WO2013083799A1 - Mutated lentiviral env proteins and their use as drugs - Google Patents

Mutated lentiviral env proteins and their use as drugs Download PDF

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Publication number
WO2013083799A1
WO2013083799A1 PCT/EP2012/074835 EP2012074835W WO2013083799A1 WO 2013083799 A1 WO2013083799 A1 WO 2013083799A1 EP 2012074835 W EP2012074835 W EP 2012074835W WO 2013083799 A1 WO2013083799 A1 WO 2013083799A1
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Prior art keywords
seq
mutated
env protein
protein
fragment
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PCT/EP2012/074835
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French (fr)
Inventor
Thierry Heidmann
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Viroxis S.A.S.
Centre National De La Recherche Scientifique
Universite Paris-Sud Xi
Institut Gustave Roussy
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Priority to BR112014013704-8A priority Critical patent/BR112014013704B1/en
Priority to JP2014545294A priority patent/JP6415324B2/en
Application filed by Viroxis S.A.S., Centre National De La Recherche Scientifique, Universite Paris-Sud Xi, Institut Gustave Roussy filed Critical Viroxis S.A.S.
Priority to RU2014127521A priority patent/RU2654673C2/en
Priority to AU2012350258A priority patent/AU2012350258B2/en
Priority to MX2014006744A priority patent/MX350689B/en
Priority to CN201280060076.2A priority patent/CN104169295B/en
Priority to US14/363,095 priority patent/US9682137B2/en
Priority to DK12806388.0T priority patent/DK2788372T3/en
Priority to IN4927CHN2014 priority patent/IN2014CN04927A/en
Priority to CA2856180A priority patent/CA2856180C/en
Priority to KR1020147018840A priority patent/KR102059918B1/en
Priority to EP12806388.0A priority patent/EP2788372B1/en
Priority to ES12806388T priority patent/ES2748864T3/en
Publication of WO2013083799A1 publication Critical patent/WO2013083799A1/en
Priority to ZA2014/03617A priority patent/ZA201403617B/en
Priority to IL232926A priority patent/IL232926B/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to mutated lentiviral ENV proteins and their use as drugs.
  • Lentivirus is a genus of slow viruses of the Retroviridae family, characterized by a long incubation period. Lentiviruses can deliver a significant amount of genetic information into the DNA of the host cell and have the unique ability among retroviruses of being able to replicate in non-dividing cells, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • Human immunodeficiency virus (HIV) is according to WHO one of the most serious health crisis the world faces today. AIDS has killed more than 25 million people since 1981. In the most severely affected countries, the average life expectancy is now declining to 49 years of age - 13 years less than in the absence of AIDS.
  • HIV-1 and HIV-2 that infect humans. They belong to a group of retroviruses called Lentiviruses and a virus similar to HIV has been found in African monkeys. Retroviruses transfer their genes from a producer cell to a target cell as a genomic RNA transcript, which is reverse-transcribed after infection and integrated into the DNA genome of the target cell.
  • the replication process for HIV-1 has an error rate of about one per 10,000 base pairs. Since the entire viral genome is just under 10,000 base pairs, it is estimated that on average one error is introduced into the HIV-1 genome at each viral replication cycle.
  • This high mutation rate contributes to extensive variability of the viruses inside any one person and an even wider variability across populations.
  • the M (for major) variant is by far the most prevalent world wide. Within the M variant are clades A, B. C, D, E, F. G H. I, J and K, with clades A through E representing the vast majority of infections globally. Clades A, C and D are dominant in Africa, while clade B is the most prevalent in Europe, North and South America and Southeast Asia. Clades E and C are dominant in Asia. These clades differ by as much as 35%. Another variant is clade O, which is observed in Cameroun isolates of HIV-1.
  • the high mutation rate is one of the mechanisms that allow the virus to escape from control by drug therapies. These new viruses represent resistant strains.
  • the high mutation rate also allows the virus to escape the patient's immune system by altering the structures that are recognized by immune components.
  • An added consequence of this extensive variability is that the virus can also escape from control by vaccines, and therefore makes it difficult to find vaccines based on envelope proteins which are effective.
  • the virus produces proteins having immunosuppressive properties, allowing to escape the patient's immune system survey.
  • cell expressing such proteins become "invisible" to the immune system.
  • the international application WO 2005/095,442 discloses mutations in the immunosuppressive domains of endogenous retroviruses (ERV) or onco retroviruses, such as HTLV or FeLV, ENV proteins.
  • ERV endogenous retroviruses
  • onco retroviruses such as HTLV or FeLV
  • ENV proteins HTLV or FeLV
  • the international application WO 2005/095, 442 never mentions or suggests that the mutations made in the immunosuppressive domain of ERV- or onco retroviruses ENV proteins can be transposable to lentiviral ENV proteins.
  • amino acid residues of the consensus sequence are selected from the groups of residues consisting of: X(l): L, S, R, P, F, A, V, M, and I; and
  • X(7) A, S, T, V, L, G, F, D, M, and E;
  • X(l 1) Y, L, F, H, C, I, T, M, and N;
  • X(12) L, I, V, M, Q, P, T, Y, and A;
  • X(17) L, F, I, R, V, P, S, M, and H, and
  • X(32) A, N, T, S, D, R, FQ, P, I, E, V, M, L, K, H, C, and B;
  • X(35) W, R, G, L, M, and P
  • X(36) N, S, D, B, K, E, R, Q, M, and G
  • X(37) S, T, A, N, D, V, I, E, Y, K, L, R, G, P, M, F, W, H, Q, B, and C;
  • X(42) K, R, N, D, S, T, G, E, I, V, Y, Q, P, H, A, W, M, and C, and
  • X(43) S, T, N, K, I, R, D, E, P, L, A, W, G, M, H, Y, F, V, and C,
  • X(44) L, Y, Q, F, E, H, S, V, K, M, T, I, W, N, D, R, P, A, and G;
  • X(45) D, E, N, S, T, K, G, L, A, Q, H, I, Y, B, R, V, P, M, F, W, Z, and C;
  • X(46) E, D, Q, Y, K, N, T, S, A, W, H, M, R, I, G, L, V, Z, F, B, and P ;
  • X(48) W, I, T, N, D, E, L, G, S, Y, R, V, K, H, A, Q, M, and F;
  • X(50) N, D, T, K, S, H, L, G, E, W, I, Q, M, R, B, Y, P, and A;
  • This consensus sequence contains 50 amino acids, in which the specific amino acids in position 10, 19, 24, 34 and 40 are defined as affecting the immunogenic properties of a HIV-1 envelope polypeptide, and the 45 remaining positions are randomly defined including the most common amino acids of wild-type HIV ENV proteins.
  • WO 2010/022,740 is a transposition from endogenous retroviruses (ERV) or onco retroviruses to lentivirus on the basis of the teaching of WO 2005/095,442 (Inventors : Renard, Mangeney & Heidmann), but said transposition is inappropriate in the case of lentivirus, as shown by the Inventor of the present invention.
  • ERP endogenous retroviruses
  • WO 2005/095,442 Inventors : Renard, Mangeney & Heidmann
  • Dr Heidmann is an Inventor in WO 2005/095,442 and in the present invention.
  • the effects of the mutations described in WO 2005/095,442 were also tested in lentivirus by the Inventor of the present invention, but no effect was observed when the mutations identified in endogenous retroviruses or onco retroviruses were transposed into ENV protein of lentivirus.
  • WO 2010/022,740 teaches that such mutations can be effective using any amino acid residue. This teaching is in contradiction with the present invention, showing that the immunosuppressive properties of HIV- 1 ENV protein are only affected by specific mutations that are defined not only by their position, but also by the nature of the substituted amino acid residues.
  • WO 2010/022,740 discloses experimental results for only one specific mutation within the immunosuppressive domain of HIV ENV protein, as defined by the 50 amino acids consensus sequence.
  • the mutation a substitution by R as the only one exemplified in the international application WO 2010/022,740, occurs at the amino acid in position 19, which again is equivalent to the position disclosed in the international application WO 2005/095,442 if one simply aligns ENV sequences (see Fig 3 in WO 2010/022,740, which is a copy of Figure 3 in Benit et al. 2001, Journal of Virology, Vol. 75, No. 23, p.l 1709-11719) (Of note not only the position of the amino acid, but also the nature of the substitution (by arginine) is similar to the one described in WO 2005/095,442).
  • WO 2010/022,740 discloses that said mutated HIV ENV protein inhibits proliferation of PBMC ex vivo, but such ex vivo result has no in vivo predictive value.
  • WO 2010/022,740 never discloses or suggests that mutants are efficient in vivo, i.e. that cell expressing mutants are detected by the immune system.
  • WO 2010/022,740 raises the same technical problem as the present invention, but does not offer an appropriate technical solution.
  • This prior art reveals the difficulties to overcome the identification of the effective mutations affecting the immunosuppressive properties of the lentiviral ENV proteins.
  • One aim of the invention is to provide new mutated ENV proteins devoid of immunosuppressive properties.
  • Another aim of the invention is to provide a new pharmaceutical composition efficient for treating lentiviral infection.
  • Another aim of the invention is to provide an efficient vaccine.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising as active substance : a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
  • said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein
  • said mutated human or simian lentiviral ENV protein having at least 70% identity, preferably at least 80% identity, to one sequence chosen from the group consisting of SEQ ID NO : 216, SEQ ID NO : 420 and SEQ ID NO : 421,
  • said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X a is A, F, G, L, R or deleted
  • X b is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
  • X a is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y
  • X b is A, F, G, R or deleted, or
  • X a is A, F, G, L, R or deleted
  • Xb is A, F, G, R or deleted
  • said fragment comprising at least 40 amino acids
  • said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid
  • X a is A, F, G, L, R or deleted
  • X b is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
  • X a is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y
  • X b is A, F, G, R or deleted, or
  • X a is A, F, G, L, R or deleted
  • Xb is A, F, G, R or deleted, in association with a pharmaceutically acceptable carrier, said substantial absence of immunosuppressive activity of the above mentioned mutated human or simian lentiviral ENV protein or of the above defined fragment being liable to be assessed by the fact that in an in vivo assay involving engrafted tumor cells rejection,
  • tumor cells being transduced either so as to express said mutated ENV protein or said fragment ("mutated ENV tumor cells"), or said tumor cells being transduced so as to express said wild type ENV protein or a fragment thereof ("wild type ENV tumor cells”),
  • tumor cells being not transduced
  • immunosuppression index of said mutated ENV protein or of said fragment ( ated env ) / immunosuppression index of wild type ENV protein (i w iid type env ) is less than 0.5
  • imutated env being defined by : (maximum area reached by mutated ENV tumor cells - maximum area reached by normal tumor cells) / (maximum area reached by normal tumor cells), and
  • iwiid type env being defined by : (maximum area reached by wild type ENV tumor cells - maximum area reached by normal tumor cells) / (maximum area reached by normal tumor cells).
  • the present application is based on the unexpected observation made by the Inventors that some specific amino acids of the immunosuppressive domain of a lentiviral ENV protein can be mutated conferring to said lentiviral ENV protein essentially no immunosuppressive properties, or no immunosuppressive properties, while retaining its antigenicity, the three- dimentional structure of the immunosuppressive domain, and its expression at the plasma membrane.
  • the mutated lentiviral ENV protein according to the invention does not alter the infectivity of a virus expressing it.
  • the "mutated simian or human lentiviral ENV proteins” means that the ENV proteins derive from the expression of an env gene of a lentivirus of human or simian.
  • Lentiviruses according to the invention encompassed by the invention are HIV-1 and 2 and Simian immunodeficiency virus (SIV).
  • SIV Simian immunodeficiency virus
  • the "mutated ENV protein" as defined in the invention, encompasses two meanings.
  • the said "mutated ENV protein” is the unnatural result of the intervention of human beings. According to the second meaning, the mutated ENV protein also encompasses naturally occurring variants for which up to now the non immunosuppressive properties remain unknown.
  • This second meaning takes into consideration the natural variability of HIV and SIV variants inside a same infected individual, wherein the said "mutated ENV protein" might be non immunosuppressive but its property is undetectable because an HIV infected patient always carries many HIV variants, the majority of which is immunosuppressive.
  • the three following proteins correspond to wild type sequences of the ENV protein of HIV-1, HIV-2 and SIV respectively. In the invention, they are considered as reference sequences of wild type ENV proteins.
  • Variant in the invention encompasses SIV, HIV-1 and HIV-2 ENV proteins.
  • Variants of the HIV-1 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the wild type amino acid sequence of the HIV-1 ENV protein, and comprises the mutations as described above, and harbour no immunosuppressive activity.
  • Variants of the HIV-2 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the wild type amino acid sequence of the HIV-2 ENV protein, and comprises the mutations as described above, and harbour no immunosuppressive activity.
  • Variants of the SIV mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the wild type amino acid sequence of the SIV ENV protein, and comprises the mutations as described above, and harbour no immunosuppressive activity.
  • the three following proteins correspond to three mutated sequences of the ENV protein of HIV- 1, HIV-2 and SIV respectively. In the invention, they are considered as reference sequences of the mutated ENV proteins.
  • SEQ ID NO : 216 corresponds to the SEQ ID NO : 417 in which the amino acid residue Y in position 5 (X a ) of the sequence A-[I/V]-E-[K/R]-X a -Xb-X-D-Q has been substituted by R.
  • SEQ ID NO : 420 corresponds to the SEQ ID NO : 418 in which the amino acid residue Y in position 5 (X a ) of the sequence A-[I/V]-E-[K/R]-X a -X b -X-D-Q has been substituted by R.
  • SEQ ID NO : 421 corresponds to the SEQ ID NO : 419 in which the amino acid residue Y in position 5 (X a ) of the sequence A-[I/V]-E-[K/R]-X a -X b -X-D-Q has been substituted by R.
  • the invention also encompasses the variants of the ' 'mutated simian or human lentiviral ENV protein", harbouring the above mentioned mutations, and conferring a lack of immunosuppressive properties to said variant.
  • Variants of the HIV-1 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the reference mutated sequence of HIV-1 ENV protein (SEQ ID NO : 216), and comprises the mutations as described above, and harbour no immunosuppressive activity.
  • Variants of the HIV-2 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the reference mutated sequence of the HIV-2 ENV protein (SEQ ID NO : 420), and comprises the mutations as described above, and harbour no immunosuppressive activity.
  • Variants of the SIV mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the reference mutated sequence of the SIV ENV protein (SEQ ID NO : 421), and comprises the mutations as described above, and harbour no immunosuppressive activity.
  • immunosuppressive domain (ISU) of the lentivirus according to the invention can be delimited by the sequence SEQ ID NO: 6,
  • SEQ ID NO: 6 corresponds to the non mutated ISU domain.
  • SEQ ID NO: 6 comprises SEQ ID NO: 1.
  • HIV-1 ENV protein comprises the amino acid sequence AVERYLKDQ (SEQ ID NO: 7)
  • HIV-2 ENV protein comprises the amino acid sequence AIEKYLKDQ (SEQ ID NO: 8)
  • SIV ENV protein comprises the amino acid sequence SEQ ID NO: 7 or 8.
  • the ISU domain looses its immunosuppressive properties.
  • the underlined amino acids are the essential amino acids.
  • the ISU domain as defined by SEQ ID NO: 6, is the minimal essential domain of the ENV protein according to the invention which harbours an immunosuppressive activity.
  • the localization of an ISU domain can be determined in all ENV proteins of viruses as described in Benit et al. 2001, Journal of Virology, Vol. 75, No. 23, p.11709-11719.
  • the ISU domain is defined by its structure and its localization, irrespective of the fact that it possesses or not an immunosuppressive activity.
  • the ISU domain refers to a specific domain in which a mutation can affect the immunosuppressive property of the ENV protein.
  • the immunosuppressive property of the ENV protein is preferably measured using in vivo procedures, which are representative of the physiological environment.
  • the immunosuppressive properties of the mutated ENV proteins according to the invention are measured according to an in vivo procedure to assay the immunosuppressive activity of a ENV protein disclosed previously [Mangeney and Heidmann Proc Natl Acad Sci USA 1998;95: 14920-14925; Mangeney et al. Proc Natl Acad Sci USA, 2007, 104(51):20534- 91
  • this in vivo procedure is performed using ENV proteins, or fragment thereof, which are not associated to another component or carrier proteins, such as BSA.
  • tumour cell lines such as MCA 205 or C18.1 cell lines by known transfection methods.
  • the tumour cells expressing the protein to be tested are then injected especially subcutaneous (s.c.) injection to a host, generally mice. Following said injection, the establishment of tumour or, to the contrary, its rejection, is determined and the tumour area is measured. Tumor establishment was determined by palpation and tumor area (mm 2 ) was determined by measuring perpendicular tumor diameters.
  • the above defined ratio relative to the immunosuppressive index can be less than 0.2, and can even have a negative value (see Figures 2 and 3).
  • In vitro assay could be carried out, using high doses of synthetic peptides but they are indirect and less convincing, since the expression "immunosuppressive" is relevant when applied to animals possessing a complete immune system and not to cell lines.
  • An additional difficulty for the functional characterization of an ISU domain relies on the fact that the ISU carried by the retroviral ENV proteins is a highly structured proteic domain, with trimer formation within the complete ENV proteins (Caffrey M., Biochimica et Biophysica Acta, 1536:116-122, 2001; Caffrey et al, The EMBO Journal, Vol. 17, No. 16, p.4572-4584, 1998).
  • Such structures are not naturally formed with ISU peptides of limited length, and this is most probably why most studies carried out with peptides provide irrelevant results and/or are dependent on specific coupling of the peptides to carrier proteins (such as BSA, e.g. Denner et al, Current Science, AIDS 1994, 8:1063- 1072).
  • the ENV proteins according to the invention are mutated. This mutation is made in vitro. Thus, the mutated ENV proteins according to the invention are isolated, and does not correspond to naturally occurring counterpart.
  • the lentiviral mutated ENV proteins are substantially devoid of immunosuppressive properties.
  • the mutated ENV proteins according to the invention have no, or have reduced immunosuppressive properties with respect to the natural non mutated ENV protein from a virus of the same species.
  • a mutated HIV ENV protein according to the invention has reduced immunosuppressive properties with respect to the wild type HIV ENV protein.
  • the terms "substantially devoid of immunosuppressive properties” means that the mutated ENV proteins according to the invention have an immunosuppressive index less than about 0.2 [Mangeney and Heidmann Proc Natl Acad Sci USA 1998;95: 14920-14925; Mangeney et al. Proc Natl Acad Sci USA, 2007, l04(51):20534-9
  • structures responsible for antigenicity relates to structures of the protein which are liable to interact with components of the immune system such as antibodies or membrane receptors of immune cells, in particular T cells.
  • the mutation(s) within the immunosuppressive domain of the lentiviral ENV proteins is (are) sufficient to decrease the immunosuppressive activity of the mutated lentiviral ENV protein with respect to the corresponding wild type ENV.
  • another amino acid be also mutated because it ensures that the structure of the mutated ENV protein is essentially conserved with respect to the corresponding wild type ENV protein.
  • the mutated lentiviral ENV protein has substantially retained the structure, especially the antigenic structure, e.g., immunogenic determinants, of the original determined lentiviral ENV protein, i.e. the wild type non mutated lentiviral ENV protein.
  • the mutated ENV protein involved in the present invention has an average length of about 700 to about 950 amino acids.
  • the invention encompasses fragments of the mutated ENV protein as defined above, provided that said fragment:
  • - preferably, comprises the extracellular parts of the ENV protein
  • the fragment of the mutated ENV protein of the invention can comprise about 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 amino acids. These values are given only in an illustrative way, as the man skilled in the art will understand that the fragment can comprise any number of amino acids comprised between 40 and 700.
  • the fragments according to the invention are such that, while retaining the antigenic structure of the full length mutated ENV protein, and thus of the wild type ENV protein, they have lost major antigenic regions that are responsible for antigenicity in another region than the region corresponding to the immunosuppressive domain.
  • the invention also relates to a pharmaceutical composition wherein the active substance is an isolated non naturally occurring mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, as above defined, or fragments thereof.
  • the invention relates to a pharmaceutical composition as defined above comprising as active substance :
  • said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein
  • said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid
  • X a is A, F, G, L or R
  • X b is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y
  • X a is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y
  • X b is A, F, G or R, or
  • X a is A, F, G, L or R
  • Xb is A, F, G or R, or
  • said fragment comprising at least 40 amino acids
  • said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid, and either
  • X a is A, F, G, L or R
  • X b is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y
  • X a is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y
  • X b is A, F, G or R
  • X a is A, F, G, L or R
  • Xb is A, F, G or R, in association with a pharmaceutically acceptable carrier.
  • the invention relates to a pharmaceutical composition as defined above comprising as active substance :
  • said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein
  • said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid
  • X a is A, F, G, L or R, and X b is L, I, V, M or P, or
  • X a is Y, I, H, C or T
  • Xb is A, F, G or R, or
  • X a is A, F, G, L or R
  • Xb is A, F, G or R
  • said fragment comprising at least 40 amino acids
  • said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid, and either
  • X a is A, F, G, L or R, and X b is L, I, V, M or P, or
  • X a is Y, I, H, C or T
  • Xb is A, F, G or R, or
  • X a is A, F, G, L or R
  • Xb is A, F, G or R, in association with a pharmaceutically acceptable carrier.
  • the invention relates to a pharmaceutical composition as defined above comprising as active substance :
  • said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein
  • said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid
  • X a is A, G, or R
  • X b is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y
  • X a is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y
  • X b is F, G or R
  • X a is F or L
  • Xb is F, G or R
  • X a is A, G or R, and Xb is F, G or R, or
  • said fragment comprising at least 40 amino acids, said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid
  • X a is A, G, or R
  • X b is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
  • X a is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and X b is F, G or R,
  • X a is F or L
  • Xb is F, G or R
  • X a is A, G or R, and Xb is F, G, R, in association with a pharmaceutically acceptable carrier.
  • the invention relates to a pharmaceutical composition as defined above comprising as active substance :
  • said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein
  • said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid
  • X a is A, G, or R, and X b is L, I, V, M or P,
  • X a is Y, I, H, C or T, and Xb is F, G or R,
  • X a is F or L
  • Xb is F, G or R
  • X a is A, G or R, and Xb is F, G or R, or
  • said fragment comprising at least 40 amino acids
  • said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid
  • X a is A, G, or R, and X b is L, I, V, M or P,
  • X a is Y, I, H, C or T, and Xb is F, G or R,
  • X a is F or L
  • Xb is F, G or R
  • X a is A, G or R, and Xb is F, G or R, in association with a pharmaceutically acceptable carrier.
  • the invention also relates to a pharmaceutical composition as defined above comprising as active substance : a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
  • said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein
  • said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid
  • X a is A, G, or R
  • X b is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
  • X a is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and X b is F, G or R, or
  • said fragment comprising at least 40 amino acids
  • said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid
  • X a is A, G, or R
  • X b is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
  • X a is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and X b is F, G or R, in association with a pharmaceutically acceptable carrier.
  • the invention relates to a pharmaceutical composition as defined above, comprising as active substance : a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
  • said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein
  • said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid
  • X a is A, G, or R, and X b is L, I, V, M or P,
  • X a is Y, I, H, C or T
  • Xb is F, G or R, or
  • said fragment comprising at least 40 amino acids
  • said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
  • X represents any amino acid
  • X a is A, G, or R, and X b is L, I, V, M or P,
  • X a is Y, I, H, C or T
  • Xb is F, G or R, in association with a pharmaceutically acceptable carrier.
  • the invention relates to a pharmaceutical composition as defined above, wherein
  • X a is R
  • X b is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y
  • X a is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y
  • X b is R, or
  • the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the following amino acid sequences :
  • A-V-E-R-X a -X b -X-DQ (SEQ ID NO: 425).
  • the invention relates to a pharmaceutical composition as defined above, wherein the resulting immunosuppressive domain, contained in the mutated lentiviral protein, comprises the amino acid sequence of the list consisting of: SEQ ID NO :9 to SEQ ID NO :37.
  • SEQ ID NO : 9 to SEQ ID NO :37 encompass SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : l l , SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 20, SEQ ID NO : 21, SEQ ID NO : 22, SEQ ID NO : 23, SEQ ID NO : 24, SEQ ID NO : 25, SEQ ID NO : 26, SEQ ID NO : 27, SEQ ID NO : 28, SEQ ID NO : 29, SEQ ID NO : 30, SEQ ID NO : 31, SEQ ID NO : 32, SEQ ID NO : 33, SEQ ID NO : 34, SEQ ID NO : 35, SEQ ID NO : 36 and SEQ ID NO : 37.
  • AVERARKD SEQ ID NO : 21 AVERLFKD SEQ ID NO : 31
  • AVERFFKD SEQ ID NO : 23 AVERLRKD SEQ ID NO : 33
  • AVERGFKD SEQ ID NO : 27 AVERRRKD SEQ ID NO : 37
  • the invention relates to a pharmaceutical composition as defined above, wherein the resulting immunosuppressive domain, contained in the mutated lentiviral protein, comprises the amino acid sequence of the list consisting of: SEQ ID NO :38 to SEQ ID NO :66.
  • SEQ ID NO : 38 to SEQ ID NO :66 encompass SEQ ID NO : 38, SEQ ID NO : 39, SEQ ID NO : 40, SEQ ID NO : 41 , SEQ ID NO : 42, SEQ ID NO : 43, SEQ ID NO : 44, SEQ ID NO : 45, SEQ ID NO : 46, SEQ ID NO : 47, SEQ ID NO : 48, SEQ ID NO : 49, SEQ ID NO : 50, SEQ ID NO : 51, SEQ ID NO : 52, SEQ ID NO : 53, SEQ ID NO : 54, SEQ ID NO : 55, SEQ ID NO : 56, SEQ ID NO : 57, SEQ ID NO : 58, SEQ ID NO : 59, SEQ ID NO : 60, SEQ ID NO : 61, SEQ ID NO : 62, SEQ ID NO : 63, SEQ ID NO : 64, SEQ ID NO : 65 and SEQ ID NO : 60
  • AIEKALKD SEQ ID NO : 38 AIEKYFKD SEQ ID NO : 44
  • AIEKFLKD SEQ ID NO : 39 AIEKYGKD SEQ ID NO : 45
  • AIEKGLKD SEQ ID NO : 40 AIEKYRKD SEQ ID NO : 46
  • AIEKLLKD SEQ ID NO : 41 AIEKAAKD SEQ ID NO : 47
  • AIEKRLKD SEQ ID NO : 42 AIEKAFKD SEQ ID NO : 48
  • AIEKARKD SEQ ID NO : 50 AIEKLAKD SEQ ID NO : 59
  • AIEKFAKD SEQ ID NO : 51 AIEKLFKD SEQ ID NO : 60
  • AIEKFFKD SEQ ID NO : 52 AIEKLGKD SEQ ID NO : 61
  • AIEKFGKD SEQ ID NO : 53 AIEKLRKD SEQ ID NO : 62
  • AIEKFRKD SEQ ID NO : 54 AIEKRAKD SEQ ID NO : 63
  • AIEKGAKD SEQ ID NO : 55 AIEKRFKD SEQ ID NO : 64
  • the invention relates to a pharmaceutical composition as defined above, wherein the resulting immunosuppressive domain, contained in the mutated lentiviral protein, comprises the amino acid sequence of the list consisting of: SEQ ID NO : 67 to SEQ ID NO : 95.
  • SEQ ID NO : 67 to SEQ ID NO :95 encompass SEQ ID NO : 67, SEQ ID NO : 68, SEQ ID NO : 69, SEQ ID NO : 70, SEQ ID NO : 71, SEQ ID NO : 72, SEQ ID NO : 73, SEQ ID NO : 74, SEQ ID NO : 75, SEQ ID NO : 76, SEQ ID NO : 77, SEQ ID NO : 78, SEQ ID NO : 79, SEQ ID NO : 80, SEQ ID NO : 81, SEQ ID NO : 82, SEQ ID NO : 83, SEQ ID NO : 84, SEQ ID NO : 85, SEQ ID NO : 86, SEQ ID NO : 87, SEQ ID NO : 88, SEQ ID NO : 89, SEQ ID NO : 90, SEQ ID NO : 91, SEQ ID NO : 92, SEQ ID NO 92, SEQ
  • AIERAGKD SEQ ID NO : 78
  • AIERYAKD SEQ ID NO : 72 AIERFAKD SEQ ID NO : 80
  • AIERYRKD SEQ ID NO : 75 AIERFGKD SEQ ID NO : 82 AIERFRKD SEQ ID NO : 83 AIERLGKD SEQ ID NO : 90
  • the previous ENV proteins having their ISU comprising the above sequence are devoid of immunosuppressive properties.
  • any ENV protein of human or simian lentivirus having in their ISU an amino acid sequence comprising of the sequences SEQ ID NO: 9 to 95, is devoid of immunosuppressive properties.
  • a simian or human lentiviral ENV protein comprising, within its ISU domain, an amino acid sequence selected from SEQ ID NO: 9 to 95 is devoid of immunosuppressive properties.
  • the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences :
  • the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences :
  • the fragments of the mutated ENV proteins according to the invention comprise or consist of the following sequences: SEQ ID NO: 67 to 211.
  • fragments are also devoid of immunosuppressive properties. However, these fragments retain the structure and the antigenicity of the corresponding immunosuppressive domain that is not mutated, i.e. the wild type immunosuppressive domain.
  • the fragments of the mutated ENV proteins according to the invention comprise or consist of the following sequences: SEQ ID NO: 96 to 211.
  • the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences : SEQ ID NO : 96, 98, 100, 102 to 108, 110 to 1 16, 1 18 to 125, 127, 129, 131 to 137, 139 to 145, 147 to 154, 156, 158, 160 to 166, 168 to 174, 176 to 183, 185, 187, 189 to 195, 197 to 203, 205 to 211.
  • the invention relates to the pharmaceutical composition as defined above, wherein said mutated protein consists of one of the following sequences SEQ ID NO : 212 to 269
  • MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 215 YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
  • VKLTPLC VSLKCTDLGNATNTNS SNTNS S SGEMMMEKGEIK
  • VKLTPLC VSLKCTDLGNATNTNS SNTNS S SGEMMMEKGEIK
  • VKLTPLC VSLKCTDLGNATNTNS SNTNS S SGEMMMEKGEIK
  • MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 231 YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS

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Abstract

The present invention relates to a pharmaceutical composition comprising, as active substance a mutated lentiviral ENV protein, substantially devoid of immunosuppressive properties or a variant of said mutated lentiviral ENV protein or a fragment of the above proteins, in association with a pharmaceutically acceptable carrier.

Description

MUTATED LENTIVIRAL ENV PROTEINS AND THEIR USE AS DRUGS
The present invention relates to mutated lentiviral ENV proteins and their use as drugs.
Lentivirus is a genus of slow viruses of the Retroviridae family, characterized by a long incubation period. Lentiviruses can deliver a significant amount of genetic information into the DNA of the host cell and have the unique ability among retroviruses of being able to replicate in non-dividing cells, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Human immunodeficiency virus (HIV) is according to WHO one of the most serious health crisis the world faces today. AIDS has killed more than 25 million people since 1981. In the most severely affected countries, the average life expectancy is now declining to 49 years of age - 13 years less than in the absence of AIDS. According to UNAIDS an estimated number of 39.5 million people were living with HIV virus in 2006 and 4.3 million were infected in 2006. In many regions new infections are heavily concentrated in the younger generations (15-24 years of age). Access to treatment and care has greatly increased in recent years. Determining real time trends to HIV incidence and in particular the impact of prevention programmes ideally requires long studies of a large number of people. Given the practical difficulties of conducting such studies focus has been placed on young women and their infants. Children living with HIV typically acquire infection through a mother-to-child-transmission (MTCT), which occur during pregnancy, delivery or during breastfeeding. Renewed efforts are urgently required to increase access to comprehensive and integrated programs to prevent HIV infection in infants and young children, which will indicate a route to HIV-free generations.
There are two known types of HIV; HIV-1 and HIV-2 that infect humans. They belong to a group of retroviruses called Lentiviruses and a virus similar to HIV has been found in African monkeys. Retroviruses transfer their genes from a producer cell to a target cell as a genomic RNA transcript, which is reverse-transcribed after infection and integrated into the DNA genome of the target cell.
The first person with a documented HIV-infection died in 1959. In the early 1980s doctors in the US become aware, that more and more patients suffered from abnormal infections and showed signs of immune failure. The syndrome was named Acquired Immune Deficiency Syndrome (AIDS) and it was soon after discovered that HIV was the causative agent for the observed destruction of the immune system. Initially patients were offered a treatment based solely on pain relief and almost all inevitably died. In mid 1990s there were two important breakthroughs in treatment. Firstly, a new group of antiretroviral agents were discovered and secondly it became possible to measure the amount of HIV virus in blood. These two advances made it possible to treat patients with a combination of different agents and doctors were able to check, whether the treatment actually worked. The result was that the immune system of infected patients gradually became normal and patients lived longer. Today infected people in Western countries are having the same level of quality of life as those not infected and they are able to have children, although the economical and psychological consequences of having HIV are huge. The situation is, however, even more severe in developing countries, where more than 95% of those people infected with HIV/ AIDS are living. Worldwide more than 25 million people have died from AIDS in the last 25 years.
Approximately 95% of the people who get infected today live in the developing countries, where expensive antiviral drugs are not available. Therefore, there is an urgent need for an effective vaccine - the only effective solution to the uncontrolled HIV pandemic. During the last few years research has brought up new knowledge on the fundamental biology of HIV-virus which is leading to new antiviral drugs and strategies for vaccine design. In spite of these substantial advances, an effective vaccine does not yet exist. Only attenuated (that is live but weakened) HIV-strains has been able to provide immunity in primate studies even though they will never reach a required safety profile suitable for mass vaccination.
The replication process for HIV-1 has an error rate of about one per 10,000 base pairs. Since the entire viral genome is just under 10,000 base pairs, it is estimated that on average one error is introduced into the HIV-1 genome at each viral replication cycle.
This high mutation rate contributes to extensive variability of the viruses inside any one person and an even wider variability across populations.
This variability has resulted in three HIV-1 variants being described, and the subspecies of virus called "clades." The distinctions are based on the structure of the envelope proteins, which are especially variable. The M (for major) variant is by far the most prevalent world wide. Within the M variant are clades A, B. C, D, E, F. G H. I, J and K, with clades A through E representing the vast majority of infections globally. Clades A, C and D are dominant in Africa, while clade B is the most prevalent in Europe, North and South America and Southeast Asia. Clades E and C are dominant in Asia. These clades differ by as much as 35%. Another variant is clade O, which is observed in Cameroun isolates of HIV-1. The greatest variation in structure is seen in the envelope proteins gpl20 and gp41. There are two important results from the very high mutation rate of HIV- 1 that have profound consequences for the epidemic. First, the high mutation rate is one of the mechanisms that allow the virus to escape from control by drug therapies. These new viruses represent resistant strains. The high mutation rate also allows the virus to escape the patient's immune system by altering the structures that are recognized by immune components. An added consequence of this extensive variability is that the virus can also escape from control by vaccines, and therefore makes it difficult to find vaccines based on envelope proteins which are effective.
Moreover, the virus produces proteins having immunosuppressive properties, allowing to escape the patient's immune system survey. Thus, cell expressing such proteins become "invisible" to the immune system.
Consequently for a vaccine, there is a need to provide proteins as antigens having lost, or substantially lost, their immunosuppressive functions, in order to generate an efficient response. This will enable the individuals once infected by the virus to allow the immune system to destroy the infected cells and prevent/cure the infection..
Prior art has already intended to provide such proteins.
For instance, the international application WO 2005/095,442 (Inventors : Renard, Mangeney & Heidmann) discloses mutations in the immunosuppressive domains of endogenous retroviruses (ERV) or onco retroviruses, such as HTLV or FeLV, ENV proteins. This document demonstrates that mutations at a specific position abolish the immunosuppressive properties of ENV proteins of ERV or onco retroviruses. However, the international application WO 2005/095, 442 never mentions or suggests that the mutations made in the immunosuppressive domain of ERV- or onco retroviruses ENV proteins can be transposable to lentiviral ENV proteins.
The international application WO 2010/022,740 discloses an extremely wide consensus sequence of a region of HIV ENV protein, described as follows:
X(l-22)-C(23)-X(24-28)-C(29)-(X30-50)
wherein the amino acid residues of the consensus sequence are selected from the groups of residues consisting of: X(l): L, S, R, P, F, A, V, M, and I; and
X(2): Q, R, K, H, L, M, and P; and
X(3): A, T, V, H, S, R, Q, G, M, and E; and
X(4): R, K, G, E, T, S, C, M, and H; and
X(5): V, I, L, D, A, S, F, M, and G; and
X(6): L, Q, V, M, P, W, T, and I; and
X(7): A, S, T, V, L, G, F, D, M, and E; and
X(8): V, L, I, M, A, W, K, G, and E; and
X(9): E, K, G, D, A, V, M, and F; and
X(10): X; and
X(l 1): Y, L, F, H, C, I, T, M, and N; and
X(12): L, I, V, M, Q, P, T, Y, and A; and
X(13): K, R, Q, G, S, E, H, W, T, V, M, N, Z, Y, A, P, and C; and
X(14): D, N, G, E, Y, V, S, H, A, M, and I; and
X(15): Q, R, H, K, P, L, M, and N; and
X(16): Q, K, R, T, H, E, S, P, M, and L; and
X(17): L, F, I, R, V, P, S, M, and H, and
X(18): L, M, P, I, H, and S; and
X(19): X; and
X(20): I, L, M, V, S, F, T, D, A, R, P, and J; and
X(21): W, R, G, F, L, M, and T; and
X(22): G, D, A, R, M, and C; and
X(24): X; and
X(25): G, R, E, N, A, M, and D; and
X(26): K, R, N, E, Q, T, S, I, M, and G; and
X(27): L, H, I, T, V, F, R, Q, S, P, A, J, M, and Y; and
X(28): I, V, T, L, R, F and M; and
X(30): T, P, Y, A, N, S, I, V, R, L, M, and H; and
X(31): T, S, P, N, M and I; and
X(32): A, N, T, S, D, R, FQ, P, I, E, V, M, L, K, H, C, and B; and
X(33): V, A, L, M, G, R, and C; and
X(34): X; and
X(35): W, R, G, L, M, and P; and X(36): N, S, D, B, K, E, R, Q, M, and G; and
X(37): S, T, A, N, D, V, I, E, Y, K, L, R, G, P, M, F, W, H, Q, B, and C; and
X(38): S, T, N, I, G, R, L, C, A, W, M and E; and
X(39): W, G, A, R, E, C, Y, V, S, M, and H; and
X(40): X; and
X(41): N, G, K, S, D, E, T, R, H, P, A, B, V, Q, Y, M, and I; and
X(42): K, R, N, D, S, T, G, E, I, V, Y, Q, P, H, A, W, M, and C, and
X(43): S, T, N, K, I, R, D, E, P, L, A, W, G, M, H, Y, F, V, and C,
X(44): L, Y, Q, F, E, H, S, V, K, M, T, I, W, N, D, R, P, A, and G; and
X(45): D, E, N, S, T, K, G, L, A, Q, H, I, Y, B, R, V, P, M, F, W, Z, and C; and
X(46): E, D, Q, Y, K, N, T, S, A, W, H, M, R, I, G, L, V, Z, F, B, and P ; and
X(47): I, D, E, M, G, T, Q, S, W, L, N, Y, K, V, R, F, A, P, and H, and
X(48): W, I, T, N, D, E, L, G, S, Y, R, V, K, H, A, Q, M, and F; and
X((49): D, N, E, G, W, Q, K, H, L, B, S, I, Y, T, A, R, M, Z, and V; and
X(50): N, D, T, K, S, H, L, G, E, W, I, Q, M, R, B, Y, P, and A;
This consensus sequence contains 50 amino acids, in which the specific amino acids in position 10, 19, 24, 34 and 40 are defined as affecting the immunogenic properties of a HIV-1 envelope polypeptide, and the 45 remaining positions are randomly defined including the most common amino acids of wild-type HIV ENV proteins.
In fact, the teaching of WO 2010/022,740 is a transposition from endogenous retroviruses (ERV) or onco retroviruses to lentivirus on the basis of the teaching of WO 2005/095,442 (Inventors : Renard, Mangeney & Heidmann), but said transposition is inappropriate in the case of lentivirus, as shown by the Inventor of the present invention.
Briefly speaking, Dr Heidmann is an Inventor in WO 2005/095,442 and in the present invention. As a matter of fact, the effects of the mutations described in WO 2005/095,442 were also tested in lentivirus by the Inventor of the present invention, but no effect was observed when the mutations identified in endogenous retroviruses or onco retroviruses were transposed into ENV protein of lentivirus.
Moreover, since any amino acid can be assigned to the positions 10, 19, 24, 34 or 40 in the consensus sequence, WO 2010/022,740 teaches that such mutations can be effective using any amino acid residue. This teaching is in contradiction with the present invention, showing that the immunosuppressive properties of HIV- 1 ENV protein are only affected by specific mutations that are defined not only by their position, but also by the nature of the substituted amino acid residues.
Furthermore, WO 2010/022,740 discloses experimental results for only one specific mutation within the immunosuppressive domain of HIV ENV protein, as defined by the 50 amino acids consensus sequence. The mutation, a substitution by R as the only one exemplified in the international application WO 2010/022,740, occurs at the amino acid in position 19, which again is equivalent to the position disclosed in the international application WO 2005/095,442 if one simply aligns ENV sequences (see Fig 3 in WO 2010/022,740, which is a copy of Figure 3 in Benit et al. 2001, Journal of Virology, Vol. 75, No. 23, p.l 1709-11719) (Of note not only the position of the amino acid, but also the nature of the substitution (by arginine) is similar to the one described in WO 2005/095,442).
More specifically, when aligning the ENV sequences respectively of an endog retrovirus or onco retrovirus and a lentivirus, according to Figure 3 of Benit et al. :
Endogenous or onco retrovirus ;
L Q N R R G L D L L F L K [ i ) G G L A A L (WO 2005/095 , 442 )
Leri fc vi i u s t
L A V E R Y L K D Q Q L W G S G K (WO 2010/022 , 740 )
Figure imgf000007_0001
19
it appears that the position 19 in lentivirus corresponds to the position in WO 2005/095,442 where a specific substitution into arginine, E→R for endogenous or onco retrovirus, results in loss of immunosuppressive activity.
The international application WO 2010/022,740 discloses that said mutated HIV ENV protein inhibits proliferation of PBMC ex vivo, but such ex vivo result has no in vivo predictive value. However, WO 2010/022,740 never discloses or suggests that mutants are efficient in vivo, i.e. that cell expressing mutants are detected by the immune system.
Moreover, as disclosed hereafter, such mutations are not efficient in vivo. Indeed, results disclosed hereafter in the example section relative to the present invention demonstrate that said substitution G19R does not inhibit in vivo the immunosuppressive properties of the ENV protein.
As a consequence, WO 2010/022,740 raises the same technical problem as the present invention, but does not offer an appropriate technical solution. This prior art reveals the difficulties to overcome the identification of the effective mutations affecting the immunosuppressive properties of the lentiviral ENV proteins.
Thus, the provision of in vivo effective non immunosuppressive lentiviral ENV proteins remains. One aim of the invention is to provide new mutated ENV proteins devoid of immunosuppressive properties.
Another aim of the invention is to provide a new pharmaceutical composition efficient for treating lentiviral infection.
Another aim of the invention is to provide an efficient vaccine.
The invention relates to a pharmaceutical composition comprising as active substance : a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein having at least 70% identity, preferably at least 80% identity, to one sequence chosen from the group consisting of SEQ ID NO : 216, SEQ ID NO : 420 and SEQ ID NO : 421,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein, X represents any amino acid,
and either
Xa is A, F, G, L, R or deleted, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G, R or deleted, or
Xa is A, F, G, L, R or deleted, and Xb is A, F, G, R or deleted, or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either
Xa is A, F, G, L, R or deleted, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G, R or deleted, or
Xa is A, F, G, L, R or deleted, and Xb is A, F, G, R or deleted, in association with a pharmaceutically acceptable carrier, said substantial absence of immunosuppressive activity of the above mentioned mutated human or simian lentiviral ENV protein or of the above defined fragment being liable to be assessed by the fact that in an in vivo assay involving engrafted tumor cells rejection,
said tumor cells being transduced either so as to express said mutated ENV protein or said fragment ("mutated ENV tumor cells"), or said tumor cells being transduced so as to express said wild type ENV protein or a fragment thereof ("wild type ENV tumor cells"),
or said tumor cells being not transduced ("normal tumor cells"),
the following ratio :
immunosuppression index of said mutated ENV protein or of said fragment ( ated env ) / immunosuppression index of wild type ENV protein (iwiid type env ) is less than 0.5,
imutated env being defined by : (maximum area reached by mutated ENV tumor cells - maximum area reached by normal tumor cells) / (maximum area reached by normal tumor cells), and
iwiid type env being defined by : (maximum area reached by wild type ENV tumor cells - maximum area reached by normal tumor cells) / (maximum area reached by normal tumor cells). The present application is based on the unexpected observation made by the Inventors that some specific amino acids of the immunosuppressive domain of a lentiviral ENV protein can be mutated conferring to said lentiviral ENV protein essentially no immunosuppressive properties, or no immunosuppressive properties, while retaining its antigenicity, the three- dimentional structure of the immunosuppressive domain, and its expression at the plasma membrane. Moreover, the mutated lentiviral ENV protein according to the invention does not alter the infectivity of a virus expressing it.
In the invention, the "mutated simian or human lentiviral ENV proteins" means that the ENV proteins derive from the expression of an env gene of a lentivirus of human or simian.
Lentiviruses according to the invention encompassed by the invention are HIV-1 and 2 and Simian immunodeficiency virus (SIV).
Because of the high mutation rate of HIV-1, HIV-2 and SIV viruses, the "mutated ENV protein", as defined in the invention, encompasses two meanings.
According to the first meaning, the said "mutated ENV protein" is the unnatural result of the intervention of human beings. According to the second meaning, the mutated ENV protein also encompasses naturally occurring variants for which up to now the non immunosuppressive properties remain unknown.
This second meaning takes into consideration the natural variability of HIV and SIV variants inside a same infected individual, wherein the said "mutated ENV protein" might be non immunosuppressive but its property is undetectable because an HIV infected patient always carries many HIV variants, the majority of which is immunosuppressive.
The three following proteins correspond to wild type sequences of the ENV protein of HIV-1, HIV-2 and SIV respectively. In the invention, they are considered as reference sequences of wild type ENV proteins.
SEQ ID NO: 417 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY wild type HIV-1 GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP
QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL
TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN
ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP
KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI
RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC
TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN
ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY
CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ
EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG
GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV
GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL
RAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCS
GKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTS
LIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIK
IFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPD
RPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLR
DLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSL
LNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
SEQ ID NO: 418 MCGRNQLFVASLLASACLIYCVQYVTVFYGVPVWRNASIPLFC Wild type HIV-2 ATKNRDTWGTIQCLPDNDDYQEIALNVTEAFDAWNNTVTEQA
VEDVWSLFETSIKPCVKLTPLCVAMRCNSTTAKNTTSTPTTTTT
ANTTIGENSSCIRTDNCTGLGEEEMVDCQFNMTGLERDKKKLY
NETWYSKDVVCESNDTKKEKTCYMNHCNTSVITESCDKHYW
DTMRFRYCAPPGFALLRCNDTNYSGFEPNCSKVVAATCTRMM
ETQTSTWFGFNGTRAENRTYIYWHGRDNRTIISLNKFYNLTVH
CKRPGNKTVVPITLMSGLVFHSQPINRRPRQAWCWFKGEWKE
AMKEVKLTLAKHPRYKGTNDTEKIRFIAPGERSDPEVAYMWT
NCRGEFLYCNMTWFLNWVENRTNQTQHNYVPCHIKQIINTWH
KVGKNVYLPPREGQLTCNSTVTSIIANIDGGENQTNITFSAEVA ELYRLELGDYKLIEVTPIGFAPTPVK YSSAPVRNK GVFVLGF
LGFLTTAGAAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKR
QQEMLRLTVWGTKNLQARVTAIEKYLKDQAQLNSWGCAFRQ
VCHTTVPWVNDTLTPDWNNMTWQEWEQRIRNLEANISESLEQ
AQIQQEKNMYELQKLNSWDVFGNWFDLTSWIKYIQYGVYIVV
GIIVLRIVIYVVQMLSRLRKGYRPVFSSPPAYFQQIHIHKDREQP
AREETEEDVGNSVGDNWWPWPIRYIHFLIRQLIRLLNRLYNICR
DLLSRSFQTLQLISQSLRRALTAVRDWLRFNTAYLQYGGEWIQ
EAFRAFARATGETLTNAWRGFWGTLGQIGRGILAVPRRIRQGA
EIALL
SEQ ID NO: 419 MGCLGNQLLIAILLLSVYGIYCTLYVTVFYGVPAWRNATIPLFC wild type SIV ATKNRDTWGTTQCLPDNGDYSEVALNVTESFDAWNNTVTEQ mac239 AIEDVWQLFETSIKPCVKLSPLCITMRCNKSETDRWGLTKSITT
TASTTSTTASAKVDMVNETSSCIAQDNCTGLEQEQMISCKFNM
TGLKRDKKKEYNETWYSADLVCEQGNNTGNESRCYMNHCNT
SVIQESCDKHYWDAIRFRYCAPPGYALLRCNDTNYSGFMPKCS
KVVVSSCTRMMETQTSTWFGFNGTRAENRTYIYWHGRDNRTI
ISLNKYYNLTMKCRRPGNKTVLPVTIMSGLVFHSQPINDRPKQ
AWCWFGGKWKDAIKEVKQTIVKHPRYTGTNNTDKINLTAPGG
GDPEVTFMWTNCRGEFLYCKMNWFLNWVEDRNTANQKPKE
QHKRNYVPCHIRQIINTWHKVGKNVYLPPREGDLTCNSTVTSLI
ANIDWIDGNQTNITMSAEVAELYRLELGDYKLVEITPIGLAPTD
VKRYTTGGTSRNKRGVFVLGFLGFLATAGSAMGAASLTLTAQ
SRTLLAGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTA
IEKYLKDQAQLNAWGCAFRQVCHTTVPWPNASLTPKWNNET
WQEWERKVDFLEENITALLEEAQIQQEKNMYELQKLNSWDVF
GNWFDLASWIKYIQYGVYIVVGVILLRIVIYIVQMLAKLRQGY
RPVFSSPPSYFQQTHIQQDPALPTREGKERDGGEGGGNSSWPW
QIEYIHFLIRQLIRLLTWLFSNCRTLLSRVYQILQPILQRLSATLQ
RIREVLRTELTYLQYGWSYFHEAVQAVWRSATETLAGAWGDL
WETLRRGGRWILAIPRRIRQGLELTLL
Variant in the invention encompasses SIV, HIV-1 and HIV-2 ENV proteins.
Variants of the HIV-1 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the wild type amino acid sequence of the HIV-1 ENV protein, and comprises the mutations as described above, and harbour no immunosuppressive activity.
Variants of the HIV-2 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the wild type amino acid sequence of the HIV-2 ENV protein, and comprises the mutations as described above, and harbour no immunosuppressive activity. Variants of the SIV mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the wild type amino acid sequence of the SIV ENV protein, and comprises the mutations as described above, and harbour no immunosuppressive activity.
The three following proteins (SEQ ID NO : 216, 420 and 421) correspond to three mutated sequences of the ENV protein of HIV- 1, HIV-2 and SIV respectively. In the invention, they are considered as reference sequences of the mutated ENV proteins.
More specifically, SEQ ID NO : 216 corresponds to the SEQ ID NO : 417 in which the amino acid residue Y in position 5 (Xa) of the sequence A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q has been substituted by R.
SEQ ID NO : 420 corresponds to the SEQ ID NO : 418 in which the amino acid residue Y in position 5 (Xa) of the sequence A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q has been substituted by R.
SEQ ID NO : 421 corresponds to the SEQ ID NO : 419 in which the amino acid residue Y in position 5 (Xa) of the sequence A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q has been substituted by R.
SEQ ID NO: 216 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY mutated HIV-1 GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP
QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL
TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN
ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP
KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI
RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC
TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN
ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY
CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ
EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG
GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV
GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL
RAIEAQQHLLQLTVWGIKQLQARILAVERRLKDQQLLGIWGCS
GKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTS
LIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIK
IFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPD
RPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLR
DLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSL
LNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
SEQ ID NO: 420 MCGRNQLFVASLLASACLIYCVQYVTVFYGVPVWRNASIPLFC mutated HIV-2 ATKNRDTWGTIQCLPDNDDYQEIALNVTEAFDAWNNTVTEQA VEDVWSLFETSIKPCVKLTPLCVAMRCNSTTAKNTTSTPTTTTT
ANTTIGENSSCIRTDNCTGLGEEEMVDCQFNMTGLERDKKKLY
NETWYSKDVVCESNDTKKEKTCYMNHCNTSVITESCDKHYW
DTMRFRYCAPPGFALLRCNDTNYSGFEPNCSKVVAATCTRMM
ETQTSTWFGFNGTRAENRTYIYWHGRDNRTIISLNKFYNLTVH
CKRPGNKTVVPITLMSGLVFHSQPINRRPRQAWCWFKGEWKE
AMKEVKLTLAKHPRYKGTNDTEKIRFIAPGERSDPEVAYMWT
NCRGEFLYCNMTWFLNWVENRTNQTQHNYVPCHIKQIINTWH
KVGKNVYLPPREGQLTCNSTVTSIIANIDGGENQTNITFSAEVA
ELYRLELGDYKLIEVTPIGFAPTPVKRYSSAPVRNKRGVFVLGF
LGFLTTAGAAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKR
QQEMLRLTVWGTKNLQARVTAIEKRLKDQAQLNSWGCAFRQ
VCHTTVPWVNDTLTPDWNNMTWQEWEQRIRNLEANISESLEQ
AQIQQEKNMYELQKLNSWDVFGNWFDLTSWIKYIQYGVYIVV
GIIVLRIVIYVVQMLSRLRKGYRPVFSSPPAYFQQIHIHKDREQP
AREETEEDVGNSVGDNWWPWPIRYIHFLIRQLIRLLNRLYNICR
DLLSRSFQTLQLISQSLRRALTAVRDWLRFNTAYLQYGGEWIQ
EAFRAFARATGETLTNAWRGFWGTLGQIGRGILAVPRRIRQGA
EIALL
SEQ ID NO: 421 MGCLGNQLLIAILLLSVYGIYCTLYVTVFYGVPAWRNATIPLFC mutated SIV ATKNRDTWGTTQCLPDNGDYSEVALNVTESFDAWNNTVTEQ mac239 AIEDVWQLFETSIKPCVKLSPLCITMRCNKSETDRWGLTKSITT
TASTTSTTASAKVDMVNETSSCIAQDNCTGLEQEQMISCKFNM
TGLKRDKKKEYNETWYSADLVCEQGNNTGNESRCYMNHCNT
SVIQESCDKHYWDAIRFRYCAPPGYALLRCNDTNYSGFMPKCS
KVVVSSCTRMMETQTSTWFGFNGTRAENRTYIYWHGRDNRTI
ISLNKYYNLTMKCRRPGNKTVLPVTIMSGLVFHSQPINDRPKQ
AWCWFGGKWKDAIKEVKQTIVKHPRYTGTNNTDKINLTAPGG
GDPEVTFMWTNCRGEFLYCKMNWFLNWVEDRNTANQKPKE
QHKRNYVPCHIRQIINTWHKVGKNVYLPPREGDLTCNSTVTSLI
ANIDWIDGNQTNITMSAEVAELYRLELGDYKLVEITPIGLAPTD
VKRYTTGGTSRNKRGVFVLGFLGFLATAGSAMGAASLTLTAQ
SRTLLAGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTA
IEKRLKDQAQLNAWGCAFRQVCHTTVPWPNASLTPKWNNET
WQEWERKVDFLEENITALLEEAQIQQEKNMYELQKLNSWDVF
GNWFDLASWIKYIQYGVYIVVGVILLRIVIYIVQMLAKLRQGY
RPVFSSPPSYFQQTHIQQDPALPTREGKERDGGEGGGNSSWPW
QIEYIHFLIRQLIRLLTWLFSNCRTLLSRVYQILQPILQRLSATLQ
RIREVLRTELTYLQYGWSYFHEAVQAVWRSATETLAGAWGDL
WETLRRGGRWILAIPRRIRQGLELTLL
The invention also encompasses the variants of the ' 'mutated simian or human lentiviral ENV protein", harbouring the above mentioned mutations, and conferring a lack of immunosuppressive properties to said variant.
Variants of the HIV-1 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the reference mutated sequence of HIV-1 ENV protein (SEQ ID NO : 216), and comprises the mutations as described above, and harbour no immunosuppressive activity.
Variants of the HIV-2 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the reference mutated sequence of the HIV-2 ENV protein (SEQ ID NO : 420), and comprises the mutations as described above, and harbour no immunosuppressive activity.
Variants of the SIV mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the reference mutated sequence of the SIV ENV protein (SEQ ID NO : 421), and comprises the mutations as described above, and harbour no immunosuppressive activity.
The immunosuppressive domain (ISU) of the lentivirus according to the invention can be delimited by the sequence SEQ ID NO: 6,
xGIVOOOxxLLxxxxxxOxxLxLxxWGxKxLOxRxxA[I/V1E[K/R1YLxDOxxLx (SEQ ID NO: 6)
in which x represents any amino acid.
SEQ ID NO: 6 corresponds to the non mutated ISU domain.
In this SEQ ID NO : 6, "x" (in small letters) is to be considered independently from "X" (in capital letters) used for the first time in SEQ ID NO : 416 of the present invention. SEQ ID NO: 6 comprises SEQ ID NO: 1.
The most advantageous immunosuppressive domains of the wild type ENV proteins according to the invention comprise the following sequences:
HIV-1 ENV protein comprises the amino acid sequence AVERYLKDQ (SEQ ID NO: 7), HIV-2 ENV protein comprises the amino acid sequence AIEKYLKDQ (SEQ ID NO: 8), and
SIV ENV protein comprises the amino acid sequence SEQ ID NO: 7 or 8.
When applying the mutations as defined above, the ISU domain looses its immunosuppressive properties.
In said SEQ ID NO: 6, the underlined amino acids are the essential amino acids. In the invention, the ISU domain, as defined by SEQ ID NO: 6, is the minimal essential domain of the ENV protein according to the invention which harbours an immunosuppressive activity.
Figure imgf000016_0001
The localization of an ISU domain can be determined in all ENV proteins of viruses as described in Benit et al. 2001, Journal of Virology, Vol. 75, No. 23, p.11709-11719. In a broad meaning, the ISU domain is defined by its structure and its localization, irrespective of the fact that it possesses or not an immunosuppressive activity.
In the invention, the ISU domain refers to a specific domain in which a mutation can affect the immunosuppressive property of the ENV protein.
The immunosuppressive property of the ENV protein is preferably measured using in vivo procedures, which are representative of the physiological environment.
The immunosuppressive properties of the mutated ENV proteins according to the invention are measured according to an in vivo procedure to assay the immunosuppressive activity of a ENV protein disclosed previously [Mangeney and Heidmann Proc Natl Acad Sci USA 1998;95: 14920-14925; Mangeney et al. Proc Natl Acad Sci USA, 2007, 104(51):20534- 91
As a physiological test, this in vivo procedure is performed using ENV proteins, or fragment thereof, which are not associated to another component or carrier proteins, such as BSA.
Briefly, a wild-type (wild type lentiviral ENV protein) or modified nucleic acid expressing the protein to be tested (mutated lentiviral ENV protein) is transfected in tumour cell lines such as MCA 205 or C18.1 cell lines by known transfection methods. The tumour cells expressing the protein to be tested are then injected especially subcutaneous (s.c.) injection to a host, generally mice. Following said injection, the establishment of tumour or, to the contrary, its rejection, is determined and the tumour area is measured. Tumor establishment was determined by palpation and tumor area (mm2) was determined by measuring perpendicular tumor diameters. Immunosuppression index is defined as i= (Senv- Snone)/Sn0ne, wherein Senv is the maximum area reached by a tumour expressing an envelope protein and Snone is the maximum area reached by a tumour not expressing ENV protein (negative control).
According to an embodiment of the invention, the above defined ratio relative to the immunosuppressive index can be less than 0.2, and can even have a negative value (see Figures 2 and 3). In vitro assay could be carried out, using high doses of synthetic peptides but they are indirect and less convincing, since the expression "immunosuppressive" is relevant when applied to animals possessing a complete immune system and not to cell lines.
An additional difficulty for the functional characterization of an ISU domain relies on the fact that the ISU carried by the retroviral ENV proteins is a highly structured proteic domain, with trimer formation within the complete ENV proteins (Caffrey M., Biochimica et Biophysica Acta, 1536:116-122, 2001; Caffrey et al, The EMBO Journal, Vol. 17, No. 16, p.4572-4584, 1998). Such structures are not naturally formed with ISU peptides of limited length, and this is most probably why most studies carried out with peptides provide irrelevant results and/or are dependent on specific coupling of the peptides to carrier proteins (such as BSA, e.g. Denner et al, Current Science, AIDS 1994, 8:1063- 1072).
As mentioned above, the ENV proteins according to the invention are mutated. This mutation is made in vitro. Thus, the mutated ENV proteins according to the invention are isolated, and does not correspond to naturally occurring counterpart.
As mentioned above, the lentiviral mutated ENV proteins are substantially devoid of immunosuppressive properties. This means that the mutated ENV proteins according to the invention have no, or have reduced immunosuppressive properties with respect to the natural non mutated ENV protein from a virus of the same species. For instance, a mutated HIV ENV protein according to the invention has reduced immunosuppressive properties with respect to the wild type HIV ENV protein.
In the invention, the terms "substantially devoid of immunosuppressive properties" means that the mutated ENV proteins according to the invention have an immunosuppressive index less than about 0.2 [Mangeney and Heidmann Proc Natl Acad Sci USA 1998;95: 14920-14925; Mangeney et al. Proc Natl Acad Sci USA, 2007, l04(51):20534-9
In the invention, structures responsible for the antigenicity of the mutated lentiviral ENV protein are essentially preserved.
As intended herein, the expression "structures responsible for antigenicity" relates to structures of the protein which are liable to interact with components of the immune system such as antibodies or membrane receptors of immune cells, in particular T cells.
The mutation(s) within the immunosuppressive domain of the lentiviral ENV proteins is (are) sufficient to decrease the immunosuppressive activity of the mutated lentiviral ENV protein with respect to the corresponding wild type ENV. However, it might be advantageous that another amino acid be also mutated because it ensures that the structure of the mutated ENV protein is essentially conserved with respect to the corresponding wild type ENV protein.
The mutated lentiviral ENV protein has substantially retained the structure, especially the antigenic structure, e.g., immunogenic determinants, of the original determined lentiviral ENV protein, i.e. the wild type non mutated lentiviral ENV protein.
These properties can be evaluated by measuring the fusion and/or infectious properties of said mutated lentiviral ENV with respect to the same properties in the wild type non mutated lentiviral ENV protein (see example).
Generally speaking, the mutated ENV protein involved in the present invention has an average length of about 700 to about 950 amino acids. The invention encompasses fragments of the mutated ENV protein as defined above, provided that said fragment:
comprises at least the sequence SEQ ID NO: 2, as defined above,
comprises at least 40 amino acids, preferably comprises at least 50 amino acids, is substantially devoid of immunosuppressive properties, as defined above,
- preferably, comprises the extracellular parts of the ENV protein,
retains the structure of the ENV protein from which it derives,
harbours the same epitopes as the corresponding fragment in the wild type ENV protein. According to a particular embodiment, the fragment of the mutated ENV protein of the invention can comprise about 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 amino acids. These values are given only in an illustrative way, as the man skilled in the art will understand that the fragment can comprise any number of amino acids comprised between 40 and 700.
Advantageously, the fragments according to the invention are such that, while retaining the antigenic structure of the full length mutated ENV protein, and thus of the wild type ENV protein, they have lost major antigenic regions that are responsible for antigenicity in another region than the region corresponding to the immunosuppressive domain.
The invention also relates to a pharmaceutical composition wherein the active substance is an isolated non naturally occurring mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, as above defined, or fragments thereof.
In a particular embodiment, the invention relates to a pharmaceutical composition as defined above comprising as active substance :
a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either
Xa is A, F, G, L or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G or R, or
Xa is A, F, G, L or R, and Xb is A, F, G or R, or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid, and either
Xa is A, F, G, L or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G or R, or Xa is A, F, G, L or R, and Xb is A, F, G or R, in association with a pharmaceutically acceptable carrier.
The invention relates to a pharmaceutical composition as defined above comprising as active substance :
a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein
X represents any amino acid,
and either
Xa is A, F, G, L or R, and Xb is L, I, V, M or P, or
Xa is Y, I, H, C or T, and Xb is A, F, G or R, or
Xa is A, F, G, L or R, and Xb is A, F, G or R,
or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein
X represents any amino acid, and either
Xa is A, F, G, L or R, and Xb is L, I, V, M or P, or
Xa is Y, I, H, C or T, and Xb is A, F, G or R, or
Xa is A, F, G, L or R, and Xb is A, F, G or R, in association with a pharmaceutically acceptable carrier.
According to a particular embodiment, the invention relates to a pharmaceutical composition as defined above comprising as active substance :
a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R,
or
Xa is F or L, and Xb is F, G or R,
or
Figure imgf000022_0001
or
Xa is A, G or R, and Xb is F, G or R, or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids, said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R,
or
Xa is F or L, and Xb is F, G or R,
or
Figure imgf000023_0001
or
Xa is A, G or R, and Xb is F, G, R, in association with a pharmaceutically acceptable carrier.
According to a particular embodiment, the invention relates to a pharmaceutical composition as defined above comprising as active substance :
a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is L, I, V, M or P,
or Xa is Y, I, H, C or T, and Xb is F, G or R,
or
Xa is F or L, and Xb is F, G or R,
or
Figure imgf000024_0001
or
Xa is A, G or R, and Xb is F, G or R, or
b) at least one fragment of said isolated mutated human or simian lenti viral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is L, I, V, M or P,
or
Xa is Y, I, H, C or T, and Xb is F, G or R,
or
Xa is F or L, and Xb is F, G or R,
or
Figure imgf000024_0002
or
Xa is A, G or R, and Xb is F, G or R, in association with a pharmaceutically acceptable carrier.
According to another particular embodiment, the invention also relates to a pharmaceutical composition as defined above comprising as active substance : a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein,
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R, or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein,
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R, in association with a pharmaceutically acceptable carrier.
According to another particular embodiment, the invention relates to a pharmaceutical composition as defined above, comprising as active substance : a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is L, I, V, M or P,
or
Xa is Y, I, H, C or T, and Xb is F, G or R, or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is L, I, V, M or P,
or
Xa is Y, I, H, C or T, and Xb is F, G or R, in association with a pharmaceutically acceptable carrier.
According to another particular embodiment, the invention relates to a pharmaceutical composition as defined above, wherein
Xa is R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is R, or
Figure imgf000027_0001
According to another particular embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the following amino acid sequences :
A-I-E-K-Xa-Xb-X-DQ (SEQ ID NO: 422),
A-I-E-R-Xa-Xb-X-DQ (SEQ ID NO: 423),
A-V-E-K-Xa-Xb-X-DQ (SEQ ID NO: 424),
A-V-E-R-Xa-Xb-X-DQ (SEQ ID NO: 425).
In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein the resulting immunosuppressive domain, contained in the mutated lentiviral protein, comprises the amino acid sequence of the list consisting of: SEQ ID NO :9 to SEQ ID NO :37.
In the invention "SEQ ID NO : 9 to SEQ ID NO :37" encompass SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : l l , SEQ ID NO : 12, SEQ ID NO : 13, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO : 17, SEQ ID NO : 18, SEQ ID NO : 19, SEQ ID NO : 20, SEQ ID NO : 21, SEQ ID NO : 22, SEQ ID NO : 23, SEQ ID NO : 24, SEQ ID NO : 25, SEQ ID NO : 26, SEQ ID NO : 27, SEQ ID NO : 28, SEQ ID NO : 29, SEQ ID NO : 30, SEQ ID NO : 31, SEQ ID NO : 32, SEQ ID NO : 33, SEQ ID NO : 34, SEQ ID NO : 35, SEQ ID NO : 36 and SEQ ID NO : 37.
The correspondence is the following one:
AVERALKD SEQ ID NO : 9 AVERYAKD SEQ ID NO : 14
AVERFLKD SEQ ID NO : 10 AVERYFKD SEQ ID NO : 15
AVERGLKD SEQ ID NO : 11 AVERYGKD SEQ ID NO : 16
AVERLLKD SEQ ID NO : 12 AVERYRKD SEQ ID NO : 17
AVERRLKD SEQ ID NO : 13 AVERAAKD SEQ ID NO : 18 AVERAFKD SEQ ID NO : 19 AVERGRKD SEQ ID NO : 29
AVERAGKD SEQ ID NO : 20 AVERLAKD SEQ ID NO : 30
AVERARKD SEQ ID NO : 21 AVERLFKD SEQ ID NO : 31
AVERFAKD SEQ ID NO : 22 AVERLGKD SEQ ID NO : 32
AVERFFKD SEQ ID NO : 23 AVERLRKD SEQ ID NO : 33
AVERFGKD SEQ ID NO : 24 AVERRAKD SEQ ID NO : 34
AVERFRKD SEQ ID NO : 25 AVERRFKD SEQ ID NO : 35
AVERGAKD SEQ ID NO : 26 AVERRGKD SEQ ID NO : 36
AVERGFKD SEQ ID NO : 27 AVERRRKD SEQ ID NO : 37
AVERGGKD SEQ ID NO : 28
In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein the resulting immunosuppressive domain, contained in the mutated lentiviral protein, comprises the amino acid sequence of the list consisting of: SEQ ID NO :38 to SEQ ID NO :66.
In the invention "SEQ ID NO : 38 to SEQ ID NO :66" encompass SEQ ID NO : 38, SEQ ID NO : 39, SEQ ID NO : 40, SEQ ID NO : 41 , SEQ ID NO : 42, SEQ ID NO : 43, SEQ ID NO : 44, SEQ ID NO : 45, SEQ ID NO : 46, SEQ ID NO : 47, SEQ ID NO : 48, SEQ ID NO : 49, SEQ ID NO : 50, SEQ ID NO : 51, SEQ ID NO : 52, SEQ ID NO : 53, SEQ ID NO : 54, SEQ ID NO : 55, SEQ ID NO : 56, SEQ ID NO : 57, SEQ ID NO : 58, SEQ ID NO : 59, SEQ ID NO : 60, SEQ ID NO : 61, SEQ ID NO : 62, SEQ ID NO : 63, SEQ ID NO : 64, SEQ ID NO : 65 and SEQ ID NO : 66.
The correspondence is the following one:
AIEKYAKD SEQ ID NO : 43
AIEKALKD SEQ ID NO : 38 AIEKYFKD SEQ ID NO : 44
AIEKFLKD SEQ ID NO : 39 AIEKYGKD SEQ ID NO : 45
AIEKGLKD SEQ ID NO : 40 AIEKYRKD SEQ ID NO : 46
AIEKLLKD SEQ ID NO : 41 AIEKAAKD SEQ ID NO : 47
AIEKRLKD SEQ ID NO : 42 AIEKAFKD SEQ ID NO : 48 AIEKAGKD SEQ ID NO : 49 AIEKGRKD SEQ ID NO : 58
AIEKARKD SEQ ID NO : 50 AIEKLAKD SEQ ID NO : 59
AIEKFAKD SEQ ID NO : 51 AIEKLFKD SEQ ID NO : 60
AIEKFFKD SEQ ID NO : 52 AIEKLGKD SEQ ID NO : 61
AIEKFGKD SEQ ID NO : 53 AIEKLRKD SEQ ID NO : 62
AIEKFRKD SEQ ID NO : 54 AIEKRAKD SEQ ID NO : 63
AIEKGAKD SEQ ID NO : 55 AIEKRFKD SEQ ID NO : 64
AIEKGFKD SEQ ID NO : 56 AIEKRGKD SEQ ID NO : 65
AIEKGGKD SEQ ID NO : 57 AIEKRRKD SEQ ID NO : 66
In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein the resulting immunosuppressive domain, contained in the mutated lentiviral protein, comprises the amino acid sequence of the list consisting of: SEQ ID NO : 67 to SEQ ID NO : 95.
In the invention "SEQ ID NO : 67 to SEQ ID NO :95" encompass SEQ ID NO : 67, SEQ ID NO : 68, SEQ ID NO : 69, SEQ ID NO : 70, SEQ ID NO : 71, SEQ ID NO : 72, SEQ ID NO : 73, SEQ ID NO : 74, SEQ ID NO : 75, SEQ ID NO : 76, SEQ ID NO : 77, SEQ ID NO : 78, SEQ ID NO : 79, SEQ ID NO : 80, SEQ ID NO : 81, SEQ ID NO : 82, SEQ ID NO : 83, SEQ ID NO : 84, SEQ ID NO : 85, SEQ ID NO : 86, SEQ ID NO : 87, SEQ ID NO : 88, SEQ ID NO : 89, SEQ ID NO : 90, SEQ ID NO : 91, SEQ ID NO : 92, SEQ ID NO : 93, SEQ ID NO : 94 and SEQ ID NO : 95.
The correspondence is the following one:
AIERALKD SEQ ID NO : 67 AIERAAKD SEQ ID NO : 76
AIERFLKD SEQ ID NO : 68 AIERAFKD SEQ ID NO : 77
AIERGLKD SEQ ID NO : 69
AIERAGKD SEQ ID NO : 78
AIERLLKD SEQ ID NO : 70
AIERARKD SEQ ID NO : 79
AIERRLKD SEQ ID NO : 71
AIERYAKD SEQ ID NO : 72 AIERFAKD SEQ ID NO : 80
AIERYFKD SEQ ID NO : 73
AIERFFKD SEQ ID NO : 81
AIERYGKD SEQ ID NO : 74
AIERYRKD SEQ ID NO : 75 AIERFGKD SEQ ID NO : 82 AIERFRKD SEQ ID NO : 83 AIERLGKD SEQ ID NO : 90
AIERGAKD SEQ ID NO : 84 AIERLRKD SEQ ID NO : 91
AIERGFKD SEQ ID NO : 85 AIERRAKD SEQ ID NO : 92
AIERGGKD SEQ ID NO : 86 AIERRFKD SEQ ID NO : 93
AIERGRKD SEQ ID NO : 87 AIERRGKD SEQ ID NO : 94
AIERLAKD SEQ ID NO : 88 AIERRRKD SEQ ID NO : 95
AIERLFKD SEQ ID NO : 89
As mentioned above, the previous ENV proteins having their ISU comprising the above sequence are devoid of immunosuppressive properties.
Thus, in other words, any ENV protein of human or simian lentivirus having in their ISU an amino acid sequence comprising of the sequences SEQ ID NO: 9 to 95, is devoid of immunosuppressive properties.
In other words, a simian or human lentiviral ENV protein comprising, within its ISU domain, an amino acid sequence selected from SEQ ID NO: 9 to 95 is devoid of immunosuppressive properties.
In particular, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences :
SEQ ID NO : 13, SEQ ID NO : 42, SEQ ID NO : 71,
SEQ ID NO : 9 to 12,
SEQ ID NO : 14 to 41,
SEQ ID NO : 43 to 70, and
SEQ ID NO : 72 to 95.
In particular, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences :
SEQ ID NO : 13, SEQ ID NO : 42, SEQ ID NO : 71,
SEQ ID NO : 9, 11, 15 to 21, 23 to 29, 31 to 38, 40, 44 to 50, 52 to 58, 60 to 67, 69, 73 to 79, 81 to 87, 89 to 95.
In the invention, the fragments of the mutated ENV proteins according to the invention comprise or consist of the following sequences: SEQ ID NO: 67 to 211.
These fragments are also devoid of immunosuppressive properties. However, these fragments retain the structure and the antigenicity of the corresponding immunosuppressive domain that is not mutated, i.e. the wild type immunosuppressive domain.
In the invention, the fragments of the mutated ENV proteins according to the invention comprise or consist of the following sequences: SEQ ID NO: 96 to 211.
The correspondences are as follows
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERAL
SEQ ID NO : 96 KDQQLLG
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERFL
SEQ ID NO : 97 KDQQLLG
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERGL
SEQ ID NO : 98 KDQQLLG
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERLL
SEQ ID NO : 99 KDQQLLG
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERRL SEQ ID NO : KDQQLLG 100
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERYA SEQ ID NO : KDQQLLG 101
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERYF SEQ ID NO : KDQQLLG 102
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERYG SEQ ID NO : KDQQLLG 103
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERYR SEQ ID NO : KDQQLLG 104
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERAA SEQ ID NO : KDQQLLG 105
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERAF SEQ ID NO : KDQQLLG 106
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERAG SEQ ID NO : KDQQLLG 107
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERAR SEQ ID NO : KDQQLLG 108
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERFA SEQ ID NO : KDQQLLG 109
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERFF SEQ ID NO : KDQQLLG 110
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERFG SEQ ID NO : KDQQLLG 111
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERFR SEQ ID NO : KDQQLLG 112
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERGA SEQ ID NO : KDQQLLG 113 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERGF SEQ ID NO : KDQQLLG 114
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERGG SEQ ID NO : KDQQLLG 115
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERGR SEQ ID NO : KDQQLLG 116
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERLA SEQ ID NO : KDQQLLG 117
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERLF SEQ ID NO : KDQQLLG 118
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERLG SEQ ID NO : KDQQLLG 119
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERLR SEQ ID NO : KDQQLLG 120
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERRA SEQ ID NO : KDQQLLG 121
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERRF SEQ ID NO : KDQQLLG 122
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERRG SEQ ID NO : KDQQLLG 123
SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERRR SEQ ID NO : KDQQLLG 124
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKA SEQ ID NO : 125 LKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKF SEQ ID NO : 126 LKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKG SEQ ID NO : 127 LKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKL SEQ ID NO : 128 LKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKR SEQ ID NO : 129 LKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKY SEQ ID NO : 130 AKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKY SEQ ID NO : 131 FKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKY SEQ ID NO : 132 GKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKY SEQ ID NO : 133 RKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKA SEQ ID NO : 134 AKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKA SEQ ID NO : 135 FKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKA SEQ ID NO : 136 GKDQAQLNSWGCAFRQ AGIVQQQQQLLDVVKRQQEMLRLTVWGTK LQARVTAIEKA SEQ ID NO : 137 RKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTK LQARVTAIEKF SEQ ID NO : 138 AKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTK LQARVTAIEKF SEQ ID NO : 139 FKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTK LQARVTAIEKF SEQ ID NO : 140 GKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTK LQARVTAIEKF SEQ ID NO : 141 RKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKG SEQ ID NO : 142 AKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKG SEQ ID NO : 143 FKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKG SEQ ID NO : 144 GKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKG SEQ ID NO : 145 RKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKL SEQ ID NO : 146 AKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKL SEQ ID NO : 147 FKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKL SEQ ID NO : 148 GKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKL SEQ ID NO : 149 RKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKR SEQ ID NO : 150 AKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKR SEQ ID NO : 151 FKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKR SEQ ID NO : 152 GKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKR SEQ ID NO : 153 RKDQAQLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKA
SEQ ID NO : 154 LQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKF
SEQ ID NO : 155 LQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKG
SEQ ID NO : 156 LQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKL
SEQ ID NO : 157 LQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKR
SEQ ID NO : 158 LQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKY
SEQ ID NO : 159 AQDQARLNSWGCAFRQ AGIVQQQQQLLDVVKRQQELLRLTVWGTK LQARVTAIEKY
SEQ ID NO : 160 FQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTK LQARVTAIEKY
SEQ ID NO : 161 GQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTK LQARVTAIEKY
SEQ ID NO : 162 RQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTK LQARVTAIEKA
SEQ ID NO : 163 AQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTK LQARVTAIEKA
SEQ ID NO : 164 FQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKA
SEQ ID NO : 165 GQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKA
SEQ ID NO : 166 RQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKF
SEQ ID NO : 167 AQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKF
SEQ ID NO : 168 FQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKF
SEQ ID NO : 169 GQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKF
SEQ ID NO : 170 RQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKG
SEQ ID NO : 171 AQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKG
SEQ ID NO : 172 FQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKG
SEQ ID NO : 173 GQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKG
SEQ ID NO : 174 RQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKL
SEQ ID NO : 175 AQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKL
SEQ ID NO : 176 FQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKL
SEQ ID NO : 177 GQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKL
SEQ ID NO : 178 RQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKR
SEQ ID NO : 179 AQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKR
SEQ ID NO : 180 FQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKR
SEQ ID NO : 181 GQDQARLNSWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKR
SEQ ID NO : 182 RQDQARLNSWGCAFRQ AGIVQQQQQLLDVVKRQQELLRLTVWGTK LQTRVTAIEKA SEQ ID NO : 183 LKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTK LQTRVTAIEKF SEQ ID NO : 184 LKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKG SEQ ID NO : 185 LKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKL SEQ ID NO : 186 LKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKR SEQ ID NO : 187 LKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKY SEQ ID NO : 188 AKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKY SEQ ID NO : 189 FKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKY SEQ ID NO : 190 GKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKY SEQ ID NO : 191 RKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKA SEQ ID NO : 192 AKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKA SEQ ID NO : 193 FKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKA SEQ ID NO : 194 GKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKA SEQ ID NO : 195 RKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKF SEQ ID NO : 196 AKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKF SEQ ID NO : 197 FKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKF SEQ ID NO : 198 GKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKF SEQ ID NO : 199 RKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKG SEQ ID NO : 200 AKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKG SEQ ID NO : 201 FKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKG SEQ ID NO : 202 GKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKG SEQ ID NO : 203 RKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKL SEQ ID NO : 204 AKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKL SEQ ID NO : 205 FKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKL SEQ ID NO : 206 GKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKL SEQ ID NO : 207 RKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKR SEQ ID NO : 208 AKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKR SEQ ID NO : 209 FKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKR SEQ ID NO : 210 GKDQAQLNAWGCAFRQ
AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKR SEQ ID NO : 211 RKDQAQLNAWGCAFRQ
In particular, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences : SEQ ID NO : 96, 98, 100, 102 to 108, 110 to 1 16, 1 18 to 125, 127, 129, 131 to 137, 139 to 145, 147 to 154, 156, 158, 160 to 166, 168 to 174, 176 to 183, 185, 187, 189 to 195, 197 to 203, 205 to 211.
Advantageously, the invention relates to the pharmaceutical composition as defined above, wherein said mutated protein consists of one of the following sequences SEQ ID NO : 212 to 269
HIV-1 LAI
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 212
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGrVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL
AVERALKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI
WN MTWMEWDREINNYTSLIHSLIEESQNQQEK EQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 213
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAJEAQQHLLQLTVWGIKQLQARIL
AVERFLKDQOI I (ilWGCSGKLICTTAVPWNASWSNKSLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 214
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGTVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL
AVERGLKDQQLLGIWGCSGKLICTTAVPWNASWSN SLEQI
WNNMTWMEWDREIN YTSL1HSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 215 YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKTLLFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINM WQE VGKAM Y APPI S GQIRC S SNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR.IL
AVERLLKDQQ L I XJIWGCSGKLICTTAVPWNAS WSNKSLEQ1
WNN TWMEWDREINNYTSLIHSLTEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 216
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNI SRAKWN ATLKQI ASKLREQFGNNK 111FKQ S S GGDPEI V
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRArEAQQHLLQLTVWGIKQLQARlL
AVERRLKDQQLLGIWGCSGKLICTTAVPWNASWSN SLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 217
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAJEAQQHLLQLTVWGIKQLQARIL
AVERYAKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI
WNNMTWM.EWDREINNYTSL1H.SLIEESQNQQE NEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 218
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLC VSLKCTDLGNATNTNS SNTNS S SGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSG1VQQQNNLL.RA1EAQQHLLQLTVWGI .QLQARIL
AVERYFKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 219
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKC NKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQ NLLRAIEAQQHLLQLTVWGI QLQARIL
AVERYGKDQQLLGIWGCSG LICTTAVPWNASWSNKSLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQE NEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 220
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLC VSLKCTDLGNATNTNS SNTNS S SGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAJEAQQHLLQLTVWGIKQLQARIL
AVERYRKD QQ L LGI WGC S G KL ICTT AVP WN A S WSNKSLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQE NEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 221
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL
AVERAAKDQQLLGIWGCSGKLICTTAVPWNAS WSNKSLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQE NEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 222 YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRP NNTR SIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKTLLFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR.IL
AVERAFKDQQLLGIWGCSGI LICTTAVPWNASWSN SLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 223
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNI SRAKWN ATLKQI ASKLREQFGNNK 111FKQ S S GGDPEI V
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARIL
AVERAGKDQQLLGIWGCSGKLICTTAVPWNASWS KSLEQI
WNN TWMEWDRETNNYTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 224
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKXREQFGNNKTllFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAJEAQQHLLQLTVWGIKQLQARIL
AVERARKD QQ L LGI WGC S G KL ICTT AVP WN A S WSNKS LEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQE NEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 225
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGI QLQAR1L
AVERFAKDQO I 1 GIWGCSGKL1CTTAVPWNASWSNKSLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEK EQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 226
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKC NKTFNGTGPCTNVS
T VQCTHGIRP VVSTQLLLNGSL AEEEVVIRS A^ F 1 DNAKTII V
QLNQSVEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR.IL
AVERFFKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI
WN MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 227
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC
VKLTPLC VSLKCTDLGNATNTNS SNTNS S SGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKTllFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARJL
AVERFGKDQQLLGIWGCSGKLICTTAVPWNASWSN SLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQE NEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 228
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKTllFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL
AVERFRKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI
WNNMTW EWDREINNYTSLIHSLIEESQNQQE EQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 229
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NC SFNI STSIRGKVQKE Y AFF YKLDIIPIDNDTT S YTLT S CNT S
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAJEAQQHLLQLTVWGIKQLQARIL
AVERGAKDQQLLGIWGCSG LICTTAVPWNASWSNKSLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEK EQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 230
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQ NLLRAIEAQQHLLQLTVWGI QLQARIL
AVERGFKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQE NEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 231 YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKTLLFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR.IL
AVERGGKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 232
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNI SRAKWN ATLKQI ASKLREQFGNNK 111FKQ S S GGDPEI V
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGI QLQARIL
AVERGRKDQQ I L GIWGCSGKLICTTAVPWNASWSNKSLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 233
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAJEAQQHLLQLTVWGIKQLQARIL
AVERLAKDQQ 1 1 GIWGCSGKLICTTAVPWNASWSNKSLEQI
WN MTWMEWDREINNYTSLIHSLIEESQNQQEK EQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 234
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLC VSLKCTDLGNATNTNS SNTNS S SGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKTllFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARIL
AVERLFKDQQ I LGIWGCSGKLICTTAVPWNASWS KSLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 235
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKC NKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKTllFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARIL
AVERLGKDQQLLGIWGCSGKLICTTAVPWNASWSN SLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 236
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAJEAQQHLLQLTVWGIKQLQARIL
AVERLRKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI
WN MTWMEWDREIN YTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 237
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFG NKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR1L
AVERRAKDQQLLG1WGCSGKLICTTAVPWNASWSN SLEQI
WNNMTWMEWDREIN YTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 238 YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRP NNTR SIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKTLLFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGS NTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
G NNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR.IL
AVERRFKDQQLLGIWGCSGI LICTTAVPWNASWSN SLEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 239
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV
QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKHIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARIL
AVERRGKDQQLLGIWGCSGKLICTTAVPWNASWS KSLEQI
WNN TW EWDRETNNYTSLIHSLIEESQNQQEK EQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY SEQ ID NO : 240
YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP
NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC
VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK
NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS
VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS
TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA
HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV
THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT
LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG
GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV
QARQLLSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARIL
AVERRRKD QQ L LGI WGC S G KL ICTT AVP WN A S WSNKS LEQI
WNNMTWMEWDREINNYTSLIHSLIEESQNQQE NEQELLEL
DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV
NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL
VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW
EALKYWWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
HIV-BH10
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 241
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSD HI LPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVERA
LKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREI NYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 242
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKC NKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKW NTLKQIDSKLREQFG NKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSD H I LPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERF
LKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 243
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI
KQIINM WQE VGKAM Y APPI S GQIRC S SNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERG
LKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 244
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKW NTLKQIDSKLREQFG NKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVERL
LKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 245
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERR
LKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM
TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 246
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVERY
AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 247 YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERY
FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMT
WMEWDREI NYTSLIHSLIEESQNQQEK EQELLELDKWASL
WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ
GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA
LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW
WNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQGAY
RAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 248
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEF CTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIF MWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVERY
GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREI NYTSLIHSLIEESQNQQEKNEQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 249
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKC NKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRP NNTR SIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERY
RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 250
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINM WQE VGKAM Y APPI S GQIRC S SNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERA
AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 251
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKC NKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKW NTLKQIDSKLREQFG NKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERA
FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMT
WMEWDREI NYTSLIHSLIEESQNQQEK EQELLELDKWASL
WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ
GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA
LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW
WNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQGAY
RAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 252
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERA
GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 253
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
C VKLTPLC V SLKCTDLKNDTNTNS S S GRMIMEKGEIK C SFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKW NTLKQIDSKLREQFG NKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVERA
RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 254
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERF
AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM
TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 255
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINM WQE VGKAM Y APPI S GQIRC S SNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERF
FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NMT
WMEWDREI NYTSLIHSLIEESQNQQEK EQELLELDKWASL
WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ
GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA
LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW
WNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQGAY
RAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 256 YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRPNNNTRKSIMQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERF
GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM
TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 257
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVERF
RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 258
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKC NKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKW NTLKQIDSKLREQFG NKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERG
AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 259
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERG
FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMT
WMEWDREI NYTSLIHSLIEESQNQQEK EQELLELDKWASL
WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ
GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA
LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW
WNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQGAY
RAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 260
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKW NTLKQIDSKLREQFG NKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVERG
GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 261
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKW NTLKQIDSKLREQFG NKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERG
RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM
TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 262
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVERL
AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 263 YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWK DMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERL
FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMT
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASL
WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ
GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA
LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW
WNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQGAY
RAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 264
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEF CTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI
KQIF MWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVERL
GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREF NYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 265
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKC NKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRP NNTR SIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERL
RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM
TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 266
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERR
AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 267
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKC NKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKW NTLKQIDSKLREQFG NKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERR
FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMT
WMEWDREI NYTSLIHSLIEESQNQQEK EQELLELDKWASL
WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ
GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA
LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW
WNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQGAY
RAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 268
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERR
GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY
WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV SEQ ID NO : 269
YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD
PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP
CVKLTPLCVSLKCTDLK DTNTNSSSGRMIMEKGEIK CSFNI
STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT
HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS
VEINCTRP NNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS
RAKW NTLKQIDSKLREQFG NKTIIFKQSSGGDPEIVTHSFN
CGGEFFYCNSTQLFNSTWFNSTWSTKGS NTEGSDTITLPCRI
KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN
NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR
RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL
LSGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVERR
RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIW NM
TWMEWDREINNYTSLIHSLIEESQNQQEK EQELLELDKWAS
LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR
QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL
ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELK SAVSLLNATAIAVAEGTDRVIEVVQG
AYRAIRHIPRRIRQGLERILL
The invention also encompasses the variants of the above sequences, harbouring the above mentioned mutations, and conferring to said variant a lack of immunosuppressive properties.
In particular, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein consists of one of the amino acid sequences : SEQ ID NO : 212, 214, 216, 218 to 224, 226 to 232, 234 to 241, 243, 245, 247 to 253, 255 to 261, 263 to 269.
The invention also relates to a pharmaceutical composition as defined above, wherein said mutated protein comprises additional mutations either downstream the C-terminal end of the sequence SEQ ID NO : 416 or upstream the N-terminal end of SEQ ID NO : 416.
These additional mutations can be advantageous to maintain the three-dimensional structure of the immunosuppressive domain and of the ENV protein (see details concerning the membrane expression of the ENV protein [see figure 4 and example] and concerning infectivity [see figure 5 and example]).
In a particular embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated protein comprises an additional mutation in one at least of the amino acids at positions 29, 36 and 37 of SEQ ID NO: 426 :
A[ ]E[K/R]XaXbXiDQX2X3LX4X5WGC[A/S][F/G]X6X7CVX8TX9VPXcXioZiZ2Z3Z4Z5 XdXe[S/T] (SEQ ID NO: 426)
wherein
Xa and Xb are as defined above,
Xi to Xio represent any amino acid,
Zi to Z5 represent no amino acid or any amino acid, independently from each other such that
o Xc is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, or deleted, preferably A, D, or N, o Xd is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, Y, W, or deleted, preferably A, G, S or Y,
o Xe is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, Y, W, or deleted, preferably A, D or N.
This sequence contains the following amino acid sequence A[I/V]E[K/R]XaXbXiDQ (SEQ ID NO : 416) elongated on its C-terminal end in which additional mutations are present. It is to be noted that "Xi" in the above mentioned sequence SEQ ID NO : 426 has the same meaning as "X" in SEQ ID NO : 416.
In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, said mutated protein comprising the amino acid sequences of the group consisting of SEQ ID NO: 271 to SEQ ID NO: 283.
SEQ ID NO: 271, contains the mutations Y41R, K72A:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER LKDQQLLGIWGCSG
KLICTTAVPWNASWSNASLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEK
NEQEL.
SEQ ID NO: 272, contains the mutations Y41R, K72G:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERMLKDQQLLGIWGCSG
KLICTTAVPWNASWSNGSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEK
NEQEL.
SEQ ID NO: 273, contains the mutations Y41R, K72S:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERMLKDQQLLGIWGCSG KLICTTAVPWNASWSNSSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEK NEQEL.
SEQ ID NO: 274, contains the mutations Y41R, W65D: SGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVER LKDQQLLGIWGCSG KLICTTAVPDNASWSNKSLEQIWNNMTWMEWDREI NYTSLIHSLIEESQNQQEK NEQEL.
SEQ ID NO: 275, contains the mutations Y41R, W65A:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERMLKDQQLLGIWGCSG KLICTTAVPANASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEK NEQEL.
SEQ ID NO: 276, contains the mutations Y41R, W65N:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERMLKDQQLLGIWGCSG KLICTTAVPNNASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEK NEQEL.
SEQ ID NO: 277, contains the mutations Y41R, S73D:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERELKDQQLLGIWGCSG
KLICTTAVPWNASWSNKDLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEK
NEQEL.
SEQ ID NO: 278, contains the mutations Y41R, S73A:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERMLKDQQLLGIWGCSG
KLICTTAVPWNASWSNKALEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEK
NEQEL.
SEQ ID NO: 279, contains the mutations Y41R, S73N:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERMLKDQQLLGIWGCSG
KLICTTAVPWNASWSNKNLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEK
NEQEL.
SEQ ID NO: 280, contains the mutations Y41R, K72Y: SGIVQQQ NLLRAIEAQQHLLQLTVWGIKQLQARILAVER LKDQQLLGIWGCSG
KLICTTAVPWNASWSNYSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEK
NEQEL.
SEQ ID NO: 281, contains the mutations R40A, Y41R, K72A:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVEAMLKDQQLLGIWGCSG KLICTTAVPWNASWSNASLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEK NEQEL.
SEQ ID NO: 282, contains the mutations Y41R, K72A, S73A:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERRLKDQQLLGIWGCSG
KLICTTAVPWNASWSNAALEOIWNNMTWMEWDREINNYTSLIHSLIEESONOOE
NEQEL.
SEQ ID NO: 283, contains the mutations Y41R, W65A, K72A, S73A:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERRLKDQQLLGIWGCSG KLICTTAVPANASWSNAALEOIWNNMTWMEWDREINNYTSLIHSLIEESONOOE NEQEL.
The mutations in the above mutated peptides are indicated by the amino acid residues which are bolded and underlined.
In another aspect, the invention relates to a pharmaceutical composition as defined above, comprising an additional mutation of one at least of the amino acids Xn, X12, X13 and X14 in the following sequence (containing the above defined SEQ ID NO: 416 sequence) : X11X12 X13X14I LA[I/V]E[K/R]XaXbXiDQ (SEQ ID NO: 428),
wherein
Xi represents any amino acid,
Xa and Xb are as defined above,
Xn is :
either deleted,
- or A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, Y or W, in particular A, G or R, and/or Xi2 is :
either deleted,
- or A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, Y or W, in particular A, G or R, and/or either deleted,
- or C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y or W, in particular R or G, and/or
Xi4 is :
either deleted,
- or A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, Y or W, in particular A or G.
This sequence contains the following amino acid sequence A[I/V]E[K/R]XaXbXiDQ (SEQ ID NO : 416) elongated on its N-terminal end in which additional mutations are present. It is to be noted that "Xi" in SEQ ID NO : 428 has exactly the same meaning as "X" in SEQ ID NO : 416.
In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated lentiviral protein, said variant or said fragment harbour a three-dimensional structure similar to the structure of the natural non mutated lentiviral ENV protein, non mutated lentiviral ENV variant or non mutated lentiviral ENV fragment thereof.
The skilled person knows how to measure the antigenicity, by using standard proceedings.
In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated lentiviral protein, said variant or said fragment are expressed at the plasma membrane at a level substantially identical to the expression at the plasma membrane of the natural non mutated lentiviral ENV protein, non mutated lentiviral ENV variant or non mutated lentiviral ENV fragment thereof.
The membrane expression of the lentiviral ENV protein according to the invention can be measured by any techniques allowing determination of a plasma membrane protein. For instance, cells can be transfected with an expression vector allowing the expression of the mutated lentiviral ENV protein according to the invention. Cells are then incubated with an antibody recognizing specifically the extracellular part of said lentiviral mutated ENV protein. The complex (antibody/ENV protein) is detected by another antibody, and the complex can be quantified by flow cytometry (see for instance figure 4, and example). If no complex is detected, the mutated ENV protein is not expressed at the plasma membrane. On the contrary, if the protein is expressed, this means that the mutated ENV protein is expressed at the plasma membrane.
In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated lentiviral ENV protein is such that a lentivirus, or a pseudotype, expressing said mutated lentiviral ENV protein, instead of the non mutated ENV protein, has a viral titer similar to the viral titer of said lentivirus, or pseudotype, expressing the non mutated lentiviral protein.
Viral titer is measured according to a commonly used protocol, and as described in the example (see figure 5).
In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated lentiviral ENV protein is a HIV-1 lentiviral protein consisting of the amino acid sequences SEQ ID NO: 284 to SEQ ID NO: 292.
SEQ ID NO MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY
Y41R GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP
QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL
TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN
ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP
KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI
RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC
TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN
ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY
CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ
EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG
GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV
GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL
RAIEAQQHLLQLTVWGIKQLQARILAVERMLKDQQLLGIWGC
SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT
SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI
KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
SEQ ID NO: 285 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY Y41R, K72A GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP
QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL
TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN
ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP
KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI
RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC
TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN
ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY
CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ
EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG
GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV
GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL
RAIEAQQHLLQLTVWGIKQLQARILAVERMLKDQQLLGIWGC
SGKLICTTAVPWNASWSNASLEQIWNNMTWMEWDREINNYT
SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI
KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP
DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL
RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS
LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
SEQ ID NO: 286 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY Y41R, K72G GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP
QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL
TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN
ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP
KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI
RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC
TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN
ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY
CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ
EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG
GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV
GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL
RAIEAQQHLLQLTVWGIKQLQARILAVERELKDQQLLGIWGC
SGKLICTTAVPWNASWSNGSLEQIWNNMTWMEWDREINNYT
SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI
KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP
DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL
RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS
LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
SEQ ID NO: 287 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY Y41R, K72S GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP
QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL
TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN
ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI
RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC
TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN
ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY
CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ
EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG
GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV
GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL
RAIEAQQHLLQLTVWGIKQLQARILAVERMLKDQQLLGIWGC
SGKLICTTAVPWNASWSNSSLEQIWNNMTWMEWDREINNYTS
LIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIK
IFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPD
RPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLR
DLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSL
LNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
SEQ ID NO: 288 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY Y41G GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP
QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL
TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN
I ST SIRGKVQKE Y AFF YKLDIIPIDNDTTS YTLT S CNT S VITQ ACP
KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI
RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC
TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN
ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY
CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ
EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG
GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV
GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL
RAIEAQQHLLQLTVWGIKQLQARILAVERGLKDQQLLGIWGC
SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT
SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI
KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP
DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL
RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS
LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
SEQ ID NO: 289 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY Y41L GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP
QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL
TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN
ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP
KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI
RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC
TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN
ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY
CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ
EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG
GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV
GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERLLKDQQLLGIWGC
SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT
SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI
KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP
DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL
RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS
LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
SEQ ID NO: 290 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY Y41A GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP
QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL
TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN
ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP
KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI
RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC
TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN
ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY
CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ
EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG
GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV
GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL
RAIEAQQHLLQLTVWGIKQLQARILAVERALKDQQLLGIWGC
SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT
SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI
KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP
DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL
RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS
LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
SEQ ID NO: 291 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY Y41F GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP
QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL
TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN
ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP
KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI
RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC
TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN
ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY
CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ
EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG
GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV
GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL
RAIEAQQHLLQLTVWGIKQLQARILAVERFLKDQQLLGIWGC
SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT
SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI
KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP
DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL
RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS
LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
SEQ ID NO: 292 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY L42R GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP
QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL
TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN
ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP
KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI
RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC
TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN
ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY
CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ
EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG
GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV
GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL
RAIEAQQHLLQLTVWGIKQLQARILAVERYMKDQQLLGIWGC
SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT
SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI
KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP
DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL
RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS
LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
Also, the following proteins are advantageous in the invention:
SEQ ID NO: 293 MGCLGNQLLIAILLLSVYGIYCTQYVTVFYGVPAWRNATIPLFC SIV MM251 ATKNRDTWGTTQCLPDNGDYSELALNVTESFDAWENTVTEQA L42R IEDVWQLFETSIKPCVKLSPLCITMRCNKSETDRWGLTKSSTTIT
TAAPTSAPVSEKIDMVNETSSCIAQNNCTGLEQEQMISCKFTMT
GLKRDKTKEYNETWYSTDLVCEQGNSTDNESRCYMNHCNTS
VIQESCDKHYWDTIRFRYCAPPGYALLRCNDTNYSGFMPKCSK
VVVSSCTRMMETQTSTWFGFNGTRAENRTYIYWHGRDNRTIIS
LNKYYNLTMKCRRPGNKTVLPVTIMSGLVFHSQPINDRPKQA
WCWFGGKWKDAIKEVKQTIVKHPRYTGTNNTDKINLTAPGGG
DPEVTFMWTNCRGEFLYCKMNWFLNWVEDRDVTTQRPKERH
RRNYVPCHIRQIINTWHKVGKNVYLPPREGDLTCNSTVTSLIAN
IDWTDGNQTSITMSAEVAELYRLELGDYKLVEITPIGLAPTDVK
RYTTGGTSRNKRGVFVLGFLGFLATAGSAMGAASLTLTAQSR
TLLAGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIE
KYRKDQAQLNAWGCAFRQVCHTTVPWPNASLTPDWNNDTW
QEWERKVDFLEENITALLEEAQIQQEKNMYELQKLNSWDVFG
NWFDLASWIKYIQYGIYVVVGVILLRIVIYIVQMLAKLRQGYRP
VFSSPPSYFQXTHTQQDPALPTREGKEGDGGEGGGNSSWPWQI
EYIHFLIRQLIRLLTWLFSNCRTLLSRAYQILQPILQRLSATLRRV
REVLRTELTYLQYGWSYFHEAVQAGWRSATETLAGAWRDLW
ETLRRGGRWILAIPRRIRQGLELTLL
SEQ ID NO: 294 MEPGRNQLFVVILLTSACLVYCSQYVTVFYGIPAWKNASIPLFC HIV2 L42R ATKNRDTWGTIQCLPDNDDYQEIILNVTEAFDAWNNTVTEQA
VEDVWHLFETSIKPCVKLTPLCVAMNCSRVQGNTTTPNPRTSS STTSRPPTSAASIINETSNCIENNTCAGLGYEEMMQCEFNMKGL EQDKKR YKDTWYLEDVVCDNTTAGTCYMRHCNTSIIKESCD
KHYWDAMRFRYCAPPGFALLRCNDTNYSGFEPKCTKVVAASC
TRMMETQTSTWFGFNGTRAENRTYIYWHGRDNRTIISLNKYY
NLTMRCKRPGNKTVLPITLMSGLVFHSQPINTRPRQAWCRFGG
RWREAMQEVKQTLVQHPRYKGINDTGKINFTKPGAGSDPEVA
FMWTNCRGEFLYCNMTWFLNWVEDKNQTRRNYCHIKQIINT
WHKVGKNVYLPPREGELACESTVTSIIANIDIDKNRTHTNITFS
AEVAELYRLELGDYKLIEITPIGFAPTDQRRYSSTPVRNKRGVF
VLGFLGFLATAGSAMGARSLTLSAQSRTLLAGIVQQQQQLLDV
VKRQQEMLRLTVWGTKNLQARVTAIEKYRKHQAQLNSWGC
AFRQ VCHTT VP WVND SL SPD WKNMT WQE WEKQ VRYLE ANI S
QSLEEAQIQQEKNMYELQKLNSWDILGNWFDLTSWVKYIQYG
VHIVVGIIALRIAIYVVQLLSRFRKGYRPVFSSPPGYLQQIHIHK
DRGQPANEGTEEDVGGDSGYDLWPWPINYVQFLIHLLTRLLIG
LYNICRDLLSKNSPTRRLISQSLTAIRDWLRLKAAQLQYGCEWI
QEAFQAFARTTRETLAGAWGWLWEAARRIGRGILAVPRRIRQ
GAELALL
Advantageously: the mutated lentiviral proteins are
1 - SEQ ID NO: 284 to 294; these mutated ENV proteins having substantially no immunosuppressive properties
advantageously,
2- SEQ ID NO: 284 to SEQ ID NO: 292; these mutated ENV proteins having substantially no immunosuppressive properties and being highly expressed at the plasma membrane, more advantageously,
3- SEQ ID NO: 284 to SEQ ID NO: 291; these mutated ENV proteins having substantially no immunosuppressive properties, being highly expressed at the plasma membrane and conferring to a virus expressing them a medium or high viral titer,
in particular
4- Y41F (SEQ ID NO: 291), Y41L (SEQ ID NO: 289), Y41A (SEQ ID NO: 290); these mutated ENV proteins having substantially no immunosuppressive properties, being highly expressed at the plasma membrane and confers to a virus expressing them a high viral titer. As mentioned above "confering to a virus expressing them a medium or high viral titer" means that, when the sequence of the wild type ENV protein is substituted by the sequence of the mutated ENV in the lentiviral or pseudotype retrovirus, the virus thus expresses a mutated lentiviral ENV protein, along with other wild type viral proteins (GAG, PRO, POL), said mutated ENV protein conferring to the virus the ability to enter in its target cell: either at a level comparable or higher to the ability of the wild type virus, i.e. high ability
or at a lower level compared to the ability of the wild type virus, i.e. medium or low ability.
The ability to enter in the target cell can be measured by the viral load. Viral load is a measure of the amount of target cells that can be infected by a given amount (1 mL) of virus (see figure 5 and example).
The invention also relates to a pharmaceutical composition comprising a nucleic acid molecule coding for a mutated lentiviral ENV protein, or variant of said protein, or fragments thereof, as defined above, in association with a pharmaceutically acceptable carrier.
In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said nucleic acid is comprised in a vector, said vector comprising means allowing the expression of said mutated lentiviral ENV protein, or variant of said protein, or fragments thereof.
In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said nucleic acid is comprised in a vector, said nucleic acid being placed under the control of sequences that allow the expression of said mutated lentiviral ENV protein, or variant of said protein, or fragments thereof.
In another embodiment, the invention relates to a pharmaceutical composition, as defined above, in particular as a vaccine, comprising a DNA molecule coding for said mutated lentiviral ENV protein, or variant of said protein, or fragments thereof.
DNA vaccines expressing ENV proteins can be produced as described in Bellier et al, (DNA vaccines expressing retrovirus- like particles are efficient immunogens to induce neutralizing antibodies, Vaccine, 27(42):5772-80, 2009).
In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, said vector being chosen among an eukaryotic or prokaryotic expression vector , in particular an eukaryotic vector which is a viral vector, in particular a pox vector, such as a fowlpox, a canarypox, or a MVA (modified vaccinia virus Ankara) vector, an adenoviral vector, a lentiviral vector, a measles vector, a Senda'i virus or a CMV (cytomegalovirus) vector.
In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, further comprising at least one nucleotide molecule coding for a GAG and/or a PRO and/or a POL protein and/or a mutated NEF protein substantially devoid of immunosuppressive properties, of a lentivirus, preferably a lentivirus of the same origin as the mutated lentiviral ENV protein.
The advantageous mutated Nef protein contains a substitution at position 93, as described in the international application WO 2006/018289 (Inventors : Renard M., Mangeney M. and Heidmann T.) and a deletion of the N-terminus of the protein involved in its myristoylation.
GAG expression will produce virus like particles (VLPs) which are particularly advantageous for a vaccine, in particular if the ENV protein is associated with the VLP (Guerbois et al, Virology, 388: 191-203, 2009).
In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains said at least one nucleic acid molecule coding for a GAG protein and/or a PRO protein and/or a POL protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains all said at least one nucleic acid molecule coding for a GAG protein and/or a PRO protein and/or a POL protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in a vector which is different from the at least one vector containing said at least one nucleic acid molecule coding for a GAG protein and/or a PRO protein and/or a POL protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, said nucleic acid molecule coding for a GAG protein, said nucleic acid molecule coding for a PRO protein, said nucleic acid molecule coding for a POL protein and said nucleic acid molecule coding for a mutated NEF protein substantially devoid of immunosuppressive properties, are all contained in vectors which are different from each other.
In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, comprising at least one nucleic acid molecule coding for a GAG protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties,
of a human or simian lentivirus, said lentivirus being preferably of the same origin as the mutated lentiviral ENV protein.
In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains said at least one nucleic acid molecule coding for a GAG protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains a nucleic acid molecule coding for a GAG protein and for a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains a nucleic acid molecule coding for a GAG protein and for a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties,
said vector being preferably a measles vector (Guerbois et al, Virology, 388: 191-203, 2009) or a canary pox vector (Poulet et al, Veterinary Record, 153(5): 141-145, 2003 ; Vaccari et al, Expert Review of Vaccines, Vol. 9, No 9, pages 997-1005, 2010).
In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in a vector which is different from the at least one vector containing said at least one nucleic acid molecule coding for a GAG protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, said nucleic acid molecule coding for a GAG protein and said nucleic acid molecule coding for a mutated NEF protein substantially devoid of immunosuppressive properties, are all contained in vectors which are different from each other.
The invention also relates to a pharmaceutical composition according the above definition, for its use as vaccine, for the treatment or the prevention of lentiviral infection, in particular HIV-1 infection, HIV-2 infection and SIV infection.
As mentioned above the pharmaceutical composition according to the invention encompasses a vaccine, comprising as active substance, a protein as defined above, or a nucleic acid as defined above, or a vector as defined above, or a combination thereof.
In a particular embodiment, the pharmaceutical composition according to the invention encompasses a vaccine, comprising a nucleic acid coding for a GAG protein as defined above, a nucleic acid coding for a mutated ENV protein as defined above and a nucleic acid coding for a mutated NEF protein as defined above.
In a particular embodiment, the pharmaceutical composition according to the invention encompasses a vaccine as defined above, comprising a nucleic acid coding for a GAG protein as defined above, a nucleic acid coding for a mutated ENV protein as defined above and a nucleic acid coding for a mutated NEF protein as defined above, which are contained in a same vector, said same vector being preferably a canary pox vector or a measles vector, more preferably a measles vector.
In a particular embodiment, the pharmaceutical composition according to the invention encompasses a vaccine, comprising a GAG protein as defined above, a mutated ENV protein as defined above and a mutated NEF protein as defined above.
In a particular embodiment, the pharmaceutical composition according to the invention encompasses a vaccine, comprising a GAG protein as defined above, a mutated ENV protein as defined above and a mutated NEF protein as defined above which are associated to at least one adjuvant. A comprehensive list of adjuvants that can be used in HIV vaccines is indicated in the FRANKLIN PIERCE LAW CENTER EDUCATIONAL REPORT: PATENT LANDSCAPE OF ADJUVANT FOR HIV VACCINES which is incorporated herein by reference. This list includes:
- Aluminum-based compounds such as aluminum phosphate and aluminum
hydroxide.
Immunostimulatory adjuvants like CpG, MPL and QS21 orMF59 which is a squalene oil-in water emulsions.
- Freunds incomplete adjuvant (FIA) or Freund's complete adjuvant (FCA), can also be used.
Calcium phosphate in particular orthophosphates, metaphosphates or
pyrophosphates and occasionally hydrogen or hydroxide ions.
Muramyl dipeptide (N- Acetyl muramyl-L-alanine-D-isoglutamine, MDP) and its synthetic analogs such as muramyl tripeptide phosphatidylethanolamine
MTPPTdEtn),
- Trehalose-6,6'-dimycolate,
- Saponins (Triterpenoid glycosides) like QS-21
- Stearyl Tyrosine (octadecyl ester hydrochloride salt of tyrosine)
Immunostimulatory adjuvants can include the following classes of compounds:
Polysaccharides like:
o Chitosan:
o Inulin: o Beta - Glucans
o Lipo-Polysaccharides or endotoxin
o MGN-3Actinidia eriantha (AEPS):
o Eldexomer.
o CpG ODN
- Liposomes like
o Dehydration-rehydration liposome vesicles (DRVs)
o Cytotoxic T lymphocyte (CTL)
o CAF01
o Liposomes containing lipid A (LA)
- Lipid Polysine Core Peptides (LCP)
- Cytokine like GM-CSF, IL-2.IL-12 or IL-15 Lipid A and Monophosphoryl Lipid A (MPL)
- Lipopeptide which are molecules consisting of lipid connected to a peptide. They are derived from the lipoprotein of bacterial cell wall.
Exogenous Immunostimulatory Adjuvants which are compounds, or proteins that are not found in the human body like:
o Proteosomes in particular Multiple Antigenic Peptides (MAP) or Exogenous Toxins
Suitably, the total amount mutated lentiviral ENV protein in a single dose of the immunogenic composition is l(^g and/or the total amount of unfused polypeptide in a single dose of the immunogenic composition is 2(^g. In one embodiment, the total amount of all antigens in a single dose of the immunogenic composition is 0.5-5(^g, 2-4(^g, 5- 3(^g, 10μ-2(^ or around 3(^g, around 2(^g or around l(^g. The amount of mutated lentiviral ENV protein in a dose of the immunogenic composition is selected as an amount which induces an immune response without significant, adverse side effects in typical recipients. Such amount will vary depending upon which specific immunogen is employed and the dosing or vaccination regimen that is selected. An optimal amount for a particular immunogenic composition can be ascertained by standard studies involving observation of relevant immune responses in subjects.
Administration of the pharmaceutical composition can take the form of one or of more than one individual dose, for example as repeat doses of the same polypeptide containing composition, or in a heterologous "prime-boost" vaccination regime, including proteins and vectors. In one embodiment, the immunogenic composition of the invention is initially administered to a subject as two or three doses, wherein the doses are separated by a period of two weeks to three months, preferably one month.
Conveniently, the composition is administered to a subject (for instance as a booster) every 6-24, or 9-18 months, for instance annually. For instance, the composition is administered to a subject (for instance as a booster) at six month or 1 year intervals. Suitably in this respect, subsequent administrations of the composition to the subject boost the immune response of earlier administrations of the composition to the same subject.
In an embodiment, the immunogenic composition of the invention is used as part of a prime -boost regimen for use in the treatment or prevention of disease or infection by HIV- 1 strains from one or more clades different from the one or more HIV-1 clades in the immunogenic composition. Conveniently, the composition is the priming dose. Alternatively, the composition is the boosting dose.
Suitably, two or more priming and/ or boosting doses are administered. A heterologous prime-boost regime uses administration of different forms of immunogenic composition or vaccine in the prime and the boost, each of which can itself include two or more administrations. The priming composition and the boosting composition will have at least one antigen in common, although it is not necessarily an identical form of the antigen, it can be a different form of the same antigen.
Prime boost immunisations according to the invention can be homologous prime-boost regimes or heterologous prime-boost regimes. Homologous prime-boost regimes utilize the same composition for prime and boost, for instance the immunogenic composition of the invention. Heterologous prime-boost regimes can be performed with a combination of protein and DNA- based formulations. Such a strategy is considered to be effective in inducing broad immune responses. Adjuvanted protein vaccines induce mainly antibodies and CD4+ T cell immune responses, while delivery of DNA as a plasmid or a recombinant vector induces strong CD8+ T cell responses. Thus, the combination of protein and DNA vaccination can provide for a wide variety of immune responses. This is particularly relevant in the context of HIV, since neutralizing antibodies, CD4+ T cells and CD8+ T cells are thought to be important for the immune defense against HIV-1.
The invention also relates to a pharmaceutical composition according the above definition, for its use for the treatment of lentiviral infection.
The invention also relates to a method for treating patient afflicted by pathologies related to lentiviral infection, comprising the administration to a patient in a need thereof of a pharmaceutically efficient amount of the pharmaceutical composition as defined above. The invention also relates to a method for inducing an immune response against a lentivirus that has infected an individual, said method comprising the administration to a patient in a need thereof of a pharmaceutically efficient amount of the pharmaceutical composition as defined above.
The invention also relates to a pharmaceutical composition as defined above, further comprising an antiviral agent.
The composition according to the invention can also be used in combination with the antiviral compositions listed in Table 1 below:
Intelence® (TMC 125/etravirine) Tibotec - Non-nucleoside reverse transcriptase inhibitor
Agenerase®(APV/amprenavir) GSK - Protease inhibitor
Aptivus (TPV/tipranavir) Boehringer - Protease inhibitor
Crixivan®(IDV/indinavir) MSD - Protease inhibitor
Invirase (SQV/saquinavir) Roche - Protease inhibitor
Kaletra®(LPV.r/lopinavir + ritonavir) Protease inhibitor
Abbott
Norvir (ritonavir) Abbott - Protease inhibitor
Prezista®(TMC 114/darunavir) Protease inhibitor Tibotec/Janssen-Cilag -
Reyataz®(ATZ/atazanavir) BMS - Protease inhibitor
Telzir®(APV/fosamprenavir) GSK - Protease inhibitor
Viracept (nelfinavir) Roche - Protease inhibitor
Fuzeon®(T20/enfuvirtide) Roche - Fusion inhibitor
Celsentri (maraviroc) Pfizer - Entry inhibitor
Isentress®(MK 0518/raltegravir) Merck - Integrase inhibitor
Rescriptor (delavirdine) Agouron - Non-nucleoside reverse transcriptase inhibitor
Sustiva®(EFV/efavirenz) BMS - Non-nucleoside reverse transcriptase inhibitor
Viramune®(nevirapine) Boehringer - Non-nucleoside reverse transcriptase inhibitor
Combivir®(Retrovir®+Epivir®) GSK - Nucleoside reverse transcriptase inhibitor
Emtriva®(FTC, emtricitabine) Gilead - Nucleoside reverse transcriptase inhibitor
Epivir®(3TC, lamivudine) GSK - Nucleoside reverse transcriptase inhibitor
Kivexa®(Ziagen®+ Epivir®) GSK - Nucleoside reverse transcriptase inhibitor
Retrovir®(AZT/zidovudine) QSK - Nucleoside reverse transcriptase inhibitor
Trizivir (Retrovir + Epivir + Ziagen ) Nucleoside reverse transcriptase inhibitor
GSK -
Videx®(ddI/didanosine) BMS - Nucleoside reverse transcriptase inhibitor
Viread®(TDF/tenofovir) Gilead - Nucleoside reverse transcriptase inhibitor
Zerit®(d4T/stavudine) BMS - Nucleoside reverse transcriptase inhibitor
Ziagen®(ABC/abacavir) GSK - Nucleoside reverse transcriptase inhibitor
Truvada®(Emtriva® + Viread®) Gilead - Nucleoside reverse transcriptase inhibitor
Atripla®(Sustiva®+ Emtriva®+ Viread®) Nucleoside and non-nucleoside reverse transcriptase inhibitor
BMS / GILEAD
Table 1 : Trade names of the different antivirals used for HIV treatment, as well as their specificity of action.
The invention also relates to a pharmaceutical composition as defined above, for simultaneous, separate or sequential use.
The invention relates to a pharmaceutical composition as defined above for its use for stimulating an immune response in a host organism.
The invention relates to a pharmaceutical composition as defined above for its use for inducing a specific immune response against the HIV ENV protein.
The invention relates to a pharmaceutical composition as defined above for its use for the prevention or the treatment of HIV infection, or pathologies related to AIDS.
The invention relates to a pharmaceutical composition as defined above, as a vaccine, for its use for inducing a specific immune response against the HIV ENV protein. The invention relates to a pharmaceutical composition as defined above, as a vaccine, for its use for the prevention or the treatment of HIV infection, or pathologies related to AIDS.
In another aspect, the invention also relates to a method to obtain the active substance of a pharmaceutical composition, as defined above, consisting of modifying the immunosuppressive property of:
a wild-type human or simian lentiviral ENV protein,
or a fragment of said wild-type human or simian lentiviral ENV protein, said fragment comprising at least 40 amino acids,
said ENV protein or fragment thereof presenting a transmembrane subunit (TM) comprising an immunosuppressive domain (ISU) containing the following amino acid sequence :
A-[I/V]-E-[K/R]-X'a-X'b-X-D-Q (SEQ ID NO: 427),
wherein
X represents any amino acid,
X'a is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and
X'b is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, said method comprising a step of introduction of at least one mutation of X'a and/or X'b, to obtain:
an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein having at least 70% identity, preferably at least 80% identity, to one sequence chosen from the group consisting of SEQ ID NO : 216, SEQ ID NO : 420 and SEQ ID NO : 421,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence : A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein
X represents any amino acid,
and either
Xa is A, F, G, L, R or deleted, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G, R or deleted, or Xa is A, F, G, L, R or deleted, and Xb is A, F, G, R or deleted, said substantial absence of immunosuppressive activity of the above mentioned mutated human or simian lentiviral ENV protein or of the above defined fragment being liable to be assessed by the fact that in an in vivo assay involving engrafted tumor cells rejection, said tumor cells being transduced either so as to express said mutated ENV protein or said fragment (mutated ENV tumor cells),
or said tumor cells being transduced so as to express said wild type ENV protein or a fragment thereof (wild type ENV tumor cells),
or said tumor cells being not transduced (normal tumor cells),
the following ratio :
immunosuppression index of said mutated ENV protein or of said fragment ( ated env ) / immunosuppression index of wild type ENV protein (iwiid type env ) is less than 0.5, imutated env being defined by : (maximum area reached by mutated ENV tumor cells - maximum area reached by normal tumor cells) / (maximum area reached by normal tumor cells), and
iwiid type env being defined by : (maximum area reached by wild type ENV tumor cells - maximum area reached by normal tumor cells) / (maximum area reached by normal tumor cells).
The invention also relates to a method to obtain the active substance of a pharmaceutical composition, according to the above defined method, consisting of modifying the immunosuppressive property of :
a wild-type human or simian lentiviral ENV protein,
or a fragment of said wild-type human or simian lentiviral ENV protein, said fragment comprising at least 40 amino acids, said ENV protein or fragment thereof presenting a transmembrane subunit (TM) comprising an immunosuppressive domain (ISU) containing the following amino acid sequence :
A-[I/V]-E-[K/R]-Y-L-X-D-Q (SEQ ID NO : 1), wherein
X represents any amino acid, said method comprising a step of introduction of at least one mutation of Y in position 5 and/or L in position 6, to obtain:
an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, F, G, L, R or deleted, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G, R or deleted, or Xa is A, F, G, L, R or deleted, and Xb is A, F, G, R or deleted.
The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,
wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, F, G, L or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G or R, or
Xa is A, F, G, L or R, and Xb is A, F, G or R.
The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition, ,
wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, F, G, L or R, and Xb is L, I, V, M or P, or
Xa is Y, I, H, C or T, and Xb is A, F, G or R, or
Xa is A, F, G, L or R, and Xb is A, F, G or R.
The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition, wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R, or
Xa is F or L, and Xb is F, G or R, or
Figure imgf000088_0001
The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,
wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is L, I, V, M or P, or
Xa is Y, I, H, C or T, and Xb is F, G or R, or
Xa is F or L, and Xb is F, G or R, or Xa is A, G or R, and Xb is A, or
Xa is A, G or R, and Xb is F, G or R.
The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,
wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R.
The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,
wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either Xa is A, G, or R, and Xb is L, I, V, M or P, or
Xa is Y, I, H, C or T, and Xb is F, G or R.
The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,
wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either
Xa is R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is R, or
Xa is R, and Xb is R.
The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,
wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutation in one at least of the amino acids at positions, 29, 36 and 37 of SEQ ID NO: 426 :
A[ ]E[K/R]XaXbXiDQX2X3LX4X5WGC[A/S][F/G]X6X7CVX8TX9VPXcXioZiZ2Z3Z4Z5 XdXe[S/T] (SEQ ID NO: 426) wherein
Xa and Xb are as defined in anyone of claims 1 to 15,
Xi to Xio represent any amino acid,
Zi to Z5 represent no amino acid or any amino acid, independently from each other such that
o Xc is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, or deleted, preferably A, D, or N,
o Xd is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, Y, W, or deleted, preferably A, G, S or Y,
o Xe is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, Y, W, or deleted, preferably A, D or N.
The present invention relates to a pharmaceutical composition comprising as active substance :
a) an isolated non naturally occurring mutated human or simian lentiviral ENV having substantially no immunosuppressive activity, said mutated human or simian lentiviral ENV resulting from mutation of the transmembrane subunit (TM) of a wild type human or simian lentiviral ENV protein, the transmembrane subunit comprising an immunosuppressive domain (ISU) comprising the following amino acid sequence:
A-[I/V]-E-[K/R]-Y-L-X-D-Q (SEQ ID NO: I),
said sequence encompassing
AIEKYLXDQ (SEQ ID NO: 2), AIERYLXDQ (SEQ ID NO: 3), A VEKYLXDQ (SEQ ID NO: 4) and A VERYLXDQ (SEQ ID NO: 5), wherein X represents any natural amino acid,
wherein the amino acids at the positions chosen among
- the position 4 of SEQ ID NO: 1,
- the position 5 of SEQ ID NO: 1,
- the position 6 of SEQ ID NO: 1,
- the positions 4 and 5 of SEQ ID NO: I,
- the position 4 and 6 of SEQ ID NO: I,
- the position 5 and 6 of SEQ ID NO: I, and - the position 4, 5 and 6 of SEQ ID NO: I,
are :
either deleted,
or substituted by another amino acid such that
o when the immunosuppressive domain comprises SEQ ID NO : 2 or 4
■ the amino acid residue at position 4 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y, and/or
■ the amino acid residue at position 5 of SEQ ID NO: I is substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T, V or W, and/or
■ the amino acid residue at position 6 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, 7, K, M, N, P, Q, R, S, T, V, W or Y,
o when the immunosuppressive domain comprises SEQ ID NO : 3 or 5,
■ the amino acid residue at position 4 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H I, K, L, M, N, P, Q, S, T, V, W or Y, and/or
■ the amino acid residue at position 5 of SEQ ID NO: I is substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T, V or W, and/or
■ the amino acid residue at position 6 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H K, M, N, P, Q, R, S, T, V, W or Y, or
b) a fragment of said mutated human or simian lentiviral ENV, said fragment comprising at least the immunosuppressive domain (ISU) of said wild type human or simian lentiviral ENV comprising the following amino acid sequence:
A-[I/V]-E-[K/R]-Y-L-X-D-Q (SEQ ID NO: 1),
wherein the amino acids at the positions chosen among
- the position 4 of SEQ ID NO: 1,
- the position 5 of SEQ ID NO: 1,
- the position 6 of SEQ ID NO: 1,
- the positions 4 and 5of SEQ ID NO: I,
- the position 4 and 6 of SEQ ID NO: I,
- the position 5 and 6 of SEQ ID NO: I, and
- the position 4, 5 and 6 of SEQ ID NO: I,
are : either deleted,
or substituted by another amino acid such that
o when the immunosuppressive domain comprises SEQ ID NO : 2 or 4
■ the amino acid residue at position 4 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S T, V, W or Y, and/or
■ the amino acid residue at position 5 of SEQ ID NO: I is substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T, V or W, and/or
■ the amino acid residue at position 6 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y,
o when the immunosuppressive domain comprises SEQ ID NO : 3 or 5,
■ the amino acid residue at position 4 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y, and/or
■ the amino acid residue at position 5 of SEQ ID NO: I is substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T, V or W, and/or
■ the amino acid residue at position 6 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y, said fragment having substantially no immunosuppressive properties,
in association of a pharmaceutically acceptable carrier.
In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein the amino acids at the positions of the group consisting of
- the position 4 of SEQ ID NO: 1,
- the position 5 of SEQ ID NO: 1,
- the position 6 of SEQ ID NO: 1,
- the positions 4 and 5 of SEQ ID NO: I,
- the position 4 and 6 of SEQ ID NO: I,
- the position 5 and 6 of SEQ ID NO: I, and
- the position 4, 5 and 6 of SEQ ID NO: I,
are substituted by another amino acid such that
o when the immunosuppressive domain comprises SEQ ID NO : 2 or 4
■ the amino acid residue at position 4 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y, and/or ■ the amino acid residue at position 5 of SEQ ID NO: I is substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T V or W, and/or
■ the amino acid residue at position 6 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T V, W or Y,
o when the immunosuppressive domain comprises SEQ ID NO : 3 or 5,
■ the amino acid residue at position 4 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T V, W or Y, and/or
■ the amino acid residue at position 5 of SEQ ID NO: I is substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T, V or W, and/or
■ the amino acid residue at position 6 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y.
Advantageously, the mutated lentiviral protein according to the invention, comprised in the above defined composition, comprises the amino acid sequence of the list consisting of: SEQ ID NO: 407 to SEQ ID NO: 414.
The correspondence is the following one:
Figure imgf000094_0001
These proteins have been mutated at position 4 of SEQ ID NO: 1, by substitution, and the amino acid at position 5 has been substituted by A.
In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein the amino acids at the positions of the group consisting of
- the position 5 of SEQ ID NO: 1,
- the position 6 of SEQ ID NO: 1, and
- the position 5 and 6 of SEQ ID NO: 1,
are substituted by another amino acid such that
■ the amino acid residue at position 5 of SEQ ID NO: 1 is substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T V or W, and/or ■ the amino acid residue at position 6 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, K, M, N, P, Q, R, S, T V, W or Y.
In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said immunosuppressive domain (ISU) comprises the amino acid sequence of the group consisting of: SEQ ID NO : 4 or 5.
In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said immunosuppressive domain (ISU) comprises the amino acid sequence SEQ ID NO : 5, and wherein
■ the amino acid residue at position 5 of SEQ ID NO: 5 is substituted by A, F, G, L or R, or
■ the amino acid residue at position 6 of SEQ ID NO: 5 is substituted by A,
F, G or R, or
■ the amino acid residues at position 5 and 6 of SEQ ID NO: 5 are substituted by A, F, G, or R.
This is the case when said ENV protein is the ENV protein of HIV I virus.
The invention also relates to a pharmaceutical composition as defined above, wherein said immunosuppressive domain (ISU) comprises the amino acid sequence SEQ ID NO : 2, and wherein
■ the amino acid residue at position 5 of SEQ ID NO: 2 is substituted by A, F,
G, L or R, or
■ the amino acid residue at position 6 of SEQ ID NO: 2 is substituted by A, F, G or R, or
■ the amino acid residues at position 5 and 6 of SEQ ID NO: 2 are substituted by A, F, G, or R.
The invention also relates to a pharmaceutical composition as defined above, wherein said immunosuppressive domain (ISU) comprises the amino acid sequence SEQ ID NO : 3, and wherein ■ the amino acid residue at position 5 of SEQ ID NO: 3 is substituted by A, F,
G, L or R, or
■ the amino acid residue at position 6 of SEQ ID NO: 3 is substituted by A, F, G or R, or
■ the amino acid residues at position 5 and 6 of SEQ ID NO: 3 are substituted by A, F, G, or R.
In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated protein further comprises the wild type amino acid sequence SEQ ID NO: 270,
XjDQX sLX WGC[A/S][F/G]XeX7CVX8TX9 VPWXwZ^ZsZ^fN/S] '[E/D/N/A JfS/TJ (SEQ ID NO: 270)
wherein Xi-Xw represents any amino acid,
wherein Zi to Z5 represent no amino acid or any natural amino acid, independently from each other,
said mutated lentiviral protein further comprising a mutation in one at least of the amino acids at positions 23, 30 and 31 of SEQ ID NO: 270
said mutation being :
either a deletion,
or a substitution such that
o the amino acid at position 25 of SEQ ID NO: 270 is substituted by A, C, D,
E, F G, H, I, K, L, M, N, P, Q, R, S, T, V, Y
o the amino acid at position 32 of SEQ ID NO: 270 is substituted by A, C, D,
E, F G, H, I, K, L, M, P, Q, R, T, V, Y W,
o the amino acid at position 33 of SEQ ID NO: 270 is substituted by C, F, G,
H, I, K, L, M, P, Q, R, S, T, V, Y W.
In one advantageous embodiment, the invention relates to a pharmaceutical composition comprising as active substance :
an isolated non naturally occurring mutated human or simian lentiviral ENV having substantially no immunosuppressive activity, said mutated human or simian lentiviral ENV resulting from mutation of a wild type human or simian lentiviral ENV protein, said wild type human or simian lentiviral ENV protein comprising the following amino acid sequence:
A[I/V]E[K/R]YLXlDQX2XsLX4X5WGC[A/S][F/G]X6X7CVX8TX9VPWXwZlZ2Z3Z4Z5[N/S][ E/D/N/AJ fS/TJ (SEQ ID NO: 415),
wherein Xi-Xio represents any amino acid,
wherein Zi to Z5 represent no amino acid or any natural amino acid, independently from each other,
wherein the amino acids at the positions chosen among
- the position 4 of SEQ ID NO: I,
- the position 5 of SEQ ID NO: 1,
- the position 6 of SEQ ID NO: 1,
- the positions 4 and 5 of SEQ ID NO: 1,
- the position 4 and 6 of SEQ ID NO: 1,
- the position 5 and 6 of SEQ ID NO: 1, and
- the position 4, 5 and 6 of SEQ ID NO: 1,
are :
either deleted,
or substituted by another amino acid such that
■ the amino acid residue at position 4 of SEQ ID NO: 1 is substituted by A, C, D, E, F, G, H, I, L, M, N, P, Q, S, T, V, W or Y, and/or
■ the amino acid residue at position 5 of SEQ ID NO: 1 is substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T, V or W, and/or
■ the amino acid residue at position 6 of SEQ ID NO: 1 is substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y, and further wherein
one at least of the amino acids at positions 29, 36 and 37 of the amino acid sequence SEQ ID NO : 415 are mutated, by
either a deletion,
or a substitution such that
o the amino acid at position 29 of SEQ ID NO: 415 is substituted by A, C, D, E, F G, H, I, K, L, M, N, P, Q, R, S, T, V, Y o the amino acid at position 36 of SEQ ID NO: 415 is substituted by A, C, D,
E, F, G, H, /, K, L, M, P, Q, R, T, V, Y W,
o the amino acid at position 37 of SEQ ID NO: 415 is substituted by C, F, G,
H, I K, L, M, P, Q, R, S, T, V, Y, W.
In another aspect, the invention relates to a pharmaceutical composition as defined above, further comprising a mutation at the position X4
X1DQX2X3LX4X5 WGC[A/S][F/G]XeX7CVX8TX9 VPWXwZlZ2Z3Z4Z5[N/S] [E/D/N/A JfS/TJ (SEQ ID NO: 270)
wherein X1-X3 andXs-Xw represents any amino acid,
Zi to Z5 represent no amino acid or any amino acid, independently from each other
said mutation being :
either a deletion,
- or a substitution by A, C, D, E, F, H, I, K, L, M, P, Q, R, S, T, V, Y or W.
In one advantageous embodiment, the invention relates to a pharmaceutical composition comprising as active substance :
an isolated non naturally occurring mutated human or simian lentiviral ENV having substantially no immunosuppressive activity, said mutated human or simian lentiviral ENV resulting from mutation of a wild type human or simian lentiviral ENV protein, said wild type human or simian lentiviral ENV protein comprising the following amino acid sequence:
A[I/V]E[K/R]YLXlDQX2X3LX4X5WGC[A/S][F/G]X6X7CVX8TX9VPWXwZlZ2Z3Z4Z5[N/S][ E/D/N/A] [S/T] (SEQ ID NO: 415),
wherein X1-X10 represents any amino acid,
wherein Zi to Z5 represent no amino acid or any natural amino acid, independently from each other,
wherein the amino acids at the positions chosen among
- the position 4 of SEQ ID NO: 1,
- the position 5 of SEQ ID NO: I,
- the position 6 of SEQ ID NO: 1,
- the positions 4 and 5 of SEQ ID NO: 1, - the position 4 and 6 of SEQ ID NO: 1,
- the position 5 and 6 of SEQ ID NO: I, and
- the position 4, 5 and 6 of SEQ ID NO: I,
are :
either deleted,
or substituted by another amino acid such that
■ the amino acid residue at position 4 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, I, L, M, N, P, Q, S, T, V, W or Y, and/or
■ the amino acid residue at position 5 of SEQ ID NO: I is substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T, V or W, and/or
■ the amino acid residue at position 6 of SEQ ID NO: I is substituted by A, C, D, E, F, G, H, 7, K, M, N, P, Q, R, S, T, V, W or Y, and possibly, further wherein
one at least of the amino acids at positions 29, 36 and 37 of the amino acid sequence SEQ ID NO : 415 are mutated, by
either a deletion,
or a substitution such that
o the amino acid at position 29 of SEQ ID NO: 415 is substituted by A, C, D,
E, F G, H, I, K, L, M, N, P, Q, R, S, T, V, Y
o the amino acid at position 36 of SEQ ID NO: 415 is substituted by A, C, D,
E, F G, H, I, K, L, M, P, Q, R, T, V, Y W,
o the amino acid at position 37 of SEQ ID NO: 415 is substituted by C, F, G, H, I, K, L, M, P, Q, R, S, T, V, Y W,
and/or possibly further wherein the amino acid at position X4 is either deleted or substituted by A, C, D, E, F, H, I, K, L, M, P, Q, R, S, T, V, Y or W, preferably by R or H.
LEGEND TO THE FIGURES
Figure 1 :
Amino acid sequences of the HIV ectodomain deletants, with the indicated length, and of single mutant (substitution are underlined) ectodomains. Figure 2:
Functional delineation of the immunosuppressive domain of the HIV envelope. The immunosuppressive activity of 115aa-long and truncated HIV envelope ectodomains (see structures on the left) was tested using the MCA205 tumor rejection in vivo assay. Immunosuppression indexes are given as histograms on the right (mean values +/- SD).
Figure 3 :
Functional identification of the aminoacid in the HIV envelope ectodomain directly involved in immunosuppressive activity, and search for aminoacid substitutions inhibiting this activity. Immunosuppresive activity was tested in vivo as in Figure 2.
Figure 4 :
Expression profile of mutated HIV envelopes using full-length HIVenv-expressing vectors and an anti-SU HIVenv(l 10H) monoclonal antibody (FACS). The data are expressed as percentages of the control HIVenv WT.
Figure 5 :
Infectivity of HIVenv-pseudotyped viral particles showing functionality of the full-length WT and of specifically mutated HIV envelopes. The results are expressed as viral titers (number of infected target cells/mL of virus supernatant).
Figure 6 :
Functional identification of the amino acid in the SIV envelope ectodomain directly involved in immunosuppressive activity, and search for aminoacid substitutions inhibiting this activity. Immunosuppressive activity was tested using the in vivo MCA205 tumor rejection assay. Immunosuppression indexes are represented as histograms (mean values +/- SD).
EXAMPLE
Immunosuppressive domains have been identified on the envelope proteins of oncoretro viruses, of either the gamma- (murine MLV) or the delta (e.g. human HTLV) type, as well as of some endogenous retroviruses (e.g. HERV-FRD). These domains have a highly conserved crystallographic structure, although their primary sequences are quite diverse, and an amino-acid (either Q, E, or K) at a definite position has previously been demonstrated to be essential for the immunosuppresive activity of the corresponding envelope protein. Mutation of this amino acid to an Arginine (R) was further demonstrated to result in inhibition of the immunosuppressive activity of the mutated envelope protein, with in some cases complete conservation of the other functional properties of the envelope protein, including its ability to be normally expressed at the cell membrane, to be captured by a nascent retroviral particle, and finally to confer infectivity of the mutant virus in vitro. Such mutants allowed an unambiguous demonstration of the essential role of the immunosuppressive activity for viremia in vivo, with the mutant IS- virus being unable to escape the host immune system and propagate in an immunocompetent animal [Schlecht-Loufet al, Proc Natl Acad Sci USA. 2010;107(8):3782-7].
On the basis of the identification of an IS domain and on the ability of the IS function to be inhibited by specific mutations within the IS domain that do not disrupt the antigenic structure of the corresponding viral protein, vaccinal approaches are being developed with vaccines containing mutated « optimized » IS-negative viral antigens, which demonstrate an increased immunognicity as compared to those using the native viral antigens. Search for similar IS domains and appropriate mutations in the case of non-oncogenic-but still pathogenic- retroviruses such as HIV is therefore of interest to tentatively develop improved vaccines.
Actually, in the case of the other major class of retroviruses, namely the lentiviruses (among which the human HIV1 and HIV2, and the SIV simian homologues), the crystallographic structure of the envelope protein discloses some similarities but also very important differences, especially in the domain corresponding to the IS domain of oncoretro viruses, with evidence in HIV and SIV for a severely extended helix-loop-helix domain, and no evidence for sequence similarities with the IS domain of oncoretro viruses. Furthermore, an IS domain with strong amino-acid similarities with the IS domain of oncoretroviruses was identified within an accessory protein of HIV and SIV, namely within the Nef protein. This protein is specifically produced by these complex retroviruses and is not encoded by oncoretroviruses. The Nef protein is essential for viremia in vivo, and it possesses several domains responsible for immune escape, among which the identified IS domain, thus strongly suggesting that lentiviruses had transferred the immunosuppressive activity found in oncoretroviruses within their envelope protein, to the accessory Nef protein. In agreement with this hypothesis, Nef-deleted or Nef-mutated retroviruses have a severely attenuated pathogenicity in in vivo macaque animal models. Here, the Inventors report on the identification of an IS activity carried by the HIV and SIV envelope proteins, and on specific mutations that inhibit this activity without major disrupt ion of the overall structure of the corresponding envelope proteins.
Results
Delination of the immunosuppressive domain of the HIV Envelope
Delineation of the immunosuppressive domain of the HIV envelope was achieved using an in vivo tumor rejection assay, that the Inventors had previously used to demonstrate the immunosuppressive activity of the Env protein of oncoretroviruses (murine MoMLV and simian MPMV). The rationale of the assay can be summarized as follows: while injection of MCA205 tumor cells (H-2b) into allogeneic Balb/c mice (H-2d) leads to the formation of no tumor or transient tumors that are rapidly rejected, injection of the same cells, but stably expressing an immunosuppressive retroviral Env protein, leads to the growth of larger tumors that persist for a longer time -in spite of the expression of the new exogenous antigen. This difference is not associated with a difference in intrinsic cell growth rate since it is not observed in syngeneic C57BL/6 mice, and is immune system-dependent. The extent of "immunosuppression" can be quantified by an index based on tumor size: (Aenv- Anone)/Anone, where Aenv and Anone are the mean areas at the peak of growth of tumors from Balb/c mice injected with env-expressing or control cells, respectively. A positive index indicates that env expression facilitates tumor growth, as a consequence of its immunosuppressive activity; a null or negative index points to no effect or even an inhibitory effect, respectively. The latter may be explained by a stimulation of the immune response of the host against the new foreign antigen, represented by a non- immunosuppressive Env protein, expressed at the surface of tumor cells.
To first delineate a minimal immunosuppressive domain (ISD) active in vivo, the Inventors analyzed the effect of a series of truncations/deletions within the HIV env gene. Assay of the series of C-term truncations in Fig. 1&2 identified a 49 residue-long domain with IS activity, with the 6 aa shorter HIV43 fragment and subsequent C-term truncated HIV37 and HIV30 fragments being IS-negative. HIV49 is embedded into the so-called ectodomain, which corresponds to a soluble part of the extracellular domain of the TM subunit, and consists in the a- helical domain involved in HIV TM trimerization, and the N- term part of the loop containing the 2 well-conserved, 6 aa-distant, cysteine residues found in most retroviral envelopes. Refine delineation of the amino acis responsible for IS activity was then performed by arginine-scanning between aa 36 and 51, i.e. in the domain associated with the transition from IS positive to negative Env subdomains. As illustrated in Fig. 3, all the X-to-Arg substitutions resulted in no significant change in the IS activity of HIV115, with one exception for Y41 and L42, which resulted in loss of IS activity of the mutant peptide.
Mutations at position 41
The Inventors then checked that the Y41R substitution did not alter the overall capacity of the HIV envelope to be expressed by an eucaryotic cell and to be exported at the cell membrane, by introducing the Y41R substitution into an expression vector for the HIV envelope. A FACS analysis of cell transfected with both the wild-type and the Y41R mutant using an anti-SU specific monoclonal antibody actually demonstrated quantitative expression of the mutant envelope at the cell surface, thus indicating that the Y41R mutation does not significantly alter the HIV Env structure and SU-TM interaction (figure 4)·
A series of distinct substitutions at the Y41 position were finally performed to tentatively identify whether other amino acids could be substituted to generate IS-negative variants: the amino acid assayed included the positively charged K and H (in addition to R), the small A and G, and the hydrophobic L and F residues. Again it was checked that these substitutions did not alter the overall structure of the HIV Env with the corresponding mutations.
Among them, substitution of Y41 by A, G, L and F resulted in loss of IS (figure 3), and did not alter the overall structure of the HIV Env with the corresponding mutations (figure 4 and 5).
Further mutation outside of the ISU domain
The effects of a set of second mutations at positions that are structurally close to Y41 position within the ENV ectodomain three-dimensional structure were also tested. Two of them (Y41R-K72A and Y41R-K78G) maintain a high level expression at cell membrane and confer infectivity (figure 5). Mutations at position 42
Interestingly, the adjacent position to the position Y41, position L42, also resulted in loss of IS activity of the mutant peptide when the mutation L42R is introduced. This mutation maintains a high level expression at cell membrane of the mutated HIV ENV protein (figure 4).
Mutations within the SIV ENV protein
Interestingly, mutation at the homologous position in SIV ENV (L42) of the position L42 in HIV-1 ENV ectodoamin also resulted in specific loss of IS activities (Figure 6).
Accordingly, the present investigation has clearly identified a definite location and a definite substitution(s) within the HIV env resulting in the loss of its IS activity. Being compatible with the conservation of the overall structure of the human and simian lentiviral Env proteins, these substitutions should be introduced in all pharmaceutical preparations which include the Env protein as a vaccine antigen.
Materials and Methods
Mice and cell lines: C57B1/6 and Balb/c mice, 6-10 weeks old, were obtained from CER Janvier (Laval, France). Mice were maintained in the animal facility of the Gustave Roussy Institute in accordance with institutional regulations. 293T (ATCC CRL 11268), and MCA205 cells were cultured in DMEM supplemented with 10% fetal calf serum (Invitrogen), streptomycin (100 μg/ml) and penicillin (100 units/ml).
Plasmid construction: The PCEL/E160 encoding the envelope protein of the BRU/LAI HIV-1 isolate is a gift from Dr Marc Sitbon. To generate the pDFG plasmids encoding the various fragments of HIV-1 envelope ectodomain, PCR fragments generated using PCEL/E160 as a template and primer pairs 1-2 (pDFG-HIVl 15), 1-3 (pDFG-HIV81), 1-4 (pDFG-HIV67), 1-5 (pDFG-HIV55), 1-6 (pDFG-HIV49), 1-7 (pDFG-HIV43), 1-8 (pDFG- HIV37), 1-9 (pDFG-HIV30) were digested with Sfil and Mlul and inserted into pDFG- ectoSyncytin-1 (Mangeney et al, 2007) opened with the same enzymes.
Mutated pDFG-HIV115 were obtained by successive PCR using appropriate primers. A first series of PCR was performed with pDFG HIV115 as template using primers 1-1 1 and 10-2, 1-13 and 12-2, 1-15 and 14-2 or 1-17 and 16-2 to introduce the E39R, Y41R, K43R or D44R mutations respectively, and primers 1-18 and 19-2, 1-20 and 21-2, 1-22 and 23-2, 1-24 and 25-2, 1-26 and 27-2, 1-28 and 29-2, 1-30 and 31-2, 1-32 and 33-2, 1-34 and 35-2, or 1-36 and 37-2 to introduce the L36R, A37R, V38R, L42R, Q45R, Q46R, L47R, L48R, G49R, or 15 OR mutations respectively. The PCR products were then used as templates in subsequent PCR using primers 1-2. These PCR fragments were digested with Sfil and Mlul and inserted into pDFG-ectoSyncytin-1 (Mangeney et al, 2007) opened with the same enzymes.
All the constructions were sequenced before
Table 1. Primer list
N° Name Primer sequence (5'-3') SEQ ID
1 TM HIV Sfi-Sens ACATggcccagccggccTCTGGTATAGTGCAGCAGC SEQ ID NO: 295
2 TM HIV115 Mlu-AS GTATacgcgtTTATAATTCTTGTTCATTCTTTTC SEQ ID NO: 296
3 TM HIV81 Mlu AS GTATacgcgtTTACATGTTATTCCAAATCTGTTCC SEQ ID NO: 297
4 TM HIV67 Mlu AS GTATacgcgtTTAAGCATTCCAAGGCACAGC SEQ ID NO: 298
5 TM HIV55 Mlu AS GTATacgcgtTTATCCAGAGCAACCCCAAATCC SEQ ID NO: 299
6 TMHIV49 Mlu AS GTATacgcgtTTACCCCAGGAGCTGTTGATCC SEQ ID NO: 300
7 TMHIV43 Mlu AS GTATacgcgtTTACTTTAGGTATCTTTCCACAGC SEQ ID NO: 301
8 TMHIV37 Mlu AS GTATacgcgtTTAAGCCAGGATTCTTGCCTGGAG SEQ ID NO: 302
9 TMHIV30 Mlu AS GTATacgcgtTTACTGCTTGATGCCCCAGAC SEQ ID NO: 303
18 TMHIV115 L36R as CTTTCCACAGCCcGGATTCTTGCCTG SEQ ID NO: 304
19 TMHIV115 L36R s CAGGCAAGAATCCgGGCTGTGGAAAG SEQ ID NO: 305
20 TMHIV115 A37R as GTATCTTTCCACAcgCAGGATTCTTGCC SEQ ID NO: 306
21 TMHIV115 A37R s GGCAAGAATCCTGcgTGTGGAAAGATAC SEQ ID NO: 307
22 TMHIV115 V38R as CTTTAGGTATCTTTCCctAGCCAGGATTCTTGCC SEQ ID NO: 308
23 TMHIV115 V38R s GGCAAGAATCCTGGCTagGGAAAGATACCTAAAG SEQ ID NO: 309
11 TMHIV115 E39R AS CCTTTAGGTATCTTctCACAGCCAGGATTC SEQ ID NO: 310
10 TMHIV115 E39R S GAATCCTGGCTGTGagAAGATACCTAAAGG SEQ ID NO: 311
13 TMHIV115 Y41R AS GTTGATCCTTTAGGcgTCTTTCCACAGCCAG SEQ ID NO: 312
12 TMHIV115 Y41R S CTGGCTGTGGAAAGAcgCCTAAAGGATCAAC SEQ ID NO: 313
24 TMHIV115 L42R as GGAGCTGTTGATCCTTTcGGTATCTTTCCACAGCC SEQ ID NO: 314
25 TMHIV115 L42R s GGCTGTGGAAAGATACCgAAAGGATCAACAGCTCC SEQ ID NO: 315
15 TMHIV115 K43R AS GAGCTGTTGATCCcTTAGGTATCTTTCCAC SEQ ID NO: 316
14 TMHIV115 K43R S GTGGAAAGATACCTAAgGGATCAACAGCTC SEQ ID NO: 317
17 TMHIV115 D44R AS AGGAGCTGTTGAcgCTTTAGGTATCTTT SEQ ID NO: 318
16 TMHIV115 D44R S AAAGATACCTAAAGcgTCAACAGCTCCT SEQ ID NO: 319
26 TMHIV115 Q45R as CCCAGGAGCTGTcGATCCTTTAGGTATC SEQ ID NO: 320
27 TMHIV115 Q45R s GATACCTAAAGGATCgACAGCTCCTGGG SEQ ID NO: 321 N° Name Primer sequence (5'-3') SEQ ID
28 TMHIV115 Q46R as CAAATCCCCAGGAGCcGTTGATCCTTTAG SEQ ID NO: 322
29 TMHIV115 Q46R s CTAAAGGATCAACgGCTCCTGGGGATTTG SEQ ID NO: 323
30 TMHIV115 L47R as CAAATCCCCAGGcGCTGTTGATCCTTTAG SEQ ID NO: 324
31 TMHIV115 L47R s CTAAAGGATCAACAGCgCCTGGGGATTTG SEQ ID NO: 325
32 TMHIV115 L48R as CCCAAATCCCCcGGAGCTGTTG SEQ ID NO: 326
33 TMHIV115 L48R s CAACAGCTCCgGGGGATTTGGG SEQ ID NO: 327
34 TMHIV115 G49R as CCCCAAATCCgCAGGAGCTGTTG SEQ ID NO: 328
35 TMHIV115 G49R s CAACAGCTCCTGcGGATTTGGGG SEQ ID NO: 329
36 TMHIV115 I50R as GCAACCCCAtcTCCCCAGGAGCTG SEQ ID NO: 330
37 TMHIV115 I50R S CAGCTCCTGGGGAgaTGGGGTTGC SEQ ID NO: 331
38 TMHIV115 W51R as GCAACCCCAtcTCCCCAGGAGCTG SEQ ID NO: 332
39 TMHIV115 W51R s CAGCTCCTGGGGAgaTGGGGTTGC SEQ ID NO: 333
HIVl 15 Y41 mutant construction: To explore the amino acid sequence necessary for the loss of immunosuppression, HIVl 15 Y41 mutants were produced by PCR using the pDFG HIVl 15 WT plasmid as template and pairs of primers 1-42 and 43-2, 1-44 and 45-2, 1-46 and 47-2, 1-48 and 49-2, 1-50 and 51-2, or 1-52 and 53-2, to introduce the Y41K, Y41H, Y41A, Y41G, Y41F, or Y41L mutations respectively. The PCR products were then used as templates in subsequent PCR using primers 1-2. These PCR fragments were digested with Sfil and Mlul and inserted into pDFG-ectoSyncytin-1 (Mangeney et al, 2007) opened with the same enzymes
All the constructions were sequenced before use.
N° Name Primer sequence (5'-3') SEQ ID NO
42 TMHIV115 Y41K as GTTGATCCTTTAGtTtTCTTTCCACAGCCAG SEQ ID NO: 334
43 TMHIV115 Y41K s CTGGCTGTGGAAAGAaAaCTAAAGGATCAAC SEQ ID NO: 335
44 TMHIV115 Y41H as GTTGATCCTTTAGGTgTCTTTCCACAGCCAG SEQ ID NO: 336
45 TMHIV115 Y41H s CTGGCTGTGGAAAGAcACCTAAAGGATCAAC SEQ ID NO: 337
46 TMHIV115 Y41A as GTTGATCCTTTAGGgcTCTTTCCACAGCCAG SEQ ID NO: 338
47 TMHIV115 Y41A s CTGGCTGTGGAAAGAgcCCTAAAGGATCAAC SEQ ID NO: 339
48 TMHIV115 Y41G as GTTGATCCTTTAGGccTCTTTCCACAGCCAG SEQ ID NO: 340
49 TMHIV115 Y41G s CTGGCTGTGGAAAGAggCCTAAAGGATCAAC SEQ ID NO: 341
50 TMHIV115 Y41F as GTTGATCCTTTAGGaATCTTTCCACAGCCAG SEQ ID NO: 342 51 TMHIV115 Y41F s CTGGCTGTGGAAAGATtCCTAAAGGATCAAC SEQ ID NO: 343
52 TMHIV115 Y41L as GTTGATCCTTTAGGagTCTTTCCACAGCCAG SEQ ID NO: 344
53 TMHIV115 Y41L s CTGGCTGTGGAAAGActCCTAAAGGATCAAC SEQ ID NO: 345
HIV115 Y41 double mutants construction: HIV1 15 Y41R K72A, G or S double mutants were produced by PCR using the pDFG HIV1 15 Y41R plasmid as template and pairs of primers 1-94 and 93-2, 1-96 and 95-2, or 1-98 and 97-2. The PCR products were then used as templates in subsequent PCR using primers 1-2. These PCR fragments were digested with Sfil and Mlul and inserted into pDFG-ectoSyncytin-1 (Mangeney et al, 2007) opened with the same enzymes. HIV1 15 R40X Y41R double-mutants were produced by PCR using the pDFG HIV1 15 Y41R as template and pairs of primers 1-54 and 55-2, 1-56 and 57-2 to introduce the R40A and R40E mutations respectively. HIV1 15 R40X Y41A double-mutants were produced by PCR using the pDFG HIV1 15 Y41A as template and pairs of primers 1-58 and 59-2, 1-60 and 61-2, 1-62 and 63-2, 1-64 and 65-2 to introduce the R40A, R40E, R40S, R40T mutations respectively. HIV1 15 R40X simple mutants were also generated using pDFG HIV1 15 WT as template and pairs of primers 1-66 and 67-2, 1- 68 and 69-2, 1-70 and 71-2, 1-72 and 73-2.
The PCR products were then used as templates in subsequent PCR using primers 1-2. These PCR fragments were digested with Sfil and Mlul and inserted into pDFG- ectoSyncytin-1 (Mangeney et al, 2007) opened with the same enzyme.
N° Name Primer sequence (5 '-3') SEQ ID
TM HIV1 15
SEQ ID
54 R40AY41R- CTGGCTGTGGAAgcAcgCCTAAAGGATCAAC
NO: 378 S
TM HIV1 15
SEQ ID
55 R40AY4 IRGTTGATCCTTTAGGcgTgcTTCCACAGCCAG
NO: 379 AS
TM HIV1 15
SEQ ID
56 R40EY41R- CTGGCTGTGGAAgaAcgCCTAAAGGATCAAC
NO: 380 S Name Primer sequence (5 '-3') SEQ ID
TM HIV115
SEQ ID
R40EY4 IRGTTGATCCTTTAGGcgTtcTTCCACAGCCAG
NO: 381 AS
TM HIV115
SEQ ID
R40AY41A- CTGGCTGTGGAAgctgcCCTAAAGGATCAAC
NO: 382 S
TM HIV115
SEQ ID
R40AY41A- GTTGATCCTTTAGGgcagcTTCCACAGCCAG
NO: 383 AS
TM HIV115
SEQ ID
R40EY41A- CTGGCTGTGGAAgaagcCCTAAAGGATCAAC
NO: 384 S
TM HIV115
SEQ ID
R40EY41A- GTTGATCCTTTAGGgcttcTTCCACAGCCAG
NO: 385 AS
TM HIV115
SEQ ID
R40SY41A- CTGGCTGTGGAAagcgcCCTAAAGGATCAAC
NO: 386 S
TM HIV115
SEQ ID
R40SY41A- GTTGATCCTTTAGGgcgctTTCCACAGCCAG
NO: 387 AS
TM HIV115
SEQ ID
R40TY41A- CTGGCTGTGGAAacagcCCTAAAGGATCAAC
NO: 388 S
TM HIV115
SEQ ID
R40TY41A- GTTGATCCTTTAGGgctgtTTCCACAGCCAG
NO: 389 AS
TM HIV115 SEQ ID
CTGGCTGTGGAAgctTACCTAAAGGATCAAC
R40A-S NO: 390
TM HIV115 SEQ ID
GTTGATCCTTTAGGTAagcTTCCACAGCCAG
R40A-AS NO: 391 N° Name Primer sequence (5 '-3') SEQ ID
TM HIV1 15 SEQ ID
68 CTGGCTGTGGAAgaaTACCTAAAGGATCAAC
R40E-S NO: 392
TM HIV1 15 SEQ ID
69 GTTGATCCTTTAGGTAttcTTCCACAGCCAG
R40E-AS NO: 393
TM HIV1 15 SEQ ID
70 CTGGCTGTGGAAagcTACCTAAAGGATCAAC
R40S-S NO: 394
TM HIV1 15 SEQ ID
71 GTTGATCCTTTAGGTAgctTTCCACAGCCAG
R40S-AS NO: 395
TM HIV1 15 SEQ ID
72 CTGGCTGTGGAAacaTACCTAAAGGATCAAC
R40T-S NO: 396
TM HIV1 15 SEQ ID
73 GTTGATCCTTTAGGTAtgtTTCCACAGCCAG
R40T-AS NO: 397
93 TMHIV1 15 GCTAGTTGGAGTAATgcATCTCTGGAACAGATTTGG SEQ ID K72A s NO: 398
94 TMHIV1 15 CCAAATCTGTTCCAGAGATgcATTACTCCAACTAGC SEQ ID K72A as NO: 399
95 TMHIV1 15 GCTAGTTGGAGTAATggATCTCTGGAACAGATTTGG SEQ ID K72G s NO: 400
96 TMHIV1 15 CCAAATCTGTTCCAGAGATccATTACTCCAACTAGC SEQ ID K72G as NO: 401
97 TMHIV1 15 GCTAGTTGGAGTAATtcATCTCTGGAACAGATTTGG SEQ ID K72S s NO: 402
98 TMHIV1 15 CCAAATCTGTTCCAGAGATgaATTACTCCAACTAGC SEQ ID K72S as NO: 403
Introduction of HIV115 mutations into an HIV Env expression vector: To introduce these HIV envelope mutations into the HIVenv pTr712 expression vector (Schnierle et al PNAS 1997), a silent mutation was first generated to create an Sfil insertion site. PCR fragments were generated using pTr712as a template and primer pairs 38-39 and 40-41. The PCR products were then used as templates in subsequent PCR using primers 38-41. The resulting PCR fragment was then digested with BsaBI and Hindlll and inserted into the pTr712 plasmid opened with the same enzymes resulting in the pTr712-Sfi plasmid. PCR fragments of each HIVl 15 mutation were then generated by using primers 40-41 and the pDFG HIVl 15 mutant plasmids as templates, digestion with Sfil and Hindlll and then insertion into the pTr712-Sfi plasmid opened with the same enzymes.
All the constructions were sequenced before use.
Figure imgf000110_0001
Establishment of env-expressing tumor cells and MCA205 tumor-rejection assay: 7.5 x 105 293T cells were cotransfected with the env-expressing pDFG retroviral vector to be tested (1.75 μg) and expression vectors for the MLV proteins (0.55 μg for the amphotropic MLV env vector and 1.75 μg for the MLV gag and pol vector). 36 hours post-transfection, supernatants were harvested for infection of MCA205 tumor cells (2.5 ml of supernatant per 5 x 105 cells with 8 μg/ml polybrene). Cells were maintained in selective medium (400 units/ml hygromycin) for 3 weeks, and then washed with PBS, trypsinized and inoculated subcutaneously in the shaved area of each mouse right flank as in Mangeney et al (1998, 2007). Tumor growth was monitored by palpation twice or thrice weekly and tumor area (mm2) determined by measuring perpendicular tumor diameters. The extent of "immunosuppression" was quantified by an index based on tumor size: (Aenv-Anone)/Anone, where Aenv and Anone are the mean areas at the peak of growth of tumors from Balb/c mice injected with env-expressing or control cells, respectively.
Analysis of HIVenv expression : 293T cells were transfected with 4mg of the expression vector for the HIV-1 envelope (pTr712) either wild-type or mutated at the indicated positions, by Calcium Phosphate precipitation. Cells are washed 16h later and then harvested 2 days post-transfection using PBS-EDTA 5mM. The 110-H monoclonal antibody (anti-V3 loop, gift from Hybridolab, Pasteur Institute) was used (1/200 dilution) to stain the HIV envelope. As a secondary antibody, the Inventors used the goat anti mouse IgG Alexa 488 (1/400) (Invitrogen). For intracellular HIVenv staining, 293T cells were fixed with a formaldehyde buffer and then permeabilized (BD cytofix /cytoperm, BD Biosciences). The isotype mouse anti IgGlKappa (BD Biosciences) was used to control non-specific staining. Fluorescence was acquired by flow cytometry using a FACS Calibur (BD Biosciences), and data analysed by the CellQuest software (BD Biosciences).
Mutated Env pseudotyping and measure of viral titer:293T cells are triple transfected with 3mg of a reporter MLV vector carrying GFP (CNCG), 1.75mg Mo-MLV gag-pol vector and 0.55mg phCMV vector encoding HIV-1 envelope wt or mutated at the indicated positions. The infectivity of Mo-MLV virions pseudotyped with HIV-1 Env, harvested 48 hours post-transfection, is measured using U87 cells (CD4+, CXCR4+) as target cells. The infectivity of the enveloppes is analysed after 72h exposure diluted 0.45mm-filtered supernatant in presence of 4mg/mL polybrene. The fluorescence (GFP) is acquired by flow cytometry using a FACS Calibur (BD Biosciences). The results are analysed by the CellQuest software (BD Biosciences). The resulting titers (number of infected cells/mL) are calculated as the following : (% GFP+cells (infected) x plated cell number)/volume of supernatant x 1000.
Env SIV mutants construction : PCR fragments were generated using p239 SPE3' (the plasmid encoding the SIV half virus containing the envelope protein) as a template and primer pairs 77-79 and 78-80, 77-81 and 78-82, 77-83 and 78-84, 77-85 and 78-86, 77-87 and 78-88, 77-89 and 78-90 to introduce the E39R, K40R, Y41R, L42R, K43R, D44R mutations respectively. The PCR products were the used as templates in subsequent PCR using primers 77-78. The resulting PCR fragment was then digested with BmgBI and Nhel and inserted into the p239 SPE3' plasmid opened with the same enzymes.
All the constructions were sequenced before use.
Introduction of SIV mutants ectodomain into pDFG : To generate the pDFG plasmids encoding the fragment of SIV envelope ectodomain-55, PCR fragments generated using p239 SPE3' WT and mutants as a template and primer pairs 91-92, were digested with Sfil and Mlul and inserted into pDFG opened with the same enzymes.
Figure imgf000112_0001
Comparative example: test of the in vivo effect of the mutation G49R, which corresponds to the mutation "G 9R in the international application WO 2010/022,740.
WO 2010/022,740 discloses a consensus sequence of 50 amino acid of the HIV ENV protein. In this sequence, it is suggested that substitution of amino acids in positions 10, 19, 24, 34 and 40 affect the immunosuppressive properties of the HIV ENV protein.
These mutations are a transposition in lentivirus of the teaching of WO 2005/095,442 limited to endogenous or onco retroviruses. The authors of WO 2005/095,442 are also the authors of the present application and they early observed that such a transposition is not effective.
Despite the fact that any amino acids can be assigned to the positions 10, 19, 24, 34 or 40 in the consensus sequence of WO 2010/022,740, only one substitution (one residue for one position) was tested by ex vivo experiments in WO 2010/022,740. It is the mutation ' 'G19R ' ', which corresponds to the mutation G49R in the present figures 1 and 3.
Because the immune response of an individual involves different organs and different cellular and non-cellular components, ex vivo results have no predictive value concerning the in vivo immunosuppressive properties of a viral protein.
To determine if the mutations previously disclosed in WO 2010/022,740 are suitable for a medical use, the mutation G49R has been tested using the MCA205 tumor rejection in vivo assay, as defined in the section "Establishment of env- expressing tumor cells and MCA205 tumor-rejection assay " of the Example.
As shown in Figure 3, the peptide G49R remains almost as immunosuppressive in vivo as the wild type HIV 115 peptide, with an immunosuppression index which is higher than 0,5. Thus, the mutation G49R does not induce a significant decrease of the in vivo immunosuppressive properties of the HIV ENV protein, with a ratio of immunosuppression index of G49R mutated ENV HIV 115 peptide ( ated env ) / immunosuppression index of wild type ENV HIV 115 peptide (iwiid type env ), which is 0,7 {i.e. higher than 0,5).
This result demonstrates the insufficiently described teaching of WO 2010/022740 since the only one mutation tested in WO 2010/022,740, using an ex vivo test, does not significantly affect the in vivo immunosuppressive properties of the HIV ENV protein.
As a consequence, WO 2010/022,740 raises the same technical problem as the present invention but does not offer a technical solution.

Claims

Revendications
1. Pharmaceutical composition comprising as active substance :
a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein having at least 70% identity, preferably at least 80%> identity, to one sequence chosen from the group consisting of SEQ ID NO : 216, SEQ ID NO : 420 and SEQ ID NO : 421,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either
Xa is A, F, G, L, R or deleted, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G, R or deleted, or
Xa is A, F, G, L, R or deleted, and Xb is A, F, G, R or deleted, or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either Xa is A, F, G, L, R or deleted, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G, R or deleted, or
Xa is A, F, G, L, R or deleted, and Xb is A, F, G, R or deleted, in association with a pharmaceutically acceptable carrier, said substantial absence of immunosuppressive activity of the above mentioned mutated human or simian lentiviral ENV protein or of the above defined fragment being liable to be assessed by the fact that in an in vivo assay involving engrafted tumor cells rejection,
said tumor cells being transduced either so as to express said mutated ENV protein or said fragment (mutated ENV tumor cells),
or said tumor cells being transduced so as to express said wild type ENV protein or a fragment thereof (wild type ENV tumor cells),
or said tumor cells being not transduced (normal tumor cells),
the following ratio :
immunosuppression index of said mutated ENV protein or of said fragment ( ated env ) / immunosuppression index of wild type ENV protein (iwiid type env ) is less than 0.5,
imutated env being defined by : (maximum area reached by mutated ENV tumor cells - maximum area reached by normal tumor cells) / (maximum area reached by normal tumor cells), and
iwiid type env being defined by : (maximum area reached by wild type ENV tumor cells - maximum area reached by normal tumor cells) / (maximum area reached by normal tumor cells).
2. Pharmaceutical composition according to claim 1 comprising as active substance : a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein,
X represents any amino acid,
and either
Xa is A, F, G, L or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G or R, or Xa is A, F, G, L or R, and Xb is A, F, G or R, or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein,
X represents any amino acid,
and either
Xa is A, F, G, L or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G or R, or Xa is A, F, G, L or R, and Xb is A, F, G or R, in association with a pharmaceutically acceptable carrier.
3. Pharmaceutical composition according to claim 1 or 2 comprising as active substance : a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, F, G, L or R, and Xb is L, I, V, M or P, or
Xa is Y, I, H, C or T, and Xb is A, F, G or R, or
Xa is A, F, G, L or R, and Xb is A, F, G or R,
or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, F, G, L or R, and Xb is L, I, V, M or P, or
Xa is Y, I, H, C or T, and Xb is A, F, G or R, or
Xa is A, F, G, L or R, and Xb is A, F, G or R, in association with a pharmaceutically acceptable carrier.
4. Pharmaceutical composition according to claim 1 or 2 comprising as active substance : a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R, or
Xa is F or L, and Xb is F, G or R,
or
Figure imgf000118_0001
or
Xa is A, G or R, and Xb is F, G or R, or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R, or
Xa is F or L, and Xb is F, G or R,
or
Figure imgf000119_0001
or
Xa is A, G or R, and Xb is F, G, R, in association with a pharmaceutically acceptable carrier.
5. Pharmaceutical composition according to claim 1 to 4 comprising as active substance :
a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is L, I, V, M or P,
or
Xa is Y, I, H, C or T, and Xb is F, G or R,
or
Xa is F or L, and Xb is F, G or R,
or
Figure imgf000119_0002
or
Xa is A, G or R, and Xb is F, G or R, or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is L, I, V, M or P,
or
Xa is Y, I, H, C or T, and Xb is F, G or R,
or
Xa is F or L, and Xb is F, G or R,
or
Figure imgf000120_0001
or
Xa is A, G or R, and Xb is F, G or R, in association with a pharmaceutically acceptable carrier.
6. Pharmaceutical composition according to claim 1, 2 or 4 comprising as active substance :
a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein,
said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence: A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein,
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R, or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R, in association with a pharmaceutically acceptable carrier.
7. Pharmaceutical composition according to claim 1 to 6 comprising as active substance :
a) an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type human or simian lentiviral ENV protein, said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is L, I, V, M or P,
or
Xa is Y, I, H, C or T, and Xb is F, G or R, or
b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said fragment comprising at least 40 amino acids,
said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is L, I, V, M or P,
or
Xa is Y, I, H, C or T, and Xb is F, G or R, in association with a pharmaceutically acceptable carrier.
8. Pharmaceutical composition according to anyone of claims 1, 2, 4 or 6,
wherein
Xa is R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is R, or Xa is R, and Xb is R.
9. Pharmaceutical composition according to anyone of claims 1 to 8, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the following amino acid sequences :
A-I-E-K-Xa-Xb-X-DQ (SEQ ID NO: 422),
A-I-E-R-Xa-Xb-X-DQ (SEQ ID NO: 423),
A-V-E-K-Xa-Xb-X-DQ (SEQ ID NO: 424),
A-V-E-Pv-Xa-Xb-X-DQ (SEQ ID NO: 425).
10. Pharmaceutical composition according to anyone of claims 1 to 9, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences :
SEQ ID NO : 13, SEQ ID NO : 42, SEQ ID NO : 71,
SEQ ID NO : 9 to 12,
SEQ ID NO : 14 to 41,
SEQ ID NO : 43 to 70, and
SEQ ID NO : 72 to 95.
11. Pharmaceutical composition according to anyone of claims 1 to 9, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences :
SEQ ID NO : 13, SEQ ID NO : 42, SEQ ID NO : 71,
SEQ ID NO : 9, 11, 15 to 21, 23 to 29, 31 to 38, 40, 44 to 50, 52 to 58, 60 to 67, 69, 73 to 79, 81 to 87, 89 to 95.
12. Pharmaceutical composition according to anyone of claims 1 to 9, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences : SEQ ID NO : 96 to 211.
13. Pharmaceutical composition according to anyone of claims 1 to 9, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences : SEQ ID NO : 96, 98, 100, 102 to 108, 110 to 116, 118 to 125, 127, 129, 131 to 137, 139 to 145, 147 to 154, 156, 158, 160 to 166, 168 to 174, 176 to 183, 185, 187, 189 to 195, 197 to 203, 205 to 211.
14. Pharmaceutical composition according to anyone of claims 1 to 9, wherein said isolated mutated human or simian lentiviral ENV protein consists of one of the amino acid sequences : SEQ ID NO : 212 to 269.
15. Pharmaceutical composition according to anyone of claims 1 to 9, wherein said isolated mutated human or simian lentiviral ENV protein consists of one of the amino acid sequences : SEQ ID NO : 212, 214, 216, 218 to 224, 226 to 232, 234 to 241, 243, 245, 247 to 253, 255 to 261, 263 to 269.
16. Pharmaceutical composition according to anyone of claims 1 to 15, wherein said mutated protein comprises an additional mutation in one at least of the amino acids at positions 29, 36 and 37 of SEQ ID NO: 426 :
A[ ]E[K/R]XaXbXiDQX2X3LX4X5WGC[A/S][F/G]X6X7CVX8TX9VPXcXioZiZ
2Z3Z4Z5XdXe[S/T] (SEQ ID NO: 426)
wherein
Xa and Xb are as defined in anyone of claims 1 to 15,
Xi to Xio represent any amino acid,
Zi to Z5 represent no amino acid or any amino acid, independently from each other such that
o Xc is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, or deleted, preferably A, D, or N,
o Xd is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, Y, W, or deleted, preferably A, G, S or Y,
o Xe is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, Y, W, or deleted, preferably A, D or N.
17. Pharmaceutical composition according to anyone of claims 1 to 16, wherein said mutated protein consists of one of the amino acid sequences of the group consisting of SEQ ID NO : 271 to 283.
18. Pharmaceutical composition comprising as active substance a nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, as defined in anyone of claims 1 to 17.
19. Pharmaceutical composition according to claim 18, wherein said nucleic acid molecule being contained in a vector, said vector comprising means allowing the expression of the mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, as defined in anyone of claims 1 to 17.
20. Pharmaceutical composition according to claim 19, wherein said vector being chosen among a measles vector, a canarypox vector, a pox vector, a fowlpox, an adenoviral vector, a lentiviral vector, a Senda'i virus, a Cytomegalovirus vector or a Modified Vaccinia Ankara vector.
21. Pharmaceutical composition, according to anyone of claims 18 to 20, comprising at least one nucleic acid molecule coding for a GAG protein and/or a PRO protein and/or a POL protein and/or a mutated NEF protein, wherein
said mutated NEF protein is substantially devoid of immunosuppressive properties, of a human or simian lentivirus, said lentivirus being preferably of the same origin as the mutated lentiviral ENV protein.
22. Pharmaceutical composition, according to anyone of claims 19 to 21, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains said at least one nucleic acid molecule coding for a GAG protein and/or a PRO protein and/or a POL protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
23. Pharmaceutical composition, according to anyone of claims 19 to 22, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains all said at least one nucleic acid molecule coding for a GAG protein and/or a PRO protein and/or a POL protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
24. Pharmaceutical composition, according to anyone of claims 19 to 21, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in a vector which is different from the at least one vector containing said at least one nucleic acid molecule coding for a GAG protein and/or a PRO protein and/or a POL protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
25. Pharmaceutical composition, according to anyone of claims 19 to 21, or 24, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, said nucleic acid molecule coding for a GAG protein, said nucleic acid molecule coding for a PRO protein, said nucleic acid molecule coding for a POL protein and said nucleic acid molecule coding for a mutated NEF protein substantially devoid of immunosuppressive properties, are all contained in vectors which are different from each other.
26. Pharmaceutical composition, according to anyone of claims 18 to 21, comprising at least one nucleic acid molecule coding for a GAG protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties,
of a human or simian lentivirus, said lentivirus being preferably of the same origin as the mutated lentiviral ENV protein.
27. Pharmaceutical composition, according to anyone of claims 18 to 21, or 26, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains said at least one nucleic acid molecule coding for a GAG protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
28. Pharmaceutical composition, according to anyone of claims 18 to 21, or 26 to 27, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains a nucleic acid molecule coding for a GAG protein and for a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
29. Pharmaceutical composition, according to anyone of claims 18 to 21, or 26 to 28, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains a nucleic acid molecule coding for a GAG protein and for a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties, said vector being a measles vector or a canary pox vector.
30. Pharmaceutical composition, according to anyone of claims 18 to 21, or 26, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in a vector which is different from the at least one vector containing said at least one nucleic acid molecule coding for a GAG protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.
31. Pharmaceutical composition, according to anyone of claims 18 to 21, 26 or 30, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, said nucleic acid molecule coding for a GAG protein and said nucleic acid molecule coding for a mutated NEF protein substantially devoid of immunosuppressive properties, are all contained in vectors which are different from each other.
32. Pharmaceutical composition according to anyone of claims 1 to 31, in association with at least one antiviral compound, preferably for a simultaneous, separated or sequential use.
33. Pharmaceutical composition according to anyone of claims 1 to 32, for its use for stimulating an immune response in a host organism.
34. Pharmaceutical composition according to anyone of claims 1 to 33, for its use for the prevention or the treatment of lentiviral infection, preferably HIV-1 infection, HIV-2 infection or SIV infection.
35. Pharmaceutical composition according to anyone of claims 1 to 34, for its use as a vaccine, in particular against HIV-1 infection, HIV-2 infection or SIV infection.
36. A method to obtain the active substance of a pharmaceutical composition, as defined in anyone of claims 1 to 35, consisting of modifying the immunosuppressive property of:
a wild-type human or simian lentiviral ENV protein,
or a fragment of said wild-type human or simian lentiviral ENV protein, said fragment comprising at least 40 amino acids, said ENV protein or fragment thereof presenting a transmembrane subunit (TM) comprising an immunosuppressive domain (ISU) containing the following amino acid sequence :
A-[I/V]-E-[K/R]-X'a-X'b-X-D-Q (SEQ ID NO: 427), wherein
X represents any amino acid,
X'a is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and
X'b is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, said method comprising a step of introduction of at least one mutation of X'a and/or
to obtain:
an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
said mutated human or simian lentiviral ENV protein having at least 70% identity, preferably at least 80%> identity, to one sequence chosen from the group consisting of SEQ ID NO : 216, SEQ ID NO : 420 and SEQ ID NO : 421,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, F, G, L, R or deleted, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G, R or deleted, or
Xa is A, F, G, L, R or deleted, and Xb is A, F, G, R or deleted, said substantial absence of immunosuppressive activity of the above mentioned mutated human or simian lentiviral ENV protein or of the above defined fragment being liable to be assessed by the fact that in an in vivo assay involving engrafted tumor cells rejection,
said tumor cells being transduced either so as to express said mutated ENV protein or said fragment (mutated ENV tumor cells),
or said tumor cells being transduced so as to express said wild type ENV protein or a fragment thereof (wild type ENV tumor cells),
or said tumor cells being not transduced (normal tumor cells),
the following ratio :
immunosuppression index of said mutated ENV protein or of said fragment ( ated env ) / immunosuppression index of wild type ENV protein (iwiid type env ) is less than 0.5,
imutated env being defined by : (maximum area reached by mutated ENV tumor cells - maximum area reached by normal tumor cells) / (maximum area reached by normal tumor cells), and
iwiid type env being defined by : (maximum area reached by wild type ENV tumor cells - maximum area reached by normal tumor cells) / (maximum area reached by normal tumor cells).
37. A method to obtain the active substance of a pharmaceutical composition, as defined in anyone of claims 1 to 35, according to claim 36, consisting of modifying the immunosuppressive property of :
a wild-type human or simian lentiviral ENV protein,
or a fragment of said wild-type human or simian lentiviral ENV protein, said fragment comprising at least 40 amino acids,
said ENV protein or fragment thereof presenting a transmembrane subunit (TM) comprising an immunosuppressive domain (ISU) containing the following amino acid sequence :
A-[I/V]-E-[K/R]-Y-L-X-D-Q (SEQ ID NO : 1),
wherein
X represents any amino acid, said method comprising a step of introduction of at least one mutation of Y in position 5 and/or L in position 6, to obtain:
an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein
X represents any amino acid,
and either
Xa is A, F, G, L, R or deleted, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G, R or deleted, or
Xa is A, F, G, L, R or deleted, and Xb is A, F, G, R or deleted.
38. A method to obtain the active substance of a pharmaceutical composition, as defined in anyone of claims 1 to 35, according to claim 36 or 37,
wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence : A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein
X represents any amino acid,
and either
Xa is A, F, G, L or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is A, F, G or R, or
Xa is A, F, G, L or R, and Xb is A, F, G or R.
39. A method to obtain the active substance of a pharmaceutical composition, as defined in anyone of claims 1 to 35, according to anyone of claims 36 to 38, wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein
X represents any amino acid,
and either
Xa is A, F, G, L or R, and Xb is L, I, V, M or P, or
Xa is Y, I, H, C or T, and Xb is A, F, G or R, or
Xa is A, F, G, L or R, and Xb is A, F, G or R.
40. A method to obtain the active substance of a pharmaceutical composition, as defined in anyone of claims 1 to 35, according to anyone of claims 36 to or 38, wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids, said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R, or
Xa is F or L, and Xb is F, G or R, or
Figure imgf000133_0001
41. A method to obtain the active substance of a pharmaceutical composition, as defined in anyone of claims 1 to 35, according to claim 36 or 40,
wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is L, I, V, M or P, or
Xa is Y, I, H, C or T, and Xb is F, G or R, or
Xa is F or L, and Xb is F, G or R, or
Xa is A, G or R, and Xb is A, or
Xa is A, G or R, and Xb is F, G or R.
42. A method to obtain the active substance of a pharmaceutical composition, as defined in anyone of claims 1 to 35, according to anyone of claims 36, 37, 38 or 40,
wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein,
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is F, G or R.
43. A method to obtain the active substance of a pharmaceutical composition, as defined in anyone of claims 1 to 35, according to anyone of claims 36 to 42, wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416), wherein,
X represents any amino acid,
and either
Xa is A, G, or R, and Xb is L, I, V, M or P, or
Xa is Y, I, H, C or T, and Xb is F, G or R.
44. A method to obtain the active substance of a pharmaceutical composition, as defined in anyone of claims 1 to 35, according to anyone of claims 36, 37, 38, 40 or 42,
wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence :
A-[I/V]-E-[K/R]-Xa-Xb-X-D-Q (SEQ ID NO: 416),
wherein,
X represents any amino acid,
and either
Xa is R, and Xb is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or
Xa is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and Xb is R, or
Figure imgf000135_0001
45. A method to obtain the active substance of a pharmaceutical composition, as defined in anyone of claims 1 to 35, according to anyone of claims 36 to 44, wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,
or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,
said mutated ENV protein and fragment thereof comprising an additional mutation in one at least of the amino acids at positions, 29, 36 and 37 of SEQ ID NO: X :
A[ ]E[K/R]XaXbXiDQX2X3LX4X5WGC[A/S][F/G]X6X7CVX8TX9VPXcXioZiZ2 Z3Z4Z5XdXe[S/T] (SEQ ID NO: 426)
wherein Xa and Xb are as defined in anyone of claims 1 to 15,
Xi to Xio represent any amino acid,
5 represent no amino acid or any amino acid, independently from each other such that
o Xc is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, or deleted, preferably A, D, or N,
o Xd is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, Y, W, or deleted, preferably A, G, S or Y,
o Xe is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, Y, W, or deleted, preferably A, D or N.
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