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  • A61P31/12—Antivirals
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  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
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  • C12N2760/18711—Rubulavirus, e.g. mumps virus, parainfluenza 2,4
  • C12N2760/18722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present application relates to mutant proteins of the Parainfluenza virus (PIV) fusion protein (F protein) which are currently listed under PIV type 5 (PIV-5 or PIV5) and PIV type 2 (PIV-2 or PIV2).
• PIV type 5 PIV-5 or PIV5
• PIV type 2 PIV-2 or PIV2
• the present application relates to products derived therefrom, such as:
• nucleic acids nucleic acids, vectors, cells, antibody-like fusion inhibitors, aptamers, interfering RNAs, myelomas, hybridomas,
• the present application also relates to these mutant and derivatized proteins for use in medical and biotechnological applications.
• PIV Parainfluen ⁇ a virus
• PIV5 is an enveloped Ruby virus of the genus Paramyxoviridae.
• PIV-5 was previously known as SV5 (Simian Virus 5) because its isolation was initially performed from primary cultures of monkey cells.
• SV5 Sesimian Virus 5
• the natural host of PIV-5 appears to be the dog, in which it causes a cough known as kennel cough.
• the PIV-5 virus is currently considered an animal virus.
• Parainfluenza virus (PIV), which is currently classified as PIV type 2 (PIV-2 or PIV2), is also an enveloped Ruby virus of the genus Paramyxoviridae.
• PIV-2 Parainfluenza virus
• hPIV-2 human isolates of PIV-2 have been identified (hPIV-2), so that PIV-2 is considered a human virus.
• the PIV-5 and PIV-2 viruses are very close to each other, both in terms of nucleic acid sequences, protein sequences, organization, structure and morphology.
• PIV-2 is the closest virus to PIV-5 that has been found in humans.
• PIV-2 can be considered, and is at least considered by the inventors, to be the human equivalent of PIV-5.
• PIV-5 and PIV-2 viruses are accomplished by fusing the viral envelope with the cell membrane.
• This fusion involves two viral glycoproteins: the hemagglutinin-neuraminidase (HN) attachment protein and the fusion protein (F).
• HN hemagglutinin-neuraminidase
• F fusion protein
• the fusion protein F of PIV-5 and PIV-2 is synthesized as a simple precursor (FO) and is in the form of a glycosylated homotrimer.
• the PIV-5 and PIV-2 F fusion protein requires proteolytic cleavage by furins from the host to generate a "pre-activated" form, consisting of two subunits linked by disulfide bridges: a large subunit carboxy terminal unit F1 and a small amino terminal subunit F2.
• the F1 subunit is composed of a hydrophobic fusion peptide (FP) as well as two repeated domains in heptads (HR-I and HR-2), having a supercoiled-type conformation ("coiled-coil").
• FP hydrophobic fusion peptide
• HR-I and HR-2 two repeated domains in heptads
• coiled-coil supercoiled-type conformation
• the fusion protein F of PIV-5 and PIV-2 requires a HN protein from the same viral type to promote fusion (Yao et al 1997). However, the precise nature of the interactions between F and HN is not well known.
• Paterson et al. 2000 indicate that the presence of the amino acid Proline at position 22 of the F protein of the W3A strain, and the presence of the amino acid 443 of the F protein of the WR strain could increase the fusogenic capacity of these viral strains.
• Russell et al. 2003 indicate that replacement of residues L447 and 1449 of F protein W3A by aromatic amino acids may increase the fusogenic activity of the F protein of this viral strain.
• Gardner and Dutch 2007 indicate that the 149 A mutation of the F protein of a wild type "SV5" (PIV-5) virus would have a pro-fusogenic effect.
• Gardner et al. 2007 indicate that the V402A mutation of the F protein of a wild-type "SV5" (PIV-5) virus would have a pro-fusogenic effect.
• fusion-capable viral proteins such as, for example, HA1 / HA2 Influenza proteins, Rhabdovirus G protein, HIV gp41 / gp120 protein.
• the inventors have considered that having a fusion protein that would be hyperfusogenic and that would also demonstrate a great deal of autonomy in its fusion capacity would make it possible to provide a solution to a certain number of medical and biotechnological situations.
• the inventors then made a selective choice with respect to the F protein of PIV-5 and / or PIV-2 with respect to all the other viral fusion proteins, such as the Inflnenza HA1 / HA2 proteins, Rhabdovints G protein, HIV gp41 / gp120 protein.
• mutant proteins of the invention exhibit a strong capacity for fusogenicity and a high degree of autonomy. They do not require the presence of the HN protein to induce cell fusion and syncytia formation.
• the inventors further show that it is possible to introduce into these mutant proteins a cleavage site different from that which the F protein exhibits in the natural state, and more particularly a tissue-specific cleavage site.
• the inventors also propose medical applications (therapeutic, preventive, palliative, but also diagnostic) and / or biotechnological mutant proteins of the invention and / or products derived or derived therefrom.
• the inventors propose using the mutant proteins of the invention or the nucleic acids encoding them, to treat, prevent or palliate, in vivo or ex vivo, the diseases or dysfunctions for which it is desired to induce or increase the formation of syncytia, such as cancers (more particularly metastatic cancers, preferentially metastatic melanomas) or impairments of placental development.
• syncytia such as cancers (more particularly metastatic cancers, preferentially metastatic melanomas) or impairments of placental development.
• the inventors furthermore propose PIV-5 and / or PIV-2 F protein blocking agents, such as antibodies, recombinant dendritic cells, antisense, siRNA, aptamer nucleic acids, to treat, prevent or mitigate, in vivo or ex vivo, the diseases or dysfunctions for which it is desired to inhibit or block the formation of syncytia, such as enveloped virus infections, allergies, autoimmune diseases, transplant rejections.
• PIV-5 and / or PIV-2 F protein blocking agents such as antibodies, recombinant dendritic cells, antisense, siRNA, aptamer nucleic acids
• the inventors also propose diagnostic means for detecting the excessive or, to the contrary, insufficient formation of syncytia.
• the inventors also propose different biotechnological means, in particular for the screening of active principles capable of reducing the formation of syncytia.
• the inventors furthermore propose cancerous cells, more particularly myelomas, which comprise a mutant protein of the invention.
• the inventors further propose hybridomas comprising a mutant protein of the invention.
• the cancer cells, more particularly the myelomas of the invention are capable of fusing with another cell, and more particularly with a B lymphocyte, without using polyethylene glycol (PEG), or electroporation, or any other means. fusion inductor.
• the cancer cells, more particularly the myelomas of the invention are capable of autonomous fusion.
• the hybridomas of the invention can be produced by a fusion (from B lymphocytes to myelomas) that does not require the use of polyethylene glycol (PEG), electroporation, or any other fusion inducing means.
• the hybridomas of the invention can be produced by implementing at least one cancer cell, more particularly at least one myeloma of the invention.
• the inventors furthermore propose recombinant stem or progenitor cells, which express a mutant protein of the invention, and their applications in the production of muscle fibers.
• Fig. 1A Reference PIV-5 Protein F Sequence (SEQ ID NO: 31, WR Isolate F Protein Sequence) and Corresponding CDS Sequence (SEQ ID NO: 30), Ia-encoding nucleic acid sequence protein of SEQ ID NO: 31).
• SEQ ID NO: 31 Reference PIV-5 Protein F Sequence (SEQ ID NO: 31, WR Isolate F Protein Sequence) and Corresponding CDS Sequence (SEQ ID NO: 30), Ia-encoding nucleic acid sequence protein of SEQ ID NO: 31).
• SEQ ID NO: 31 In bold and underlined within the sequence of SEQ ID NO: 31, are indicated the amino acids of positions: - 22 (L),
• Figure 1B sequence of the reference PIV-2 protein F (SEQ ID NO: 33, sequence of the F protein of the Greer isolate) and corresponding CDS sequence (SEQ ID NO: 32, nucleic acid sequence encoding the protein of SEQ ID NO: 33).
• Figure 2A alignment of the PIV-5 F protein (amino acids 4 to 529 of SEQ ID NO: 31) to that of PIV-2 (amino acids 8 to 533 of SEQ ID NO: 33): the protein identity is 47.7% between these two proteins. The consensus sequence resulting from this alignment is referenced under SEQ ID NO: 34.
• Figure 2B amino acid (s) and mutation (s) within the PIV-2 F protein that correspond to those of the PIV-5 F protein
• Figure 3 illustrates the substitution of the natural cleavage site (SEQ ID NO: 23) of the F protein of PIV-5 by a tissue-specific cleavage site, in this case by the site of an enzyme specifically expressed by metastatic tumor tissue, ie matrix metalloprotease 9 (MMP-9).
• SEQ ID NO: 23 the natural cleavage site of the F protein of PIV-5
• MMP-9 metastatic tumor tissue
• Illustrative sequence of an MMP-9 site sequence of SEQ ID NO: 28, sequence of SEQ ID NO: 29.
• Figure 4 Structure of the plasmid pcDNA3.1 on which was cloned the PIV-5 protein F coding sequence.
• PIV5 PIV-5 BGHpolyA
• F protein poly-adenylation signal of bovine growth hormone (Bovine)
• FIGS. 5A, 5B, 5C, 5D Visualization of Mutations Realized by the Inventors in PIV-5 Protein F
• FIG. 6A illustration of observations made under a microscope during semi-quantitative fusion tests (large panel of mutants made by the inventors)
• FIG. 6B diagram presenting the fusion scores obtained after the semi-quantitative fusion tests (broad panel of mutants made by the inventors)
• FIG. 7A illustration of observations made under a microscope during semi-quantitative fusion tests (selection of mutants made by the inventors)
• FIG. 7B diagram presenting the fusion scores obtained after the semi-quantitative fusion tests (selection of mutants made by the inventors)
• protein encompasses in its scope the term glycoprotein. This is particularly the case for F and HN proteins which are in fact glycoproteins.
• Protein F of PIV-5 and PIV-2 (non-mutant protein):
• FIG. 1A protein sequence of SEQ ID NO: 31, coding nucleic acid sequence of SEQ ID NO: 30. This is the sequence of isolate WR, which is a simian isolate. These sequences are those available in the Genbank database under number AB021962.
• This alternative sequence of the F protein of the isolate WR is identical to the sequence of SEQ ID NO : 31, with the exception of the amino acid at position 443 which is S and not P. For the sake of brevity, this alternative sequence will here be denoted "SEQ ID NO: 31 with S at 443".
• sequence of SEQ ID NO: 31 and the alternative sequence "SEQ ID NO: 31 with S at 443", preferentially the alternative sequence "SEQ ID NO: 31 with S at 443", serve as a reference for the sequence (s) of protein.
• F of PIV-5 in the context of the present patent application.
• PIV-5 isolates other than the WR isolate and especially: other simian isolates, such as, for example, the W3A isolate, isolates of other non-human animals, such as that: o canine isolates, eg CPI +, CPI-, H221, 78524, Tl isolates, o porcine isolates, for example the SER porcine isolate, so-called "human" isolates which are derived from samples taken from human beings but which have been placed in culture on animal cells (cf. introductory part above), such as the MIL isolate, the DEN isolate, the LN isolate, the MEL isolate 5 and the isolate which, in WO 02 077211, is described as being a
• the F protein of the W3A isolate presents:
• this cytoplasmic extension contains from two to seven amino acids .
• F protein sequences of these isolates vary by less than 5% (more particularly by up to 3%) relative to the F protein sequence of the WR strain (regardless of the extension cytoplasmic, i.e., calculating this percentage over the length of the F protein of WR); cf. end of page 85 of the article Chatziandreou et al. 2004.
• a protein F of PIV-5 can therefore consist of:
• the variant sequences of the PIV-5 F protein of SEQ ID NO: 31 notably comprise the F protein sequences of the W3A, MIL, DEN, LN, MEL, cryptovirus, CPI +, CPI-, H221, 78524, Tl and SER isolates. mentioned above (see Table 1 above and article Chatziandreou et al., 2004).
• the sequence which in the present application serves as a reference for the PIV-2 F protein is the sequence of the Gréer strain which is presented in FIG. 1B (protein sequence of SEQ ID NO: 33, coding nucleic acid sequence of SEQ ID NO: 32).
• PIV-2 isolates other than the Greer isolate such as for example the Toshiba, V98, V94 isolates.
• PTV-2 F protein may therefore consist of:
• a variant sequence of this sequence of SEQ ID NO: 33 is able to be defined as: o being of a size identical to that of SEQ ID NO: 33 or less than a maximum of two amino acids to that of SEQ ID NO: 33 or greater than a maximum of two amino acids to that of SEQ ID NO: 33, preferentially in an identical size to that of SEQ ID NO: 33, 'preferably being identical in size to that of SEQ ID NO: 33, and o having a greater than 95% sequence identity, preferentially at least 96%, more preferably at least 97%, relative to the sequence of SEQ ID NO: 33 (this identity being calculated over the length of the sequence of SEQ ID NO: 33).
• the consensus sequence (SEQ ID NO: 34) resulting from the alignment of the PIV-5 F protein sequence (SEQ ID NO: 31) to that of PIV-2 (SEQ ID NO: 33) is as follows. read in Figure 2A. This consensus sequence can be re-transcribed as follows:
• the F protein sequence of the PIV-5 WR isolate is the sequence of SEQ ID NO: 34 preceded by the MGT amino acids at the N-terminus (cf. Figures 1A and 2A).
• the Pev-2 Greer isolate F protein sequence is the sequence of SEQ ID NO: 34, preceded by amino acids MHHLHPM (SEQ ID NO: 86) at the N-terminus followed by amino acids ENPAFFSKNNHGNIYGIS (SEQ ID NO: 87) at the C-terminal end (cf. Figures IB and 2A).
• the PIV-5 and PIV-2 F protein sequence may be considered to be a sequence comprising the sequence of SEQ ID NO: 34, preferably as the sequence of a PIV virus F protein that comprises the sequence of SEQ ID NO: 34. More particularly, the sequence of F proteins of PIV-5 and PIV-2 can be considered as being: a) the sequence of SEQ ID NO: 34:
• variant sequence of the sequence described under a) above, said variant sequence being :
• sequence identity having a sequence identity of greater than 95%, preferably at least 96%, more preferably at least 97%, relative to the sequence of SEQ ID NO: 31 or to said alternative sequence "SEQ ID NO: 31 with S in 443 "(this identity being calculated over the length of the sequence of SEQ ID NO: 31 or, if appropriate, of said alternative sequence” SEQ ID NO: 31 with S at 443 ");
• the present application relates to a mutant protein whose amino acid sequence comprises a sequence which is likely to derive from that of the F protein of a PIV-5 or PIV-2 virus: by replacement:
• amino acid hydrophobic selected from V, I, L, preferentially V (147Hy mutation in F of PIV-5; 151Hy mutation in
• amino acid hydrophobic selected from V, I, L, preferably V (158Hy mutation in F of PIV-5; 162Hy mutation in
• Said amino acid positions are calculated with respect to the sequence of the precursor form (FO) of said F protein (i.e., the F protein sequence before cleavage), counting the positions of the N-terminus towards the C-terminus.
• FO precursor form
• the positions indicated in the PIV-2 F protein are the positions corresponding to those indicated in the PIV-5 F protein: cf. Figure 2B, giving the table of positional matches.
• sequence of said PIV-5 or PIV-2 virus F protein is as defined above. It can therefore be defined in particular as comprising the sequence of SEQ ID NO: 34 (consensus sequence of F proteins of PIV-5 and PIV-2).
• a mutant protein of the invention comprises a sequence that is derivable from that of the F protein of a PIV-5 virus.
• this variant sequence: o being of a size identical to that of SEQ ID NO: 31 (that is, 529 amino acids), or being greater than 7 amino acids in size greater than that of SEQ ID NO: 31 (i.e., 530, 531 , 532, 533, 534, 535 or 536 amino acids), or being up to 7 amino acids in size less than that of SEQ ID NO:
• sequence of said PIV-5 F protein consists of:
• variant sequences include, in particular, the F protein sequence of one of the W3A, MIL, DEN, LN, MEL, Cryptovirus, CPI +, CPI-, H221, 78524, Tl and SER isolates shown in Table 1 herein. request (see above), that is to say in one of the sequences of SEQ ID NO: 35 to 46.
• the sequence of said PIV-5 protein F consists of the sequence of SEQ ID NO: 31 (PIV-5 WR isolate F protein sequence shown in FIG. 1A), or said alternative sequence " SEQ ID NO: 31 with S at 443 ", very preferentially in said alternative sequence” SEQ ID NO: 31 with S at 443 ".
• said sequence which is derivable from that of said PIV-5 F protein is derivable from this F protein sequence by at least said mutations 22P, 132E 5 290A, 447P and 158Hy mentioned above.
• said sequence which is likely to derive from that of said PIV-5 F protein is capable of deriving from this F protein sequence by at least said mutations 22P, 132E, 290A, 447P and 147Hy mentioned above.
• said sequence which is capable of deriving from that of said PIV-5 F protein is capable of deriving from this F protein sequence by at least said mutations 22P, 132E, 290A, 447P, 147Hy and 158Hy mentioned above.
• Said sequence likely to derive from that of the PIV-5 F protein may not comprise a mutation other than the mutations 22P, 132E, 290A, 447P and 147Hy / 158Hy mentioned above, with respect to said F protein sequence of PIV-5.
• said sequence likely to derive from that of the PIV-5 F protein may be capable of deriving from this F protein sequence by said 22P, 132E, 290A, 447P and 147Hy / 158F mutations mentioned above and by at least one mutation other than these mutations 22P, 132E, 290A, 447P mentioned above, preferentially by: at least one pre-fusion mutation chosen from:
• At least one post-fusion mutation chosen from:
• said sequence which is capable of deriving from that of said PIV-5 protein F is capable of deriving from this protein F sequence by said mutations 22P, 132E, 290A, 447P and 147Hy / 158Hy mentioned above, and by :
• At least one post-fusion mutation chosen from:
• said sequence which is derivable from that of said PIV-5 F protein is derivable from the protein sequence F by said 22P mutations, 132E, 290A 5 447P and 147Hy / 158Hy mentioned above, and by at least one post-fusion mutation chosen from:
• Said hydrophobic amino acid chosen to replace the amino acid in position 463 is advantageously chosen from V, I 5 L, preferentially V.
• the mutations shown in Table 4 can be introduced into the F protein of any PIV-5 isolate, i.e., the WR isolate or variant isolate. They can therefore more particularly be introduced into the Protein F sequence of SEQ ID NO: 31 shown in Figure IA (WR protein F available in the Genbank database under number AB021962).
• a mutant protein of the invention comprises a sequence which is likely to derive from that of the F protein of a PIV-2 virus.
• the sequence of said PIV-2 F protein is as defined above. It may especially consist of: the sequence of SEQ ID NO: 33 (Pev-2 Greer isolate F protein sequence shown in FIG. 1B), or a variant sequence of this sequence of SEQ ID NO: 33 , this variant sequence: o being of a size identical to that of SEQ ID NO: 33 (that is to say consisting of 551 amino acids), or being of a size greater than a maximum of 2 amino acids to that of SEQ ID NO: 33 (i.e., consisting of 552 or 553 amino acids), or being of a size less than a maximum of 2 amino acids to that of SEQ ID NO: 33 (c that is, consisting of 549 or 550 amino acids), and o having a sequence identity of greater than 95%, preferably at least 96%, more preferably at least 97%, relative to the sequence of SEQ ID NO: 33, this identity being calculated over the length of the sequence of SEQ ID NO: 33.
• sequence of said PIV-2 F protein consists of: the sequence of SEQ ID NO: 33 (PEG-2 Greer Isolate F protein sequence shown in FIG. 1B), or
• this variant sequence: o being of a size identical to that of SEQ ID NO: 33 (that is to say consisting of 551 amino acids), or being greater than 2 amino acids in size to that of SEQ ID NO: 33 (i.e., 552 or 553 amino acids), and o having more than 95% sequence identity preferably at least 96%, more preferably at least 97%, relative to the sequence of SEQ ID NO: 33, this identity being calculated over the length of the sequence of SEQ ID NO: 33.
• sequence of said PIV-2 F protein consists of: the sequence of SEQ ID NO: 33 (sequence of the F protein of the Greer isolate of
• PrV-2 shown in FIG. 1B or in a variant sequence of this sequence of SEQ ID NO: 33, this variant sequence: o being of a size identical to that of SEQ ID NO: 33 (i.e. ie consisting of 551 amino acids), and o having a sequence identity of greater than 95%, preferably at least 96%, more preferably at least 97%, relative to the sequence of SEQ ID NO: 33, this identity being calculated over the length of the sequence of SEQ ID NO: 33.
• the sequence of said F protein consists of the sequence of SEQ ID NO: 33 (F protein sequence of the Piv -2 Greer isolate shown in FIG. 1B).
• said sequence which is likely to derive from that of said PIV-2 F protein is capable of deriving from this F protein sequence by at least said mutations 24P, 133E, 294A, 445P and 162Hy mentioned above.
• said sequence which is likely to derive from that of said PIV-2 F protein is capable of deriving from this F protein sequence by at least said mutations 24P, 133E, 294A, 445P and 151Hy mentioned above.
• said sequence which is capable of deriving from that of said PIV-2 F protein is capable of deriving from this F protein sequence by at least said mutations 24P, 133E, 294A, 445P, 162Hy and 51Hy mentioned above.
• Said sequence likely to derive from that of the PIV-5 F protein may not comprise a mutation other than the mutations 24P, 133E, 294A, 445P and 151Hy / 162Hy mentioned above, with respect to said F protein sequence of PIV-2.
• said sequence likely to derive from that of the PIV-2 F protein may be capable of deriving from this F protein sequence by said mutations 24P, 133E, 294A, 445P and 151Hy / 162Hy mentioned above and by at least a mutation other than these mutations 24P, 133E, 294A, 445P mentioned above, preferentially by:
• At least one post-fusion mutation chosen from: replacing the amino acid at position 474 with a hydrophobic amino acid.
• said sequence which is derivable from that of said F protein of PIV-2 is derivable from the protein sequence F by said 24P mutations, 133E 5 294A, 445P and 15 LHY / 162Hy mentioned above, and by:
• said sequence which is capable of deriving from that of said PIV-2 F protein is capable of deriving from this F protein sequence by said mutations 24P, 133E, 294A, 445P and 151Hy / 162Hy mentioned above, and by at least one post-fusion mutation chosen from:
• Said hydrophobic amino acid chosen to replace the amino acid at position 474 is advantageously chosen from V, I, L, preferentially V.
• the mutations shown in Table 5 can be introduced into the F protein of any PIV-2 isolate, that is, Greer isolate or variant isolate. They can therefore more particularly be introduced into the protein sequence of SEQ ID NO: 33 shown in FIGURE 1B (Gréer protein F, SEQ ID NO: 88 to 90). SEQ ID NO: 88
• VNGITSASCR AHDALIGSIL NLYLTELTTI FHNQITNPAL TPLSIQALRI LLGSTLPIVI 240
• KLQEVVVQVP NR [LEYANEL QNYPANDCVV TPNSVFCRYN EGSPIPESQY QCLRGNLNSC 360
• VNGITSASCR AHDALIGSIL NLYLTELTTI FHNQITNPAL TPLSIQALRI LLGSTLPIVI 240
• VNGITSASCR AHDALIGSIL NLYLTELTTI FHNQITNPAL TPLSIQALRI LLGSTLPIVI 240
• a mutant protein of the invention may comprise a sequence which is capable of deriving from that of the F protein of a PIV-5 OR PIV-2 virus by: the mutations mentioned above, and further by substituting the native cleavage site of said F protein with another enzyme cleavage site, and / or inserting into said F protein an enzyme cleavage site other than the native cleavage site of that F protein, preferably by substituting the native cleavage site of said F protein with another enzyme cleavage site.
• the cleavage site of a PIV-5 or PIV-2 F protein is the site of cleavage of the two subunits (F1 and F2) of this F protein.
• this cleavage site is a cleaved site by forms.
• this cleavage site consists of the sequence
• RRRRR SEQ ID NO: 23. It is in positions 98 to 102 of the native form of PIV-5 protein F (cf. Figure IA).
• An example of a PIV-5 F protein sequence fragment comprising the native (or natural) cleavage site of PIV-5 F protein is:
• the cleavage site In the native form of the PIV-2 F protein, the cleavage site consists of the sequence KTRQKR (SEQ ID NO: 25). It is located at positions 101 to 106 of the native form of the PIV-2 protein F (cf. Figure IB).
• PIV-2 is LTPLIENLSKISTVTDTKTRQKRFAGVVVGLAALGVA (SEQ ID NO: 26).
• said cleavage site other than the native cleavage site is a tissue-specific cleavage site.
• said cleavage site other than the native cleavage site is a cleavage site for an enzyme expressed specifically by one or more tumor tissues, very preferentially a cleavage site for an enzyme expressed specifically by one or more metastatic tissues.
• said cleavage site other than the native cleavage site may be a cleavage site for a metalloprotease, such as the cleavage site for matrix metalloprotease 9 (MMP-9); cf. Example 2 below.
• MMP-9 matrix metalloprotease 9
• a cleavage site for matrix metalloprotease 9 may comprise or consist of the sequence PRRIT (SEQ ID NO: 28) and / or the sequence
• the present application also relates to a nucleic acid, DNA or RNA, which encodes a mutant protein according to the invention (according to the universal genetic code and taking into account the degeneracy of this code), and to a nucleic acid complementary to such a nucleic acid (nucleic acid of the same length perfectly complementary).
• nucleic acids are derived from the sequence of SEQ ID NO: 30 (sequence encoding native PIV-5 F protein), or from an alternative sequence encoding said alternative sequence "SEQ ID NO: 31 with S at 443", or a variant sequence encoding a variant F protein, or the sequence of SEQ ID NO: 32 (sequence encoding the native PIV-2 F protein) or a variant sequence encoding a variant F protein.
• the present application also relates to a nucleic acid vector, more particularly to a vector for transfection, transduction or transformation, comprising at least one nucleic acid according to the invention.
• Such a vector may advantageously be an expression vector.
• it is a vector allowing the expression of said at least one nucleic acid in an animal cell (non-human animal cell and / or human cell), more preferably:
• a human cell advantageously in a pathological human cell, more particularly a human tumor cell, more preferably a metastatic melanoma cell, or
• Such an expression vector may advantageously be an adenoviral vector.
• This adenoviral vector may comprise elements for regulating the expression of said nucleic acid, preferentially a promoter, allowing an expression of said nucleic acid in tumor cells, preferably in metastatic cells, more preferably in metastatic melanoma cells.
• this expression is specific.
• this expression is sufficiently specific to allow the expression of said nucleic acid in said tumor or metastatic cells, without there being any significant expression in non-tumor cells (even, non-metastatic).
• such an adenoviral vector is an oncolytic adenoviral vector.
• an expression vector of the invention may be an adenoviral vector which comprises elements for regulating the expression of said nucleic acid, preferentially a promoter, allowing an expression of said nucleic acid in placental cells, preferably in cells Pathological placents that are deficient in fusogenicity.
• this expression is specific.
• this expression is sufficiently specific to allow the expression of said nucleic acid in said placental cells, without there being any significant expression in non-placental cells.
• a vector of the invention may alternatively or complementarily be a vector allowing the insertion of said at least one nucleic acid into the genome of an animal cell (non-human animal cell and / or human cell), more preferably a cell.
• human advantageously from a pathological human cell, more particularly of a human tumor cell, preferably a metastatic human cell, more preferably in metastatic melanoma cells.
• Such a vector is more particularly intended for gene therapy of tumors, especially 'Metastatic Tumors, "in particular metastatic melanoma.
• the present application also relates to a vector which comprises at least one nucleic acid of the invention, and which allows the insertion of said at least one nucleic acid into the genome of an animal cell (non-human animal cell and / or human cell), more preferably a human cell, preferably a placental cell, preferably a human placental cell.
• a vector is more particularly intended for the gene therapy of diseases or conditions involving a deficiency of placental development.
• the present application also relates to a cell, which comprises at least one mutant protein according to the invention, and / or at least one nucleic acid, DNA or AKNf according to the invention, and / or at least one vector according to the invention. .
• Such a cell may be a human cell or a non-human animal cell.
• such a cell is a tumor cell, preferably a metastatic cell, more preferably a metastatic melanoma cell.
• a tumor cell preferably a metastatic cell, more preferably a metastatic melanoma cell.
• a metastatic cell finds particular applications as a fusogenic capacity cell capable of inducing the formation of syncytia, as described below.
• a cell of the invention may be a non-tumor cell of the human or non-human animal immune system, preferably a non-human or non-human non-tumor dendritic cell, said cell expressing at least one mutant protein on its surface. according to the invention.
• a cell finds particular applications as an agent capable of inducing the production of inhibitor of cell fusion, for example by active immunization, as described below.
• a mutant protein of the invention, and / or a nucleic acid, DNA or RNA of the invention, and / or a vector of the invention, and / or a cell of the invention can be used in the treatment and / or the prevention and / or palliation of a disease or condition that involves the presence and / or proliferation of cells that are pathological and / or not favorable to the state of health of the body, more particularly in the treatment and / or prevention and / or palliation of a disease or condition that involves an insufficiency of cellular fusogenicity, preferably in the treatment and / or prevention and / or palliation of a disease or a neoplastic state, such as a tumor, a metastatic tumor, preferably a metastatic melanoma.
• Such diseases or conditions can be treated and / or prevented and / or palliated by decreasing or eliminating these pathological and / or unfavorable cells.
• a mutant protein of the invention expressed on the surface of such cells will induce the fusion of these cells, and hence the formation of syncytia, leading to the destruction (or at least decreasing number) of these cells.
• a mutant protein of the invention, and / or a nucleic acid, DNA or RNA of the invention, and / or a vector of the invention, and / or a cell of the invention can be used in the treatment and / or the prevention and / or palliation of a disease or condition that involves a deficiency of placental development.
• Such diseases or conditions can be treated and / or prevented and / or palliated by induction or stimulation of placental cell fusion.
• the present application therefore also relates to a pharmaceutical composition or a drug which comprises at least one mutant protein of the invention and / or at least one nucleic acid, DNA, RNA of the invention, and / or at least one vector of the invention. the invention and / or a cell of the invention.
• Such a pharmaceutical composition or such a medicament may in particular be intended for the treatment and / or prevention and / or palliation of a disease or a condition which involves the presence and / or proliferation of pathological and / or non-favorable cells.
• the state of health of the organism as indicated above (for example, tumor, metastatic tumor, metastatic melanoma), or the treatment and / or prevention and / or palliation of a disease or condition a condition that involves a deficiency of cellular fusogenicity (eg, impaired placental development).
• Such a pharmaceutical composition or such a medicament may further comprise at least one pharmaceutically and / or physiologically acceptable vehicle.
• a mutant protein of the invention (or a nucleic acid, DNA, RNA, or an expression vector of the invention), can be used to be expressed by a human cell or a non-human animal cell, preferably to be expressed on the surface of such a cell.
• This cell may be a pathological cell, preferably a tumor cell, more preferably a metastatic cell, more preferably a metastatic melanoma cell, or may be a non-tumor cell, for example a healthy cell.
• pathological cells that have been removed from a human patient or from a diseased non-human animal subject may be treated ex vivo (or in vitro) by contacting at least one mutant protein of the invention and / or at least one nucleic acid, DNA or RNA of the invention and / or at least one expression vector of the invention so as to make them express a mutant protein of the invention.
• non-pathological cells but localized near the pathological cells of the patient or subject can be taken to undergo this treatment.
• Cells thus treated ex vivo (or in vitro) can then be intended to be readmitted to said patient or subject.
• Such cells are useful for the treatment and / or prevention and / or palliation of the pathology affected by said patient or subject, for example a tumor, a metastatic tumor, a metastatic melanoma.
• this cell may be a placental cell, more particularly a placental cell suffering from a deficiency of fusogenicity.
• a mutant protein of the invention Once treated by expression of a mutant protein of the invention on its surface, such a cell can be intended for the treatment and / or prevention and / or palliation of a deficiency of placental development.
• the present application is therefore more particularly relative to a mutant protein according to the invention, a nucleic acid, DNA or RNA according to the invention, a vector according to the invention, a cell according to the invention, for use in the treatment and / or prevention and / or palliation of a disease or a neoplastic state, preferentially a tumor, more preferably a metastatic tumor very preferably of a metastatic melanoma.
• the present application is also more particularly relating to a mutant protein according to the invention, a nucleic acid, DNA or RNA according to the invention, a vector according to the invention, a cell according to the invention, for use in the treatment and / or the prevention and / or palliation of a deficiency of placental development.
• the present application also relates to products that have the ability to decrease or block cell fusion. These products are inhibitors of one or more of the mutant proteins of the invention.
• Such inhibitors may be used in the treatment and / or prevention and / or palliation of a disease or condition that involves an excess of cellular fusogenicity, preferentially in the treatment and / or prevention and / or palliation enveloped virus infection (such as HIV, Influenza, Parainfluenza, Rhabdovirus), allergy, autoimmune disease, transplant rejection.
• enveloped virus infection such as HIV, Influenza, Parainfluenza, Rhabdovirus
• an inhibitor according to the invention is:
• an antibody directed against a mutant protein according to the invention or an Fab or F (ab ') 2 fragment of such an antibody, or
• an aptamer nucleic acid or an aptamer peptide which specifically binds to at least one mutant protein according to the invention or to a nucleic acid, DNA, RNA according to the invention, or
• a cell of the recombinant immune system preferably a recombinant dendritic cell, which expresses on its surface at least one mutant protein according to the invention, or
• an antisense nucleic acid of a nucleic acid according to the invention or a small interfering RNA (siRNA) comprising a double-stranded RNA of 19 to 22 nucleotides, capable of binding (to hybridize) to a nucleic acid according to the invention.
• siRNA small interfering RNA
• the present application therefore also relates to a non-tumor cell of the human or non-human animal immune system, preferably a non-human human or animal dendritic cell, said cell expressing on its surface at least one mutant protein according to the invention, and the use of this cell in the treatment and / or prevention and / or palliation of a disease or condition that involves an excess of cellular fusogenicity, preferentially in the treatment and / or prevention and / or the palliation of an enveloped virus infection (such as an HIV infection, Influenza, Parainfluenza, Rhabdovirus), an allergy, an autoimmune disease, a transplant rejection.
• an enveloped virus infection such as an HIV infection, Influenza, Parainfluenza, Rhabdovirus
• the present application also relates to an antibody directed against a mutant protein according to the invention, or against several of the mutant proteins of the invention.
• this antibody is an antibody specific for said mutant protein (s) of the invention.
• this antibody is a monoclonal antibody.
• An inhibitor of the invention may be a conservative fragment of such an antibody, such as an Fab or F (ab ') 2 fragment.
• Such an antibody or antibody fragment may be for blocking or inhibiting a cell fusion mechanism, for example by administering this antibody or antibody fragment to a patient or subject in need thereof.
• a mutant protein of the invention may be intended to be itself administered to said patient or subject so as to induce active immunization against this protein, that is to say so as to induce production by said patient or subject of anti-mutant protein antibody.
• one or more vaccine adjuvants may be administered together or delayed in time with this or these mutant proteins.
• the present application therefore also relates to a therapeutic and / or preventive and / or palliative vaccine, which comprises at least one mutant protein of the invention as an immunogenic agent, and advantageously at least one immunizing adjuvant.
• Such a vaccine may be intended for the treatment and / or prevention and / or palliation of a disease or condition that involves an excess of cellular fusogenicity, preferentially in the treatment and / or prevention and / or palliation of enveloped virus infection (such as HIV, Influenza, Parainfluenza, Rhabdovims), allergy, autoimmune disease, transplant rejection.
• enveloped virus infection such as HIV, Influenza, Parainfluenza, Rhabdovims
• This application also relates to:
• an aptamer nucleic acid or an aptamer peptide which specifically binds to at least one mutant protein of the invention or to a nucleic acid, DNA, RNA of the invention,
• a cell of the recombinant immune system preferentially a recombinant dendritic cell, which expresses on its surface at least one mutant protein of the invention
• small interfering RNA small interfering RNA (small interfering RNA, siRNA) comprising a double-stranded RNA of 19 to 22 nucleotides, capable of binding (to hybridize) with a nucleic acid of the invention, and advantageously of blocking or inhibiting the transcription of this nucleic acid.
• siRNA small interfering RNA
• Such products are also inhibitors of the invention. They can therefore be intended for the treatment and / or the prevention and / or palliation of a disease or condition that involves an excess of cellular fusogenicity, as indicated above. They are more particularly intended for the treatment and / or the prevention and / or palliation of a disease or condition that involves at least one expressing gene or protein expression hyper-expressor F.
• the present application therefore also relates to a pharmaceutical composition or a medicament which comprises at least one inhibitor of the invention.
• Such a pharmaceutical composition or such a medicament may in particular be intended for the treatment and / or prevention and / or palliation of a disease or condition which involves an excess of cellular fusogenicity, as indicated above.
• Such a pharmaceutical composition or such a medicament may further comprise at least one pharmaceutically and / or physiologically acceptable vehicle.
• the present application relates more particularly to an inhibitor according to the invention, for use in the treatment and / or prevention and / or palliation of a disease or condition involving an excess of cellular fusogenicity, said disease or state being a enveloped virus infection (preferentially an HIV infection and / or Influenza and / or Parainfluenza and / or Rhabdovirus), an allergy, an autoimmune disease, a transplant rejection.
• a enveloped virus infection preferentially an HIV infection and / or Influenza and / or Parainfluenza and / or Rhabdovirus
• the present application relates more particularly to a mutant protein according to the invention, for use as an immunogenic agent in the treatment and / or prevention and / or palliation of a disease or condition involving an excess of cellular fusogenicity, said disease or condition being an enveloped virus infection (preferentially an HIV and / or Influenza and / or Parainfluenza and / or Rhabdovirus infection), an allergy, an autoimmune disease, a transplant rejection .
• an enveloped virus infection preferentially an HIV and / or Influenza and / or Parainfluenza and / or Rhabdovirus infection
• an allergy an autoimmune disease
• transplant rejection a transplant rejection
• the present application relates more particularly to a vaccine or vaccine composition, more particularly a vaccine or vaccine composition intended for the treatment and / or prevention and / or palliation of a disease or condition involving an excess of fusogenicity.
• a disease or condition being an enveloped virus infection (preferentially an HIV infection and / or Influenza and / or Parainfluenza and / or Rhabdovirus), an allergy, an autoimmune disease, a transplant rejection.
• a vaccine or vaccine composition comprises at least one mutant protein according to the invention, and optionally at least one physiologically acceptable adjuvant.
• the present application also relates to a method, more particularly an in vitro method, for the diagnosis or prognosis of a disease or condition involving:
• syncytia such as a tumor, a metastatic tumor, a metastatic melanoma or a deficiency of placental development, or on the contrary an excessive formation of syncytia, such as an enveloped virus infection (preferentially an HIV infection and / or Influenza and / or Parainfluenza and / or
• the diagnostic or prognostic method of the invention comprises the detection of at least one mutant protein according to the invention or at least one nucleic acid according to the invention, for example in a biological sample such as a biological sample that has been taken from the patient or subject object of said diagnosis or prognosis.
• This detection may for example be carried out by sequencing the proteins or nucleic acids contained in said sample.
• This detection may for example be carried out by detecting said at least one mutant protein of the invention using an antibody, an aptamer peptide, or an aptamer oligonucleotide binding to said at least one mutant protein. , more particularly using an antibody, aptamer peptide, or aptamer oligonucleotide of the invention.
• This detection may for example be carried out by detecting said at least one nucleic acid of the invention using a nucleic acid, an aptamer peptide, or an aptamer oligonucleotide binding to said at least one nucleic acid, more particularly using a nucleic acid complementary to a nucleic acid of the invention, an aptamer peptide, or an aptamer oligonucleotide of the invention.
• the present application also relates to said antibody, aptamer peptide, aptamer oligonucleotide, complementary nucleic acid, for use in a method of diagnosis or prognosis of insufficient formation, or excessive syncytia.
• the present application also relates to a method, more particularly an in vitro method, for the screening of a compound capable of decreasing or blocking the formation of syncytia.
• the method of the invention comprises contacting a candidate compound with cells expressing at least one mutant protein according to the invention, so as to determine whether this candidate compound decreases or blocks the fusion of said cells (for example, in comparing the melting level reached in the presence of said candidate compound to that reached in its absence).
• Such compounds are candidate active ingredients for the treatment and / or prevention and / or palliation of a disease or condition involving an excess of cellular fusogenicity, such as enveloped virus infections, allergies, diseases autoimmune, graft rejection.
• Biotechnological applications myeloma, hybridoma:
• the present application also relates to a tumor cell, more particularly myeloma, comprising at least one mutant protein according to the invention, preferentially comprising at least one such mutant protein on its surface, and / or comprising at least one nucleic acid according to the invention.
• invention and / or comprising at least one vector according to the invention, more particularly an expression vector according to the invention.
• a tumor cell more particularly such a myeloma
• a hybridoma by fusion of this tumor cell with a B lymphocyte
• the present application also relates to a hybridoma, more particularly an antibody producing hybridoma, which comprises at least one mutant protein according to the invention, and / or at least one nucleic acid according to the invention, and / or at least one vector according to the invention.
• a hybridoma may in particular be produced by contacting at least one B lymphocyte with at least one tumor cell, more particularly myeloma, comprising at least one mutant protein according to the invention, preferably comprising at least one such mutant protein at its surface, and / or comprising at least one nucleic acid according to the invention, and / or comprising at least one vector according to the invention.
• Such a tumor cell has an intrinsic fusogenic capacity: it is therefore capable of fusing with said at least one B lymphocyte, without the use of polyethylene glycol (PEG) or electroporation means or any other means which, in the prior art, are conventionally used to induce the fusion of a tumor cell to a B lymphocyte for the purpose of producing a hybridoma.
• PEG polyethylene glycol
• Biotechnological applications stem or progenitor cells:
• the present application also relates to a stem or progenitor cell, comprising at least one mutant protein according to the invention, preferentially comprising at least one such mutant protein on its surface, and / or comprising at least one nucleic acid according to the invention, and / or comprising at least one vector according to the invention, more particularly an expression vector according to the invention.
• a stem or progenitor cell has an intrinsic fusogenic capacity: it is therefore capable of forming syncytia by fusion.
• this stem or progenitor cell has, in addition, a differentiating capacity in a muscle cell, it is then capable of forming a muscle fiber (by cell fusion and formation of a syncytium).
• the present application is therefore also related to such a stem or progenitor cell for use in the production, for example in vitro production, of a muscle fiber.
• This production can, for example, be carried out by placing a plurality of said stem or progenitor cells in contact with each other on or in a culture medium allowing the proliferation of stem cells, or, where appropriate, progenitors, so that the fusogenic capacity said stem or progenitor cells can be exerted therein, thereby inducing the formation of a syncytia, more particularly a muscle fiber.
• Examples of culture media for the proliferation of stem cells, or where appropriate progenitor cells, which are furthermore suitable for the expression of their possible capacity to differentiate into a muscle cell, more particularly into a muscle fiber, are known from the art. a person skilled in the art, for example the MCDB medium. Examples of cell markers making it possible to observe the differentiation of a stem or progenitor cell into a muscle cell, more particularly into a muscle fiber, are also known to those skilled in the art, for example CD56.
• the term “comprising”, with which "including” or “containing” is synonymous, is an open term, and does not exclude the presence of one or more element (s), ingredient (s) or additional method step (s) that would not be explicitly stated, while the term “consistent” or “constituted” is a closed term, which excludes the presence of any additional element, step , or ingredient that would not be explicitly exposed.
• the term “substantially consisting of” or “essentially consisting of” is a partially open term, which does not exclude the presence of one or more element (s), ingredient (s) or additional step (s) in the to the extent that such element (s), ingredient (s) or additional step (s) do not materially affect the basic properties of the invention.
• the LLC-MK2 cell line (Macaca mulattd kidney cell line) is available from the American Type Culture Collection (ATCC) under the number CCL-7.
• the A549 cell line (human lung carcinoma cell line) is available from ATCC under the number CCL-185.
• the recombinant HuH7-Tat line (human hepatoma cell line) is available by transducing cells of the HuH-7 line with HIV-I Tat.
• the HuH-7 line is available from the Japanese Collection of Bioresources
• the transduction of the HuH-7 line by HIV-Tat was carried out using the retro viral LXSN-tat vector transducing the Tat plasmid.
• LLC-MK2, A549 and HuH7-Tat cells were cultured in Eagle's Minimum Essential Medium (EMEM) or DMEM (Dulbecco / Vogt Modified Eagles Essential Minimum Medium) with 5% fetal calf serum.
• EMEM Eagle's Minimum Essential Medium
• DMEM Dulbecco / Vogt Modified Eagles Essential Minimum Medium
• the PIV-5 WR strain was obtained from ATCC (ATCC number VR-288), and was grown on LLC-MK2 cells as described in Terrier et al. 2008.
• the viral RNA was extracted from the supernatant obtained from an infection of LLC-MK2 cells by PIV-5, using the kit Absolutely RNA® microprep kit (Stratagene, USA), following the instructions given by the provider.
• Reverse transcription was performed using random hexamers pd (N) 6 (Amersham Biosciences, GB) and a transcriptase reverse ⁇ Reverse Transcriptase; RT) of avian myeloblastosis virus ⁇ Avion Myeloblastosis Virus; AMV) (AMV-RT reverse transcriptase available from Promega).
• Amplification of the complete PIV-5 sequence was performed with a primer pair designed from the PIV-5 nucleotide sequence available in the databases (GenBank accession number AB021962).
• the pair of primers used was as follows:
• Antisense primer SEQ ID NO: 2
• the amplification was carried out according to the following protocol: 95 ° C. for 2 min, then 39 cycles (95 ° C./30 sec 55 ° C./1 min 72 ° C./3 min) and a final elongation of 10 min at 72 ° C. ° C.
• the F PIV-5 complement DNA was cloned into the expression plasmid pcDNA3.1 (+) at the NotI and XhoI sites at the multiple cloning site (cf. Figure 4).
• PIV-5 F protein mutant proteins were made by directed mutation within the pcDNA3.1 plasmid encoding the PIV-5 F fusion protein.
• the mutation (s) was (are) generated by PCR using complementary primers, following the protocol indicated by the supplier (QuickChange® Site-Directed Mutagenesis System available from Stratagene). The list of primers used is given in Table 2 below. All plasmids were monitored by sequencing. Table 2: list of primers
• the 443P mutation is theoretically pre-existing in the F protein of the WR isolate. However, in the sample of this isolate that the inventors received from ATCC, this mutation was in fact not present. It had to be introduced by the inventors.
• the cells were transfected with the plasmids using the ExGen500 reagent (Fermentas), following the instructions given by the supplier.
• One to three micrograms of plasmid DNA was added to the cells (at 70-80% confluency) for 48h.
• the efficiency of transfection was estimated using a plasmid encoding fluorescent green protein (Green Fluorescence Protein; GFP).
• the transfected cells were fixed with paraformaldehyde (1% v / v) in phosphate buffer (phosphate buffered saline; PBS) then washed twice.
• the cell mats were incubated in the presence of a monoclonal antibody directed against the fusion protein F
• PIV-5 in this case the monoclonal antibody Fia described in Randall et al. 1987, diluted 1/10 in PBS for 3h.
• the monoclonal antibody Fia was obtained by immunizing mice against an isolate of PIV-5 (in this case the isolate LN) 5 preparation of hybridomas and selection of anti-F antibodies.
• the cell mats were then washed and incubated with an IgG-Alexa Fluor® 633 anti-mouse secondary antibody (Invitrogen) diluted 1/200 in PBS for 30 minutes. After rinsing, the cells were incubated for 10 minutes with Dapi (4 ', 6'-Di-Amidino-2-phenyl Indole) at 1/1000 mixed or not with wheat germ Pagglutinin (Wheat Germ Agglutinin; WGA) coupled. to Alexa Fluor® 488 (WGA-Alexa Fluor® available from Invitrogen) at 1/200 in phosphate buffer (Phosphate Buffer Saline; PBS). The images were acquired using a TCS SP2 confocal microscope (Leica).
• A549 cells were transfected with the plasmids encoding the different Fus and were deposited on ice. The cell mats were rinsed with phosphate buffer PBS (Phosphate Buffer Saline; PBS) with 1% azide sodium.
• PBS Phosphate Buffer Saline
• a PIV-5 monoclonal anti-F protein monoclonal antibody was then added to the carpets (1/500 in 1% PBS phosphate buffer in fetal calf serum), and incubated. minutes at 4 ° C.
• the mats were then rinsed and incubated in the presence of a secondary anti-mouse antibody coupled to Alexa Fluor® 488 1/1000 (Invitrogen). After rinsing, the cells were gently peeled off using 500 ⁇ l of PBS phosphate buffer 0.5 mM EDTA (ethylene-diamine tetraacetic acid). The cells were transferred into dedicated flow cytometry tubes containing 500 ⁇ l of a 1% paraformaldehyde solution. The fluorescence intensity of 5000 cells was measured on a fluorescence activated cell sorting (FACS) cytometer, in this case on Becton Dickinson's FACSVantage TM SEflow.
• FACS fluorescence activated cell sorting
• the score thus calculated is the sum of the two notes; the theoretical maximum score is 10 and corresponds to a syncytium of maximum size with a maximum number of nuclei.
• Quantitative fusion test (measurement of luciferase activity)
• donor cells were detached with 0.5 mM EDTA phosphate buffer (PBS), and were counted and then new 6-well plates (10 5 cells / well).
• HuH7-Tat "indicator” cells (4.10 5 cells / well) were removed by means of PBS-EDTA buffer, then rinsed and added to the "donor" cells.
• the luciferase activity was measured at 72 hours of co-culture using a kit for measuring luciferase activity, in this case the Luciferase Assay System (E1500) kit of Promega, following the indications given by supplier.
• E1500 Luciferase Assay System
• FIGS. 5A, 5B, 5C and 5D the positions of the mutations of Table 3 are illustrated. Mutant proteins were thus constructed, produced and tested by the inventors.
• the PIV-5 F protein sequence that was implemented during the construction and production of these mutant proteins was an alternative F protein sequence of WR isolate.
• This alternative sequence was identical to the sequence of SEQ ID NO: 31 (Genbank sequence), except for the amino acid at position 443 which was S and not P (alternative sequence SEQ ID NO: 31 with S at 443). ").
• sequences of SEQ ID NO: 47 to 79 are therefore the sequences which result from the replacement, within said alternative sequence "SEQ ID NO: 31 with S at 443", of the amino acids indicated for each of these sequences in Table 3 above.
• mutant proteins which have in common the understanding of the three autonomy mutations and the 449P pre-fusion mutation such as the mutant Fus8, Fus10, Fus10.4, Fus10.5, Fus1, Fus ⁇ .l proteins. , Fus8.2, Fus8.4, Fus8.5, Fus8.6, Fus8.7, FuslO.l, Fusl0.2, FuslO.3, and the group of mutant proteins which have in common to understand the three autonomy mutations, the pre-fusion mutation 447P and at least one post-fusion mutation (147V or 158V), such as Fus7.1, Fus7.2 and Fus7.3.
• Figures 7A and 7B show an illustration of the observations made under the microscope and show the fusion scores for a selection of mutant proteins tested, namely the group of mutant proteins which have in common to include:
• a hydrophobic amino acid such as V, I or L 5 e.g. 147V post-fusion mutation and / or the 158V post-fusion mutation, such as Fus7.1, Fus7.2 and Fus7.3 mutant proteins.
• mutant proteins of the invention were further modified by substitution of the natural cleavage site of the native F protein, for example to replace it with a tissue-cleavage site. specific.
• the natural cleavage site of PIV-5 F protein may be substituted by the site of an enzyme specifically expressed by metastatic tumor tissue, ie, matrix metalloprotease 9 (MMP-9).
• MMP-9 matrix metalloprotease 9
• the natural cleavage site of the PIV-5 F protein is: RRRRR (SEQ ID NO: 23).
• F protein sequence fragment comprising the natural cleavage site of PIV-5 F protein is:
• An example of an MMP-9 cleavage site is: PRRIT (SEQ ID NO: 28).
• mutant F protein sequence fragment of the invention comprising an MMP-9 cleavage site is: IGENLETIRNQLIPTPRRITFAGVVIGL (SEQ ID NO: 29).
• Mutant proteins of PIV-5 F protein were produced as described in Example 1 above.
• the replacement of the cleavage site was carried out by 3 successive directed mutageneses within the plasmid pcDNA3.1 encoding the F PIV-5 fusion protein.
• the mutations were generated by PCR using complementary primers, following the protocol indicated by the supplier (QuickChange® Site-Directed Mutagenesis System available from Stratagene). All plasmids were monitored by sequencing.
• the cells were transfected with the plasmids using the ExGen500 reagent
• plasmid DNA was added to the cells (at 70-80% confluency) for 48h.
• the efficiency of transfection was estimated using a plasmid encoding fluorescent green protein (Green Fluorescence Protein; GFP).
• the transfected cells were fixed with paraformaldehyde (1% v / v) in phosphate buffer (phosphate buffered saline; PBS) then washed twice.
• the cell mats were incubated in the presence of a monoclonal antibody directed against the F PIV-5 fusion protein, in this case the Fia monoclonal antibody described in Randall et al. 1987, diluted 1/10 in PBS for 3h.
• the Fia monoclonal antibody was obtained by immunizing mice against a PIV-5 isolate (in this case the LN isolate), preparing hybridomas and selecting specific anti-F antibodies.
• the cell mats were then washed and incubated with an IgG-Alexa Fluor® 633 anti-mouse secondary antibody (Invitrogen) diluted 1/200 in PBS for 30 minutes. After rinsing, the cells were incubated for 10 minutes with Dapi (4 ', 6'-Di-Amidino-2-phenyl Indole) 1/1000 mixed or not with wheat germ agglutinin (Wheat Germ Agglutinin; WGA ) coupled to Alexa Fluor® 488 (WGA-Alexa Fluor® available from Invitrogen) at 1/200 in phosphate buffer (Phosphate Buffer Saline, PBS). The images were acquired using a TCS SP2 confocal microscope (Leica). Bibliographical references

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• 2008
  • 2008-11-21 FR FR0806548A patent/FR2938841B1/fr not_active Expired - Fee Related
• 2009
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