# Patent

## Title
Chimeric poly peptides and the therapeutic use thereof against a Flaviviridae  infection

## Abstract

The invention relates to building a chimeric polypeptide used for preventing or treating a Flaviviridae infection. The use of the inventive chimeric polypeptide for producing recombinant viral vectors such as a measles living viral vector is also disclosed.

## Background
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 20, 2012, is named DI20521A.txt and is 85,853 bytes in size.
FIELD OF THE INVENTION
The present invention relates to the construction of a chimeric polypeptide for use in the prevention or treatment of a Flaviviridae infection. The present invention also relates to the use of the chimeric polypeptide in the production of recombinant viral vectors, such as a live measles viral vector to act as a vaccine.
BRIEF DESCRIPTION OF THE PRIOR ART
Flaviviruses (the Flaviviridae family) are envelope viruses the genome of which is a RNA molecule with a positive polarity. The flavivirion is composed of three structural proteins designated C (core protein), M (membrane protein) and E (envelope protein). The E protein, which is exposed on the virus surface, is responsible for the principal biological functions of the virion, including attachment of the virus and specific membrane fusion. Translation of genomic RNA produces a precursor polyprotein which is simultaneously cleaved by cellular and viral proteases to generate individual viral proteins: three structural proteins C, prM (the glycosylated precursor of the M protein), E, and seven non-structural proteins NS1 to NS5. Replication of flaviviruses occurs in the cytoplasm of infected cells. Flaviviruses have complex natural transmission cycles which involve several natural hosts (mainly mammals and birds) and vectors, the latter being hematophage arthropods such as ticks and mosquitoes. These viruses are the principal cause of severe human diseases such as hemorrhagic manifestations or meningo-encephalitic syndromes. The dengue virus (DEN) is one of the two major flaviviruses known to be the cause of severe hemorrhagic diseases across the world.
Dengue is an emerging disease and a bane to public health. Development of live-attenuated vaccine candidates against the four serotypes of the dengue virus has been carried out for more than 50 years. The search for a vaccine is hindered by the absence of an animal model which mimics the disease caused by the dengue virus, namely the hemorrhagic fever of dengue and the shock syndrome associated therewith. Vaccine effectiveness can be evaluated in the mouse using a neurovirulent strain of DEN virus adapted to the mouse, which kills adult animals after intracerebral inoculation. However, studies based on that type of murine model do not reflect the activity of the dengue virus in man. In the monkey, protection can only be demonstrated by measuring the reduction in viremia after an experimental infectious challenge. Thus, the ultimate test for a vaccine against dengue must be based on clinical trials carried out in man. The majority of attenuated vaccine candidates which have been developed over the last 50 years have been either too reactogenic or insufficiently immunogenic in clinical trials. Several attenuated tetravalent candidates are currently in phase I or II clinical trials. The difficulty in the development of this type of live-attenuated tetravalent vaccines appears to be in obtaining a balanced mixture of four valencies in order to generate protective immunity against the four serotypes.
Various live chimeric vaccine candidates are also being developed. They are based on the exchange of homologous structural genes between different flaviviruses. Several types of intertype chimeric viruses have been constructed and tested in the mouse or monkey. Chimeric viruses have been constructed with Chimerivax® techniques based on the yellow fever vaccinal virus (YF-17D). Tetravalent mixtures of those YF-17D/DEN chimeras have been tested in the rhesus monkey. Here again, the balance between the four chimeric viruses is difficult to obtain for uniform immunization. A phase I/II clinical test is currently being prepared to test a chimerivax-dengue 2 vaccine. Other approaches include subunit candidate vaccines produced from an expression vector, and naked DNA type candidates which are in pre-clinical development. Thus, the development of a tetravalent vaccine against the dengue virus is still an unresolved problem, one of the most important on the list of the World Health Organization.
Thus, there is a need to develop novel effective vaccine candidates which are easy to produce and to formulate, for the prevention or treatment of an infection by a virus of the Flaviviridae family such as the dengue virus.
SUMMARY OF THE INVENTION
The present invention relates to a chimeric polypeptide and its applications in the prevention or treatment of an infection by Flaviviridae.
More particularly, an object of the present invention is a chimeric polypeptide comprising a peptide of a subdomain the E protein of Flaviviridae bound to a peptide of a subdomain of the Membrane M protein of Flaviviridae.
An object of the present invention is also an isolated or purified polynucleotide coding for a chimeric polypeptide of the invention.
An object of the present invention is also a recombinant measles viral vector into the genome of which a polynucleotide of the invention has been inserted.
An object of the invention also pertains to purified monoclonal or polyclonal antibodies specifically recognizing at least one polynucleotide as defined above and/or the chimeric polypeptide of the invention.
The invention also relates to the use of a viral vector of the invention for the preparation of an immunogenic composition intended for the prevention or treatment of a Flaviviridae infection in a sensitive species.
The present invention also relates to cloning or expression vectors comprising a polynucleotide of the invention.
Another object of the present invention is to provide an immunogenic composition intended for the prevention and/or treatment of a Flaviviridae infection in a sensitive species, characterized in that it comprises at least one of the following elements:

    
    
        a chimeric polypeptide as defined above;
        a polynucleotide according to the invention;
        a recombinant viral vector according to the invention;
        an antibody according to the invention; and
        a cloning and/or expression vector according to the invention.
    
    


The present invention also proposes a method for preventing and/or treating a Flaviviridae infection in a sensitive species, comprising administering a pharmaceutically effective amount of at least one of the following elements:

    
    
        a chimeric polypeptide as defined above;
        a polynucleotide according to the invention;
        a recombinant viral vector according to the invention;
        an antibody according to the invention; and
        a cloning and/or expression vector according to the invention.
    
    





BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the 384 nucleotide (nt) sequence coding for the [EDIII]DV-1 domain of the FGA/89 viral strain fused via the Arg-Ser dipeptide to the signal peptide of calreticulin (ssCRT) flanked by the two BsiWI (5′) and BssHII (3′) sites necessary for insertion into the MVSchw vector;
FIG. 2 shows the 516 nucleotide (nt) sequence coding for the [EDIII+M1-40]DV-1 fusion protein of the FGA/89 viral strain fused via the Arg-Ser dipeptide to the signal peptide of calreticulin (ssCRT) flanked by the two BsiWI (5′) and BssHII (3′) sites necessary for insertion into the MVSchw vector;
FIG. 3 shows Vero cells infected with recombinant measles viral vectors according to the invention. Multiplicity of infection 10 TCIP50/cell for 30 h. Immunofluorescence carried out with a specific anti-DV-1 HMAF antibody;
FIG. 4 shows the expression and secretion of the [EDIII]DV-1 and [EDIII+M1-40]DV-1 antigenic domains in cytoplasmatic lysates (C) and supernatants (S) from Vero cells infected with recombined MVSchw-DV-1 viruses;
FIG. 5 shows the expression and secretion of the [EDIII]DV-1 and [EDIII+M1-40]DV-1 antigenic domains in filtered and concentrated supernatants from drosophila S2 cells inducibly expressing these antigens;
FIG. 6 shows a nucleotide sequence coding for a chimeric polypeptide according to a preferred mode of the invention;
FIG. 7 shows an amino acid sequence of a chimeric polypeptide according to a preferred mode of the invention and the nucleotide sequence coding for it;
FIG. 8 shows an amino acid sequence of a dimer of the ectodomain III (EDIII) according to a preferred mode of the invention, and the nucleotide sequence coding for it;
FIG. 9 shows an amino acid sequence of a dimer of the ectodomain III (EDIII) according to a preferred mode of the invention, and the nucleotide sequence coding for it;
FIG. 10 shows an amino acid sequence of a chimeric polypeptide according to a preferred mode of the invention, and the nucleotide sequence coding for it;
FIG. 11 shows an amino acid sequence of the ectodomain III (EDIII) according to a preferred mode of the invention, and the nucleotide sequence coding for it;
FIG. 12 shows an amino acid sequence of a dimer of the ectodomain III (EDIII) according to a preferred mode of the invention, and the nucleotide sequence coding for it;
FIG. 13 shows a nucleic acid sequence coding for a peptide of the ectodomain III (EDIII) peptide according to a preferred mode of the invention;
FIG. 14 shows a nucleotide sequence coding for a chimeric polypeptide according to a preferred mode of the invention;
FIGS. 15 to 19 show an amino acid sequence of chimeric polypeptides according to a preferred mode of the invention;
FIGS. 20 to 24 show a nucleotide sequence coding respectively for the chimeric polypeptides of FIGS. 15 to 19;
FIGS. 25 to 29 respectively show a representative diagram of the chimeric polypeptides of FIGS. 15 to 19;
FIG. 30 shows the peptide sequence of the apoptoM sequence of four serotypes of the dengue virus used in the construction of a chimeric precursor according to the invention.



DETAILED DESCRIPTION OF THE INVENTION
The originality of the present invention is based on the construction of a chimeric polypeptide and its applications in the prevention or treatment of an Flaviviridae infection. The term “Flaviviridae” means a virus selected from the group constituted by the West Nile virus, dengue virus, Japanese encephalitis virus and yellow fever virus.
1. Polypeptide and Polynucleotide
In a first aspect, the present invention relates to a chimeric polypeptide comprising a peptide of a subdomain of the E protein of Flaviviridae linked to a peptide of a subdomain of the membrane M protein of Flaviviridae. The invention also pertains to a polypeptide which consists of said peptides.
The term “polypeptide” as used in the present invention means both proteins and peptides. By “chimeric polypeptide” it is meant any protein or peptide comprising sub-portions of different origins, for example a polypeptide A deriving from a first species and a polypeptide (protein or peptide) deriving from a second species. A protein or peptide is also considered to be a chimeric polypeptide if it includes sub-portions deriving from different proteins or peptides from the same species, or even from the same protein or peptide from different species.
Preferably, the peptide of a subdomain of the E protein consists of the ectodomain III comprising an amino acid sequence as defined in any one of the following sequences:

    
    
        amino acids 19 to 120 of SEQ ID NO: 1;
        amino acids 19 to 119 of SEQ ID NO: 3;
        amino acids 21 to 123 of SEQ ID NO: 6;
        amino acids 21 to 121 of SEQ ID NO: 12; and
        amino acids 21 to 123 of SEQ ID NO: 14.
    
    


More particularly, the peptide of a subdomain of the E protein consists of
a dimer of the ectodomain III of the dengue 1, 2, 3 or 4 virus comprising an amino acid sequence as defined in any one of the following sequences:

    
    
        amino acids 21 to 262 of SEQ ID NO: 8; and
        amino acids 21 to 262 of SEQ ID NO: 10;
    
    


or a tetramer of the ectodomain III of the dengue 1, 2, 3, 4 virus comprising a sequence ranging from amino acid 21 to 494 of SEQ ID NO: 16 or ranging from amino acid 18 to 429 of SEQ ID NO: 24.
Regarding the peptide of a subdomain of the M protein, this in particular consists of the ectodomain 1-40 comprising an amino acid sequence ranging from position 123 to 170 of SEQ ID NO: 3 or in the apoptoM sequence comprising an amino acid sequence ranging from position 154 to 170 of SEQ ID NO: 3 or ranging from position 122 to 132 of SEQ ID NO: 12.
In a preferred mode, the chimeric polypeptide of the invention further comprises a binding segment binding the peptide of a subdomain of the E protein to the peptide of a subdomain of the M protein. Said binding segment is preferably a pentapeptide with sequence: RRDKR (SEQ ID NO: 34) or RREKR (SEQ ID NO: 35).
Highly preferably, the chimeric polypeptide of the invention comprises an amino acid sequence as defined in any one of the following sequences:

    
    
        a) amino acids 19 to 162 of SEQ ID NO: 3;
        b) amino acids 21 to 168 of SEQ ID NO: 6;
        c) amino acids 21 to 132 of SEQ ID NO: 12;
        d) amino acids 18 to 624 of SEQ ID NO: 20;
        e) amino acids 18 to 609 of SEQ ID NO: 21;
        f) amino acids 18 to 624 of SEQ ID NO: 22;
        g) amino acids 18 to 489 of SEQ ID NO: 23; and
        h) amino acids 21 to 474 of SEQ ID NO: 24.
    
    


The invention also relates to polypeptides (and fragments thereof) which are coded by the nucleotide sequences mentioned below.
Highly preferably, the polypeptide of the invention has a percentage identity of at least 80% after optimum alignment with a sequence as defined in any one of amino acid sequences a) to h) defined above, preferably at least 90%, more preferably at least 98% and still more preferably at least 100%.
In a connected aspect, the invention relates to an isolated or purified polynucleotide coding for a chimeric polypeptide as defined above.
By “isolated or purified”, it is meant molecules which have been altered by man from their native state, i.e. if such molecules exist in nature, they have been changed and/or removed from their initial environment. As an example, a polynucleotide or a polypeptide naturally present and found in the biological environment of a living organism which naturally expresses it is not “isolated” in this context. However, the same polynucleotide or polypeptide when separated from its natural environment and/or obtained by cloning, amplification and/or chemical synthesis is considered in the present invention to be “isolated”. Further, a polynucleotide or polypeptide which is introduced into an organism by transformation, gene manipulation or any other recombination method is “isolated” even if it is present in said organism.
By the terms “nucleotide sequence”, “nucleic acid”, “nucleic sequence or nucleic acid sequence”, “polynucleotide”, oligo nucleotide”, “polynucleotide sequence”, which will be used indiscriminately in the present description, it is meant a precise chain of nucleotides, which may or may not be modified, which allows to define a fragment or a region of a nucleic acid, which may or may not comprise non-natural nucleotides and which can correspond both to a double-stranded DNA, a single-stranded DNA or to transcription products of said DNAs. Thus, the nucleic sequences of the invention also encompass PNAs (peptide nucleic acid) or the like. The polynucleotide fragments of the invention comprise at least 15 consecutive nucleotides. Preferably, they comprise at least 20 consecutive nucleotides and more preferably they comprise at least 30 consecutive nucleotides.
The invention also pertains to fragments of polynucleotides, which consist of fragments of 15, 20 or 30 successive nucleotides.
Any polynucleotide which has been chemically, enzymatically or metabolically modified but which has retained the biochemical properties of the original chimeric polypeptide is included within the scope of the present invention.
In a preferred embodiment, the polynucleotide of the present invention, when it codes for a chimeric polypeptide of the invention, advantageously comprises a nucleotide sequence as defined in any one of the following sequences:
a) nucleotides 7 to 492 of SEQ ID NO: 4;
b) SEQ ID NO: 5;
c) Nucleotides 7 to 504 of SEQ ID NO: 7;
d) Nucleotides 7 to 504 of SEQ ID NO: 13; and
e) SEQ ID NOS: 25 to 29.
Highly preferably, the polynucleotide of the invention has a percentage identity of at least 80% after optimum alignment with a sequence as defined in any one of the nucleotide sequences a) to e) defined above, preferably at least 90%, more preferably at least 98% and most preferably at least 100% identity.
The term “percentage identity” between two nucleic acid or amino acid sequences as used in the present invention means a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after best alignment, that percentage being purely statistical and the differences between the two sequences being randomly distributed and over their entire length. By “best alignment” or “optimum alignment”, it is meant the alignment at which the percentage identity determined as below is the highest. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having aligned them in an optimum manner, said comparison being carried out using comparison segments or windows to identify and compare local regions with sequence similarity. The optimum sequence alignment for comparison may be carried out manually or using a Smith and Waterman (1981) local homology algorithm, using the Neddleman and Wunsch (1970) local homology algorithm, using the Pearson and Lipman (1988) sequence similarity search method, or using software employing these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr, Madison, Wis.). To obtain an optimal alignment, the BLAST program is preferably used, with the BLOSUM 62 matrix. It is also possible to use PAM or PAM250 matrices.
The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences aligned in an optimum manner, the nucleic acid or amino acid sequence to be compared possibly comprising additions or deletions compared with the reference sequence for optimum alignment between these two sequences. The percentage identity is calculated by determining the number of identical positions for which the nucleotide or amino acid residue is identical between the two sequences, dividing this number of identical positions by the total number of compared positions and multiplying the result obtained by 100 to obtain the percentage identity between these two sequences.
The term “nucleic sequences having a percentage identity of at least 80%, preferably at least 90%, more preferably at least 98% after optimum alignment with a reference sequence”, is intended to designate nucleic sequences having, with respect to the reference nucleic acid, certain modifications, in particular a deletion, truncation, extension, chimeric fusion, and/or a substitution, in particular a point substitution, and for which the nucleic sequence has at least 80%, preferably at least 90%, more preferably at least 98% identity after optimum alignment with the reference nucleic sequence. Preferably, the specific hybridization conditions or high stringency conditions will be such that they ensure at least 80%, preferably at least 90%, more preferably at least 98% identity after optimum alignment between one of the two sequences and the complementary sequence of the other.
A hybridization under high stringency conditions means that the temperature and ionic strength conditions are selected so that they can maintain the hybridization between two complementary nucleic acid fragments. By way of illustration, the highly stringent conditions for the hybridization step with the aim of defining the nucleotide sequences described above are advantageously as follows.
DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) pre-hybridization at 42° C. for 3 hours in a phosphate buffer (20 mM, ph 7.5) containing 5×SSC (1×SSC corresponds to a solution of 0.15 M NaCl+0.015 M of sodium citrate), 50% formamide, 7% sodium dodecyl sulphate (SDS), 10× Denhardt's solution, 5% dextran sulphate and 1% salmon sperm DNA; (2) hybridization proper for 20 hours at a temperature depending on the probe size (i.e.: 42° C. for a probe size >100 nucleotides), followed by 2 washings of 20 minutes at 20° C. in 2×SSC+2% SDS, 1 washing of 20 minutes at 20° C. in 0.1×SSC+0.1% SDS. The final washing is carried out in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for a probe size >100 nucleotides. The high stringency hybridization conditions described above for a polynucleotide of a defined size may be adapted by the skilled person for larger or smaller oligonucleotides, as discussed by Sambrook et al, 1989.
The polypeptides and polynucleotides of the present invention may be prepared using any appropriate process. They may in particular be obtained by chemical synthesis, but it is also possible to obtain them via a biological route pathway, especially using different vectors in appropriate cell cultures. The peptides of the present invention may, if necessary, be in the deglycosylated or glycosylated form. A person skilled in the field of the invention will be able to obtain different polynucleotides/polypeptides and will also be able to determine which of the polynucleotides/polypeptides obtained have a suitable biological activity.
2. Recombinant Viral Vector
In a further aspect, the invention concerns a recombinant measles viral vector with a polynucleotide of the invention inserted into the genome thereof. Such vectors are prepared using methods which are routinely used by the skilled person and the resulting clones may be introduced into a suitable host using standard methods which are known to the skilled person.
In a preferred mode, the recombinant viral vector of the invention is advantageously a live measles (Schwarz strain) viral vector, such as those selected from the group of viral vectors constituted by those deposited at the CNCM under numbers I-3440, I-3442, I-3452, I-3453, I-3454, I-3455, I-3619, I-3620, I-3621, I-3622 and I-3623. The complete antigenome sequence of the measles Schwarz strain virus can, for example, be obtained from pTM plasmids of strains deposited under numbers I-3440 and I-3442.
The vector pTM-MVSchw2-[EDIII+M1-40]WNV(IS-98-ST1) was deposited at the CNCM (Paris, France) on 26 May 2005 under number 1-3440.
This vector is a pTM plasmid containing the complete antigenome of the measles virus, Schwarz strain, and an additional expression unit placed between the P and M genes containing the sequence of the domain III of the envelope E protein of West Nile virus (WNV IS-98-ST1) fused to the sequence 1-40 of the membrane protein.
The vector may be cultivated in 100 μ/ml LB ampicillin medium after inoculating small colonies.
It is preserved long-term by freezing at −80° C.
The vector pTM-MVSchw2-[EDIII+ApoptoM]DV-1(FGA89) was deposited at the CNCM (Paris, France) on 26 May 2005 under number I-3442.
This vector is a pTM plasmid containing the complete antigenome of the measles virus, Schwarz strain, and an additional expression unit placed between the P and M genes containing the sequence of the domain III of the envelope E protein of the dengue 1 virus, FGA89 strain, fused to the apoptotic sequence of the Membrane M protein.
The vector may be cultivated in 100 μ/ml LB ampicillin medium after inoculating small colonies.
It is preserved long-term by freezing at −80° C.
The vector pTRE2-SSCRT-ApoptoMden1/DIII-Hα1-2 was deposited at the CNCM (Paris, France) on 16 Jun. 2006 under number 1-3452.
This vector is a pTRE2 plasmid containing the sequence of the signal peptide of human calreticulin fused to the pro-apoptotic sequences of the M protein and to the ectodomains III of the E protein with the α-helice sequences of the dengue 1 and 2 viruses.
The sequence of the insert contained in the plasmid and constituting a polynucleotide of the invention is SEQ ID NO: 9 shown in FIG. 8.
The vector may be cultivated in 100 μ/ml LB ampicillin medium.
It is preserved long-term by freezing at −80° C.
The vector pTRE2-ssCRT-ApoptoMden1/DIII-Hα3-4 was deposited at the CNCM (Paris, France) on 16 Jun. 2006 under number I-3453.
This vector is a pTRE2 plasmid containing the sequence of the signal peptide of human calreticulin fused to the pro-apoptotic sequences of the M protein of dengue 1 virus and to the sequences of the ectodomains III and Hα of the dengue 3-4 viruses.
The sequence of the insert contained in the plasmid and constituting a polynucleotide of the invention is SEQ ID NO: 11 shown in FIG. 9.
The vector may be cultivated in LB ampicillin 100 μ/ml medium.
It is preserved long-term by freezing at −80° C.
The vector pTRE2-ssCRT-ApoptoMden1/DIII-Hα1-2-3-4 was deposited at the CNCM (Paris, France) on 16 Jun. 2005 under number I-3454.
This vector is a pTRE2 plasmid containing the signal peptide sequence of human calreticulin fused to the pro-apoptotic sequences of the M protein of dengue 1 virus and to the sequences of the ectodomains III with α helices (Hα) of the E proteins of the dengue 1, 2, 3, 4 viruses.
The sequence of the insert contained in the plasmid and constituting a polynucleotide of the invention is SEQ ID NO: 4 shown in FIG. 2.
The vector may be cultivated in 100 μ/ml LB ampicillin medium.
It is preserved long-term by freezing at −80° C.
The vector pTRE2-ssCRT-EctoM40den1/DIII-Hα1-2-3-4 was deposited at the CNCM (Paris, France) on 16 Jun. 2005 under number 1-3455.
This vector is a pTRE2 plasmid containing the signal peptide sequence of human calreticulin fused to the sequences of the ectodomain 1-40 of the M protein of the dengue 1 virus and to the sequences of the ectodomains III with α helices (Hα) of the E proteins of the dengue 1, 2, 3, 4 viruses.
The sequence of the insert contained in the plasmid and constituting a polynucleotide of the invention is SEQ ID NO: 17 shown in FIG. 12.
The vector may be cultivated in 100 μ/ml LB ampicillin medium.
It is preserved long-term by freezing at −80° C.
The vector pUC57-TetraDVA (introduced into an E. coli strain) was deposited at the CNCM (Paris, France) on 14 Jun. 2006 under number I-3619.
This vector is a pUC plasmid containing a nucleotide sequence optimized for the expression in mammalian cells of a tetrameric construct of the ectodomains III of the envelope E protein of the 4 serotypes (1, 2, 3, 4) of the dengue virus, fused to the ectodomain of the Membrane M protein.
The insert contained in the plasmid vector is shown in FIG. 25.
The strain containing the vector may be cultivated in 100 μ/ml LB ampicillin medium.
The vector pUC57-TetraDVB (introduced into an E. coli strain) was deposited at the CNCM (Paris, France) on 14 Jun. 2006 under number I-3620.
This vector is a pUC plasmid containing a nucleotide sequence optimized for the expression in mammalian cells of a tetrameric construct of the ectodomains III of the envelope E protein of the 4 serotypes (1, 2, 3, 4) of the dengue virus, fused to the ectodomain of the Membrane M protein.
The insert contained in the plasmid vector is shown in FIG. 26.
The strain containing the vector may be cultivated in 100 μ/ml LB ampicillin medium.
The vector pUC57-TetraDVC (introduced into an E. coli strain) was deposited at the CNCM (Paris, France) on 14 Jun. 2006 under number I-3621.
This vector is a pUC plasmid containing a nucleotide sequence optimized for the expression in mammalian cells of a tetrameric construct of the ectodomains III of the Envelope E protein of the 4 serotypes (1, 2, 3, 4) of the dengue virus, fused to the ectodomain of the Membrane M protein.
The insert contained in the plasmid vector is shown in FIG. 27.
The strain containing the vector may be cultivated in 100 μ/ml LB ampicillin medium.
The vector pUC57-TetraDVD (introduced into an E. coli strain) was deposited at the CNCM (Paris, France) on 14 Jun. 2006 under number I-3622.
This vector is a pUC plasmid containing a nucleotide sequence optimized for the expression in mammalian cells of a tetrameric construct of the ectodomains III of the Envelope E protein of the 4 serotypes (1, 2, 3, 4) of the dengue virus, fused to the ectodomain of the Membrane M protein.
The insert contained in the plasmid vector is shown in FIG. 28.
The strain containing the vector may be cultivated in 100 μ/ml LB ampicillin medium.
The vector pUC57-TetraDVE (introduced into an E. coli strain) was deposited at the CNCM (Paris, France) on 14 Jun. 2006 under number I-3623.
This vector is a pUC plasmid containing a nucleotide sequence optimized for the expression in mammalian cells of a tetrameric construct of the ectodomains III of the Envelope E protein of the 4 serotypes (1, 2, 3, 4) of the dengue virus, fused to the ectodomain of the Membrane M protein.
The insert contained in the plasmid vector is shown in FIG. 29.
The strain containing the vector may be cultivated in 100 μ/ml LB ampicillin medium.
3. Cloning and/or Expression Vectors and Antibodies
In a further aspect, the invention concerns any cloning and/or expression vector and any cell host (prokaryotic or eukaryotic) transformed by such a vector, and comprising regulation elements allowing the expression of the nucleotide sequence coding for a chimeric polypeptide of the invention. Such vectors are prepared using methods which are routinely used by the skilled person, and the resulting clones may be introduced into an appropriate host using standard methods such as, for example, lipofection, electroporation, thermal shock, transformation after chemical permeabilization of the membrane, or cell fusion.
The invention also encompasses host cells, in particular eukaryotic and prokaryotic cells, transformed by the vectors of the invention as well as transgenic animals, preferably mammals, with the exception of man, comprising one of said transformed cells of the invention. These animals may be used as models, to study the etiology of inflammatory and/or immune diseases, and in particular inflammatory diseases of the digestive tract, or in the study of cancers.
Of the cells which may be used in the present invention, the following may be cited: bacterial cells (Olins and Lee (1993), Curr Op Biotechnology 4: 520), but also yeast cells (Buckholz (1993), Curr Op Biotechnology 4, 538), as well as animal cells, in particular mammalian cell cultures (Edwards and Aruffo (1993), Curr Op Biotechnology 4, 558).
In the context of the present invention, the term “cloning and/or expression vector” refers to a polynucleotide construct designed to be transfected into different cell types. For this reason, these vectors encompass expression vectors designed for the expression of a nucleotide sequence in a host cell; cloning vectors designed for the isolation, propagation and replication of inserted nucleotides or shuttle vectors which comprise the attributes of more than one vector.
4. Polyclonal or Monoclonal Antibodies
The polypeptides and polynucleotides of the present invention may also be used to prepared polyclonal or monoclonal antibodies capable of binding (preferably in a specific manner) to at least one chimeric polypeptide/polynucleotide of the invention. The present invention thus also relates to such purified antibodies which may be obtained by very well known techniques such as, for example, the technique described by Kolher and Milstein (continuous cultures of fused cells secreting antibody of predefined specificity, Nature (1975), 262: 495-497). In a preferred embodiment of the invention, the antibodies are of the “humanized” type. A person skilled in the art would be able to use his general knowledge to prepare these types of antibodies.
5. Methods and Use
In a further aspect, the invention concerns the prevention and/or treatment of a Flaviviridae infection in a sensitive species. More particularly, the invention relates to the use of a recombinant viral vector of the invention for the preparation of an immunogenic composition intended for the prevention or treatment of a Flaviviridae infection in a sensitive species. The term “sensitive species” means any animal which is susceptible to a Flaviviridae infection, for example a human being.
The invention also relates to a method for preventing and/or treating a Flaviviridae infection in a sensitive species, comprising administering a pharmaceutically effective amount of at least one of the following elements:

    
    
        a chimeric polypeptide of the invention;
        a polynucleotide of the invention;
        a recombinant viral vector of the invention;
        an antibody of the invention; and
        a cloning and/or expression vector of the invention.
    
    


The term “Flaviviridae infection” means, for example, flaviviroses such as dengue, yellow fever, Japanese encephalitis and West Nile fever. The means for preparing and administering the elements of the present invention will not be described in more detail as they are already known to the skilled person.
6. Compositions
The present invention also concerns immunogenic compositions useful in the prevention and/or treatment of a Flaviviridae infection. The term “immunogenic composition” means a composition which contains elements having the capacity to induce, in vivo or in vitro, a cellular and/or humoral type immune response.
In a preferred embodiment, the composition of the present invention further contains a pharmaceutically acceptable vehicle and an element selected from the group constituted by:

    
    
        a polynucleotide of the invention;
        a chimeric polypeptide of the invention;
        a recombinant viral vector of the invention;
        an antibody of the invention; and
        a cloning and/or expression vector of the invention.
    
    


The compositions of the present invention may be in any solid or liquid form which is normal for pharmaceutical administration, examples of forms of administration being a liquid, a gel, or any other support which can allow controlled release, for example. Examples of compositions which may be used which may be cited are compositions which can be injected into human beings.
The compositions of the invention may also comprise components which increase or are capable of increasing the immunogenicity of the chimeric polypeptides of the invention, in particular other immunogenic peptides, specific or non-specific immunity adjuvants such as alun, QS21, Freund's adjuvant, SBA2 adjuvant, montanide, polysaccharides or equivalent compounds.
A person versed in the art will be able to prepare pharmaceutically acceptable compositions and to determine, as a function of several factors, the preferred mode of administration and the amount which has to be administered. Factors which may influence the choice include: the nature of the treatment, the exact nature of the ingredients, active or non active, in the composition; the stage of the disease; the condition, age and weight of the patient, etc.
EXAMPLES
The following examples demonstrate other characteristics and advantages of the present invention and serve to illustrate rather than limit the scope of the present invention. Modifications and variations may be made without departing from the spirit and scope of the invention. Although other methods or products equivalent to those discussed below may be used to test or implement the present invention, preferred equipment and methods are described.
Example 1
The Recombined Measles Virus MVSchw-[EDIII+M1-40]DV-1 as a Prototype for a Candidate Vaccine Against Dengue (PTR156)
Described in FIGS. 1 and 2, two immunogenic constructs based on the one hand on the capacity of domain III of the envelope E glycoprotein of the dengue virus to induce neutralizing antibodies and on the other hand on the involvement of the ectodomain of the membrane M protein (M1-40) in viral pathogenicity (apoptosis) were inserted into the genome of the attenuated measles virus (Schwarz strain) (MVSchw). The viral sequences were under the dependency of the signal peptide of calreticulin to allow them to be targeted into the secretion pathway. The expression of the [EDIII]DV-1 and [EDIII+M1-40]DV-1 sequences by the recombined viruses MVSchw was verified in infected Vero cells by indirect immunofluorescence using a polyclonal HMAF mouse serum directed against DV-1 (FIG. 3). Secretion of the EDIII and EDIII antigenic domains fused to the M1-40 sequence (with the intercalating pentapeptide RRDKR) of the dengue virus, type-1 (DV-1) of the FGA/89 strain (Holmes E C et al, Mol Biol Evol 16(3): 405-409, 1999 and Desprès P et al, Virology 196: 209-219) was observed by Western blot analysis in the supernatants of cells infected with the MVSchw-[EDIII]DV-1 and MVSchw-[EDIII+M1-40]DV-1 virus respectively (FIG. 4). Similarly, the production and secretion in the culture supernatants of a S2/[EDIII+M1-40]DV-1 drosophila cell line induced for expression of these proteins has been demonstrated (FIG. 5).
Two groups of four adult CD46/FNAR mice were immunized with 104 TCID50 of each of the recombined MVSchw viruses (Tables 1 and 2). The empty MVSchw virus was used as a negative control. We determined the production of antibodies directed against the MVSchw virus (MV Ag) and the DV-1 virus (DV-1 Ag) including those which were specific to EDIII (Thullier P et al, 2001, J Gen Virol 82: 1885-1892) and neutralizing (Anti-DV -1 FRNT75) in immunized mice (Tables 1 and 2). We observed that the MVSchw-[EDIII]DV-1 virus is less effective at producing specific antibodies to DV-1 or EDIII alone after two inoculations spaced by one month (Table 1). Using the same immunization protocol, we observed that the MVSchw-[EDIII+M1-40]DV-1 virus induced significant titers of antibodies directed against DV-1 and against EDIII alone (Table 2). Anti-DV-1 antibodies including those which were neutralizing were still detected four months after the end of immunization.
When mice immunized more than 6 months previously with MVSchw-[EDIII+M1-40]DV-1 were given a booster with a single dose of 5 μg of total proteins of a concentrate of supernatant of S2/[EDIII+M1-40]DV-1 drosophila cells induced for expression of the fusion protein [EDIII+M1-40]DV-1 and in the presence of Alugel as an adjuvant, large amounts of anti-DV-1, anti-EDIII and neutralizing DV-1 antibodies were observed, which means that there was a well established humoral memory response in the vaccinated animals (Table 2). The anti-DV-1 antibodies were still present several months after the antigenic booster (Table 2).
Three months after the antigenic booster with r[EDIII+M1-40]DV-1, mice immunized with MVschw-[EDIII+M1-40]DV-1 were inoculated intraperitoneally with 107 FFU of FGA/NA dld strain DV-1 (DV-1 is responsible for an asymptomatic infection in the IFNAR mouse). Large titers of anti-DV-1 antibody (in particular directed against EDIII) including those which neutralized DV-1 Hawaii strain (neutralizing titer # 4000) were observed after 3 weeks of viral challenge (Table 2). It should be noted that mice immunized 9 months previously with MVSchw-[EDIII+M1-40]DV-1 (this construct not inducing anti-EDIII antibody or neutralizing DV-1 antibody) then challenged intraperitoneally with 107 FFU of FGA/NA did strain DV-1 produced anti-DV-1, anti-EDIII and neutralizing titers of 20000, 5000 and 80 respectively; these were values equivalent to those observed in BALB/c mice or IFNAR mice challenged under the same experimental conditions.
In conclusion, the fusion sequence [EDIII+M1-40]DV-1 secreted by the MVSchw-[EDIII+M1-40]DV-1 virus is capable of generating neutralizing anti-DV-1 antibodies and of inducing a long term humoral memory response which is effectively stimulated on the one hand by the soluble antigen r[EDIII+M1-40]DV-1 and on the other hand in response to a viral infection. In contrast, the only antigenic EDIII domain secreted by the MVSchw-[EDIII+M1-40]DV-1 virus is of low immunogenicity in CD46+-FINAR mice. Our complementary work demonstrates that the pro-apoptotic ApoptoM (M32-40)peptide at the C-terminal end of M determines the immunogenic power of the [EDIII+M1-40]DV-1 fusion sequence since the MVSchw-[EDIII+M1-40]DV-1 virus without ApoptoM induces a production of anti-DV-1 antibody equivalent to that obtained after immunization with the MVSchw-[EDIII]DV-1 virus.
As a general methodology for pediatric vaccination against dengue, we propose to immunize young individuals with the MVSchw-[EDIII+M1-40]DV-1 virus using a double inoculation spaced by one month, then to restimulate them later with the soluble r[EDIII+M1-40]DV-1 antigen as a vaccine booster or prophylactically against the risk of an infection by the dengue virus. This immunization strategy is being validated for the four serotypes of dengue based on an antigen composed of a tetramer of the four EDIII domains of DV-1, -2, -3 and -4 fused to the cytotoxic ApoptoM sequence.
Our first experimental results underline the importance of a reduced size immunogen derived from the envelope protein of the DV-1 virus in combination with the immunostimulating capacity of the live measles vector, a strategy which allows the induction of a neutralizing humoral response which is effective against the dengue virus. They define a proof of concept for the design of the tetrameric constructs of the antigenic domains of DV which will allow simultaneous and long term immunization against the four dengue serotypes.
Example 2
Construction of Chimeric Polypeptides Optimized for Expression in Mammals
In the present example, the inventors have developed novel antigenic constructs useful against a Flaviviridae infection.
These novel chimeric polypeptides are based on an antigen composed of a tetramer of the four EDIII domains of the four serotypes of the dengue virus, fused together and to one or the other of the cytotoxic apoptoM sequences shown in FIG. 30.
Thereafter, these chimeric polypeptides optimized for expression in a mammal were inserted into the genome of the attenuated measles virus, Schwarz strain, (MVSchw) viral strain. FIGS. 15 to 29 show the amino acid sequences of five preferred chimeric polypeptides and the polynucleotides encoding them.
These chimeric polypeptides were constructed using the following optimization conditions:
Optimization information for these sequences:

    
    
        (Cai No=0.806, mean % GC=53.46, GC distribution: homogeneous about 50%);
        elimination of internal “TATA box” sequences, a portion rich in AT or GC, elements with sequences ARE, INS and CRS, repeat sequences, sequences with secondary RNA structure, cryptic splicing sequences;
        elimination of the following restriction sites: NheI, BamHI, XhoI, EcoRI, KpnI, SalI, BspEI, BgIII, NotI, BssHII, BsiWI, with the exception of BsiWI in the first position and BssHII in the last position;
        Elimination of TTTT, TTTAA, AAAGGG, AAAAGG, GGGAAA, GGGGAA motifs and their complements TTCCCC, TTTCCC, CCTTTT, CCCTTT.
    
    









TABLE 1







Antibody response directed against the MVSchw virus in CD46


+/IFNAR mice inoculated i.p. with MVSchw-[EDIII]DV-1











Immunization
MV Ag
DV-1 Ag
DV-1 rEDIII
Anti-DV-1


(month)
titerc
titerd
titere
FRNT75f














1a
15000
<100
<100
ND


2b
40000
100
10
<10





a104 TCIP50 of MV-DV-1 [EDIII + M1-40]DV1was given i.p. to four adult mice;


bThe individuals received a booster injection with 104 TCIP50 of MV-DV-1 [EDIII + M1-40]DV-1;


cDetermined by ELISA (Trinity Biotech) on pooled serums inactivated by heating;


dDetermined by ELISA on pooled serums inactivated by heating. Microtitration plates were coated with 5 × 105 FFU of purified sucrose, FGA/NA d1d as viral antigen;


eDetermined by ELISA on pooled serums inactivated by heating. Microtitration plates were coated with 50 ng of highly purified recombinant DV-1 EDIII as viral antigen;


fAnti-DV-1 neutralization antibodies were detected using a focus reduction neutralization test (FRNT). Pooled serums inactivated by heating were incubated with the DV-1 Hawaii line and titrating the virus on Vero cells using the plaque immunoassay test. FRNT75, the highest serum dilution tested, reduced the number of FFU by at least 75%.













TABLE 2







Antibody response directed against the MVSchw virus in CD46+/IFNAR


mice inoculated i.p. with MVSchw-[EDIII + M1-40]DV-1











Immunization
MV Ag
DV-1 Ag
DV-1 rEDIII
Anti-DV-1


(months)
titere
titerf
titerg
FRNT75h














 1a
15000
<100
≦100
ND


 2b
40000
1600
400
10


3
30000
1000
600
10


6
20000
500
100
40


 7c
20000
20000
100000
800


9
10000
2000
10000
100


10d 
10000
200000
800000
4000





a104 TCIP50 of MV-DV-1 [EDIII + M1-40]DV1was given i.p. to four adult mice;


bThe individuals received a booster injection with 104 TCIP50 of MV-DV-1 [EDIII + M1-40]DV-1;


cThe immunized mice received a booster injection of 5 μg of total secreted proteins deriving from a supernatant of S2 cells expressing rDV1[EDIII + M1-40]DV-1 in the presence of Alugel adjuvant;


dThe immunized mice were inoculated i.p. with 107 FFU of FGA/NA d1d from the DV-1 line for three weeks;


eDetermined by ELISA (Trinity Biotech) on pooled serums inactivated by heating;


fDetermined by ELISA on pooled serums inactivated by heating. Microtitration plates were coated with 5 × 105 FFU of purified sucrose, FGA/NA d1d as viral antigen;


gDetermined by ELISA on pooled serums inactivated by heating. Microtitration plates were coated with 50 ng of highly purified recombinant DV-1 EDIII as viral antigen;


hAnti-DV-1 neutralization antibodies were detected using FRNT. Pooled serums inactivated by heating were incubated with the DV-1 Hawaii line and titrating the virus on Vero cells using the plaque immunoassay test. FRNT75, the highest serum dilution tested, reduced the number of FFU by at least 75%.

## Claims

1. A chimeric polypeptide comprising a subdomain of the E protein of Flaviviridae bound to a subdomain of the membrane M protein of Flaviviridae;
wherein said chimeric polypeptide comprises an amino acid sequence selected from the group consisting of:
amino acids 19 to 162 of SEQ ID NO: 3;
amino acids 21 to 168 of SEQ ID NO: 6;
amino acids 21 to 132 of SEQ ID NO: 12;
amino acids 18 to 624 of SEQ ID NO: 20;
amino acids 18 to 609 of SEQ ID NO: 21;
amino acids 18 to 624 of SEQ ID NO: 22;
amino acids 18 to 489 of SEQ ID NO: 23; and
amino acids 21 to 474 of SEQ ID NO: 24.

2. The chimeric polypeptide according to claim 1, wherein the subdomain of the E protein consists of the ectodomain III selected from the group consisting of:
amino acids 19 to 119 of SEQ ID NO: 3;
amino acids 21 to 123 of SEQ ID NO: 6; and
amino acids 21 to 121 of SEQ ID NO: 12.

3. The chimeric polypeptide according to claim 1, wherein the subdomain of the E protein consists of a dimer of the ectodomain III of the dengue virus serotypes 1, 2, 3 or 4.

4. The chimeric polypeptide according to claim 1, wherein the subdomain of the E protein consists of a tetramer of the ectodomain III of the dengue virus serotypes 1, 2, 3, or 4.

5. The chimeric polypeptide according to claim 1, wherein the subdomain of the M protein consists of the ectodomain 1-40 of the M protein.

6. The chimeric polypeptide according to claim 1, wherein the subdomain of the M protein consists of an apoptoM sequence.

7. The chimeric polypeptide according to claim 1, further comprising a binding segment binding the subdomain of the E protein to the peptide of the subdomain of the M protein.

8. The chimeric polypeptide according to claim 7, wherein the binding segment is a pentapeptide having the amino acid sequence RRDKR (SEQ ID NO: 33) or RREKR (SEQ ID NO: 34).

9. The chimeric polypeptide according to claim 1, wherein the Flaviviridae is selected from the group consisting of West Nile virus, dengue virus, Japanese encephalitis virus and yellow fever virus.

10. An immunogenic composition comprising the chimeric polypeptide of claim 1 and a pharmaceutically acceptable vehicle.

11. A method for immunizing a subject, comprising administering a pharmaceutically effective amount of the chimeric polypeptide of claim 1 to a subject in need thereof.

12. The method according to claim 11, further comprising administering a booster immunization of a chimeric polypeptide comprising a subdomain of the E protein of Flaviviridae bound to a peptide of a subdomain of the membrane M protein of Flaviviridae in the presence of an adjuvant.

13. The method according to claim 11, wherein the Flaviviridae is selected from the group consisting of a dengue virus, yellow fever virus, Japanese encephalitis virus and West Nile fever virus.

14. The chimeric polypeptide of claim 1 wherein the polypeptide comprises amino acids 19 to 162 of SEQ ID NO: 3.

15. The chimeric polypeptide of claim 1 wherein the polypeptide comprises amino acids 21 to 168 of SEQ ID NO: 6.

16. The chimeric polypeptide of claim 1 wherein the polypeptide comprises amino acids 21 to 132 of SEQ ID NO: 12.

17. The chimeric polypeptide of claim 1 wherein the polypeptide comprises amino acids 18 to 624 of SEQ ID NO: 20.

18. The chimeric polypeptide of claim 1 wherein the polypeptide comprises amino acids 18 to 609 of SEQ ID NO: 21.

19. The chimeric polypeptide of claim 1 wherein the polypeptide comprises amino acids 18 to 624 of SEQ ID NO: 22.

20. The chimeric polypeptide of claim 1 wherein the polypeptide comprises amino acids 18 to 489 of SEQ ID NO: 23.

21. The chimeric polypeptide of claim 1 wherein the polypeptide comprises amino acids 21 to 474 of SEQ ID NO: 24.