Use of a HIV derived accessory protein for the reactivation of latent HIV

Abstract

The present invention concerns the use of a protein comprising at least a HIV-derived accessory protein tat (trans-activator of transcription) or any derivative thereof for the reactivation of latent human immunodeficiency virus (HIV) from cells present in a HIV-infected patient.

Claims

1. A method of reversing HIV-1 latency in an HIV infected cell, comprising exposing the HIV infected cell to a mutant protein of an HIV Tat which consists of the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

2. A method of reversing HIV-1 latency in an HIV infected cell, comprising exposing the HIV infected cell to a fusion protein which consists of mutant protein fused to a transactivation domain, wherein the mutant protein consists of either SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; and wherein the transactivation domain consists of the transactivation domain of either NFκB p65 or NFAT2.

3. The method of claim 1, wherein said amino acid sequence is SEQ ID NO:1.

4. The method of claim 1, wherein said amino acid sequence is SEQ ID NO:2.

5. The method of claim 1, wherein said amino acid sequence is SEQ ID NO:3.

6. The method of claim 2, wherein said mutant protein consists of SEQ ID NO:1.

7. The method of claim 6, wherein said transactivation domain is the transactivation domain of NFκB p65.

8. The method of claim 6, wherein said transactivation domain is the transactivation domain of NFAT2.

9. The method of claim 2, wherein said mutant protein consists of SEQ ID NO:2.

10. The method of claim 9, wherein said transactivation domain is the transactivation domain of NFκB p65.

11. The method of claim 9, wherein said transactivation domain is the transactivation domain of NFAT2.

12. The method of claim 2, wherein said mutant protein consists of SEQ ID NO:3.

13. The method of claim 12, wherein said transactivation domain is the transactivation domain of NFκB p65.

14. The method of claim 12, wherein said transactivation domain is the transactivation domain of NFAT2.

Description

(1) The current invention thus relates to the use of a protein comprising at least a HIV-derived accessory protein tat (trans-activator of transcription) or any derivative thereof for the reactivation of latent human immunodeficiency virus (HIV) from cells present in a HIV-infected patient.

(2) Alternatively it can be expressed that the invention concerns a method of reactivating latent HIV present in a host cell by exposing a HIV infected cell with a HIV-derived accessory protein tat (trans-activator of transcription) or any derivative thereof.

(3) Tat stands for, as mentioned above, “Trans-Activator of Transcription” and Tat consists of between 86 and 101 amino acids depending on the subtype.

(4) Also, in molecular biology, tat is a protein which is encoded for by the tat gene in HIV-1. Tat is a regulatory protein that drastically enhances the efficiency of viral transcription.

(5) Preferably said protein comprises at least a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV but more preferably said protein comprises at least the first 57 N-terminal amino acids of wild-type tat (86-101 aa) for the reactivation of latent HIV.

(6) Said protein may also comprise at least the first 60 N-terminal amino acids of wild-type tat (86-101 aa) for the reactivation of latent HIV.

(7) Said 57 amino acids are represented by the following SEQ ID No. 1:

(8) TABLE-US-00001 MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRK KRRQRRR.

(9) The mentioned 60 amino acids are represented by the following SEQ ID No; 2:

(10) TABLE-US-00002 MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRK KRRQRRRAHQ

(11) The 66 amino acid deletion mutant [T66] according to the invention with a reactivation capacity close to full length exon 1 Tat72 has the amino acid sequence of SEQ ID NO 3:

(12) TABLE-US-00003 MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRK KRRQRRRAHQNSQTHQ.

(13) In Table 1 below substitutions are provided for each position in these SEQ ID NO: 1, 2 and 3 respectively which are feasible in order to obtain a protein falling within the scope of the present invention.

(14) TABLE-US-00004 TABLE 1 Primary T66 amino acid  sequence with indicated substitutions No T66 Substitutions  1. M  2. E D  3. P L  4. V I  5. D N  6. P H  7. R N/S/K  8. L I  9. E D 10. P 11. W 12. K N/E/Q/H 13. H Q/R 14. P S 15. G 16. S 17. Q R/K 18. P 19. K R/T/A/I/S/Q/N/E/G/P 20. T 21. A P/D/N/E/S 22. C 23. T N/S 24. N K/P/T/S/A/Q/R/G 25. C 26. Y F 27. C 28. K 29. K R/H/A/M/V/Q/E/S/I/Y 30. C 31. C S 32. F Y/W/L/M 33. H 34. C 35. Q L/P/Y/I/V/M/A/T 36. V L/W/A/I/S/Y/M/D/K/H/N/R/T/F/C 37. C 38. F L 39. M T/Q/I/L/H/V/A/S 40. T K/N/H/Q/S/R/A/T/D 41. K 42. A G 43. L 44. G S/R 45. I T/V/L 46. S F/Y/V/I/C/P/L/H/R 47. Y H/N 48. G 49. R K 50. K R 51. K R 52. R W/Q 53. R K/S/G/T/Q/N 54. Q H/R/P/L/K/S 55. R Q/H 56. R H/P/Q/T/S 57. R G/S/T/N/K/A/P/Q 58. A T/P/S 59. H P/S/A/T 60. Q P/H/R/E/K/N/Y/L 61. N S/D/G/R/C/A/H 62. S N/G/Y/R/D/C/H 63. Q K/E/G/P/S/T/A/R 64. T D/A/I/N/S/P/G/V/H/E/L 65. H N/D/Y/R 66. Q K
(SEQ ID NO: 4)

Definitions

(15) By the term “amino acid” is meant, for purposes of the specification and claims and in reference to the protein according to the present invention, to refer to a molecule that has at least one free amine group and at least one free carboxyl group and may further comprise one or more free chemical reactive group other than an amine or a carboxyl group (e.g., a hydroxyl, a sulfhydryl, etc). The amino acid may be a naturally occurring amino acid (e.g., L-amino acid and is depicted in this specification as a capital letter in the sequence), a non-naturally occurring amino acid (e.g., D-amino acid and is depicted in this specification as a small letter in the sequence), a synthetic amino acid, a modified amino acid, an amino acid derivative, an amino acid precursor, and a conservative substitution.

(16) A person skilled in the art would know that the choice of amino acids incorporated into a protein will depend, in part, on the specific physical, chemical or biological characteristics required of the protein. Such characteristics are determined, in part, by determination of helicity and activity. For example, a skilled person would know that amino acids in a synthetic protein may be comprised of one or more of naturally occurring (L-) amino acid and non-naturally occurring (D-) amino acid.

(17) A “conservative substitution” is used in this specification to mean one or more amino acids substitution in the sequence of the protein such that the protein still demonstrate the unexpected, improved biological activity. This includes substitutions of amino acids having substantially the same charge, size, hydrophilicity, and/or aromaticity as the amino acid replaced.

Nomenclature Used in this Specification

(18) For the L-natural amino acids, as known in the art, the following abbreviations were used:

(19) TABLE-US-00005 Symbol Name 3-Letter 1-Letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

(20) Furthermore part of the invention is the use of any of the above mentioned proteins wherein said protein comprises in addition another protein forming a fusion protein. Examples of such domains fused to HIV tat are transactivation domains (TAD) of host transcription factors such as NFkB or NFAT. By using the specific recruitment of Tat to the HIV LTR, additional transactivation domains are delivered in close proximity to the HIV promoter and surprisingly result in additional activation. Table 5 shows the increased activity of T66 linked to TADs from NFkB p65 and NFAT2. Another option would be to add a cell-targeting moiety to HIV tat such as anti-CD3 antibody or IL7 to target the transactivator more specifically to cells that are known to harbor latent HIV.

(21) The T66 protein is used in a concentration of 1 μM to 10 μM, preferably of 2 μM to 5 μM.

(22) After the use of the protein according to the invention HIV can be eradicated by addition of an antiviral agent such as a small molecule and/or antibody directed towards HIV in order to realize a cure of HIV.

(23) Part of the invention is also a pharmaceutical composition comprising the protein above referenced and a pharmaceutically accepted carrier.

(24) To the present invention also belongs a method for treating a subject with human immunodeficiency virus (HIV) comprising the steps of: a) administering to said subject an effective amount of the pharmaceutical composition comprising the protein according to the invention and a pharmaceutically accepted carrier; and b) administering to said subject an effective amount of one or more anti-viral agent.

Experimental Part

(25) TABLE-US-00006 TABLE 2 HIV LTR transactivation by Tat C-terminal deletion mutants. Tat variant % Tat72 activity 1-66 92.2 (±12.7) 1-64 77.2 (±10.8) 1-60 77.2 (±10.8) 1-57 40.2 (±2.3) 1-50  0.0 (NA)

(26) Activity is expressed in % with respect to the activity achieved by full length Tat72 exon-1 set to 100%. Shown data are mean values [n 4] with indicated (standard deviation).

(27) Tat-mediated transactivation was determined by triple plasmid transfection of HEK293 cells with pEF-T, a Tat expression plasmid, LTR-FLuc, a HIV LTR-controlled firefly luciferase reporter plasmid and pEF1-RLuc, a EF1α promoter driven Renilla luciferase reporter plasmid. Luciferase reporter activities were assessed 24 h post transfection using the Dual-Glo luciferase assay [Promega]. The measured HIV LTR luciferase data were normalized by Renilla luciferase data retrieved from the pEF1-RLuc plasmid. LTR activity is expressed as percentage of wild type exon-1 Tat72 activity.

(28) TABLE-US-00007 TABLE 3 Titration of tat protein variants on latent HIV LTR-GFP reporter cell line. Tat [μg] 100 80 70 60 50 40 30 0 T72 93.7 93.8 88.8 76.3 60.4 47.2 18.6 1.2 T66 94.5 93.9 87.6 88.7 87.0 53.8 45.7 1.0 T60 93.3 95.8 95.2 92.1 87.0 33.4 28.5 1.1

(29) MT4-LTR-GFP cells were incubated overnight with different Tat variants at the indicated concentration [μg/ml]. LTR activation was determined by flow cytometry.

(30) TABLE-US-00008 TABLE 4 Ex vivo activation of CD4 cells with Tat protein T60, T66, and T86. % Activity (PMA/PHA) Tat Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 T60 405/328 ND 213/196 ND ND/254 T66 838/680 1121/986 530/487 ND/479 ND/636 T86 ND ND ND ND/436 ND/408

(31) Activation of latent HIV from primary CD4.sup.+ T cells by overnight incubation with different Tat proteins. Indicated numbers represent HIV activation in percentage to reference compounds PMA and PHA set to 100%. The shown % increase compared to PMA/PHA is statistically significant (p<0.01).

(32) CD4.sup.+ T cells were isolated from 200 ml whole blood using CD4 microbeads (Miltenyi Biotec) according to manufacturer's protocol. Blood originates from HIV-infected individuals under long-term HAART with undetectable plasma virus. Ten to twenty replicates of cell pools plated at 1×10.sup.6 CD4.sup.+ T cells/well were incubated overnight with compounds or mock controls (DMSO/PBS). Total RNA was isolated from each replicate using the Magmax 96 Total RNA isolation kit (Ambion) following the manufacturer's protocol. Duplicate cDNA reactions were performed on each RNA replicate using SuperScript III First-Strand Synthesis kit (Invitrogen) according to the manufacturer's protocol. Quantitative real-time PCR (QPCR) was conducted on each cDNA applying gag-specific primers and the nucleic acid detection dye Sybr Green I [Invitrogen]. Standard curves were generated using cDNA synthesized from in vitro transcribed RNA. The detection limit of the QPCR assay was determined to be within 1-10 copies/reaction. Cycle threshold (ct) values 40 were excluded from the analysis. The Wilcoxon rank sum test was used to calculate the statistical significance of the relative HIV-1 gag RNA copy number between different conditions.

(33) TABLE-US-00009 TABLE 5 T66 fusion proteins result in increased activation when compared to T66. pEF-T ID FLUC RLUC FLUC.sub.norm Fold % T66 T66 15437 267 37049 26 100 T66-NFK114 25480 380 42918 30 116 T66-NFAT2C 22614 304 47674 33 129 Cell control 1450 641 1448 1 4

(34) Triple plasmid transfection of HEK293 cells with pEF-T (Tat expression plasmid), LTR-FLuc (HIV LTR-controlled firefly luciferase reporter plasmid) and pEF1-RLuc (EF1α promoter driven Renilla luciferase reporter plasmid). Luciferase reporter activities were assessed 24 h post transfection using the Dual-Glo luciferase assay [Promega]. The measured HIV LTR luciferase data were normalized by Renilla luciferase data collected from the co-transfected pEF1-RLuc signal. LTR activation is either expressed as fold increase over cell control or as percentage of T66 activity.