequence encoding a signal peptide of glycoprotein is used as the DNA sequence encoding a signal peptide of secretory protein. Examples of the glycoprotein include herpes simplex virus glycoprotein gD, varicella zoster virus (VZV) gB, human cytomegalovirus (HCMV) gH, gL, gO, vesicular stomatitis virus (VSV) G protein, rotavirus outer capsid glycoprotein, VP7 and the like.
In a preferred embodiment, another immunogenic plasmid of 4.3 kb is constructed by inserting exon 1 of the SIVmac239 tat gene, and a DNA sequence encoding a signal peptide of HSV gD and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively, into the MCS (multi-cloning site) of the vector pGXIO according to the present invention, in which the genes and the signal sequences are operably linked to the vector. This plasmid is used as a third or fourth SIV immunogenic plasmid in the DNA vaccine against the SIVmac239/rhesus macaques monkey according to the present invention. The detailed procedure for constructing this plasmid is described in Example 5 and illustrated Fig. 8. This plasmid is designated pGX-SIV/TV and was also deposited with Korean Collection of Type Cultures (KCTC), one of international depository authorities, on April, 2002, as Accession No. KCTC 10216BP, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
To be concrete, the fourth SIV immunogenic plasmid pGXIO-SIV/TV comprises a gene (TV) formed by fusing exon 1 of the SIVmac239 tat gene and vpx gene. Thus, this plasmid was formed by fusing the signal sequence encoding 33 N- terminal amino acids of HSV gD to the 5'end of the TV gene so that the gene TV comes under direct transcriptional control of CMV promoter.
Meanwhile, it should be understood that various changes and modifications of methods for mutagenesis, types of promoter, and types and lengths of the glycoprotein signal sequence, other than those described above, can be made according to the purpose for practicing the present invention, and such changes and modifications will be apparent to those skilled in the art.
(5) Immunogenic plasmid comprising the SIVmac239 regulatory genes vif, nef, tat and vpx In. a preferred embodiment of the present invention, a plasmid is constructed by inserting (i) the SIVmac239 vif gene, and a DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 nef gene fused to the 3'and 5'ends of the SIVmac239 vif gene, respectively, and (ii) a DNA sequence comprising any one of genes having from exon 1 to a full-length sequence of the SIVmac239 tat gene, and a DNA sequence encoding a signal peptide of secretory protein and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively, into a vector, in which the genes and signal sequences are operably linked to the vector. Concrete examples of the vector which can be used in the present invention include, but are not limited to, pGXIO and pTV2 ; pVXI, pRC/CMV, pREP4 and pREP10 (though including RSV promoter), pcDNAl, pcDNAl. 1, pcDNA3, pcDNA3/CAT, pRC/CMV and pRC/CMV2 supplied by Invitrogen Corp.; pCMV-script supplied by Stratagene; and pSI, pCI and pCI-neo supplied by Promega. The most preferred is pGXIO.
The plasmid thus constructed is delivered along with the first SIV immunogenic plasmid pGXIO-SIV/GE and the second SIV immunogenic plasmid (for example, pGXIO-SIV/dpol) to enhance the protection induced by the first and second immunogenic plasmids. Also, this plasmid can reduce the number of immunogenic plasmid which should be prepared for a DNA vaccine, thereby lowering the production cost.
In a preferred embodiment of the present invention, the SIVmac239 vif regulatory gene and the SIVmac239 nef regulatory gene, which is fused at the 5'end of the SIVmac239 vif regulatory gene, may be modified to remove its immunosuppressive effects. The modification can be effected by various methods. For example, Serl 14- Leul50 in the gene vif can be modified (Fields Virology Third edition, pl901, Lippincott-Raven Col, 1996) and Argl37, Argl38 and Gly2 (involved in myristylation) in the gene nef can be modified.
In another preferred embodiment of the present invention, the tat gene is preferably used in a modified form since it may bring about immune disturbance, though it can be used in its full-length form. A region in the tat gene which can be modified comprises the entire gene except exon 1. The exon 1 of the tat gene expresses the enzyme activity (immune disturbance) of the Tat but the effect is less than that of exon 1+exon 2. The immune epitope included in exon 2 of the tat gene cannot be used. Therefore, it is the. most preferable to use only exon 1 site of the tat gene.
The nef and tat genes can be independently modified. For example, there can be a case where the nef gene is modified while the tat gene is not modified, and where the tat gene is modified while the nef gene is not modified, or where both the nef and tat genes are modified.
To each 3'end of the SIVmac239 vif and tat genes, a DNA sequence encoding a signal peptide of secretory protein is fused. As a result of such fusion, the transcription of the vif and tat genes is directly controlled by each CMV promoter, thereby increasing expression levels of the Vif and Nef proteins. Preferably, the DNA sequence encoding a signal peptide of glycoprotein is used as a signal sequence of secretory protein. Examples of the glycoprotein include herpes simplex virus glycoprotein gD, varicella zoster virus (VZV) gB, human cytomegalovirus (HCMV) gH, gL, gO, vesicular stomatitis virus (VSV) G protein, rotavirus outer capsid glycoprotein, VP7 and the like.
In one embodiment, an immunogenic plasmid of 7.5 kb is constructed by inserting exon 1 of the SIVmac239 tat gene, and a DNA sequence encoding signal peptide of HSV gD and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively, into a third SIV immunogenic plasmid comprising the SIVmac239 vif gene, and a DNA sequence encoding a signal peptide of HSV gD and the modified SIVmac239 nef gene (having codons for Argl37 and Argl38 deleted) fused to the 3'and 5'ends of the SIVmac239 vif gene, respectively, to be operably linked to MCS (multi-cloning site) of the vector pGXIO according to the invention, in which the genes and the signal sequences are operably linked to the third SIV immunogenic plasmid. This plasmid is used as an additional SIV immunogenic plasmid in the DNA vaccine against the SIVmac239/rhesus macaques monkey according to the present invention. The detailed procedure for constructing this plasmid is described in Example 6 and illustrated Fig. 9. This plasmid is designated pGX-SIV/VNTV.
In another preferred embodiment, an immunogenic plasmid of 7.5 kb is constructed by inserting the SIVmac239 vif gene, and a DNA sequence encoding signal peptide of HSV gD and the modified SIVmac239 nef gene fused to the 3'and 5'ends of the SIVmac239 vif gene, respectively, into a fourth SIV immunogenic plasmid comprising exon 1 of the SIVmac239 tat gene, and a DNA sequence encoding signal peptide of HSV gD and the SIVmac239 vpx gene fused to the 3'and 5'ends of the SIVmac239 tat gene, respectively, to be operably linked to MCS (multi-cloning site) of the vector pGX10 according to the present invention, in which the genes and the signal sequences are operably linked to the fourth SIV immunogenic plasmid. This plasmid is used as an additional SIV immunogenic plasmid in the DNA vaccine against the SIVmac239/rhesus macaques monkey according to the present invention. The detailed procedure for constructing this plasmid is described in Example 7 and illustrated Fig. 10. This plasmid is designated pGX-SIV/TVVN Meanwhile, it should be understood that various changes and modifications of methods for mutagenesis, types of promoter, and types and lengths of the glycoprotein signal sequence, other than those described above, can be made according to the purpose for practicing the present invention, and such changes and modifications will be apparent to those skilled in the art.
It is obvious the present invention is effective for treating HIV-1 infection in humans. HIV-1 and SIV both belong to lentivirus species, and structures and functions of their structural genes are not exactly the same, but are very similar. Also, within some genes commonly found in HIV-1 and SIV, such as the gag gene, the DNA sequences have a high homology and their polyclonal antibodies are cross-reactive.
Above all, the conditions appearing in humans infected with HIV are similar to the conditions appearing in monkeys infected with SIV. For these reasons, when substituting SIV genes with HIV genes while using a human model, it may be expected to achieve efficacy similar to that in a SIV/monkey model. Of course, because no AIDS vaccine has been reported which succeeds in humans, it is not guaranteed that the assessment in SIVmac/monkey model will be perfectly replicated in a human model.
Since there may exist factors which act only on human beings, it is possible that a substance which are effective in primate models such as the SIVmac239/monkey model is not effective at all in humans. However, the present invention (using a primate model) can be effectively used to assess a candidate substance before testing the efficacy of a vaccine in human beings and thereby, to reduce the risk of the clinical demonstration. Moreover, it can be readily expected that a substance showing excellent effects in primate models has a higher possibility to be applied to human beings, as compared to a substance showing excellent effects in non-primate models such as mouse, cat, etc.
HIV immunogenic plasmids. According to the present invention, the following four types of basic immunogenic plasmids were constructed for use as AIDS DNA vaccines to be examined for their efficacy in humans: 1) An immunogenic plasmid (hereinafter referred to as"first HIV immunogenic plasmid) comprising: the vector pGXIO, and (i) the HIV-1 gag gene encoding matrix protein (MA), capsid protein (CA) and nucleocapsid protein (NC); (ii) the HIV-1 dpol sequence in the pol gene, encoding protease; (iii) the HIV-1 env gene encoding envelope protein; and (iv) the HIV-1 regulatory gene rev, encoding the protein Rev, operably linked thereto; 2) An immunogenic plasmid (hereinafter referred to as"second HIV immunogenic plasmid) comprising: the vector pGXIO, and the HIV-1 pol gene encoding reverse transcriptase (RT) and integrase (INT) and a DNA sequence encoding a signal peptide of secretory protein fused to the 3'end of the HIV-1 pol gene, operably linked thereto; 3) An immunogenic plasmid (hereinafter referred to as"third HIV immunogenic plasmid) comprising: the HIV vif gene, and a DNA sequence encoding signal peptide of secretory protein and the HIV-1 nef gene, fused to the 3'and 5'ends of the HIV-1 vif gene, respectively; and 4) An immunogenic plasmid (hereinafter referred to as"fourth HIV immunogenic plasmid) comprising: a DNA sequence comprising any one of genes having from exon 1 to a full-length sequence of the HIV-1 tat gene, and a DNA sequence encoding signal peptide of secretory protein and the HIV-1 vpx gene fused to the 3'and 5'ends of the HIV-1 tat gene, respectively.
Now, the above four types of HIV immunogenic plasmids will be described in detail.
(1) First HIV immunogenic plasmid This novel immunogenic plasmid of 8.7 kb is formed by introducing the gag, dpol (protease) and env genes, and the HIV rev regulatory gene to be operably linked to the MCS (multi-cloning site) of the vector pGXIO according to the present invention, which is used in the DNA vaccine against AIDS human patients. The detailed procedure for constructing this plasmid is described in Example 8 and illustrated Fig. 11. This plasmid is designated pGXIO-HIV/GE.
The plasmid pGXIO-SIV/GE shows a superior expression efficiency, as compared to the immunogenic plasmid pTV-HIV/GE of 11.0 kb disclosed in Korean Patent Application Laid-open No. 2001-0054338 and its corresponding US Patent Publication No. 2001004531, which were filed by the present inventors.
(2) Second HIV immunogenic plasmid. This novel immunogenic plasmid is formed by introducing the HIV pol gene encoding reverse transcriptase and integrase and the DNA sequence encoding signal peptide of secretory protein fused to the 3'end of the HIV pol gene into the MCS (multi-cloning site) to be operably linked to the vector pGXIO according to the present invention, which is used in the DNA vaccine against AIDS human patients, together with the first HIV immunogenic plasmid.
In a preferred embodiment of the present invention, the pol gene may be mutated so that its integrase activity is suppressed. When using such mutated gene in a DNA vaccine, possibility of production of a virus which can proliferate in the subject inoculated with the DNA vaccine is further lowered, thereby leading to improvement in safety. For example, nucleotides 5130-5135 site in the integrase region is known to be very important for the enzyme activity of integrase (codon for Asp 116, Fields Virology, Third edition pl893, Lippincott-Raven Co. , 1996). Therefore, it is possible to inhibit the activity of integrase by modifying nucleotides 5130-5135 site so as to prevent proliferation of the virus in host cells. Particularly, nucleotides 5130-5132 site of integrase is deleted and/or nucleotides 5133-5135 site is substituted with codon for serine. Consequently, the gene was mutated to express Ser117, instead of Asn117.
In our own experiments, it was confirmed that such mutated HIV-1 virus did not proliferate in host cells. The above-described position numbers of the nucleotide sequence followed the HIV-1 JR-CSF clone of GeneBank Accession Number M38429.
In addition, it is possible to inhibit the activity of integrase by modifying coding sequences in which Aspll6, Asp64 and Glu152 are conserved. These amino acids are known to stabilize transition state by coupling with a divalent metal ion such as Mg2+ at the active site of the integrase. For HIV-1 integrase, Hisl2, Hixl6, Cys40 and Cys43 are important amino acids to form DNA binding structure (metal- finger) (Field Virology supra, pi 893).
To the 3'end of the HIV-1 pol gene encoding reverse transcriptase and integrase (expressed as RT-INT in the drawings), a DNA sequence encoding signal peptide of secretory protein is fused. As a result of such fusion, transcription of transcriptase and integrase is directly controlled by CMV promoter, thereby increasing expression levels of the enzymes. Preferably, a signal peptide of glycoprotein is used as a signal sequence of secretory protein. Examples of the glycoprotein include herpes simplex virus glycoprotein gD, varicella zoster virus (VZV) gB, human cytomegalovirus (HCMV) gH, gL, gO, vesicular stomatitis virus (VSV) G protein, rotavirus outer capsid glycoprotein, VP7 and the like.
In one embodiment, a second HIV immunogenic plasmid of 6.2 kb is formed by inserting the HIV pol gene encoding reverse transcriptase and integrase in which the nucleotides 5130-5132 site is deleted and the nucleotides 5133-5135 site is substituted with serine codon, and a DNA sequence encoding a signal peptide of glycoprotein D (gD) derived from herpes simplex virus (HSV) which is fused to the 3'end of the HIV- 1 pol gene, into the MCS (multi-cloning site) to be operably linked to the vector pGXIO according to the present invention. The pol gene comes under direct transcriptional control of CMV promoter due to the DNA signal sequence encoding 33 N-terminal amino acids of HSV gD, which is fused to the 3'end of the pol gene, thereby increasing expression strength of reverse transcriptase and integrase. This plasmid is used as a second HIV immunogenic plasmid in the DNA vaccine against AIDS human patients. The detailed procedure for constructing this plasmid is described in Example 9 and illustrated Fig. 12. This plasmid is designated pGX- HIV/dpol.
Meanwhile, it should be understood that various changes and modifications of methods for mutagenesis, types of promoter, and types and lengths of the glycoprotein signal sequence, other than those described above, can be made according to the purpose for practicing the present invention, and such changes and modifications will be apparent to those skilled in the art.
The plasmid pGXIO-HIV/dpol shows a superior expression efficiency (data not shown), as compared to the immunogenic plasmid pTV-HIV/dpol of 7.5 kb disclosed in Korean Patent Application Laid-open No. 2001-0054338 and its corresponding US Patent Publication No. 2001004531, which were filed by the present inventors.
(3) Third SIV immunogenic plasmid This novel plasmid is constructed by inserting the HIV-1 vif gene, and a DNA sequence encoding a signal peptide of secretory protein and the HIV-1 nef gene fused to the 3'and 5'ends of the HIV-1 vif gene, respectively, to a vector, in which the genes and signal sequence are operably linked to the vector. The vector which can be used includes any mammalian cell expression vectors, preferably, a DNA vaccine vector optimized to induce immune response upon expression in muscle cells, DC, and T cells.
More preferably, the vector is a vector including CMV promoter and optionally TPL sequence SV40 pA. Concrete examples of vectors which can be used in the present invention include, but are not limited to, pGXIO and pTV2; pVXl, pRC/CMV, pREP4 and pREP10 (though including RSV promoter), pcDNAI, pcDNAl. 1, pcDNA3, pcDNA3/CAT, pRC/CMV and pRC/CMV2 supplied by Invitrogen Corp.; pCMV-script supplied by Stratagene; and pSI, pCI and pCI-neo supplied by Promega. The most preferred is pGXIO.
The third immunogenic plasmid thus constructed is delivered along with the first SIV immunogenic plasmid pGXIO-HIV/GE and the second HIV immunogenic plasmid (for example, pGXIO-HIV/dpol) to enhance the protection induced by the first and second immunogenic plasmids.
In addition, the third immunogenic plasmid is delivered along with the first HIV immunogenic plasmid pGXIO-HIV/GE and the second SIV immunogenic plasmid (for example, pGXIO-HIV/pol) and the fourth HIV immunogenic plasmid (for example, pGXIO-HIV/TV) to enhance the protection induced by the first and second immunogenic plasmids.
In a preferred embodiment of the present invention, the HIV-1 vif regulatory gene and the HIV-1 nef regulatory gene, which is fused to the 5'end of the HIV-1 vif regulatory gene, may be modified to remove immunosuppressive effects. The modification can be effected by various methods. For example, Serll4-Leul50 in the vif gene can be modified (Fields Virology Third edition, pl901, Lippincott-Raven Col, 1996) and Argl37, Argl38 and Gly2 (involved in myristylation) in the nef gene can be modified.
To the 3'end of the HIV vif gene, a DNA sequence encoding a signal peptide of secretory protein is fused. As a result of such fusion, the transcription of the vif gene is directly controlled by CMV promoter, thereby increasing expression levels of the Vif and Nef proteins. Preferably, the DNA sequence encoding a signal peptide of glycoprotein is used as the DNA sequence encoding a signal peptide of secretory protein. Examples of the glycoprotein include herpes simplex virus glycoprotein gD, varicella zoster virus (VZV) gB, human cytomegalovirus (HCMV) gH, gL, gO, vesicular stomatitis virus (VSV) G protein, rotavirus outer capsid glycoprotein, VP7 and the like.
In one embodiment, another immunogenic plasmid of 5.1 kb is constructed by inserting the HIV-1 vif gene, and a DNA sequence encoding a signal peptide of HSV gD and the modified HIV-1 nef gene fused to the 3'and 5'ends of the SIVmac239 vif gene, respectively, to the MCS (multi-cloning site) of the vector pGXIO according to the present invention, in which the genes and the signal sequence are operably linked to the vector. This plasmid is used as a third or fourth HIV immunogenic plasmid in the DNA vaccine against AIDS human patients. The detailed procedure for constructing this plasmid is described in Example 10 and illustrated Fig. 13. This plasmid is designated pGX-HIV/VN To be concrete, the third SIV immunogenic plasmid pGX10-HIV/VN comprises a gene (VN) formed by binding the HIV-1 vif and nef. The nef gene is modified by the deletion of codons for Argl37 and Argl38 which are known to play an important role in the downregulation activity of CD4 (J. Biol. Chem. 270 ; 15307,1995) so as to prevent the immunosuppressive effects of the Nef protein. Thus, this plasmid was devised to increase expression of fused Vif-Nef by fusing the signal sequence encoding 33 N-terminal amino acids of HSV gD to the 3'end of the VN gene so that the VN gene comes under direct transcription control of CMV promoter.
Meanwhile, it should be understood that various changes and modifications of methods for mutagenesis, types of promoter, and types and lengths of the glycoprotein signal sequence, other than those described above, can be made according to the purpose for practicing the present invention, and such changes and modifications will be apparent to those skilled in the art.
4) Forth SIV immunogenic plasmid. This novel plasmid is constructed by inserting a DNA sequence comprising any one of genes having from exon 1 to a full-length sequence of the HIV-1 tat gene, and a DNA sequence encoding signal peptide of secretory protein and the HIV-1 vpx gene fused to the 3'and 5'ends of the HIV-1 tat gene, respectively, into a vector, in which the genes and signal sequence are operably linked to the vector. The vector which can be used includes any mammalian cell expression vectors, preferably, a DNA vaccine vector optimized to induce immune response upon expression in muscle cells, DC, and T cells. More preferably, the vector is a vector including CMV promoter and optionally TPL sequence SV40 pA. Concrete examples of vectors which can be used in the present invention include, but are not limited to, pGXIO and pTV2; pVXl, pRC/CMV, pREP4 and pREP 10 (though including RSV promoter), pcDNAl, pcDNAl. 1, pcDNA3, pcDNA3/CAT, pRC/CMV and pRC/CMV2 supplied by Invitrogen Corp.; pCMV-script supplied by Stratagene; and pSI, pCI and pCI-neo supplied by Promega. The most preferred is pGXIO.
The fourth plasmid thus constructed is delivered along with the first HIV immunogenic plasmid pGXIO-HIV/GE and the second HIV immunogenic plasmid (for example, pGXIO-HIV/dpol) to enhance the protection induced by the first and second immunogenic plasmids.
Additionally, the fourth HIV immunogeinc plasmid is delivered along with the first HIV immunogenic plasmid pGXIO-HIV/GE, the second HIV immunogenic plasmid (for example, pGXIO-HIV/dpol) and the third HIV immunogenic plasmid (for example, pGXIO-HIV/VN) to enhance the protection induced by the first and second immunogenic plasmids.
The tat gene is preferably used in a modified form since it may bring about immune disturbance, though it can be used in its full-length form. A region in the tat gene which can be modified comprises the entire gene except exon 1. The exon 1 of the tat gene expresses the enzyme activity (immune disturbance) of the Tat but the effect is less than that of exon 1+exon 2. The immune epitope included in exon 2 of the tat gene cannot be used. Therefore, it is the most preferable to use only the exon 1 site of the tat gene.
To the 3'end of the HIV-1 tat regulatory gene, a DNA sequence encoding a signal peptide of secretory protein is fused. As a result of such fusion, the transcription of the tat gene is directly controlled by the CMV promoter, thereby increasing expression levels of the Tat and Vpx proteins. Preferably, the DNA sequence encoding a signal peptide of glycoprotein is used as the DNA sequence encoding a signal peptide of secretory protein. Examples of the glycoprotein include herpes simplex virus glycoprotein gD, varicella zoster virus (VZV) gB, human cytomegalovirus (HCMV) gH, gL, gO, vesicular stomatitis virus (VSV) G protein, rotavirus outer capsid glycoprotein, VP7 and the like.
In one embodiment, another immunogenic plasmid of 4.2 kb is constructed by inserting exon 1 of the HIV-1 tat gene, and a DNA sequence encoding a signal peptide of HSV gD and the HIV-1 vpx gene fused to the 3'and 5'ends of the HIV-1 tat gene, respectively, into the MCS (multi-cloning site) of the vector pGXIO according to the present invention, in which the genes and the signal sequences are operably linked to the vector. This plasmid is used as a third or fourth HIV immunogenic plasmid in the DNA vaccine against AIDS human patients. The detailed procedure for constructing this plasmid is described in Example 11 and illustrated Fig. 14. This plasmid is designated pGX-HIV/TV.
To be concrete, the fourth HIV immunogenic plasmid pGXIO-HIV/TV comprises a gene (TV) formed by fusing exon 1 of the SIVmac239 tat gene and vpx gene. Thus, this plasmid was formed by fusing the signal sequence encoding 33 N- terminal amino acids of HSV gD to the 5'end of the TV gene so that the gene TV comes under direct transcriptional control of CMV promoter.
Meanwhile, it should be understood that various changes and modifications of methods for mutagenesis, types of promoter, and types and lengths of the glycoprotein signal sequence, other than those described above, can be made according to the purpose for practicing the present invention, and such changes and modifications will be apparent to those skilled in the art.
(5) Immunogenic plasmid comprising the HIV-1 regulatory genes vif, nef, tat and vpx In a preferred embodiment of the present invention, a plasmid is constructed by inserting (i) the HIV-1 vif gene, and a DNA sequence encoding a signal peptide of secretory protein and the HIV-1 nef gene fused to the 3'and 5'ends of the HIV-1 vif gene, respectively, and (ii) a DNA sequence comprising any one of genes having from exon 1 to a full-length sequence of the HIV-1 tat gene, and a DNA sequence encoding a signal peptide of secretory protein and the HIV-1 vpx gene fused to the 3'and 5'ends of the HIV-1 tat gene, respectively, into a vector, in which the genes and signal sequences are operably linked to the vector. Concrete examples of the vector which can be used in the present invention include, but are not limited to, pGXIO and pTV2; pVXI, pRC/CMV, pREP4 and pREP10 (though including RSV promoter), pcDNAI, pcDNAl. 1, pcDNA3, pcDNA3/CAT, pRC/CMV and pRC/CMV2 supplied by Invitrogen Corp.; pCMV-script supplied by Stratagene; and pSI, pCI and pCI-neo supplied by Promega. The most preferred ispGXIO.
The plasmid thus constructed is delivered along with the first HIV immunogenic plasmid pGXIO-HIV/GE and the second HIV immunogenic plasmid (for example, pGXIO-HIV/dpol) to enhance the protection induced by the first and second immunogenic plasmids. Also, this plasmid can reduce the number of immunogenic plasmid which should be prepared for a DNA vaccine, thereby lowering the production cost.
In a preferred embodiment of the present invention, the HIV-1 vif regulatory gene and the HIV-1 nef regulatory gene, which is fused at the 5'end of the HIV-1 vif regulatory gene, may be modified to remove its immunosuppressive effects. The modification can be effected by various methods. For example, Serll4-Leul50 in the gene vif can be modified (Fields Virology Third edition, pl901, Lippincott-Raven Col, 1996) and Argl37, Argl38 and Gly2 (involved in myristylation) in the gene nef can be modified.
In another preferred embodiment of the present invention, the tat gene is preferably used in a modified form since it may bring about immune disturbance, though it can be used in its full-length form. A region in the tat gene which can be modified comprises the entire gene except exon 1. The exon 1 of the tat gene expresses the enzyme activity (immune disturbance) of the Tat but the effect is less than that of exon 1+exon 2. The immune epitope included in exon 2 of the tat gene cannot be used. Therefore, it is the most preferable to use only exon 1 site of the tat gene. The nef and tat genes can be independently modified. For example, there can be a case where the nef gene is modified while the tat gene is not modified, and where the tat gene is modified while the nef gene is not modified, or where both the nef and tat genes are modified.
To each 3'end of the HIV-1 vif and tat genes, a DNA sequence encoding a signal peptide of secretory protein is fused. As a result of such fusion, the transcription of the vif and tat genes is directly controlled by each CMV promoter, thereby increasing expression levels of the Vif and Nef proteins. Preferably, the DNA sequence encoding a signal peptide of glycoprotein is used as a signal sequence of secretory protein. Examples of the glycoprotein include herpes simplex virus glycoprotein gD, varicella zoster virus (VZV) gB, human cytomegalovirus (HCMV) gH, gL, gO, vesicular stomatitis virus (VSV) G protein, rotavirus outer capsid glycoprotein, VP7 and the like.
In one embodiment, an immunogenic plasmid of 7.5 kb is. constructed by inserting exon 1 of the HIV-1 tat gene, and a DNA sequence encoding signal peptide of HSV gD and the HIV-1 vpx gene fused to the 3'and 5'ends of the HIV-1 tat gene, respectively, into a third HIV immunogenic plasmid comprising the HIV-1 vif gene, and a DNA sequence encoding a signal peptide of HSV gD and the modified HIV-1 nef gene (having codons for Argl37 and Argl38 deleted) fused to the 3'and 5'ends of the HIV-1 vif gene, respectively, to be operably linked to MCS (multi-cloning site) of the vector pGXIO according to the invention, in which the genes and the signal sequences are operably linked to the third HIV immunogenic plasmid. This plasmid is used as an additional HIV immunogenic plasmid in the DNA vaccine against AIDS human patients. The detailed procedure for constructing this plasmid is described in Example 12 and illustrated Fig. 15. This plasmid is designated pGX-SIV/VNTV.
In another preferred embodiment, an immunogenic plasmid of 7.5 kb is constructed by inserting the HIV-1 vif gene, and a DNA sequence encoding signal peptide of HSV gD and the modified HIV-1 nef gene fused to the 3'and 5'ends of the HIV-1 vif gene, respectively, into a fourth HIV immunogenic plasmid comprising exon 1 of the HIV-1 tat gene, and a DNA sequence encoding signal peptide of HSV gD and the HIV-1 vpx gene fused to the 3'and 5'ends of the HIV-1 tat gene, respectively, to be operably linked to MCS (multi-cloning site) of the vector pGXIO according to the present invention, in which the genes and the signal sequences are operably linked to the fourth HIV immunogenic plasmid. This plasmid is used as an additional HIV immunogenic plasmid in the DNA vaccine against AIDS human patients. The detailed procedure for constructing this plasmid is described in Example 13 and illustrated Fig. 16. This plasmid is designated pGX-SIV/TVVN Meanwhile, it should be understood that various changes and modifications of methods for mutagenesis, types of promoter, and types and lengths of the glycoprotein signal sequence, other than those described above, can be made according to the purpose for practicing the present invention, and such changes and modifications will be apparent to those skilled in the art.
DNA VACCINE
The experiments by the inventors revealed that combined administration of immunogenic plasmids pGXIO-SIV/GE and pGXIO-SIV/dpol or immunogenic plasmids pGXIO-SIV/GE, pGXIO-SIV/dpol, pGXIO-SIV/VN and pGXIO-SIV/TV to rhesus monkey elicited higher inhibition of both the proliferation of SIVmac239 in rhesus monkey and the reduction in the number of CD4+ cells, a typical symptom of AIDS development, as compared to combined administration of immunogenic plasmids pTV-SIV/GE and pTV-SIV/dpol. Therefore, compositions containing immunogenic plasmids of the invention are useful as vaccines for prophylaxis of AIDS.
Immunotherapy is a method for inhibiting virus proliferation by enhancing immune response to virus, rather than a method for introducing chemical substances which can inhibit enzymes necessary for proliferation of virus such as reverse transcriptase and protease. DNA immunotherapy has been applied to HIV infected chimpanzees (Boyer J D. , et al., AIDS 14: 1515-22,2000 ; and Boyer J D. , et al. , J. Infect. Dis. 176: 1501-9,1997) or HIV infected humans (MacGregor RR J. Infect. Dis. 178: 92-100,1998). Since the therapy efficacy of vaccine correlates with its ability to elicit immune response to inhibit proliferation of virus, it is accepted by those in the art that vaccines showing good protection efficacy can be used in therapy. Therefore, compositions containing immunogenic plasmids of the invention can be used as vaccines for treatment of AIDS.
The administration manner and formulation of DNA vaccines of the invention for AIDS protection are in accordance with practices used for common vaccines, especially DNA vaccines for protection. DNA vaccines for AIDS therapy can be administered and formulated the same as DNA vaccines for AIDS protection in that they are used to increase immune response to AIDS virus.
DNA vaccine of the invention will preferably be administered by direct (in vivo) gene transfer. Naked DNA can be given by intramuscular, subcutaneous, intravenous, intraarterial or buccal injection. Plasmid DNA may be coated onto gold particles and introduced biolistically with a"gene-gun"into the epidermis of the skin or the oral or vaginal mucosae (Fynan et al., Proc. Natl. Acad. Sci. USA 90: 11478,1993 ; Tang et al., Nature 356: 152,1992 ; Fuller et al., J. Med. Primatol. 25 : 236, 1996; Keller et al. , Cancer Gene Ther. , 3: 186,1996). DNA vaccine vectors may also be used in conjunction with various delivery systems. Liposomes have been used to deliver DNA vaccines by intramuscular injection (Gregoriadis et al, FEBS Lett. 402: 107,1997) or into the respiratory system by non-invasive means such as intranasal inhalation (Fynan et al. , supra). Other potential delivery systems include microencapsulation (Jones et al., 1998; Mathiowitz et al. , 1997) or cochleates (Mannino et al. , 1995, Lipid matrix-based vaccines for mucosal and systemic immunization. Vaccine Designs: The Subunit and Adjuvant Approach, M. F. Powell and M. J. Newman, eds. , Pleum Press, New York, 363-387), which can be used for parenteral, intranasal (e. g. , nasal spray) or oral (e. g., liquid, gelatin capsule, solid in food) delivery. DNA vaccines can also be injected directly into tumors or directly into lymphoid tissues (e. g. , Peyer's patches in the gut wall). It is also possible to formulate the vector to target delivery to certain cell types, for example, to APC. Targeting to APC such as dendritic cells is possible through attachment of a mannose moiety (dendritic cells have a high density of mannose receptors) or a ligand for one of the other receptors found preferentially on APC. There is no limitation as to the route by which the DNA vaccine is delivered, nor the manner in which it is formulated, as long as the cells that are transfected can express antigen in such a way that an immune response is induced.
Where DNA vaccine of the present invention is administered to mucosa, it may be placed into a pharmaceutically acceptable suspension, solution or emulsion for administration to mucosa. Suitable mediums include saline and liposomal preparations.
More specifically, pharmaceutically acceptable carriers preferred for use with the gene expression plasmids of the invention may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions suitable for ingestion, inhalation, or administration as a suppository to the rectum or vagina. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and certain organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or. suspensions, including saline and buffered media. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. One skilled in the art will select among these available compounds depending upon the particular mucosal inductor site targeted, i. e. , whether for ingestion or inhalation. Further, a composition of antigen-encoding polynucleotide preparations comprising gene expression plasmids may be lyophilized using means well known in the art, for administration by inhalation as an aerosol or subsequent reconstitution and use according to the invention.
Isotonic buffered solution is the preferred medium for maximal uptake of the gene plasmids contained in DNA vaccines of the invention. Further, use of absorption promoters, detergents, and mild chemical irritants is also preferred to enhance transmission of antigen-encoding polynucleotide preparation compositions through the point of entry and into contact with tissue adjacent to or containing a mucosal inductor site. For reference concerning general principles regarding promoters and detergents which have been used with success in mucosal delivery of organic and peptide-based drugs, see Chien, Novel Drug Delivery Systems, Ch. 4 (Marcel Dekker, 1992). Specific information concerning known means and principles of nasal drug delivery are discussed in Chien, supra at Ch 5. Examples of suitable nasal absorption promoters are set forth at Ch. 5, Tables 2 and 3; milder agents are preferred. Suitable agents for use in the method of this invention for mucosal/nasal delivery are also described in Chang, et al. , Nasal Drug Delivery, "Treatise on Controlled Drug Delivery", Ch. 9 and Table 3-4B thereof, (Marcel Dekker, 1992). Suitable agents which are known to enhance absorption of drugs through skin are described in Sloan, Use of Solubility Parameters from Regular Solution Theory to Describe Partitioning-Driven Processes, Ch. 5, "Prodrugs: Topical and Ocular Drug Delivery" (Marcel Dekker, 1992), and at places elsewhere in the text."Detergents/Absorption Promoters"refers to chemical agents which are presently known in the art to facilitate absorption and transfection of certain small molecules, as well as peptides. "Mucosa" refers to mucosal tissues of a host wherever they may be located in the body including, but not limited to, respiratory passages (including bronchial passages, lung epithelia and nasal epithelia), genital passages (including vaginal, penile and anal mucosa), urinary passages (e. g., urethra, bladder), the mouth, eyes and vocal cords. "Point of Entry" refers to the site of introduction of the polynucleotide into a host, including immediately adjacent tissue.
"Mucosal Inductor Site" refers to a site on the mucosa where uptake of the antigen- encoding polynucleotide preparation is sought, including, but not limited to, Waldeyer's ring, Peyer's patches, gut-associated lymphoid tissues, bronchial-associated lymphoid tissues, nasal-associated lymphoid tissues, genital-associated lymphoid tissues, and tonsils.
The dosage of DNA vaccine according to the present invention can be varied depending on administration manner, tissues to which DNA vaccine is administered, such as skeletal muscle and skin, desired antibody titer, particular treatment requirement for immunization subject, etc. The effective amount of each plasmid contained in DNA vaccine of the present invention is from 0.01 to 0.2 mg/kg of weight, preferably 0. 01 to 0.1 mg/kg of weight. The use of coated projectiles enables a smaller amount of the vaccine to be administered.
Lyophilized DNA Vaccine Formulations. The DNA vaccine composition can be lyophilized to increase its stability at room temperature, to reduce the requirement for costly cold storage, and to extend product shelf-life. The lyophilization process consists of three successive steps of freezing, primary drying and secondary drying. After freezing the product, the primary drying step involves lowering pressure and supplying heat for water vapor sublimation.
During the secondary drying step, the residual absorbed moisture evaporates from the dried material.
In one embodiment, DNA vaccine of the present invention can be lyophilized as follows: (1) Determine the collapse temperature of the formulation by using freeze- drying microscopic analysis; (2) Place the vials on the freeze-drier shelves at room temperature and subsequently equilibrate at-1C for about 30 minutes; (3) Cool the shelves to-55C and hold that temperature for 2 hours; (4) Carry out primary drying at a product temperature of about-32C or 5C below the collapse temperature; (5) Carry out secondary drying at 35C (Complete the drying after adjusting the chamber pressure to