U.S. patent application number 12/087893 was filed with the patent office on 2009-06-11 for peptide domain required for interaction between the envelope of a virus pertaining to the herv-w interference group and an hasct receptor.
This patent application is currently assigned to BIOMERIEUX. Invention is credited to Valerie Cheynet, Francois Mallet, Guy Oriol.
Application Number | 20090148454 12/087893 |
Document ID | / |
Family ID | 37037521 |
Filed Date | 2009-06-11 |
United States Patent
Application |
20090148454 |
Kind Code |
A1 |
Mallet; Francois ; et
al. |
June 11, 2009 |
Peptide Domain Required For Interaction Between The Envelope of a
Virus Pertaining to The Herv-W Interference Group and an Hasct
Receptor
Abstract
The invention relates to a peptide domain required for
interaction between the envelope of a virus pertaining to the
HERV-W interference group and a hASCT receptor, comprising an N end
point and a C end point. Said peptide domain is defined, at the N
end point thereof, by a pattern formed by the amino acids L
(Z)-proline-cysteine-X-cysteine in which Z is any amino acid, is a
whole number between 2 and 30, and X is any amino acid, and at the
C end point thereof, by a pattern formed by the amino acids
serine-aspartic acid-Xa-Xb-Xc-Xd-Xe-aspartic acid-Xf-Xg-(Z) in
which Xa, Xb, Xc, Xd, Xe, Xf, Xg are any amino acids, Z is any
amino acid, B is a whole number between 15 and 25, preferably 20.
The peptide domain comprises, between the N end point and the C end
point, at least one pattern selected from the following patterns: a
pattern formed by the amino acids cysteine-X2-X3-X4-X5-X6-cysteine
in which X2, X3, X4, X5, X6 are any amino acids, and a pattern
formed by the amino acids
cysteine-X7-X8-X9-X10-X11-X12-X13-X14-X15-cysteine-trytophane in
which X7, X8, X9, X10, X11, X12, X13, X14, X15 are any amino
acids.
Inventors: |
Mallet; Francois;
(Villeurbanne, FR) ; Oriol; Guy; (Saint Chamond,
FR) ; Cheynet; Valerie; (Verin, FR) |
Correspondence
Address: |
OLIFF & BERRIDGE, PLC
P.O. BOX 320850
ALEXANDRIA
VA
22320-4850
US
|
Assignee: |
BIOMERIEUX
MARCY-L'ETOILE
FR
|
Family ID: |
37037521 |
Appl. No.: |
12/087893 |
Filed: |
February 9, 2007 |
PCT Filed: |
February 9, 2007 |
PCT NO: |
PCT/FR2007/000236 |
371 Date: |
October 20, 2008 |
Current U.S.
Class: |
424/139.1 ;
435/320.1; 435/369; 435/5; 435/69.1; 514/1.1; 514/44R; 530/350;
530/387.9; 536/23.72 |
Current CPC
Class: |
C12N 2740/10063
20130101; C07K 2317/34 20130101; C07K 16/1036 20130101; A61K 39/21
20130101; A61K 39/12 20130101; C12N 2740/10022 20130101; C12N
2740/10034 20130101; A61K 38/162 20130101; A61P 31/14 20180101;
A61P 31/12 20180101; G01N 2333/15 20130101; C12N 7/00 20130101;
C07K 14/005 20130101 |
Class at
Publication: |
424/139.1 ;
530/350; 536/23.72; 435/320.1; 435/369; 514/12; 435/5; 530/387.9;
435/69.1; 514/44 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 14/00 20060101 C07K014/00; C12N 15/11 20060101
C12N015/11; C12N 15/00 20060101 C12N015/00; C12N 5/08 20060101
C12N005/08; A61K 31/7088 20060101 A61K031/7088; A61K 38/16 20060101
A61K038/16; C12Q 1/70 20060101 C12Q001/70; C07K 16/18 20060101
C07K016/18; C12P 21/00 20060101 C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 2006 |
FR |
0650468 |
Claims
1. A peptide domain necessary for an interaction between an
envelope of a virus belonging to an HERV-W interference group and
an hASCT receptor, the peptide domain comprising: an N-terminus
motif having an amino acid sequence selected from the group
consisting of: SEQ ID No. 1 to SEQ ID No. 29, 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 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.
2. A nucleotide sequence encoding a peptide domain as defined in
claim 1.
3. An epitope derived from the peptide domain of claim 1, wherein
the epitope induces an immune response against a virus belonging to
the HERV-W interference group.
4. A nucleotide sequence encoding an epitope as defined in claim
3.
5. An expression vector comprising: the nucleotide sequence of
claim 2, and elements necessary for expressing the nucleotide
sequence.
6. A microorganism or host cell transformed with at least one
expression vector as defined in claim 5.
7. An antibody directed against a peptide domain as defined in
claim 1.
8. A method of inhibiting, preventing and/or treating in an animal
an infection caused by a virus belonging to an HERV-W interference
group, the method comprising: administering to the animal, in an
effective amount, a composition comprising as an active substance
at least one peptide domain of claim 1 and elements necessary for a
constitutive and/or inducible expression of said peptide
domain.
9. A pharmaceutical composition comprising, as an active substance,
at least one peptide domain of claim 1 and elements necessary for a
constitutive and/or inducible expression of said peptide domain,
and a pharmaceutically acceptable vehicle.
10. A diagnostic composition for detecting and/or quantifying a
virus belonging to an HERV-W interference group, and/or for
quantifying an immune response against said virus, the composition
comprising at least one peptide domain of claim 1.
11. A method for detecting and/or quantifying virus belonging to an
HERV-W interference group in a biological sample taken from an
individual liable to be infected by said virus, the method
comprising: contacting said biological sample with at least one
antibody of claim 7 under conditions allowing formation of a
complex between the virus and the antibody, and detecting and/or
quantifying the formation of said complex.
12. A method of in vitro screening of a virus belonging to an
HERV-W interference group in a biological sample or specimen, the
method comprising: contacting the composition of claim 10 with the
biological sample or specimen, and determining whether an immune
response to the virus is produced.
13. A method of inhibiting an interaction between an envelope of a
virus belonging to an HERV-W interference group and an hASCT
receptor, the method comprising contacting the peptide domain of
claim 1 with said hASCT receptor.
14. A method of identifying chemical or biological molecules whose
interaction with all or part of the peptide domain of claim 1
blocks an interaction between an envelope of a virus belonging to
an HERV-W interference group and an hASCT receptor, the method
comprising: contacting at least one chemical or biological molecule
with the peptide domain of claim 1; and detecting whether
interaction occurs between the envelope of the virus belonging to
the HERV-W interference group and the hASCT receptor.
15. A method of generating antibodies which block an interaction
between an envelope of a virus belonging to an HERV-W interference
group and an hASCT receptor, the method comprising inoculating an
animal or cultured cells with the peptide domain of claim 1,
thereby inducing the animal or cultured cells to generate
antibodies directed against the peptide domain of claim 1.
16. A method of determining a peptide domain necessary for an
interaction between an envelope of a virus belonging to an HERV-W
interference group and an hASCT receptor, the method comprising:
identifying a nucleotide and/or peptide sequence of a precursor
envelope of said virus, excluding a signal part of said sequence,
detecting an amino acid motif having an amino acid sequence
corresponding to SEQ ID No. 43, and excluding a C-terminus in the
amino acid sequence between 15 and 25 amino acid positions after
the amino acid motif having an amino acid sequence corresponding to
SEQ ID No. 43.
17. A peptide domain determined by the method of claim 16.
18. The method of claim 8, wherein the animal is a human.
19. An antibody produced by the method of claim 15.
20. A method of producing a peptide domain necessary for an
interaction between an envelope of a virus belonging to an HERV-W
interference group and an hASCT receptor, the method comprising:
transforming a microorganism or a host cell with at least one
expression vector of claim 5, culturing the transformed
microorganism or host cell, and recovering the peptide domain
produced by the microorganism or host cell.
21. An expression vector comprising: the nucleotide sequence of
claim 4, and elements necessary for expressing the nucleotide
sequence.
22. A microorganism or host cell transformed with at least one
expression vector of claim 21.
23. A method of producing a peptide domain necessary for an
interaction between an envelope of a virus belonging to an HERV-W
interference group and an hASCT receptor, the method comprising:
transforming a microorganism or host cell with at least one
expression vector of claim 21, culturing the transformed
microorganism or host cell, and recovering the peptide domain
produced by the microorganism or host cell.
24. An antibody directed against the epitope of claim 3.
25. A method of inhibiting, preventing and/or treating in an animal
an infection caused by a virus belonging to an HERV-W interference
group, the method comprising: administering to the animal, in an
effective amount, a composition comprising as an active substance
at least one epitope of claim 3 and elements necessary for a
constitutive and/or inducible expression of said epitope.
26. The method of claim 25, wherein the animal is a human.
27. A method of inhibiting, preventing and/or treating in an animal
an infection caused by a virus belonging to an HERV-W interference
group, the method comprising: administering to the animal, in an
effective amount, a composition comprising as an active substance
at least one antibody of claim 7.
28. The method of claim 27, wherein the animal is a human.
29. A method of inhibiting, preventing and/or treating in an animal
an infection caused by a virus belonging to an HERV-W interference
group, the method comprising: administering to the animal, in an
effective amount, a composition comprising as an active substance
at least one nucleotide sequence of claim 2.
30. The method of claim 29, wherein the animal is a human.
31. A method of inhibiting, preventing and/or treating in an animal
an infection caused by a virus belonging to an HERV-W interference
group, the method comprising: administering to the animal, in an
effective amount, a composition comprising as an active substance
at least one nucleotide sequence of claim 4.
32. The method of claim 31, wherein the animal is a human.
33. A pharmaceutical composition comprising, as an active
substance, at least one epitope of claim 3 and elements necessary
for a constitutive and/or inducible expression of said epitope, and
a pharmaceutically acceptable vehicle.
34. A pharmaceutical composition comprising, as an active
substance, at least one nucleotide sequence of claim 2, and a
pharmaceutically acceptable vehicle.
35. A pharmaceutical composition comprising, as an active
substance, at least one nucleotide sequence of claim 4, and a
pharmaceutically acceptable vehicle.
36. A pharmaceutical composition comprising, as an active
substance, at least one antibody of claim 7, and a pharmaceutically
acceptable vehicle.
37. A diagnostic composition for detecting and/or quantifying a
virus belonging to an HERV-W interference group, and/or for
quantifying an immune response against said virus, the composition
comprising at least one epitope of claim 3.
38. A diagnostic composition for detecting and/or quantifying a
virus belonging to an HERV-W interference group, and/or for
quantifying an immune response against said virus, the composition
comprising at least one nucleotide sequence of claim 2.
39. A diagnostic composition for detecting and/or quantifying a
virus belonging to an HERV-W interference group, and/or for
quantifying an immune response against said virus, the composition
comprising at least one nucleotide sequence of claim 4.
40. A diagnostic composition for detecting and/or quantifying a
virus belonging to an HERV-W interference group, and/or for
quantifying an immune response against said virus, the composition
comprising at least one antibody of claim 7.
41. A method of inhibiting an interaction between an envelope of a
virus belonging to an HERV-W interference group and an hASCT
receptor, the method comprising contacting the epitope of claim 3
with said hASCT receptor.
42. A method of inhibiting an interaction between an envelope of a
virus belonging to an HERV-W interference group and an hASCT
receptor, the method comprising contacting the antibody of claim 7
with said envelope.
Description
[0001] The present invention relates to a polypeptide domain
responsible for interactions between a retroviral envelope of the
HERV-W interference group and the receptors of the hASCT
family.
[0002] Human endogenous retroviruses (HERVs) constitute 8% of the
human genome and are involved both in pathologies and in
nonpathological phenomena.
[0003] The human endogenous retrovirus family W (HERV-W) is derived
from an infectious retroviral element that was integrated into the
germ line 25 to 40 million years ago. The HERV-W envelope protein,
also called syncytin, is a fusogenic glycoprotein involved in the
formation of the syncytiotrophoblastic layer of the placenta. It is
encoded by the env gene of the proviral locus ERVW1 and synthesized
in the form of a gPr73 precursor which is specifically cleaved into
two mature proteins, a surface subunit gp50 (SU) and a
transmembrane subunit gp24 (TM).
[0004] In vitro, syncytin of the HERV-W family induces a cell to
cell fusion that is dependent on its interaction with a
receptor-transporter of amino acids of the ASCT family (h-ASCT2,
hASCT1). Phylogenic studies then showed that syncytin is related to
a group of retroviruses comprising in particular the cat endogenous
virus RD114, the monkey endogenous virus BaEV, simian retroviruses
and avian retroviruses: avian reticuloendotheliosis virus REV-A and
spleen necrosis virus SNV, all having in common the type 2
sodium-dependant neutral amino acid receptor-transporter or hASCT2
(Rasko et al, 1999, Proc. Natl. Acad. Sci. USA Vol. 96, pp.
2129-2134; Tailor et al, 1999 Journal of Virology, vol. 73(5) May
1999, P. 4470-4474). Thus, the infection of cells with viruses of
this retrovirus group leads to a specific reduction in the
transport of amino acids (Rasko et al., 1999). The infection of a
cell with one of these retroviruses (or the expression of one of
these envelopes in the cell) prevents, through interference
(interaction) in relation to a receptor of the ASCT family, the
infection of this same cell by another of these retroviruses or the
fusion with another cell expressing another envelope. Through
interference in relation to a receptor of the ASCT family, the
infection of a cell by one of these retro-viruses prevents the
infection by another of these retroviruses. All these retroviruses
belong to the same HERV-W virus interference group.
[0005] The mechanisms of binding between the envelope and the ASCT
receptor remain obscure, and to date no domain for binding to an
ASCT receptor has been identified and defined either in the SU of
the HERV-W envelope protein or in the SUs of retroviruses of the
same interference group. This theme is nevertheless essential since
the inhibition of the envelope/ASCT receptor interaction would in
addition make it possible to prevent the entry of a retrovirus into
the cell, and therefore to block its replication cycle, to block
the phenomenon of envelope/ASCT receptor interaction and/or of cell
fusion which may be involved in the formation of tumors, in the
proliferation of metastasic cells or in drug resistance phenomena
(see by way of illustration the publication "Cell fusion: A hidden
enemy?, Cancer Cell: May 2003, vol. 3), to block the phenomenon of
envelope/ASCT receptor interaction and/or of cell fusion which may
be involved in nervous system diseases and even to inhibit the
cell-cell fusion involved in trophoblastic differentiation
(contraceptive vaccination). Furthermore, the inhibition of the
envelope/hASCT receptor interaction could prevent tumor propagation
by counteracting a local immunosuppression which may result from
the envelope/hASCT receptor interaction. Indeed, it has been shown,
on the one hand, that the infection of cells with viruses of this
retrovirus group (in particular those inducing immunodeficiencies)
leads to a specific reduction in the transport of amino acids
(Rasko et al, PNAS, vol. 96. pp 2129-2134 (1999)), and on the other
hand, a direct link is proposed between the impairment of the
transport of amino acids and immunosuppression (Espinosa A,
Villarreal L P., T-Ag inhibits implantation by EC cell derived
embryoid bodies. Virus Genes. 2000; 20(3): 195-200; J E, Battini J
L, Gottschalk R J, Mazo I, Miller A D., The RD114/simian type D
retrovirus receptor is a neutral amino acid transporter. Proc Natl
Acad Sci USA, 1999, Mar. 2; 96(5): 2129-34). Thus, as regards
nervous system diseases, it is known that the hASCT receptors are
involved in the specific transport of neutral amino acids and that
neuronal cells, for the transmission of information, predominantly
use neuromediators of a polypeptide nature. Thus, the binding of
the Env-HERV-W protein to receptors which normally have to
transport the amino acids required for the synthesis of
neuromediators can affect the capacity of the neurons to synthesize
the neuromediators by reducing the entry of the physiological
agonists such as amino acids via the ASCT receptors. Moreover, if
neurons whose intercellular networks form connections which are
essential for the transmission of information circulating in the
brain and the spinal cord, form syncytia following a fusion of
several neurons which is induced by the Env-HERV-W protein, all the
networks for transmission of information become disrupted and
connected to the same fused "cellular package" and, furthermore,
the neuromediator production activity of each cell is no longer
individualized or connected to the upstream or downstream
conduction pathways (dendrites and axons) which are specific to
it.
[0006] Surprisingly, the inventors have identified the polypeptide
region responsible for the interactions between the envelope of a
virus belonging to the HERV-W interference group and an hASCT
receptor.
[0007] To this effect, the present invention relates to a peptide
domain necessary for the interaction between the envelope of a
virus belonging to the HERV-W interference group and an hASCT
receptor, defined in that it starts with an N-terminus and ends
with a C-terminus, and in that: [0008] the N-terminus is defined by
a motif, consisting of the amino acids
(Z).sub..alpha.-proline-cysteine-X-cysteine in which [0009] Z is
any amino acid [0010] .alpha. is an integer between 2 and 30 [0011]
X is any amino acid, [0012] said motif being chosen from SEQ ID No.
1 to SEQ ID No. 29 and SEQ ID No. 44 to SEQ ID No. 72 [0013] the
C-terminus is defined by a motif consisting the amino acids
serine-aspartic acid-X.sub.a-X.sub.b-X.sub.c-X.sub.d-X.sub.e
aspartic acid-X.sub.f-X.sub.g-(Z).sub.p in which [0014] X.sub.a,
X.sub.b, X.sub.c, X.sub.d, X.sub.e, X.sub.f, X.sub.g are any amino
acids [0015] Z is any amino acid [0016] .beta. is an integer
between 15 and 25, preferably 20 [0017] said motif being chosen
from SEQ ID No. 30 to SEQ ID No. 40, [0018] and in that said
peptide domain comprises, between the N-terminus and the
C-terminus, at least one motif chosen from the following motifs:
[0019] a motif consisting of the amino acids
cysteine-tyrosine-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-cysteine,
in which X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6 are any amino
acids said motif corresponding to SEQ ID No. 41 [0020] a motif
consisting of the amino acids
cysteine-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.su-
b.14-X.sub.15-cysteine-tryptophan, [0021] in which X.sub.7,
X.sub.8, X.sub.9, X.sub.10, X.sub.11, X.sub.12, X.sub.13, X.sub.14,
X.sub.15 are any amino acids [0022] said motif corresponding to SEQ
ID No. 42 or SEQ ID No. 73.
[0023] The expression peptide domain according to the invention is
understood to mean a minimum region of the envelope of a virus of
the HERV-W interference group necessary for the recognition of an
hASCT receptor.
[0024] The peptide domains of the invention may be obtained by the
genetic engineering technique which comprises the steps of: [0025]
culturing a microorganism or eukaryotic cells transformed with the
aid of a nucleotide sequence according to the invention, and [0026]
recovering the peptide domain produced by said microorganism or
said eukaryotic cells.
[0027] This technique is well known to a person skilled in the art.
For further details concerning it, reference may be made to the
book below: Recombinant DNA Technology I, Editors Ales Prokop,
Raskesh K Bajpai; Annals of the New York Academy of Sciences,
Volume 646, 1991. The peptide domains of the invention may also be
prepared by conventional peptide syntheses well known to a person
skilled in the art.
[0028] The expression interference group is understood to mean all
the viruses for which the infection (expression) of a cell by one
of its members prevents infection by another member of the group by
receptor interference.
[0029] The expression any amino acid is understood to mean in
particular an amino acid chosen from arginine, histidine, lysine,
aspartic acid, glutamic acid, asparagine, glutamine, serine,
threonine, asparagine, threonine, alanine, isoleucine, leucine,
methionine, phenylalanine, tryptophan, tyrosine, valine, cysteine,
glycine, proline.
[0030] The expression hASCT receptor is understood to mean any
sodium-dependent neutral amino acid transporter.
[0031] The expression motifs is understood to mean a succession of
amino acids corresponding to a particular region of interest of the
peptide domain according to the invention, which is expressed by
all the viruses of the HERV-W virus interference group.
[0032] Preferably, .alpha. is an integer between 3 and 18.
[0033] Preferably X or Xaa of the Pro Cys Xaa Cys motif in SEQ ID
No. 1 to SEQ ID No. 29 is an amino acid chosen from aspartic acid,
glutamic acid, arginine: these sequences are those preferably
chosen from SEQ ID No. 44 to SEQ ID No. 72.
[0034] Preferably, X.sub.a, X.sub.b, X.sub.c or the amino acids at
positions 3, 4 and 5 of SEQ ID Nos. 30 to 40 are a glycine, X.sub.d
or the amino acid Xaa at position 6 of SEQ ID Nos. 30 to 40 is an
amino acid chosen from proline and valine; Xe or the amino acid Xaa
at position 7 of SEQ ID Nos. 30 to 40 is an amino acid chosen from
glutamine, leucine and threonine; X.sub.f or the amino acid Xaa at
position 9 of SEQ ID Nos. 30 to 40 is an amino acid chosen from
lysine, threonine, methionine and glutamine, X.sub.g or the amino
acid at position 10 of SEQ ID Nos. 30 to 40 is an amino acid chosen
from alanine, lysine, isoleucine, threonine and valine.
[0035] Preferably, .beta. is an integer equal to 20.
[0036] Preferably, X.sub.2 or the amino acid Xaa at position 3 of
SEQ ID No. 41 is an amino acid chosen from asparagine, threonine,
glutamic acid, histidine, X.sub.3 or the amino acid Xaa at position
4 of SEQ ID No. 41 is an amino acid chosen from histidine, alanine,
serine, lysine, glutamic acid; X.sub.4 or the amino acid Xaa at
position 5 of SEQ ID No. 41 is an amino acid chosen from tyrosine,
threonine, alanine, X.sub.5 or the amino acid Xaa at position 6 of
SEQ ID No. 41 is an amino acid chosen from glutamine, arginine,
threonine, X.sub.6 or the amino acid Xaa at position 7 of SEQ ID
No. 41 is an amino acid chosen from leucine, glutamine, glutamic
acid.
[0037] Preferably X.sub.7 or the amino acid Xaa at position 2 of
SEQ ID No. 42 is an amino acid chosen from proline, threonine,
arginine and asparagine; X.sub.8 or the amino acid Xaa at position
3 of SEQ ID No. 42 is an amino acid chosen from glycine, glutamic
acid, asparagine, Xg or the amino acid Xaa at position 4 of SEQ ID
No. 42 is an amino acid chosen from glycine, asparagine,
isoleucine, threonine, serine, X.sub.10 or the amino acid Xaa at
position 5 of SEQ ID No. 42 is lysine or is deleted; X.sub.11 or
the amino acid xaa at position 6 of SEQ ID No. 42 is an amino acid
chosen from lysine, valine, isoleucine, leucine, X.sub.12 or the
amino acid Xaa at position 7 of SEQ ID No. 42 is an amino acid
chosen from glycine, asparagine; X.sub.13 or the amino acid Xaa at
position 8 of SEQ ID No. 42 is an amino acid chosen from glutamine,
lysine, valine, X.sub.14 or the amino acid Xaa at position 9 of SEQ
ID No. 42 is an amino acid chosen from valine, proline, serine,
threonine, X.sub.15 or the amino acid Xaa at position 10 of SEQ ID
No. 42 is an amino acid chosen from valine, isoleucine.
[0038] Preferably,
the amino acid Xaa at position 2 of SEQ ID No. 73 is chosen from
proline, threonine, arginine and asparagine, the amino acid Xaa at
position 3 of SEQ ID No. 73 is chosen from glycine, glutamic acid,
asparagine, the amino acid Xaa at position 4 of SEQ ID No. 73 is
chosen from glycine, asparagine, isoleucine, threonine, serine, the
amino acid Xaa at position 5 of SEQ ID No. 73 is chosen from
lysine, valine, isoleucine, leucine, the amino acid Xaa at position
6 of SEQ ID No. 73 is chosen from glycine, asparagine, the amino
acid Xaa at position 7 of SEQ ID No. 73 is chosen from glutamine,
lysine, valine, the amino acid Xaa at position 8 of SEQ ID No. 73
is chosen from valine, proline, serine, threonine, the amino acid
Xaa at position 9 of SEQ ID No. 73 is chosen from valine,
isoleucine.
[0039] Deletions are possible in the domains according to the
invention indicated above. According to a particular embodiment of
the invention, X.sub.10 is deleted.
[0040] The invention also relates to a nucleotide sequence encoding
a peptide domain according to the invention above.
[0041] Such sequences may be prepared by chemical synthesis and
genetic engineering using techniques well known to a person skilled
in the art and described, for example, in Sambrook, J. et al.,
Molecular Cloning: A Laboratory Manual, 1989.
[0042] The invention also relates to an epitope derived from the
peptide domain according to the invention, characterized in that it
induces an immune response against a virus belonging to the HERV-W
interference group.
[0043] The expression epitope is understood to mean all or part of
the peptide domain according to the invention recognized by a
receptor located at the surface of a B or T lymphocyte or of a
circulating antibody.
[0044] The expression immune response is understood to mean all the
biological mechanisms which allow a pluricellular organism to
maintain the coherence of the cells and tissues which constitute it
and to ensure its integrity in response to any attack which
modifies the molecular structures of its constituents or which
introduces foreign molecules into the organism.
[0045] The invention also relates to a nucleotide sequence encoding
an epitope as defined above. As indicated above, such sequences may
be prepared by chemical synthesis and genetic engineering using
techniques well known to a person skilled in the art and described,
for example, in Sambrook J. et al., Molecular Cloning: A Laboratory
Manual, 1989.
[0046] The invention also relates to an expression vector
characterized in that it comprises a nucleotide sequence according
to the invention, and the means necessary for its expression.
[0047] By way of expression vector, there may be mentioned, for
example, plasmids, viral vectors of the vaccinia virus, adenovirus,
baculovirus, poxvirus or retrovirus type, bacterial vectors of the
salmonella or BCG type.
[0048] The expression means necessary for its expression is
understood to mean any means which make it possible to obtain a
peptide from a nucleotide sequence, such as in particular a
promoter, a transcription terminator, a replication origin and
preferably a selectable marker.
[0049] The vectors of the invention may also comprise sequences
necessary for targeting peptides to particular cell
compartments.
[0050] The invention also relates to a host microorganism or cell
transformed with at least one expression vector according to the
invention.
[0051] By way of examples of microorganisms which are suitable for
the purposes of the invention, there may be mentioned yeasts, such
as those of the following families: Saccharomyces,
Schizosaccharoyces, Kluveromyces, Pichia, Hanseluna, Yarowia,
Schwani omyces, Zygosaccharomyces; Saccharomyces cerevisiae,
Saccharomyces carlsbergensis and Kluveromyces lactis being
preferred; and bacteria such as E. coli and those of the following
families: Lactobacillus, Lactococcus, Salmonella, Streptococcus,
Bacillus and Streptomyces.
[0052] By way of examples of transformed host cells, there may be
mentioned cells derived from animals such as mammals, reptiles,
insects and the like. The preferred eukaryotic cells are cells
derived from the Chinese hamster (CHO cells), from monkeys (COS and
Vero cells), from young hamster kidney (BHK cells), from pig kidney
(PK 15 cells) and from rabbit kidney (RK13 cells), human
osteosarcoma cell lines (143 B cells), human HeLa cell lines and
human hepatoma cell lines (of the Hep G2 cell type), and insect
cell lines (for example Spodoptera frugiperda), a human embryonic
kidney cell line (for example HEK293T). The host cells may be
provided in cultures in suspension or in flasks, in tissue
cultures, organ cultures and the like.
[0053] The invention also relates to an antibody directed against a
peptide domain according to the invention or against an epitope
according to the invention.
[0054] The expression antibody is understood to mean both a whole
antibody and an antibody fragment.
[0055] The recombinant antibodies may be obtained according to
conventional methods known to a person skilled in the art, from
prokaryotic organisms, such as bacteria, or from eukaryotic
organisms, such as yeasts, mammalian, plant, insect or animal
cells, or by extracellular production systems.
[0056] The monoclonal antibodies may be prepared according to
conventional techniques known to a person skilled in the art such
as the hybridoma technique whose general principle is recalled
below.
[0057] In a first instance, an animal, generally a mouse (or
cultured cells in the context of in vitro immunizations), is
immunized with a target antigen of interest, whose B lymphocytes
are then capable of producing antibodies against said antigen.
These antibody-producing lymphocytes are then fused with "immortal"
myelomatous cells (murine in the example) in order to give rise to
hybridomas. From the heterogenous mixture of cells thus obtained, a
selection of the cells capable of producing a particular antibody
and of multiplying it indefinitely is then carried out. Each
hybridoma is multiplied in clone form, each leading to the
production of a monoclonal antibody whose recognition properties in
relation to the antigen of interest may be tested, for example by
ELISA, by one- or two-dimensional immunotransfer, by
immuno-fluorescence or with the aid of a biosensor. The monoclonal
antibodies thus selected are subsequently purified in particular
according to the affinity chromatography technique.
[0058] The expression antibody fragment is understood to mean any
antibody fragment following an immune response against a virus
belonging to the HERV-W interference group. These antibody
fragments may, for example, be obtained by proteolysis. Thus, they
may be obtained by enzymatic digestion, resulting in fragments of
the Fab type (treatment with papain; Porter R R, 1959, Biochem. J.,
73: 199-126) or of the F(ab)'.sub.2 type (treatment with pepsin;
Nisonoff A. et al., 1960, Science, 132: 1770-1771). They may also
be prepared by the recombinant route (Skerra A., 1993, Curr. Opin.
Immunol., 5: 256-262). Another antibody fragment which is suitable
for the proposals of the invention comprises an Fv fragment which
is a dimer consisting of the noncovalent combination of the
variable light (VL) domain and of the variable heavy (VH) domain of
the Fab fragment, and therefore the combination of two polypeptide
chains. In order to improve the stability of the Fv fragment due to
the dissociation of the two polypeptide chains, this Fv fragment
may be modified by genetic engineering by inserting a suitable
linker peptide between the VL domain and the VH domain (Huston P.
et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 5879-5883). The
expression scFv fragment ("single chain Fragment variable") is then
used because it consists of a single polypeptide chain. The use of
a linker peptide preferably composed of 15 to 25 amino acids makes
it possible to link the C-terminus of one domain to the N-terminus
of the other domain, thus constituting a monomeric molecule endowed
with binding properties similar to those of the antibody in its
complete form. Both orientations of the VL and VH domains are
suitable (VL-linker-VH and VH-linker-VL) because they exhibit
identical functional properties. Of course, any fragment known to a
person skilled in the art and exhibiting the immunological
characteristics defined above are suitable for the purposes of the
invention.
[0059] The invention also relates to the use of at least one
peptide domain according to the invention, of at least one epitope
according to the invention, of at least one antibody according to
the invention or of at least one nucleotide sequence according to
the invention, for the preparation of a medicament intended for the
inhibition, prevention or treatment of an infection caused by a
virus belonging to the HERV-W interference group in an animal,
preferably humans. The peptide domain according to the invention
may be used in particular for targeting cells expressing a receptor
of the hASCT family in order to transduce a signal, and modulate
the flow of amino acids (cancer treatments).
[0060] The expression elements necessary for a constitutive
expression of peptides is understood to mean a ubiquitous promoter
specific to eukaryotic cells. By way of elements necessary for an
inducible expression of the peptides, there may be mentioned the
elements for regulating the E. coli operon for resistance to
tetracycline (Gossen M. et al., Proc. Natl. Acad. Sci. USA, 89:
5547-5551 (1992)).
[0061] The use of at least one peptide domain according to the
invention, of at least one epitope according to the invention, or
of at least one nucleotide sequence according to the invention is
particularly suitable for the preparation of a medicament intended
for the prevention of an infection caused by a virus belonging to
the HERV-W interference group in an animal, preferably humans. The
use of at least one antibody according to the invention is
particularly suitable for the preparation of a medicament intended
for the inhibition or treatment of an infection or a pathology
induced by a virus belonging to the HERV-W interference group in an
animal, preferably humans.
[0062] The invention also relates to a pharmaceutical composition
comprising, by way of active substance, at least one peptide domain
according to the invention, at least one epitope according to the
invention, or alternatively at least one of the nucleotide
sequences according to the invention, in particular placed under
the control of elements necessary for a constitutive and/or
inducible expression of said peptide domains or epitopes, in
combination with a pharmaceutically appropriate vehicle. The
invention also relates to a pharmaceutical composition comprising,
by way of active substance, at least one antibody according to the
invention, in combination with a pharmaceutically appropriate
vehicle.
[0063] Of course, persons skilled in the art will easily determine
the pharmaceutically appropriate vehicle and the quantity of
peptide domains, of epitopes, of nucleotide acids or of antibodies
to be used according to the constituents of the pharmaceutical
composition.
[0064] In the pharmaceutical compositions according to the
invention, for oral, sublingual, subcutaneous, intramuscular,
intravenous, topical, intratracheal, rectal or transdermal
administration, the active substance may be administered in unit
forms for administration or as a mixture with conventional
pharmaceutical supports and intended for administration by the oral
route, for example in the form of a tablet, a gelatin capsule, an
oral solution, and the like, or by the rectal route, in the form of
a suppository, or by the parentral route, in particular in the form
of a solution for injection, in particular by the intravenous,
intradermal or subcutaneous route, and the like, according to
conventional protocols well known to persons skilled in the art.
For topical application, the active substance may be used in
creams, ointments, lotions, eyedrops.
[0065] When a solid composition in tablet form is prepared, the
active substance is mixed with the pharmaceutically acceptable
excipient, also called a pharmaceutical vehicle, such as gelatin,
starch, lactose, magnesium stearate, talc, gum Arabic or the like.
The tablets may be coated with sucrose, with a cellulose derivative
or with other appropriate materials. It is also possible to treat
them such that they have a prolonged or delayed activity and they
continuously release a predetermined quantity of the active
substance. It is also possible to obtain a preparation as gelatin
capsules by mixing the active substance with a diluent and pouring
the mixture into soft or hard gelatin capsules. It is also possible
to obtain a preparation in syrup form or for administration in the
form of drops, in which the active substance is present together
with a sweetener, an antiseptic, such as in particular
methylparaben and propylparaben, and a taste enhancer or an
appropriate colorant. The powders or water-dispersible granules may
contain the active substance in the form of a mixture with
dispersing agents or wetting agents, or suspending agents, well
known to persons skilled in the art. For parentral administration,
aqueous suspensions, isotonic saline solutions or sterile solutions
or solutions for injection which contain dispersing agents,
pharmacologically compatible wetting agents, such as in particular
polyethylene glycol or butylene glycol, are used.
[0066] The medicament or the pharmaceutical composition according
to the invention may additionally comprise an activating agent
which induces the effects of a medication or reinforces or
supplements the effects of the principal medication, by increasing
in particular the bioavailability of the principal medication.
[0067] The dosage depends on the seriousness of the condition and
will be adapted according to a conventional protocol. As a guide,
when the active substance is a monoclonal antibody, the weekly dose
is from 1 to 10 mg/kg, in combination with a pharmaceutically
acceptable excipient.
[0068] The invention also relates to a diagnostic composition for
the detection and/or quantification of a virus belonging to the
HERV-W interference group, or the detection and/or quantification
of an immune response against said virus, comprising at least one
peptide domain according to the invention, at least one epitope
according to the invention, at least one of the nucleotide
sequences according to the invention, or at least one antibody
according to the invention.
[0069] A diagnostic composition comprising at least one peptide
domain according to the invention, at least one epitope according
to the invention, at least one of the nucleotide sequences
according to the invention, is particularly suitable if it is
desired to determine if a patient has an immune response against a
virus belonging to the HERV-W interference group while a diagnostic
composition comprising at least one antibody according to the
invention is particularly suitable for the detection and/or
quantification of a virus belonging to the HERV-W interference
group.
[0070] The invention also relates to a method for the detection
and/or quantification of a virus belonging to the HERV-W
interference group in a biological sample taken from an individual
liable to be infected by said virus, characterized in that it
comprises the steps consisting in: [0071] bringing said biological
sample into contact with at least one antibody according to the
invention under conditions allowing the formation of a complex
between the virus and the antibody, and [0072] detecting and/or
quantifying the formation of said complex by any appropriate
means.
[0073] The expression biological sample is understood to mean a
biological sample of human or animal origin liable to contain said
virus, such as a sample of blood, plasma, serum, urine,
cerebrospinal fluid, or of tissues, such as placenta, testicles,
prostate and breast.
[0074] The step of bringing into contact is a step that is
conventionally known to a person skilled in the art.
[0075] The detection/quantification step may be carried out by any
detection means known in the field of immunological assays of very
small molecules, such as direct detection, that is to say without
the intermediary of a binding partner or of binding partners, and
indirect detection, that is to say through the intermediary of a
binding partner or of binding partners.
[0076] The direct detection of the binding between the antibody or
antibody fragment of the invention and the virus may be carried out
for example by surface plasmon resonance or by cyclic voltammetry
on an electrode bearing a conducting polymer. In this case, the
antibody of the invention serves to immunocapture all or part of
the virus, which is then eluted. The elution may be carried out by
any elution method known to a person skilled in the art, such as a
pH shock.
[0077] In the case of indirect detection, the second step of the
method of the invention may be carried out according to the
conventional ELISA competition assay technique. The antibody of the
invention then serves as binding partner serving to capture all or
part of the virus in the sample. The detection may then be
performed by competition between all or part of the virus which may
be contained in the sample to be tested and a previously labeled
known quantity of virus.
[0078] The expression labeling is understood to mean the attachment
of a marker capable of directly or indirectly generating a
detectable signal. A nonlimiting list of these markers consists of:
[0079] enzymes which produce a detectable signal, for example, by
colorimetry, fluorescence, luminescence, such as horseradish
peroxidase, alkaline phosphatase, acetylcholine esterase,
.beta.-galactosidase, glucose-6-phosphate dehydrogenase, [0080]
chromophores such as luminescent, coloring compounds, [0081]
radioactive molecules such as 32P, 35S or 125I, [0082] fluorescent
molecules such as fluorescein, rhodomine, alexa or phycocyanins,
and [0083] particles such as gold or magnetic latex particles,
liposomes.
[0084] Indirect labeling systems may also be used, such as, for
example, via another ligand/anti-ligand pair. The
ligand/anti-ligand pairs are well known to a person skilled in the
art, and the following pairs may be mentioned for example:
biotin/streptavidin, biotin/avidin, hapten/antibody,
antigen/antibody, peptide/antibody, sugar/lectin,
polynucleotide/complementary strand for the polynucleotide. In this
case, it is the ligand which is bound to the binding partner. The
anti-ligand may be detected directly by the markers described in
the proceeding paragraph or may itself be detected by a
ligand/anti-ligand.
[0085] These indirect systems may lead, under certain conditions,
to an amplification of the signal. This signal amplification
technique is well known to a person skilled in the art, and
reference may be made to the previous patent applications
FR98/10084 or WO95/08000 by the applicant or to the article J.
Histochem. Cytochem., (1997), 45: 481-491.
[0086] The labeling of molecules is widely known to a person
skilled in the art and is described for example by Greg T.
Hermanson in Bioconjugate Techniques, 1996, Academic Press Inc.,
525B Street, San Diego, Calif. 92101 USA.
[0087] Depending on the type of labeling used, such as for example
using an enzyme, a person skilled in the art will add reagents
which allow visualization of the labeling.
[0088] Such reagents are widely known to a person skilled in the
art and are described in particular in Principles and Practice of
Immunoessay, 2nd edition, Edited by C. Price, D. G. Newman,
Stockton Press, 1997, 345 Park Avenue South, N.Y.
[0089] The invention also relates to the use of the above
composition for the in vitro screening of a virus belonging to the
HERV-W interference group in a biological sample or specimen. In
particular, the early screening of a virus such as SRV1 and SRV2,
viruses that are involved in immunodeficiency mechanisms in
monkeys, makes it possible to provide a treatment suitable for the
host before the appearance of an immunodeficiency. Moreover, the
early screening of HERV-W, involved in placental pathologies, makes
it possible to modulate its expression, for example, during a
preeclampsia.
[0090] The invention also relates to the use of a peptide domain
according to the invention or of an epitope according to the
invention, or of an antibody according to the invention, for
inhibiting the interaction between the envelope of a virus
belonging to the HERV-W interference group and an ASCT receptor.
This makes it possible, in particular, to obtain a contraceptive
immunotherapy.
[0091] The invention also relates to the use of a peptide domain
according to the invention for identifying chemical or biological
molecules whose interaction with all or part of this peptide domain
blocks the interaction between the envelope of a virus belonging to
the HERV-W interference group and an ASCT receptor. For example,
when a peptide domain according to the invention of HERV-W is used,
this makes it possible to obtain in particular chemical or
biological molecules that are highly suitable in order to obtain a
contraceptive treatment. The use of such chemical molecules to
inhibit the interaction between the envelope of a virus belonging
to the HERV-W interference group and an ASCT receptor is of
therapeutic interest.
[0092] As a guide, two generic methods allowing the screening of
chemical or biological molecules capable of inhibiting the
env/receptor interaction are described below.
[0093] In a context where it is possible to produce a soluble
envelope, it is advisable to determine if a chemical or biological
molecule alters the env/receptor interaction according to an ELISA
type method using cells expressing at least one hASCT receptor in a
capture phase. Thus, on a 96-well plate, cells expressing an hASCT
receptor of interest are cultured or adsorbed, and an env/receptor
interaction is detected via the use of a labeled soluble envelope
(histine tag, GPF fusion), and therefore capable of generating a
reference signal that can be assayed. If a signal reduction is
observed after preincubation of said soluble envelope with a
chemical or biological molecule, that means that the chemical or
biological molecule alters the env/receptor interaction.
Alternatively, it is possible to use a retroviral vector
pseudotyped by the envelope of interest and expressing a detectable
marker (LacZ) and to carry out the same test. It is also possible
to select molecules of interest via a measurement of fusion
inhibition. Cells expressing the receptor of interest
(cell-recept), for example HeLa or XC-RDR cells, and cells
constitutively expressing a marker (for example, LacZ) and
transiently or stably expressing the envelope of interest
(cell-env-LacZ) are used; the envelope of interest was modified
beforehand at the level of its intracytoplasmic tail by exchange
with the intracytoplasmic domain of HERV-W env so as to make it
constitutively fusogenic (Cheynet et al, 79(9): 5586-5593, 2005).
The bringing into contact of the two cell types, "cell-recept" in
excess and "cell-env-LacZ" in insufficient amount, leads to the
formation of multinucleated giant cells or syncytia, containing one
or two blue nuclei derived from "cell-env-LacZ" and tens of white
nuclei derived from "cell-recept". An identical co-culture
performed in the presence of a chemical or biological molecule
altering the env/receptor interaction leads to a reduction in the
number of syncytia and their nucleus content. Automation of such
measurements with the aid of a CCD camera is possible.
[0094] The invention also relates to the use of a peptide domain in
accordance with the invention for generating antibodies blocking
the interaction between the envelope of a virus belonging to the
HERV-W interference group and an HASCT receptor.
[0095] The invention also relates to a method for determining a
polypeptide region necessary for the interaction between the
envelope of a virus belonging to the HERV-W interference group and
an hASCT receptor, characterized in that: [0096] the nucleotide
and/or peptide sequence of the precursor envelope of said virus is
identified [0097] the signal part is excluded [0098] a
serine-aspartic
acid-X.sub.a-X.sub.b-X.sub.c-X.sub.d-X.sub.e-aspartic
acid-X.sub.f-X.sub.g is detected domain in which [0099] X.sub.a,
X.sub.b, X.sub.c, X.sub.d, X.sub.e, X.sub.f, X.sub.g, are any amino
acids which correspond to SEQ ID No. 43 [0100] the C-terminus is
excluded between 15 and 25 amino acids, preferably 20 amino acids,
after said serine-aspartic
acid-X.sub.a-X.sub.b-X.sub.c-X.sub.d-X.sub.e-aspartic
acid-X.sub.f-X.sub.g domain, which corresponds to SEQ ID No.
43.
[0101] Preferably, X.sub.a, X.sub.b, X.sub.c is an amino acid which
is glycine, X.sub.d is an amino acid chosen from proline and
valine, X.sub.e is an amino acid chosen from glutamine, leucine and
threonine, X.sub.f is an amino acid chosen from lysine, threonine,
methionine and glutamine, X.sub.g is an amino acid chosen from
alanine, lysine, isoleucine, threonine and valine.
[0102] For the purposes of the present invention, the nucleotide
sequence and/or peptide consequent of the precursor envelope of
said virus is identified by any means known to a person skilled in
the art, who may refer in particular to Maniatis (ed. 1989).
[0103] The signal part is excluded by any means known to a person
skilled in the art, as described in particular in "Improved
Prediction of Signal Peptide: SignalP 3.0" Jannick Dyrlov Bendtsen,
Henrik Nielsen, Gunnar von Hiejne and Soren Brunak, J. Mol. Biol.,
340: 783-795, 2004.
[0104] Said domain is detected by any means known to a person
skilled in the art, that is to say using Blast or Fasta type
software (see, in particular, Altschul S F, Gish W, Miller W, Myers
E W, Lipman D J., Basic local alignment search tool. J. Mol. Biol.
1990 Oct. 5; 215(3): 403-10).
[0105] The invention also relates to a peptide domain capable of
being obtained by the above method.
[0106] 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.
[0107] FIG. 1 illustrates the phenotypical characteristics and
properties of soluble recombinant proteins derived from Env-W. In
particular, FIG. 1a illustrates the largest soluble recombinant
protein comprising all or part of the SU and TM subunits (Env-Gp60)
and the soluble recombinant protein corresponding to the SU subunit
(EnvSU). FIG. 1b represents the flow cytometry analysis of the test
for binding of the EnvSU recombinant protein to XC hASCT2 and XC
hASCT1 cells expressing the hASCT2 and hASCT1 receptors,
respectively. FIG. 1c illustrates the test of interference of
binding to the TE671 cells (control hASCT2), TE671RD cells (blocked
hASCT2) and TE671galv cells (blocked Pit1).
[0108] FIG. 2 illustrates the definition of the minimum binding
domain of the ERV-W envelope to the hASCT2 receptor (RBD for
receptor binding domain). In particular, FIG. 2a describes all the
deletion mutants designed from EnvSU. FIG. 2b represents the flow
cytometry analysis of the test of binding of the recombinant
proteins derived from EnvSU to the XC hASCT2 cells expressing the
hASCT2 receptor, in particular the binding of the Env197, Env168
and Env144 mutants and the binding defect of the Env69-317,
Env169-317 and Env117 mutants.
[0109] FIG. 3a illustrates the definition of an immunogenic peptide
inside the domain according to the invention (RBD) corresponding to
region 21-144 of the precursor of the HERV-W envelope protein. FIG.
3b shows the inhibition of the binding of the RBD to its receptor
with the aid of an antibody produced from the immunogenic peptide
(antiSU-EnvW) and the absence of inhibition of RBD-receptor binding
in the presence of a nonspecific antibody (antiTM-EnvW).
[0110] FIG. 4 represents the alignment of the retroviral envelope
sequences belonging to the same interference group and shows the
boundaries of the signal peptide, of the SU (surface unit) subunit
and of the TM (Trans membrane) subunit and the receptor binding
site. The sequences are HERV-W (Human Endogenous Retroviral Family
W), RD114 (Cat Endogenous retrovirus), REV (Avian
Reticuloendotheliosis Virus), BAEV (Baboon endogenous virus (strain
M7)), SRV1 (Simian retrovirus SRV-1), SRV2 (Simian retrovirus
SRV-2) and MPMV (Simian Mason-Pfizer virus).
[0111] 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
Construction and Production of the HERV-W Envelope SU Subunit
[0112] 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).
[0113] The soluble envelope (Gp60, 1-435) was constructed as
described below: [0114] (1) The native cleavage site RNKR (AA 314
to 317) between the SU and TM subunits was mutated to AAAR 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. [0115] (2) The transmembrane (tm) and intracytoplasmic
(CYT) regions corresponding to amino acids 436 to 538 were deleted
in order to obtain a soluble protein. [0116] (3) A spacer arm
having the composition (GGGS).sub.3, followed by a polyhistidine
tail (RGS-HHHHHH), 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).
[0117] 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 immediately downstream of the sequence AAAR, in order to
allow the purification of this protein by IMAC and the detection by
an anti-histidine monoclonal antibody (Qiagen, RGS H6).
[0118] The schematic structure of the various proteins produced
from the vectors phCMV-Env-W, phCMV-EnvGp60 and phCMV-EnvSU is
illustrated in FIG. 1a.
Production of the Soluble Envelope
[0119] 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 .mu.m membranes in order to remove the cellular
debris. 20 .mu.l of supernatant are directly analyzed on a
polyacrylamide gel and by Western blotting with an anti-histidine
monoclonal antibody (Quiagen, RGS H6). The GP60 and SU proteins are
correctly expressed in soluble form.
Binding Test and Analysis by Flow Cytometry
[0120] 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 PiTl receptor.
[0121] 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.degree. 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.degree. C. with an anti-histidine
monoclonal antibody (RGSH6, Quiagen). The cells were washed once
with PBA and incubated with a secondary antibody coupled to
fluorescein isocyanate for 1 hour at 4.degree. C. The cells were
washed twice with PBA and analyzed by flow cytometry.
[0122] 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.
[0123] 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 (FIG. 1b). Furthermore, this protein
was tested in relation to human cells TE671 and derived cells
TE671RD and TE671galv. The soluble SU protein bound to the TE6781
cells expressing the hASCT2 receptor and the TE671galv cells
blocked for the PiT1 receptor, but did not bind to the TE671RD
cells blocked for the hASCT2 receptor (FIG. 1c). The SU recombinant
protein and the envelope of the RD114 retrovirus specifically
interfered.
EXAMPLE 2
Identification of the Domains for Interaction of the SU Part of the
W Envelope with its hASCT2 Receptor
[0124] 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 (FIG. 2a) were transfected into the HEK293T cells by
precipitation with calcium phosphate. The conditions for production
and analysis of the proteins are identical to those detailed in
example 1.
[0125] 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.
[0126] 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.
[0127] Consequently, the boundaries of the domains for interaction
of the W envelope with its receptor are defined by amino acids 21
to 144.
[0128] 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.
[0129] 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)
[0130] A peptide (112-129, TGMSDGGGVQDQAREKHV+C, 19 amino acids)
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.
[0131] The Env144 protein is preincubated at 37.degree. 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
[0132] Immunization of Mice with DNA
[0133] 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 .mu.g of DNA were first performed for each mouse
followed by a booster with two injections of 4 .mu.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.
Preparation of the Cellular Lysate
[0134] The rhabdomyosarcoma cells TelCeB6 (ATCC CRL8805) were
transfected with the plasmid phCMV-env-W. After about 20 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
.mu.g/.mu.l of total proteins.
Immunization of Mice with an Extract of Cellular Lysate
[0135] The same mice first of all received an injection of 10 .mu.g
of cellular lysate by the intraperitoneal route followed by a
booster injection of 2.times.100 .mu.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 .mu.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 (Quiagen)
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 .mu.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
immuno-fluorescence 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 .mu.g/.mu.l of total
proteins and an Env AS protein as negative control at a
concentration of 13.3 .mu.g/.mu.l of total proteins and the most
effective antibodies were selected.
[0136] 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)
[0137] 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.degree. 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
(0.4.times.10.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
2.times.10.sup.5 cells per well and the co-culture is continued for
24 h. An XGal
(5-bromo-4-chloro-3-indolyl-.beta.-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-Grunwald 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.
[0138] The results presented in table 1 below express the number of
fused cells counted.
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
[0139] 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
[0140] 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 PLUSH kit (Gibco Invitrogen), with the DNA
corresponding to the HERV-W env gene cloned in the sense
orientation at the concentration of 2 .mu.g/.mu.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 .mu.g/.mu.l (DNA
410) and with the DNA corresponding to a mutated HERV-W env gene at
the concentration of 1.3 .mu.g/.mu.l (DNA LQMV) according to the
protocol detailed below. The cells transfected with the DNA 409are
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.
1). Protocol:
[0141] 1st Day: Culture of the TelCeB6 cells: [0142] inoculation of
the dishes 100 mm in diameter .about.50-70% confluence; [0143]
incubation in supplemented DMEM medium (6 ml per dish) for 24 hours
at 37.degree. C. under 5% CO.sub.2. 2nd Day: Transfection with the
LipofectAMINE PLUS.TM. kit:
1 Precomplexing of the DNA
[0143] [0144] mixing of 750 .mu.l of medium not supplemented with
the abovementioned DNAs in a 15 ml falcon tube (reference 2096),
that is 2 .mu.l of 409 or 3 .mu.l of 410 or 3 .mu.l LQMV; [0145]
stirring under vortex of PLUS reagent and adding 20 .mu.l thereof
to the DNA solution; [0146] stirring under vortex immediately 10
seconds at 1400 rpm; [0147] incubation 15 minutes at room
temperature.
2 Preparation of the Cells
[0147] [0148] replacement with 5 ml of nonsupplemented medium.
3 Dilution of the LipofectAMINE
[0149] In a tube for a dish, mix 30 .mu.l of LipofectAMINE Reagent
with 750 .mu.l of nonsupplemented medium.
4 Complexing of the DNA
[0150] Mixing of 780 .mu.l of dilute LipofectAMINE and 772 .mu.l of
solution of precomplexed DNA (total: 1552 .mu.l); stirring under
vortex immediately 10 sec at 1400 rpm, incubation 15 minutes at
room temperature.
5 Transfection and Production of Recipient Animals Transplanted
with the Target Cells, Treated or Not Treated by Injection of
Anti-Env Antibody Deposition of 1552 .mu.l in a dish; incubation
2-3 hours at 37.degree. C. under 5% CO.sub.2; replacement of the
transfection medium with 6 ml of supplemented medium; incubation 1
hour at 37.degree. C. under 5% CO.sub.2; injection by the
intraperitoneal route (IP) to SCID mice (in a volume of 1 ml),
1/5th 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).
[0151] 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.
3rd Day:
[0152] 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);
observation under an "inverted phase" microscope with counting of
the syncytia and/or after staining on a slide; 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.
[0153] A cellular aliquot of each sample is fixed in the presence
of methanol/acetone (v/v) and then stored at -20.degree. C. until a
crystal violet staining is obtained (1%). Photographs were taken of
the stained slides.
2) Mice:
[0154] Groups of 2 mice are inoculated with:
the cells transfected with the three types of plasmid (DNAs 409,
410 and LQMV) with no antibody (3.times.2=6 mice) the cells
transfected with the three types of plasmid (DNAs 409, 410 and
LQMV) with the monoclonal antibody 2H1H8 (3.times.2=6 mice).
3) Results:
[0155] The number of fused cells determined by direct reading on a
gridded counting chamber per 100 cells is indicated in Table 2
below:
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
[0156] 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.
[0157] To statistically objectify the primary analysis represented
by the numbers indicated in Table 2, a Chi-2 test was performed in
order to compare the data in Table 2.
[0158] 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:
i) Statistical validation of the specificity of the pathogenic
effect in vivo: Env expressed (409): 20.5 positives counted on
average out of 100 cells, anti-sense Env (410): 9.5 positives
counted on average out of 100 cells, mutated Env (LQMV): 6.5
positives counted on average out of 100 cells, mean of the controls
(410 and LQMV): 9.5+6.5/2=8%. Env versus control 410: Chi-2=5.89
(p<0.02) Env versus control LQMV: Chi-2=9.83 (p<0.002) Env
versus the two controls (410 and LQMV): Chi-2=7.69 (p<0.01)
Control 410 versus control LQMV: Chi-2=0.61 (difference not
significant).
[0159] The controls are therefore statistically equivalent and
there is no "real" difference linked to the type of control.
[0160] 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.
ii) Statistical validation of the therapeutic activity of the
antibodies tested on the pathogenic effect in vivo: Env expressed
(409): 20.5 positives counted on average out of 100 cells, Env
expressed (409)+monoclonal antibody 2H1H8: 3.5 positives counted on
average out of 100 cells. Env alone versus injection of the
antibody 2H1H8: Chi-2=15.38 (p<0.001)
[0161] 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
1) Materials
[0162] Soluble protein: supernatant filtered on 0.45 .mu.m
containing the soluble protein (293T cells transfected with the
plasmid 460 (envelope-spacer-His6).
[0163] Expression verified by Western blotting with an anti-RGS-His
antibody.
[0164] Antibody: monoclonal antibody 2H1H8 (IgG, 5.50 mg/ml).
[0165] Cells: XChASCT2, cellular clone XC (ATCC CCL-165, rat cells)
expressing the hASCT2 receptor.
[0166] DMEM medium (Gibco Invitrogen 41966-029) with South American
serum.
[0167] Preincubation, incubation, labeling in a 1.5 ml Eppendorf
tube.
2) Protocol
[0168] 1 IP (Intraperitoneal) Inoculation of the XChASCT1, XChASCT2
Cells and Control Cells XChASCT--into SCID Mice
[0169] Injection into mice of 1/5th of the flask at 70% confluence
in a volume of 2 ml.
2 Preincubation
[0170] Incubation of the soluble protein supernatant (filtered
supernatant of the 293T line) with the monoclonal antibody 2H1H8
(990 .mu.l of supernatant with 10 .mu.l of antibody ( 1/100th
dilution)) for 1 hour at 37.degree. C. in the cell incubator with
occasional stirring (every 15 minutes).
3 Inoculation
[0171] IP (intraperitoneal) inoculation of the proteins alone or
with the antibody into mice transplanted with the cells
(1.times.10.sup.6 cells per point, that is 1/5th of a confluent
dish 100 mm in diameter).
[0172] After injection of the antibody (200 microliters),
maintained as IP, for 6 hours, with an occasional peritoneal
massage (every 30-60 minutes).
4 Recovery of the Cells by Peritoneal Lavage of the Transplanted
Mice
[0173] Centrifugation 3000 revolutions for 5 minutes at +4.degree.
C. [0174] Recovery of the cellular pellet and dilution in the
labeling media (maintained at +4.degree. C. until fixing).
5 Labeling
[0174] [0175] Primary antibody:
[0176] The pellet is taken up in 100 .mu.l of anti-RGS His antibody
(100th dilution--Quiagen) in a PBA buffer (PBS with 2% fetal calf
serum and 0.1% sodium azide), maintained at +4.degree. C.
[0177] 1 hour in ice with occasional stirring (every 15
minutes).
[0178] Washing in PBA buffer (1 ml per tube), maintained at
+4.degree. C. [0179] Secondary Antibody:
[0180] Centrifugation 3000 revolutions for 5 minutes at +4.degree.
C. The pellet is taken up in 100 .mu.l of anti-mouse antibody-FITC
( 1/20th dilution--DAKO, reference F0479 in a PBA buffer)
maintained at +4.degree. C.
[0181] 1 hour in ice with occasional stirring (every 15
minutes).
[0182] 2 washes in PBA buffer (1 ml per tube), maintained at
+4.degree. C.
[0183] Pellet taken up in 500 .mu.l of PBA, maintained at
+4.degree. C., and analyzed by FACS.
[0184] Alternatively, analysis by IF after fixing on a slide in
acetone/methanol (50%/50%) at -20.degree. C. and counter-staining
with Evans blue.
3) Nice:
[0185] Groups of 2 mice are inoculated with:
each type of cell (expressing the 2 types of receptor hASCT1 and
hASCT2 and one not expressing the receptor hASCT--as a control)
with no antibody (3.times.2=6 mice) the three types of cell with
the Env protein and the monoclonal antibody 2H1H8 (3.times.2=6
mice).
4) Results
[0186] The results by immunofluorescence (IF) reading with a
microscope are presented in Table 3 below:
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
[0187] 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.
[0188] 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.
[0189] The results of the statistical analysis are presented
below:
i) Statistical validation of the specificity of the pathogenic
effect in vivo: control hASCT1: 24 positives counted on average out
of 77 cells, control hASCT2: 23 positives counted on average out of
57 cells, hASCT- cells: 1 positive counted on average out of 28
cells. Env+grafts hASCT1 versus hASCT-: Chi-2=8.62 (p<0.01)
Env+grafts hASCT2 versus HASCT-: Chi-2=12.53 (p<0.001)
Env+grafts hASCT1 versus env+grafts hASCT2: Chi-2=1.21 (difference
not significant).
[0190] 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.
[0191] 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 trans-planted with the cells expressing none of
these receptors at their surface.
[0192] These results confirm the specificity of the effect obtained
in vivo in the presence of the Env protein in the animal
models.
ii) Statistical validation of the therapeutic activity of the
antibodies tested on the pathogenic effect in vivo: Env+grafts
control hASCT1: 24 positives counted on average out of 77 cells
Env+grafts hASCT1+monoclonal antibody 2H1H8: 2 positives counted on
average out of 73 cells. Env+grafts hASCT1 alone versus injection
antibody 2H1H8: Chi-2=21.14 (p<0.001)
[0193] The results obtained show a statistically significant effect
for the monoclonal antibody (probability of the result due to
chance (p) less than 0.001).
Env+grafts hASCT2: 23 positives counted on average out of 57 cells
Env+grafts hASCT2+monoclonal antibody 2H1H8: 1 positive counted on
average out of 45 cells. Env+grafts hASCT2 alone versus injection
antibody 2H1H8: Chi-2=20.31 (p<0.001).
[0194] 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).
[0195] 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
[0196] 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.
* * * * *