Title of Invention

MUTATED HIV NEF PROTEIN DEVOID OF IMMUNOSUPPRESSION AVTIVITY

Abstract The present invention relates to a mutated Nef protein or fragment thereof which is completely devoid of immunosuppression activity in comparison to the NEF protein and having ability to down regulate the CD4 and MHC1 proteins similar to that of the natural NEF protein.
Full Text Field of Invention
The field of invention is biotechnology. More specifically, the invention pertains to a process for modulating the immunosuppressive activity of a specific protein secreted by human or simian immunodeficiency virus. This has applications in vaccine preparation for prevention and/or treatment of viral diseases; in preparation of pharmaceutical compositions having enhanced immunosuppressive activity for use in prevention or treatment of pathologies requiring an inhibition of the immune system such as allergies, autoimmune diseases or graft rejections; and also for developing screening methods for identification of anti-viral agents.
Background of the Invention
Although more than 20 years of scientific research have been devoted to finding a vaccine against HIV (Human Immunodeficiency Virus), a convincing prophylactic means to fight HIV is still awaiting to be discovered. Thus, more than 20 clinical trials of anti-HIV vaccines have been launched, and as of today, none of them has shown sufficient efficacy in preventing infections. In the present invention, the inventors have been able to overcome the barrier in development of an effective vaccine against HIV (Human Immunodeficiency Virus) /SIV (Simian immunodeficiency virus). This has been made possible due to the identification by the inventors, of a specific domain in an accessory protein called Nef (Negative regulatory factor) secreted by HIV/SIV, in which mutation of at least one amino acid results' in modulation of the immunosuppressive activity. This has important implications in development of vaccines and pharmaceutical preparations for preventing and /or treating infections caused by HIV/SIV.
Significance of Nef protein
Nef (negative regulatory factor), is a 27 to 35 kDa regulatory protein of HIV or SIV. In vivo,
the Nef protein is in particular found in HIV (such as H1V-1 or HIV-2) infected individuals or
in SIV infected apes.
Among its various functions, Nef is in particular involved in the down-regulation of the expression of Class I MHC (Major Histocompatibility Complex) molecules (MHC-I) of the A and B types in humans (HLA-A and HLA-B). This property of Nef has been shown to be sensitive to mutations. However, mutations of HIV-1 Nef at the amino acid position 93 have proved inefficient at modulating its MHC-I down-regulation properties (AH et al. (2003) J. Immunol. 171:3999-4005).

Nef is also involved in the down-regulation of CD4 molecules normally expressed at the surface of T helper cells.
Furthermore, Nef also down-regulates the expression of mature Class II MHC molecules and
up-regulates the expression of immature Class II MHC molecules.
All these regulations are a consequence of Nef interference with normal cellular trafficking
and in particular with the endocytosis-degradation pathway (Le Gall el al. (1998) Immunity
8:483-495).
Limitations of existing vaccines using Nef
Nef, as one of the antigens of HIV or SIV, has been included in several vaccine compositions,
alone or in combination with other antigens, such as described, for example, in WO 01/00232
or in WO 03/011334. However, these approaches have not been demonstrated to be effective
either. The lack of an effective immune response against HIV or SIV, as a result of Nef
administration, might relate to an as yet unidentified function of Nef, whereas generation of
an active vaccine against HIV or SIV most probably requires an effective immune response to
be raised against Nef.
Advantages of vaccines based on present invention
In the present invention, the inventors have been able to identify a specific domain in the Nef
protein, which is responsible for immunosuppression. Modification of at least one amino acid
in this domain, results in a mutated Nef protein, which does not possess any
immunosuppressive activity, but whose other intracellular functions are intact. Hence, such a
mutated Nef protein can be used as an effective and safe agent for manufacture of vaccines.
The antibodies against such a mutated protein (without immunosuppressive activity) also bind
to the natural protein (with immunosuppressive activity), rendering it ineffective.
Objects of the invention
It is an object of the present invention to relate immunosuppressive properties of HIV or SIV to the Nef protein. Another object is to disclose a specific domain in the Nef protein, which is responsible for immunosuppression. A further object of the invention is to provide a pharmaceutical or vaccine composition comprising a modified Nef protein. Yet another object of the invention is to disclose a specific immunosuppressive domain in the Nef protein, which can be used for testing the binding of drugs and thus act as a tool for the screening of antiviral compounds.

Summary of the invention
The present invention discloses a specific domain in an accessory protein, Nef secreted by HIV or SIV viruses, which is involved in immuno-suppression. Mutation of at least one amino acid in this domain can be used for modulating (increasing, decreasing or keeping unchanged) the immunosuppressive property of the Nef protein. This aspect has got practical applications in the manufacture of vaccines and also development of screening assays for antiviral compounds. A mutated Nef protein which does not possess immunosuppressive activity can be safely used in vaccines, as the antibodies generated against it will bind the Nef protein also and thus prevent immunosuppression. In addition, the domain can be used as a tool for identification of compounds having anti-viral activity, by checking their binding to the domain.
Detailed Description of the invention
The present invention relates to the use of the immunosuppressive function of an accessory protein, Nef of the human or simian immunodeficiency virus for the preparation of a vaccine. In particular, the present invention relates to vaccine compositions comprising a mutated Nef protein, in which at least one amino acid has been modified in a specific domain of the protein, associated with immunosuppression. As intended herein, a mutation either relates to the substitution, the insertion or the deletion of at least one amino acid purposely brought to a Nef protein, or to the naturally occurring substitution, insertion or deletion of at least one amino acid in a given Nef protein with respect to the majority of Nef proteins (i.e. at least about 80% of the identified Nef proteins).
According to the invention, a given protein is said to hold an immunosuppressive property, if it is liable to inhibit the immune system of an organism in which it is present. In particular, the immunosuppressive property of said given protein can be measured by following the general procedure described in Mangeney & Heidmann (1998) Proc. Natl. Acad. Sci. U.S.A. 95:14920-5 and Mangeney et al. (2001) J. Gen.Virol. 82:2515-8. Stable tumor cell lines expressing, or in particular excreting, said given protein in the intra- or extracellular space are established and engrafted onto mice, and the size of the tumors (Aprotein) is compared, after several days, to the size of tumors (Anone) obtained from mice engrafted with tumor cell lines which do not express, or in particular excrete, said given protein. If the size of the tumors which express, or in particular excrete, the given protein is significantly greater than the size of the non-expressing, or in particular the non-excreting

tumors, the given protein is said to be immunosuppressive. The immunosuppressive property of a given protein can also be characterized by its immunosuppression index [(Aprotein-AnoneVAnone], If the immunosuppression index of a given protein is positive then the given protein is said to be immunosuppressive, and if its immunosuppression index is equal to zero or negative, the given protein is said to have essentially no immunosuppressive activity.
The present invention results from the relation which has been established by the Inventors between the immunosuppressive properties of HJV or SIV and the Nef protein. In other terms, the present invention results from the identification of the immunosuppressive function of the Nef protein, which is furthermore shown to be both an intra and an extracellular function. Further, the Inventors have shown that the immunosuppressive function of Nef is independent from the Nef-induced downregulation of CD4 or MHC-I.
Thus, in a preferred embodiment, the invention relates to the use of a mutation of at least one amino acid in the immunosuppressive domain of a Nef protein, for modulating the immunosuppressive property of said protein, provided that the resulting mutated protein presents substantially preserved CD4 and/or MHC-I down-regulation functions with respect to the non-mutated Nef protein.
As intended herein the expression "substantially preserved CD4 and/or MHC-I down-regulation functions" means that at least 60%, in particular at least 80% of the CD4 and/or MHC-I downregulation functions of a given Nef protein is preserved in the corresponding Nef protein carrying a mutation according to the invention.
The donwregulation of CD4 and MHC-I can be determined by measuring the fluorescence of CD4 or MHC-I expressing cells transformed with increasing amounts of nucleic acids encoding said given protein and contacted with fluorescent anti-CD4 or anti-MHC-I antibodies. Such methods are well known to the man skilled in the art and are in particular described in the following examples.
The immunosuppressive domain of an immunosuppressive protein is defined as being the region of said protein which is responsible for conferring its immunosuppressive activity to said protein and, in particular, it is constituted of all the amino acids, the mutation of which is liable to modulate the immunosuppressive property of said protein.
As intended herein, the expression "modulating the immunosuppressive property" of a given protein relates to an increase or a decrease in the immunosuppressive property of said protein.
In a preferred embodiment, the invention relates to the above defined use of a mutation of at least one amino acid in the immunosuppressive domain of a Nef protein, tor

inhibiting the immunosuppressive property of said protein. According to this embodiment the Nef protein presents an imunosuppressive property.
As intended in the present invention, the inhibiting of the immunosuppressive property of a given protein, yields a protein with substantially no immunosuppressive activity, that is having an immunosuppression index equal to zero or negative.
Nef proteins devoid of immunosuppressivity are particularly advantageous for the manufacture of anti-HlV or anti-SIV vaccines. Indeed, vaccine compositions containing Nef proteins devoid of immunosuppressivity according to the present invention are particularly effective at preventing HIV or SIV infections since they potently stimulate the immune response and in particular the production of antibodies directed against the Nef protein and the elicitation of a cellular immune response against infected cells which express the Nef protein. This stimulation of the immune response therefore prevents the subsequent immunosuppressive action of Nef when it is liberated in the organism or expressed by infected cells during the initial steps of HIV or SIV infection. Thus, the absence of immunosuppression conveyed by the Nef protein, which results from the immune response elicited against Nef, prevents the HIV or SIV precocious infectious cycles from being effective and favour the elimination of the virus by the immune system.
In particular, vaccine compositions according to the invention are more effective than Nef-containing compositions of the prior art to induce an anti-HIV or anti-SIV response from the immune system, since it is herein disclosed that non-mutated Nef is in itself an inhibitor of the immune system.
In another preferred embodiment, the invention relates to the above defined use, to obtain a Nef protein mutated in its immunosuppressive domain, or a fragment thereof, provided said fragment comprises the mutated immunosuppressive domain of said Nef protein, for the manufacture of a medicament or a vaccine intended for the prevention and/or the treatment of viral diseases.
As intended herein, viral diseases encompass all diseases or syndromes resulting from a viral infection, such as AIDS for instance. Besides, vaccines according to the invention are meant to be used prophylactically or therapeutically.
In yet another preferred embodiment, the invention relates to the above defined use, wherein the structure of the Nef protein is substantially preserved.
The substantial preservation of the structure of a Nef protein mutated in its immunosuppressive domain with respect to its natural counterpart can be for instance determined by comparing the circular dichroism spectra, the RMN spectra, the X-ray

diffraction pattern, or any other physicochemical property of said mutated Nef protein with that of the natural Nef protein from which it derives, according to methods well known to the man skilled in the art. It is to be noted that, as intended herein, the natural Nef protein from which the mutated Nef protein is deriving presents an immunosuppressive activity.
In a further preferred embodiment, the invention relates to the above defined use, wherein the epitopes, in particular the conformational epitopes, of the Nef protein are substantially preserved. In particular, B-cell epitopes as well as T-cell epitopes are preserved.
More particularly, the invention relates to the above defined use, wherein the epitopes, in particular the conformational epitopes, located outside of the immunosuppressive domain of the Nef protein are substantially preserved.
The substantial preservation of the epitopes for a Nef protein mutated in its immunosuppressive domain, with respect to its natural counterpart, can be for instance determined by checking that antibodies known to bind to a natural Nef protein, also bind to the corresponding Nef mutant.
In another further preferred embodiment, the invention relates to the above defined use, wherein the intracellular functional properties of the Nef protein other than its immunosuppressive properties are substantially preserved.
More preferably, the invention relates to the above defined use, wherein the CD4 and/or MHC-I down-regulation functions of the Nef protein are substantially preserved.
The intracellular functional properties of the Nef protein other than its immunosuppressive properties relate to the non-immunosuppressive functions of the protein which are only operative when said protein is hosted inside a cell. Such functions notably comprise the down-regulation of CD4 and MHC-I expression.
The down-regulation of CD4 and MHC-I expression by a given protein can be determined by measuring the fluorescence of CD4 or MHC-I expressing cells transformed with increasing amounts of nucleic acids encoding said given protein and contacted with fluorescent anti-CD4 or anti-MHC-1 antibodies. Such methods are well known to the man skilled in the art and are in particular described in the following examples.
The present invention also relates to a process for cancelling the immunosuppressive property of a Nef protein, comprising:
- mutating the immunosuppressive domain of said Nef protein by deletion, substitution or
insertion of at least one amino acid,
- checking the cancelling of said immunosuppressive activity by an in vivo
immunosupressivity assay.

The in vivo immunosuppressivity assay corresponds to the above described assay.
A preferred embodiment of the above mentioned process comprises a further step of checking that the structure and/or the epitopes, in particular the epitopes located outside the immunosuppressive domain, of the Nef protein are substantially preserved.
Another preferred embodiment of the above mentioned process comprises a further step of checking that the structure and/or the epitopes, in particular the epitopes located outside the immunosuppressive domain, and/or the CD4 and/or MHC-I down-regulation functions, of the Nef protein are substantially preserved.
The substantial preservation of the structure and/or the epitopes, and/or the CD4 and/or MHC-I down-regulation functions, of the Nef protein can be determined as described above.
The present invention relates in particular to a pharmaceutical or vaccine composition, comprising as active substance, a protein or a polypeptide comprising or being constituted of a Nef protein or a fragment thereof, wherein
• the immunosuppressive domain of said Nef protein is mutated by deletion, substitution
and/or insertion of at least one amino acid, provided that said Nef protein has
substantially no immunosuppressive activity, and
• said fragment comprises the mutated immunosuppressive domain of said Nef protein
and has substantially no immunosuppressive activity, in association with a
pharmaceutically acceptable carrier.
In particular, the sequences adjacent to the respective N-terminal and C-terminal ends of said fragment can be identical to the sequences adjacent to the respective N-terminal end and C-terminal end of said fragment in the Nef protein from which it derives.
In a particular embodiment of the above mentioned pharmaceutical or vaccine composition the protein or polypeptide comprising a fragment of Nef protein is such that the sequences adjacent to the respective N-terminal and/or C-terminal end of said fragment are different from the sequences adjacent to the respective N-terminal end and/or C-terminal end of said fragment in the Nef protein from which it derives.
More particularly, in another embodiment of the above mentioned pharmaceutical or vaccine composition, the protein or polypeptide comprising a fragment of Nef protein is such that:
• the sequence adjacent to the N-terminal end of said fragment is different from the sequences adjacent to the N-terminal end of said fragment in the Nef protein from which it derives, or

• the sequence adjacent to the C-terminal end of said fragment is different from
the sequences adjacent to the C-terminal end of said fragment in the Nef
protein from which it derives, or
• the sequence adjacent to the respective N-terminal and C-terminal ends of said
fragment are different from the sequences adjacent to the respective N-terminal
and C-terminal ends of said fragment in the Nef protein from which it derives.
The mutated Nef protein or fragment thereof according to the invention is said to be immunosuppressive deficient.
The present invention also relates to a pharmaceutical or vaccine composition, comprising as active substance, a protein or a polypeptide comprising or being constituted of a Nef protein or a fragment thereof, wherein :
• the immunosuppressive domain of said Nef protein is mutated by deletion, substitution
and/or insertion of at least one amino acid, provided that said Nef protein has
substantially no immunosuppressive activity and that the CD4 and/or MHC-1 down-
regulation functions, of the Nef protein are substantially preserved, and
• said fragment comprises the mutated immunosuppressive domain of said Nef protein
and has substantially no immunosuppressive activity, in association with a
pharmaceutical ly acceptable carrier.
In a preferred embodiment of the above defined pharmaceutical or vaccine composition, the sequence of the mutated immunosuppressive domain of the Nef protein is comprised in the amino acid sequence extending from the N-terminus of the first a helix to the C-terminus of the second a helix of the Nef protein.
The structure of the Nef protein is in particular described in Arold et al. (1997) Structure 5:1361-72 and in Grzesiek el al. (1997) Protein Science 6:1248-63. The nomenclature of the secondary structure elements of the Nef protein, and in particular of its a helices, is based on the structural description of the core domain of the Nef protein, according to Arold el al (1997) and Grzesiek et al. (1997).
In another preferred embodiment of the above defined pharmaceutical or vaccine composition, the sequence of the mutated immunosuppressive domain of the Nef protein is comprised in a sequence ranging from the amino acid at position 80 to the amino acid at position 150, particularly from the amino acid at position 81 to the amino acid at position 140, of the sequence of said Nef protein, and in particular:

• in a sequence ranging from the amino acid at position 80 to the amino acid at position
120, more particularly from the amino acid at position 81 to the amino acid at position
118, of the sequence of a HIV-1 Nef protein, or
• in a sequence ranging from the amino acid at position 104 to the amino acid at
position 150, in particular from the amino acid at position 104 to the amino acid at
position 140, of the sequence of a H1V-2 Nef protein.
Nef protein sequences can be easily accessed by the man skilled in the art. By way of example, several HIV-1, H1V-2 or SIV Nef protein sequences are presented in Figure 4.
More preferably, in the above defined pharmaceutical or vaccine composition, the sequence of the mutated immunosuppressive domain of the HIV-1 Nef protein is comprised in a sequence ranging from the amino acid at position 90 to the amino acid at position 113, in particular from the amino acid at position 90 to the amino acid at position 112, of the sequence of said Nef protein.
In a particular embodiment of the above defined pharmaceutical or vaccine composition, the sequence of the mutated immunosuppressive domain of the Nef protein is comprised in a sequence which is homologous to the amino acid sequence ranging from the amino acid at position 80 to the amino acid at position 120 of SEQ ID NO: 1, in particular from the amino acid at position 81 to the amino acid at position 117 of SEQ ID NO: 1, more particularly from the amino acid at position 90 to the amino acid at position 112 of SEQ ID NO: 1.
SEQ ID NO: 1 corresponds to the amino acid sequence of the Nef protein described by Wain-Hobson et al. (1985) Cell 40:9-17 (HIV-1 strain LAI).
According to the invention, two sequences are said to be homologous if they can be aligned by using an algorithm such as defined in Altschul et al. Nucleic Acids Res. (1997) 25:3389 or by using the Clustal W software, well known from the man skilled in the art and described in Thompson et al., Nucleic Acids Res. (1994) 22:4673-4680, for instance.
In particular, two sequences are said to be homologous if the amino acid identity percentage between said two sequences is equal to or larger than about 35%.
By way of example, Figure 4 represents a sequence alignment of several Nef proteins originating from HIV-1, HIV-2 or SIV, as obtained with the Clustal W software. Sequences homologous to the amino acid sequence ranging from the amino acid at position 81 to the amino acid at position 118 of SEQ ID NO: 1 are boxed.
In another particular embodiment of the above defined pharmaceutical or vaccine composition, the sequence of the mutated immunosuppressive domain of the Nef protein is

comprised in a 26 or 27 amino acid-long sequence of said Nef protein, the N-terminal end of said 26 or 27 amino acid-long sequence being the pentapeptide AX,DX2S and the C-terminal end of said 26 or 27 amino acid-long sequence being the amino acid L, in which X, represents any amino acid, and in particular I, V, L, F, or R, and X2 represents any amino acid, and in particular M, L, or F.
Examples of such sequences are presented in Figure 4.
In yet another particular embodiment of the invention, the above defined pharmaceutical or vaccine composition comprises as active substance a protein or polypeptide comprising or being constituted of a Nef protein or a fragment thereof comprising the following sequence:
AX,DX2SX3X4X5KX6X7GX8LX9G
wherein
X| represents I, L, V, F, or R, X2 represents M, L, or F, XT, represents H, D, or F, X,} represents F or L, Xs represents I or L,
X6 represents any amino acid different from E, in particular R, X7 represents K, Q, or R, Xg represents G or no amino acid, Xg represents E, D, or R.
SEQ ID NO: 3 comprises the immunosuppressive domain of Nef. Examples of such sequences are presented in Figure 4.
According to a particularly preferred embodiment of the invention, the above defined pharmaceutical or vaccine composition comprises as active substance a protein or polypeptide comprising or being constituted of a Nef protein or a fragment thereof, wherein the amino acid homologous to the amino acid at position 93 of SEQ ID NO: 1 is replaced by any amino acid different from E, in particular by W, F, M, Y, R, H or K, more particularly by R, H, or K, and preferably by R.
The amino acid homologous to the amino acid at position 93 of SEQ ID NO: 1 can be determined by aligning the sequence of the above mentioned protein or polypeptide with SEQ ID NO: 1 (for instance using the Clutal W software) and by selecting the amino acid which is aligned with the amino acid at position 93 of SEQ ID NO: 1. By way of example Figure 4

represents the amino acid homologous to the amino acid at position 93 of SEQ ID NO: 1 for several Nef proteins originating from HIV-1, HIV-2 or SIV. Advantageously, the single substitution of the amino acid homologous to the amino acid at position 93 of SEQ ID NO: 1 amino acid yields Nef mutants substantially devoid of immunosuppressivity.
According to another particularly preferred embodiment of the invention, the above defined pharmaceutical or vaccine composition comprises as active substance, a protein or polypeptide comprising or being constituted of a HIV-1 Nef protein or a fragment thereof, wherein the amino acid at position 93 of the sequence of said HIV-1 Nef protein is replaced by any amino acid different from E, in particular by W, F, M, Y, R, H or K, more particularly by R, H, or K, and preferably by R.
According to yet another particularly preferred embodiment of the invention, the above defined pharmaceutical or vaccine composition comprises as active substance a Nef protein, wherein the amino acid homologous to the amino acid at position 93 of SEQ ID NO: 1 is replaced by any amino acid different from E, in particular by W, F, M, Y, R, H or K, more particularly by R, H, or K, and preferably by R.
By way of example, as depicted in Figure 6B, the position homologous to the position 93 of HIV-1 Nef corresponds to position 125 in SIV strain mac239 Nef (SEQ ID NO: 22) and the substitution of the E at position 125 in SIV strain mac239 Nef by R yields an immunosuppressive-deficient Nef mutant (SEQ ID NO: 23).
According to a most preferred embodiment of the invention, the above defined pharmaceutical or vaccine composition comprises as active substance a mutated Nef protein corresponding to SEQ ID NO: 2.
In yet another preferred embodiment of the invention, the above defined pharmaceutical or vaccine composition is characterized in that when a Nef protein is comprised in said pharmaceutical or vaccine composition, the structure of the Nef protein is substantially preserved.
In a further preferred embodiment of the invention, the above defined pharmaceutical or vaccine composition is characterized in that when a Nef protein is comprised in said pharmaceutical or vaccine composition, the epitopes, such as B cell or T cell epitopes, in particular the contbrmational epitopes, of the Nef protein are substantially preserved.
More particularly, the invention relates to the above defined pharmaceutical or vaccine composition, wherein the epitopes, in particular the conformational epitopes, located outside of the immunosuppressive domain of the Nef protein are substantially preserved.

In another further preferred embodiment of the invention, the above defined pharmaceutical or vaccine composition is characterized in that when a Nef protein is comprised in said pharmaceutical or vaccine composition, the intracellular functional properties other than the immunosuppressive properties of the Nef protein are substantially preserved.
More preferably, the invention relates to the above defined pharmaceutical or vaccine composition, wherein the CD4 and/or MHC-I down-regulation functions of the Nef protein are substantially preserved.
The present invention also relates to the protein or polypeptide as defined in the above mentioned pharmaceutical or vaccine composition.
In particular, the present invention relates to a protein represented by SEQ ID NO: 2 or SEQIDNO: 31.
The present invention also relates to a pharmaceutical or vaccine composition, comprising as active substance a nucleic acid encoding a protein or polypeptide such as defined above.
The present invention also relates to the nucleic acid sequences coding for the protein or the polypeptide as defined in the above mentioned pharmaceutical or vaccine composition.
in particular, the present invention relates to a nucleic acid sequence coding for a protein represented by SEQ ID NO: 2 or SEQ ID NO: 31.
The present invention also relates to the use of a protein or a polypeptide as defined above, or of a nucleic acid as defined above, for the manufacture of a medicament or a vaccine intended for the prevention and/or the treatment of viral diseases, such as HIV infections.
In a preferred embodiment of the invention, the above defined medicament, or the above defined pharmaceutical or vaccine composition comprising a protein or a polypeptide as defined above as active substance, also comprise at least one HIV protein or lipopeptide, or a fragment thereof, in particular selected from gp41, gp!20, gp!40, gp!60, Env, Gag, Pol, Rev, RT, Vpu or Tat.
In a preferred embodiment of the invention, the above defined medicament, or the above defined pharmaceutical or vaccine composition comprising as active substance a nucleic acid encoding a protein or polypeptide such as defined above, also comprise at least one nucleic acid encoding a HIV protein, or a fragment thereof, in particular selected from a nucleic acid encoding gp!20, gp!40, gp!60, Env, Gag, Pol, Rev, RT, Vpu or Tat.

or
In another preferred embodiment of the above defined medicament, or the above defined pharmaceutical or vaccine composition comprising as active substance a nucleic acid encoding a protein or polypeptide such as defined above, the nucleic acid is naked comprised in a vector, in particular selected from a canarypox viral vector, an adenoviral vector, or a measles viral vector.
The present invention also relates to the use of a protein or a polypeptide as defined above, for the preparation of:
- polyclonal or monoclonal antibodies, or fragments thereof, such as Fab or F(ab)'2
fragments, directed against said protein or polypeptide as defined above,
- scFv polypeptides directed against said protein or polypeptide as defined above,
- aptamers directed against said protein or polypeptide as defined above,
- binding peptides directed against said protein or polypeptide as defined above.
The procedures for the preparation of the above mentioned antibodies or fragments of antibodies, scFv polypeptides, aptamers, or binding peptides, are particularly well known to the man skilled in the art. As regards binding peptides, they can also be prepared according to methods well known to the man skilled in the art, such as ribosome or phage display methods.
The present invention also relates to antibodies or fragments thereof, scFv polypeptides, aptamers, or binding peptides, directed against the above defined proteins or polypeptides involved in the invention, provided that said antibodies or fragments thereof, scFv polypeptides, or aptamers do not bind to proteins or polypeptides different from the above defined proteins or polypeptides involved in the invention.
As intended herein, the above defined antibodies or fragments thereof, scFv polypeptides, aptamers, or binding peptides, bind specifically to the proteins or the polypeptides according to the invention, in other words they are specific ligands for the proteins or the polypeptides according to the invention. In particular, the specificity of these ligands is such that they bind to the proteins or polypeptides according to the invention, but not to the proteins from which said proteins or polypeptides according to the invention are derived by mutation.
The present invention also relates to a method for preparing mutants of a Nef protein,
wherein:
in a first step, the sequence ranging from the amino acid at position 80 to the amino acid at position 150 of the sequence said Nef protein is mutated by deletion, insertion or substitution of at least one amino acid,

in a second step, the immunosuppressive properties of the mutated Nef protein obtained in the first step are checked and mutants lacking immunosuppressive properties are selected.
The mutants obtained according to this method are immunosuppressive-deficient mutants.
In a preferred embodiment of the above defined method for preparing mutants of a Nef protein, in a third step the CD4 and/or MHC-I downregulation functions of the mutated Nef protein obtained in the second step are checked and mutated Nef proteins having substantially preserved CD4 and/or MHC-I downregulation functions with respect to said Nef protein are selected.
In another preferred embodiment of the above defined method for preparing mutants of a Nef protein, the mutated sequence ranges:
from the amino acid at position 80 to the amino acid at position 120, more particularly from the amino acid at position 90 to the amino acid at position 112, of the sequence of a HIV-1 protein, or
from the amino acid at position 104 to the amino acid at position 150 of the sequence of a HIV-2 Nef protein.
In another preferred embodiment of the above defined method for preparing mutants of a Nef protein, the sequence of the Nef protein is mutated by directed mutagenesis of the nucleic acid sequence coding for said Nef protein.
In another preferred embodiment of the above defined method for preparing mutants of a Nef protein, the immunosuppressive properties of the mutated Nef protein are checked according to the general procedure described in Mangeney & Heidmann (1998) Proc. Natl. Acad. Sci. U.S.A. 95:14920-5 and Mangeney et al. (2001) J. Gen. Virol. 82:2515-8 as defined above, in particular the above-defined immunosuppression index is measured and mutated Nef protein having immunosuppression indexes equal to zero or negative are selected.
The down-regulation of CD4 and MHC-I expression by a given protein can be determined by measuring the fluorescence of CD4 or MHC-I expressing cells transformed with increasing amounts of nucleic acids encoding said given protein and contacted with fluorescent anti-CD4 or anti-MHC-I antibodies. Such methods are well known to the man skilled in the art and are in particular described in the following examples.
The present invention also relates to the mutants of a Nef protein liable to be prepared by the above defined method and to pharmaceutical compositions comprising said mutants of a Nef protein in association with a pharmaceutically acceptable carrier.

The present invention also relates to a new protein or polypeptide comprising or being constituted by the immunosuppressive domain of a Nef protein, provided that if present, the sequences adjacent to the respective N-terminal end and/or C-terminal end of the immunosuppressive domain in said protein or polypeptide are different from the sequences adjacent to the respective N-terminal end and/or C-terminal end of the immunosuppressive domain in the Nef protein from which it derives.
More particularly, in an embodiment of the above defined new protein or polypeptide:
• the sequence adjacent to the N-terminal end of the immunosuppressive domain
is different from the sequences adjacent to the N-terminal end of the
immunosuppressive domain in the Nef protein from which it derives, or
• the sequence adjacent to the C-terminal end of the immunosuppressive domain
is different from the sequences adjacent to the C-terminal end of the
immunosuppressive domain in the Nef protein from which it derives, or
• the sequence adjacent to the respective N-terminal and C-terminal ends of the
immunosuppressive domain are different from the sequences adjacent to the
respective N-terminal and C-terminal ends of the immunosuppressive domain
in the Nef protein from which it derives.
In a particular embodiment of the present invention, the new protein or polypeptide as defined above presents CD4 and/or MHC-I down-regulation functions.
The Nef immunosuppressive domain which constitutes or is comprised in the above defined new protein or polypeptide can be either mutated or not with respect to the immunosuppressive domain of naturally occurring Nef proteins.
Thus, the new protein or polypeptide as defined above can be immunosuppressive, in the general case, if the Nef immunosuppressive domain which it comprises or which it is constituted of, derives without mutations from a naturally occuring Nef protein, which generally presents immunosuppressive properties.
The new protein or polypeptide can also comprise or be constituted of a Nef immunosuppressive domain which is mutated with respect to its natural form. This mutation can either be silent as concerns the immunosuppressive properties of the Nef immunosuppressive domain, which means, in the general case, that it does not affect the immunosuppressive properties of the Nef immunosuppressive domain, or the mutation can render the immunosuppressive domain immunosuppressive-deficient, as is the case for the above-mentioned mutations affecting Nef immunosuppressive domain.

Further, in certain particular cases, the immunosuppressive domain can also derive from naturally occuring Nef variants devoid of immunosuppressive properties.
In a preferred embodiment of the above defined new protein or polypeptide, the sequence of the immunosuppressive domain of the Nef protein is comprised in the amino acid sequence extending from the N-terminus of the first a helix to the C-terminus of the second a helix of the Nef protein.
In another preferred embodiment of the above defined new protein or polypeptide, the sequence of the immunosuppressive domain of the Nef protein is comprised in a sequence ranging from the amino acid at position 80 to the amino acid at position 150, particularly from the amino acid at position 81 to the amino acid at position 140, of the sequence of said Nef protein, and in particular:
• in a sequence ranging from the amino acid at position 80 to the amino acid at position
120, more particularly from the amino acid at position 81 to the amino acid at position
118, of the sequence of a HIV-1 Nef protein
• in a sequence ranging from the amino acid at position 104 to the amino acid at
position 150, in particular from the amino acid at position 104 to the amino acid at
position 140, of the sequence of a HIV-2 Nef protein.
More preferably, in the above defined new protein or polypeptide, the sequence of the immunosuppressive domain of the HIV-1 Nef protein is comprised in a sequence ranging from the amino acid at position 90 to the amino acid at position 113, in particular from the amino acid at position 90 to the amino acid at position 112, of the sequence of said Nef protein.
In another preferred embodiment of the above defined new protein or polypeptide, the sequence of the immunosuppressive domain of the Nef protein is comprised in a sequence which is homologous to the amino acid sequence ranging from the amino acid at position 80 to the amino acid at position 120 of SEQ ID NO: 1, in particular from the amino acid at position 81 to the amino acid at position 117 of SEQ ID NO: 1, more particularly from the amino acid at position 90 to the amino acid at position 112 of SEQ ID NO: 1.
In a particularly preferred embodiment of the above defined new protein or polypeptide, the sequence of the immunosuppressive domain of the Nef protein is comprised in a 26 or 27 amino acid-long sequence, the N-terminal end of said Nef protein, the N-terminal end of said 26 or 27 amino acid-long sequence being the pentapeptide AX,DX2S and the C-terminal end of said 26 or 27 amino acid-long sequence being the amino acid L, in

which X, represents any amino acid, and in particular I, V, L, F, or R, and X2 represents any amino acid, and in particular M, L, or F.
In a preferred embodiment of the present invention, the Nef immunosuppressive domain which constitutes or is comprised in the above defined new protein or polypeptide is not mutated, such a domain is said to be non-mutated, and is derived from a naturally immunosuppressive Nef protein. Advantageously, the new protein or polypeptide which comprises or is constituted of such a non-mutated domain is immunosuppressive.
Jn another preferred embodiment, the new protein or polypeptide is constituted of one of the following HIV-1 Nef fragments :
80-120 TYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO- 32)
81-120 YKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO- 33)
82-120 KAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO- 34)
83-120 AAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO: 35)
84-120 AVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO: 36)
85-120 VDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO: 37)
86-120 DLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO: 38)
87-120 LSHFLKEK.GGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO: 39)
88-120 SHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO: 40)
89-120 HFLK.EKGGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO: 41)
90-120 FLKEKGGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO: 42)
80-119 TYKAAVDLSHFLKEKGGLEGL1HSQRRQDILDLWIYHTQG (SEQ ID NO: 43)
81-119 YKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQG (SEQ ID NO: 44)
82-119 KAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQG (SEQ ID NO: 45)
83-119 AAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQG (SEQ ID NO: 46)
84-119 AVDLSHFLKEKGGLEGL1HSQRRQDILDLW1YHTQG (SEQ ID NO: 47)
85-119 VDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQG (SEQ ID NO: 48)
86-119 DLSHFLK.EKGGLEGLIHSQRRQDILDLWIYHTQG (SEQ ID NO: 49)
87-119 LSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQG (SEQ ID NO: 50)
88-119 SHFLKEKGGLEGLIHSQRRQDILDLWIYHTQG (SEQ ID NO: 51)
89-119 HFLKEKGGLEGLIHSQRRQDILDLWIYHTQG (SEQ ID NO: 52)
90-119 FLKEKGGLEGLIHSQRRQDILDLWIYHTQG (SEQ ID NO: 53)
80-118 TYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQ (SEQ ID NO: 54)
81-118 YKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQ (SEQ ID NO: 55)
82-118 KAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQ (SEQ ID NO: 56)
83-118 AAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQ (SEQ ID NO: 57)
84-118 AVDLSHFLKEKGGLEGL1HSQRRQDILDLWIYHTQ (SEQ ID NO: 58)
85-118 VDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQ (SEQ ID NO: 59)
86-118 DLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQ (SEQ ID NO: 60)
87-118 LSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQ (SEQ ID NO: 61)
88-118 SHFLKEK.GGLEGLIHSQRRQDILDLWIYHTQ (SEQ ID NO: 62)
89-118 HFLKEKGGLEGL1HSQRRQDILDLWIYHTQ (SEQ ID NO: 63)
90-118FLKEKGGLEGLIHSQRRQDILDLWIYHTQ(SEQIDNO:64)
80-117 TYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHT (SEQ ID NO: 65)
81-117 YKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHT (SEQ ID NO: 66)
82-117 (CAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHT (SEQ ID NO: 67)
83-117 AAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHT (SEQ ID NO: 68)
84-117 AVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHT (SEQ ID NO: 69)
85-117 VDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHT (SEQ ID NO: 70)
86-117 DLSHFLKEKGGLEGLIHSQRRQDILDLWIYHT (SEQ ID NO: 71)
87-117 LSHFLKEK.GGLEGL1HSQRRQDILDLWIYHT (SEQ ID NO: 72)
88-117 SHFLK.EKGGLEGLIHSQRRQDILDLWIYHT (SEQ ID NO: 73)
89-117 HFLK.EKGGLEGLIHSQRRQDILDLWIYHT (SEQ ID NO: 74)
90-117 FLKEKGGLEGL1HSQRRQDILDLWIYHT(SEQ ID NO: 75)

«?" 81-116 YKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYH (SEQ ID NO- 77)
82-116 KAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYH (SEQ ID NO- 78)
83-116 AAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYH (SEQ ID NO' 79)
84-116 AVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYH (SEQ ID NO- 80)
85-116 VDLSHFLKEKGGLEGLIHSQRRQDILDLWIYH (SEQ ID NO- 81)
86-116 DLSHFLKEKGGLEGLIHSQRRQDILDLWIYH (SEQ ID NO- 82)
87-116 LSHFLKEKGGLEGLIHSQRRQDILDLWIYH (SEQ ID NO' 83)
88-116 SHFLKEKGGLEGLIHSQRRQDILDLWIYH (SEQ ID NO- 84)
89-116 HFLKEKGGLEGLIHSQRRQDILDLWIYH (SEQ ID NO- 85)
90-116 FLKEKGGLEGLIHSQRRQDILDLWIYH (SEQ ID NO' 86)
80-115 TYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIY (SEQ ID NO- 87) 8l-H5YKAAVDLSHFLKEKGGLEGLIHSQRRQDlLDLWIY(SEQIDNO-88)
82-115 KAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIY (SEQ ID NO: 89)
83-115 AAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIY (SEQ ID NO: 90)
84-115 AVDLSHFLKEKGGLEGL1HSQRRQDILDLWIY (SEQ ID NO: 91)
85-115 VDLSHFLKEKGGLEGLIHSQRRQDILDLWIY (SEQ ID NO: 92)
86-115 DLSHFLKEKGGLEGLIHSQRRQDILDLWIY (SEQ ID NO: 93)
87-115 LSHFLKEKGGLEGIJHSQRRQDILDLWIY (SEQ ID NO: 94)
88-115 SHFLKEKGGLEGLIHSQRRQDILDLWIY (SEQ ID NO: 95)
89-115 HFLKEKGGLEGLIHSQRRQDILDLWIY (SEQ ID NO: 96)
90-115 FLKEKGGLEGLIHSQRRQDILDLWIY (SEQ ID NO: 97)
80-114 TYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWI (SEQ ID NO: 98)
81-114 YKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWI (SEQ ID NO: 99)
82-114KAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWI(SEQIDNO: 100)
83-114 AAVDLSHFLKEKGGLEGLIHSQRRQDILDLWI (SEQ ID NO: 101)
84-114 AVDLSHFLKEKGGLEGLIHSQRRQDILDLWI (SEQ ID NO: 102)
85-114 VDLSHFLKEKGGLEGLIHSQRRQDILDLWI (SEQ ID NO: 103)
86-114 DLSHFLKEKGGLEGLIHSQRRQDILDLWI (SEQ ID NO: 104)
87-114 LSHFLKEKGGLEGLIHSQRRQDILDLWI (SEQ ID NO: 105)
88-114 SHFLKEKGGLEGLIHSQRRQDILDLWI (SEQ ID NO: 106)
89-114 HFLKEKGGLEGL1HSQRRQDILDLWI (SEQ ID NO: 107)
90-114 FLKEKGGLEGLIHSQRRQDILDLWI (SEQ ID NO: 108)
80-113 TYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLW (SEQ ID NO: 109)
81-113 YKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLW (SEQ ID NO: 110)
82-113 KAA VDLSHFLKEKGGLEGLIHSQRRQDILDLW (SEQ ID NO: 111)
83-113 AAVDLSHFLKEKGGLEGLIHSQRRQDILDLW (SEQ ID NO: 112)
84-113 AVDLSHFLKEKGGLEGLIHSQRRQDILDLW (SEQ ID NO: 113)
85-113 VDLSHFLKEKGGLEGLIHSQRRQDILDLW (SEQ ID NO: 114)
86-113 DLSHFLKEKGGLEGLIHSQRRQDILDLW (SEQ ID NO: 115)
87-113 LSHFLKEKGGLEGLIHSQRRQDILDLW (SEQ ID NO: 116)
88-113 SHFLKEKGGLEGLIHSQRRQDILDLW (SEQ ID NO: 117)
89-113 HFLKEKGGLEGLIHSQRRQDILDLW (SEQ ID NO: 118)
90-113 FLKEKGGLEGLIHSQRRQDILDLW (SEQ ID NO: 119)
80-112 TYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDL (SEQ ID NO: 120)
81-112 YKAAVDLSHFLKEKGGLEGLIHSQRRQDILDL (SEQ ID NO: 121)
82-112 KAAVDLSHFLKEKGGLEGLIHSQRRQDILDL (SEQ ID NO: 122)
83-112 AAVDLSHFLKEKGGLEGLIHSQRRQDILDL (SEQ ID NO: 123)
84-112 AVDLSHFLKEKGGLEGL1HSQRRQDILDL (SEQ ID NO: 124)
85-112 VDLSHFLKEKGGLEGLIHSQRRQDILDL (SEQ ID NO: 125)
86-112 DLSHFLKEKGGLEGLIHSQRRQDILDL (SEQ ID NO: 126)
87-112 LSHFLKEKGGLEGLIHSQRRQD1LDL (SEQ ID NO: 127)
88-112 SHFLKEKGGLEGLIHSQRRQDILDL (SEQ ID NO: 128)
89-112 HFLKEKGGLEGLIHSQRRQDILDL (SEQ ID NO: 129)
90-112 FLKEKGGLEGLIHSQRRQDILDL (SEQ ID NO: 130)
or of homologous peptide sequences presenting at least 80% sequence identity, preferably
90% identity, with said HIV-1 Nef fragments.

The present invention also relates to new proteins or polypeptides comprising said UlV-i Nef fragments or homologous peptide sequences, provided that if present, the sequences adjacent to the respective N-terminal end and/or C-terminal end of the HIV-1 Nef fragments in said protein or polypeptide are different from the sequences adjacent to the respective N-terminal end and/or C-terminal end of the HIV-1 Nef fragments in the Nef proteins from which they derive.
In another preferred embodiment, the new protein or polypeptide is constituted of one of the following HIV-2 Nef fragments:
104-150 RVPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLD1YLEKEEG (SEQ ID NO- 131)
105-150 VPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO- 132)
106-150 PLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO- 133)
107-150 LREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO- 134)
108-150 REMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 135)
109-150 EMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 136)
110-150 MTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 137)
111-150 TYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 138)
112-150 YRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 139)
113-150RLARDMSHLIKEKGGLEGLYYSDRRRRVLD1YLEKEEG(SEQIDNO: 140)
114-150LARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK.EEG(SEQIDNO: 141)
115-150 ARDMSHLIK.EKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 142)
116-150 RDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 143)
117-150 DMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 144)
118-150 MSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 145)
119-150 SHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 146)
120-150 HLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 147)
121-150 LIKEKGGLEGLYYSDRRRRVLDIYLEKEEG (SEQ ID NO: 148)
104-149 RVPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 149)
105-149 VPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 150)
106-149 PLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 151)
107-149 LREMTYRLARDMSHL1KEKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 152)
108-149 REMTYRLARDMSHLIKEK.GGLEGLYYSDRRRRVLDIYLEK.EE (SEQ ID NO: 153)
109-149 EMTYRLARDMSHLIK.EKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 154)
110-149 MTYRLARDMSHL1K.EKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 155)
111-149 TYRLARDMSHLIK.EKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 156)
112-149 YRLARDMSHLIKEK.GGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 157)
113-149 RLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 158)
114-149 LARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 159)
115-149 ARDMSHL1KEKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 160)
116-149 RDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK.EE (SEQ ID NO: 161)
117-149 DMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 162)
118-149 MSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 163)
119-149 SHL1KEK.GGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 164)
120-149 HLIKEKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 165)
121-149 LIKEKGGLEGLYYSDRRRRVLDIYLEKEE (SEQ ID NO: 166)
104-148 RVPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO: 167)
105-148 VPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO: 168)
106-148 PLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO: 169)
107-148 LREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO: 170)
108-148 REMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE(SEQ ID NO: 171)
109-148 EMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO: 172)
110-148 MTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO: 173)
111-148 TYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO: 174)
112-148 YR1.ARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO: 175)
113-148 RLARDMSHLIKI'KGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO: 176)

114-148 LARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO- 177)
115-148 ARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO- 178)
116-148 RDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO- 179)
117-148 DMSHLIKEK.GGLKGLYYSDRRRRVLDIYLEKE (SEQ ID NO- 180)
118-148MSHLIKEKGGLEGLYYSDRRRRVLDIYLEKE(SEQIDNO- 181)
119-148 SHLIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO' 182)
120-148 HL1KEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO- 183)
121-148 LIKEKGGLEGLYYSDRRRRVLDIYLEKE (SEQ ID NO- 184)
104-147 RVPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO- 185)
105-147 VPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO- 186)
106-147 PLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO- 187)
107-147 LREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO- 188)
108-147 REMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO- 189)
109-147 EMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 190)
110-147 MTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 191)
111-147 TYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 192)
112-147 YRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 193)
113-147 RLARDMSHLIK.EK.GGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 194)
114-147 LARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 195)
115-147 ARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 196)
116-147 RDMSHUKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 197)
117-147 DMSHLIKEKGGLEGLYYSDRRRRVLDIYLEK(SEQ ID NO: 198)
118-147 MSHLIK.EKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 199)
119-147 SHLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 200)
120-147 HLIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 201)
121-147 LIKEKGGLEGLYYSDRRRRVLDIYLEK (SEQ ID NO: 202)
104-146 RVPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 203)
105-146 VPLREMTYRLARDMSHUKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 204)
106-146 PLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 205)
107-146 LREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 206)
108-146 REMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 207)
109-146 EMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 208)
110-146 MTYRLARDMSHLIKEK.GGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 209)
111-146 TYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 210)
112-146 YRLARDMSHL1KEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 211)
113-146 RLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 212)
114-146 LARDMSHLIKEK.GGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 213)
115-146 ARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 214)
116-146 RDMSHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 215)
117-146 DMSHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 216)
118-146 MSHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 217)
119-146 SHLIKEKGGLEGLYYSDRRRRVLDIYLE (SEQ ID NO: 218)
120-146 HLIK.EK.GGLEGLYYSDRRRRVLD1YLE (SEQ ID NO: 219)
121-146 L1KEKGGLEGLYYSDRRRRVLDIYLE(SEQ ID NO: 220)
104-145 RVPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 221)
105-145 VPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 222)
106-145 PLREMTYRLARDMSHL1KEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 223)
107-145 LREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 224)
108-145 REMTYRLARDMSHL1KEK.GGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 225)
109-145 EMTYRLARDMSHLIKEK.GGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 226)
110-145 MTYRLARDMSHUKEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 227)
111-145 TYRLARDMSHL1KEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 228)
112-145 YRLARDMSHL1KEK.GGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 229)
113-145 RLARDMSHL1KEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 230)
114-145 LARDMSHLIKEK.GGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 231)
115-145 ARDMSHLIKEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 232)
116-145 RDMSHLIKEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 233)
117-145 DMSHLIKEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 234)
118-145 MSHLIKEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO: 235)
119-145 SHL1KEK.GGLEGLYYSDRRRRVLD1YL (SEQ ID NO: 236)

120-145 HLIKEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO- 237)
121-145 LIKEKGGLEGLYYSDRRRRVLDIYL (SEQ ID NO- 238)
104-144 RVPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO- 239)
105-144 VPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO- 240)
106-144 PLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO- 241)
107-144 LREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO- 242)
108-144 REMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO" 243)
109-144 EMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO' 244)
110-144 MTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO' 245)
111-144 TYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO- 246)
112-144 YRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO: 247)
113-144 RLARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO: 248) *
114-144 LARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO: 249)
115-144 ARDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO: 250)
116-144 RDMSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO: 251)
117-144 DMSHLIKEK.GGLEGLYYSDRRRRVLDIY (SEQ ID NO: 252)
118-144 MSHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO: 253)
119-144 SHLIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO: 254)
120-144 HL1KEKGGLEGLYYSDRRRRVLD1Y (SEQ ID NO: 255)
121-144 LIKEKGGLEGLYYSDRRRRVLDIY (SEQ ID NO: 256)
104-143 RVPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 257)
105-143 VPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 258)
106-143 PLREMTYRLARDMSHLIK.EKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 259)
107-143 LREMTYRLARDMSHL1KEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 260)
108-143 REMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 261)
109-143 EMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 262)
110-143 MTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 263)
111-143 TYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 264)
112-143 YRLARDMSHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 265)
113-143 RLARDMSHLIK.EKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 266)
114-143 LARDMSHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 267)
115-143 ARDMSHLIKEK.GGLEGLYYSDRRRRVLDI (SEQ ID NO: 268)
116-143 RDMSHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 269)
117-143 DMSHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 270)
118-143 MSHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 271)
119-143 SHLIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 272)
120-143 HL1KEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 273)
121-143 LIKEKGGLEGLYYSDRRRRVLDI (SEQ ID NO: 274)
or of homologous peptide sequences presenting at least 80% sequence identity, preferably
90% identity, with said H1V-2 Nef fragments.
The present invention also relates to new proteins or polypeptides comprising said HIV-2 Nef fragments or homologous peptide sequences, provided that if present, the sequences adjacent to the respective N-terminal end and/or C-terminal end of the HIV-2 Nef fragments in said protein or polypeptide are different from the sequences adjacent to the respective N-terminal end and/or C-terminal end of the HIV-2 Nef fragments in the Nef proteins from which they derive.
In another particularly preferred embodiment of the above defined new protein or polypeptide, the immunosuppressive domain is mutated by deletion, substitution and/or insertion of at least one amino acid, and in particular the amino acid homologous to the amino acid at position 93 of SEQ ID NO: 1 is replaced by any amino acid different from E, in

particular by W, F, M, Y, R, H or K, more particularly by R, H, or K, and preferably by R. Advantageously, such a new protein or polypeptide is devoid of immunosuppressive activity.
In yet another particularly preferred embodiment of the above defined new protein or polypeptide, the immunosuppressive domain of a HIV-1 Nef protein is mutated by the substitution of the amino acid at position 93 of the sequence of said HIV-1 Nef protein by any amino acid different from E, in particular by W, F, M, Y, R, H or K, more particularly by R, H, or K, and preferably by R.
The present invention also relates to a nucleic acid, characterized in that it codes for a new protein or polypeptide as defined above.
The present invention also relates to peptidomimetics of the new proteins or polypeptides as defined above.
The present invention also relates to antibodies or fragments thereof, scFv polypeptides, aptamers, or binding peptides, directed against a sequence ranging from the amino acid at position 80 to the amino acid at position 150 of a Nef protein, and in particular:
• against a sequence ranging from the amino acid at position 80 to the amino acid at
position 120, more particularly from the amino acid at position 90 to the amino acid at
position 120, of the sequence of a HIV-1 Nef protein
• against a sequence ranging from the amino acid at position 104 to the amino acid at
position 150 of the sequence of a HIV-2 Nef protein.
The present invention also relates to antibodies or fragments thereof, scFv polypeptides, aptamers, or binding peptides, directed against the new proteins or polypeptides as defined above, provided that said antibodies or fragments thereof, scFv polypeptides, or aptamers do not bind to Nef proteins wherein the immunosuppressive domains corresponds to that of said new proteins or polypeptides as defined above.
As intended herein the above defined antibodies or fragments thereof, scFv polypeptides, aptamers, or binding peptides, are specific for the new proteins or polypeptides according to the invention (i.e. proteins or polypeptides comprising the immunosuppressive domain of the Nef protein according to the invention). In particular, these antibodies or fragments thereof, scFv polypeptides, or aptamers do not bind to the immunosuppressive domain of the Nef protein in its natural setting, that is, these antibodies or fragments thereof, scFv polypeptides, or aptamers do not bind to natural Nef proteins.
The present invention also relates to a pharmaceutical or vaccine composition, comprising as active substance an above defined new protein or polypeptide, or an above

defined nucleic acid encoding said new protein or polypeptide, in association with a pharmaceutically acceptable carrier.
The present invention also relates to the use of a protein or a polypeptide comprising or being constituted of a Nef protein or fragments thereof, wherein said protein or polypeptide presents an immunosuppressive activity, for the manufacture of a medicament intended for the prevention or the treatment of pathologies requiring an inhibition of the immune system, such as allergies, autoimmune diseases or graft rejections.
In an advantageous embodiment, the invention relates to the above mentioned use of a new protein or polypeptide as defined above, wherein said new protein or polypeptide presents an immunosuppressive property, for the manufacture of a medicament intended for the prevention or the treatment of pathologies requiring an inhibition of the immune system, such as allergies, autoimmune diseases or graft rejections.
The present invention also relates to the use of a protein or a polypeptide comprising or being constituted of a Nef protein or fragments thereof, for screening compounds liable to inhibit the immunosuppressive activity of Nef proteins.
The present invention also relates to the use of a new protein or polypeptide as defined above, for screening compounds liable to inhibit the immunosuppressive activity of Nef proteins.
Advantageously, such compounds which are liable to inhibit the immunosuppressive activity of Nef proteins, can be used as anti-viral agents.
As intended herein the compounds to screen can be of any chemical nature. In particular, the compounds to screen are included in chemical compound libraries.
In a preferred embodiment of the above defined use of a new protein or polypeptide as defined above, the sequence of the immunosuppressive domain of the Nef protein corresponds to a non-mutated sequence.
The present invention also relates to compounds liable to inhibit the immunosuppressive activity of Nef proteins. Such compounds may be useful for the manufacture of pharmaceutical compositions, in particular intended for the prevention or the treatment of viral diseases, such as HIV or SIV infections.
The immunosuppressive-inhibitory activity of these compounds can be determined by measuring the immunosuppression index of a given Nef protein in the absence or in the presence of the compounds. A compound will be said to possess an immunosuppressive-inhibitory activity when the immunosuppression index of a given Nef protein in the presence

of said compound is decreased with respect to the the immunosuppression index of the same Nef protein in the absence of said compound.
The present invention also relates to the use of ligands of the immunosuppressive domain of Nef proteins, such as antibodies or fragments thereof, scFv poiypeptides, aptamers, or binding peptides, to screen for compounds liable to inhibit the immunosuppressive activity of Nef proteins.
The present invention also relates to a method to screen for compounds liable to inhibit the immunosuppressive activity of Nef proteins, comprising the following steps:
- contacting a Nef protein, or a fragment thereof comprising the immunosuppressive domain
of a Nef protein, or a new protein or polypeptide as defined above, with compounds to screen,
- selecting the compounds which bind to the immunosuppressive domain of the Nef protein,
or of the fragment thereof comprising the immunosuppressive domain of said Nef protein, or
of the new protein or polypeptide as defined above,
- optionally checking that the selected compounds inhibit the immunosuppressive activity of
Nef proteins.
In a preferred embodiment of the invention, the above mentioned screening method comprises the following steps:
- contacting a Nef protein, or a fragment thereof comprising the immunosuppressive domain
of a Nef protein, or a new protein or polypeptide as defined above, with a compounds to
screen and with a ligand of the immunosuppressive domain, such as an antibody, a scFv
polypeptide, an aptamer, or a binding peptide,
- selecting the compounds which prevent the binding of the ligand to the Nef protein, or to the
Nef fragment, or to a new protein or polypeptide as defined above and which do not bind to
said ligand,
- optionally checking that the selected compounds inhibit the immunosuppressive activity of
Nef proteins.
The present invention also relates to a screening method for compounds liable to inhibit the immunosuppressive activity of Nef proteins, wherein compounds which bind to a Nef protein or a fragment thereof are selected and it is checked that said selected compounds inhibit the immunosuppressive activity of said Nef protein.
The present invention also relates to the compounds which are selected according to the above defined screening method of the invention.

DESCRIPTION OF THE
Figure 1
Figure 1 represents the immunosuppression index (vertical axis) of wild type Nef (white column) and of its E93R mutant (grey column).
Figure 2
Figure 2 represents the downregulation of CD4 expression (vertical axis, arbitrary units) by HeLa cells transformed with the indicated amount (horizontal axis, in ug) of wild type Nef expressing vectors (black circles, plain lines) or E93R Nef mutant expressing vectors (white circles, dotted lines).
Figure 3
Figure 3 represents the downregulation of MHC-1 expression (vertical axis, arbitrary units, left for Nef, right for the E93R Nef mutant) by 293T cells transformed with the indicated amount (horizontal axis, in ug) of wild type Nef expressing vectors (black circles, plain lines) or E93R Nef mutant expressing vectors (white circles, dotted lines).
Figure 4
Figure 4 represents a sequence alignment generated by the Clustal W software of Nef amino-acid sequences from independent HIV-1, HIV-2 and SIV isolates. The part of the sequences of the Nef proteins comprising the immunosuppressive domain is boxed. The amino acids corresponding or homologous to E93 of SEQ ID NO: 1 (NEF_HIVB1) are in bold. The stars represent positions for which amino acids are conserved; single points, positions for which amino acids are substantially conserved; and double points, positions for which amino acids possess similar physicochemical properties.
Figure 5
Figure 5 represents the immunosuppression index of HIV-1 strain LAI Nef and of three of its fragments (1-89, 80-120 and 113-206). The partial sequence of HIV-1 strain LAI is presented on top of the figure with the position of several amino acids as well as the positions of the fragments. The presence (+) or absence (-) of an immunosuppressive activity for wild type Nef and for each fragment is indicated on the right (immunosuppression index).

Figure 6A and Figure 6R
Figure 6A represents the immunosuppression index (vertical axis) of HIV-1 strain Al Nef (left column) and for HIV-2 strain ST Nef (right column).
Figure 6B represents the immunosuppression index (vertical axis) of SIV strain mac239 Nef (left column) and of the corresponding E125R mutant (right column).
EXAMPLES
EXAMPLE 1
Cloning of the genes encoding wild type Nef and the E93R Nef mutant
H1V-1 strain LAI Nef was retrieved from pCDNA3-Nef (Peden K., Emerman M. and Montagnier L. 1991, Virology 185(2):661-672) (gift from O. Schwartz, Institut Pasteur, France) by PCR with high-fidelity Pfx Platinum polymerase (Invitrogen) and the following primers:
5'-ATACATGGCCCAGCCGGCCGGTGGCAAGTGGTCAAAAAGTAGT-3' (SEQ ID NO: 4) and 5'-ATACATGGATCCACGCGTTCAGCAGTTCTTGAAGTACTCCGG-3' (SEQ ID NO: 5).
The amplification product was digested with Sfi] and Bam\M and ligated in the pSecTag2A vector (Invitrogen) opened with the same enzymes. Nef preceded with the export signal sequence of the vector was then amplified with the following primers: 5'-ATACATACCGGTATGGAGACAGACACACTCCTGCTATG-3' (SEQ ID NO: 6), and 5'-ATACATGGATCCACGCGTTCAGCAGTTCTTGAAGTACTCCGG-3' (SEQ ID NO: 7).
The product was digested with Age\ and Mlul and ligated into the retroviral vector pDFG-MoTMtag (Mangeney & Heidmann (1998) Proc. Natl. Acad. Sci. U.S.A. 95:14920-5) digested with the same enzymes to obtain pDFG-expNef (SEQ ID NO: 16), expressing the exported version of HIV-Nef (SEQ ID NO: 17).
The mutation E93R was then introduced in pDFG-expNef by ligation of the three following fragments to yield pDFG-expNefE93R (SEQ ID NO: 18), expressing the exported version of F.93R Nef (SEQ ID NO: 19):
1) the Age\-Mlu\ fragment of the vector;
2) a PCR product obtained with primers
5'-ATACATACCGGTATGGAGACAGACACACTC-3' (SEQ ID NO: 8) and

5'-ATACATCTTAAGAAAGTGGCTAAGATCTACAGCTGCC-3' (SEQ ID NO: 9)
and digested with A/Ill; 3) a PCR product obtained with primers
5'-ATACATCTTAAGCGAAAGGGGGGACTGGAAGGG-3' (SEQ ID NO: 10) and
5f-ATACATACGCGTTCAGCAGTTCTTGAA-3' (SEQ ID NO: 11) digested with Afl\\ and Mlu\.
Nef and its mutant E93R were then retrieved from the pDFG-expNef vectors with the following primers:
5'-ATACATGTCGACCCAACTAGAACCATGGGTGGCAAGTGGTCAAAAAGTAG-3'(SEQ
ID NO: 12), and
5'-ATACATACGCGTTCAGCAGTTCTTGAA-3' (SEQ ID NO: 13).
The product was digested with Sail and MM and ligated into phCMV-envT (Blaise et al. (2003) Proc. Natl Acad. Sci. 100:13013-8) digested with Xho\ and Mul, to yield respectively phCMV-Nef (SEQ ID NO: 20), expressing Nef (SEQ ID NO: 1), and phCMV-NefE93R (SEQ ID NO: 21), expressing E93R Nef (SEQ ID NO: 2).
Similarly, the sequences coding for Nef and the E93R Nef mutant with the export signal sequence were respectively extracted from pDFG-expNef and pDFG-expNefE93R and inserted into phCMV to yield phCMV-expNef (SEQ ID NO: 14) and phCMV-expNefE93R (SEQ ID NO: 15).
EXAMPLE 2
Determination of the immunosuppression index of wild type Nef and of the E93R Nef
mutant
The immunosuppression index of Nef and of its E93R mutant were measured following the general procedure described in Mangeney & Heidmann (1998) Proc. Natl. Acad Sci. U.S.A. 95:14920-5 and Mangeney et al. (2001) J. Gen.Virol. 82:2515-8.
Briefly, MCA205 cells were stably transformed by plasmids pDFG-expNef and pDFG-expNefE93R, or optionally by plasmids phCMV-expNef and phCMV-expNefE93R, respectively. 106 MCA cells expressing either wild type Nef, the E93R Nef mutant or no exogenous protein were then injected into Balb/c mice and tumor areas were measured every other day. After 7 to 8 days the immunosuppression index was determined.

The immunosuppression index of a protein was calculated as (A -A VA
i — ^ \, 'protein ' ^none/'^none'
where Aprotein and Anone are the peak tumor areas obtained with MCA cells expressing the proteins of interest (i.e. Nef or the E93R Nef mutant) and no exogenous protein, respectively. The results are presented in Figure 1. As can be seen, the immunosuppression index of Nef is approximately 0.6, which indicates that the size of the Nef expressing tumors is 1.6 times bigger than normal tumors, thus demonstrating that Nef is an immunosuppressive protein which inhibits the anti-tumor immune response. In contrast, the immunosuppressive index of the E93R Nef mutant is negative, thus demonstrating that this mutant has no immunosuppressive activity, and that tumors expressing this Nef mutant are more easily recognized and eliminated by the immune system than normal tumors.
EXAMPLE 3
Down-regulation of CD4 expression by Nef and its E93R mutant
HeLa cells were cotransfected with 1 |ig of CMV-CD4 (Janvier et al. (2001) J. Virol. 75:3971-6) and the indicated amount of phCMV-Nef or phCMV-NefE93R. CD4 expression was then measured by FACS using a PC5-coupled anti-human CD4 antibody (IM2636, Immunotech). The results presented in Figure 2 indicate that wild type Nef and the E93R Nef mutant downregulate CD4 expression to a similar extent. This implies that the structure of the E93R Nef mutant is unchanged with respect to that of wild type Nef.
EXAMPLE 4
Down-regulation of MHC-I expression by Nef and its E93R mutant
293T cells were cotransfected with 1 ng of CMV-HLA A2 (Le Gall et al. (2000) J. Virol. 74:9256-66) and the indicated amount of phCMV-Nef or phCMV-NefE93R. MHC-l expression was measured by FACS with PE-coupled anti human MHC-I antibody W6/32 (eBioscience).
The results presented in Figure 3 indicate that wild type Nef and the E93R Nef mutant downregulate MHC-I expression to a similar extent. This also implies that the structure of the E93R Nef mutant is unchanged with respect to that of wild type Nef.
EXAMPLE 5
Determination of the localization of the immunosuppressive domain of wild type Nef

Based on the three-dimensional structure of Nef, three fragments of the Nef protein of HIV-1 strain LAI have been designed in order to determine the localization of the immunosuppressive domain of Nef:
1) a fragment extending from residue number 1 to residue number 89
2) a fragment extending from residue number 80 to residue number 120
3) a fragment extending from residue number 113 to residue number 206
Fragment 2 comprises the putative immunosuppressive domain, while fragments number 1 and 3 do not comprise this domain. Fragment 2 extents both ways from the putative immunosuppressive domain of Nef in order to include the whole two alpha-helical domains containing the putative immunosuppressive domain of Nef, according to the known core structure of the HIV-1 Nef protein (PBD entry 1EFN).
The DNAs coding for those fragments were generated by PCR using the Nef gene cloned into the pCDNA3 vector (Peden K., Emerman M. and Montagnier L. 1991, Virology 185(2):661-672) as a template and the following primers pairs:
• for fragment 1:
atacatggcccagccggccggtggcaagtggtcaaaaagtagt (SEQ ID NO: 22) atacatacgcgttcagtggctaagatctacagctgcctt (SEQ ID NO: 23)
• for fragment 2:
- atacatggcccagccggccacttacaaggcagctgtagatcttagc (SEQ ID NO:
24)
- atacatacgtcgttcagccttgtgtgtggtagatccac (SEQ ID NO: 25)
• for fragment 3:
- atacatggcccagccggccgatatccttgatctgtggatctaccac (SEQ ID NO:
26)
- atacataacgcgttcagcagttcttgaagtactccgg (SEQ ID NO: 27)
The PCR products were digested with Sfil and MM and cloned into pDFG-expNef opened with the same enzymes, resulting in the genetic fusion of the fragments with the

extracellular exportation signal peptide of the human IgK light chain. Thus, the obtained constructs expressed extracellularly localized fragments of HIV-1 Nef. They were used in an in vivo immunosuppression assay as described in Example 2. A positive index (+) indicates that the considered fragment has in vivo immunosuppressive properties, whereas an index inferior or equal to zero (-) indicates that the considered fragment is devoid of such properties.
As illustrated in Figure 5, the fragment extending from residue 90 to residue 120 of HIV-1 Nef protein displays an immunosuppressive property in vivo. This fragment thus comprises the immunosuppressive domain of Nef. Fragments 1 and 3 indicate that this immunosuppressive domain could be further reduced to amino acids 90 to 112.
EXAMPLE 6
Determination of the immunosuppression index of additional wild type Nef proteins
The immunosuppression index of Nef was determined as described in Example 2 for HIV-1 strain Al Nef (SEQ ID NO: 28) and for HIV-2 strain ST Nef (SEQ ID NO: 29).
As expected, the results indicate that these Nef proteins are also immunosuppressive (Figure 6A).
EXAMPLE 7
Determination of the immunosuppression index of a SIV Nef and of its E125R mutant
The immunosuppression index was determined for the SIV strain mac239 wild type Nef (SEQ ID NO: 30) and its E125R mutant (SEQ ID NO: 31) as described in Example 2. The E125R mutation in SIV mac239 Nef is homologous to the above defined E93R mutation of HIV-1 LAI and was introduced following a procedure similar to that described in Example i
The results indicate that the SIV Nef protein possesses an immunosuppressive activity while the E-R mutant is completely devoid of such an activity (Figure 6B).








We claim:
1. A mutated Nef protein or fragment thereof which is completely devoid
of immunosuppression activity in comparison to the NEF protein and
having ability to down regulate the CD4 and MHC1 proteins similar to
that of the natural NEF protein wherein :
In the mutated protein or fragment thereof, glutamic acid (E) at position 93 is substituted with arginine (R) in the immunosuppressive domain comprising amino acids from position 80-120 as represented by sequence given below TYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGY(SEQ ID NO: 32) in the NEF protein produced by HIV1 and at homologous positions in the NEF protein produced by HIV2 and SIV .
2. The mutated NEF protein as claimed in claim I wherein the mutation
involves substitution of glutamic acid (E) at the homologous position
125 with arginine (R) in the natural NEF protein produced by SIV.
3. An immunosuppressive protein domain corresponding to the amino
acid sequence of the mutated NEF protein as claimed in claim 1 ,
produced by HIV-1 from position 80-120 as represented by the
sequence ID NO: 32 given below
TYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGY (SEQ ID NO: 32) or homologous position in NEF protein produced by HIV-2 corresponding to the amino acid sequence from position 104-150 as represented by the sequence SEQ ID NO: 131 given below RVPLREMTYRLARDMSHLIKEKGGLEGLYYSDRRRRVLDIYLEKEEG(SEQ ID NO: 131)
4. A nucleic acid coding for a protein as claimed in claim 1 .


Documents:

723-delnp-2007-Abstract-(24-11-2010).pdf

723-delnp-2007-abstract.pdf

723-DELNP-2007-Claims-(15-11-2010).pdf

723-delnp-2007-Claims-(24-11-2010).pdf

723-delnp-2007-claims.pdf

723-DELNP-2007-Correspondence-Others (20-01-2010).pdf

723-DELNP-2007-Correspondence-Others-(15-07-2010).pdf

723-DELNP-2007-Correspondence-Others-(15-11-2010).pdf

723-DELNP-2007-Correspondence-Others-(18-10-2010).pdf

723-DELNP-2007-Correspondence-Others-(22-07-2010).pdf

723-delnp-2007-Correspondence-Others-(24-11-2010).pdf

723-DELNP-2007-Correspondence-Others.pdf

723-delnp-2007-description (complete).pdf

723-delnp-2007-drawings.pdf

723-delnp-2007-Form-1-(24-11-2010).pdf

723-delnp-2007-form-1.pdf

723-DELNP-2007-Form-13-(18-10-2010).pdf

723-delnp-2007-form-18.pdf

723-delnp-2007-Form-2-(24-11-2010).pdf

723-delnp-2007-form-2.pdf

723-delnp-2007-form-26.pdf

723-delnp-2007-form-3.pdf

723-delnp-2007-form-5.pdf

723-DELNP-2007-GPA-(15-07-2010).pdf

723-DELNP-2007-GPA-(18-10-2010).pdf

723-DELNP-2007-GPA-(22-07-2010).pdf

723-delnp-2007-pct-210.pdf

723-delnp-2007-pct-237.pdf


Patent Number 245377
Indian Patent Application Number 723/DELNP/2007
PG Journal Number 03/2011
Publication Date 21-Jan-2011
Grant Date 17-Jan-2011
Date of Filing 29-Jan-2007
Name of Patentee INSTITUT GUSTAVE ROUSSY
Applicant Address 39 RUE CAMILLE DESMOULINS F-94805, VILLEJUIF CEDEX FRANCE.
Inventors:
# Inventor's Name Inventor's Address
1 RENARD, MARTIAL 14 RUE DES MEUNIERS F-75012 PARIS FRANCE.
2 MANGENEY, MARIANNE 4 RUE PAUL FORT F-75014 PARIS FRANCE.
3 HEIDMANN, THIERRY 11 RUE EDOUARD DETAILLE F-75017 PARIS FRANCE.
PCT International Classification Number A61K 39/21
PCT International Application Number PCT/EPR2005/008907
PCT International Filing date 2005-08-17
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA