HIV pre-immunization and immunotherapy
The present invention relates generally to immunization and immunotherapy for the treatment or prevention of HIV. In particular, the methods include in vivo and/or ex vivo enrichment of HIV-specific CD4+ T cells.
1. A lentiviral vector comprising at least one encoded genetic element, wherein the at least one encoded genetic element comprises
each of: (i) a sequence comprising at least 80% sequence identity with SEQ ID NO: 2 and (ii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 108; or
two of: (iii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 97, (iv) a sequence comprising at least 80% sequence identity with SEQ ID NO: 6, and (v) a sequence comprising at least 80% sequence identity with SEQ ID NO: 7.
2. The lentiviral vector of claim 1 , wherein the at least one encoded genetic element comprises: each of: SEQ ID NO: 2 and SEQ ID NO: 108 or two of: SEQ ID NO: 97, SEQ ID NO: 6, and SEQ ID NO: 7.
3. A lentiviral vector system for expressing a lentiviral particle, the system comprising:
a lentiviral vector according to claim 1 ;
an envelope plasmid for expressing an envelope protein optimized for infecting a cell; and
at least one helper plasmid for expressing gag, pol, and rev genes,
wherein the lentiviral vector, the envelope plasmid, and the at least one helper plasmid are capable of transfecting a packaging cell line, and wherein, upon such transfection, the packaging cell line is capable of producing a lentiviral particle capable of inhibiting production of chemokine receptor CCR5 or targeting an HIV RNA sequence.
4. The lentiviral vector system of claim 3 , wherein the at least one helper plasmid comprises a first plasmid for expressing the gag and pol genes, and a second plasmid for expressing the rev gene.
5. A lentiviral particle capable of infecting a cell, the lentiviral particle comprising an envelope protein optimized for infecting a cell, and a lentiviral vector according to claim 1 .
6. The lentiviral particle of claim 5 , wherein the envelope protein is optimized for infecting a T cell.
7. The lentiviral particle of claim 6 , wherein the envelope protein is optimized for infecting a CD4+ T cell.
8. A modified cell comprising a CD4+ T cell, wherein the CD4+ T cell has been transfected with a lentiviral particle according to claim 5 .
9. The modified cell of claim 8 , wherein the CD4+ T cell is capable of recognizing an HIV antigen.
10. The modified cell of claim 9 , wherein the HIV antigen comprises a gag antigen.
11. The modified cell of claim 8 , wherein the CD4+ T cell expresses a decreased level of CCR5 following infection with the lentiviral particle.
12. A method of treating cells infected with HIV, the method comprising:
(a) obtaining, or having obtained, peripheral blood mononuclear cells (PBMC) from a subject infected with HIV, wherein the subject has been previously immunized with a therapeutically effective amount of a first stimulatory agent;
(b) contacting, or having contacted, the PBMC with a therapeutically effective amount of a second stimulatory agent, wherein the contacting is carried out ex vivo;
(c) transducing, or having transduced, the PBMC ex vivo with a viral delivery system encoding at least one genetic element, wherein at least one encoded genetic element comprises:
each of: (i) a sequence comprising at least 80% sequence identity with SEQ ID NO: 2 and (ii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 108; or
two of: (iii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 97, (iv) a sequence comprising at least 80% sequence identity with SEQ ID NO: 6, and (v) a sequence comprising at least 80% sequence identity with SEQ ID NO: 7; and
(d) culturing, or having cultured, the transduced PBMC for at least 1 day.
13. The method of claim 12 , wherein the transduced PBMC are cultured from about 1 to about 35 days.
14. The method of claim 12 , further comprising infusing, or having infused, the transduced PBMC into a subject.
15. The method of claim 14 , wherein the subject is a human.
16. The method of claim 12 , wherein at least one of the first stimulatory agent and the second stimulatory agent comprises a peptide.
17. The method of claim 16 , wherein the peptide comprises a gag peptide.
18. The method of claim 12 , wherein at least one of the first stimulatory agent and the second stimulatory agent comprises a vaccine.
19. The method of claim 18 , wherein the vaccine comprises an HIV vaccine.
20. The method of claim 19 , wherein the HIV vaccine comprises a MVA/HIV62B vaccine or a variant thereof.
21. The method of claim 12 , wherein the first stimulatory agent and the second stimulatory agent are the same.
22. The method of claim 12 , wherein the viral delivery system comprises a lentiviral particle.
23. The method of claim 12 , wherein the at least one genetic element, when expressed, is capable of targeting an HIV RNA sequence.
24. The method of claim 23 , wherein, when the at least one encoded genetic element comprises a sequence having at least 80% sequence identity with SEQ ID NO: 97, the at least one genetic element, when expressed, is also capable of inhibiting production of chemokine receptor CCR5.
25. The method of claim 12 , wherein the at least one encoded genetic element comprises:
each of: SEQ ID NO: 2 and SEQ ID NO: 108 or
two of: SEQ ID NO: 97, SEQ ID NO: 6, and SEQ ID NO: 7.
26. A method of treating HIV infection in a subject, the method comprising:
(a) removing, or having removed, leukocytes from the subject, wherein the subject has been previously immunized with a therapeutically effective amount of a first stimulatory agent;
(b) purifying, or having purified, peripheral blood mononuclear cells (PBMC) ex vivo from the leukocytes;
(c) contacting, or having contacted, the PBMC ex vivo with a therapeutically effective amount of a second stimulatory agent;
(d) transducing, of having transduced, the PBMC ex vivo with a viral delivery system encoding at least one genetic element, wherein the at least one genetic element comprises:
each of: (i) a sequence comprising at least 80% sequence identity with SEQ ID NO: 2 and (ii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 108; or
two of: (iii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 97, (iv) a sequence comprising at least 80% sequence identity with SEQ ID NO: 6, and (v) a sequence comprising at least 80% sequence identity with SEQ ID NO: 7; and
(e) culturing, or having cultured, the transduced PBMC for at least 1 day.
27. The method of claim 26 , wherein the transduced PBMC are cultured from about 1 to about 35 days.
28. The method of claim 26 , further comprising infusing, or having infused, the transduced PBMC into the subject.
29. The method of claim 28 , wherein the subject is a human.
30. The method of claim 26 , wherein at least one of the first stimulatory agent and the second stimulatory agent comprises a peptide.
31. The method of claim 30 , wherein the peptide comprises a gag peptide.
32. The method of claim 26 , wherein at least one of the first stimulatory agent and the second stimulatory agent comprises a vaccine.
33. The method of claim 32 , wherein the vaccine comprises an HIV vaccine.
34. The method of claim 33 , wherein the HIV vaccine comprises a MVA/HIV62B vaccine or a variant thereof.
35. The method of claim 26 , wherein the first stimulatory agent and the second stimulatory agent are the same.
36. The method of claim 26 , wherein the viral delivery system comprises a lentiviral particle.
37. The method of claim 26 , wherein the at least one genetic element comprises at least one small RNA capable of targeting an HIV RNA sequence.
38. The method of claim 26 , wherein the at least one genetic element comprises:
each of: SEQ ID NO: 2, and SEQ ID NO: 108; or
two of: SEQ ID NO: 97, SEQ ID NO: 6, and SEQ ID NO: 7.
39. A lentiviral vector comprising an encoded microRNA cluster, wherein the encoded microRNA cluster comprises a sequence comprising (i) at least 90% sequence identity with SEQ ID NO: 1, (ii) at least 90% sequence identity with SEQ ID NO: 2, and (iii) at least 90% sequence identity with SEQ ID NO: 108.
40. The lentiviral vector of claim 39 , wherein the encoded microRNA cluster comprises a sequence comprising (i) at least 95% sequence identity with SEQ ID NO: 1, (ii) at least 95% sequence identity with SEQ ID NO: 2, or (iii) at least 95% sequence identity with SEQ ID NO: 108.
41. The lentiviral vector of claim 39 , wherein the encoded microRNA cluster comprises a sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 108.
42. A lentiviral particle capable of infecting a target cell, the lentiviral particle comprising:
(a) an envelope protein capable of infecting the target cell; and
(b) an encoded microRNA cluster, wherein the encoded microRNA cluster comprises a sequence comprising (i) at least 90% sequence identity with SEQ ID NO: 1, (ii) at least 90% sequence identity with SEQ ID NO: 2, and (iii) at least 90% sequence identity with SEQ ID NO: 108.
43. The lentiviral particle of claim 42 , wherein the encoded microRNA cluster comprises a sequence comprising (i) at least 95% sequence identity with SEQ ID NO: 1, (ii) at least 95% sequence identity with SEQ ID NO: 2, or (iii) at least 95% sequence identity with SEQ ID NO: 108.
44. The lentiviral particle of claim 42 , wherein the encoded microRNA cluster comprises a sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 108.
45. The lentiviral particle of claim 42 , wherein the target cell is a CD4+ T cell.
46. A modified cell comprising a primary T cell infected with a lentiviral particle, wherein the lentiviral particle comprises:
(a) an envelope protein capable of infecting the target cell; and
(b) an encoded microRNA cluster, wherein the encoded microRNA cluster comprises a sequence comprising (i) at least 90% sequence identity with SEQ ID NO: 1, (ii) at least 90% sequence identity with SEQ ID NO: 2, and (iii) at least 90% sequence identity with SEQ ID NO: 108.
47. The modified cell of claim 46 , wherein the encoded microRNA cluster comprises a sequence comprising (i) at least 95% sequence identity with SEQ ID NO: 1, (ii) at least 95% sequence identity with SEQ ID NO: 2, or (iii) at least 95% sequence identity with SEQ ID NO: 108.
48. The modified cell of claim 46 , wherein the encoded microRNA cluster comprises a sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 108.
49. A method of treating cells infected with HIV, the method comprising:
(a) contacting or having contacted peripheral blood mononuclear cells (PBMC) isolated from a subject infected with HIV with a therapeutically effective amount of an ex vivo stimulatory agent, wherein the contacting is conducted ex vivo;
(b) transducing or having transduced the PBMC ex vivo with a lentiviral particle, wherein the lentiviral particle comprises:
(i) an envelope protein capable of infecting the PBMC; and
(ii) an encoded microRNA cluster, wherein the encoded microRNA cluster comprises a sequence comprising (a) at least 90% sequence identity with SEQ ID NO: 1, (b) at least 90% sequence identity with SEQ ID NO: 2, and (c) at least 90% sequence identity with SEQ ID NO: 108.
50. The method of claim 49 , wherein the encoded microRNA cluster comprises a sequence comprising (i) at least 95% sequence identity with SEQ ID NO: 1, (ii) at least 95% sequence identity with SEQ ID NO: 2, or (iii) at least 95% sequence identity with SEQ ID NO: 108.
51. The method of claim 49 , wherein the encoded microRNA cluster comprises a sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 108.
52. The method of claim 49 , further comprising infusing or having infused the transduced PBMC into a subject.
53. The method of claim 49 , further comprising positively selecting or having positively selected HIV-specific CD4+ T cells from the PBMC.
54. The method of claim 49 , further comprising immunizing or having immunized the subject with an effective amount of an in vivo stimulatory agent, wherein the immunization occurs prior to contacting the peripheral blood mononuclear cells (PBMC) with the ex vivo stimulatory agent.
55. The method of claim 54 , wherein each of the in vivo stimulatory agent and ex vivo stimulatory agent is independently selected from a peptide and a vaccine.
56. A lentiviral vector comprising at least one encoded genetic element, wherein the at least one encoded genetic element comprises
at least two of: (i) a sequence comprising at least 80% sequence identity with SEQ ID NO: 1, (ii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 2, and (iii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 108; or
each of: (iv) a sequence comprising at least 80% sequence identity with SEQ ID NO: 97, (v) a sequence comprising at least 80% sequence identity with SEQ ID NO: 6, and (vi) a sequence comprising at least 80% sequence identity with SEQ ID NO: 7.
57. The lentiviral vector of claim 56 , wherein the at least one encoded genetic element comprises:
at least two of: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 108; or
each of: SEQ ID NO: 97, SEQ ID NO: 6, and SEQ ID NO: 7.
58. A lentiviral vector system for expressing a lentiviral particle, the system comprising:
a lentiviral vector according to claim 56 ;
an envelope plasmid for expressing an envelope protein optimized for infecting a cell; and
at least one helper plasmid for expressing gag, pol, and rev genes,
wherein the lentiviral vector, the envelope plasmid, and the at least one helper plasmid are capable of transfecting a packaging cell line, and wherein, upon such transfection, the packaging cell line is capable of producing a lentiviral particle capable of inhibiting production of chemokine receptor CCR5 or targeting an HIV RNA sequence.
59. The lentiviral vector system of claim 58 , wherein the at least one helper plasmid comprises a first plasmid for expressing the gag and pol genes, and a second plasmid for expressing the rev gene.
60. A lentiviral particle capable of infecting a cell, the lentiviral particle comprising an envelope protein optimized for infecting a cell, and a lentiviral vector according to claim 56 .
61. The lentiviral particle of claim 60 , wherein the envelope protein is optimized for infecting a T cell.
62. The lentiviral particle of claim 61 , wherein the envelope protein is optimized for infecting a CD4+ T cell.
63. A modified cell comprising a CD4+ T cell, wherein the CD4+ T cell has been transfected with a lentiviral particle according to claim 60 .
64. The modified cell of claim 63 , wherein the CD4+ T cell is capable of recognizing an HIV antigen.
65. The modified cell of claim 64 , wherein the HIV antigen comprises a gag antigen.
66. The modified cell of claim 63 , wherein the CD4+ T cell expresses a decreased level of CCR5 following infection with the lentiviral particle.
67. A method of treating cells infected with HIV, the method comprising:
(a) obtaining, or having obtained, peripheral blood mononuclear cells (PBMC) from a subject infected with HIV, wherein the subject has been previously immunized with a therapeutically effective amount of a first stimulatory agent;
(b) contacting, or having contacted, the PBMC with a therapeutically effective amount of a second stimulatory agent, wherein the contacting is carried out ex vivo;
(c) transducing, or having transduced, the PBMC ex vivo with a viral delivery system encoding at least one genetic element, wherein at least one encoded genetic element comprises:
at least two of: (i) a sequence comprising at least 80% sequence identity with SEQ ID NO: 1, (ii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 2, and (iii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 108; or
each of: (iv) a sequence comprising at least 80% sequence identity with SEQ ID NO: 97, (v) a sequence comprising at least 80% sequence identity with SEQ ID NO: 6, and (vi) a sequence comprising at least 80% sequence identity with SEQ ID NO: 7; and
(d) culturing, or having cultured, the transduced PBMC for at least 1 day.
68. The method of claim 67 , wherein the transduced PBMC are cultured from about 1 to about 35 days.
69. The method of claim 67 , further comprising infusing, or having infused, the transduced PBMC into a subject.
70. The method of claim 69 , wherein the subject is a human.
71. The method of claim 67 , wherein at least one of the first stimulatory agent and the second stimulatory agent comprises a peptide.
72. The method of claim 71 , wherein the peptide comprises a gag peptide.
73. The method of claim 67 , wherein at least one of the first stimulatory agent and the second stimulatory agent comprises a vaccine.
74. The method of claim 73 , wherein the vaccine comprises an HIV vaccine.
75. The method of claim 74 , wherein the HIV vaccine comprises a MVA/HIV62B vaccine or a variant thereof.
76. The method of claim 67 , wherein the first stimulatory agent and the second stimulatory agent are the same.
77. The method of claim 67 , wherein the viral delivery system comprises a lentiviral particle.
78. The method of claim 67 , wherein the at least one genetic element, when expressed, is capable of targeting an HIV RNA sequence.
79. The method of claim 78 , wherein, when the at least one encoded genetic element comprises a sequence having at least 80% sequence identity with SEQ ID NO: 1, or a sequence having at least 80% sequence identity with SEQ ID NO: 97, the at least one genetic element, when expressed, is also capable of inhibiting production of chemokine receptor CCR5.
80. The method of claim 67 , wherein the at least one encoded genetic element comprises:
at least two of: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 108; or
each of: SEQ ID NO: 97, SEQ ID NO: 6, and SEQ ID NO: 7.
81. A method of treating HIV infection in a subject, the method comprising:
(a) removing, or having removed, leukocytes from the subject, wherein the subject has been previously immunized with a therapeutically effective amount of a first stimulatory agent;
(b) purifying, or having purified, peripheral blood mononuclear cells (PBMC) ex vivo from the leukocytes;
(c) contacting, or having contacted, the PBMC ex vivo with a therapeutically effective amount of a second stimulatory agent;
(d) transducing, of having transduced, the PBMC ex vivo with a viral delivery system encoding at least one genetic element, wherein the at least one genetic element comprises:
at least two of: (i) a sequence comprising at least 80% sequence identity with SEQ ID NO: 1, (ii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 2, and (iii) a sequence comprising at least 80% sequence identity with SEQ ID NO: 108; or
each of: (iv) a sequence comprising at least 80% sequence identity with SEQ ID NO: 97, (v) a sequence comprising at least 80% sequence identity with SEQ ID NO: 6, and (vi) a sequence comprising at least 80% sequence identity with SEQ ID NO: 7; and
(e) culturing, or having cultured, the transduced PBMC for at least 1 day.
82. The method of claim 81 , wherein the transduced PBMC are cultured from about 1 to about 35 days.
83. The method of claim 81 , further comprising infusing, or having infused, the transduced PBMC into the subject.
84. The method of claim 81 , wherein the subject is a human.
85. The method of claim 81 , wherein at least one of the first stimulatory agent and the second stimulatory agent comprises a peptide.
86. The method of claim 85 , wherein the peptide comprises a gag peptide.
87. The method of claim 81 , wherein at least one of the first stimulatory agent and the second stimulatory agent comprises a vaccine.
88. The method of claim 87 , wherein the vaccine comprises an HIV vaccine.
89. The method of claim 88 , wherein the HIV vaccine comprises a MVA/HIV62B vaccine or a variant thereof.
90. The method of claim 81 , wherein the first stimulatory agent and the second stimulatory agent are the same.
91. The method of claim 81 , wherein the viral delivery system comprises a lentiviral particle.
92. The method of claim 81 , wherein the at least one genetic element comprises at least one small RNA capable of targeting an HIV RNA sequence.
93. The method of claim 92 , wherein, when the at least one genetic element comprises a sequence having at least 80% sequence identity with SEQ ID NO: 1, or a sequence having at least 80% sequence identity with SEQ ID NO: 97, the at least one genetic element, when expressed, is also capable of inhibiting production of chemokine receptor CCR5.
94. The method of claim 81 , wherein the at least one genetic element comprises:
at least two of: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 108; or
each of: SEQ ID NO: 97, SEQ ID NO: 6, and SEQ ID NO: 7.