Mutated non-primate lentiviral Env proteins and their use as drugs

Abstract

A pharmaceutical composition includes, as active substance a mutated non-primate lentiviral Env protein having decreased immunosuppressive properties, substantially no immunosuppressive properties or no immunosuppressive properties, or a variant of the mutated lentiviral Env protein, or a fragment of the above proteins, in association with a pharmaceutically acceptable carrier.

Claims

1. A pharmaceutical composition comprising as an active substance an isolated mutated feline lentiviral ENV protein that has lost at least 50%, at least 75% or 100% of its immunosuppressive activity in comparison to immunosuppressive activity of a wild type ENV protein, or a fragment thereof, said fragment of said isolated mutated feline lentiviral ENV protein comprising at least 40 amino acids, said mutated feline lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type feline lentiviral ENV protein, said mutated ENV protein having at least 80% identity to the sequence SEQ ID NO: 5, said mutated feline lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence: TABLE-US-00054 (SEQIDNO:1) A-[I/M/T/L]-X1-X2-X3-X4-X5-T-A, wherein, X.sub.1 is A or R, and X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.2 is A or R, and X.sub.1, X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.3 is A or R, and X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are any amino acid, or X.sub.4 is A or R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.5 are any amino acid, or X.sub.5 is A or R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are any amino acid, in association with a pharmaceutically acceptable carrier.

2. A pharmaceutical composition comprising as an active substance an isolated mutated feline lentiviral ENV protein that has lost at least 50%, at least 75% or 100% of its immunosuppressive activity in comparison to immunosuppressive activity of a wild type ENV protein, or a fragment thereof, said fragment of said isolated mutated feline lentiviral ENV protein comprising at least 40 amino acids, said mutated feline lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type feline lentiviral ENV protein, said mutated ENV protein having at least 80% identity to the sequence SEQ ID NO: 5, said mutated feline lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence: TABLE-US-00055 (SEQIDNO:3) [V/I]-[E/R]-A-[I/M/T/L]-X1-X2-X3-X4-X5-T-A-[F/L]- A-M, wherein, X.sub.1 is A or R, and X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.2 is A or R, and X.sub.1, X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.3 is A or R, and X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are any amino acid, or X.sub.4 is A or R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.5 are any amino acid, or X.sub.5 is A or R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are any amino acid, in association with a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 1, wherein said loss of at least 50%, at least 75% or 100% of the immunosuppressive activity of said mutated feline lentiviral ENV protein or of said fragment thereof being liable to be assessed by the fact that in an in vivo assay involving engrafted tumor cells rejection, in animals excluding human beings, said tumor cells being transduced either so as to express said mutated ENV protein or said fragment (mutated ENV tumor cells), or said tumor cells being transduced so as to express said wild type ENV protein or a fragment thereof (wild type ENV tumor cells), or said tumor cells being not transduced (normal tumor cells), the following ratio: immunosuppression index of said mutated ENV protein or of said fragment (i.sub.mutated env)/immunosuppression index of wild type ENV protein (i.sub.wild type env) is less than 0.5, i.sub.mutated env being defined by: (maximum area reached by mutated ENV tumor cellsmaximum area reached by normal tumor cells)/(maximum area reached by normal tumor cells), and i.sub.wild type env being defined by: (maximum area reached by wild type ENV tumor cellsmaximum area reached by normal tumor cells)/(maximum area reached by normal tumor cells).

4. The pharmaceutical composition according to claim 1, wherein: X.sub.1 is A or R, X.sub.2 is A or R, and X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.1 is A or R, X.sub.3 is A or R, and X.sub.2, X.sub.4 and X.sub.5 are any amino acid, or X.sub.1 is A or R, X.sub.4 is A or R, and X.sub.2, X.sub.3 and X.sub.5 are any amino acid, or X.sub.1 is A or R, X.sub.5 is A or R, and X.sub.2, X.sub.3 and X.sub.4 are any amino acid, or X.sub.2 is A or R, X.sub.3 is A or R, and X.sub.1, X.sub.4 and X.sub.5 are any amino acid, or X.sub.2 is A or R, X.sub.4 is A or R, and X.sub.1, X.sub.3 and X.sub.5 are any amino acid, or X.sub.2 is A or R, X.sub.5 is A or R, and X.sub.1, X.sub.3 and X.sub.4 are any amino acid, or X.sub.3 is A or R, X.sub.4 is A or R, and X.sub.1, X.sub.2 and X.sub.5 are any amino acid, or X.sub.3 is A or R, X.sub.5 is A or R, and X.sub.1, X.sub.2 and X.sub.4 are any amino acid, or X.sub.4 is A or R, X.sub.5 is A or R, and X.sub.1, X.sub.2 and X.sub.3 are any amino acid.

5. The pharmaceutical composition according to claim 1, wherein: X.sub.1 is R, and X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.2 is R, and X.sub.1, X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.3 is R, and X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are any amino acid, or X.sub.4 is R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.5 are any amino acid, or X.sub.5 is R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are any amino acid.

6. The pharmaceutical composition according to claim 1, wherein: X.sub.1 is R, X.sub.2 is R, and X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.1 is R, X.sub.3 is R, and X.sub.2, X.sub.4 and X.sub.5 are any amino acid, or X.sub.1 is R, X.sub.4 is R, and X.sub.2, X.sub.3 and X.sub.5 are any amino acid, or X.sub.1 is R, X.sub.5 is R, and X.sub.2, X.sub.3 and X.sub.4 are any amino acid, or X.sub.2 is R, X.sub.3 is R, and X.sub.1, X.sub.4 and X.sub.5 are any amino acid, or X.sub.2 is R, X.sub.4 is R, and X.sub.1, X.sub.3 and X.sub.5 are any amino acid, or X.sub.2 is R, X.sub.5 is R, and X.sub.1, X.sub.3 and X.sub.4 are any amino acid, or X.sub.3 is R, X.sub.4 is R, and X.sub.1, X.sub.2 and X.sub.5 are any amino acid, or X.sub.3 is R, X.sub.5 is R, and X.sub.1, X.sub.2 and X.sub.4 are any amino acid, or X.sub.4 is R, X.sub.5 is R, and X.sub.1, X.sub.2 and X.sub.3 are any amino acid.

7. The pharmaceutical composition according to claim 1, wherein: X.sub.1 is A or R, and X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.2 is A or R, and X.sub.1, X.sub.3, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.3 is A or R, and X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.4 is A or R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.5 are any amino acid different from A or R, or X.sub.5 is A or R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are any amino acid different from A, G or R.

8. The pharmaceutical composition according to claim 1, wherein: X.sub.1 is A or R, X.sub.2 is A, G or R, and X.sub.3, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.1 is A or R, X.sub.3 is A, G or R, and X.sub.2, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.1 is A or R, X.sub.4 is A, G or R, and X.sub.2, X.sub.3 and X.sub.5 are any amino acid different from A, G or R, or X.sub.1 is A or R, X.sub.5 is A, G or R, and X.sub.2, X.sub.3 and X.sub.4 are any amino acid different from A, G or R, or X.sub.2 is A or R, X.sub.3 is A, G or R, and X.sub.1, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.2 is A or R, X.sub.4 is A, G or R, and X.sub.1, X.sub.3 and X.sub.5 are any amino acid different from A, G or R, or X.sub.2 is A or R, X.sub.5 is A, G or R, and X.sub.1, X.sub.3 and X.sub.4 are any amino acid different from A, G or R, or X.sub.3 is A or R, X.sub.4 is A, G or R, and X.sub.1, X.sub.2 and X.sub.5 are any amino acid different from A, G or R, or X.sub.3 is A or R, X.sub.5 is A, G or R, and X.sub.1, X.sub.2 and X.sub.4 are any amino acid different from A, G or R, or X.sub.4 is A or R, X.sub.5 is A, G or R, and X.sub.1, X.sub.2 and X.sub.3 are any amino acid different from A, G or R.

9. The pharmaceutical composition according to claim 1, wherein: TABLE-US-00056 (SEQIDNO:489) A-[I/M/T/L]-[A/G/R]-K-[F/P]-[L/V/I]-Y-T-A, or (SEQIDNO:490) A-[I/M/T/L]-E-[A/G/R]-[F/P]-[L/V/I]-Y-T-A, or (SEQIDNO:491) A-[I/M/T/L]-E-K-[A/G/R]-[L/V/I]-Y-T-A, or (SEQIDNO:492) A-[I/M/T/L]-E-K-[F/P]-[A/G/R]-Y-T-A, or (SEQIDNO:493) A-[I/M/T/L]-E-K-[F/P]-[L/V/I]-[A/G/R]-T-A, or (SEQIDNO:494) A-[I/M/T/L]-E-K-[A/G/R]-[A/G/R]-Y-T-A, inparticular: (SEQIDNO:489) A-[I/M/T/L]-[A/G/R]-K-[F/P]-[L/V/I]-Y-T-A, or (SEQIDNO:495) A-[I/M/T/L]-E-G-[F/P]-[L/V/I]-Y-T-A, or (SEQIDNO:491) A-[I/M/T/L]-E-K-[A/G/R]-[L/V/I]-Y-T-A, or (SEQIDNO:492) A-[I/M/T/L]-E-K-[F/P]-[A/G/R]-Y-T-A, or (SEQIDNO:496) A-[I/M/T/L]-E-K-[F/P]-[L/V/I]-G-T-A, or (SEQIDNO:494) A-[I/M/T/L]-E-K-[A/G/R]-[A/G/R]-Y-T-A.

10. The pharmaceutical composition according to claim 1, wherein: TABLE-US-00057 (SEQIDNO:497) A-[I/M/T/L]-R-K-[F/P]-[L/V/I]-Y-T-A, or (SEQIDNO:498) A-[I/M/T/L]-E-R-[F/P]-[L/V/I]-Y-T-A, or (SEQIDNO:499) A-[I/M/T/L]-E-K-R-[L/V/I]-Y-T-A, or (SEQIDNO:500) A-[I/M/T/L]-E-K-[F/P]-R-Y-T-A, or (SEQIDNO:501) A-[I/M/T/L]-E-K-[F/P]-[L/V/I]-R-T-A, or (SEQIDNO:502) A-[I/M/T/L]-E-K-R-R-Y-T-A.

11. The pharmaceutical composition according to claim 1, wherein said isolated mutated feline lentiviral ENV protein or said fragment thereof, comprises one of the amino acid sequences SEQ ID NO: 28 to 171.

12. The pharmaceutical composition according to claim 1, wherein said isolated mutated feline lentiviral ENV protein consists of one of the amino acid sequences: SEQ ID NO: 5 and SEQ ID NO: 317 to 342 and 374 to 419.

13. The pharmaceutical composition as defined in claim 1, wherein the composition stimulates an immune response in a host organism.

14. The pharmaceutical composition according to claim 2, wherein said loss of at least 50%, at least 75% or 100% of the immunosuppressive activity of said mutated feline lentiviral ENV protein or of said fragment thereof being liable to be assessed by the fact that in an in vivo assay involving engrafted tumor cells rejection, in animals excluding human beings, said tumor cells being transduced either so as to express said mutated ENV protein or said fragment (mutated ENV tumor cells), or said tumor cells being transduced so as to express said wild type ENV protein or a fragment thereof (wild type ENV tumor cells), or said tumor cells being not transduced (normal tumor cells), the following ratio: immunosuppression index of said mutated ENV protein or of said fragment (i.sub.mutated env)/immunosuppression index of wild type ENV protein (i.sub.wild type env) is less than 0.5, or even less than 0.25, i.sub.mutated env being defined by: (maximum area reached by mutated ENV tumor cellsmaximum area reached by normal tumor cells)/(maximum area reached by normal tumor cells), and i.sub.wild type env being defined by: (maximum area reached by wild type ENV tumor cellsmaximum area reached by normal tumor cells)/(maximum area reached by normal tumor cells).

15. The pharmaceutical composition according to claim 2, wherein: X.sub.1 is A or R, X.sub.2 is A or R, and X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.1 is A or R, X.sub.3 is A or R, and X.sub.2, X.sub.4 and X.sub.5 are any amino acid, or X.sub.1 is A or R, X.sub.4 is A or R, and X.sub.2, X.sub.3 and X.sub.5 are any amino acid, or X.sub.1 is A or R, X.sub.5 is A or R, and X.sub.2, X.sub.3 and X.sub.4 are any amino acid, or X.sub.2 is A or R, X.sub.3 is A or R, and X.sub.1, X.sub.4 and X.sub.5 are any amino acid, or X.sub.2 is A or R, X.sub.4 is A or R, and X.sub.1, X.sub.3 and X.sub.5 are any amino acid, or X.sub.2 is A or R, X.sub.5 is A or R, and X.sub.1, X.sub.3 and X.sub.4 are any amino acid, or X.sub.3 is A or R, X.sub.4 is A or R, and X.sub.1, X.sub.2 and X.sub.5 are any amino acid, or X.sub.3 is A or R, X.sub.5 is A or R, and X.sub.1, X.sub.2 and X.sub.4 are any amino acid, or X.sub.4 is A or R, X.sub.5 is A or R, and X.sub.1, X.sub.2 and X.sub.3 are any amino acid.

16. The pharmaceutical composition according to claim 2, wherein: X.sub.1 is R, and X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.2 is R, and X.sub.1, X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.3 is R, and X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are any amino acid, or X.sub.4 is R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.5 are any amino acid, or X.sub.5 is R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are any amino acid.

17. The pharmaceutical composition according to claim 1, wherein said mutated ENV protein has at least 90% identity to the sequence SEQ ID NO: 5.

18. The pharmaceutical composition according to claim 1, wherein said fragment of said isolated mutated feline lentiviral ENV protein comprises at least 60 amino acids.

19. The pharmaceutical composition according to claim 2, wherein said fragment of said isolated mutated feline lentiviral ENV protein comprises at least 60 amino acids.

20. The pharmaceutical composition according to claim 2, wherein said mutated ENV protein has at least 90% identity to the sequence SEQ ID NO: 5.

21. The pharmaceutical composition according to claim 7, wherein: X.sub.1 is R, and X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.2 is R, and X.sub.1, X.sub.3, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.3 is R, and X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.4 is R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.5 are any amino acid different from A, G or R, or X.sub.5 is R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are any amino acid different from A, G or R.

22. The pharmaceutical composition according to claim 8, wherein: X.sub.1 is R, X.sub.2 is R, and X.sub.3, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.1 is R, X.sub.3 is R, and X.sub.2, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.1 is R, X.sub.4 is R, and X.sub.2, X.sub.3 and X.sub.5 are any amino acid different from A, G or R, or X.sub.1 is R, X.sub.5 is R, and X.sub.2, X.sub.3 and X.sub.4 are any amino acid different from A, G or R, or X.sub.2 is R, X.sub.3 is R, and X.sub.1, X.sub.4 and X.sub.5 are any amino acid different from A, G or R, or X.sub.2 is R, X.sub.4 is R, and X.sub.1, X.sub.3 and X.sub.5 are any amino acid different from A, G or R, or X.sub.2 is R, X.sub.5 is R, and X.sub.1, X.sub.3 and X.sub.4 are any amino acid different from A, G or R, or X.sub.3 is R, X.sub.4 is R, and X.sub.1, X.sub.2 and X.sub.5 are any amino acid different from A, G or R, or X.sub.3 is R, X.sub.5 is R, and X.sub.1, X.sub.2 and X.sub.4 are any amino acid, different from A, G or R or X.sub.4 is R, X.sub.5 is R, and X.sub.1, X.sub.2 and X.sub.3 are any amino acid different from A, G or R.

23. A pharmaceutical composition comprising as active substance an isolated mutated feline lentiviral ENV protein that has lost at least 50%, at least 75%, or 100% of its immunosuppressive activity in comparison to immunosuppressive activity of a wild type ENV protein, or a fragment thereof comprising at least 40 amino acids, said mutated feline lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type feline lentiviral ENV protein, said mutated ENV protein having 77% identity to sequence SEQ ID NO: 5, said mutated feline lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence: TABLE-US-00058 (SEQIDNO:1) A-[I/M/T/L]-X1-X2-X3-X4-X5-T-A, wherein, X.sub.1 is R, and X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.2 is R, and X.sub.1, X.sub.3, X.sub.4 and X.sub.5 are any amino acid, or X.sub.3 is R, and X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are any amino acid, or X.sub.4 is R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.5 are any amino acid, or X.sub.5 is R, and X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are any amino acid, in association with a pharmaceutically acceptable carrier.

Description

LEGEND TO THE FIGURES

(1) FIG. 1:

(2) (A) FIV Envelope phylogeny, with the A-D subtypes indicated and the reference Petaluma strain boxed (from Pu et al, Journal of Feline Medicine and Surgery, 2005, 7:65-70).

(3) (B) Structure of the FIV Envelope protein, delineation of the characteristic functional domains and alignment of the 64-aa immunosuppressive-containing domains from selected FIV Env proteins. The SU and TM subunits of the FIV Env are indicated, together with the fusion peptide, the transmembrane anchoring domain of the TM subunit, and the immunosuppressive domain (ISD).

(4) The aligned sequences of the 64-aa immunosuppressive-containing domains correspond to fragments of the Envelope protein from 33 distinct strains (i.e. 33 different accession numbers), only 20 of these 64-aa fragments are different (i.e. 20 SEQ ID numbers have been given).

(5) FIG. 2:

(6) Immunosuppressive activity of the full-length FIV envelope protein (FIV Env) and of the 64 aa-long FIV envelope subdomain delineated in FIG. 1 (FIV64 Env). Immunosuppression was tested using the in vivo MCA205 tumor rejection assay (see scheme on top and Materials and Methods): MCA205 tumor cells are transduced with an expression vector (containing a selectable hygromycin gene) for the indicated Env protein or fragment, stably transduced cells are then selected for expression of the corresponding Env, and are finally engrafted into Balb/c mice (allogenic graft, normally resulting in tumor rejection); tumor growth/rejection is then monitored and provides an immunosuppressive index (calculated as indicated in Materials and Methods). Y-axis corresponds to the value of the immunosuppression index. Immunosuppression indexes are means of at least 3 independent experiments, with standard deviation. The Murine Leukemia Virus (MLV) envelope ectodomains, wild-type and mutant (Schlecht-Louf et al., Proc Natl Acad Sci USA. 2010, 107(8):3782-7) are used as an internal control.

(7) FIG. 3:

(8) Functional identification of the aminoacids in the FIV envelope 64 aa domain directly involved in immunosuppressive activity, and search for aminoacid substitutions inhibiting this activity. Immunosuppressive activity was tested as in FIG. 2, using the in vivo MCA205 tumor rejection assay (see Materials and Methods). The mutated aminoacids are indicated with their position (see sequence of FIV64 on top) and the nature of the substitution. Wwt and Wmut are controls (from the human endogenous retrovirus HERV-W Envelope, Mangeney et al., Proc Natl Acad Sci USA. 2007, 104(51):20534-9). Immunosuppression indexes correspond to 3 independent experiments, with standard deviation.

(9) FIG. 4:

(10) Antibody response of mice inoculated with cells expressing wild-type and mutant FIV64 Env domains (see scheme on top): MCA205 tumor cells are stably transduced as in FIG. 2 with expression vectors for the indicated FIV64 Env wt and mutants, and cells engrafted into C57Bl/6 mice (syngenic graft, resulting in constant tumor growth without rejection); specific anti-FIV64 antibodies are quantitated from the collected blood samples as indicated in Materials and Methods. Results are representative of 2 independent experiments.

(11) FIG. 5:

(12) Antibody response of mice injected with MBP-FIV64, wt and mutants, recombinant proteins (see scheme on top): FIV64 Env, wt and mutants, fused with MBP are i.v. injected three times with a 1-week interval into Balb/C mice (10 g per injection); mice are then blood-sampled and anti-MBP IgG levels are determined by ELISA using plates coated with MBP-LacZ (MBP-LacZ is a control MBP fusion with the 83aa LacZ fragment (-subunit of E. coli -galactosidase, see Materials and Methods). Results are representative of 2 independent experiments.

(13) FIG. 6:

(14) Expression of the FIV Env proteins, wt and mutants, at the surface of cells transfected with the indicated expression vectors (see scheme on top).

(15) Expression profile of the wild-type and mutant FIV Env proteins was assayed by FACS analysis using full-length FIVenv-expressing vectors and an anti-SU FIVenv monoclonal antibody (Antibodies-online, BmbH, Germany). The data correspond to the Mean Fluorescence Intensity, from three independent experiments, with standard deviation.

(16) FIG. 7:

(17) Functional characterization of the FIV Env proteins, wild-type (wt) and mutants. Fusogenic activity of the FIVenv, wild-type and mutants, are measured by a cell-cell fusion assay and quantified by a fusion index (see scheme on top and Materials and Methods). The data correspond to three independent experiments, with standard deviation).

EXAMPLES

(18) The FIV Envelope and a Delineated Subdomain of the TM Subunit are Immunosuppressive In Vivo

(19) Identification of an immunosuppressive activity of the FIV envelope and of the domain within the FIV envelope responsible for this activity was achieved using an in vivo tumor rejection assay that we had previously used to demonstrate the immunosuppressive activity of the Env protein of oncoretroviruses (e.g. murine MoMLV and simian MPMV, see Mangeney and Heidmann, Proc Natl Acad Sci USA, 1998, 95:14920-14925; Mangeney et al., Proc Natl Acad Sci USA, 2007, 104(51):20534-9).

(20) The rationale of the assay can be summarized as follows: while injection of MCA205 tumor cells (H-2.sup.b) into allogeneic Balb/c mice (H-2.sup.d) leads to the formation of no tumor or transient tumors that are rapidly rejected, injection of the same cells, but stably expressing an immunosuppressive retroviral Env protein, leads to the growth of larger tumors that persist for a longer time-in spite of the expression of the new exogenous antigen.

(21) This difference is not associated with a difference in intrinsic cell growth rate since it is not observed in syngeneic C57BL/6 mice, and is immune system-dependent.

(22) The extent of immunosuppression can be quantified by an index based on tumor size: (A.sub.envA.sub.none)/A.sub.none, where A.sub.env and A.sub.none are the mean areas at the peak of growth of tumors from Balb/c mice injected with env-expressing or control cells, respectively. A positive index indicates that env expression facilitates tumor growth, as a consequence of its immunosuppressive activity; a null or negative index points to no effect or even an inhibitory effect, respectively. The latter can be explained by a stimulation of the immune response of the host against the new foreign antigen, represented by a non-immunosuppressive Env protein, expressed at the surface of tumor cells. Accordingly, as illustrated in FIG. 2, it can be observed that the FIV env is immunosuppressive, and that this activity is carried by a 64-mer peptide of the transmembrane (TM) subunit of the envelope protein. The immunosuppression indexes are close to those found for non-lentiviral env proteins (such as that of the murine MLV retrovirus shown in the figure as a control, together with a non-immunosuppressive mutant).

(23) Identification of the FIV Env Amino-Acids Critical for Immunosuppression and its Inhibition, Via R-Scanning

(24) To characterize further the immunosuppressive domain (ISD) of the FIV env active in vivo, we analyzed the effect of a series of amino-acid substitutions within the FIV env 64 aa domain shown to carry the IS activity (see above). The 64-aa FIV env domain is embedded into the so-called ectodomain, which corresponds to the extracellular domain of the TM subunit, and consists in the -helical domain involved in FIV TM trimerization, and the N-term part of the loop containing the 2 well-conserved, 6/7 aa-distant, cysteine residues found in most retroviral envelopes. Refine delineation of the amino acids responsible for IS activity was thus performed by arginine-scanning within this domain. As illustrated in FIG. 3, X-to-Arg substitutions resulted in a significant change in the IS activity of the FIVenv 64 aa domain, with maximum effect observed for the F687 position and to a lesserbut significantextent for the adjacent E685, K686, L688 and Y689 positions. A series of other substitutions are performed at the F687 position, substitutions for the small A and G, and the hydrophobic L and F residues.

(25) We also analyzed the effect of this series of amino-acid substitutions within the FIVenv 64 aa domain by measuring the production of anti-FIV env IgG antibodies in mice inoculated under syngenic conditions (C57Bl/6 mice) with the cells expressing the wild-type and mutant FIVenv 64 aa domains. As illustrated in FIG. 4, X-to-Arg substitutions resulted in an increase in the antibody response, consistent with an increased immunogenicity of FIVenv 64, with again a more important effect observed for the F687 position.

(26) Additionally, a series of experiments were performed with recombinant proteins containing the FIVenv 64 domain, to assay the relative immunogenicity of the wt and mutants proteins. The domains were fused with the carrier MBP (Maltose Binding Protein), and the proteins were injected i.v. into Balb/C mice (see Materials and Methods). The immunosuppressive effects of the FIVenv 64 wt and mutants were assayed by measuring the inhibition of the anti-MBP antibody response raised in the injected mice. As illustrated in FIG. 5, a significant decreaserelative to the control (MBP-LacZ)is observed with FIVenv 64 wt, consistent with its immunosuppressive activity acting in cis, whereas almost no reduction is observed for the F687R and L688R mutants, indicative of the loss of its IS activity. A similar effect is observed for the other mutants, although at a lesser extent.

(27) Impact of the Identified Mutations on Env Protein Folding and Proper Expression as a Functional Transmembrane Protein

(28) We then tested whether the above-mentioned substitutions alter the overall capacity of the FIV envelope to be expressed by an eucaryotic cell and to be exported at the cell membrane, by introducing the mutants into an expression vector for the full-length FIV envelope. A FACS analysis of cells transfected with the wild-type and the mutant FIV envelope genes inserted within a CMV-driven expression vector (see Materials and Methods) and using an anti-SU specific monoclonal antibody, demonstrated quantitative expression of the mutant envelopes at the cell surface (FIG. 6). In particular, this analysis indicated that the F687R and L688R mutations did not significantly altered the FIV Env structure and/or SU-TM interaction. Finally, the fusogenic activity of the FIV full-length envelopes was tested by a cell-cell fusion assay, in which cells prone to FIVenv-mediated fusion were transfected as above with the corresponding expression vectors, and cell-cell fusion monitored 24-48 h post transfection by measuring the amount of multi-nucleated syncytia formed, with the process being quantified by a fusion index (see Materials and Methods). As illustrated in FIG. 7 and despite the conservation of the amount of env protein expressed at the cell surface (cf FIG. 6), variations in the fusion index exist among the various env mutants tested, although all of them remain fusion-positive,

(29) Accordingly, the present investigation has clearly identified definite positions and definite substitutions within the FIV env resulting in the loss of its IS activity.

(30) Being compatible with the conservation of the overall structure of the FIV Env protein, these substitutions should be introduced in all pharmaceutical preparations which include the Env protein as a vaccine antigen.

(31) Materials and Methods

(32) Mice and Cell Lines:

(33) C57Bl/6 and Balb/c mice, 6-10 weeks old, were obtained from Harlan (France). Mice were maintained in the animal facility of the Gustave Roussy Institute in accordance with institutional regulations. 293T (ATCC CRL11268), and MCA205 cells were cultured in DMEM supplemented with 10% fetal calf serum (Invitrogen), streptomycin (100 g/ml) and penicillin (100 units/ml).

(34) Plasmids Constructions: phCMV-FIVenv: The pET34TF10 full feline immunodeficiency virus (FIV) genome clone (strain petaluma) (Talbott et al., Proc Natl Acad Sci USA, 1989, 86(15):5743-4747) served as template to generate full-length FIVenv PCR fragment using primers 1-2. This PCR fragment was digested with XhoI and MluI restriction enzymes to be ligated with phCMV vector opened with the same enzymes to generate a phCMV-FIVenv expression vector. phCMV-FIVenv mutants: Mutated phCMV-FIVenv was obtained by successive PCR using appropriate primers. A first series of PCRs was performed with phCMV-FIVenv as template with primer 3 and the reverse primer designed with the mutation (i.e. primers 5, 7, 9, 11, 13, 15, 17, 19, 21, 23); and primer 2 and the forward primer bearing the mutation (i.e. primers 4, 6, 8, 10, 12, 13, 14, 16, 18, 20, 22) to introduce the mutations E685A, E685R, K686A, K686R, F687A, F687R, L688A, L688R, Y689A and Y689R, respectively. The two PCR fragments bearing the same mutation were purified and mixed to be used as template for a subsequent PCR with primers 3 and 2. These PCR fragments were digested by SpeI and MluI restriction enzymes and purified. In the meantime the FIVenv fragment obtained after digestion of the phCMV-FIVenv with the XhoI and SpeI restriction enzymes was purified. A three-fragment ligation with phCMV opened with XhoI and MluI, the FIVenv fragment digested with XhoI and SpeI, and the SpeI-MluI-restricted PCR fragment with the desired mutation, was performed to obtain the phCMV-FIVenv mutants. Mutations of the amino acids E685, K686 and Y689 into G, L or F, mutations of the amino acid F687 into G or L, and mutations of the amino acid L688 into G or F are similarly performed using appropriate primers pairs (i.e. primers 31-58). pDFG-FIVenv and pDFG-FIVenv mutants: FIVenv and FIVenv mutant inserts were obtained by restriction with AgeI and MluI of the phCMV-FIVenv and mutants. These inserts were ligated with pDFG-ectoSyncytin-1 (see above Mangeney et al, PNAS, 2007) opened with the same enzymes. pDFG-FIV64 and pDFG-FIV112, wild-type and mutants: fragments of the FIVenv, wild-type and mutants, were PCR-amplified from phCMV-FIVenv using primers 24-25 or 24-26 to obtain FIV64 and FIV112 fragments, respectively. These PCR fragments were digested with SfiI and MluI and inserted into pDFG-ectoSyncytin-1 (see above Mangeney et al, PNAS, 2007) opened with the same enzymes. pSIN-FIV64: This vector was based on the lentiviral vector pHR'SIN-cPPT-SEW described in Demaison et al. (Human Gene Therapy, 2002, 13:803-813). The pSIN-FIV64 vectors were obtained by insertion of the hygromycin resistance gene under the control of the phosphoglycerate kinase promoter (PGK) at the end of WPRE sequence of pHR'SIN-cPPT-SEW. Then the FIV-64 fragments, wt and mutants, were PCR-amplified from pDFG-FIV64 wt and mutants, using primers 27-28, digested with Bam HI and Not I, and inserted downstream the pSec sequence under control of the SFFV promoter. pMal-MBP-FIV: Bacterial expression vectors for fusion proteins with E. coli maltose-binding protein (MBP) were constructed by ligation of a modified pMal-c2x (described in Center et al., Protein Sci, 1998, 7(7):1612-1619) opened with PstI and HindIII, with the FIV-env ectodomain, wt and mutants, obtained by PCR from phCMV Env wt and mutants, with primer pair 29-30. These pMal-MBP-FIV64 vectors encode a 64-residue long FIV-ectodomain fused to the C terminus of MBP through a trialanine linker. The empty pMal-c2x vector encodes the 85-residue long -subunit of E. coli -galactosidase fused to the C terminus of MBP and was used as a control (pMal-LacZa).

(35) All the constructions were sequenced before use.

(36) TABLE-US-00053 TABLE2 Primerlist No Name Primersequence(5-3) SEQIDNO: 1 FIVenvXhoAge atacatCTCGAGACCGGTccaactagaaccATGGCAGA 348 KozakATG(FOR) AGGATTTGCAGCC 2 FIVenvStopMlu ATACATacgcgtTCATTCCTCCTCTTTTTCAGAC 349 (REV) ATGCCAC 3 FIV-env-2000-FOR TACTGCTATAGGGATGGTAACACAATACCAC 350 CAAG 4 FIV-env-E685A- GATTAAAAGTAGAAGCTATGGCAAAATTTTT 351 FOR GTATACAGC 5 FIV-env-E685A- GCTGTATACAAAAATTTTGCCATAGCTTCTA 352 REV CTTTTAATC 6 FIV-env-E685R- GATTAAAAGTAGAAGCTATGAGAAAATTTTT 353 FOR GTATACAGC 7 FIV-env-E685R- GCTGTATACAAAAATTTTCTCATAGCTTCTAC 354 REV TTTTAATC 8 FIV-env-K686A- GTAGAAGCTATGGAAGCATTTTTGTATACAG 355 FOR CTTTC 9 FIV-env-K686A- GAAAGCTGTATACAAAAATGCTTCCATAGCT 356 REV TCTAC 10 FIV-env-K686R- GTAGAAGCTATGGAAAGATTTTTGTATACAG 357 FOR CTTTC 11 FIV-env-K686R- GAAAGCTGTATACAAAAATCTTTCCATAGCT 358 REV TCTAC 12 FIV-env-F687A- GAAGCTATGGAAAAAGCCTTGTATACAGCTT 359 FOR TC 13 FIV-env-F687A- GAAAGCTGTATACAAGGCTTTTTCCATAGCT 360 REV TC 14 FIV-env-F687R- GAAGCTATGGAAAAAAGATTGTATACAGCTT 361 FOR TC 15 FIV-env-F687R- GAAAGCTGTATACAATCTTTTTTCCATAGCTTC 362 REV 16 FIV-env-L688A- GCTATGGAAAAATTTGCCTATACAGCTTTCG 363 FOR CTATG 17 FIV-env-L688A- CATAGCGAAAGCTGTATAGGCAAATTTTTCC 364 REV ATAGC 18 FIV-env-L688R- GCTATGGAAAAATTTAGGTATACAGCTTTCG 365 FOR CTATG 19 FIV-env-L688R- CATAGCGAAAGCTGTATACCTAAATTTTTCC 366 REV ATAGC 20 FIV-env-Y689A- CTATGGAAAAATTTTTGGCCACAGCTTTCGC 367 FOR TATGC 21 FIV-env-Y689A- GCATAGCGAAAGCTGTGGCCAAAAATTTTTC 368 REV CATAG 22 FIV-env-Y689R- CTATGGAAAAATTTTTGCGGACAGCTTTCGC 369 FOR TATGC 23 FIV-env-Y689R- GCATAGCGAAAGCTGTCCGCAAAAATTTTTC 370 REV CATAG 24 FIV64-Sfi-FOR GGTGACGCGGCCCAGCCGGCCgctatagaaaaggtga 371 ctggagcc 25 FIV64-Mlu-REV ATACATACGCGTTTAccttgtccacaactcaagagg 372 26 FIVeq112-MLU- ATACATACGCGTTTAccctgttttcccttgtacattattttg 373 REV 27 BamHIpsec-FOR ATAGGATCCAGAACCATGGAGACAGAC 435 ACACTC 28 NotIFIV-REV TATGCGGCCGCTTACCTTGTCCACAA 436 CTCAAG 29 PstIFIV72-FOR ATAGCTGCAGCCCAAGTTCTGGCAACCCAT 437 30 HindIIIFIV72-REV TATAAGCTTTTACCTTGTCCACAACTCAAG 438 31 FIV-env-G678A- CATCAAGTACTAGTAATAGCATTAAAAGTAG 439 FOR AAGCTATG 32 FIV-env-G678A- CATAGCTTCTACTTTTAATGCTATTACTAGTA 440 REV CTTGATG 33 FIV-env-E685G- GATTAAAAGTAGAAGCTATGGGAAAATTTTT 441 FOR GTATACAGC 34 FIV-env-E685G- GCTGTATACAAAAATTTTCCCATAGCTTCTAC 442 REV TTTTAATC 35 FIV-env-E685L- GATTAAAAGTAGAAGCTATGTTAAAATTTTT 443 FOR GTATACAGC 36 FIV-env-E685L- GCTGTATACAAAAATTTTAACATAGCTTCTA 444 REV CTTTTAATC 37 FIV-env-K686G- GTAGAAGCTATGGAAGGATTTTTGTATACAG 445 FOR CTTTC 38 FIV-env-K686G- GAAAGCTGTATACAAAAATCCTTCCATAGCT 446 REV TCTAC 39 FIV-env-K686L- GTAGAAGCTATGGAATTATTTTTGTATACAG 447 FOR CTTTC 40 FIV-env-K686L- GAAAGCTGTATACAAAAATAATTCCATAGCT 448 REV TCTAC 41 FIV-env-F687G- GAAGCTATGGAAAAAGGATTGTATACAGCTT 449 FOR TC 42 FIV-env-F687G- GAAAGCTGTATACAATCCTTTTTCCATAGCTTC 450 REV 43 FIV-env-F687L- GAAGCTATGGAAAAATTATTGTATACAGCTT 451 FOR TC 44 FIV-env-F687L- GAAAGCTGTATACAATAATTTTTCCATAGCTTC 452 REV 45 FIV-env-L688G- GCTATGGAAAAATTTGGATATACAGCTTTCG 453 FOR CTATG 46 FIV-env-L688G- CATAGCGAAAGCTGTATATCCAAATTTTTCC 454 REV ATAGC 47 FIV-env-Y689G- CTATGGAAAAATTTTTGGGAACAGCTTTCGC 455 FOR TATGC 48 FIV-env-Y689G- GCATAGCGAAAGCTGTTCCCAAAAATTTTTC 456 REV CATAG 49 FIV-env-Y689L- CTATGGAAAAATTTTTGTTAACAGCTTTCGCT 457 FOR ATGC 50 FIV-env-Y689L- GCATAGCGAAAGCTGTTAACAAAAATTTTTC 458 REV CATAG 51 FIV-env-E685F- GATTAAAAGTAGAAGCTATGTTTAAATTTTT 459 FOR GTATACAGC 52 FIV-env-E685F- GCTGTATACAAAAATTTAAACATAGCTTCTA 460 REV CTTTTAATC 53 FIV-env-K686F- GTAGAAGCTATGGAATTTTTTTTGTATACAGC 461 FOR TTTC 54 FIV-env-K686F- GAAAGCTGTATACAAAAAAAATTCCATAGCT 462 REV TCTAC 55 FIV-env-L688F- GCTATGGAAAAATTTTTTTATACAGCTTTCGC 463 FOR TATG 56 FIV-env-L688F- CATAGCGAAAGCTGTATAAAAAAATTTTTCC 464 REV ATAGC 57 FIV-env-Y689F- CTATGGAAAAATTTTTGTTTACAGCTTTCGCT 465 FOR ATGC 58 FIV-env-Y689F- GCATAGCGAAAGCTGTAAACAAAAATTTTTC 466 REV CATAG

(37) Recombinant Proteins:

(38) Recombinant proteins were produced using BL21 (DE3) Escherichia coli cells (Stratagene) and pMal-derived expression vectors (New England Biolabs, France). Recombinant WT and mutants TM subunit ectodomains were soluble and were purified on cross-linked Amylose Resin (New England Biolabs, France) packed in column with PBS as a binding and washing buffer and 20 mM Tris-C1, 5 mM maltose, pH 7.5, as an elution buffer. Proteins were then dialysed against phosphate-buffered saline pH 7.4 (PBS), and endotoxins were removed using Endotrap Blue Resin (Hyglos GmbH, Germany) according to manufacturer's protocol.

(39) Establishment of Env-Expressing Tumor Cells and MCA205 Tumor-Rejection Assay:

(40) 7.510.sup.5 293 T cells were cotransfected with the env-expressing pDFG retroviral vector to be tested (1.75 g) and expression vectors for the MLV proteins (0.55 g for the amphotropic MLV env vector and 1.75 g for the MLV gag and pol vector). 36 hours post-transfection, supernatants were harvested for infection of MCA205 tumor cells (2.5 ml of supernatant per 510.sup.5 cells with 4 g/ml polybrene). Cells were maintained in selective medium (400 units/ml hygromycin) for 3 weeks, and then washed with PBS, trypsinized and inoculated subcutaneously in the shaved area of each mouse right flank as in Mangeney et al (see above PNAS 1998, PNAS 2007). Tumor growth was monitored by palpation twice or thrice weekly and tumor area (mm.sup.2) determined by measuring perpendicular tumor diameters. The extent of immunosuppression was quantified by an index based on tumor size: (A.sub.envA.sub.none)/A.sub.none, where A.sub.env and A.sub.none are the mean areas at the peak of growth of tumors from Balb/c mice injected with env-expressing or control cells, respectively.

(41) Analysis of the Antibody Response of Mice Inoculated with MCA205-Transduced Cells Expressing FIV64 Env Wild-Type and Mutants:

(42) the MCA205-transduced cells were also inoculated as above into syngenic mice (C57Bl/6) and sera were collected 1, 2, and 3 weeks after injection. The antibody response against FIV64 env was assayed by ELISA. Briefly, several dilutions of the sera were incubated 1 h at RT on a plate pre-coated with 1 ng/ml of MBP-FIV64, and antibody binding was analysed by using a labeled anti-mouse IgG secondary antibody (GE Healthcare, UK).

(43) Analysis of FIVenv Expression:

(44) 310.sup.5 293 T cells were transfected with 2 g of the expression vector for the FIV envelope (phCMV) either wild-type or mutated at the indicated positions using Fugene HD (Roche). Cells were washed 16 h later and then harvested 2 days post-transfection using PBS-EDTA 5 mM. The SU1-30 monoclonal antibody (Antibodies online, BmbH, Germany) was used (1/200 dilution) to stain the FIV envelope. As a secondary antibody, we used the goat anti mouse IgG Alexa 488 (1/400) (Invitrogen). Fluorescence was acquired by flow cytometry using a FACS Calibur (BD Biosciences), and data analysed by the CellQuest software (BD Biosciences).

(45) Cell-Cell Fusion Assays:

(46) For cell-cell fusion assays, 510.sup.4 to 110.sup.5 cells seeded in 24-well plates were transfected by using lipofectamine LTX (Life technologies) with 250 ng of env expression plasmid. Fusion activity of each envelope protein was visualized 24 to 48 h after transfection by May-Grunwald and Giemsa staining, according to the manufacturer's instructions (Sigma). The fusion index, which represents the percentage of fusion events in a cell population, is defined as [(NS)/T]100), where N is the number of nuclei in the syncytia, S is the number of syncytia, and T is the total number of nuclei counted