Patent Grant
8765143
This is a 371 national stage application of PCT/FR2009/051142 filed Jun. 16, 2009 which claims priority of the foreign application France 0803377 filed Jun. 17, 2008.
The object of the invention is novel fusion proteins and use thereof especially for preparing vaccines intended for the prevention and/or the prophylactic and/or therapeutic treatment of hepatitis C, or the prevention and/or the prophylactic and/or therapeutic treatment of hepatitis C and of hepatitis B. The present invention also relates to obtaining chimeric subviral envelope particles between the envelope proteins of the hepatitis B virus (HBV, for hepatitis B virus) and of the hepatitis C virus (HCV, for hepatitis C virus).
The hepatitis C virus, identified in 1989, then cloned and sequenced, still represents at the present time a real problem for public health on account of its widespread distribution throughout the world and the frequent evolution of the illness towards chronicity. In 2000, the WHO estimated that around 3% of the world's population, i.e. around 170 million people, were infected by HCV.
HCV induces chronic hepatitis, which can evolve into cirrhosis and hepatocellular carcinoma. Interferon and ribavirin form the basic treatment of chronic hepatitis induced by the HCV, but these treatments are not sufficiently efficacious and have important secondary effects. The available treatments remain costly, relatively toxic and are only efficacious in half of the cases of infection.
Even though the whole of the genome and the viral proteins have been known for many years, its structure and is morphogenesis remain hypothetical. No vaccine yet exists against hepatitis C and the search for such a vaccine candidate is currently very active, [Houghton, M., and Abrignani, S. (2005). Prospects for a vaccine against the hepatitis C virus. Nature 436 (7053), 961-6].
In the case of a prophylactic vaccine, the quasi-totality of the potential candidates are based on the use of one of the two or both envelope proteins of the HCV, commonly known as the proteins E1 and E2, and capable of inducing both a cellular immune response and a neutralising humoral response.
However, the proteins E1 and E2 of the HCV do not self-assemble into subviral particles as may be the case for other viruses. Furthermore, on account of a high retention of their transmembrane region in the endoplasmic reticulum, their purification necessitates solubilising than with detergents. Disappointing yields ensue and the purity of the fractions obtained is mediocre [Fours, X., Bukh, J., and Purcell, R. H. (2002). The challenge of developing a vaccine against hepatitis C virus. J Hepatol 37(5), 684-95].
The alternative, consisting in resorting to the envelope proteins deleted of their transmembrane domain, makes it possible to favour the secretion of E1 and E2 on the outside of the cell, but the change of three dimensional conformation that ensues may turn out to be undesirable on account of a diminished antigenic capacity with regard to wild-type proteins.
The HBV virus exists in two forms: in the form of infectious virions and in the form of excess of envelope particles that are found in the blood of infected subjects in much higher quantity than the virus itself. This phenomenon is due to the capacity of the S protein of the HBV to self-assemble and break out into subviral particles (or excess of envelope) which do not contain capsid protein or nucleic acid [Moriarty, A. M., Hoyer, B. H., Shih, J. W., Gerin, J. L., and Hamer, D. H. (1981). Expression of the hepatitis B virus surface antigen gene in cell culture by using a simian virus 40 vector. Proc Natl Acad Sci USA 78(4), 2606-10]. The wild-type S protein of the HBV comprises four transmembrane domains. The subviral particles that result from the self-assembly of the wild-type S protein are non-infectious but very immunogenic: they have been considered as a good vaccine candidate against hepatitis B since the mid 1970s. They have in fact served as the foundation for the elaboration of vaccines having proven their efficaciousness to induce a protective immune response of infection by HBV.
The use of subviral envelope particles of HBV as vector for proteins foreign to the hepatitis B virus has already been the object of prior work. Thus, patent application n°US2004/0146529 entitled “HBV/HCV virus like particle” discloses obtaining HBV-HCV envelope chimeras comprising, on the one hand, systematically the whole of the S protein of the HBV virus, and, on the other hand, according to the examples, a fragment of the ectodomain of one of the E1 or E2 envelope proteins of the HCV, grafted at the N-terminal end of the S protein of the HBV virus [Mark Selby, Edward Glazer and Michael Houghton “HBV/HCV virus-like particle” US patent n° 2004/014655291.
However, it is not demonstrated that these constructions can induce the formation of well structured subviral particles, and that they are capable of inducing a quality antigenic response.
In response of the drawbacks of the prior art, the aim of the present invention is to provide a HCV-HBV fusion protein capable of forming non infectious, well structured and efficiently secreted subviral particles, and containing the quasi-totality of E1 and/or E2.
One of the objectives of the invention is also to provide a vaccine against hepatitis C and/or against hepatitis B.
An additional interest of the invention stems from the fact that it can be easily adapted to existing industrial production lines for currently commercialised vaccines against hepatitis B.
The object of the invention is an immunogenic fusion protein comprising at least the following two peptides:
a)—on the C-terminal side, a first peptide composed:
b)—on the N-terminal side, a second peptide composed:
said second peptide being chosen from the protein E1, the protein E2 or a fusion peptide comprising the protein E1 and the protein E2.
The invention also relates to an immunogenic fusion protein having an amino acid sequence having a percentage of identity of at least 83%, especially of at least 85%, particularly of at least 90%, and more particularly of at least 95%, with the amino acid sequence composed of the S protein deleted of the N-terminal transmembrane domain thereof (Sd), of the transmembrane domain and of the ectodomain of a protein of a hepatitis C virus isolate (E1 or E2).
“Fusion protein” is taken to mean any protein comprising at least the S protein deleted of the transmembrane domain thereof located at the N-terminal end thereof of an HBV (Sd) isolate, and the quasi-integrality of an envelope protein E1 and/or E2 of an HCV isolate, the transmembrane domain of which replaces that deleted at the N-terminal of S of the HBV (
“Immunogenic protein” is taken to mean any protein, especially any fusion protein according to the invention as well as any fragment of any fusion protein disclosed herein, provided with antigenic properties and capable of inducing a reaction or an immune response. In particular, a protein is considered as immunogenic towards HCV and/or HBV if it induces respectively after immunisation an anti-E1, anti-E2 and/or anti-S humoral response, detected for example according to the protocol described:
“Capacity to form subviral particles” is taken to mean the capacity of any protein, especially the S protein of the HBV, particularly of the S protein deleted of the N-terminal transmembrane domain thereof, and more particularly of a fusion protein of the invention comprising the N-terminal deleted S protein, to self-assemble in the presence of the wild-type S protein or, if appropriate, to self-assemble into filamentous or spherical subviral particles; said capacity to form subviral particles can at least be highlighted by observation according to especially electron microscope analysis, and especially by the test described in examples 1 and 2 (§I-5 and §II-7) of this document and especially illustrated by
“Non infectious subviral particle” is taken to mean any filamentous or spherical particle resulting from the assembly of the wild-type S protein of the HBV (
and the result of which is negative.
“Isolate of the HCV virus” is taken to mean any isolate member of the family of Flaviviridae and belonging to the genus Hepacivirus and composed of a polyprotein of more than 3 000 amino acids of the HCV virus, schematised in
“Isolate of the HBV virus” is taken to mean any isolate member of the Hepadnaviridae family and belonging to the genus Orthohepadnavirus, or any isolate classified by the International Committee for the Taxonomy of Viruses (ICTV) as being related to the HBV [Schaefer S. Hepatitis B virus taxonomy and hepatitis B virus genotypes. World J. Gastroenterol. 2007 Jan. 7: 13(1):14-21].
“S proteins” or “wild-type S protein” (5) is taken to mean:
“Assembly” is taken to mean the capacity of a protein to form subviral particles by associating with the wild-type S protein. “Self assembly” is taken to mean the capacity of a protein to form by itself subviral particles.
“S protein deleted of the transmembrane domain thereof located at the N-terminal end thereof” or “N-terminal deleted S protein” or “deleted S protein” (Sd) is taken to mean:
“Envelope protein of a hepatitis C virus isolate” is taken to mean the quasi-integrality of one of the two proteins E1 and E2, composed of their ectodomain and their transmembrane domain (
“Ectodomain of the envelope proteins of the hepatitis C virus” is taken to mean,
“Transmembrane domains of envelope proteins of the hepatitis C virus” is taken to mean,
“Percentage of identity” is taken to mean the percentage determined by direct comparison of two sequences of polypeptide molecules, by determining the number of residues of amino acids at the two sequences, then by dividing it by the number of residues of amino acids of the longest sequence of the two, and by multiplying the result by 100.
An object of the present invention relates to a fusion protein comprising any nucleotide and/or peptide sequence of any isolate of the HCV virus and/or of the HBV virus, whatever the aforementioned percentage of identity of said sequence with regard to the specific sequences disclosed herein.
The term “natural variant” refers to any variability, any polymorphism, any diversity, of a sequence of DNA, of an allele, or of a protein sequence, between isolates of a same species or of a same population. The “percentage of natural variability” is determined by direct comparison of two polypeptide or polynucleotide molecules, derived from a wild-type reference molecule and provided with biological properties of interest, such as immunogenic properties and/or the capacity to form subviral particles. It is quantified by determining the exact number of identical residues of amino acids, or of nucleic acids, between the two sequences, then by dividing than by the number of residues of amino acids, or of nucleic acids, of the shorter sequence of the two, and by multiplying the result by 100.
Said percentage of variability between two sequences is particularly versatile because it depends especially on the virus considered, the genotype considered, the fragment of sequence considered—the region of the ectodomain of the HCV being for example more variable than the region of the transmembrane domain—, etc. . . . . Thus, the percentage of variability of the proteins E1 and E2 of the HCV is 88% of nucleotides and 90% of amino acids, between strains of a same genotype. But it falls to 55% of nucleotides and 59% of amino acids between strains of different genotypes. HBV being a DNA virus, it is much less variable than HCV [Zhang M, Gaschen B, Blay W, Foley B, Haigwood N, Kuiken C, Korber B. Tracking global patterns of N-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin. Glycobiology. December 14(12):1229-46].
An object of the present invention relates to a fusion protein, and/or a hybrid nucleic acid molecule, comprising any natural variant or any fragment of natural variant, provided with a peptide and/or nucleotide sequence derived from any isolate of the HCV virus and/or of the HBV virus, whatever the aforementioned percentage of natural and/or synthetic variability of said sequence with regard to the specific sequences disclosed herein.
The term “synthetic variant” refers to any polypeptide or polynucleotide molecule according to the invention, or any fragment of polypeptide or polynucleotide molecule disclosed herein, derived by recombination of a reference wild-type molecule, either by addition, deletion or substitution, to said reference wild-type molecule, on condition that it conserves the biological properties of interest, such as the immunogenic properties and/or the capacity to form subviral particles. The “percentage of synthetic variability” is determined by direct comparison of said derived molecule with said reference wild-type molecule, and by determining the exact number of identical residues of amino acids, or of nucleic acids, between the two sequences, with regard to their position and their nature, then by dividing them by the number of residues of amino acids, or of nucleic acids, of the shorter sequence of the two, and by multiplying the result by 100.
An object of the present invention relates to a fusion protein comprising any synthetic variant or any fragment of synthetic variant, provided with a nucleotide and/or peptide sequence derived from any isolate of the HCV virus and/or of the HBV virus.
According to a particularly advantageous aspect of the invention, the transmembrane domains of E1 and/or of E2, and constituting a part of the fusion protein, are deleted of at least one of the last three amino acids, and especially of the last three amino acids, located at the C-terminal position, so that said transmembrane domains deleted of E1 and/or of E2 have a percentage of identity with the transmembrane domains of wild-type E1 and/or E2, of at least 91%, as regards E1 and of at least 90%, as regards E2.
Said deletion of at least one of the three amino acids, and especially of the last three amino acids, at the C-terminal position has the advantage of inactivating the cleavage site of the peptidases represented in , and which is necessary for the maturation of the polyprotein of the HCV, but which is undesirable within the scope of the chimeric constructions of the present invention.
[1]. The object of the invention is an immunogenic fusion protein comprising at least the following two peptides:
a)—on the C-terminal side, a first peptide composed:
b)—on the N-terminal side, a second peptide composed:
The object of the present invention especially relates to a fusion protein comprising at least:
[1a]. Advantageously, the object of the present invention is an aforementioned immunogenic fusion protein, comprising at the N-terminal end of said second peptide (E1 or E2), a third peptide composed of the sequence of amino acids of a transfer initiation peptide (PIT) of a hepatitis C virus isolate.
“Transfer initiation peptide” is taken to means a protein E1 or E2 (respectively PIT1 and PIT2) or a fusion protein according to the invention,
[2]. According to another particularly advantageous aspect, the object of the present invention is an aforementioned immunogenic fusion protein, in which the second peptide located on the N-terminal side, is composed:
A particular object of the invention resides in the fusion protein E1-Sd, a schematic representation of which is given in
[3]. Advantageously, the object of the present invention is particularly an aforementioned immunogenic fusion protein in which the second peptide located on the N-terminal side, is composed:
A particular object of the invention resides in the fusion protein E2-Sd, a schematic representation of which is given in
[3b]. In this respect, the invention relates more particularly to an immunogenic fusion protein, comprising the following three peptides:
a)—on the C-terminal side, a first peptide composed:
b)—a second peptide of sequence composed:
c)—on the N-terminal side, a third peptide of sequence composed:
said second peptide being located between the first and the third peptide, the first, second and third peptides being preferably contiguous.
A particular object of the invention resides in the fusion protein E1-E2-Sd, which comprises the ectodomain of E1 of the HCV grafted at the N-terminal of the quasi-integrality of the protein E2, itself grafted at the N-terminal of the deleted S protein of HBV.
A particular object of the invention also resides in the fusion protein E2-E1-Sd, and which comprises the ectodomain of E2 of the HCV grafted at the N-terminal of the quasi-integrality of the protein E1, itself grafted at the N-terminal of the deleted S protein of HBV.
[4]. According to a particularly advantageous aspect of the invention, the first and the second peptide constituting the immunogenic fusion protein are contiguous, and the C-terminal end of the second peptide is bonded in a covalent manner to the N-terminal end of the first peptide.
Advantageously, according to the invention, the proteins E1 or E2 of the HCV virus, or the fragments of a fusion protein of the invention PIT1-E1, PIT2-E2, E1-E2, or PIT1-E1-E2 are bonded in a covalent and contiguous manner to the deleted S protein of the HBV virus.
According to another advantageous aspect of the invention, a binding peptide links the first and the second peptide constituting the aforementioned fusion protein, said binding peptide being composed of 1 amino acid, or 2 amino acids, or 3 amino acids, or 4 amino acids, or 5 amino acids; under the condition that said immunogenic fusion protein maintains the ability for self-assembling into subviral particles, in the presence of the wild-type S protein, and the immunogenic properties vis-à-vis the HCV virus, or the HBV virus, or, the HCV and HBV viruses.
[5]. Another particular object of the invention is an aforementioned immunogenic fusion protein, in which the first peptide in C-terminal position is composed:
and especially of the amino acid sequence represented by the SEQ ID NO: 2, or
A more particular object of the invention resides in the fusion protein E1-Sd or E2-Sd, or E1-E2-Sd, for which the deleted S protein is that of the HBVadw isolate and has for sequence the SEQ ID NO: 2 (cf. table 1).
[6]. Another particular object of the invention is an aforementioned immunogenic fusion protein, in which the second peptide in N-terminal position is composed:
and especially of the amino acid sequence represented by the SEQ ID NO: 4, or
Another particular object of the invention is a fusion protein for which the sequence of protein E1 is derived from the HCV-1a isolate, and corresponds specifically to the aforementioned region, such as especially the fusion proteins E1-Sd of SEQ ID NO: 4 or E1-E2-Sd, PIT1-E1-E2-Sd (cf. table 1).
[7]. The invention particularly relates to an aforementioned immunogenic fusion protein, in which the first and the second peptide are contiguous, said fusion protein being composed of:
[7b]. In this respect, the object of the invention is more particularly an aforementioned immunogenic fusion protein, comprising a third transfer initiation peptide located at the N-terminal side of the second peptide, said fusion protein being represented by:
Another object of the invention is the fusion protein PIT1-E1-Sd, for which the sequence of protein E1 is derived from HCV, and particularly from the HCV-1a isolate, and is provided with the aforementioned sequence SEQ ID NO: 12, said sequence corresponding SEQ ID NO: 2 of Sd, grafted at the N-terminal of the SEQ ID NO: 4 of E1, itself grafted at the N-terminal of the amino acid sequence of the transfer initiation peptide of the protein E1 (PIT1) included in the region extending from the amino acid in position 166 to that in position 191 of the HCV.
The insertion of a transfer initiation peptide at the N-terminal of the aforementioned fusion protein E1-Sd has the particular advantage of addressing the latter once translated to the endoplasmic reticulum, so that it is correctly glycosylated and that its three dimensional conformation and/or that its antigenic characteristics do not show any substantial alteration with regard to wild-type proteins.
[8]. Another object of the invention is an aforementioned immunogenic fusion protein, in which the second peptide in N-terminal position is composed:
and especially the amino acid sequence represented by the SEQ ID NO: 6, or
Another object of the invention is the fusion protein E2-Sd or E1-E2-Sd, for which the sequence of protein E2 is derived from the HCV, and particularly from the HCV-1a isolate, and corresponds specifically to the aforementioned region of the sequence of the protein E2.
[9]. The invention particularly relates to an aforementioned immunogenic fusion protein, in which the first and the second peptide are contiguous, said fusion protein being composed of:
Another object of the invention is the fusion protein E2-Sd, for which the sequence of the protein E2 is derived from the HCV-1a isolate, and is provided with the aforementioned sequence SEQ ID NO: 10, said sequence corresponding to SEQ ID NO: 2 of Sd, grafted ahead of the SEQ ID NO: 6 of E2.
[9b]. In this respect, the object of the invention is more particularly an aforementioned immunogenic fusion protein (PIT2-E2-Sd), comprising a third transfer initiation peptide located on the N-terminal side of the second peptide, said fusion protein being represented by:
Another object of the invention is the fusion protein PIT2-E2-Sd, for which the sequence of protein E2 is derived from the HCV-1a isolate, and is provided with the aforementioned sequence SEQ ID NO: 14, said sequence corresponding to the SEQ ID NO: 2 of Sd, grafted at the N-terminal of the SEQ ID NO: 6 of E2, itself grafted at the N-terminal of the amino acid sequence of the transfer initiation peptide of the protein E2 (PIT2) included in the region composed of the amino acid in position 366 to that in position 383 of HCV.
Another object of the invention is the fusion protein E1-E2-Sd or PIT1-E1-E2-Sd. Another object of the invention is the aforementioned fusion proteins in purified form.
The invention also relates to a hybrid nucleic acid molecule encoding for any of the aforementioned fusion proteins.
“Hybrid nucleic acid molecule” is taken to mean any nucleic acid molecule comprising at least one sequence encoding for the S protein deleted of the transmembrane domain thereof located at the N-terminal end thereof of a hepatitis B virus (HBV) isolate, and at least one sequence encoding for the quasi-integrality of an envelope protein of a hepatitis C virus isolate, or any molecule derived from a molecule defined above, and modified following the natural degeneration of its genetic code.
Advantageously, the invention relates to an aforementioned hybrid nucleic acid molecule, encoding for a fusion protein defined above, comprising the following three nucleic acid sequences:
a)—on the 3′ side, a first nucleic acid sequence encoding for the S protein deleted of the transmembrane domain thereof located at the N-terminal end thereof, of a hepatitis B virus (HBV) isolate, or
b)—on the 5′ side of the first sequence, a second nucleic acid sequence encoding for the transmembrane domain and the ectodomain of at least one envelope protein E1 or E2 of a hepatitis C virus isolate, or
c)—on the 5′ side of the second sequence, a third nucleic acid sequence encoding for a transfer initiation peptide of an envelope protein E1 or E2 of a hepatitis C virus isolate (PIT), or
Advantageously, the object of the present invention is an aforementioned hybrid nucleic acid molecule (especially the molecules e1-sd or e2-sd, or e1-e2-sd, pit1-e1-sd or pit2-e2-sd, as defined in table 1 below), encoding for an aforementioned fusion protein (especially E1-Sd or E2-Sd, or E1-E2-Sd, PIT1-E1-Sd or PIT2-E2-Sd), comprising respectively from its 5′ end to its 3′ end, a nucleic acid sequence encoding for a transfer initiation peptide (PIT) of an isolate of the hepatitis C virus, a nucleic acid sequence encoding for a protein E1 or E2 of an isolate of the HCV virus, a nucleic acid sequence encoding for the deleted S protein of an isolate of the HBV virus.
The insertion of a nucleic acid sequence encoding for said transfer initiation peptide grafted at the N-terminal of the aforementioned fusion protein (especially E1-Sd or E2-Sd, or E1-E2-Sd) has the particular advantage that the latter once translated is addressed to the endoplasmic reticulum, so that it is correctly glycosylated and that its three dimensional conformation and/or that its antigenic characteristics do not show substantial alteration with regard to wild-type S proteins.
Another object of the present invention is an aforementioned hybrid nucleic acid molecule only comprising the first two aforementioned nucleic acid sequences and devoid of nucleic acid sequence encoding for a transfer initiation peptide.
The object the present invention especially relates to an aforementioned hybrid nucleic acid molecule encoding for a fusion protein of the invention, comprising at least:
so that the fusion protein encoded by said aforementioned hybrid nucleic acid molecule maintains the ability to form subviral particles, and the immunogenic properties vis-à-vis the HBV and/or HCV virus, and especially the property of inducing a double immunisation against the HBV and HCV viruses.
“Percentage of homology” is taken to mean the percentage determined by direct comparison of two sequences of polynucleotide molecules, by determining the number of residues of nucleic acids of the two sequences, then by dividing it by the number of residues of nucleic acids of the longest sequence of the two, and by multiplying the result by 100.
According to a particularly advantageous aspect of the invention, the nucleic acid sequences encoding for the transmembrane domains of E1 and/or of E2, are deleted of at least one of the last nine nucleic acids, and preferentially of the last nine nucleic acids, located in 3′ position, so that the nucleic acid sequences encoding for the transmembrane domains of E1 and/or of E2 correspond:
The expression “transmembrane domains of E1 and/or of E2 deleted of at least one of the last nine nucleic acids located in 3′” position relates to deletions of 3 nucleic acids, or of 6 nucleic acids, or of 9 nucleic acids located in 3′ position of one of said transmembrane domains.
Advantageously, the first and the second nucleic acid sequence of the aforementioned hybrid nucleic acid molecule and encoding for the aforementioned immunogenic fusion protein are contiguous, and the 5′ end of the first nucleic acid sequence is bonded in a covalent manner to the 3′ end of the second nucleic acid sequence.
According to a particularly advantageous aspect, the hybrid nucleic acid molecule defined above corresponds for example to the following molecules: e1-sd, e2-sd, or pit1-e1-sd or pit 2-e2-sd, encoding respectively for the following fusion proteins: E1-Sd, E2-Sd, PIT1-E1-Sd or PIT2-E2-Sd).
According to another advantageous aspect of the invention, a nucleotide sequence encoding for a binding peptide, links said aforementioned first and second nucleotide sequences, said nucleotide sequence being composed:
under the condition that the immunogenic fusion protein encoded by the hybrid nucleic acid molecule and which comprises said binding peptide, itself encoded by said nucleotide sequence, maintains the ability for self-assembling into non infectious subviral particles, in the presence of the wild-type S protein, and conserves the immunogenic properties vis-à-vis the HCV virus, and/or, the HBV virus.
In this respect, the object of the invention is particularly an aforementioned hybrid nucleic acid molecule, in which the first nucleic acid sequence encoding for the N-terminal deleted S protein, of HBV, and particularly of the HBVadw isolate, is located on the 3′ side of said hybrid nucleic acid molecule, and is composed:
A more particular object of the invention relates to an aforementioned hybrid nucleic acid molecule (especially e1-sd or e2-sd, or e1-e2-sd, pit1-e1-sd or pit2-e2-sd, or pit1-e1-e2-sd) encoding for an aforementioned fusion protein (especially E1-Sd or E2-Sd, or E1-E2-Sd, PIT1-E1-Sd or PIT2-E2-Sd, or PIT1-E1-E2-Sd) for which said nucleic acid sequence encoding for the deleted S protein of the HCV is that of the HBVadw isolate, and has for sequence SEQ ID NO: 1.
In this respect again, the invention relates more particularly to an aforementioned hybrid nucleic acid molecule, in which the second nucleic acid sequence encoding for the transmembrane domain and the ectodomain of the protein E1 of HCV, and particularly of the HCV-1a isolate, is located on the 5′ side of said hybrid nucleic acid molecule, and is composed:
and especially the nucleic acid sequence represented by the SEQ ID NO: 3, or
The invention particularly relates to an aforementioned hybrid nucleic acid molecule, encoding for an aforementioned fusion protein, comprising a transfer initiation peptide located on the N-terminal side, said hybrid nucleic acid molecule being represented by:
The nucleic acid sequence SEQ ID NO: 11, also named pit1-e1-sd, encodes for the fusion protein PIT1-E1-Sd of SEQ ID NO: 12 (cf. table 1).
Another object of the invention is a hybrid nucleic acid molecule (especially pit1-e1-sd of SEQ ID NO: 11) for which:
In this respect, the object of the invention relates more particularly to an aforementioned hybrid nucleic acid molecule, in which the second nucleic acid sequence encoding for the transmembrane domain and the ectodomain of the protein E2 of HCV, and particularly of the HCV-1a isolate, is located on the 5′ side of said molecule, and is composed:
and especially the nucleic acid sequence represented by the SEQ ID NO: 5, or
The invention relates particularly to an aforementioned hybrid nucleic acid molecule, encoding for a fusion protein of the invention, comprising a transfer initiation peptide located on the N-terminal side, said hybrid nucleic acid molecule being represented by:
The nucleic acid sequence SEQ ID NO: 13, also named pit2-e2-sd, encodes for the fusion protein PIT2-E2-Sd of SEQ ID NO: 14 (cf. table 1).
Another object of the invention is a hybrid nucleic acid molecule (especially pit2-e2-sd de SEQ ID NO: 3) for which:
Another object of the invention is a vector comprising an aforementioned hybrid nucleic acid molecule encoding for an aforementioned fusion protein, as well as the means necessary for their expression linked in an operational manner to said hybrid nucleic acid molecule.
As regards expression vectors that are suitable for the purposes of the invention, plasmids, viral vectors of lentiviral type, Semliki, adenovirus, poxvirus, vaccine virus, baculovirus, bacterial vectors of the salmonella type and BCG may for example be cited.
“Necessary means for the expression” is taken to mean a protein, the term protein being used for any molecule of amino acids, such as protein, fusion protein, fragment of protein, peptide, polyprotein, polypeptide, etc., any means which make it possible to obtain the protein, such as especially a promoter, a transcription terminator, an origin of replication and preferably a selection marker. The means necessary for the expression of a peptide are linked in an operational manner to the nucleic acid sequence encoding for the peptide of interest.
“Linked in an operational manner” is taken to mean a juxtaposition of said necessary components for the expression and the gene encoding for the peptide of interest, which are in a relation such that it allows them to function in an expected manner. For example, there may exist supplementary bases between the promoter and the gene of interest as long as their functional relation is preserved.
The necessary means for the expression of a peptide may be homologous means, in other words included in the genome of the vector used, or instead may be heterologous. In the latter case, said means are cloned with the peptide of interest to be expressed.
Preferably, the promoter used in the Semliki vector and the lentiviral vector used as expression vector is a homologous promoter and is a cytomegolovirus (CMV) promoter.
Non limiting examples of heterologous promoters comprise especially (i) viral promoters such as the SV40 (Simian Virus 40) promoter, the promoter of the gene of the thymidine-kinase of the Herpes simplex virus (TK-HSV-1), the LTR of the Rous sarcoma virus (RSV), the cytomegolovirus (CMV) promoter and the adenovirus major late promoter (MLP), as well as (ii) any cell promoter which controls the transcription of genes encoding for peptides in higher eukaryotes, such as the promoter of the constitutive gene of phosphoglycerate-kinase (PGK) (Adra et al., 1987, Gene, 60: 65-74), the promoter of specific genes of the liver alpha1-antitrypsine and FIX and the specific promoter SM22 of the cells of the smooth muscle (Moessler et al., 1996, Development, 122: 2415-2425).
The invention particularly relates to an aforementioned vector, further comprising a lentiviral vector.
The object of the invention is more particularly an aforementioned vector, in which the lentiviral vector is the vector pLenti.
The latter has been disclosed previously, especially by Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M., and Trono, D. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272(5259), 263-7.
Another object of the invention is an aforementioned vector, further comprising a defective viral vector derived from the genome of the Semliki Forest virus.
The latter have been disclosed previously, especially by Schlesinger, S., and T. M. Dubensky, Jr. 1999. Alphavirus vectors for gene expression and vaccines. Curr. Opin. Biotechnol. 10:434-439.
The object of the invention is especially an aforementioned vector, in which the defective viral vector derived from the genome of the Semliki Forest virus is the vector pSFV1, the latter being especially commercialised by Invitrogen.
In this respect, the object of the invention is more particularly an aforementioned vector, in which the hybrid nucleic acid molecule is composed:
Another object of the invention is a vector derived from pSFV1 of sequence SEQ ID NO: 15, comprising a hybrid nucleic acid molecule (especially pit1-e1-sd of SEQ ID NO: 11) encoding for the aforementioned fusion protein (especially PIT1-E1-Sd of SEQ ID NO: 12), and for which:
In this respect, the object of the invention is more particularly one of the aforementioned vectors, in which the hybrid nucleic acid molecule is composed:
Another object of the invention is a vector of SEQ ID NO: 16, comprising a hybrid nucleic acid molecule (especially pit1-e1-sd of SEQ ID NO: 13) encoding for the aforementioned fusion protein (especially PIT1-E1-Sd of SEQ ID NO: 14), and for which:
The invention also relates to an aforementioned vector, comprising a hybrid nucleic acid molecule composed of the following three nucleic acid sequences:
a)—on the 3′ side, a first nucleic acid sequence encoding for the S protein deleted of the transmembrane domain thereof located at the N-terminal end thereof (Sd), of a hepatitis B virus (HBV) isolate, or
b)—at the 5′ end of said first nucleic acid sequence, a second nucleic acid sequence encoding for the transmembrane domain and the ectodomain of at least one envelope protein E1 or E2, of a hepatitis C virus isolate, or
said second sequence being chosen from the sequences encoding for the protein E1, for the protein E2 or for a fusion peptide comprising the protein E1 and the protein E2, and,
c)—at the 5′ end of said second nucleic acid sequence, a third nucleic acid sequence encoding for a transfer initiation peptide (PIT) of a hepatitis C virus isolate, or
Advantageously, another object of the invention is an aforementioned vector, in which the first and the second nucleic acid sequence of the aforementioned hybrid nucleic acid molecule (especially e1-sd or e1-e2-sd) and encoding for the aforementioned immunogenic fusion protein (especially E1-Sd or E2-Sd) are contiguous, and the 5′ end of the first nucleic acid sequence is bonded in a covalent manner to the 3′ end of the second nucleic acid sequence.
According to another advantageous aspect, the invention relates to the aforementioned vector (especially a lentiviral vector, such as the vector pLenti, or a defective viral vector derived from the genome of the Semliki Forest virus, such as the vector pSFV1) for which a nucleotide sequence, encoding for a binding peptide, links said first and second nucleotide sequences encoding for the aforementioned immunogenic fusion protein (especially E1-Sd or E2-Sd), said sequence nucleotide being composed
of 3 nucleic acids, or 6 nucleic acids, or 9 nucleic acids, or 12 nucleic acids, or 15 nucleic acids, under the condition that said nucleotide sequence, encoding for a binding peptide, does not alter the capacity of the aforementioned immunogenic fusion protein (especially E1-Sd or E2-Sd), to self-assembly into subviral particles, in the presence of the wild-type S protein, and that the latter also conserves the immunogenic properties vis-à-vis the HCV virus, and/or, the HBV virus.
Advantageously, the aforementioned vector comprises the first nucleic acid sequence encoding for the N-terminal deleted S protein, of HBV, and especially of the HBVadw isolate of the hepatitis B virus, said sequence being:
1630 to 2241 of HBV, and particularly of the HBVadw isolate,
and especially the nucleic acid sequence represented by the SEQ ID NO: 1, or
A more particular object of the invention resides in the aforementioned vector (especially a lentiviral vector, such as the vector pLenti, or a defective viral vector derived from the genome of the Semliki Forest virus, such as the vector pSFV1) comprising an aforementioned hybrid nucleic acid molecule (especially e1-sd or e2-sd, or e1-e2-sd, pit1-e1-sd or pit2-e2-sd) encoding for a fusion protein (especially E1-Sd or E2-Sd, or E1-E2-Sd, PIT1-E1-Sd or PIT2-E2-Sd) for which said nucleic acid sequence encoding for the deleted S protein of the HBV is that of the HBVadw isolate, and has for sequence SEQ ID NO: 1.
According to a particular aspect of the invention, the aforementioned vector comprises the second nucleic acid sequence encoding for the transmembrane domain and the ectodomain of the protein E1 of HCV, and particularly of the HCV-1a isolate, which is located on the 5′ side of said molecule, and which is composed:
and especially the nucleic acid sequence represented by the SEQ ID NO: 3, or
A more particular object of the invention resides in the aforementioned vector (especially a lentiviral vector, such as the vector pLenti, or a defective viral vector derived from the genome of the Semliki Forest virus, such as the vector pSFV1) comprising an aforementioned hybrid nucleic acid molecule (especially e1-sd, or e1-e2-sd, pit1-e1-sd) encoding for a fusion protein (especially E1-Sd, or E1-E2-Sd, PIT1-E1-Sd) for which said nucleic acid sequence encoding for the protein E1 of the HCV is that of the HCV-1a isolate, and has for sequence SEQ ID NO: 3.
According to an advantageous aspect of the invention, the aforementioned vector comprises the second nucleic acid sequence encoding for the transmembrane domain and the ectodomain of the protein E2 of HCV, and particularly of the HCV-1a isolate, which is located on the 5′ side of said molecule, and which is composed:
and especially the nucleic acid sequence represented by the SEQ ID NO: 5, or
A more particular object of the invention resides in the aforementioned vector (especially a lentiviral vector, such as the vector pLenti, or a defective viral vector derived from the genome of the Semliki Forest virus, such as the vector pSFV1) comprising an aforementioned hybrid nucleic acid molecule (especially e2-sd, or e1-e2-sd, pit2-e2-sd) encoding for a fusion protein (especially E2-Sd, or E1-E2-Sd, PIT2-E2-Sd) for which said nucleic acid sequence encoding for the protein E2 of the HCV is that of the HCV-1a isolate, and has for sequence SEQ ID NO: 5.
Another object of the invention is a subviral, chimeric, immunogenic and non infectious envelope particle, comprising the following proteins:
The term “chimeric subviral particle” is taken to mean any subviral particle comprising at least the wild-type S protein of HBV and an aforementioned immunogenic fusion protein resulting from the self-assembly of the wild-type S protein, or the assembly of an aforementioned fusion protein, in the presence of the wild-type S protein.
Advantageously, the immunogenic fusion protein of the aforementioned immunogenic chimeric subviral particle is:
Another object of the invention is an aforementioned immunogenic chimeric subviral particle comprising an aforementioned fusion protein, and especially the fusion protein E1-Sd of SEQ ID NO: 8, or E1-E2-Sd, and for which the sequence of the protein E1 is especially the aforementioned SEQ ID NO: 4, and is especially grafted at the C-terminal of the amino acid sequence of the S protein (Sd) deleted of the sequence SEQ ID NO: 2.
According to a particular aspect of the invention, the immunogenic fusion protein of the aforementioned immunogenic chimeric subviral particle is composed of:
Another object of the invention is an aforementioned immunogenic chimeric subviral particle comprising an aforementioned fusion protein, and especially the fusion protein E1-Sd of SEQ ID NO: 10, or E1-E2-Sd, and for which the sequence of the protein E1 is especially the aforementioned SEQ ID NO: 6, and is especially grafted at the C-terminal of the amino acid sequence of the S protein (Sd) deleted of sequence SEQ ID NO: 2.
According to an advantageous aspect of the invention, the aforementioned immunogenic chimeric subviral particle comprises the following two fusion proteins:
Another object of the invention is an aforementioned immunogenic chimeric subviral particle comprising two aforementioned fusion proteins (especially E1-Sd of SEQ ID NO: 8, or E1-E2-Sd; and E2-Sd of SEQ ID NO: 10).
The invention also relates to an aforementioned immunogenic chimeric subviral particle, in which the immunogenic fusion protein comprises at least one of the envelope proteins (E1) of a hepatitis C virus isolate, said protein being composed:
Another object of the invention is an aforementioned immunogenic chimeric subviral particle comprising an aforementioned fusion protein (especially E1-Sd, or E1-E2-Sd or PIT1-E1-E2-Sd, or PIT2-E2-E1-Sd), for which the sequence of the protein E1 is derived from the HCV-1a isolate.
Another object of the invention is an aforementioned immunogenic chimeric subviral particle, in which the immunogenic fusion protein comprises at least one of the envelope proteins of a hepatitis C virus isolate, said protein being composed of sequences of amino acids chosen from the following:
Another object of the invention is an aforementioned immunogenic chimeric subviral particle comprising an aforementioned fusion protein (especially E2-Sd, or E1-E2-Sd or PIT1-E1-E2-Sd), for which the sequence of the protein E2 is derived from the HCV-1a isolate.
According to a particular aspect, the invention also relates to an aforementioned immunogenic chimeric subviral particle, comprising the following two fusion proteins:
Another object of the invention is an aforementioned immunogenic chimeric subviral particle comprising two aforementioned fusion proteins (especially E1-Sd, or PIT1-E1-Sd or E2-Sd or PIT1-E1-Sd or E1-E2-Sd or PIT1-E1-E2-Sd).
Another object of the invention is an immunogenic composition comprising as active ingredient at least one compound chosen from:
and, a pharmaceutically acceptable vehicle.
According to a particular embodiment of the invention, the pharmaceutical composition also contains a pharmaceutically acceptable vehicle, of which those skilled in the art will easily determine the nature and the quantity to use as a function of usual parameters and the constituents of the desired pharmaceutical composition, the pharmaceutical form and the mode of administration.
The pharmaceutical compositions of the invention are suitable for oral, sublingual, sub-cutaneous, intramuscular, intravenous, topic, local, intra-tracheal, intra-nasal, transdermal, rectal, intraocular, intra-aulicular administration, said active ingredient being able to be administered in unit administration form.
The unit administration forms may for example be tablets, capsules, granules, powders, injectable solutions or oral suspensions, transdermal patches, sublingual, buccal, intra-tracheal, intraocular, intra-nasal, intra-auricular administration forms, by inhalation, topical forms of administration, transdermal, sub-cutaneous, intramuscular or intravenous, rectal forms of administration or implants. For topical administration, creams, gels, ointments, lotions or eye drops may be envisaged.
Advantageously, the aforementioned immunogenic composition comprises an active ingredient chosen from at least one of the following three compounds:
a)—an aforementioned fusion protein composed of:
A particular object of the invention relates to one of the three following compounds:
a)—the aforementioned fusion protein comprising the protein E1 (especially E1-Sd, PIT1-E1-Sd, E1-E2-Sd, PIT1-E1-E2-Sd), a schematic representation of which is given in
b)—the aforementioned hybrid nucleic acid molecule comprising a nucleic acid sequence encoding for the protein E1 (especially e1-sd, pit1-e1-sd, e1-e2-sd, pit1-e1-e2-sd), and which comprises the quasi-integrality of the nucleic acid sequence encoding for the protein E1 of the HCV, grafted at the 5′ side of the nucleic acid sequence encoding for the deleted S protein of HBV, Of
c)—the aforementioned subviral particle comprising the protein E1 (especially E1-Sd, PIT1-E1-Sd, E1-E2-Sd, PIT1-E1-E2-Sd), a schematic representation of which is given in
According to a particular aspect of the invention, the aforementioned immunogenic composition comprises an active ingredient chosen from at least one of the following compounds:
a)—a fusion protein composed of:
the amino acid sequence of a synthetic variant, derived from the SEQ ID NO: 10, under the condition that said amino acid sequence maintains the ability to form immunogenic non infectious subviral particles, vis-à-vis the hepatitis B virus and/or vis-à-vis the hepatitis C virus,
b)—a hybrid nucleic acid molecule composed of:
A particular object of the invention relates to one of the following three compounds:
a)—the aforementioned fusion protein comprising the protein E2 (especially E2-Sd, PIT2-E2-Sd, E1-E2-Sd, PIT1-E1-E2-Sd), a schematic representation of which is given in
b)—the aforementioned hybrid nucleic acid molecule comprising a nucleic acid sequence encoding for the protein E2 (especially e2-sd, pit2-e2-sd, e1-e2-sd, pit1-e1-e2-sd), and which comprises the quasi-integrality of the nucleic acid sequence encoding for the protein E1 of the HCV, grafted at the 5′ side of the nucleic acid sequence encoding for the deleted S protein of HBV, or
c)—the aforementioned subviral particle comprising the protein E1 (especially E1-Sd, PIT-E1-Sd, E1-E2-Sd, PIT-E1-E2-Sd), and which comprises the quasi-integrality of the protein E2 of the HCV, grafted at the N-terminal of the deleted S protein of HBV.
According to an advantageous aspect of the invention, the aforementioned immunogenic composition comprises for active ingredient a chimeric subviral particle, comprising at least the two immunogenic fusion proteins composed of:
A particular object of the invention relates to the aforementioned subviral particle comprising two aforementioned fusion proteins, one comprising:
[27]. According to a particularly advantageous aspect of the invention, the active ingredient of the aforementioned immunogenic composition is composed of the mixture comprising:
A particular object of the invention relates to the mixture comprising the following compounds:
a)—at least two aforementioned fusion proteins, one comprising the protein E1 (especially E1-Sd of sequence SEQ ID NO: 8, or PIT1-E1-Sd, or E1-E2-Sd, or PIT1-E1-E2-Sd) the other comprising the protein E2 (especially E2-Sd of sequence SEQ ID NO: 10, or PIT2-E2-Sd, or E1-E2-Sd, or PIT1-E1-E2-Sd) of the HCV, the latter being grafted at the N-terminal of the deleted S protein of HBV, or
b)—at least two aforementioned hybrid nucleic acid molecules, one comprising the nucleic acid sequence encoding for the protein E1 (especially pit1-e1-sd of sequence SEQ ID NO: 11, or e1-sd, or e1-e2-Sd, or pit1-e1-e2-sd), the other comprising the nucleic acid sequence encoding for the protein E1 (especially pit2-e2-sd of sequence SEQ ID NO: 13, or e2-sd, or e1-e2-Sd, or pit1-e1-e2-sd), the latter being grafted at the 5′ side of the nucleic acid sequence encoding for the deleted S protein of HBV, or
c)—at least two aforementioned subviral particles, one comprising the protein E1 (especially E1-Sd of sequence SEQ ID NO: 8, or PIT1-E1-Sd, or E1-E2-Sd, or PIT1-E1-E2-Sd) the other comprising the protein E2 (especially E2-Sd of sequence SEQ ID NO: 10, or PIT2-E2-Sd, or E1-E2-Sd, or PIT1-E1-E2-Sd) of HCV, the latter being grafted at the N-terminal of the deleted S protein of HBV.
Another object of the invention is the use of an aforementioned immunogenic composition for the manufacture of a medicine for the prophylactic and/or therapeutic treatment and/or for the prevention of hepatitis C.
The present invention also relates to the use of an aforementioned immunogenic composition for the manufacture of a medicine for the prophylactic and/or therapeutic treatment and/or for the prevention of hepatitis B.
Advantageously, the object of the present invention relates to the use of an aforementioned immunogenic composition for the manufacture of a medicine for the prophylactic and/or therapeutic treatment and/or for the prevention of hepatitis B and of hepatitis C.
Another object of the invention is a cell line that expresses the previously described chimeric, immunogenic non infectious subviral particles.
By way of examples of micro-organisms that are suitable for the purposes of the invention, may be cited yeasts, such as the following families: Saccharomyces, Schizosaccharomyces, Kluveromyces, Pichia, Hanseluna, Yarowia, Schwaniomyces, Zygosaccharomyces, Saccharomyces cerevisiae, Saccharomyces carlsbergensis and Kluveromyces lactis being preferred; and bacteria, such as E. coli and those of the following families: Lactobacillus, Lactococcus, Salmonella, Streptococcus, Bacillus and Streptomyces.
As examples of eukaryote cells, cells from animals such as mammals, reptiles, insects and equivalent may be cited. The preferred eukaryote cells are cells from the Chinese hamster (CHO cells), from the monkey (COS and Vero cells), the kidney of the dwarf hamster (BHK cells), the kidney of the pig (PK 15 cells) and the kidney of the rabbit (RK13 cells), human cell lines of the osteosacorma (143 B cells), human HeLa cell lines and human cell lines of the hepatoma (Hep G2 type cells), as well as insect cell lines (for example Spodoptera frugiperda).
Advantageously, the object of the present invention relates to an aforementioned cell line which is Chinese hamster ovary line known as CHO.
These have been previously disclosed, especially by Michel M L Pontisso P, Sobezak E, Malpiéce Y, Streeck R E, Tiollais P. Synthesis in animal cells of hepatitis B surface antigen particles carrying a receptor for polymerized human serum albumin. Proc Natl Acad Sci USA. 1984 December; 81(24):7708-12.
The present invention also relates to an aforementioned cell line, which is a yeast, said yeast may be especially Saccharomyces cerevisae.
Another object of the invention is a cell line disclosed above, which is the new-born hamster kidney cell line (BHK), and is particularly the new-born hamster kidney cell line (BHK-21).
The latter have been disclosed previously, especially by Goldman R D, Follett E A. Birefringent filamentous organelle in BHK-21 cells and its possible role in cell spreading and motility. Science. 1970 Jul. 17; 169(942):286-8.
According to a particular aspect, the invention also relates to a method of production of aforementioned chimeric, immunogenic non infectious subviral particles from an aforementioned cell line, comprising the following steps:
1—a step of TRANSDUCTION of the cells of the cell line with a lentiviral vector comprising a nucleic acid sequence encoding for the wild-type S protein of a hepatitis B virus (HBV) isolate,
2—a step of CULTURE of said cells in order to produce a cell line capable of expressing the wild-type subviral envelope particles of the hepatitis B virus.
3—a step of SELECTION of a clone having an optimal secretion of wild-type subviral envelope particles of the hepatitis B virus,
4—a step of OVER-TRANSDUCTION of said clone with an aforementioned vector,
5—a step of CULTURE of said cells in order to produce a cell line capable of expressing the aforementioned chimeric, immunogenic non infectious subviral particles,
6—a step of SELECTION of said cells capable of secreting in an optimal manner the aforementioned chimeric, immunogenic non infectious subviral particles,
7—a step of CULTURE of said clone for the production of the aforementioned chimeric, immunogenic non infectious subviral particles, and
8—a step of PURIFICATION of the subviral particles from the culture medium collected (centrifugation, ultra-centrifugation on gradients, collection of the positive fractions for the chimeric subviral particles, dialysis).
Table 1 provides a list of nucleic acid sequences and of amino acids disclosed in the present invention. By convention, lower case letters are used for nucleic acid sequences and upper case letters for amino acids sequences.
Other characteristics and advantages of the invention will become clearer on reading the following description of a preferential embodiment, given by way of simple illustrative and non limiting example, and by referring to the appended figures, among which:
) result in the maturation of said polyprotein especially into structural proteins (capsid, envelope E1 and E2). The numbers indicate the position of the residues of amino acids bounding the domains of the different structural proteins of the polyprotein.
According to the particular but non limiting embodiment of the invention, the fusion protein E1-Sd of around 50 kD comprises 393 residues of amino acids derived from the protein E1 and of the S domain. The residues 192 to 380 of the complete protein E1 are linked to the N-terminal end of the deleted S protein composed of residues 23 to 226 of the wild-type S protein.
According to the particular but non limitative embodiment of the invention, the fusion protein E2-Sd of around 85 kD comprises 564 residues of amino acids derived from the protein E2 and of the domain S. The residues 384 to 743 of the complete protein E2 are linked to the N-terminal end of the deleted S protein composed of residues 23 to 226 of the wild-type S protein.
Caption: M: molecular weight marker (kD); the β-gal, E1E2 or E1E2 and S tracks represent control cellular lysates derived from transfections of BHK-21 cells by the corresponding SFV RNA.
By way of non limiting example, a particular embodiment of the invention is given hereafter, for obtaining subviral envelope particles of the hepatitis C virus (HCV) in Semliki system (cf.
This embodiment is based on obtaining immunogenic fusion proteins of the invention and in particular the fusion proteins E1-Sd or E2-Sd comprising, on the one hand, the S envelope protein deleted, at the N-terminal, of the transmembrane domain thereof (Sd), of the hepatitis B virus (HBV) and, on the other hand, the quasi integrality of one of the envelope proteins E1 or E2 of the HCV. This transitory embodiment demonstrates the capacity of assembling said deleted S protein (Sd), in the presence of the wild-type S protein, making it possible to obtain the aforementioned chimeric subviral particles, thanks especially to the high level of expression of the vector based on the replication properties of the Semliki forest virus (pSFV vector), before proceeding to obtaining stable clones that are producers of said particles by using lentiviral vectors.
I.1) Construction of Plasmids p SFV 1-E1-Sd and pSFV1-E2-Sd.
The pSFV1 vector (Invitrogen) having a bicistronic structure of 11033 pb is used for the following constructions. This vector is provided with a promoter sequence of SP6-RNA-polymerase, inserted at the 5′ side of the first cistron, in order to be able to initiate the synthesis of a complete RNA of positive polarity (RNA named 42S(+)) by transcription in vitro. After transfection into mammal cells, these recombinant RNA capped in vitro self-replicate in the presence of the nsP1-4 replicase of the SFV and serving for the production of proteins of interest by the intermediary of a secondary RNAm named 26S(+).
Wild-type complementary DNA (DNAc) or a hybrid nucleic acid molecule encoding for a fusion protein of the invention is obtained by carrying out several series of amplifications by polymerase chain reactions (PCR). Starting with the plasmid pSFV1-E1E2 (consisting in a SFV vector containing the sequence encoding for the two proteins E1 and E2 of the HCV), a first PCR, named PCR A, enabled the amplification of the sequences encoding for the envelope proteins E1 or E2 of the HCV with their transmembrane domain and preceded by sequences encoding for their respective transfer initiation peptide of addressing to the endoplasmic reticulum. Starting with the plasmid pHBV1.5 disclosed previously |Patient, R., Hourioux, C., Sizaret, P. Y., Trassard, S., Sureau, C., and Roingeard, Y., (2007) Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking J Virol, 81(8), 3842-51], a second PCR, named PCR B, enabled the amplification of the sequence encoding for the deleted S protein of the HBV. The “HCV” parts of the sequences encoding for the fusion proteins according to the invention have been named “A” and the “HBV” parts have been named “B” (
I.1.1) Amplification of DNAc Sequences Encoding for the Fusion Proteins of the Invention (Especially E1-Sd and E2-Sd):
The amplifications by PCR are carried out in a reaction medium of 50 μL containing 50 ng of DNA, 15 μmol of each pair of primers (Proligo), 200 μM of each of the desoxyribonucleotides dNTP (Invitrogen) and 10 units of Taq-DNA-polymerase (Takara) comprising a correcting activity so as to minimise errors linked to successive amplifications (
Table 2 “Oligonucleotides used for the genetic amplification of the sequences A and B constituting chimeric sequences” below indicates the sequences of primers used.
The sequences corresponding to the HCV are represented therein in black type, those corresponding to the HBV in black underlined type, those corresponding to the BamHI restriction site in italic underlined type, those corresponding to the ATG initiation codon of the translation in bold type, those corresponding to the ATG termination codon of the translation in bold type.
These amplification reactions, carried out in an “iCycler” thermal cycler (Biorad), consisted in an initial denaturation of 5 minutes at 95° C., followed by 25 cycles comprising a denaturation of 1 minute at 95° C., an hybridisation of 1 minute at 60° C. and an elongation of 1 minute at 69° C. The 25 cycles are followed by a final elongation of 10 minutes at 69° C. The fragments amplified by the PCR A and B are purified on 1% agarose gel by means of the “Wizard SV Gel and PCR Clean-Up System” system (Promega) in accordance with the manufacturer's recommendations. The purified fragments of the PCR A and B are then hybridised by a PCR named PCRHYB in the absence of primers for 10 cycles then are amplified by a final PCR named PCRFIN for 25 cycles in the presence of “forward” primers of sequences A and “reverse” primers of sequences B. The programmes of cycles of the latter two PCR are similar to those used for the PCR A and B. The amplified fragments of molecules of hybrid nucleic acids of the invention thereby obtained are purified on gel as above.
I.1.2) Cloning of Hybrid Sequences of DNAc in the pSFV1 Vector
The PCRFIN products purified are cloned in the pGEM-T® vector (“pGEM-T Easy Vector System”, Promega) in accordance with the manufacturer's recommendations. The fragments of hybrid nucleic acids molecules of the invention are freed of the pGEM-T® by restriction with the enzyme BamHI (Biolabs) then cloned at the BamHI site of the plasmid pSFV1. The different plasmids comprising the fusion proteins of the invention (especially the plasmids pSFV1-E1-Sd and pSFV1-E2-Sd) are amplified by bacterial transformation then purified by maxi-preparation of DNA in phenol/chloroform. The orientation of the insert is verified by enzymatic restriction and all of the constructions are verified by sequencing.
I-2) Obtaining the Cell Line Temporarily Transfected by the RNA of Different SFV Constructions
The culture procedures for new-born hamster kidney cells (BHK-21) as well as the in vitro transcription protocols of SFV matrix plasmids and transfection of self-replicating recombinant RNA were identical to those described previously |Patient, R., Hourioux, C., Sizaret, P. Y., Trassard, S., Sureau, C., and Roingeard, P., (2007) Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking J Virol, 81(8), 3842-51]. The pSFV-S construction, expressing the wild-type S protein of the HBV and previously disclosed in the preceding article, was used as control.
I-3) Analysis of the Intracellular Production of Wild-Type Chimeric Envelope Proteins
The biochemical procedures for analysing the proteins of interest (especially 5, Sd, E1, E2, E1-Sd, E2-Sd etc.) by immunofluorescence confocal microscopy and by Western Blot, the ultra-structural analysis procedures of cells transfected in transmission electron microscopy as well as the procedures of quantification (ELISA)/purification (sucrose gradient then affinity chromatography) of HCV-HBV chimeric subviral envelope particles are those disclosed previously |Patient, R., Hourioux, C., Sizaret, P. Y., Trassard, S., Sureau, C., and Roingeard, P., (2007) Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking I Virol, 81(8), 3842-51].
The detection of wild-type S proteins E1 and E2 of HCV and fusion proteins of the invention is carried out by means of a monoclonal murine anti-E1 antibody (A4, provided by Dr Harry Greenberg, University of Stanford, Calif.) [Dubuisson, J., Hsu, H. H., Cheung, R. C., Greenberg, H. B., Russell, D. G., and Rice, C. M. (1994). Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses. J Virol 68(10), 6147-60)] or anti-E2 antibody (H52, provided by Dr Jean Dubuisson, Institut Pasteur de Lille) [Deleersnyder, V., Pillez, A., Wychowski, C., Blight, K., Xu, J., Hahn, Y. S., Rice, C. M., and Dubuisson, J. (1997). Formation of native hepatitis C virus glycoprotein complexes. J Virol 71(1), 697-704].
I-4) Analysis of the Culture Supernatant.
After transfection, the culture supernatant of around 107 cells transfected is cleaned by a centrifugation of 10 minutes at 1500 g then is ultra-centrifuged at 4° C. for 16 hours at 35,000 rpm by means of a SW41 rotor (L70 Ultracentrifuge, Beckman). The residue is taken up in 50 μL of the lysis buffer then analysed by Western-blot.
I.5) Production of Fusion Proteins E1-Sd and E2-Sd of the Invention
Sixteen hours after the transfection by the plasmids pSFV1 comprising the hybrid nucleic acid molecules of the invention, pit1-e1-sd, pit2-e2-sd, the BHK-21 cells were lysed then analysed by Western-blot by means of monoclonal anti-E1 and anti-E2 antibodies. After transitory production in BHK-21 cells, the sizes of the fusion proteins E1-Sd and E2-Sd are around 50 kD for the protein E1-Sd and around 85 kD for the protein E2-Sd. These results, correlated with the intense immunofluorescence obtained by the detection of said fusion proteins of the invention with the anti-E1, anti-E2 and anti-S antibodies, show that they are correctly produced, correctly glycosylated, and thus laid out according to the desired transmembrane topology (
So as to restore the secretion capacity of the different fusion proteins of the invention, co-transfections are carried out by introducing in trans the wild-type form of the S protein of the HBV to each of the fusion proteins of the invention (
Transmission electron microscopy images show that in all of these experiments of co-production of the wild-type S protein with one of the fusion proteins of the invention it is possible to produce an important quantity of spherical and filamentous subviral particles. Western-blot analyses show that these more or less filamentous subviral particles are rich in fusion proteins of the invention.
The implementation according to example 1 of the present invention in “Semliki” system shows that the fusion proteins of the invention containing the quasi-integrality of the proteins E1 or E2 of the HCV (their transmembrane domain replacing that located at the N-terminal of the S protein of the HBV) assembly themselves into chimeric subviral particles of same nature as the subviral particles used in the production of vaccines against hepatitis B, thereby facilitating the purification of said chimeric subviral particles of the invention, and potentially, the development of an industrial application of a vaccine against HCV, reproducing that of the vaccine against HBV.
The implementation according to example 1 of the present invention also shows the production of fusion proteins comprising the non truncated proteins E1 and/or E2 of HCV. This characteristic may prove to be determining in inducing an optimal neutralising and cellular immune response.
However, in “Semliki” system, the chimeric subviral particles are produced by temporary transfection of cells. Indeed, the high cytotoxicity of these vectors does not make it possible to obtain an efficient secretion in the long term of chimeric subviral particles. Furthermore, the purification of said particles from the homogenate of the cells co-transfected necessitates a relatively cumbersome implementation, ill suited to industrial production.
By way of non limiting example, an embodiment is disclosed with reference to
One of the objectives of this embodiment is also to obtain a cellular system of production of chimeric subviral particles of the invention similar to that used for the industrial manufacture of the vaccine against hepatitis B.
The use of lentiviral vectors has made it possible to develop cellular clones producing in a stable manner the wild-type S protein of the HBV associated with one of the fusion proteins E1-Sd and/or E2-Sd.)
(II-1) pLENTI Plasmid Lentiviral Vectors.
The pLENTIhph plasmid of 9955 pb, comprising the selection gene hph, and encoding for a hygromycin resistance protein as selection marker was used for the following constructions
[Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M., and Trono, D. (1996). In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector. Science 272(5259), 263-7].
Firstly, the nucleic acid sequence encoding for the wild-type S protein is released from pSFV1-S as disclosed previously [Patient, R., Hourioux, C., Sizaret, P. Y., Trassard, S., Sureau, C., and Roingeard, P., (2007) Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking J Virol, 81(8), 3842-51] by restriction with the enzyme BamHI (Biolabs) then purified on 1% agarose gel by means of the “Wizard SV Gel and PCR Clean-Up System” system (Promega) in accordance with the manufacturer's recommendations. This purified fragment was then cloned at the BamHI site included in the multiple cloning site of the pLENTIhph plasmid by means of T4-DNA-ligase (Biolabs) in accordance with the manufacturer's recommendations. The pLENTIhph-S plasmid was finally amplified by bacterial transformation then purified by maxi-preparation of DNA by means of the “Nucleobond PC 500 Kit” system (Qiagen) in accordance with the manufacturer's recommendations. The orientation of the insert was then verified by enzymatic restriction.
In the same way, the different hybrid nucleic acids molecules of the invention of DNAc encoding for the fusion proteins of the invention (especially E1-Sd and E2-Sd) are cloned at the BamHI site of the pLENTIgfp plasmid. The plasmids thereby obtained (especially pLENTIgfp-E1-Sd and E2-Sd) are then amplified, purified and sequenced (cf. II-1).
II-2) Production of Recombinant Lentivirus.
Twenty four hours before the transfection of the lentiviral plasmids, HEK 293T cells were seeded at a rate of 3.106 cells per 75 cm2 flask (Falcon) in DMEM-glutamax medium (Invitrogen) supplemented with 10% of decomplemented foetal bovine serum (ATGC), 100 UI/mL of penicillin and 100 μg/mL of streptomycin. These cells were cultivated under 5% of CO2 and the culture medium was changed 4 hours before the transfection. One pmole of each plasmid p8.74, pVSV-G and pLENTI (pLENTIhph-S, pLENTIgfp-E1-Sd, E2-Sd) was transfected simultaneously in the cells HEK 293T by means of the “Calcium Phosphate. Transfection Kit” system (Invitrogen) in accordance with the manufacturer's recommendations. The culture medium is changed 24 hours after the transfection and collected 48 hours and 72 hours after the transfection. The media collected are filtered at 0.45 μm then concentrated by ultracentrifugation on a 20% sucrose cushion at 4° C. for 90 minutes at 100.000 g. The residue containing the recombinant lentiviruses is taken up in 500 μL of phosphate buffer (PBS) and conserved at −80° C. Batches of recombinant lentiviral vectors of the invention are generated, of which especially the lentiviral vectors LVhph-S, LVgfp-E1-Sd, LVgfp-E2-Sd. The titration of the lentiviral transducing units (TU) of each batch is determined from the assay of the p24 protein and by means of the “Innotest HIV Kit” (Innogenetics) system in accordance with the manufacturer's recommendations.)
II-3) Cellular Culture and Transduction.
The cell line used for the stable and constitutive production of HCV-HBV chimeric subviral particles is a Chinese hamster ovary line named CHO. This line has already been perfectly validated for the production of recombinant proteins of medical interest, and especially for the vaccine against hepatitis B “GenHevac B Pasteur®” (Sanofi Pasteur MSD). These CHO cells are cultivated under 5% CO2 in DMEM-F12 medium (Invitrogen) supplemented with 10% decomplemented foetal bovine serum (ATGC), 100 UI/mL of penicillin and 100 μg/mL of streptomycin. Twenty four hours before the transduction, the CHO cells are cultured in a 6-well plate (Falcon) at a rate of 105 cells per well. They are then transduced in a new medium with a multiplicity of infection of 2.5 (i.e. a ratio of transducing units (TU) per cell of 2.5) in the presence of 4 μg/mL of polybrene (Sigma-Aldrich) then cultivated for 24 hours. The following day, the transduction medium is removed, the cells obtained according to the implementation of the present invention are rinsed in PBS buffer then are maintained normally in culture for 2 more days.
II-3.1) Generation of a Stable CHO Clone Producer of the Wild-Type S Protein
The CHO line used is transduced with the lentivirus LVhph-S according to the protocol described above, and schematised in
II-3.2) Generation of Stable CHO Clones Producers of the Wild-Type S Protein of the HBV Virus and Fusion Proteins of the Invention E1-Sd and E2-Sd.
The CHO-S cellular clone is later over-transduced with recombinant lentivirus LVgfp encoding for the GFP protein as selection marker, and comprising a hybrid nucleic acid molecule of the invention, especially the vector lentivirus LVgfp-PIT1-E1-Sd or LVgfp-PIT2-E2-Sd according to the protocol described above in §II-3.1. (
II-4) Analysis of the Intracellular Production of Chimeric and Wild-Type Envelope Proteins of the HBV and the HCV.
The biochemical analysis procedures by immunofluorescence and by Western Blot of the proteins of interest S, E1-Sd or E2-Sd of each clone cellular have already been disclosed previously, for example I in §1-4.
II-5) Purification and Analysis of Chimeric Subviral Particles Derived from the Culture Supernatant.
Around 200 mL of culture supernatant for each clone selected is clarified by centrifugation at 4° C. for 10 minutes at 1500 g. The proteins are precipitated by addition of a solution of (NH4)2SO4 (pH 7.5; 45% final) then centrifugation at 4° C. for 15 minutes at 10.000 g. The residue is taken up in a minimum volume of TNE buffer (10 mM Tris/HCl pH 7.5/100 mM NaCl/1 mM EDTA). After a series of dialyses in TNE buffer, CsCl is added until a density of around 1.22 g/cm3 is obtained, then two successive isopycnal ultra-centrifugations are carried out at 15° C. for 48 hours at 40.000 rpm by means of a 45Ti rotor (L70 Ultracentrifuge, Beckman). Fractions are collected from the summit of the gradient and the wild-type S protein is quantified by ELISA [Patient, R., Hourioux, C., Sizaret, P. Y., Trassard, S., Sureau, C., and Roingeard, P., (2007) Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking J Virol, 81(8), 3842-51]. The positive fractions were mixed and dialysed at 4° C. in THE buffer.
The purified preparations were finally analysed by Western Blot and by transmission electron microscopy as disclosed previously in example I au §1-4.
II-6) Intracellular Production of the S Protein and Fusion Proteins.
Following their expansion, the CHO-S, CHO-E1-Sd and CH0-E2-Sd cellular clones were analysed by immunofluorescence then Western-blot by means of antibodies directed against the proteins Sd, E1 or E2 (cf.: example I in §1-4 and
II-7) Purification of Particles
A first purification test was carried out from 200 mL of culture supernatant of the CHO-5, CHO-E1-Sd or CHO-E2-Sd clone. These supernatants, the concentration in wild-type S protein of which is evaluated by ELISA at around 100 ng/mL for the three clones, are ultra-centrifuged on an isopycnal gradient of CsCl. The fractions of each gradient formed are then analysed by a quantification of the wild-type S protein (
A deposition corresponding to one μg of wild-type S protein for each purification is carried out for the analysis in acrylamide gel. This makes it possible to show that the fusion proteins of the invention E1-Sd and E2-Sd are detected at the expected size and in respectable quantity in their respective preparation by the antibodies anti-E1 or anti-E2. Similarly, the two expected forms of glycosylation of the wild-type S protein are detected in each preparation. The analysis by negative coloration in transmission electron microscopy made it possible to confirm the presence of spherical subviral envelope particles of around 20 nm diameter in the three preparations studied.
A new pLENTIgluc plasmid comprising the selection gene glue of Gaussia princeps, in place of the gene gfp, is formed and encodes for a luciferase (GLuc) secreted as selection marker. The fragment of DNAc encoding for the fusion protein E1-Sd is cloned at the BamHI site of the pLENTIgluc plasmid. The pLENTIgluc-E1-Sd plasmid thereby obtained is amplified, purified and sequenced (cf. II-1).
III.1 Production of Recombinant Lentivirus.
Twenty four hours before the transfection of the lentiviral plasmids, HEK 293T cells were cultured at a rate of 3.106 cells per 75 cm2 flask (Falcon) in DMEM-glutamax medium (Invitrogen) supplemented with 10% decomplemented foetal bovine serum (ATGC), 100 UI/mL of penicillin and 100 μg/mL of streptomycin. These cells are cultivated under 5% CO, and the culture medium is changed 4 hours before the transfection. One pmole of each p8.74, pVSV-G and pLENTIgluc-E1-Sd plasmid is transfected simultaneously into the HEK 293T cells by means of the “Calcium Phosphate Transfection Kit” system (Invitrogen) in accordance with the manufacturer's recommendations. The culture medium is changed 24 hours after the transfection and collected 48 hours and 72 hours after the transfection. The media collected are filtered at 0.45 μm then concentrated by ultracentrifugation on a 20% sucrose cushion at 4° C. for 90 minutes at 100.000 g. The residue containing the recombinant lentivirus is taken up in 500 μL of phosphate buffer (PBS) and conserved at −80° C. A new batch of recombinant lentivirus is thereby generated: LVgluc-E1-Sd. The titration of the lentiviral transducing units (TU) of this batch is determined from the assay of the p24 protein and by means of the “Innotest HIV Kit” systems (Innogenetics) in accordance with the manufacturer's recommendations.
III.2. Generation of a Stable CHO Clone Producer of the Wild-Type S Protein of the HBV Virus, Fusion Protein of the Invention E2-Sd and E1-Sd.
The culture of the CHO cells and the method of transduction disclosed and used for example II is adapted to this example. After a first transduction with the lentivirus enabling the CHO-S cellular clone to be obtained, a second transduction is carried out with the lentivirus LVgfp-E2-Sd according to the protocol disclosed for example II, enabling the CHO-E2-Sd cellular clone to be obtained.
The latter is again transduced with the lentivirus LVgluc-E1-Sd according to the protocol disclosed above. Three days after the transduction, the cells are trypsinized and re-cultured at limit dilution in a 96-well plate (Falcon) at a rate of 1 cell per well. The cells are maintained under culture for three weeks, at the end of which the supernatant of the emerging cellular clones is collected. In these supernatants, the presence of luciferase is detected by the measurement of its enzymatic activity by means of the “Gaussia Luciferase Assay Kit” system (Biolabs) in accordance with the manufacturer's recommendations. The light emitted by the enzymatic reaction is measured by means of a Centro LB 960 luminometer (Berthold Technologies). The cellular clones corresponding to the supernatants GLuc+ are recovered then amplified in large quantity. The clone thereby obtained is named CHO-S/E2-Sd/E1-Sd.
5 to 10 milligrammes of chimeric subviral particles for each type of particle (S+E1-Sd, S+E2-Sd, S only of the HBV) are purified from the supernatant of cellular culture according to the method described above.
Four groups of mice and rabbits are made use of for an immunisation series.
The immunisation is carried out by three injections of 10 micrograms of the immunogenic according to the classical method.
The first group is immunised by the particles S+E1-Sd.
The second group is immunised by the particles S+E2-Sd.
The third group is immunised by the particles HBV S alone.
The fourth group is immunised by the mixture of chimeric subviral particles S+E1-Sd and chimeric subviral particles S+E2-Sd.
The global humoral response produced in these animals is detected by ELISA and Western blot analysis. For the anti-S antibodies, commercial ELISA (Abbott, Roche) make it possible to determine the number of international units (UI) of anti-S antibodies, which are known to have neutralising properties against HBV. A concentration at least equal to 10 UI is considered as protective against HBV. (Jilg W, et al. Hepatitis B-vaccination: Strategy for booster doses in high risk population groups. Progress in Hepatitis B Immunization. Eds. P. Coursaget et al. Collogue Inserm. 1990; 190: 419-427.) For the anti-E1 and ant-E2 antibodies, the Western blot is used to evaluate whether an anti-E1 and anti-E2 antibody response is generated. If this is the case, this analysis is completed by an analysis of the presence of anti-E1 and anti-E2 antibodies neutralising the virus. The analysis of the neutralisation of HCV by these antibodies is performed in the JFH-1 system (Wakita et al., Nat Med 2005) which makes it possible to propagate an HCV strain of genotype 2 in vitro, or by the use of chimeric viral strains comprising the structural proteins of the HCV of genotype 1 or 3.
| Number | Date | Country | Kind |
|---|---|---|---|
| 08 03377 | Jun 2008 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR2009/051142 | 6/16/2009 | WO | 00 | 3/3/2011 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2009/153518 | 12/23/2009 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20040146529 | Selby et al. | Jul 2004 | A1 |
| Number | Date | Country |
|---|---|---|
| 2008025067 | Mar 2008 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20110150921 A1 | Jun 2011 | US |
