Methods for producing peptides including HERV-W envelope motifs and for producing antibodies specific for the peptides
10040829 ยท 2018-08-07
Assignee
Inventors
Cpc classification
C12N2740/10034
CHEMISTRY; METALLURGY
C12N7/00
CHEMISTRY; METALLURGY
G01N2333/15
PHYSICS
C07K2317/34
CHEMISTRY; METALLURGY
A61K39/21
HUMAN NECESSITIES
C12N2740/10022
CHEMISTRY; METALLURGY
International classification
A61K39/21
HUMAN NECESSITIES
A61K38/16
HUMAN NECESSITIES
Abstract
A peptide domain necessary for an interaction between an envelope of a virus belonging to an HERV-W interference group and an hASCT receptor comprises (i) an N-terminus motif having an amino acid sequence selected from the group consisting of: SEQ ID No. 1 to SEQ ID No. 29, (ii) a C-terminus motif having an amino acid sequence selected from the group consisting of: SEQ ID No. 30 to SEQ ID No. 40, and (iii) at least one motif between the N-terminus and the C-terminus, and having an amino acid sequence selected from the group consisting of: SEQ ID No. 41, SEQ ID No. 42 and SEQ ID No. 73.
Claims
1. A method comprising: chemically synthesizing a peptide; or performing genetic engineering to produce the peptide, wherein the peptide is no longer than the receptor binding domain of the HERV-W envelope protein and comprises: an N-terminus motif including an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 29; a C-terminus motif including an amino acid sequence selected from the group consisting of SEQ ID NO: 30 to SEQ ID NO: 40; and at least one motif between the N-terminus and the C-terminus including an amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 73.
2. The method of claim 1, wherein the Xaa of the Pro Cys Xaa Cys motif in SEQ ID NO: 1 to SEQ ID NO: 29 is aspartic acid, glutamic acid, or arginine.
3. The method of claim 1, wherein the amino acids at positions 3, 4, and 5 of SEQ ID NO: 30 to SEQ ID NO: 40 are glycine.
4. The method of claim 1, wherein the amino acid at position 6 of SEQ ID NO: 30 to SEQ ID NO: 40 is proline or valine.
5. The method of claim 1, wherein the amino acid at position 7 of SEQ ID NO: 30 to SEQ ID NO: 40 is glutamine, leucine, or threonine.
6. The method of claim 1, wherein the amino acid at position 9 of SEQ ID NO: 30 to SEQ ID NO: 40 is lysine, threonine, methionine, or glutamine.
7. The method of claim 1, wherein the amino acid at position 10 of SEQ ID NO: 30 to SEQ ID NO: 40 is alanine, lysine, isoleucine, threonine, or valine.
8. The method of claim 1, wherein the peptide comprises SEQ ID NO: 41 in which the amino acid at: position 3 is asparagine, threonine, glutamic acid, or histidine; position 4 is histidine, alanine, serine, lysine, or glutamic acid; position 5 is tyrosine, threonine, or alanine; position 6 is glutamine, arginine, or threonine; and position 7 is leucine, glutamine, or glutamic acid.
9. The method of claim 1, wherein the peptide comprises SEQ ID NO: 42 in which the amino acid at: position 2 is proline, threonine, arginine, or asparagine; position 3 is glycine, glutamic acid, or asparagine; position 4 is glycine, asparagine, isoleucine, threonine, or serine; position 5 is lysine or not present; position 6 is lysine, valine, isoleucine, or leucine; position 7 is glycine or asparagine; position 8 is glutamine, lysine, or valine; position 9 is valine, proline, serine, or threonine; and position 10 is valine or isoleucine.
10. The method of claim 1, wherein the peptide comprises SEQ ID NO: 73 in which the amino acid at: position 2 is proline, threonine, arginine, or asparagine; position 3 is glycine, glutamic acid, or asparagine; position 4 is glycine, asparagine, isoleucine, threonine, or serine; position 5 is lysine, valine, isoleucine, or leucine; position 6 is glycine or asparagine; position 7 is glutamine, lysine, or valine; position 8 is valine, proline, serine, or threonine; and position 9 is valine or isoleucine.
11. The method of claim 1, wherein the peptide has the amino acid sequence of SEQ ID NO: 74.
12. The method of claim 1, wherein the peptide is chemically synthesized.
13. The method of claim 1, wherein the peptide is produced by genetic engineering.
14. The method of claim 13, wherein the peptide is produced by a genetic engineering technique comprising: culturing a microorganism or eukaryotic cells that include a nucleotide sequence encoding the peptide so as to express the peptide; and recovering the peptide expressed by the microorganism or eukaryotic cells.
15. The method of claim 14, wherein the microorganism or eukaryotic cells include the nucleotide sequence and elements necessary for expressing the nucleotide sequence.
16. The method of claim 1, further comprising: immunizing an animal with the peptide to produce an antibody-producing lymphocyte; fusing the antibody-producing lymphocyte with an immortal cell to produce a hybridoma; and multiplying the hybridoma to produce a monoclonal antibody.
17. The method of claim 1, wherein the peptide is no longer than SEQ ID NO: 74.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) The accompanying figures are given by way of explanatory example and have no limiting character. They will make it possible to better understand the invention.
(2)
(3)
(4)
(5)
EXAMPLES
(6) The following examples are given by way of illustration and have no limiting character. They will make it possible to better understand the invention.
Example 1: Molecular and Phenotypical Characterization of Recombinant Envelopes
(7) Construction and Production of the HERV-W Envelope SU Subunit
(8) A vector phCMVEnv-Gp60 allowing the expression of a soluble recombinant envelope protein was designed from the expression vector phCMV-Env-W (Blond J Virol, Vol 74(7): 3321-3329, 2000) containing the HERV-W envelope gene (538 amino acids) (clone PH74, Blond et al. J Virol Vol 73(2): 1175-1185, 1999).
(9) The soluble envelope (Gp60,1-435) was constructed as described below: (1) The native cleavage site RNKR (positions 314 to 317 of SEQ ID NO: 75) between the SU and TM subunits was mutated to AAAR (SEQ ID NO: 82) in order to allow the production of a fusion protein that was stable and not of two SU-TM subunits cleaved and then recombined by a disulfide bridge. (2) The transmembrane (tm) and intracytoplasmic (CYT) regions corresponding to amino acids 436 to 538 were deleted in order to obtain a soluble protein. (3) A spacer arm having the composition (GGGS)3 (SEQ ID NO: 83), followed by a polyhistidine tail (RGS-HHHHHH) (SEQ ID NO: 84), were added at the C-terminal position in order to allow the purification of this protein by IMAC and the detection by an anti-histidine monoclonal antibody (QIAGEN, RGS H6).
(10) Starting with the vector phCMVEnv-Gp60 expressing the soluble envelope, the vector phCMV-EnvSU was constructed, allowing the production of an SU protein. The soluble SU is a fusion protein containing a C-terminal polyhistidine tail having the sequence RGS-HHHHHH (SEQ ID NO: 84) immediately downstream of the sequence AAAR (SEQ ID NO: 82), in order to allow the purification of this protein by IMAC and the detection by an anti-histidine monoclonal antibody (QIAGEN, RGS H6).
(11) The schematic structure of the various proteins produced from the vectors phCMV-Env-W, phCMV-EnvGp60 and phCMV-EnvSU is illustrated in
(12) Production of the Soluble Envelope
(13) The expression plasmid phCMV-EnvGp60 or phCMV-EnvSU is transfected into the HEK293T cells by precipitation with calcium phosphate. The supernatant containing the GP60 or SU envelope is collected after 48 hours of production in a serum-free medium and filtered on 0.45 m membranes in order to remove the cellular debris. 20 l of supernatant are directly analyzed on a polyacrylamide gel and by Western blotting with an anti-histidine monoclonal antibody (QIAGEN, RGS H6). The GP60 and SU proteins are correctly expressed in soluble form.
(14) Binding Test and Analysis by Flow Cytometry
(15) The stable lines XChASCT2 and XChASCT1 constitutively expressing the hASCT2 (XChASCT2) or hASCT1 (XChASCT1) receptors were established after transfection of XC cells (rat sarcoma) with vectors expressing either human receptor hASCT followed by selection of a clone as described above (Frendo et al., Mol. Cell Biol., Vol 23(10): 3566-3574, 2003). The following human cells are described in Blond J Virol, Vol 74(7): 3321-3329, 2000. The TE671 cells express hASCT2. The TE671RD cells constitutively express the RD114 envelope (cat endogenous retrovirus) belonging to the same interference group and therefore recognizing the hASCT2 receptor. TE671galv cells constitutively express the GALV (gibbon ape leukemia virus) envelope belonging to another interference group and recognizing the PiT1 receptor.
(16) The cells were washed in PBS and harvested by detaching with 0.02% versene in PBS. A total of 10.sup.6 cells were incubated with 1 ml of filtered supernatant containing the soluble envelope (Gp60 or SU) for 1 hour at 37 C. The cells were washed with PBA (PBS and 0.5% sodium azide) containing 2% fetal calf serum and were labeled for 1 hour at 4 C. with an anti-histidine monoclonal antibody (RGSH6, QIAGEN). The cells were washed once with PBA and incubated with a secondary antibody coupled to fluorescein isocyanate for 1 hour at 4 C. The cells were washed twice with PBA and analyzed by flow cytometry.
(17) Using the target cells XChASCT2, the inventors demonstrated that the recombinant protein Gp60 corresponding to a soluble form of the envelope has a phenotypical characteristic identical to that of the wild-type envelope, namely that it is capable of binding to XC cells expressing the hASCT2 receptor.
(18) Using the target cells XChASCT2, XChASCT1, TE671, TE671RD, TE671 galv, the inventors demonstrated that the recombinant protein corresponding to the SU subunit of the envelope exhibits phenotypical characteristics identical to those of the wild-type envelope. First of all, the SU subunit is capable of binding to two receptors hASCT1 and hASCT2 (
Example 2: Identification of the Domains for Interaction of the SU Part of the W Envelope with its hASCT2 Receptor
(19) In order to identify the boundaries of the region of the envelope binding to the hASCT2 receptor, the inventors constructed a set of deletion mutants from the N- and C-terminal ends. The domains of the SU subunit were obtained by PCR and subcloned into the expression vector pHCMV-EnvSU and sequenced. The expression plasmids phCMV-EnvSU, Env69-317, Env197, Env168, Env169-317, Env117 and Env144 (
(20) EnvSU, Env69-317 and Env197 proteins were correctly expressed in soluble form. Using the XC cells constitutively expressing hASCT2, the inventors demonstrated that the Env197 protein was capable of binding to the receptor expressed at the surface of the cells like the SU subunit (1-317). Thus, the first 176 residues of the mature SU subunit (and therefore lacking its signal peptide) were sufficient for binding to the surface of the cells expressing the hASCT2 receptor. The deletion of the 21-68 region resulted in a loss of binding to the receptor also indicating its involvement in the receptor binding domain (RBD). On the other hand, the truncated Env168 protein showed a lower capacity for binding to the hASCT2 receptor.
(21) In order to obtain the equivalent quantities in the supernatants between the various truncated proteins, the inventors fused two smaller domains of the N-terminal region of SU (Env117 and Env144) to the C-terminal region of the SU subunit (Env169-317), the latter domain not binding to hASCT2. The level of expression of the Env117 and Env144 proteins was similar and the proteins were expressed in soluble form. The binding test showed that only the Env144 protein was capable of binding to the cells expressing the hASCT2 receptor. The absence of binding of the Env117 protein to the surface of the cells indicated the loss of at least one determinant of binding inside the 117-144 region.
(22) Consequently, the boundaries of the domains for interaction of the W envelope with its receptor are defined by amino acids 21 to 144.
(23) It should be noted that, in general, proteins (including the envelope proteins) intended for secretion or for membrane expression are synthesized in the granular endoplasmic reticulum (ER). The translocation of the neosynthesized proteins in the ER is conditioned by an N-terminal signal peptide (Walter and Lingappa 1986). The hydrophobic region of the signal peptide initiates penetration into the membrane of the reticulum, bringing behind it the remainder of the neosynthesized peptide. Since the translocation starts at the same time as the synthesis, it is the peptide being translated which crosses the ER membrane. While the protein passes into the lumene of the ER, the signal sequence is cleaved by a specific cellular enzyme, signal peptidase (Walter P, Johnson A E: Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu. Rev. Cell Biol. 1994, 10: 87-119). The translocation of Env into the ER is stopped by the (hydrophobic) transmembrane domain of the glycoprotein which becomes anchored at the phospholipid membranes. In the ER lumene, the regions (SU and part of TM) intended to become extracytoplasmic are folded (the disulfide bridges formed), glycosylated and oligomerized. After oligomerization, the proteins undergoing maturation are transported into the Golgi apparatus where they undergo new glycan maturation processes and cleavage by furin-type endoproteases recognizing a motif R/KXXR leading to two SU and TM subunits.
(24) The mature protein is targeted to the plasma membrane by virtue of a motif present on the intracytoplasmic tail containing a tyrosine (aliphatic/aromatic(Y-X-X)).
Example 3: Test of In Vitro Inhibition of the Binding of the Envelope to its Receptor (Definition of a Peptide and Generation of a Rabbit Antibody)
(25) A peptide (112-129, TGMSDGGGVQDQAREKHV+C, 19 amino acids) (SEQ ID NO: 85) was defined from the region defined in example 2 and from a determination of the potentially antigenic regions of the SU subunit. A cysteine was added at the C-terminal position for the KLH (keyhole limpet hemocyanin, cf. Frendo et al., Mol. Cell Biol. Vol 23(10): 3566-3574, 2003) coupling. This peptide was used to immunize a rabbit and then to affinity purify the polyclonal antibody directed against the region 112-119 contained in the serum of this rabbit.
(26) The Env144 protein is preincubated at 37 C. for one hour with either the anti-SU polyclonal antibody or with an anti-TM polyclonal antibody. The formation of the Env144 protein-anti-SU antibody complex drastically reduced the binding of the envelope to the cells expressing the hASCT2 receptor. By contrast, the use of an antibody not directed against the RBD did not adversely affect its binding to the hASCT2 receptor.
Example 4: Production of Monoclonal Antibodies Directed Against the HERV-W Envelope Protein
(27) Immunization of Mice with DNA
(28) Three female six-week-old BALB/c mice (IFFA-Credo) were immunized by direct injection of naked plasmid DNA (phCMV-env-W) containing the gene for the HERV-W envelope. The injections were performed by the intradermal route with the aid of a gene gun. Five injections of 2 g of DNA were first performed for each mouse followed by a booster with two injections of 4 g of DNA. The sera were collected and the antibody titer for each serum was determined. Since the antibody titer was too low, a cellular lysate was prepared.
(29) Preparation of the Cellular Lysate
(30) The rhabdomyosarcoma cells TelCeB6 (ATCC CRL8805) were transfected with the plasmid phCMV-env-W. After about hours and the presence of syncytia, a cellular extract was prepared in PBS buffer containing 0.5% TRITON. The protein extracts were assayed by Bradford. The env-W antigen concentration corresponded to 9.5 g/l of total proteins.
(31) Immunization of Mice with an Extract of Cellular Lysate
(32) The same mice first of all received an injection of 10 g of cellular lysate by the intraperitoneal route followed by a booster injection of 2100 g of cellular lysate by the intraperitoneal route. Three days before the fusion with myeloma cells, another injection was performed, by the intravenous route, with 22 g of soluble envelope protein Gp60 obtained from the plasmid phCMV-Env-Gp60 as described in example 1, purified beforehand, before injection, on to an Ni-NTA resin (QIAGEN) according to the following conditions: binding in phosphate buffer pH 8, washes in phosphate buffer pH 8 and in ammonium acetate pH 6, elution in ammonium acetate buffer pH 3.5 and concentration with speed vac. 47 g of the eukaryotic Gp60 protein thus obtained were reserved for the injection by the intravenous route described above. After fusion, the hybridoma supernatants were tested by immunofluorescence on the transfected and bound cells (TeLCeb6), the antibodies were screened by a functional ELISA test using the Env-W protein at a concentration of 9.2 g/l of total proteins and an Env AS protein as negative control at a concentration of 13.3 g/l of total proteins and the most effective antibodies were selected.
(33) The monoclonal antibodies 2H1H8, 12C7A3 and 1F11B10 were thus obtained. The monoclonal antibodies 2H1H8 and 12C7A3 are directed against the nonglycosylated N-terminal part of the SU region of the Env-HERV-W proteins. They are directed against the RDB as shown by a Western blot assay with the aid of Env 144. The monoclonal antibody 1F11B10 is directed against the glycosylated C-terminal part of the SU region of the Env-HERV-W protein as shown by a Western blot assay with the aid of Env 169-317. It does not recognize Env 144.
Example 5: Test of In Vitro Inhibition of the Binding of the Envelope to its Receptor and Inhibition of the Formation of Syncytia with the Aid of Monoclonal Antibodies by a Cell to Cell Fusion Test (Coculture)
(34) The plasmid for expressing the envelope glycoprotein is transfected into the cells TELCeB6 by precipitation with calcium phosphate at two quantities 100 and 500 ng (Cosset et al., Journal of Virology, 69 (10): 6314-6322 (1995)). The cells expressing the envelope are detached from the support 20 hours after transfection and are preincubated at 37 C. for one hour, respectively, with the anti-HIV 23A5 monoclonal antibody, the anti-TM Env-HERV-W 6A2B2 monoclonal antibody (previously obtained), the anti-SU Env-HERV-W 2H1H8 12C7A3 and 1F11B10 monoclonal antibodies (dilution 1/50th). Next, they are reinoculated at equal concentration (0.410.sup.5 cells/well) into 12-well plates. Epithelioid-carcinoma-indicating human cells (Hela, ATCC CCL-2) are then added to the transfected cells in an amount of 210.sup.5 cells per well and the co-culture is continued for 24 h. An XGal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) staining may then be performed in order to stain the nucleus of the cells TELCeB6 (Cosset et al., Journal of Virology, 69 (10): 6314-6322 (1995)). It is followed by staining with May-Grnwald and Giemsa solutions (MERCK) performed according to the suppliers' recommendations. The fusion observed corresponds to a fusion from within, that is to say a cell to cell fusion, starting with a cell expressing the envelope, in contrast to a fusion from without which corresponds to a formation of syncytia following a virion-cell(s) fusion.
(35) The results presented in table 1 below express the number of fused cells counted.
(36) TABLE-US-00001 TABLE 1 Antibody 23A5 6A2B2 2H1H8 1F11B10 12C7A3 100 ng 261 231 130 223 ND 500 ng 217 273 73 210 11 ND: not determined
(37) The results presented above show that the formation of the Env protein-anti-SU 2H1H8 and 12C7A3 antibody complex dramatically reduces the binding of the envelope to cells expressing the hASCT2 receptor and cell fusion. By contrast, the use of an antibody not directed against rdb (6A2B2 or 1F11B10) does not adversely affect the binding of the envelope and the fusion of the cells significantly, as may be observed by comparing to the results obtained with the anti-HIV 23A5 control antibody.
Example 6: Study of the In Vivo Interaction Between the Envelope Protein and the hASCT2 Receptor and the In Vivo Formation of Syncytia
(38) To verify the results obtained in vitro, an animal model was designed. Rhabdomyosarcoma cells TelCeB6 (ATCC CRL8805), in culture in DMEM medium (GIBCO INVITROGEN 41966-029) supplemented with South American serum, were respectively transfected, with the aid of the LIPOFECTAMINE PLUS kit (GIBCO INVITROGEN), with the DNA corresponding to the HERV-W env gene cloned in the sense orientation at the concentration of 2 g/l (DNA 409), with the DNA corresponding to the HERV-W env gene cloned in the anti-sense orientation at the concentration of 1.5 g/l (DNA 410) and with the DNA corresponding to a mutated HERV-W env gene at the concentration of 1.3 g/l (DNA LQMV) according to the protocol detailed below. The cells transfected with the DNA 409 are capable of expressing the fusogenic W envelope protein, the cells transfected with the DNA 410 are not capable of expressing the envelope protein and the cells transfected with the DNA LQMV are capable of expressing a nonfusogenic mutated W envelope protein.
(39) 1). Protocol:
(40) 1st Day: Culture of the TelCeB6 Cells: inoculation of the dishes 100 mm in diameter.fwdarw.50-70% confluence; incubation in supplemented DMEM medium (6 ml per dish) for 24 hours at 37 C. under 5% CO.sub.2.
(41) 2nd Day: Transfection with the LIPOFECTAMINE PLUS Kit:
(42) 1 Precomplexing of the DNA mixing of 750 l of medium not supplemented with the abovementioned DNAs in a 15 ml falcon tube (reference 2096), that is 2 l of 409 or 3 l of 410 or 3 l LQMV; stirring under vortex of PLUS reagent and adding 20 l thereof to the DNA solution; stirring under vortex immediately 10 seconds at 1400 rpm; incubation 15 minutes at room temperature.
(43) 2 Preparation of the Cells replacement with 5 ml of nonsupplemented medium.
(44) 3 Dilution of the LIPOFECTAMINE
(45) In a tube for a dish, mix 30 l of LIPOFECTAMINE
(46) Reagent with 750 l of nonsupplemented medium.
(47) 4 Complexing of the DNA
(48) Mixing of 780 l of dilute LIPOFECTAMINE and 772 l of solution of precomplexed DNA (total: 1552 l);
(49) stirring under vortex immediately 10 sec at 1400 rpm, incubation 15 minutes at room temperature.
(50) 5 Transfection and Production of Recipient Animals Transplanted with the Target Cells, Treated or not Treated by Injection of Anti-Env Antibody
(51) Deposition of 1552 l in a dish;
(52) incubation 2-3 hours at 37 C. under 5% CO.sub.2;
(53) replacement of the transfection medium with 6 ml of supplemented medium;
(54) incubation 1 hour at 37 C. under 5% CO.sub.2;
(55) injection by the intraperitoneal route (IP) to SCID mice (in a volume of 1 ml), th of each dish at 70% confluence, with or without additional injection of anti-Env protein antibody (monoclonal antibody 2H1H8, polyclonal antibody 69 (anti-SU) and 71 (anti-TM) at 1/100).
(56) Production of animals tolerating the transplant and allowing dissemination of the transplanted cells in the body, in parallel with the establishment of pseudo-ascites in the peritoneal cavity.
(57) 3rd Day:
(58) Collection of the cells from each animal by peritoneal lavage: injection of 2 ml of air followed by 2 ml of physiological saline and then massaging and recovering the 2 ml of peritoneal fluid (protocol developed for the recovery, in transplanted animals, of the cells implanted in the peritoneal cavity);
(59) observation under an inverted phase microscope with counting of the syncytia and/or after staining on a slide;
(60) immediate reading performed after spreading on slides with a gridded chamber in the presence of Trypan blue (exclusion of the dead cells). The number of cells which have fused to each other was counted per field with a wide angle lens (40) which makes it possible to establish the count on more than about one hundred cells so as to have a series of statistically representative counts.
(61) A cellular aliquot of each sample is fixed in the presence of methanol/acetone (v/v) and then stored at 20 C. until a crystal violet staining is obtained (1%). Photographs were taken of the stained slides.
(62) 2) Mice:
(63) Groups of 2 mice are inoculated with:
(64) the cells transfected with the three types of plasmid (DNAs 409, 410 and LQMV) with no antibody (32=6 mice) the cells transfected with the three types of plasmid (DNAs 409, 410 and LQMV) with the monoclonal antibody 2H1H8 (32=6 mice).
(65) 3) Results:
(66) The number of fused cells determined by direct reading on a gridded counting chamber per 100 cells is indicated in Table 2 below:
(67) TABLE-US-00002 TABLE 2 ECP (Tryptan blue) reading: direct reading of the syncytia Lines S1* count S2* count Mean 409 control 19 22 20.5 409 + 2H1H8 3 4 3.5 410 control 8 11 9.5 410 + 2H1H8 2 3 2.5 LQMV control 8 5 6.5 LQMV + 2H1H8 1 1 1 S1* and S2* = mouse 1 and mouse 2
(68) Each number represents the number of fused and visualized cells per field studied. As some cells may be superposed in the optical path, the count for the cells appearing fused in the controls is greater than zero. The reality of the syncytia and the discrimination with stacks of cells were then verified by staining the cells on a slide, with visualization of multiple cell nuclei contained in a space delimited by the continuation of a single and sole cell membrane. Moreover, photos showing cells in the course of fusion made it possible to objectify the reality of the fusion upon analysis by phase contrast microscopy and the total absence of an equivalent phenomenon in the controls.
(69) To statistically objectify the primary analysis represented by the numbers indicated in Table 2, a Chi-test was performed in order to compare the data in Table 2.
(70) The results of the statistical analysis taking into account the background noise of the primary reading, without secondary analysis after staining on a slide or a search for typical cells in the course of fusion which are never seen in the controls, are the following:
(71) i) Statistical validation of the specificity of the pathogenic effect in vivo:
(72) Env expressed (409): 20.5 positives counted on average out of 100 cells,
(73) anti-sense Env (410): 9.5 positives counted on average out of 100 cells,
(74) mutated Env (LQMV): 6.5 positives counted on average out of 100 cells,
(75) mean of the controls (410 and LQMV): 9.5+6.5/2=8%.
(76) Env versus control 410: Chi-2=5.89 (p<0.02)
(77) Env versus control LQMV: Chi-2=9.83 (p<0.002)
(78) Env versus the two controls (410 and LQMV): Chi-2=7.69 (p<0.01)
(79) Control 410 versus control LQMV: Chi-2=0.61 (difference not significant).
(80) The controls are therefore statistically equivalent and there is no real difference linked to the type of control.
(81) The results obtained from this stage of analysis (not excluding the background noise linked to the artefactual images and by comparing the two types of control with each other (which prove to be equivalent)) was statistically very significant (overall p<0.01). Subsequent analysis, by staining, of the specificity of the effects therefore merely confirm the specificity of the effect obtained in vivo in the presence of the Env protein, thereby validating the animal model of the in vivo study of syncytia whose fusion was induced by HERV-W Env.
(82) ii) Statistical validation of the therapeutic activity of the antibodies tested on the pathogenic effect in vivo:
(83) Env expressed (409): 20.5 positives counted on average out of 100 cells,
(84) Env expressed (409)+monoclonal antibody 2H1H8: 3.5 positives counted on average out of 100 cells.
(85) Env alone versus injection of the antibody 2H1H8: Chi-2=15.38 (p<0.001)
(86) The results obtained show a statistically significant effect for the monoclonal antibody (probability of result due to chance (p) less than 0.001). Subsequent analyses, by staining, of the specificity of the effects merely confirm the specificity of the effect obtained in vivo in the presence of the Env protein and of antibody, thereby validating the therapeutic effect on the animal model.
Example 7: Study In Vivo of the Binding of the HERV-W Env Protein to Cells Possessing or not Possessing Type 1 or 2 hASCT Receptors and of the Inhibition of this Binding by Injection of Antibodies Directed Against HERV-W Env
(87) 1) Materials
(88) Soluble protein: supernatant filtered on 0.45 m containing the soluble protein (293T cells transfected with the plasmid 460 (envelope-spacer-His6).
(89) Expression verified by Western blotting with an anti-RGS-His antibody.
(90) Antibody: monoclonal antibody 2H1H8 (IgG, 5.50 mg/ml).
(91) Cells: XChASCT2, cellular clone XC (ATCC CCL-165, rat cells) expressing the hASCT2 receptor.
(92) DMEM medium (GIBCO INVITROGEN 41966-029) with South American serum.
(93) Preincubation, incubation, labeling in a 1.5 ml EPPENDORF tube.
(94) 2) Protocol
(95) 1 IP (intraperitoneal) inoculation of the XChASCT1, XChASCT2 cells and control cells XChASCTinto SCID mice
(96) Injection into mice of th of the flask at 70% confluence in a volume of 2 ml.
(97) 2 Preincubation
(98) Incubation of the soluble protein supernatant (filtered supernatant of the 293T line) with the monoclonal antibody 2H1H8 (990 l of supernatant with 10 l of antibody ( 1/100th dilution)) for 1 hour at 37 C. in the cell incubator with occasional stirring (every 15 minutes).
(99) 3 Inoculation
(100) IP (intraperitoneal) inoculation of the proteins alone or with the antibody into mice transplanted with the cells (110.sup.6 cells per point, that is th of a confluent dish 100 mm in diameter).
(101) After injection of the antibody (200 microliters), maintained as IP, for 6 hours, with an occasional peritoneal massage (every 30-60 minutes).
(102) 4 Recovery of the Cells by Peritoneal Lavage of the Transplanted Mice Centrifugation 3000 revolutions for 5 minutes at +4 C. Recovery of the cellular pellet and dilution in the labeling media (maintained at +4 C. until fixing).
(103) 5 Labeling Primary antibody:
(104) The pellet is taken up in 100 l of anti-RGS His antibody (100th dilutionQIAGEN) in a PBA buffer (PBS with 2% fetal calf serum and 0.1% sodium azide), maintained at +4 C.
(105) 1 hour in ice with occasional stirring (every 15 minutes).
(106) Washing in PBA buffer (1 ml per tube), maintained at +4 C. Secondary antibody:
(107) Centrifugation 3000 revolutions for 5 minutes at +4 C. The pellet is taken up in 100 l of anti-mouse antibody-FITC ( 1/20th dilutionDAKO, reference F0479 in a PBA buffer) maintained at +4 C.
(108) 1 hour in ice with occasional stirring (every 15 minutes).
(109) 2 washes in PBA buffer (1 ml per tube), maintained at +4 C.
(110) Pellet taken up in 500 l of PBA, maintained at +4 C., and analyzed by FACS.
(111) Alternatively, analysis by IF after fixing on a slide in acetone/methanol (50%/50%) at 20 C. and counter-staining with Evans blue.
(112) 3) Mice:
(113) Groups of 2 mice are inoculated with:
(114) each type of cell (expressing the 2 types of receptor hASCT1 and hASCT2 and one not expressing the receptor hASCTas a control) with no antibody (32=6 mice)
(115) the three types of cell with the Env protein and the monoclonal antibody 2H1H8 (32=6 mice).
(116) 4) Results
(117) The results by immunofluorescence (IF) reading with a microscope are presented in Table 3 below:
(118) TABLE-US-00003 TABLE 3 IF reading: number of fluorescent cells/total number of cells (NF/NT) in the same field Lines NF/NT Mean XC control 1/18, 0/10 1/28 XC + 2H1H8 0/20 0/20 XChASCT1 control 12/40, 3/12, 9/25 24/77 XChASCT1 + 2H1H8 1/25, 0/18, 1/30 2/73 XChASCT2 control 8/22, 15/35 23/57 XChASCT1 + 2H1H8 1/45 1/45
(119) Each number represents the number of cells visualized as fluorescent per field studied. As some cells may have bound fluorescence in a non-specific manner, the count for the cells appearing fluorescent under the control conditions is therefore greater than zero in one of the two fields counted (mean of the two fields= 1/28, that is 0.036%, which is entirely reasonable for the background noise of such a reading technique). The reality of the cells that bound the Env protein to their hASCT1 or hASCT2 receptor was then verified by cytofluorometric analysis.
(120) In order to statistically objectify the analysis presented in Table 3, a Chi-2 test was performed in order to compare the data obtained under the conditions according to which (i) the Env protein can bind to a receptor hASCT1 (control hASCT1) or hASCT2 (control hASCT2) present at the surface of the cells transplanted into SCID mice versus the grafted control cells which have no receptor (control X) to which the Env protein injected into the corresponding animals cannot bind and thus does not give membrane fluorescence in the presence of an anti-Env antibody and (ii) the Env protein can bind to a receptor hASCT1 (control hASCT1) or hASCT2 (control hASCT2) present at the surface of the cells transplanted into SCID mice versus the injection of a monoclonal antibody directed against Env-SU.
(121) The results of the statistical analysis are presented below:
(122) i) Statistical validation of the specificity of the pathogenic effect in vivo:
(123) control hASCT1: 24 positives counted on average out of 77 cells,
(124) control hASCT2: 23 positives counted on average out of 57 cells,
(125) hASCT cells: 1 positive counted on average out of 28 cells.
(126) Env+grafts hASCT1 versus hASCT: Chi-2=8.62 (p<0.01)
(127) Env+grafts hASCT2 versus hASCT: Chi-2=12.53 (p<0.001)
(128) Env+grafts hASCT1 versus env+grafts hASCT2: Chi-2=1.21 (difference not significant).
(129) The cells expressing the hASCT1 or hASCT2 receptors at their surface are therefore indeed statistically equivalent and there is no difference in the Env binding to the receptor linked to subtype 1 or 2, under the conditions of the experiment.
(130) The results obtained with the animals transplanted with the cells expressing the membrane receptors hASCT1 or hASCT2 are statistically significant in the light of the results obtained with the control animals transplanted with the cells expressing none of these receptors at their surface.
(131) These results confirm the specificity of the effect obtained in vivo in the presence of the Env protein in the animal models.
(132) ii) Statistical validation of the therapeutic activity of the antibodies tested on the pathogenic effect in vivo:
(133) Env+grafts control hASCT1: 24 positives counted on average out of 77 cells
(134) Env+grafts hASCT1+monoclonal antibody 2H1H8: 2 positives counted on average out of 73 cells.
(135) Env+grafts hASCT1 alone versus injection antibody 2H1H8: Chi-2=21.14 (p<0.001)
(136) The results obtained show a statistically significant effect for the monoclonal antibody (probability of the result due to chance (p) less than 0.001).
(137) Env+grafts hASCT2: 23 positives counted on average out of 57 cells
(138) Env+grafts hASCT2+monoclonal antibody 2H1H8: 1 positive counted on average out of 45 cells.
(139) Env+grafts hASCT2 alone versus injection antibody 2H1H8: Chi-2=20.31 (p<0.001).
(140) The results obtained show a statistically significant effect for the monoclonal antibody (probability of the result due to chance (p) overall less than 0.01).
(141) The validation of the animal models against the appropriate controls makes it possible to demonstrate that antibodies may have a therapeutic activity by significantly inhibiting the pathogenic effects of the HERV-W Env protein.
Example 8: Alignment of the Sequences of the Interference Group
(142) The protein sequences of the envelopes of the retroviruses HERV-W (swiss-prot Q9UQF0), RD114 (swiss-prot Q98654), REV (swiss-prot P31796), BAEV (swiss-prot P10269), SRV1 (swiss-prot P04027), SRV2 (swiss-prot P51515) and MPMV (swiss-prot P07575) were aligned with the aid of the MACVECTOR software with the CLUSTALW procedure. The signal peptide, the SU (surface unit) subunit and the TM (Trans membrane) subunit are indicated. The receptor binding site is underlined.