Patent Grant
8795685
The present invention relates to the use of the immunosuppressive function of an accessory protein of the human or simian immunodeficiency virus for the preparation of a vaccine. In particular, the present invention relates to vaccine compositions comprising a Nef protein.
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.
Nef (negative regulatory factor), is a 27 to 35 kDa regulatory protein of HIV or SIV. 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 (Ali 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 et al. (1998) Immunity 8:483-495).
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.
Thus, an object of the present invention is to relate immunosuppressive properties of HIV or SIV to the Nef protein.
Another object of the invention relates to the identification of an immunosuppressive domain in the Nef protein.
A further object of the invention is to provide pharmaceutical or vaccine compositions comprising a modified Nef protein.
The present invention relates to the use, in particular to the in vitro or to the ex vivo 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.
In vivo, the Nef protein is in particular found in HIV (such as HIV-1 or HIV-2) infected individuals or in SIV infected apes.
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. That is, 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−Anone)/Anone]. 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 HIV 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, for 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-HIV 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-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 a process for cancelling the immunosuppressive property of a Nef protein, comprising:
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
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 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
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 α helix to the C-terminus of the second α 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 et al. (1997) Protein Science 6:1248-63. The nomenclature of the secondary structure elements of the Nef protein, and in particular of its α helices, is based on the structural description of the core domain of the Nef protein, according to Arold et 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:
Nef protein sequences can be easily accessed by the man skilled in the art. By way of example, several HIV-1, HIV-2 or SIV Nef protein sequences are presented in
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,
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 AX1DX2S and the C-terminal end of said 26 or 27 amino acid-long sequence being the amino acid L, in which X1 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
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:
wherein
X1 represents I, L, V, F, or R,
X2 represents M, L, or F,
X3 represents H, D, or F,
X4 represents F or L,
X5 represents I or L,
X6 represents any amino acid different from E, in particular R,
X7 represents K, Q, or R,
X8 represents G or no amino acid,
X9 represents E, D, or R.
SEQ ID NO: 3 comprises the immunosuppressive domain of Nef.
Examples of such sequences are presented in
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 Clustal 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
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
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 conformational 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 imnmunosuppressive 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 SEQ ID NO: 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, gp120, gp140, gp160, 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 gp120, gp140, gp160, Env, Gag, Pol, Rev, RT, Vpu or Tat.
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 or 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:
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:
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:
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:
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 occurring 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 α helix to the C-terminus of the second α 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:
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 AX1DX2S and the C-terminal end of said 26 or 27 amino acid-long sequence being the amino acid L, in which X1 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.
In another preferred embodiment, the new protein or polypeptide is constituted of one of the following HIV-1 Nef fragments:
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 HIV-1 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:
or of homologous peptide sequences presenting at least 80% sequence identity, preferably 90% identity, with said HIV-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 anino 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:
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 polypeptides, 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:
In a preferred embodiment of the invention, the above mentioned screening method comprises the following steps:
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.
HIV-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:
The amplification product was digested with SfiI and BamHI 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:
The product was digested with AgeI and MluI 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 E93R Nef (SEQ ID NO: 19):
Nef and its mutant E93R were then retrieved from the pDFG-expNef vectors with the following primers:
The product was digested with SalI and MluI and ligated into phCMV-envT (Blaise et al. (2003) Proc. Natl. Acad. Sci. 100:13013-8) digested with XhoI and MluI, 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).
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 (Aprotein−Anone)/Anone, 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
HeLa cells were cotransfected with 1 μg 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 (IM636, Immunotech). The results presented in
293T cells were cotransfected with 1 μg 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-I expression was measured by FACS with PE-coupled anti human MHC-I antibody W6/32 (eBioscience).
The results presented in
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 IEFN).
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:
The PCR products were digested with SfiI and MluI 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 Igκ 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
The immunosuppression index of Nef was determined as described in Example 2 for HIV-1 strain A1 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 (
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 1.
The results indicate that the SIV Nef protein possesses an immunosuppressive activity while the E→R mutant is completely devoid of such an activity (
| Number | Date | Country | Kind |
|---|---|---|---|
| 04292056 | Aug 2004 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2005/008907 | 8/17/2005 | WO | 00 | 10/1/2007 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2006/018289 | 2/23/2006 | WO | A |
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| Number | Date | Country | |
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| 20080220008 A1 | Sep 2008 | US |
