Patent Grant
9974852
The present invention relates to mutated non-primate 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 have the unique ability among retroviruses of being able to integrate into the DNA of the host and replicate in non-dividing cells. They are in most cases pathogenic, infection resulting in a severe immunodeficiency syndrome (AIDS).
Lentiviruses can be found in primates including humans (HIV1, HIV2) and monkeys (SIV), as well as in other animals.
As examples of animal lentiviruses, one may cite feline lentiviruses such as Feline Immunodeficiency Virus (FIV) which can be found in cats, as well as in other felidae (e.g. lion, cougar).
Other examples of animal lentiviruses are bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV, BIV from Indonesia), equine infectious anaemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), Visna/Maedi virus (VMV) and ovine Visna/Maedi virus (OMVV).
Feline immunodeficiency virus (FIV) causes an AIDS-like syndrome in the domestic cat, with marked similarity to AIDS caused by human immunodeficiency virus (HIV) in humans. FIV in cats typically manifests itself as a transient acute-phase syndrome, characterized by febrile episodes, lymphadenopathy, neutropenia, and weight loss. This initial phase is followed by a protracted asymptomatic period with progressive loss of CD4+ T lymphocytes and a terminal AIDS phase characterized by succumbing to opportunistic infections. Variability exists among FIV strains, not only in sequence relatedness but also in pathogenicity in vivo. FIV has been reported worldwide with a prevalence rate ranging from 1 to 28%. It affects 2-4% of cats in the US.
Following criteria similar to those for HIV subgroups distinction, FIV has been classified into subgroups according to the envelop protein (Env) diversity. Sodora et al. defined the original Petaluma strain as a prototype subgroup A and the divergent Japanese TM2 strain as prototype subgroup B. A majority of FIV sequences obtained worldwide were categorized in the A and B subgroups. A subtype C was also defined and is the least prevalent FIV subgroup, as well as two other subtypes D and E.
As in the case of HIV, the development of an effective vaccine against FIV is difficult because of the variations of the virus strains, most probably associated with the high intrinsic mutation rate of its replicative machinery. A dual-subtype vaccine for FIV released in 2002 called Fel-O-Vax was shown to have some protective effect against subtypes A and B FIV, but a more effective vaccine is still needed for an efficient protection of cats.
A key feature that could result in higher efficacy of a FIV dedicated vaccine is to increase the immunogenicity of the FIV antigens that are introduced in the vaccine. In this respect, it is essential to ascertain that the antigens do not carry an immunosuppressive activity, which would severely decrease the immunogenicity of the vaccine. In fact, most vaccines against retroviruses contain the Env protein as it is the first exposed viral protein to be «seen» by the immune system of an infected animal, and it is important for a vaccinated animal to be able to mount an efficient line of defense against this protein. Yet, it has been shown in the case of the non-lentiviral retroviruses—including oncoretroviruses such as the feline FeLV or the human HTLV retroviruses—that their Env protein carries an immunosuppressive activity which drastically decreases the immunogenicity of the corresponding proteins, and severely impairs the efficacy of a vaccine expressing the Env protein.
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 animals once infected by the virus to allow the immune system to destroy the infected cells and prevent/cure the infection.
One aim of the invention is to provide new mutated Env proteins devoid of immunosuppressive properties.
Another aim of the invention is to provide new mutated animal Env proteins.
Another aim of the invention is to provide a new pharmaceutical composition efficient for preventing/treating lentiviral infection.
Another aim of the invention is to provide an efficient vaccine against animal lentiviruses, in particular feline lentiviruses.
The invention relates to a pharmaceutical composition comprising as active substance an isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated feline lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type feline lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 5,
said mutated feline lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In particular, the invention relates to a pharmaceutical composition comprising as active substance an isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
in particular said fragment of said isolated mutated feline lentiviral ENV protein comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated feline lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type feline lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 5,
said mutated feline lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
The present application is based on the unexpected observation made by the Inventors that some specific amino acids of the immunosuppressive domain (ISU) of a lentiviral Env protein can be mutated conferring to said lentiviral Env protein decreased immunosuppressive properties, substantially no immunosuppressive properties or no immunosuppressive properties, while retaining its antigenicity, the three-dimensional structure of the immunosuppressive domain, and its expression at the plasma membrane.
The expression “having decreased immunosuppressive properties” means that the mutated Env protein has lost at least 50% of its immunosuppressive activity in comparison to the immunosuppressive activity of the wild type Env protein.
The expression “having substantially no immunosuppressive properties” means that the mutated Env protein has lost at least 75% of its immunosuppressive activity in comparison to the immunosuppressive activity of the wild type Env protein.
The expression “having no immunosuppressive properties” means that the mutated Env protein has lost 100% of its immunosuppressive activity in comparison to the immunosuppressive activity of the wild type Env protein.
Moreover, the mutated lentiviral Env protein according to the invention has retained a part or the totality of its fusogenic activity.
In the invention, the expression “mutated feline lentiviral ENV proteins” means that the ENV proteins derive from the expression of an env gene of a lentivirus of a feline.
Feline lentiviruses according to the invention encompass the FIV which can be found in cats, as well as in other felidae (e.g. lion, cougar).
Because of the natural variability of feline lentiviruses, 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 FIV variants inside a same infected animal, wherein the said “mutated Env protein” might be non immunosuppressive but its property is undetectable because an FIV infected animal carries many FIV variants, the majority of which is immunosuppressive.
The following protein (Accession No. P16090) corresponds to the wild type sequence of the Env protein of FIV (Petaluma strain). In the invention, it is considered as a reference sequence of the wild type ENV protein.
Variants of the FIV 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 FIV Env protein, and comprise the mutations as described above, and harbour a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity.
A region of the feline lentiviral Env protein containing the amino acids which may be mutated according to the invention can be delimited by the sequence SEQ ID NO: 2:
SEQ ID NO: 2 corresponds to the consensus sequence of a specific region of the ISU domain. In the invention, the ISU domain comprises SEQ ID NO: 2.
When applying the mutations as defined above, the ISU domain of the Env protein loses partially or totally its immunosuppressive properties.
Examples of fragments of the wild type ISU domains of FIV:
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.
As an example of a mutated feline lentiviral Env protein, SEQ ID NO: 5 corresponds to the sequence of a mutated Env protein of the FIV Petaluma strain. In the invention, it is considered as a reference sequence of the mutated Env protein.
More specifically, SEQ ID NO: 5 corresponds to the SEQ ID NO: 4 in which the amino acid residue F in position 5 (X3) of the sequence A-[I/M/T/L]-X1-X2-X3-X4-X5-T-A (SEQ ID NO: 1) has been substituted by R.
The invention also encompasses the variants of the “mutated feline lentiviral Env protein”, harbouring the above mentioned mutations, and conferring a decrease or a lack of immunosuppressive properties to said variant.
Variants of the FIV 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 FIV env protein (SEQ ID NO: 5), and comprise the mutations as described above, and harbour a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity.
Variants of the FIV mutated Env proteins according to the invention have at least 90% of identity with the transmembrane region of SEQ ID NO 5
According to a particular embodiment of the invention, variants of the FIV mutated Env proteins according to the invention have a transmembrane region which has at least 90% of identity with the transmembrane region of the FIV mutated Env protein, said variants comprising a mutated immunosuppressive domain (ISU) containing the sequence SEQ ID NO: 1.
According to another particular embodiment of the invention, variants of the FIV mutated Env proteins according to the invention have a 64 amino acid sequence, corresponding to a fragment of the ISU domain, which has at least 95% of identity with a 64 amino acid sequence of the FIV mutated Env protein comprising the sequence SEQ ID NO: 1.
In a particular embodiment, the invention relates to a pharmaceutical composition as defined above comprising as active substance an isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof, said mutated feline lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type feline lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 5,
said mutated feline lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in particular, wherein,
in association with a pharmaceutically acceptable carrier.
SEQ ID NO: 3 (14 amino acid long) is a consensus sequence containing SEQ ID NO: 1 (9 amino acid long).
The invention relates to a pharmaceutical composition as defined above comprising as active substance an isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated feline lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type feline lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 5,
said mutated feline lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in particular, wherein,
in association with a pharmaceutically acceptable carrier,
said decrease, substantial absence or absence of immunosuppressive activity of the above mentioned mutated feline 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, in animals excluding human beings,
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 (imutated env)/immunosuppression index of wild type ENV protein (iwild type env) is less than 0.5, or even less than 0.25,
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
iwild 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 immunosuppressive property of the ENV protein is preferably measured using in vivo procedures, which are representative of the physiological environment.
The immunosuppressive property of the mutated ENV proteins according to the invention is 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):20534-9].
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 transduced in tumour cell lines such as MCA 205 or C18.1 cell lines by known transduction 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. Tumour establishment is determined by palpation and tumour area (mm2) is determined by measuring perpendicular tumour diameters. Immunosuppression index is defined as i=(Senv−Snone)/Snone, 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 immunosuppression index (imutated env/iwild type env) can be less than 0.25, and can even have a negative value (see
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 do not correspond to naturally occurring counterpart.
As mentioned above, the lentiviral mutated Env proteins have decreased immunosuppressive properties, substantially no immunosuppressive properties or no 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 proteins from a virus of the same species. For instance, a mutated FIV Env protein according to the invention has reduced immunosuppressive properties with respect to the wild type FIV Env protein.
In the invention, the terms “a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity” means that the mutated Env proteins according to the invention have an immunosuppressive index less than about 50 or 25% of that of the wild type Env protein [Mangeney and Heidmann Proc Natl Acad Sci USA 1998; 95: 14920-14925; Mangeney et al. Proc Natl Acad Sci USA, 2007, 104(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 testing the ability of the Env proteins to be normally expressed at the cell membrane under conditions which preserve the surface and transmembrane subunits (SU:TM) interactions and recognition by specific anti-Env antibodies, and by measuring the functional fusogenic activity of said mutated lentiviral Env with respect to the same properties in the wild type non mutated lentiviral Env protein (see examples).
Generally speaking, the mutated ENV protein involved in the present invention has an average length of about 750 to about 1000 amino acids.
The invention encompasses fragments of the mutated ENV protein as defined above, provided that said fragment:
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, 700, 750, 800, 850, 900, 950 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 from about 40 to about 950 amino acids.
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, because said regions could be detrimental for targeting an immune response against the immunosuppressive domain.
The invention also relates to a pharmaceutical composition wherein the active substance is an isolated non naturally occurring feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, as above defined, or fragments thereof.
In a particular embodiment, the invention relates to a pharmaceutical composition as defined above wherein:
In a particular embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated immunosuppressive domain contains the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 3, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated immunosuppressive domain contains the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 3, wherein:
in particular:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated immunosuppressive domain contains the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 3, wherein:
in particular:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated immunosuppressive domain contains the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 3, wherein: X1 is A, G or R, and X2, X3, X4 and X5 are any amino acid different from A, G or R, or
in particular:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated immunosuppressive domain contains the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 3, wherein:
in particular:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated feline lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) contains the following amino acid sequence:
in particular:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated feline lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) contains the following amino acid sequence:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated feline lentiviral ENV protein or said fragment thereof, comprises one of the amino acid sequences SEQ ID NO: 28 to 171.
In the invention “SEQ ID NO: 28 to 171” encompasses SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170 and 171.
The correspondence is the following one:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said fragment of said isolated mutated feline lentiviral ENV protein comprises at least 40 amino acids, in particular at least 60 amino acids.
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated feline lentiviral ENV protein or said fragment thereof, comprises one of the amino acid sequences SEQ ID NO: 172 to 315.
In the invention “SEQ ID NO: 172 to 315” encompasses SEQ ID NO: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314 and 315.
The correspondence is the following one:
As mentioned above, the previous ENV proteins having their ISU comprising the above sequence have decreased immunosuppressive properties, substantially no immunosuppressive properties or no immunosuppressive properties.
Thus, in other words, any feline lentiviral Env protein having in their ISU an amino acid sequence comprising the sequences SEQ ID NO: 28 to 315, have decreased immunosuppressive properties, substantially no immunosuppressive properties or no immunosuppressive properties.
In other words, a feline lentiviral ENV protein comprising, within its ISU domain, an amino acid sequence selected from SEQ ID NO: 28 to 315 have decreased immunosuppressive properties, substantially no immunosuppressive properties or no immunosuppressive properties.
In the invention, the fragments of the mutated ENV proteins according to the invention have decreased immunosuppressive properties, substantially no immunosuppressive properties or no 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 another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein mutated feline lentiviral ENV protein or fragment thereof further comprises an additional mutation of one at least of the amino acids X1*, X2*, X3*, X4* and X5* in the following sequence:
wherein
in association with a pharmaceutically acceptable carrier.
In such a pharmaceutical composition, the mutated feline lentiviral ENV protein, or fragment thereof, comprises a mutation of one at least of the amino acids X1, X2, X3, X4 and X5 in association with an additional mutation of one at least of the amino acids X1*, X2*, X3*, X4*and X5*.
In the invention, “a mutation of one at least of the amino acids X1*, X2*, X3*, X4* and X5*” means that said mutated acids are:
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein mutated feline lentiviral ENV protein or fragment thereof further comprises an additional mutation of the amino acids X3* in the following sequence:
wherein
in association with a pharmaceutically acceptable carrier.
In such a pharmaceutical composition, the mutated feline lentiviral ENV protein, or fragment thereof, comprises a mutation of one at least of the amino acids X1, X2, X3, X4 and X5 in association with an additional mutation of X3*.
The sequences SEQ ID NO: 316 and SEQ ID NO: 434 contain the following amino acid sequence A[I/M/T/L]X1X2X3X4X5TA (SEQ ID NO: 1) elongated on its N-terminal end, in which an additional mutation is present.
In another aspect, the invention relates to a pharmaceutical composition comprising as active substance an isolated mutated feline lentiviral ENV protein having substantially no immunosuppressive activity, or a fragment thereof,
said mutated feline lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type feline lentiviral ENV protein,
said mutated feline lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein
in association with a pharmaceutically acceptable carrier.
In another aspect, the invention relates to a pharmaceutical composition comprising as active substance an isolated mutated feline lentiviral ENV protein having substantially no immunosuppressive activity, or a fragment thereof,
said mutated feline lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type feline lentiviral ENV protein,
said mutated feline lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein
in association with a pharmaceutically acceptable carrier.
In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated feline lentiviral protein, said variant or said fragment harbour a three-dimensional structure similar to the structure of the natural non mutated feline lentiviral ENV protein, non mutated variant or non 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 feline lentiviral protein or said fragment thereof 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 or non mutated 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 examples).
If no complex is detected, the mutated ENV protein is not expressed at the plasma membrane. On the contrary, if the complex is detected, this means that the mutated ENV protein is expressed at the plasma membrane and in an appropriate conformation so as to be detected by the antibody recognizing the extracellular part of the protein.
In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated feline lentiviral ENV protein is such that it has conserved, totally or partially, its fusogenic activity, which is responsible for virus-cell or cell-cell membrane fusion and can be measured by appropriate assays.
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated feline lentiviral ENV protein consists of one of the amino acid sequences: SEQ ID NO: 5 and SEQ ID NO: 317 to 342 and 374 to 419.
The invention also encompasses the variants of the above sequences, harbouring the above mentioned mutations, and conferring to said variant a decrease of immunosuppressive properties, a substantial absence of immunosuppressive properties or an absence of immunosuppressive properties.
Advantageously: the mutated feline lentiviral proteins are:
1—SEQ ID NO: 5, SEQ ID NO: 317 to 342 and SEQ ID NO: 374 to 419; these mutated ENV proteins having decreased immunosuppressive properties, substantially no immunosuppressive properties or no immunosuppressive properties.
advantageously,
2—SEQ ID NO: 5, SEQ ID NO: 317 to 342 and SEQ ID NO: 374 to 419; these mutated ENV proteins having decreased immunosuppressive properties, substantially no immunosuppressive properties or no immunosuppressive properties, and being highly expressed at the plasma membrane,
more advantageously,
3—SEQ ID NO: 5, SEQ ID NO: 317 to 342, and SEQ ID NO: 374 to 419; these mutated ENV proteins having decreased immunosuppressive properties, substantially no immunosuppressive properties or no immunosuppressive properties, being highly expressed at the plasma membrane and possessing a medium or high fusogenic activity.
As mentioned above “possessing a medium or high fusogenic activity” means that when the ENV proteins are expressed at a cell plasma membrane, by using appropriate expression vectors the fusogenic activity of said proteins as measured by quantitating the ENV-mediated cell-cell fusion (see Materials and Methods) is:
In another aspect, the invention relates to a pharmaceutical composition comprising as active substance a nucleic acid molecule coding for a mutated feline lentiviral ENV protein, or a fragment of said mutated feline lentiviral ENV protein, as defined above.
In a particular embodiment, the invention concerns a pharmaceutical as defined above, wherein said nucleic acid molecule is contained in a vector, said vector comprising means allowing the expression of the mutated feline lentiviral ENV protein, or a fragment of said mutated feline lentiviral ENV protein, as defined above.
In another embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said nucleic acid molecule is comprised in a vector, said nucleic acid molecule 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 feline lentiviral ENV protein or a fragment thereof.
DNA vaccines expressing ENV proteins can be produced as described in Bellier et al. (Vaccine, 2009, 27(42):5772-80).
In another embodiment, the invention concerns a pharmaceutical as defined above, wherein said vector is chosen among a canarypox vector, a pox vector, a fowlpox, an adenoviral vector, a lentiviral vector, a measles vector, a Sendaï virus vector, a Cytomegalovirus vector or a Modified Vaccinia Ankara vector.
In another embodiment, the invention concerns a pharmaceutical as defined above, comprising at least one nucleic acid molecule coding for a GAG protein and/or a PRO protein and/or a POL protein of a feline lentivirus, said lentivirus being preferably of the same origin as the mutated lentiviral ENV protein.
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, 2009, 388:191-203).
In another embodiment, the invention concerns a pharmaceutical as defined above, wherein said nucleic acid molecule coding for a mutated feline lentiviral ENV protein, or a fragment of said mutated feline 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.
In another embodiment, the invention concerns a pharmaceutical as defined above, wherein said nucleic acid molecule coding for a mutated feline lentiviral ENV protein, or a fragment of said mutated feline 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.
In another embodiment, the invention concerns a pharmaceutical as defined above, wherein said nucleic acid molecule coding for a mutated feline lentiviral ENV protein, or a fragment of said mutated feline 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.
In another embodiment, the invention concerns a pharmaceutical as defined above, wherein said nucleic acid molecule coding for a mutated feline lentiviral ENV protein, or a fragment of said mutated feline lentiviral ENV protein, said nucleic acid molecule coding for a GAG protein, said nucleic acid molecule coding for a PRO protein and said nucleic acid molecule coding for a POL protein, are all contained in vectors which are different from each other.
In another embodiment, the invention concerns a pharmaceutical as defined above, comprising at least one nucleic acid molecule coding for a GAG protein of a feline lentivirus, said lentivirus being preferably of the same origin as the mutated lentiviral ENV protein.
In another embodiment, the invention concerns a pharmaceutical as defined above, wherein said nucleic acid molecule coding for a mutated feline lentiviral ENV protein, or a fragment of said mutated feline 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.
In another embodiment, the invention concerns a pharmaceutical as defined above, wherein said nucleic acid molecule coding for a mutated feline lentiviral ENV protein, or a fragment of said mutated feline lentiviral ENV protein, is contained in the same vector as the one which also contains a nucleic acid molecule coding for a GAG protein.
In another embodiment, the invention concerns a pharmaceutical as defined above, wherein said nucleic acid molecule coding for a mutated feline lentiviral ENV protein, or a fragment of said mutated feline lentiviral ENV protein, is contained in the same vector as the one which also contains a nucleic acid molecule coding for a GAG protein,
said vector being preferably a canary pox vector (Poulet et al., Veterinary Record, 2003, 153(5):141-145; Vaccari et al., Expert Review of Vaccines, 2010, Vol. 9, No 9: 997-1005).
In another embodiment, the invention concerns a pharmaceutical as defined above, wherein said nucleic acid molecule coding for a mutated feline lentiviral ENV protein, or a fragment of said mutated feline 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.
In another embodiment, the invention concerns a pharmaceutical as defined above, wherein said nucleic acid molecule coding for a mutated feline lentiviral ENV protein, or a fragment of said mutated feline lentiviral ENV protein, and said nucleic acid molecule coding for a GAG protein are contained in vectors which are different from each other.
In another aspect, the invention concerns a pharmaceutical composition as defined above, in association with at least one antiviral compound, preferably for a simultaneous, separated or sequential use.
The composition according to the invention can also be used in combination with the antiviral compositions listed in Table 1 below:
In another aspect, the invention concerns a pharmaceutical composition as defined above, for its use for stimulating an immune response in a host organism.
In another aspect, the invention concerns a pharmaceutical composition as defined above, for its use for the prevention or the treatment of lentiviral infection, preferably FIV infection.
In another aspect, the invention concerns a pharmaceutical composition as defined above, for its use as a vaccine, in particular against FIV 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 molecule 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 as defined above, comprising a nucleic acid coding for a GAG protein as defined above and a nucleic acid coding for a mutated ENV protein as defined above, which are contained in a same vector, said same vector being preferably a canary pox vector.
In a particular embodiment, the pharmaceutical composition according to the invention encompasses a vaccine, comprising a GAG protein as defined above and a mutated ENV 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 and a mutated ENV protein as defined above which are associated to at least one adjuvant.
As examples of adjuvants, a non limitative list is given below:
Suitably, the total amount of mutated lentiviral ENV protein in a single dose of the immunogenic composition is 1-500 μg and/or the total amount of unfused polypeptides in a single dose of the immunogenic composition is 1-500 μg. In one embodiment, the total amount of all antigens in a single dose of the immunogenic composition is 1-1000 μg, 10-500 μg, 100-200 μg, or around 100 μg.
The amount of mutated feline 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 infected animals.
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 an animal (for instance as a booster) every 6-24, or 9-18 months, for instance annually. For instance, the composition is administered to an animal (for instance as a booster) at six month or 1 year intervals. Suitably in this respect, subsequent administrations of the composition to the animal boost the immune response of earlier administrations of the composition to the same animal.
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 FIV strains from one or more clades different from the one or more FIV 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 FIV, since neutralizing antibodies, CD4+ T cells and CD8+ T cells are thought to be important for the immune defense against FIV.
The invention also relates to a method for treating animals afflicted by pathologies related to lentiviral infections, comprising the administration to an animal 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, as a vaccine, for its use for the prevention or the treatment of FIV infection, or pathologies related to feline immunodeficiency.
In another aspect, the invention relates to a pharmaceutical composition comprising as active substance:
an isolated non naturally occurring mutated feline lentiviral ENV having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said mutated feline lentiviral ENV resulting from mutation of the transmembrane subunit (TM) of a wild type feline lentiviral ENV protein, the transmembrane subunit comprising an immunosuppressive domain (ISU) comprising the following amino acid sequence:
wherein the amino acids at the positions chosen among
are:
In another aspect, the invention relates to a method to obtain the active substance of a pharmaceutical composition, as defined above, consisting in modifying the immunosuppressive property of:
a wild-type feline lentiviral ENV protein,
or a fragment of said wild-type feline lentiviral ENV protein, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said ENV protein or fragment thereof presenting a transmembrane subunit (TM) comprising an immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein
said method comprising a step of introduction of at least one mutation of XA and/or XB and/or XC and/or XD and/or XE,
to obtain:
an isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
said mutated feline lentiviral ENV protein having at least 70% identity, preferably at least 80% identity, to one sequence SEQ ID NO: 5,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In a particular embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, consisting in modifying the immunosuppressive property of a wild-type feline lentiviral ENV protein, or a fragment of said wild-type feline lentiviral ENV protein, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said ENV protein or fragment thereof presenting a transmembrane subunit (TM) comprising an immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein
said method comprising a step of introduction of at least one mutation of XA and/or XB and/or XC and/or XD and/or XE,
to obtain:
an isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
said mutated feline lentiviral ENV protein having at least 70% identity, preferably at least 80% identity, to one sequence SEQ ID NO: 5,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, consisting in modifying the immunosuppressive property of a wild-type feline lentiviral ENV protein, or a fragment of said wild-type feline lentiviral ENV protein, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said ENV protein or fragment thereof presenting a transmembrane subunit (TM) comprising an immunosuppressive domain (ISU) containing the following amino acid sequence:
said method comprising a step of introduction of at least one mutation of E in position 3 and/or K in position 4 and/or [F/P] in position 5 and/or [L/V/I] in position 6 and/or Y in position 7,
to obtain:
an isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, consisting in modifying the immunosuppressive property of a wild-type feline lentiviral ENV protein, or a fragment of said wild-type feline lentiviral ENV protein, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said ENV protein or fragment thereof presenting a transmembrane subunit (TM) comprising an immunosuppressive domain (ISU) containing the following amino acid sequence:
said method comprising a step of introduction of at least one mutation of E in position 3 and/or K in position 4 and/or [F/P] in position 5 and/or [L/V/I] in position 6 and/or Y in position 7,
to obtain:
an isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, wherein said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, wherein said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, wherein said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, wherein said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, wherein said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, wherein said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, wherein said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, wherein said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, wherein said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
X3 is R, and X1, X2, X4, X5 are A, F, G, L, R, C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y.
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above
wherein said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
In another embodiment, the invention concerns a method to obtain the active substance of a pharmaceutical composition, as defined above, consisting in modifying the immunosuppressive property of:
a wild-type feline lentiviral ENV protein,
or a fragment of said wild-type feline lentiviral ENV protein, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said ENV protein or fragment thereof presenting a transmembrane subunit (TM) comprising an immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein
said method comprising a step of introduction of at least one mutation of XA and/or XB and/or XC and/or XD and/or XE,
to obtain:
an isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity,
said mutated feline lentiviral ENV protein having at least 70% identity, preferably at least 80% identity, to one sequence SEQ ID NO: 5,
or a fragment of said isolated mutated feline lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, said fragment comprising at least 40 amino acids, in particular at least 60 amino acids,
said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:
wherein
in particular, wherein
X5 is R, G, L, A or F and X1, X2, X3 and X4 are any amino acid.
In other aspect, the invention also relates to pharmaceutical compositions comprising as active substance a mutated lentiviral protein isolated from other animal lentiviruses, infecting bovine, equine, ovine or caprine animal species.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated bovine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated bovine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type bovine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 420,
said mutated bovine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated bovine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated bovine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type bovine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: SEQ ID NO: 420,
said mutated bovine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated bovine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated bovine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type bovine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 423,
said mutated bovine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated bovine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated bovine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type bovine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 423,
said mutated bovine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In a particular embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated bovine lentiviral Env protein, or said fragment thereof, further comprises additional mutations of at least one amino acid chosen among the 5th, 6th, 7th, 8th and 9th amino acids, in particular the 7th amino acid, located upstream the amino acid ZA1 of SEQ ID NO: 421, SEQ ID NO: 422, SEQ ID NO: 424 or SEQ ID NO: 425.
In another aspect, the invention relates to a pharmaceutical composition comprising as active substance a nucleic acid molecule coding for a mutated bovine lentiviral ENV protein, or a fragment of said mutated bovine lentiviral ENV protein, as defined above.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated equine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated equine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type equine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 426,
said mutated equine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated equine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated equine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type equine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 426,
said mutated equine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In a particular embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated equine lentiviral ENV protein, or said fragment thereof, further comprises additional mutations of at least one amino acid chosen among the 5th, 6th, 7th, 8th and 9th amino acids, in particular the 7th amino acid, located upstream the amino acid ZA1 of SEQ ID NO: 427 or SEQ ID NO: 428.
In another aspect, the invention relates to a pharmaceutical composition comprising as active substance a nucleic acid molecule coding for a mutated equine lentiviral ENV protein, or a fragment of said mutated equine lentiviral ENV protein, as defined above.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated caprine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated caprine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type caprine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 429,
said mutated caprine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated caprine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated caprine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type caprine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 429,
said mutated caprine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In a particular embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated caprine lentiviral ENV protein, or said fragment thereof, further comprises additional mutations of at least one amino acid chosen among the 5th, 6th, 7th, 8th and 9th amino acids, in particular the 7th amino acid, located upstream the amino acid ZA1 of SEQ ID NO: 432 or SEQ ID NO: 433.
In another aspect, the invention relates to a pharmaceutical composition comprising as active substance a nucleic acid molecule coding for a mutated caprine lentiviral ENV protein, or a fragment of said mutated caprine lentiviral ENV protein, as defined above.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated ovine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated ovine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type ovine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 430,
said mutated ovine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated ovine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated ovine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type ovine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 430
said mutated ovine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated ovine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated ovine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type ovine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 431
said mutated ovine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In another aspect, the invention also relates to a pharmaceutical composition comprising as active substance an isolated mutated ovine lentiviral ENV protein having a decreased immunosuppressive activity, substantially no immunosuppressive activity or no immunosuppressive activity, or a fragment thereof,
said mutated ovine lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of a wild type ovine lentiviral ENV protein,
said mutated ENV protein having at least 70% identity, preferably at least 80% identity, to the sequence SEQ ID NO: 431
said mutated ovine lentiviral ENV protein or fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence:
wherein,
in association with a pharmaceutically acceptable carrier.
In a particular embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated ovine lentiviral ENV protein, or said fragment thereof, further comprises additional mutations of at least one amino acid chosen among the 5th, 6th, 7th, 8th and 9th amino acids, in particular the 7th amino acid, located upstream the amino acid ZA1 of SEQ ID NO: 432 or SEQ ID NO: 433.
In another aspect, the invention relates to a pharmaceutical composition comprising as active substance a nucleic acid molecule coding for a mutated ovine lentiviral ENV protein, or a fragment of said mutated ovine lentiviral ENV protein, as defined above.
(A) FIV Envelope phylogeny, with the A-D subtypes indicated and the reference Petaluma strain boxed (from Pu et al, Journal of Feline Medicine and Surgery, 2005, 7:65-70).
(B) Structure of the FIV Envelope protein, delineation of the characteristic functional domains and alignment of the 64-aa immunosuppressive-containing domains from selected FIV Env proteins. The SU and TM subunits of the FIV Env are indicated, together with the fusion peptide, the transmembrane anchoring domain of the TM subunit, and the immunosuppressive domain (ISD).
The aligned sequences of the 64-aa immunosuppressive-containing domains correspond to fragments of the Envelope protein from 33 distinct strains (i.e. 33 different accession numbers), only 20 of these 64-aa fragments are different (i.e. 20 SEQ ID numbers have been given).
Immunosuppressive activity of the full-length FIV envelope protein (FIV Env) and of the 64 aa-long FIV envelope subdomain delineated in
Functional identification of the aminoacids in the FIV envelope 64 aa domain directly involved in immunosuppressive activity, and search for aminoacid substitutions inhibiting this activity. Immunosuppressive activity was tested as in
Antibody response of mice inoculated with cells expressing wild-type and mutant FIV64 Env domains (see scheme on top): MCA205 tumor cells are stably transduced as in
Antibody response of mice injected with MBP-FIV64, wt and mutants, recombinant proteins (see scheme on top): FIV64 Env, wt and mutants, fused with MBP are i.v. injected three times with a 1-week interval into Balb/C mice (10 μg per injection); mice are then blood-sampled and anti-MBP IgG levels are determined by ELISA using plates coated with MBP-LacZα (MBP-LacZα is a control MBP fusion with the 83aa LacZα fragment (α-subunit of E. coli β-galactosidase, see Materials and Methods). Results are representative of 2 independent experiments.
Expression of the FIV Env proteins, wt and mutants, at the surface of cells transfected with the indicated expression vectors (see scheme on top).
Expression profile of the wild-type and mutant FIV Env proteins was assayed by FACS analysis using full-length FIVenv-expressing vectors and an anti-SU FIVenv monoclonal antibody (Antibodies-online, BmbH, Germany). The data correspond to the Mean Fluorescence Intensity, from three independent experiments, with standard deviation.
Functional characterization of the FIV Env proteins, wild-type (wt) and mutants. Fusogenic activity of the FIVenv, wild-type and mutants, are measured by a cell-cell fusion assay and quantified by a fusion index (see scheme on top and Materials and Methods). The data correspond to three independent experiments, with standard deviation).
The FIV Envelope and a Delineated Subdomain of the TM Subunit are Immunosuppressive In Vivo
Identification of an immunosuppressive activity of the FIV envelope and of the domain within the FIV envelope responsible for this activity was achieved using an in vivo tumor rejection assay that we had previously used to demonstrate the immunosuppressive activity of the Env protein of oncoretroviruses (e.g. murine MoMLV and simian MPMV, see Mangeney and Heidmann, Proc Natl Acad Sci USA, 1998, 95:14920-14925; Mangeney et al., Proc Natl Acad Sci USA, 2007, 104(51):20534-9).
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 can 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. Accordingly, as illustrated in
Identification of the FIV Env Amino-Acids Critical for Immunosuppression and its Inhibition, Via R-Scanning
To characterize further the immunosuppressive domain (ISD) of the FIV env active in vivo, we analyzed the effect of a series of amino-acid substitutions within the FIV env 64 aa domain shown to carry the IS activity (see above). The 64-aa FIV env domain is embedded into the so-called ectodomain, which corresponds to the extracellular domain of the TM subunit, and consists in the α-helical domain involved in FIV TM trimerization, and the N-term part of the loop containing the 2 well-conserved, 6/7 aa-distant, cysteine residues found in most retroviral envelopes. Refine delineation of the amino acids responsible for IS activity was thus performed by arginine-scanning within this domain. As illustrated in
We also analyzed the effect of this series of amino-acid substitutions within the FIVenv 64 aa domain by measuring the production of anti-FIV env IgG antibodies in mice inoculated under syngenic conditions (C57Bl/6 mice) with the cells expressing the wild-type and mutant FIVenv 64 aa domains. As illustrated in
Additionally, a series of experiments were performed with recombinant proteins containing the FIVenv 64 domain, to assay the relative immunogenicity of the wt and mutants proteins. The domains were fused with the carrier MBP (Maltose Binding Protein), and the proteins were injected i.v. into Balb/C mice (see Materials and Methods). The immunosuppressive effects of the FIVenv 64 wt and mutants were assayed by measuring the inhibition of the anti-MBP antibody response raised in the injected mice. As illustrated in
Impact of the Identified Mutations on Env Protein Folding and Proper Expression as a Functional Transmembrane Protein
We then tested whether the above-mentioned substitutions alter the overall capacity of the FIV envelope to be expressed by an eucaryotic cell and to be exported at the cell membrane, by introducing the mutants into an expression vector for the full-length FIV envelope. A FACS analysis of cells transfected with the wild-type and the mutant FIV envelope genes inserted within a CMV-driven expression vector (see Materials and Methods) and using an anti-SU specific monoclonal antibody, demonstrated quantitative expression of the mutant envelopes at the cell surface (
Accordingly, the present investigation has clearly identified definite positions and definite substitutions within the FIV env resulting in the loss of its IS activity.
Being compatible with the conservation of the overall structure of the FIV Env protein, 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:
C57Bl/6 and Balb/c mice, 6-10 weeks old, were obtained from Harlan (France). Mice were maintained in the animal facility of the Gustave Roussy Institute in accordance with institutional regulations. 293T (ATCC CRL11268), and MCA205 cells were cultured in DMEM supplemented with 10% fetal calf serum (Invitrogen), streptomycin (100 μg/ml) and penicillin (100 units/ml).
Plasmids Constructions:
All the constructions were sequenced before use.
Recombinant Proteins:
Recombinant proteins were produced using BL21 (DE3) Escherichia coli cells (Stratagene) and pMal-derived expression vectors (New England Biolabs, France). Recombinant WT and mutants TM subunit ectodomains were soluble and were purified on cross-linked Amylose Resin (New England Biolabs, France) packed in column with PBS as a binding and washing buffer and 20 mM Tris-C1, 5 mM maltose, pH 7.5, as an elution buffer. Proteins were then dialysed against phosphate-buffered saline pH 7.4 (PBS), and endotoxins were removed using Endotrap Blue Resin (Hyglos GmbH, Germany) according to manufacturer's protocol.
Establishment of Env-Expressing Tumor Cells and MCA205 Tumor-Rejection Assay:
7.5×105 293 T 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 5×105 cells with 4 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 (see above PNAS 1998, PNAS 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 the Antibody Response of Mice Inoculated with MCA205-Transduced Cells Expressing FIV64 Env Wild-Type and Mutants:
the MCA205-transduced cells were also inoculated as above into syngenic mice (C57Bl/6) and sera were collected 1, 2, and 3 weeks after injection. The antibody response against FIV64 env was assayed by ELISA. Briefly, several dilutions of the sera were incubated 1 h at RT on a plate pre-coated with 1 ng/ml of MBP-FIV64, and antibody binding was analysed by using a labeled anti-mouse IgG secondary antibody (GE Healthcare, UK).
Analysis of FIVenv Expression:
3×105 293 T cells were transfected with 2 μg of the expression vector for the FIV envelope (phCMV) either wild-type or mutated at the indicated positions using Fugene HD (Roche). Cells were washed 16 h later and then harvested 2 days post-transfection using PBS-EDTA 5 mM. The SU1-30 monoclonal antibody (Antibodies online, BmbH, Germany) was used (1/200 dilution) to stain the FIV envelope. As a secondary antibody, we used the goat anti mouse IgG Alexa 488 (1/400) (Invitrogen). Fluorescence was acquired by flow cytometry using a FACS Calibur (BD Biosciences), and data analysed by the CellQuest software (BD Biosciences).
Cell-Cell Fusion Assays:
For cell-cell fusion assays, 5×104 to 1×105 cells seeded in 24-well plates were transfected by using lipofectamine LTX (Life technologies) with 250 ng of env expression plasmid. Fusion activity of each envelope protein was visualized 24 to 48 h after transfection by May-Grunwald and Giemsa staining, according to the manufacturer's instructions (Sigma). The fusion index, which represents the percentage of fusion events in a cell population, is defined as [(N−S)/T]×100), where N is the number of nuclei in the syncytia, S is the number of syncytia, and T is the total number of nuclei counted
| Number | Date | Country | Kind |
|---|---|---|---|
| 13305767 | Jun 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/061924 | 6/6/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/195510 | 12/11/2014 | WO | A |
| Number | Date | Country |
|---|---|---|
| 2005095442 | Oct 2005 | WO |
| 2010022740 | Mar 2010 | WO |
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| Number | Date | Country | |
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| 20160129107 A1 | May 2016 | US |
