EP0861324A1 - Polyvalent anti-dengue vaccine - Google Patents
Polyvalent anti-dengue vaccineInfo
- Publication number
- EP0861324A1 EP0861324A1 EP96939121A EP96939121A EP0861324A1 EP 0861324 A1 EP0861324 A1 EP 0861324A1 EP 96939121 A EP96939121 A EP 96939121A EP 96939121 A EP96939121 A EP 96939121A EP 0861324 A1 EP0861324 A1 EP 0861324A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- dengue
- protein
- dengue virus
- polypeptide
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
- C07K14/18—Togaviridae; Flaviviridae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2770/24011—Flaviviridae
- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
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Definitions
- the present invention relates to a vaccine in polyvalent units against viruses of the family of Flaviviridiae and in particular against flaviviruses such as Dengue viruses, yellow fever, Japanese encephalitis, or other Flaviviridiae, or "Flavi- like "such as hepatitis C virus, and mixtures thereof (18)
- Flaviviruses are viruses of the arbovirus family (old group B arboviruses). They are vertebrate viruses generally transmitted by hematophagous arthropods (mosquitoes, ticks, sandflies), in which they multiply before transmission. so intervene an arthropod vertebrate and a reservoir Some arboviruses can affect humans and induce pathologies, the arboviruses which are found especially in tropical regions If there has been a vaccine against yellow fever since 1930, or more recently against encephalitis Japanese and tick, the other flaviviruses currently have no vaccine, this is particularly the case for Dengue
- the virus responsible for Dengue spreads concomitantly with its vector, the mosquito Aedes aegypti
- the disease is characterized by a high fever accompanied by headache, nausea and joint and muscle pain which disappears after a few days Symptoms may, however, intensify leading to hemorrhagic manifestations which are sometimes fatal when the disease is associated with hypovolemic shock, especially in children
- the agent responsible for Dengue is an RNA flavivirus from the Flavivmdae family, just like the yellow fever virus or that of Japanese encephalitis We know 4 serotypes Dengue hemorrhagic fever could be a consequence of a poorly understood immunological phenomenon, probably involving a cascade of e cytokines linked to the induction of cytotoxic cellular immunity which may result from sequential infection by several serotypes.
- a first vaccine against Dengue is currently being tested. It is a vaccine based on 4 living serotypes, attenuated by successive passages of the virus in cell culture.
- the major drawback of this type of vaccine is the possibility of attenuated viruses reversing towards virulence, by genetic reversion or recombination between genomes. It also presents a potential risk for newborns and for immunocompromised people. In addition, some children vaccinated with the tetravalent vaccine seem not to be protected against the 4 serotypes.
- the structure of the genome is the amino acid sequence of the Dengue virus, in particular that of serotype 2, has been described by
- the Dengue virus is an enveloped RNA virus of about 50 nanometers. Like the yellow fever virus, all viral proteins are coded by a segment of approximately 10 ⁇ e kilobases, the structure of which is as follows:
- the structural proteins are C (capsids), pre-matrix / matrix (preM / M) and the envelope protein (E).
- proteins are encoded by the 5 'fragment, followed by non-structural proteins (NS) encoded by the rest of the genome. Proteins are produced by a proteolytic process induced by cellular signalase and other cellular and viral proteinases.
- the envelope glycoprotein (E) is the majority surface protein.
- the structure of sterotype 1 has been described by Mason et al. (2).
- Serotype 2 has been described in (1), and is the best known of the 4 serotypes; type 3 serotype has been described by Osatomi K. al. (3) and the type 4 serotype has been described by Zhao et al., (4).
- baculoviruses are very attractive for this type of production thanks to the high level of genetic expression they can achieve and for their absence of pathogenicity for humans.
- Baculovirus has already been used for the expression of flavivirus proteins. Dengue serotype 1 (5), Dengue serotype 4 (DEN-4) (6) and serotype 2 (DEN-2) have been described. (7), (8), (9) and serotypes 2 and 3 have been described by C. Delenda et al. (10).
- sequence homology between the 4 genomes corresponding to the 4 serotypes of the virus is relatively large; by way of example, protein E of these viruses has a percentage of homology as presented in table 1 below:
- GST glutathione-S-transferase
- the present invention results from the discovery that a polypeptide or a glycopeptide consisting of a polypeptide or glycoprotein derived from a protein of a Flaviviridiae, namely the Dengue virus in which a deletion of its carboxylated end has been substituted by an amino acid residue chosen from histidine, tryptophan or cysteine exhibited a much immunogenic character more important than the same polypeptide or glycopeptide not having this residue of added amino acids, this residue having the effect of improving the antigen presentation and consequently of inducing a cellular and humoral response much more important than in the absence of this residue
- polypeptide must be understood in the broad sense, that is to say including sequences of at least 15 amino acids comprising or not glycosylated units or glycohpids, and which whether its structure, primary, secondary or tertiary
- the present invention relates more generally to polypeptides, with increased immunogenic power, derived from Flaviviridiae, characterized by the existence of an amino acid residue at their carboxy-terminal end, which amino acid residue contains from 2 to 8 amino acids selected from histidine, tryptophan or cysteine
- the amino acid residue can be homogeneous in the sense that it consists of 2 to 8 histidines, 2 to 8 tryptophanes or 2 to 8 cysteines it can also consist of a mixture of 2 or 3 of these amino acids, it may finally contain a few amino acids other than the histidme, tryptophan or cysteine as long as their number does not exceed 25% of the residue
- the present invention relates to such immunogenic polypeptides, consisting of a polypeptide derived from protein E, from protein preM, from protein NS1 or NS3 of the Dengue virus and in which for protein E the carboxyl end has been deleted from 70 to 105 amino acids to be substituted by a residue of 2 to 8 amino acids chosen from histidine, cysteine or tryptophan II can be the same for the preM protein deleted from 30 to 50 amino acids in its carboxy-terminal end for be substituted by the same type of residue For the other proteins this deletion is not necessary, and the polypeptide of the invention derived from NS1 or NS3 comprises a residue of 2 to 8 amino acids chosen from histidm, cysteine or tryptophan
- the polypeptide is derived from protein E and the deletion is approximately 100 amino acids, if in addition, the amino acid residue contains 2 to 8 histidines, this gives the peptide the property of binding to cations divalent or trivalent or equivalent
- the polypeptides or peptides of the invention can be derived from each of the 4 serotypes of Dengue, knowing that the percentage of homology between the 4 serotypes is high and is between 67% and 80% for the protein E as indicated in table 1 above
- the present invention also relates to compositions consisting of mixtures of polypeptides of the invention. These mixtures can constitute an active principle of a polyvalent vaccine, either against different serotypes of the same virus, such as Dengue, or against different viruses of the same family. Flaviviridiae or Flavi-hke and likely to affect the same populations
- the present invention also relates to a composition capable of stimulating the immunity of individuals liable to be affected by the Dengue virus, characterized in that it contains at least 3 polypeptides or glycopeptides of the invention
- a preferred composition will be that containing at least one polypeptide of the invention derived from a Dengue protein originating from each of the 4 serotypes, protein E is a good candidate it has an essential function in viral infectivity and is the intermediary between the protein and cell receptors, protein E also appears to be a key target of the protective immune response in infected hosts insofar as it induces neutralizing antibodies and an immunocellular response
- composition of the invention could be a mixture of 3 or 4 polypeptides or glycopeptides described above, part of which is derived from protein E of each of
- each peptide or glycopeptide is present in a proportion of 10 to 50% in weight relative to all the peptides of the composition, and preferably a substantially equal percentage for each of the peptides
- the immunogenic compositions of the invention may also consist of a mixture of polypeptides derived from protein E, preM, NS1 or NS3 and comprising at least 3 antigens of 3 subtypes of the Dengue virus, each being provided with the amino acid residue at the carboxy-terminal end.
- the invention relates to immunogenic compositions consisting of a mixture of polypeptides or glycopeptides derived from protein E, preM, NS1 or NS3 of the Dengue virus and comprising at least 3 antigens of 3 serotypes of this virus and more generally an antigen corresponding to each of the known serotypes, not necessarily comprising the amino acid residue at their carboxy-terminal end, but characterized in that each of the peptides, polypeptides or glycopeptides has the capacity to induce an immune reaction in humans. 'host
- composition according to the invention may also contain polypeptides, glycopeptides or proteins originating from other flaviviruses such as those of yellow fever from Japanese encephalitis, from encephalitis with ticks or hepatitis C or more generally other Flaviviridiae haemorrhagic fevers present in the tropical zones of Africa, Asia or America, as well as if necessary from other viral, bacterial or parasitic antigens , from the moment they are provided with the amino acid residue as defined above at their carboxy-terminal end
- the invention also relates to the recombinant nucleic acid sequences coding for immunogenic peptides of Flaviviridiae and in particular of the Dengue virus, deleted in 3 'from 210 to 315 nucleotides, to which has been added in 3' a sequence coding for 2 with 8 amino acids chosen from histidme, tryptophan or cysteine in the sense defined above
- the nucleic acid sequences according to the invention more particularly code for glycopeptides derived from protein E of Dengue serotype 1, 2 , 3 or 4, and the sequence added in 3 ′ preferably codes for a sequence containing 6 histidines
- the invention also relates to nucleic acid vectors suitable for use in the expression of exogenous genes in a eukaryote, these vectors carry , in addition to a nucleic acid sequence as described above, a signal sequence homologous or heterologous to that of the encoded protein, the set being dependent on a promoter capable of being activated in said euk
- Recombinant baculoviruses can be obtained by various techniques known to those skilled in the art, but a particularly advantageous technique is that of homologous recombination using shuttle vectors between E Coli and the baculovirus.
- Expression vectors as obtained by this technique, and described in more detail in the experimental part below, have been deposited at the CNCM, these are
- a recombinant baculovirus deposited at the CNCM under the number 1-1624, on October 11, 1995 and carrying a sequence coding for a peptide constitutes the E gene of the Dengue virus serotype No. 4 in which the COOH end has been deleted from 100 amino acids and substituted by 6 histidines,
- the invention also relates to compositions capable of inducing an immune, cellular or humoral response in a host capable of being infected with the Dengue virus and comprising a mixture of nucleic acids coding respectively for the polypeptides of the invention, namely those derived from the Dengue protein, such as protein E, preM, NS1 or NS3, and carrying at their carboxy-terminal end a residue of 2 to 8 amino acids chosen from histidm, cysteine or tryptophan, or more generally coding for polypeptides from other Flaviviridiae such as yellow fever, Japanese encephalitis, tick-borne encephalitis or other "Flav ⁇ -l ⁇ e" such as the hepatitis C virus
- Flaviviridiae such as yellow fever, Japanese encephalitis, tick-borne encephalitis or other "Flav ⁇ -l ⁇ e”
- hepatitis C virus it has been shown that injection into animals d a plasmid containing the sequences necessary for the expression of proteins or
- the invention also relates to polyvalent subunit vaccines against the Dengue virus in particular against the 4 serotypes of said virus and comprising an immunogenic composition as described above, with, where appropriate, a vaccination adjuvant which may in particular be an adjuvant carrying divalent or valent ions such as aluminum hydroxide or calcium phosphate or an adjuvant of Freund's adjuvant type, a derivative of muramylpeptide or an "iscom" (Immunostimulating Complex), (19) L ' immunogenic effect of the compositions of the invention may be the consequence of uptake of the polypeptides or glycoproteins of the invention by divalent or trivalent ions and the possible formation of multimers which would have an increased immunogenic power compared to the polypeptides derived from Dengue and already described in the literature (references 3, 7, 8, 9)
- the subunit vaccines of the invention may also consist of compositions capable of inducing an immunogenic reaction against other Flaviviridiae They may be intended for the prophylaxis or therapy of infection by one or more viral species simultaneously
- FIG. 1 represents the electrophoretic profile of the recombinant polypeptides E DEN1 to DEN 4 secreted in the supernatant of the infected Sf9 cells
- the radioimmunoprecipitated proteins were treated with endoglycosidase F (F) or H (H) or not treated (O)
- F endoglycosidase F
- H H
- O not treated
- the preparation of the E genes was carried out by reverse transcription / polymerase chain reaction (RT / PCR) from the RNA extracted from Aedes pseudoscutella ⁇ s AP61 mosquito cells infected with the various viruses
- RT / PCR reverse transcription / polymerase chain reaction
- the ohgonucleotide primer located 5 ′ of the gene to be amplified has a restriction site (except for Dengue 2), the ATG initiation codon FOLLOWED by nucleotides coding for the first 6 amino acids among the 15 corresponding to the signal sequence of each protein
- the ohgonucleotide primer located 3 ′ of the gene to be amplified has the sequence corresponding to 6 amino acids of protein E of the virus followed by 6 codons for the histidme, a stop codon and a restriction site
- La oligonucleotide sequence is as shown below 5 'end (positive direction)
- the fragments amplified from viral RNA extracted from Aedes pseudoscutellaris AP61 cells were then cloned into the shuttle vector pVL-poly (17).
- E ⁇ 100H ⁇ s6 corresponds to protein E of serotype 1, 2, 3, 4 deleted by 100 amino acids at its carboxyl end and to which 6 histidine residues have been added
- the genes coding for the proteins D. (1, 2,3 or 4) E ⁇ 100 His6 and their signal sequence are thus placed under the control of the polyhedrin promoter.
- the number of nucleotides between the end of the promoter and the initiation codon is 42 (Dengue 1, Dengue 3,
- Dengue 4 Dengue 4 or 50 (Dengue 2) nucleodides.
- the recombinant baculoviruses Ac. D (1, 2,3 or 4) E ⁇ 100H ⁇ s6 were obtained by homologous recombination in Spodoptera frugiperda Sf9 cells between the shuttle vectors pVL.D (1, 2,3 or 4) E ⁇ 100H ⁇ s6 and the linearized bacRovirus AcRP23lacZ at the unique site Bsu36l Les recombinant baculoviruses were selected by 3 successive cloning by the beach method. 3 ° Structural characteristics of the recombinant proteins obtained:
- Figure 1 shows the electrophoretic profile of the 4 recombinant proteins corresponding to Dengue viruses 1, 2, 3 or 4.
- the profile of proteins treated with endoglycosidase F or H indicates that they are all glycosylated, probably with only one of the two glycosylation sites because the difference in molecular weight does not exceed 4000 daltons
- Immunogens The viral proteins with a concentration of 5 ⁇ g of pure protein are mixed with 100 ⁇ g of aluminum hydroxide (Alu-gel-S, Serva) and inoculated into SWISS mice by mtrapé ⁇ tonéale o
- the viral strains of Dengue having served to immunization are titrated on AP61 cells (detection of foci by immunocytochemistry) and inoculated at the rate of 10 3 plaque-forming particles (PFU) per mouse
- the control antigen injected into the mice corresponds to a peptide containing the last three amino acids of protein E (KKG) followed of six histidines (KKG H ⁇ s6)
- the inoculum of each injection contains 100 ⁇ g of peptide mixed with Alu-gel
- mice Three-week-old mice are vaccinated by an intracourt ⁇ tonéale injection of a mixture of products corresponding to the 4 serotypes of Dengue (DE ⁇ 100H ⁇ s6 or virus) at 3 weeks, 4 weeks and 6 weeks, then bled individually one week after the last injection
- the vaccinating peptide of the negative control is identical for the 4 serotypes
- the neutralizing antibodies of these sera are titrated against each homologous virus
- the sera are also used for the protection test after passive transfer to the mouse Neutralization test:
- Antibodies vaccinated mice are titrated by seroneutralisatio ⁇ in 24-well plates with 50 PFU of homologous virus on Vero cells
- the neutralizing antibody titer corresponds to the dilution neutralizing 80% of the foci of virus detected by immunocytochemistry using antibodies anti-viral proteins and anti-mouse IgG conjugate labeled with peroxidase
- Mouse protection test 100 LD50 of virus are inoculated into groups of 5-day-old mice that received 0.05 ml miterperitoneal injection the day before of immunized mouse serum (passive transfer) Each group of passive transfer mice is subjected to a test dose by one of the 4 virus serotypes The sou ⁇ s are observed for ⁇ _. weeks and signs of paralysis or death are noted
- the neutralization titer was measured on sera taken at 7 weeks after the 3 antigen injections
- the neutralization titer corresponds to the reverse of the dilution of serum leading to an 80% reduction in focal units (UFF) of the homologous virus strain on VERO cells Viruses counterparts were used for the neutralization and challenge test
- mice were immunized with the tetravalent mixture in the 3 cases of figures
- This mixture can therefore constitute the basis of a composition or of an active principle of a vaccine in polyvalent units against Dengue
- mice Groups of 10 three-week BALB / c mice were immunized on days D1, D7 and D21 using 5 ⁇ g of recombinant antigen coupled to 100 ⁇ g of aluminum hydroxide taken as an adjuvant. The mice were bled on day D28 and tested in seroneutralization with respect to 200 Home Formative Units of each of the reference strains (Dengue 1: Hawai 1944; Dengue 2: New Guinea C, 1944;
- Recombinant proteins from serotypes D2, D3 and D4 therefore appear to be particularly advantageous and the titer of neutralizing antibody in monkey serum vaccinated with a purified recombinant envelope protein was compared with that obtained with an attenuated virus; the results are presented in table 5 below.
- the preparations were injected on day D0 (A and E) or on days D0, D28, D56 after sampling the plasma. o
- the monkeys were then subjected to a test dose on day D83 by intradermal injection of 2 ⁇ 10 5 infectious particles of dengue virus 2, strain Jamaica 1983. The plasmas were then collected on D98.
- the monkeys receive three inoculations of 100 ⁇ g of adjuvant peptides with 1 mg of aluminum adjuvant (group B) monthly.
- the sera are collected four weeks after the last inoculation and tested against their neutralizing antibody against 200 plaque-forming units (PFU) of the homologous strain of Dengue 2 Janaque.
- the titles correspond to the dilutions of serum which neutralize 50% of the plates.
- Table 5 indicates the specificity of induction of neutralizing antibodies of the recombinant protein resulting from serotype 2 of Dengue in cynomolgus monkeys, with a response at least four times more important than that obtained with the attenuated virus
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Abstract
Polyvalent subunit vaccine against the Flaviviridiae particularly against the Dengue virus, comprising recombinant peptides issued from each serotype, of which the carboxylated extremity is substituted by a peptide containing from 2 to 8 histidines, tryptophanes or cysteines.
Description
VACCIN POLYVALENT ANTI-DENGUE VERSATILE ANTI-DENGUE VACCINE
La présente invention est relative à un vaccin sous unîtes polyvalent contre des virus de la famille des Flaviviridiae et notamment contre des flavivirus tels les virus de la Dengue, de la fièvre jaune, de l'encéphalite japonaise, ou autres Flaviviridiae, ou « Flavi-like » comme le virus de I' hépatite C, ainsi que des mélanges de ceux-ci (18)The present invention relates to a vaccine in polyvalent units against viruses of the family of Flaviviridiae and in particular against flaviviruses such as Dengue viruses, yellow fever, Japanese encephalitis, or other Flaviviridiae, or "Flavi- like "such as hepatitis C virus, and mixtures thereof (18)
Les flavivirus sont des virus de la famille des arbovirus (anciens arbovirus du groupe B) Ce sont des virus de vertébrés transmis généralement par des arthropodes hématophages (moustiques, tiques, phlébotomes), chez qui ils se multiplient avant la transmission Le cycle de maintien fait donc intervenir un vertébré arthropode et un réservoir Certains arbovirus peuvent concerner l'homme et induire des pathologies, les arboviroses que l'on trouve surtout dans les régions tropicales Si il existe un vaccin contre la fièvre jaune depuis 1930, ou plus récemment contre les encéphalites japonaises et à tique, les autres flavivirus n'ont actuellement aucun vaccin , c'est le cas notamment de la DengueFlaviviruses are viruses of the arbovirus family (old group B arboviruses). They are vertebrate viruses generally transmitted by hematophagous arthropods (mosquitoes, ticks, sandflies), in which they multiply before transmission. so intervene an arthropod vertebrate and a reservoir Some arboviruses can affect humans and induce pathologies, the arboviruses which are found especially in tropical regions If there has been a vaccine against yellow fever since 1930, or more recently against encephalitis Japanese and tick, the other flaviviruses currently have no vaccine, this is particularly the case for Dengue
Des dizaines de millions d'individus sont affectés annuellement par la Dengue, en particulier dans les régions intertropicales où la maladie sévit à l'état de pandémie ininterrompue Le virus responsable de la Dengue s'est propagé de façon concomitante à son vecteur, le moustique Aedes aegypti La maladie se caractérise par une forte fièvre accompagnée de maux de tête, de nausées et de douleurs articulaires et musculaires qui s'effacent après quelques jours Les symptômes peuvent cependant s'intensifier pour conduire à des manifestations hémorragiques parfois mortelles lorsque la maladie est associée à un choc hypovolémique, surtout chez l'enfant L'agent responsable de la Dengue est un flavivirus à ARN de la famille des Flavivmdae, tout comme le virus de la fièvre jaune ou celui de l'encéphalite japonaise On en connaît 4 sérotypes La Dengue hémorragique pourrait être une conséquence d'un phénomène immunologique mal connu, impliquant sans doute une cascade de cytokines liées à l'induction d'une
immunité cellulaire cytotoxique pouvant résulter d'une infection séquentielle par plusieurs sérotypes. L'absence de modèle animal freine la conception d'un vaccin car il n'est pas possible d'explorer en détail les facteurs impliqués dans les manifestations graves de la maladie. Un premier vaccin contre la Dengue est actuellement expérimenté. II s'agit d'un vaccin à base des 4 sérotypes vivants, atténués par passages successifs du virus en culture de cellules. L'inconvénient majeur de ce type de vaccin est ld possibilité des virus atténués de reverser vers la virulence, par réversion génétique ou recombinaison entre génomes. II présente également un risque potentiel pour les nouveaux-nés et pour les personnes immunodéprimées. De plus, certains enfants vaccinés au moyen du vaccin tétravalent semblent ne pas être protégés contre les 4 sérotypes.Tens of millions of people are affected each year by Dengue, in particular in the intertropical regions where the disease prevails with the state of uninterrupted pandemic. The virus responsible for Dengue spreads concomitantly with its vector, the mosquito Aedes aegypti The disease is characterized by a high fever accompanied by headache, nausea and joint and muscle pain which disappears after a few days Symptoms may, however, intensify leading to hemorrhagic manifestations which are sometimes fatal when the disease is associated with hypovolemic shock, especially in children The agent responsible for Dengue is an RNA flavivirus from the Flavivmdae family, just like the yellow fever virus or that of Japanese encephalitis We know 4 serotypes Dengue hemorrhagic fever could be a consequence of a poorly understood immunological phenomenon, probably involving a cascade of e cytokines linked to the induction of cytotoxic cellular immunity which may result from sequential infection by several serotypes. The absence of an animal model hinders the design of a vaccine because it is not possible to explore in detail the factors involved in serious manifestations of the disease. A first vaccine against Dengue is currently being tested. It is a vaccine based on 4 living serotypes, attenuated by successive passages of the virus in cell culture. The major drawback of this type of vaccine is the possibility of attenuated viruses reversing towards virulence, by genetic reversion or recombination between genomes. It also presents a potential risk for newborns and for immunocompromised people. In addition, some children vaccinated with the tetravalent vaccine seem not to be protected against the 4 serotypes.
La structure du génome est ia séquence d'amino-acides du virus de la Dengue notamment celui du sérotype 2 a été décrite parThe structure of the genome is the amino acid sequence of the Dengue virus, in particular that of serotype 2, has been described by
V. Deubel et al. (1 ).V. Deubel et al. (1).
Le virus de la Dengue est un virus à ARN enveloppé d'environ 50 nanomètres. Comme le virus de la fièvre jaune, toutes les protéines virales sont codées par un segment d'environ 10τé kilobases dont la structure est la suivante :The Dengue virus is an enveloped RNA virus of about 50 nanometers. Like the yellow fever virus, all viral proteins are coded by a segment of approximately 10τe kilobases, the structure of which is as follows:
5' C-preM/M-E-NS1 -NS2A-NS2B-NS3-NS4A-NS4B-NS5 3'.5 'C-preM / M-E-NS1 -NS2A-NS2B-NS3-NS4A-NS4B-NS5 3'.
Les protéines structurales sont C (capsides), pré- matrix/matrix(preM/M) et la protéine d'enveloppe (E).The structural proteins are C (capsids), pre-matrix / matrix (preM / M) and the envelope protein (E).
Ces protéines sont codées par le fragment en 5', suivi par des protéines non structurales (NS) codées par le reste du génome. Les protéines sont produites par un processus protéolytique induit par une signalase cellulaire et d'autres protéinases cellulaires et virales.These proteins are encoded by the 5 'fragment, followed by non-structural proteins (NS) encoded by the rest of the genome. Proteins are produced by a proteolytic process induced by cellular signalase and other cellular and viral proteinases.
La glycoprotéine d'enveloppe (E) est la protéine de surface majoritaire. La structure du stérotype 1 a été décrite par Mason et al. (2).
Le sérotype 2 a été décrit dans (1 ), et est le mieux connu des 4 sérotypes ; le sérotype de type 3 a été décrit par Osatomi K. al. (3) et le sérotype de type 4 a été décrit par Zhao et al., (4).The envelope glycoprotein (E) is the majority surface protein. The structure of sterotype 1 has been described by Mason et al. (2). Serotype 2 has been described in (1), and is the best known of the 4 serotypes; type 3 serotype has been described by Osatomi K. al. (3) and the type 4 serotype has been described by Zhao et al., (4).
De nombreuses tentatives ont été faites pour développer des vaccins sous unités généralement exprimés en tant que peptides ou protéines recombindntes par utilisation d'un vecteur baculoviral exprimé dans des cellules d'insectesNumerous attempts have been made to develop vaccines in units generally expressed as peptides or recombinant proteins by the use of a baculoviral vector expressed in insect cells.
En effet, les baculovirus sont très attractifs pour ce type de production grâce au haut niveau d'expression génétique qu'ils peuvent atteindre et pour leur absence de pathogénicité pour l'homme. Le baculovirus a déjà été utilisé pour l'expression de protéines de flavivirus le sérotype 1 de la Dengue (5), le sérotype 4 de la Dengue (DEN-4) (6) et le sérotype 2 (DEN-2) ont été décrits (7), (8), (9) et les sérotypes 2 et 3 ont été décrits par C. Delenda et al. (10).In fact, baculoviruses are very attractive for this type of production thanks to the high level of genetic expression they can achieve and for their absence of pathogenicity for humans. Baculovirus has already been used for the expression of flavivirus proteins. Dengue serotype 1 (5), Dengue serotype 4 (DEN-4) (6) and serotype 2 (DEN-2) have been described. (7), (8), (9) and serotypes 2 and 3 have been described by C. Delenda et al. (10).
L'homologie de séquence entre les 4 génomes correspondant aux 4 sérotypes du virus est relativement importante ; à titre d'exemple, la protéine E de ces virus présente un pourcentage d'homologie tel que présenté dans le tableau 1 ci-dessous :The sequence homology between the 4 genomes corresponding to the 4 serotypes of the virus is relatively large; by way of example, protein E of these viruses has a percentage of homology as presented in table 1 below:
Malgré cela aucune des constructions décrites dans l'état de la technique ne permet d'induire une réponse immunologique efficace contre les 4 sérotypes : généralement le titre obtenu est bas, et la résistance induite à un challenge contre une infection par le virus virulent ne peut être considérée comme suffisante.
Certaines constructions ont permis néanmoins une bonne induction de l'immunogmicité, et notamment dans le cas décrit dans (11 ) où une glycoprotéine d'enveloppe délétée dans sa partie carboxy- termmale de 100 acides aminés, permet d'obtenir une immunogénicité augmentée Mais celle-ci n'est pas efficace contre les autres sérotypes de la DengueDespite this, none of the constructions described in the state of the art makes it possible to induce an effective immunological response against the 4 serotypes: generally the titer obtained is low, and the resistance induced to a challenge against infection by the virulent virus cannot be considered sufficient. Certain constructions have nevertheless allowed good induction of immunogmicity, and in particular in the case described in (11) where an envelope glycoprotein deleted in its carboxy-termmal part of 100 amino acids, makes it possible to obtain an increased immunogenicity But that -It is not effective against other Dengue serotypes
Enfin, Delenda et al (9, 10) ont effectivement montré qu'une troncature dans la partie carboxy-terminale de la protéine était susceptible d'augmenter la réponse immunitaire La purification des protéines recombinantes obtenues par l'expression dans un système hétérologue pose toujours un problème de compatibilité entre le rendement recherché et la préservation de la structure de ladite protéine et notamment de ses motifs glycosylés Une technique performante est la purification d'affinité de peptides ou protéines couplés de façon covalente à une courte séquence d'amino-acides qui joue le rôle de marquage (tag) Des exemples de techniques exploitant cette approche sont la glutathione-S-transférase (GST) purifiée sur des billes de sépharose-glutathion (12), l'association de la protéine Mal E au maltose et à ses dérivés (13), une fusion de la protéine A avec des colonnes d'immunoglobulmes (14), ou le marquage à l'histidine qui présente une affinité pour des colonnes de métal chélate (15) Schmidt et al (16) décrit dans un article récent les avantages et les limites de cette technique Mais il est à noter que l'homme du métier cherche systématiquement à éliminer les résidus histidine après l'étape de purification notamment en utilisant la carboxypeptidase AFinally, Delenda et al (9, 10) have actually shown that a truncation in the carboxy-terminal part of the protein is capable of increasing the immune response. The purification of the recombinant proteins obtained by expression in a heterologous system always poses a problem of compatibility between the desired yield and the preservation of the structure of said protein and in particular of its glycosylated units. An effective technique is the affinity purification of peptides or proteins covalently coupled to a short sequence of amino acids which plays the role of labeling Examples of techniques exploiting this approach are glutathione-S-transferase (GST) purified on sepharose-glutathione beads (12), the association of the protein Mal E with maltose and its derivatives (13), a fusion of protein A with immunoglobulin columns (14), or histidine labeling which has an affinity for co lons of metal chelate (15) Schmidt et al (16) describes in a recent article the advantages and the limits of this technique But it should be noted that a person skilled in the art systematically seeks to remove the histidine residues after the purification step in particular by using carboxypeptidase A
La présente invention résulte de la découverte selon laquelle un polypeptide ou un glycopeptide constitué d'un polypeptide ou glycoprotéine issu d'une protéine d'un Flaviviridiae, à savoir le virus de la Dengue dans laquelle une délétion de son extrémité carboxylée a été substituée par un résidu d'acides aminés choisi parmi l'histidine, le tryptophane ou la cystéine présentait un caractère immunogène beaucoup
plus important que le même polypeptide ou glycopeptide n'ayant pas ce résidu d'acides aminés ajoutés, ce résidu ayant pour effet d'améliorer la présentation antigénique et par conséquent d'induire une réponse cellulaire et humorale beaucoup plus importante qu'en l'absence de ce résiduThe present invention results from the discovery that a polypeptide or a glycopeptide consisting of a polypeptide or glycoprotein derived from a protein of a Flaviviridiae, namely the Dengue virus in which a deletion of its carboxylated end has been substituted by an amino acid residue chosen from histidine, tryptophan or cysteine exhibited a much immunogenic character more important than the same polypeptide or glycopeptide not having this residue of added amino acids, this residue having the effect of improving the antigen presentation and consequently of inducing a cellular and humoral response much more important than in the absence of this residue
Dans tout ce qui précède et suit, l'expression « polypeptide » doit être comprise au sens large, c'est-à-dire incluant des séquences d'au moins 15 acides aminés comprenant ou non des motifs glycosylés ou des glycohpides, et quelle que soit sa structure, primaire, secondaire ou tertiaireIn all of the above and following, the expression “polypeptide” must be understood in the broad sense, that is to say including sequences of at least 15 amino acids comprising or not glycosylated units or glycohpids, and which whether its structure, primary, secondary or tertiary
La présente invention porte plus généralement sur des polypeptides, à pouvoir immunogène accru, issus de Flaviviridiae, caractérisés par l'existence d'un résidu d'acides aminés à leur extrémité carboxy-terminale, lequel résidu d'acides aminés contient de 2 à 8 acides aminés choisis parmi l'histidine, le tryptophane ou la cystéineThe present invention relates more generally to polypeptides, with increased immunogenic power, derived from Flaviviridiae, characterized by the existence of an amino acid residue at their carboxy-terminal end, which amino acid residue contains from 2 to 8 amino acids selected from histidine, tryptophan or cysteine
Dans le texte de la présente demande, le résidu d'acides aminés peut être homogène dans le sens où il est constitué de 2 à 8 histidines, 2 à 8 tryptophanes ou 2 à 8 cystéines il peut être également constitué d'un mélange de 2 ou 3 de ces acides aminés , il peut enfin comporter quelques acides aminés autres que l'histidme, le tryptophane ou la cystéine à partir du moment où leur nombre n'excède pas 25 % du résiduIn the text of the present application, the amino acid residue can be homogeneous in the sense that it consists of 2 to 8 histidines, 2 to 8 tryptophanes or 2 to 8 cysteines it can also consist of a mixture of 2 or 3 of these amino acids, it may finally contain a few amino acids other than the histidme, tryptophan or cysteine as long as their number does not exceed 25% of the residue
Ces constructions permettent de résoudre le problème du manque d'immunogénicité de certains peptides issus de ces virus, comme par exemple certains sérotypes du virus de la Dengue contre lesquels les techniques actuelles ne permettaient pas de faire des vaccins de type sous-unitéThese constructions make it possible to solve the problem of the lack of immunogenicity of certain peptides derived from these viruses, such as for example certain serotypes of the Dengue virus against which current techniques did not make it possible to make vaccines of the subunit type.
La présente invention porte sur de tels polypeptides immunogènes, constitués d'un polypeptide issu de la protéine E, de la protéine preM, de la protéine NS1 ou NS3 du virus de la Dengue et dans laquelle pour la protéine E l'extrémité carboxylée a été délétée de 70 à
105 acides aminés pour être substituée par un résidu de 2 à 8 acides aminés choisis parmi l'histidine, la cystéine ou le tryptophane II peut en être de même pour la protéine preM délétée de 30 à 50 acides aminés dans son extrémité carboxy-terminale pour être substituée par le même type de résidu Pour les autres protéines cette délétion n'est pas nécessaire , et le polypeptide de l'invention issu de NS1 ou NS3 comporte un résidu de 2 à 8 acides aminés choisis parmi l'histidme, la cystéine ou le tryptophaneThe present invention relates to such immunogenic polypeptides, consisting of a polypeptide derived from protein E, from protein preM, from protein NS1 or NS3 of the Dengue virus and in which for protein E the carboxyl end has been deleted from 70 to 105 amino acids to be substituted by a residue of 2 to 8 amino acids chosen from histidine, cysteine or tryptophan II can be the same for the preM protein deleted from 30 to 50 amino acids in its carboxy-terminal end for be substituted by the same type of residue For the other proteins this deletion is not necessary, and the polypeptide of the invention derived from NS1 or NS3 comprises a residue of 2 to 8 amino acids chosen from histidm, cysteine or tryptophan
De façon avantageuse, le polypeptide est issu de la protéine E et la délétion est de environ 100 acides aminés , si en outre, le résidu d'acides aminés contient 2 à 8 histidines, cela confère au peptide la propriété de se fixer sur des cations divalents ou trivalents ou tnvalents Les polypeptides ou les peptides de l'invention peuvent être issus de chacun des 4 sérotypes de la Dengue, sachant que le pourcentage d'homologie entre les 4 sérotypes est élevé et se situe entre 67 % et 80 % pour la protéine E comme cela est indiqué dans le tableau 1 ci-dessusAdvantageously, the polypeptide is derived from protein E and the deletion is approximately 100 amino acids, if in addition, the amino acid residue contains 2 to 8 histidines, this gives the peptide the property of binding to cations divalent or trivalent or equivalent The polypeptides or peptides of the invention can be derived from each of the 4 serotypes of Dengue, knowing that the percentage of homology between the 4 serotypes is high and is between 67% and 80% for the protein E as indicated in table 1 above
La présente invention porte également sur des compositions constituées de mélanges de polypeptides de l'invention Ces mélanges peuvent constituer un principe actif de vaccin polyvalent, soit contre différents sérotypes d'un même virus, comme la Dengue, soit contre différents virus de la même famille des Flaviviridiae ou Flavi-hke et susceptibles d'affecter les mêmes populationsThe present invention also relates to compositions consisting of mixtures of polypeptides of the invention. These mixtures can constitute an active principle of a polyvalent vaccine, either against different serotypes of the same virus, such as Dengue, or against different viruses of the same family. Flaviviridiae or Flavi-hke and likely to affect the same populations
La présente invention est également relative à une composition apte à stimuler l'immunité d'individus susceptibles d'être affectés par le virus de la Dengue, caractérisée en ce qu'elle contient au moins 3 polypeptides ou glycopeptides de l'invention Une composition préférée sera celle contenant au moins un polypeptide de l'invention issu d'une protéine de la Dengue provenant de chacun des 4 sérotypes , la protéine E est un bon candidat elle a une fonction essentielle dans l'infectiosité virale et est l'intermédiaire entre le virus et les récepteurs cellulaires , la protéine E semble également être une cible essentielle de
la réponse immunitaire protectrice dans les hôtes infectés dans la mesure où elle induit des anticorps neutralisants et une réponse immunocellulaireThe present invention also relates to a composition capable of stimulating the immunity of individuals liable to be affected by the Dengue virus, characterized in that it contains at least 3 polypeptides or glycopeptides of the invention A preferred composition will be that containing at least one polypeptide of the invention derived from a Dengue protein originating from each of the 4 serotypes, protein E is a good candidate it has an essential function in viral infectivity and is the intermediary between the protein and cell receptors, protein E also appears to be a key target of the protective immune response in infected hosts insofar as it induces neutralizing antibodies and an immunocellular response
Une composition de l'invention particulièrement avantageuse pourrait être un mélange de 3 ou 4 polypeptides ou glycopeptides décrits ci-dessus dont une partie est issue de la protéine E de chacun desA particularly advantageous composition of the invention could be a mixture of 3 or 4 polypeptides or glycopeptides described above, part of which is derived from protein E of each of
4 sérotypes, délétée d'une centaine d'acides aminés, à laquelle est greffé de façon covalente un résidu contenant 2 à 8 histidines, et de préférence de chacun des sérotypes connus du virus4 serotypes, deleted from a hundred amino acids, to which is covalently grafted a residue containing 2 to 8 histidines, and preferably each of the known serotypes of the virus
Afin que la composition immunogène de l'invention soit efficace vis-à-vis des 3 ou 4 sérotypes en question ou plus si des nouveaux sérotypes étaient amenés à apparaître, chaque peptide ou glycopeptide y est présent dans une proportion de 10 à 50 % en poids par rapport à l'ensemble des peptides de la composition, et de préférence un pourcentage sensiblement égal pour chacun des peptides Les compositions immunogènes de l'invention peuvent également être constituées, d'un mélange de polypeptides issus de la protéine E, preM, NS1 ou NS3 et comportant au moins 3 antigènes de 3 sous-types du virus de la Dengue, chacun étant muni du résidu d'acides aminés à l'extrémité carboxy-terminale De façon plus générale, l'invention porte sur des compositions immunogènes constituées d'un mélange de polypeptides ou glycopeptides issus de la protéine E, preM, NS1 ou NS3 du virus de la Dengue et comportant au moins 3 antigènes de 3 sérotypes de ce virus et plus généralement un antigène correspondant à chacun des sérotypes connus, ne comportant pas nécessairement le résidu d'acides aminés à leur extrémité carboxy-terminale, mais caractérisés en ce que chacun des peptides, polypeptides ou glycopeptides présente la capacité d'induire une réaction immunitaire chez l'hôteSo that the immunogenic composition of the invention is effective vis-à-vis the 3 or 4 serotypes in question or more if new serotypes were caused to appear, each peptide or glycopeptide is present in a proportion of 10 to 50% in weight relative to all the peptides of the composition, and preferably a substantially equal percentage for each of the peptides The immunogenic compositions of the invention may also consist of a mixture of polypeptides derived from protein E, preM, NS1 or NS3 and comprising at least 3 antigens of 3 subtypes of the Dengue virus, each being provided with the amino acid residue at the carboxy-terminal end. More generally, the invention relates to immunogenic compositions consisting of a mixture of polypeptides or glycopeptides derived from protein E, preM, NS1 or NS3 of the Dengue virus and comprising at least 3 antigens of 3 serotypes of this virus and more generally an antigen corresponding to each of the known serotypes, not necessarily comprising the amino acid residue at their carboxy-terminal end, but characterized in that each of the peptides, polypeptides or glycopeptides has the capacity to induce an immune reaction in humans. 'host
La composition selon l'invention peut en outre contenir des polypeptides, glycopeptides ou protéines provenant d'autres flavivirus comme ceux de la fièvre jaune de l'encéphalite japonaise, de l'encéphalite
à tiques ou de l'hépatite C ou plus généralement d'autres Flaviviridiae des fièvres hémorragiques présents dans les zones tropicales d'Afrique, d'Asie ou d'Amérique, ainsi que le cas échéant issus d'autres antigènes viraux, bactériens ou parasitaires , à partir du moment où ils sont munis du résidu d'acides aminés tel que défini ci-dessus à leur extrémité carboxy-terminaleThe composition according to the invention may also contain polypeptides, glycopeptides or proteins originating from other flaviviruses such as those of yellow fever from Japanese encephalitis, from encephalitis with ticks or hepatitis C or more generally other Flaviviridiae haemorrhagic fevers present in the tropical zones of Africa, Asia or America, as well as if necessary from other viral, bacterial or parasitic antigens , from the moment they are provided with the amino acid residue as defined above at their carboxy-terminal end
L'invention porte également sur les séquences d'acides nucléiques recombinants codant pour des peptides immunogènes des Flaviviridiae et notamment du virus de la Dengue, délétés en 3' de 210 à 315 nucléotides, auxquels a été ajoutée en 3' une séquence codant pour 2 à 8 acides aminés choisis parmi l'histidme, le tryptophane ou la cystéine dans le sens défini plus haut Les séquences d'acides nucléiques selon l'invention codent plus particulièrement pour des glycopeptides issus de la protéine E de la Dengue de sérotype 1 , 2, 3 ou 4, et la séquence ajoutée en 3' code préférentiellement pour une séquence contenant 6 histidines L'invention porte également sur des vecteurs d'acides nucléiques appropriés pour une utilisation dans l'expression de gènes exogènes chez un eucaryote , ces vecteurs portent, outre une séquence d'acides nucléiques telle que décrite ci-dessus, une séquence signal homologue ou hétérologue de celle de la protéine codée, l'ensemble étant sous la dépendance d'un promoteur susceptible d'être activé dans ladite cellule eucaryote Un type de cellule eucaryote préféré est celui des cellules d'insectes et notamment les cellules de Spodoptera frugiperda SF 9 Le vecteur est un vecteur navette pVLd susceptible de réaliser une recombinaison homologue avec un baculovirus, et porteur, en amont de la séquence signal, d'un promoteur de gènes endogènes au baculovirus par exemple celui de la polyédπneThe invention also relates to the recombinant nucleic acid sequences coding for immunogenic peptides of Flaviviridiae and in particular of the Dengue virus, deleted in 3 'from 210 to 315 nucleotides, to which has been added in 3' a sequence coding for 2 with 8 amino acids chosen from histidme, tryptophan or cysteine in the sense defined above The nucleic acid sequences according to the invention more particularly code for glycopeptides derived from protein E of Dengue serotype 1, 2 , 3 or 4, and the sequence added in 3 ′ preferably codes for a sequence containing 6 histidines The invention also relates to nucleic acid vectors suitable for use in the expression of exogenous genes in a eukaryote, these vectors carry , in addition to a nucleic acid sequence as described above, a signal sequence homologous or heterologous to that of the encoded protein, the set being dependent on a promoter capable of being activated in said eukaryotic cell A preferred type of eukaryotic cell is that of insect cells and in particular the cells of Spodoptera frugiperda SF 9 The vector is a shuttle vector pVLd capable of carrying out a homologous recombination with a baculovirus, and carrying, upstream of the signal sequence, a promoter of genes endogenous to baculovirus, for example that of polyhedron
Les baculovirus recombinants peuvent être obtenus par différentes techniques connues de l'homme du métier mais une technique particulièrement avantageuse est celle de la recombinaison homologue en utilisant des vecteurs navettes entre E Coli et le baculovirus
Des vecteurs d'expressions tels qu'obtenus par cette technique, et décrite plus en détails dans la partie expérimentale ci- dessous ont été déposés à la CNCM , il s'agitRecombinant baculoviruses can be obtained by various techniques known to those skilled in the art, but a particularly advantageous technique is that of homologous recombination using shuttle vectors between E Coli and the baculovirus. Expression vectors as obtained by this technique, and described in more detail in the experimental part below, have been deposited at the CNCM, these are
- d'un baculovirus recombinant déposé à la CNCM sous le numéro 1-1497, le 1er décembre 1994 et porteur d'une séquence codant pour un peptide constitué du gène E du virus de la Dengue sérotype n° 2 dans laquelle l'extrémité COOH a été délétée de 100 acides aminés et substituée par 6 histidines ,- a recombinant baculovirus deposited at the CNCM under the number 1-1497, on December 1, 1994 and carrying a sequence coding for a peptide consisting of the E gene of the Dengue virus serotype No. 2 in which the COOH end has been deleted from 100 amino acids and substituted by 6 histidines,
- d'un baculovirus recombinant déposé à la CNCM sous le numéro 1-1624, le 11 octobre 1995 et porteur d'une séquence codant pour un peptide constitue du gène E du virus de la Dengue sérotype n° 4 dans laquelle l'extrémité COOH a été délétée de 100 acides aminés et substituée par 6 histidines ,- a recombinant baculovirus deposited at the CNCM under the number 1-1624, on October 11, 1995 and carrying a sequence coding for a peptide constitutes the E gene of the Dengue virus serotype No. 4 in which the COOH end has been deleted from 100 amino acids and substituted by 6 histidines,
- d'un baculovirus recombinant déposé à la CNCM sous le numéro 1-1625, le 11 octobre 1995 et porteur d'une séquence codant pour un peptide constitué du gène E du virus de la Dengue sérotype n° 3 dans laquelle l'extrémité COOH a été délétée de 100 acides aminés et substituée par 6 histidines ,- a recombinant baculovirus deposited at the CNCM under the number 1-1625, on October 11, 1995 and carrying a sequence coding for a peptide consisting of the E gene of the Dengue virus serotype No. 3 in which the COOH end has been deleted from 100 amino acids and substituted by 6 histidines,
- d'un baculovirus recombinant déposé à la CNCM sous le numéro 1-1626, le 1 1 octobre 1995 et porteur d'une séquence codant pour un peptide constitué du gène E du virus de la Dengue sérotype n° 1 dans laquelle l'extrémité COOH a été délétée de 100 acides aminés et substituée par 6 histidines- a recombinant baculovirus deposited at the CNCM under the number 1-1626, on October 11, 1995 and carrying a sequence coding for a peptide consisting of the E gene of the Dengue virus serotype No. 1 in which the end COOH has been deleted from 100 amino acids and substituted by 6 histidines
L'invention porte également sur des compositions aptes à induire une réponse immunitaire, cellulaire ou humorale chez un hôte susceptible d'être infecté par le virus de la Dengue et comprenant un mélange d'acides nucléiques codant respectivement pour les polypeptides de l'invention, à savoir ceux issus de la protéine de la Dengue, telle la protéine E, preM, NS1 ou NS3, et portant à leur extrémité carboxy- terminale un résidu de 2 à 8 acides aminés choisis parmi l'histidme, la cystéine ou le tryptophane, ou plus généralement codant pour des
polypeptides issus d'autres Flaviviridiae comme la fièvre jaune, l'encéphalite japonaise, encéphalite à tiques ou autres « Flavι-lικe » comme le virus de l'hépatite C En effet, il a été montré que l'injection chez l'animal d'un plasmide contenant les séquences nécessaires à l'expression de protéines ou de polypeptides déclenche une réponse immunitaire cellulaire et humorale chez l'hôte permettant dans certains modèles animaux de protéger ces derniers contre une charge virale létale , de tels résultats ont été décrits par J A Wolff et al (1990), Science 247 1465- 1468 ou par E.F Fynan et al (1993), DNA cell biol 12 785-789 Les compositions immunogènes comprenant de telles séquences d'acides nucléiques et les vaccins constitués de ces compositions, avec le cas échéant un adjuvant, font également partie de l'inventionThe invention also relates to compositions capable of inducing an immune, cellular or humoral response in a host capable of being infected with the Dengue virus and comprising a mixture of nucleic acids coding respectively for the polypeptides of the invention, namely those derived from the Dengue protein, such as protein E, preM, NS1 or NS3, and carrying at their carboxy-terminal end a residue of 2 to 8 amino acids chosen from histidm, cysteine or tryptophan, or more generally coding for polypeptides from other Flaviviridiae such as yellow fever, Japanese encephalitis, tick-borne encephalitis or other "Flavι-lικe" such as the hepatitis C virus Indeed, it has been shown that injection into animals d a plasmid containing the sequences necessary for the expression of proteins or polypeptides triggers a cellular and humoral immune response in the host making it possible in certain animal models to protect the latter against a lethal viral load, such results have been described by JA Wolff et al (1990), Science 247 1465-1468 or by EF Fynan et al (1993), DNA cell biol 12 785-789 Immunogenic compositions comprising such nucleic acid sequences and the vaccines made up of these compositions, with the optionally an adjuvant, are also part of the invention
L'invention est également relative à des vaccins sous-unités polyvalents contre le virus de la Dengue notamment contre les 4 sérotypes dudit virus et comprenant une composition immunogène telle que décrite ci-dessus, avec le cas échéant un adjuvant de vaccination qui peut être notamment un adjuvant porteur d'ions divalents ou tπvalents tels l'hydroxide d'aluminium ou le phosphate de calcium ou encore un adjuvant de type adjuvant de Freund, un dérivé du muramylpeptide ou un « iscom » (Immunostimulating Complex), (19) L'effet immunogène des compositions de l'invention peut être la conséquence d'un captage des polypeptides ou glycoprotéines de l'invention par les ions divalents ou trivalents et la formation éventuelle de multimères qui présenteraient un pouvoir immunogène accru par rapport aux polypeptides issus de la Dengue et déjà décrit dans la littérature (références 3, 7, 8, 9)The invention also relates to polyvalent subunit vaccines against the Dengue virus in particular against the 4 serotypes of said virus and comprising an immunogenic composition as described above, with, where appropriate, a vaccination adjuvant which may in particular be an adjuvant carrying divalent or valent ions such as aluminum hydroxide or calcium phosphate or an adjuvant of Freund's adjuvant type, a derivative of muramylpeptide or an "iscom" (Immunostimulating Complex), (19) L ' immunogenic effect of the compositions of the invention may be the consequence of uptake of the polypeptides or glycoproteins of the invention by divalent or trivalent ions and the possible formation of multimers which would have an increased immunogenic power compared to the polypeptides derived from Dengue and already described in the literature (references 3, 7, 8, 9)
Les vaccins sous-unités de l'invention peuvent être également constitués de compositions aptes à induire une réaction immunogène contre d'autres Flaviviridiae Ils peuvent être destinés à la prophylaxie ou la thérapie d'infection par une ou plusieurs espèces virales simultanément
Un vaccin polyvalent sous unités selon l'invention, et contenant de 1 à 4 glycopeptides ou polypeptides immunogènes de l'invention, le cas échéant associés à des glycopeptides immunogènes provenant d'autres flavivirus ou virus de fièvres hémorragiques, présente à la fois l'avantage de la polyvalence liée à l'effet immunogène du principe actif contre les différents sérotypes du virus et celui de l'innocuité totale En effet, il existe actuellement des vaccins contre la Dengue à base des 4 sérotypes vivants atténués par passage successifs du virus en culture , mais comme nous l'avons vu plus haut, ce vaccin présente parfois un risque pour des personnes immuno-dépπmées ou des nouveaux-nés, et n'est pas toujours efficace contre les différents sérotypes et notamment sur les 4 sérotypes de la Dengue II peut être également envisagé dans les vaccins de l'invention d'additionner les vaccins actuellement en expérimentation clinique d'1 , 2, 3 ou 4 polypeptides selon l'invention La description détaillée ci-après d'un mode de réalisation particulier utilisant la protéine E des sérotypes 1 à 4 (DEN1 à DEN4) illustre, sans être limitative, les propriétés structurelles et fonctionnelles des polypeptides, compositions polypeptidiques et vaccins de l'inventionThe subunit vaccines of the invention may also consist of compositions capable of inducing an immunogenic reaction against other Flaviviridiae They may be intended for the prophylaxis or therapy of infection by one or more viral species simultaneously A polyvalent vaccine in units according to the invention, and containing from 1 to 4 immunogenic glycopeptides or polypeptides of the invention, if appropriate associated with immunogenic glycopeptides originating from other flaviviruses or haemorrhagic fever viruses, presents both advantage of the versatility linked to the immunogenic effect of the active principle against the various serotypes of the virus and that of total safety Indeed, there are currently vaccines against Dengue based on the 4 living serotypes attenuated by successive passage of the virus in culture, but as we have seen above, this vaccine sometimes poses a risk for immunocompromised people or newborns, and is not always effective against the various serotypes and in particular on the 4 serotypes of Dengue It may also be envisaged in the vaccines of the invention to add the vaccines currently in clinical experimentation with 1, 2, 3 or 4 polypeptides se The invention The detailed description below of a particular embodiment using protein E of serotypes 1 to 4 (DEN1 to DEN4) illustrates, without being limiting, the structural and functional properties of polypeptides, polypeptide compositions and vaccines. the invention
La figure 1 représente le profil électrophorétique des polypeptides E recombinés DEN1 à DEN 4 sécrétés dans le surnageant des cellules Sf9 infectées Les protéines radioimmunoprécipitées ont été traitées par de l'endoglycosidase F (F) ou H (H) ou non traitées (O) I Construction des baculovirus recombinantsFIG. 1 represents the electrophoretic profile of the recombinant polypeptides E DEN1 to DEN 4 secreted in the supernatant of the infected Sf9 cells The radioimmunoprecipitated proteins were treated with endoglycosidase F (F) or H (H) or not treated (O) I Construction of recombinant baculoviruses
1 ° Préparation des fragments de gène codant pour la protéine d'enveloppe E des virus de la Dengue1 ° Preparation of the gene fragments coding for the envelope protein E of the Dengue viruses
Les souches de virus de la Dengue ayant servi au clonage des gènes E sont inscrites dans le tableau 2 ci-dessous
tableau 2The Dengue virus strains that were used to clone the E genes are listed in Table 2 below. table 2
2° Préparation des baculovirus recombinants exprimant les protéines tronquées du virus de la Dengue :2 ° Preparation of the recombinant baculoviruses expressing the truncated proteins of the Dengue virus:
La préparation des gènes de E a été réalisée par transcription inverse/réaction en chaîne par polymérase (RT/PCR) à partir des ARN extraits de cellules de moustiques Aedes pseudoscutellaπs AP61 infectées par les différents virus La taille en acides aminés attendue des protéines E délétées de 100 acides aminés à leur extrémité C-terminale (EΔ100) est indiquée dans la colonne de droite du tableau 2The preparation of the E genes was carried out by reverse transcription / polymerase chain reaction (RT / PCR) from the RNA extracted from Aedes pseudoscutellaπs AP61 mosquito cells infected with the various viruses The expected amino acid size of the deleted E proteins of 100 amino acids at their C-terminal end (EΔ100) is indicated in the right column of Table 2
L'amorce ohgonucleotidique située en 5' du gène à amplifier possède un site de restriction (sauf pour la Dengue 2), le codon d'initiation ATG SUIVI des nucléotides codant pour les 6 premiers acides aminés parmi les 15 correspondant à la séquence signal de chaque protéine L'amorce ohgonucleotidique située en 3' du gène à amplifier possède la séquence correspondant à 6 acides aminés de la protéine E du virus suivie de 6 codons pour l'histidme, d'un codon stop et d'un site de restriction La séquence des oligonucléotides est celle indiquée ci-dessous
Extrémité 5' (sens positif)The ohgonucleotide primer located 5 ′ of the gene to be amplified has a restriction site (except for Dengue 2), the ATG initiation codon FOLLOWED by nucleotides coding for the first 6 amino acids among the 15 corresponding to the signal sequence of each protein The ohgonucleotide primer located 3 ′ of the gene to be amplified has the sequence corresponding to 6 amino acids of protein E of the virus followed by 6 codons for the histidme, a stop codon and a restriction site La oligonucleotide sequence is as shown below 5 'end (positive direction)
Dengue 1 ;Dengue 1;
5'CGGGATCCATGGGGATCATTTTCATπTGCTGATG-3'5'CGGGATCCATGGGGATCATTTTCATπTGCTGATG-3 '
Dengue 2 :Dengue 2:
5'-GGCCTTGATTTTCATCTTAC-3'5'-GGCCTTGATTTTCATCTTAC-3 '
Dengue 3 :Dengue 3:
5'-CGGGATCCATGGTGGTTA I I M I ATACTATTAATG-3'5'-CGGGATCCATGGTGGTTA I I M I ATACTATTAATG-3 '
Dengue 4 : δ'-CGGGATCCATGACTGTCTTCTTTGTCCTAATG-S'Dengue 4: δ'-CGGGATCCATGACTGTCTTCTTTGTCCTAATG-S '
Extrémité 3' (sens négatif)3 'end (negative direction)
Dengue 1 :Dengue 1:
5'-CGGAGCTCAATGATGATGATGATGATGTCCTTTCTTGAACCAGCTTAG-3'5'-CGGAGCTCAATGATGATGATGATGATGTCCTTTCTTGAACCAGCTTAG-3 '
Dengue 2 :Dengue 2:
S'-GGAGCTCAATGATGATGATGATGATGTCCTTTCTTAAACCAGTTG-S'S'-GGAGCTCAATGATGATGATGATGATGTCCTTTCTTAAACCAGTTG-S '
Dengue 3 :Dengue 3:
5'-CGGAGCTCAATGATGATGATGATGATGTCCCTTCTTGTACCAGTTAAT-3'5'-CGGAGCTCAATGATGATGATGATGATGTCCCTTCTTGTACCAGTTAAT-3 '
Dengue 4 :Dengue 4:
5'-CGGAGCTCAATGATAATGATAATGATACCCTTTCCTGAACCAATGGAG-3'5'-CGGAGCTCAATGATAATGATAATGATACCCTTTCCTGAACCAATGGAG-3 '
Les fragments amplifiés à partir d'ARN viral extrait de cellules d'Aedes pseudoscutellaris AP61 ont ensuite été clones dans le vecteur navette pVL-poly (17).The fragments amplified from viral RNA extracted from Aedes pseudoscutellaris AP61 cells were then cloned into the shuttle vector pVL-poly (17).
D (1 ,2,3 ou 4)EΔ100Hιs6 correspond à la protéine E du sérotype 1 , 2, 3, 4 délétée de 100 acides aminés à son extrémité carboxyle et à laquelle ont été ajoutés 6 résidus histidine D (1, 2,3 or 4) EΔ100Hιs6 corresponds to protein E of serotype 1, 2, 3, 4 deleted by 100 amino acids at its carboxyl end and to which 6 histidine residues have been added
Les gènes codant pour les protéines D. (1 ,2,3 ou 4)EΔ100 His6 et leur séquence signal sont ainsi placés sous le contrôle du promoteur de la polyédrine. Le nombre de nucléotides compris entre la fin du promoteur et le codon d'initiation est de 42 (Dengue 1 , Dengue 3,The genes coding for the proteins D. (1, 2,3 or 4) EΔ100 His6 and their signal sequence are thus placed under the control of the polyhedrin promoter. The number of nucleotides between the end of the promoter and the initiation codon is 42 (Dengue 1, Dengue 3,
Dengue 4) ou de 50 (Dengue 2) nucléodides.Dengue 4) or 50 (Dengue 2) nucleodides.
Les baculovirus recombinés Ac. D(1 ,2,3 ou 4)EΔ100Hιs6 ont été obtenus par recombinaison homologue dans les cellules Spodoptera frugiperda Sf9 entre les vecteurs navettes pVL.D(1 ,2,3 ou 4)EΔ100Hιs6 et le baculovirus AcRP23lacZ linéarisé au site unique Bsu36l Les baculovirus recombinés ont été sélectionnés par 3 clonages successifs par la méthode des plages. 3° Caractéristiques structurelles des protéines recombinées obtenues :The recombinant baculoviruses Ac. D (1, 2,3 or 4) EΔ100Hιs6 were obtained by homologous recombination in Spodoptera frugiperda Sf9 cells between the shuttle vectors pVL.D (1, 2,3 or 4) EΔ100Hιs6 and the linearized bacRovirus AcRP23lacZ at the unique site Bsu36l Les recombinant baculoviruses were selected by 3 successive cloning by the beach method. 3 ° Structural characteristics of the recombinant proteins obtained:
La figure 1 montre le profil électrophorétique des 4 protéines recombinées correspondant aux virus de la Dengue 1 , 2, 3 ou 4. Le profil des protéines traitées à l'endoglycosidase F ou H indique qu'elles sont toutes glycosylées, sans doute à un seul des deux sites de glycosylation car la différence de poids moléculaire n'excède pas 4000 daltonsFigure 1 shows the electrophoretic profile of the 4 recombinant proteins corresponding to Dengue viruses 1, 2, 3 or 4. The profile of proteins treated with endoglycosidase F or H indicates that they are all glycosylated, probably with only one of the two glycosylation sites because the difference in molecular weight does not exceed 4000 daltons
II. Expression et purification des protéines DEΔ100Hιs6II. Expression and purification of DEΔ100Hιs6 proteins
1 ) Infection des cellules Sf9 : dix milliards de cellules Sf9 en suspension sont infectées par le baculovirus recombiné AcD(1 ,2,3 ou 4)EΔ100Hιs6 à une multiplicité d'infection de 2 à 5 La durée de l'infection est d'une heure à 28°C. L'inoculum est ensuite retiré et les cellules sontincubées pendant 3 jours à 28°C dans 2 litres de milieu TC100 (GIBCO) sans sérum de veau foetal1) Infection of Sf9 cells: ten billion Sf9 cells in suspension are infected with the recombinant baculovirus AcD (1, 2,3 or 4) EΔ100Hιs6 at a multiplicity of infection from 2 to 5 The duration of the infection is one hour at 28 ° C. The inoculum is then removed and the cells are incubated for 3 days at 28 ° C. in 2 liters of TC100 medium (GIBCO) without fetal calf serum
2) Préparation des surnageants de cellules infectées : 2 litres de surnageant de culture cellulaire sont récupérés 3 jours après
l'infection, centrifugés à 2500 rpm à 4°C pendant 30 minutes Le surnageant est additionné de 400 g/htre d'acétate d'ammonium sous agitation entre 3 et 14 heures à 4°C Le précipité est centrifugé à 10 000 rpm pendant 20 minutes à 4°C et redissous dans 1/20ème du volume initial 5 (100 ml) de tampon chromatographie (0,5M NaCl , 20mM Tπs-HCI pH 7,9) contenant de l'aprotmine à une concentration finale de 20μg/ml et du phenyi methyl sulfonyl fluoπde à 1 mM final Le surnageant concentré est ensuite dialyse deux fois contre 100 volumes de tampon de chromatographie io 3) Chromatographie : le milieu dialyse est additionné d'imidazole 5mM final puis incubé 30 mm (sous agitation douce) à température du laboratoire en présence de 5 ml de résine TALON (Clontech) La résine est lavée trois fois avec 30 ml de tampon de chromatographie contenant de l'imidazole à 10mM La résine est ensuite i s placée dans une colonne, lavée avec 10 volumes de tampon de chromatographie, et la protéine est éluée avec 10 ml de tampon de chromatographie contenant de l'imidazole à 50mM Des fractions de 1 ml sont récupérées avec un collecteur de fractions Une deuxième élution est pratiquée à l'aide de tampon de chromatographie contenant de l'imidazole 0 à 100 mM Les fractions contenant la protéine E recombinée sont déterminées par dot-blot à l'aide d'anticorps de souris anti-E et de conjugué anti-souris couplé à la phosphatase alcaline, ou à l'aide de conjugué Ni-NTA couplé à la phosphatase alcaline (Qiagen)2) Preparation of the supernatants of infected cells: 2 liters of cell culture supernatant are recovered 3 days after infection, centrifuged at 2,500 rpm at 4 ° C for 30 minutes The supernatant is added with 400 g / hour of ammonium acetate with stirring between 3 and 14 hours at 4 ° C The precipitate is centrifuged at 10,000 rpm for 20 minutes at 4 ° C and redissolved in 1 / 20th of the initial volume 5 (100 ml) of chromatography buffer (0.5M NaCl, 20mM Tπs-HCl pH 7.9) containing aprotmine at a final concentration of 20μg / ml and phenyi methyl sulfonyl fluoπde at 1 mM final The concentrated supernatant is then dialyzed twice against 100 volumes of chromatography buffer io 3) Chromatography: the dialysis medium is supplemented with 5mM imidazole final then incubated 30 mm (with gentle shaking) at laboratory temperature in the presence of 5 ml of TALON resin (Clontech) The resin is washed three times with 30 ml of chromatography buffer containing imidazole at 10 mM The resin is then placed in a column, washed with 10 volumes of chromatography buffer, and the protein is eluted with 10 ml of chromatography buffer containing imidazole at 50 mM Fractions of 1 ml are recovered with a fraction collector A second elution is carried out using chromatography buffer containing imidazole 0 to 100 mM The fractions containing the recombinant protein E are determined by dot-blot using anti-E mouse antibodies and anti-mouse conjugate coupled to alkaline phosphatase, or using coupled Ni-NTA conjugate with alkaline phosphatase (Qiagen)
4) Contrôle de purification : Un aliquote de chaque fraction 5 est déposé sur gel de polyacrylamide La présence de ia protéine d'intérêt est vérifiée par Westem-blot et après coloration au nitrate d'argent4) Purification control: An aliquot of each fraction 5 is deposited on polyacrylamide gel The presence of the protein of interest is checked by Westem-blot and after staining with silver nitrate
Immunogènes : Les protéines virales à concentration de 5μg de protéine pure sont mélangées à 100 μg d'hydroxyde d'aluminium (Alu- gel-S, Serva) et inoculées à des souris SWISS par voie mtrapéπtonéale o Les souches virales de Dengue ayant servi à l'immunisation sont titrées
sur cellules AP61 (détection des foyers par immunocytochimie) et inoculées à raison de 103 particules formant plage (PFU) par souris L'antigène contrôle injecté aux souris correspond à un peptide contenant les trois derniers acides aminés de la protéine E (KKG) suivis de six histidines (KKG Hιs6) L'inoculum de chaque injection contient 100 μg de peptide mélangé à de l'Alu-gelImmunogens: The viral proteins with a concentration of 5 μg of pure protein are mixed with 100 μg of aluminum hydroxide (Alu-gel-S, Serva) and inoculated into SWISS mice by mtrapéπtonéale o The viral strains of Dengue having served to immunization are titrated on AP61 cells (detection of foci by immunocytochemistry) and inoculated at the rate of 10 3 plaque-forming particles (PFU) per mouse The control antigen injected into the mice corresponds to a peptide containing the last three amino acids of protein E (KKG) followed of six histidines (KKG Hιs6) The inoculum of each injection contains 100 μg of peptide mixed with Alu-gel
Calcul de la dose d'épreuve : l'activité létale des virus de la Dengue pour le souriceau de 5 jours est analysée par inoculation intracérébrale de 0,02 ml de suspension virale à différentes dilutions La dose létale tuant 50 % des souris (DL50) est calculée par la méthode deCalculation of the test dose: the lethal activity of the Dengue viruses for the 5-day old mouse is analyzed by intracerebral inoculation of 0.02 ml of viral suspension at different dilutions The lethal dose killing 50% of the mice (LD50) is calculated by the method of
Reed et MuenchReed and Muench
Immunisation des souris : Les souris de trois semaines sont vaccinées par une injection intrapéπtonéale d'un mélange de produits correspondant aux 4 sérotypes de Dengue (DEΔ100Hιs6 ou virus) à 3 semaines, 4 semaines et 6 semaines, puis saignées individuellement une semaine après la dernière injection Le peptide vaccinant du contrôle négatif est identique pour les 4 sérotypes Les anticorps neutralisants de ces sérums sont titrés vis-à-vis de chaque virus homologue Les sérums servent aussi au test de protection après transfert passif au souriceau Test de neutralisation : Les anticorps des souris vaccinées sont titrés par séroneutralisatioπ dans des plaques 24 cupules en présende de 50 PFU de virus homologue sur des cellules Vero Le titre d'anticorps neutralisants correspond à la dilution neutralisant 80 % des foyers de virus détectés par immunocytochimie à l'aide d'anticorps anti- protéines virales et de conjugué anti-lgG de souris marqué à la peroxidaseImmunization of mice: Three-week-old mice are vaccinated by an intrapéπtonéale injection of a mixture of products corresponding to the 4 serotypes of Dengue (DEΔ100Hιs6 or virus) at 3 weeks, 4 weeks and 6 weeks, then bled individually one week after the last injection The vaccinating peptide of the negative control is identical for the 4 serotypes The neutralizing antibodies of these sera are titrated against each homologous virus The sera are also used for the protection test after passive transfer to the mouse Neutralization test: Antibodies vaccinated mice are titrated by seroneutralisatioπ in 24-well plates with 50 PFU of homologous virus on Vero cells The neutralizing antibody titer corresponds to the dilution neutralizing 80% of the foci of virus detected by immunocytochemistry using antibodies anti-viral proteins and anti-mouse IgG conjugate labeled with peroxidase
Test de protection des souriceaux : 100 DL50 de virus sont inoculées à des groupes de souriceaux de 5 jours ayant reçu la veille une injection mtrapéritonéale de 0,05 ml de sérum de souris immunisées (transfert passif) Chaque groupe de souriceaux du transfert passif est
soumis à une dose d'épreuve par un des 4 sérotypes de virus Les souπs sont observées pendant <_. semaines et les signes de paralysie ou la mort sont notésMouse protection test: 100 LD50 of virus are inoculated into groups of 5-day-old mice that received 0.05 ml miterperitoneal injection the day before of immunized mouse serum (passive transfer) Each group of passive transfer mice is subjected to a test dose by one of the 4 virus serotypes The souπs are observed for <_. weeks and signs of paralysis or death are noted
Résultats : Les résultats obtenus sont présentés dans le tableau 3 ci-dessousResults: The results obtained are presented in Table 3 below
Ce tableau rassemble les résultats obtenus quant au titre d'anticorps neutralisant d'une part et quant au pourcentage de souriceaux survivants après un challenge par des virus virulents selon les conditions décrites ci-dessus Pour chacun des sérotypes sont comparés les résultats obtenus avec les peptides de l'invention comprenant le résidu histidine, avec les virus vivants, et avec les peptidesKKG Hιs6 comprenant le résidu histidine, et ce aux doses respectives d'antigènes de 5μg 103 Pfu et 100 μg Le titre de neutralisation a été mesuré sur des sérums prélevés à 7 semaines après les 3 injections d'antigène Le titre de neutralisation correspond à l'inverse de la dilution de sérum conduisant à une réduction de 80 % des unités formant foyers (UFF) de la souche de virus homologue sur cellules VERO Les virus homologues ont été utilisés pour le test de neutralisation et de challengeThis table brings together the results obtained with regard to the neutralizing antibody titer on the one hand and as to the percentage of mice surviving after a challenge with virulent viruses according to the conditions described above. For each of the serotypes, the results obtained with the peptides are compared. of the invention comprising the histidine residue, with live viruses, and with the KKG Hιs6 peptides comprising the histidine residue, and this at respective doses of antigens of 5 μg 10 3 Pfu and 100 μg The neutralization titer was measured on sera taken at 7 weeks after the 3 antigen injections The neutralization titer corresponds to the reverse of the dilution of serum leading to an 80% reduction in focal units (UFF) of the homologous virus strain on VERO cells Viruses counterparts were used for the neutralization and challenge test
Les souris ont été immunisées par le mélange tétravalent dans les 3 cas de figures
The mice were immunized with the tetravalent mixture in the 3 cases of figures
tableau 3table 3
Ces résultats indiquent que la formulation contenant les 4 protéines recombinées induit des anticorps neutralisant dirigés contre les 4 sérotypes de virusThese results indicate that the formulation containing the 4 recombinant proteins induces neutralizing antibodies directed against the 4 virus serotypes.
Ce mélange peut donc constituer la base d'une composition ou d'un principe actif d'un vaccin sous unités polyvalent contre la DengueThis mixture can therefore constitute the basis of a composition or of an active principle of a vaccine in polyvalent units against Dengue
Le titre élevé d'anticorps neutralisants produits après 3 injections de 5 μg de protéines est remarquable Aucune des protéines recombinées produites précédemment sans histidtne n'avait abouti à l'induction de titres aussi élevés d'anticorps neutralisants. Deux hypothèses sont avancées pour expliquer ces résultats soit la purification imparfaite des protéines précédentes contenait des constituants immunosuppresseurs, soit la séquence de 6 histidines contenue dans les protéines actuelles en présence de particules formées d'ions divalents ou tπvalents lui confère une présentation spatiale multiméπque et en conséquence des propriétés immunogènes particulières
III. Induction des anticorps neutralisants par les protéines d'enveloppe recombinante de chacun des quatre sérotypes :The high titer of neutralizing antibodies produced after 3 injections of 5 μg of protein is remarkable. None of the recombinant proteins produced previously without histide had resulted in the induction of such high titers of neutralizing antibodies. Two hypotheses are put forward to explain these results either the imperfect purification of the preceding proteins contained immunosuppressive constituents, or the sequence of 6 histidines contained in the current proteins in the presence of particles formed of divalent or tπvalent ions gives it a multi-spatial presentation and in consequence of specific immunogenic properties III. Induction of neutralizing antibodies by the recombinant envelope proteins of each of the four serotypes:
1 ° Les protéines d'enveloppe de chaque sérotype viral délétées (Δ) de 103 acides aminés à leur extrémité C-terminal ont été exprimées par le système du baculo-virus et purifiées par chromatographie sur colonne de cobalt grâce au segment de six histidines (His6) qui composent leur extrémité C-terminal. L'antigène vaccinant a été injecté aux souris sous forme monotypique et à titre de comparaison les résultats obtenus après l'injection de la forme tétravalente tels que présentés dans le tableau III ci-dessus sont rappelés au niveau de la ligne 4 x DEΔ100His6. La ligne PBS représente un témoin représenté par le tampon du même nom.1 ° The envelope proteins of each deleted viral serotype (Δ) of 103 amino acids at their C-terminal end were expressed by the baculovirus system and purified by chromatography on a cobalt column using the segment of six histidines ( His6) which make up their C-terminal end. The vaccinating antigen was injected into mice in monotypic form and by way of comparison the results obtained after the injection of the tetravalent form as presented in table III above are recalled at the level of the line 4 × DEΔ100His6. The PBS line represents a witness represented by the buffer of the same name.
Immunisation des souris : Des groupes de 10 souris BALB/c de trois semaines ont été immunisées aux jours J1 , J7 et J21 à l'aide de 5 μg d'antigène recombinant couplé à 100 μg d'hydroxide d'aluminium pris comme adjuvant. Les souris ont été saignées au jour J28 et testés en séroneutralisation vis-à-vis de 200 Unités Formatrices de Foyer de chacune des souches de référence (Dengue 1 : Hawai 1944 ; Dengue 2 : Nouvelle Guinée C, 1944 ;Immunization of the mice: Groups of 10 three-week BALB / c mice were immunized on days D1, D7 and D21 using 5 μg of recombinant antigen coupled to 100 μg of aluminum hydroxide taken as an adjuvant. The mice were bled on day D28 and tested in seroneutralization with respect to 200 Home Formative Units of each of the reference strains (Dengue 1: Hawai 1944; Dengue 2: New Guinea C, 1944;
Dengue 3 : H87 Philippines 1956 ; Dengue 4 : H251 , Philippines 1951 ). Le titre correspond à l'inverse de la dilution neutralisant plus de 50 % des foyers.Dengue 3: H87 Philippines 1956; Dengue 4: H251, Philippines 1951). The title corresponds to the reverse of the dilution neutralizing more than 50% of the outbreaks.
Les résultats indiqués dans le tableau 4 ci-dessous indiquent que tant l'immunogène D2EΔ100His6 que celui D3EΔ100His6 induisent la formation d'anticorps neutralisant contre le sérotype Dengue 2 alors que les sérotypes Dengue 3 et Dengue 4 ne sont neutralisés respectivement que par la protéine recombinante D3EΔ100His6 et D4EΔ100His6. Enfin le D1 EΔ100His6 n'a aucun effet neutralisant.
TABLEAU 4 :The results indicated in table 4 below indicate that both the immunogen D2EΔ100His6 and that D3EΔ100His6 induce the formation of neutralizing antibodies against the serotype Dengue 2 while the serotypes Dengue 3 and Dengue 4 are only neutralized respectively by the recombinant protein D3EΔ100His6 and D4EΔ100His6. Finally, D1 EΔ100His6 has no neutralizing effect. TABLE 4:
TITRE NEUTRA LISANTNEUTRAL READING TITLE
Immunogène Dengue 1 Dengue 2 Dengue 3 Dengue 4Immunogen Dengue 1 Dengue 2 Dengue 3 Dengue 4
D1EΔ100His6 20 <20 <20 <20D1EΔ100His6 20 <20 <20 <20
D2EΔ100His6 <20 >100 20 <20D2EΔ100His6 <20> 100 20 <20
D3EΔ100His6 <20 100 100 <20D3EΔ100His6 <20 100 100 <20
D4EΔ100His6 <20 <20 <20 100D4EΔ100His6 <20 <20 <20 100
4xDEΔ100His6 100 >100 100 >1004xDEΔ100His6 100> 100 100> 100
PBS <20 <20 <20 <20PBS <20 <20 <20 <20
Les protéines recombinantes issues des sérotypes D2, D3 et D4 paraissent donc particulièrement intéressantes et le titre d'anticorps neutralisant dans le sérum de singe vacciné avec une protéine d'enveloppe recombinante purifée a été comparée à celle obtenue avec un virus atténué ; les résultats sont présentés dans le tableau 5 ci-dessous.
Recombinant proteins from serotypes D2, D3 and D4 therefore appear to be particularly advantageous and the titer of neutralizing antibody in monkey serum vaccinated with a purified recombinant envelope protein was compared with that obtained with an attenuated virus; the results are presented in table 5 below.
TABLEAU 5 : Réponse immune des singes cynomolgus vaccinés contre la dengue 2 et infectés par une souche sauvage de virus -1TABLE 5: Immune response of cynomolgus monkeys vaccinated against dengue 2 and infected with a wild strain of virus -1
00 Ul00 Ul
Singes J28 J56 J70 J98Monkeys J28 J56 J70 J98
ELISA PRNT ELISA PRNT ELISA PRNT ELISA PRNTELISA PRNT ELISA PRNT ELISA PRNT ELISA PRNT
A1 <100 <20 <100 ND <100 <20 400 160A1 <100 <20 <100 ND <100 <20 400 160
A2 <100 <20 <100 ND <100 20 630 160A2 <100 <20 <100 ND <100 20 630 160
B1 <100 <20 <100 ND <100 <20 250 80B1 <100 <20 <100 ND <100 <20 250 80
B2 <100 <20 <100 ND <100 <20 630 >640B2 <100 <20 <100 ND <100 <20 630> 640
C1 100 <20 800 20 1000 160 12000 2560C1 100 <20 800 20 1000 160 12000 2560
C2 <100 <20 300 20 800 160 400 320C2 <100 <20 300 20 800 160 400 320
D1 <100 <20 <100 ND <100 <20 100 80D1 <100 <20 <100 ND <100 <20 100 80
D2 <100 <20 <100 ND <100 <20 300 80D2 <100 <20 <100 ND <100 <20 300 80
E1 500 80 600 20 400 40 5000 >1280E1 500 80 600 20 400 40 5000> 1280
E2 160 20 200 20 300 20 <100 <40E2 160 20 200 20 300 20 <100 <40
TJ πTJ π
HH
Des groupes de 2 singes ont reçu différentes préparations immunogènes:Groups of 2 monkeys received different immunogenic preparations:
Vaccin de l'encéphalite japonaise (A); nonapeptide KKGH6 (B); Protein d'enveloppe recombinante D2EΔ100His6 (C); PBSJapanese encephalitis vaccine (A); nonapeptide KKGH6 (B); Recombinant envelope protein D2EΔ100His6 (C); PBS
(D); vaccin dengue 2 atténué (E). PRNT : Plaque réduction neutralisation test (réf. 10). o oo(D); dengue 2 vaccine attenuated (E). PRNT: Test neutralization reduction plate (ref. 10). o oo
Les préparations ont été injectées au jour J0 (A and E) ou aux jours J0, J28, J56 après échantillonnage du plasma. oThe preparations were injected on day D0 (A and E) or on days D0, D28, D56 after sampling the plasma. o
Les singes ont ensuite été soumis à une dose d'épreuve au jour J83 par injection intradermique de 2X105 particules infectieuses de virus dengue 2, souche Jamaïque 1983. Les plasmas ont été ensuite collectés à J98.
The monkeys were then subjected to a test dose on day D83 by intradermal injection of 2 × 10 5 infectious particles of dengue virus 2, strain Jamaica 1983. The plasmas were then collected on D98.
Obtention des immunogènes ' Obtaining immunogens '
Les singes reçoivent mensuellement trois inoculations de 100 μg de peptides adjuvantes avec 1 mg d'adjuvant aluminium (groupe B). Trois inoculations de 100 μg de protéines d'enveloppe recombinante de Dengue 2 D2EΔ100His6 mélangés avec 1 mg d'adjuvant alum (groupe C) ; trois inoculations de tampon PBS avec l'adjuvant (groupe D) ou une inoculation de 0,5 ml de vaccins vivants Dengue 2 atténués de type THAÏ préparés par PASTEUR MERIEUX SERUMS & VACCINS (groupe E).The monkeys receive three inoculations of 100 μg of adjuvant peptides with 1 mg of aluminum adjuvant (group B) monthly. Three inoculations of 100 μg of recombinant Dengue 2 envelope proteins D2EΔ100His6 mixed with 1 mg of alum adjuvant (group C); three inoculations of PBS buffer with the adjuvant (group D) or an inoculation of 0.5 ml of live attenuated Dengue 2 vaccines of the THAI type prepared by PASTEUR MERIEUX SERUMS & VACCINES (group E).
Les sérums sont collectés quatre semaines après la dernière inoculation et testes vis-à-vis de leur anticorps neutralisant contre 200 unités formant plaques (PFU) de la souche homologue de Dengue 2 Janaque. Les titres correspondent aux dilutions de sérum qui neutralisent 50 % des plaques Le tableau 5 indique la spécificité d'induction d'anticorps neutralisant de la protéine recombinante issue du sérotype 2 de la Dengue chez les singes cynomolgus, avec une réponse au moins quatre fois plus importante que celle obtenue avec le virus atténué
The sera are collected four weeks after the last inoculation and tested against their neutralizing antibody against 200 plaque-forming units (PFU) of the homologous strain of Dengue 2 Janaque. The titles correspond to the dilutions of serum which neutralize 50% of the plates. Table 5 indicates the specificity of induction of neutralizing antibodies of the recombinant protein resulting from serotype 2 of Dengue in cynomolgus monkeys, with a response at least four times more important than that obtained with the attenuated virus
REFERENCESREFERENCES
(1 ) V. Deubel, R.M. Kinney, D.W. Trent (1986) Nucléotide séquence and deduced amino acid séquence of the structural proteins of Dengue type 2 virus, Jamaica génotype Virology, 155 365-377(1) V. Deubel, R.M. Kinney, D.W. Trent (1986) Nucleotide sequence and deduced amino acid sequence of the structural proteins of Dengue type 2 virus, Jamaica genotype Virology, 155 365-377
(2) (Mason P.W., McAda P.C., Mason T.L., Fournier M.J. (1987) Virology 161 262-267(2) (Mason P.W., McAda P.C., Mason T.L., Fournier M.J. (1987) Virology 161 262-267
(3) OSATOMI K. et al. (1990) Virology 176 643-647(3) OSATOMI K. et al. (1990) Virology 176 643-647
(4) Zhao B. et al. (1986) Cloning full cength Dengue type 4 virol DNA séquences analysis of gènes coding for structural proteins(4) Zhao B. et al. (1986) Cloning full cength Dengue type 4 virol DNA sequences analysis of genes coding for structural proteins
Virology, 155 77-88Virology, 155 77-88
(5) Putnak R. et al (1991 ) Dengue 1 virus envelop glycoprotein gène expressed in recombinant baculovirus ehcits virus- neutralizing antiboding in mice and products them from virus challenge Am J trop Med thig , 45 159-167(5) Putnak R. et al (1991) Dengue 1 virus envelop glycoprotein gene expressed in recombinant baculovirus ehcits virus- neutralizing antiboding in mice and products them from virus challenge Am J trop Med thig, 45 159-167
(6) Y.M. Zhang, E.P. Hayes, T.C. McCarty, D.R. Dubois, P.L. Summers, K.H. Eckels, R.M. Chanock, C.J. Lai (1988) Immunization of mice with Dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces résistance to Dengue virus encephalitis J Virol , 62 3027-3031(6) YM Zhang, EP Hayes, TC McCarty, DR Dubois, PL Summers, KH Eckels, RM Chanock, CJ Lai (1988) Immunization of mice with Dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to Dengue virus encephalitis J Virol, 62 3027-3031
(7) Deubel V. et al. (1991 ) Processing sécrétion and immunoreactivity of carboxyterminally truncated Dengue 2 enveloppe proteins expressed in insect cells by recombinant baculoviruses Virology 180 442-447 (8) R. Feighny, J. Burrous, R. Putnak (1994) Dengue type -(7) Deubel V. et al. (1991) Processing secretion and immunoreactivity of carboxyterminally truncated Dengue 2 envelope proteins expressed in insect cells by recombinant baculoviruses Virology 180 442-447 (8) R. Feighny, J. Burrous, R. Putnak (1994) Dengue type -
2 virus envelope protein made using recombinant baculovirus protects mice against virus challenge Am J Trop Med Hyg , 50 322-3282 virus envelope protein made using recombinant baculovirus protects mice against virus challenge Am J Trop Med Hyg, 50 322-328
(9) C. Delenda, M.P. Frenkiel, V. Deubel (1994) Protective efficacy in mice of a secreted form of recombinant Dengue-2 virus
envelope protein produced in baculovirus infected insect cells Arch Virol , 139 197-207(9) C. Delenda, MP Frenkiel, V. Deubel (1994) Protective efficacy in mice of a secreted form of recombinant Dengue-2 virus envelope protein produced in baculovirus infected insect cells Arch Virol, 139 197-207
(10) C. Delenda, I. Staropoli, M.P. Frenkiel, L. Cabanié, V. Deubel (1994) Analysis of C terminally truncated Dengue 2 and Dengue 3 virus envelope glycoproteins processing in insect cells and immunogemc properties in mice J Gen Virol , 75 , 1569-1578(10) C. Delenda, I. Staropoli, MP Frenkiel, L. Cabanié, V. Deubel (1994) Analysis of C terminally truncated Dengue 2 and Dengue 3 virus envelope glycoproteins processing in insect cells and immunogemc properties in mice J Gen Virol, 75, 1569-1578
(11) R. Men, M. Bray, CJ. Lai (1991 ) Carboxy-terminally truncated Dengue virus envelope glycoproteins expressed on the cell surface and secreted extracellularly exhibit increased immunogenicity in mice J Virol , 65, 1400-1407(11) R. Men, M. Bray, CJ. Lai (1991) Carboxy-terminally truncated Dengue virus envelope glycoproteins expressed on the cell surface and secreted extracellularly exhibit increased immunogenicity in mice J Virol, 65, 1400-1407
(12) Nikolow D.B. et al. (1992) Nature 360 4046(12) Nikolow D.B. et al. (1992) Nature 360 4046
(13) Maina E. et al. (1988) A vector to express and puπfy foreign proteins m E Çgh by fusion to, and séparation from, maltose bmgtng protein Gène 74 365-373 ( 14) Moks T et al . (1987) Biotechnology 5 379-382(13) Maina E. et al. (1988) A vector to express and puπfy foreign proteins m E Çgh by fusion to, and separation from, maltose bmgtng protein Gene 74 365-373 (14) Moks T et al. (1987) Biotechnology 5 379-382
(15) Hochuli E. et al. (1987) J chromatography 41± 177- 184(15) Hochuli E. et al. (1987) J chromatography 41 ± 177-184
(16) J. Schmitt, H. Hess, H.G. Stunnenberg. Affinity purification of histidine-tagged proteins Mol Biol Reports, 18 223-230 (17) Flamand M. et al. (1992) Expression and sécrétion of(16) J. Schmitt, H. Hess, H.G. Stunnenberg. Affinity purification of histidine-tagged proteins Mol Biol Reports, 18 223-230 (17) Flamand M. et al. (1992) Expression and secretion of
Japanese encéphalites virus nonstructural protein NS1 by insect cells using a recombinant baculovirus Virology 191 826-836Japanese encephalitis virus nonstructural protein NS1 by insect cells using a recombinant baculovirus Virology 191 826-836
(18) Brevet européen n° 0 318 216(18) European patent n ° 0 318 216
(19) Morein B. (1987) Potentiation of immune response by immunization with antigens in defined multimeπc physical forms Vet(19) Morein B. (1987) Potentiation of immune response by immunization with antigens in defined multimeπc physical forms Vet
Immunol Immunopathol 17 153-159
Immunol Immunopathol 17 153-159
Claims
REVENDICATIONS
1. Polypeptide ou glycopeptide immunogène caractérisé en ce qu'il est constitué d'un polypeptide ou glycoprotéine issu d'un Flaviviridiae notamment de la protéine E, ou preM, ou NS1 ou NS3 du virus de la Dengue, ledit polypeptide portant à son extrémité carboxylée un résidu de 2 à 8 acides aminés choisis parmi l'histidine, le tryptophane ou la cystéine.1. Immunogenic polypeptide or glycopeptide characterized in that it consists of a polypeptide or glycoprotein derived from a Flaviviridiae in particular from protein E, or preM, or NS1 or NS3 of the Dengue virus, said polypeptide carrying at its end carboxylated a residue of 2 to 8 amino acids chosen from histidine, tryptophan or cysteine.
2. Polypeptide ou glycopeptide selon la revendication 1 caractérisé en ce que le glycopeptide est constitué de la protéine d'enveloppe E délétée de 70 à 105 acides aminés à son extrémité carboxylée.2. Polypeptide or glycopeptide according to claim 1 characterized in that the glycopeptide consists of the envelope protein E deleted from 70 to 105 amino acids at its carboxylated end.
3. Polypeptide ou glycopeptide selon les revendications 1 ou3. Polypeptide or glycopeptide according to claims 1 or
2 caractérisé en ce que le séquence à l'extrémité carboxylée est constituée d'un résidu de 6 histidines lui conférant ainsi la propriété de se fixer sur des cations divalents ou trivalents.2 characterized in that the sequence at the carboxylated end consists of a residue of 6 histidines thus giving it the property of fixing on divalent or trivalent cations.
4. Composition apte à stimuler l'immunité d'individus susceptibles d'être infectés par le virus de la Dengue caractérisée en ce qu'elle contient au moins un polypeptide ou glycopeptide selon l'une des revendications 1 à 3. 5. Composition selon la revendication 4 caractérisée en ce que le glycopeptide est issu de la glycoprotéine E du virus de la Dengue.4. Composition capable of stimulating the immunity of individuals liable to be infected with the Dengue virus, characterized in that it contains at least one polypeptide or glycopeptide according to one of claims 1 to 3. 5. Composition according to claim 4 characterized in that the glycopeptide is derived from the glycoprotein E of the Dengue virus.
6. Composition selon les revendications 4 ou 5 caractérisée en ce que les polypeptides ou glycopeptides sont un mélange d'au moins6. Composition according to claims 4 or 5 characterized in that the polypeptides or glycopeptides are a mixture of at least
3 polypeptides selon l'une des revendications 1 à 3 issus de au moins 3 sérotypes du virus de la Dengue, et de préférence de chacun des sérotypes connus.3 polypeptides according to one of claims 1 to 3 from at least 3 serotypes of the Dengue virus, and preferably from each of the known serotypes.
7. Composition selon les revendications 4 à 6 caractérisée en ce qu'elle contient le polypeptide D3EΔ100His6 et/ou le polypeptide D2EΔ100His6.
8 Composition selon les revendications 4 à 6 7 constituée de polypeptides ou glycopeptides issus de la protéine E ou preM ou NS1 ou NS3 du virus de la Dengue, et de préférence la protéine E7. Composition according to Claims 4 to 6 characterized in that it contains the D3EΔ100His6 polypeptide and / or the D2EΔ100His6 polypeptide. 8 Composition according to claims 4 to 6 7 consisting of polypeptides or glycopeptides derived from protein E or preM or NS1 or NS3 of the Dengue virus, and preferably protein E
9 Composition selon la revendication 6 ou 7, caractérisée en ce que chaque peptide est présent dans une proportion de 10 à 50 % en poids par rapport à l'ensemble des peptides de ladite composition9 Composition according to claim 6 or 7, characterized in that each peptide is present in a proportion of 10 to 50% by weight relative to the set of peptides of said composition
10 Composition selon l'une des revendications 4 à 9 caractérisée en ce qu'elle contient en outre des polypeptides, glycopeptides ou protéines provenant d'autres flavivirus comme la fièvre jaune ou l'encéphalite japonaise, ou l'hépatite C10 Composition according to one of claims 4 to 9 characterized in that it also contains polypeptides, glycopeptides or proteins from other flaviviruses such as yellow fever or Japanese encephalitis, or hepatitis C
11 Composition, selon l'une des revendications 4 à 9 caractérisée en ce qu'elle contient en outre des antigènes viraux, bactériens ou parasitaires portant à leur extrémité carboxy-terminale un résidu de 2 à 8 acides aminés choisis par l'histidine, le tryptophane et la cystéine11 Composition according to one of claims 4 to 9 characterized in that it also contains viral, bacterial or parasitic antigens carrying at their carboxy-terminal end a residue of 2 to 8 amino acids chosen by histidine, the tryptophan and cysteine
12 Séquence d'acides nucléiques constituée d'une séquence codant pour une protéine du virus de la Dengue, délétée en 3' de 210 à 315 nucléotides, auxquelles a été ajoutée en 3' une séquence codant pour 2 à 8 acides aminés choisis parmi l'histidine, le tryptophane ou la cystéine 13 Séquence d'acides nucléiques selon la revendication 12 caractérisée en ce que la séquence code pour une protéine E de la Dengue de sérotype 1 , 2, 3 ou 412 Nucleic acid sequence consisting of a sequence coding for a Dengue virus protein, deleted in 3 'from 210 to 315 nucleotides, to which has been added in 3' a sequence coding for 2 to 8 amino acids chosen from histidine, tryptophan or cysteine 13 Nucleic acid sequence according to claim 12 characterized in that the sequence codes for a Dengue protein E of serotype 1, 2, 3 or 4
14 Séquence d'acides nucléiques selon les revendications 12 ou 13 caractérisée en ce que la séquence en 3' code pour 6 histidines 15 Vecteur d'acides nucléiques approprié pour une utilisation dans l'expression de gènes exogènes chez un eucaryote, caractérisé en ce qu'il contient une séquence d'acides nucléiques selon l'une des revendications 12 à 14, pourvue en 5' d'une séquence signal homologue et/ou hétérologue de celle de la protéine codée par ladite séquence, l'ensemble étant sous la dépendance d'un promoteur susceptible d'être activé dans ladite cellule eucaryote
16. Vecteur d'expression selon la revendication 15 caractérisé en ce que la cellule eucaryote est une cellule d'insecte notamment de Spodoptera frugiperda SF9, le vecteur est un vecteur navette susceptible de réaliser une recombinaison homologue avec un baculovirus, et porteur, en amont de la séquence signal, d'un promoteur d'un gène endogène au baculovirus notamment celui de la polyédrine,14 Nucleic acid sequence according to claims 12 or 13 characterized in that the 3 'sequence codes for 6 histidines 15 Nucleic acid vector suitable for use in the expression of exogenous genes in a eukaryote, characterized in that 'it contains a nucleic acid sequence according to one of claims 12 to 14, provided at 5' with a signal sequence homologous and / or heterologous to that of the protein coded by said sequence, the whole being under dependence a promoter capable of being activated in said eukaryotic cell 16. Expression vector according to claim 15 characterized in that the eukaryotic cell is an insect cell in particular of Spodoptera frugiperda SF9, the vector is a shuttle vector capable of carrying out homologous recombination with a baculovirus, and carrier, upstream of the signal sequence, of a promoter of a gene endogenous to baculovirus, in particular that of polyhedrin,
17. Vecteur d'expression selon les revendications 15 ou 16 caractérisé en ce qu'il s'agit d'un baculovirus recombinant déposé à la CNCM sous le numéro 1-1497, le 1er décembre 1994 et porteur d'une séquence codant pour un peptide constitué du gène E du virus de la17. Expression vector according to claims 15 or 16 characterized in that it is a recombinant baculovirus deposited at the CNCM under the number 1-1497, on December 1, 1994 and carrying a sequence coding for a peptide consisting of the E virus gene
Dengue sérotype r° 2 dans laquelle l'extrémité COOH a été délétée de 100 acides aminés et substituée par 6 histidines.Dengue serotype r ° 2 in which the COOH end has been deleted from 100 amino acids and substituted by 6 histidines.
18. Vecteur d'expression selon les revendications 15 ou 16 caractérisé en ce qu'il s'agit d'un baculovirus recombinant déposé à la CNCM sous le numéro 1-1624, le 11 octobre 1995 et porteur d'une séquence codant pour un peptide constitué du gène E du virus de la Dengue sérotype n° 4 dans laquelle l'extrémité COOH a été délétée de 100 acides aminés et substituée par 6 histidines.18. Expression vector according to claims 15 or 16 characterized in that it is a recombinant baculovirus deposited at the CNCM under the number 1-1624, on October 11, 1995 and carrying a sequence coding for a peptide consisting of the Dengue virus E serotype No. 4 gene in which the COOH end has been deleted from 100 amino acids and substituted with 6 histidines.
19. Vecteur d'expression selon les revendications 15 ou 16 caractérisé en ce qu'il s'agit d'un baculovirus recombinant déposé à la19. Expression vector according to claims 15 or 16 characterized in that it is a recombinant baculovirus deposited at the
CNCM sous le numéro 1-1625, le 11 octobre 1995 et porteur d'une séquence codant pour un peptide constitué du gène E du virus de la Dengue sérotype n° 3 dans laquelle l'extrémité COOH a été délétée de 100 acides aminés et substituée par 6 histidines. 20. Vecteur d'expression selon les revendications 15 ou 16 caractérisé en ce qu'il s'agit d'un baculovirus recombinant déposé à la CNCM sous le numéro 1-1626, le 1 1 octobre 1995 et porteur d'une séquence codant pour un peptide constitué du gène E du virus de la Dengue sérotype n° 1 dans laquelle l'extrémité COOH a été délétée de 100 acides aminés et substituée par 6 histidines.
21. Composition apte à induire une réponse immunitaire cellulaire eu humorale chez un hôte susceptible d'être infecté par le virus de la Dengue caractérisée en ce qu'elle contient un acide nucléique codant pour un polypeptide selon l'une des revendications 1 à 3. 22. Procédé de préparation d'un polypeptide ou d'un glycopeptide selon l'une des revendications 1 à 4 caractérisé en ce qu'il comprend une étape de chromatographie par affinité sur un support présentant une affinité pour la séquence de 2 à 8 acides aminés.CNCM under number 1-1625, October 11, 1995 and carrying a sequence coding for a peptide consisting of the E gene of the Dengue virus serotype No. 3 in which the COOH end has been deleted from 100 amino acids and substituted by 6 histidines. 20. Expression vector according to claims 15 or 16 characterized in that it is a recombinant baculovirus deposited at the CNCM under the number 1-1626, on October 11, 1995 and carrying a sequence coding for a peptide consisting of the E gene of the Dengue virus serotype No. 1 in which the COOH end has been deleted from 100 amino acids and substituted with 6 histidines. 21. Composition capable of inducing a cellular or humoral immune response in a host capable of being infected with the Dengue virus, characterized in that it contains a nucleic acid coding for a polypeptide according to one of claims 1 to 3. 22. Method for preparing a polypeptide or a glycopeptide according to one of claims 1 to 4 characterized in that it comprises a step of affinity chromatography on a support having an affinity for the sequence of 2 to 8 acids amines.
23. Procédé selon la revendication 22 caractérisé en ce que le support est une colonne contenant des ions divalents ou trivalents notamment du nickel, du cuivre, du cobalt ou du zinc présentant une affinité pour l'histidine, le tryptophane ou la cystéine riches en électrons.23. The method of claim 22 characterized in that the support is a column containing divalent or trivalent ions including nickel, copper, cobalt or zinc having an affinity for histidine, tryptophan or cysteine rich in electrons .
24. Vaccin polyvalent contre le virus de la Dengue caractérisé en ce qu'il comprend une composition immunogène selon l'une des revendications 4 à 1 1 et 21 , avec le cas échéant un adjuvant de vaccination qui peut être notamment un adjuvant porteur d'ions divalents ou trivalents tels l'hydroxide d'aluminium ou du phosphate de calcium, ou un adjuvant de type adjuvant de Freund, ou un dérivé du muramylpeptide ou un iscom. 25. Vaccin polyvalent selon la revendication 24 caractérisé en ce qu'il contient en outre des polypeptides ou glycopeptides immunogènes provenant d'autres flavivirus comme la fièvre jaune ou l'encéphalite japonaise, ou l'encéphalite à tiques ou l'hépatite C.24. Multipurpose vaccine against the Dengue virus characterized in that it comprises an immunogenic composition according to one of claims 4 to 1 1 and 21, with if necessary a vaccination adjuvant which can be in particular an adjuvant carrying divalent or trivalent ions such as aluminum hydroxide or calcium phosphate, or an adjuvant of Freund's adjuvant type, or a derivative of muramylpeptide or an iscom. 25. Multipurpose vaccine according to claim 24 characterized in that it also contains immunogenic polypeptides or glycopeptides from other flaviviruses such as yellow fever or Japanese encephalitis, or tick-borne encephalitis or hepatitis C.
26. Vaccin polyvalent selon la revendication 24 caractérisé en ce qu'il contient 4 peptipes ou glycopeptides correspondant à chacun des sérotypes du virus de la Dengue avec un adjuvant contenant des ions divalents ou trivalents.26. Multipurpose vaccine according to claim 24 characterized in that it contains 4 peptipes or glycopeptides corresponding to each of the serotypes of the Dengue virus with an adjuvant containing divalent or trivalent ions.
27. Vaccin polyvalent selon la revendication 24, caractérisé en ce qu'il contient 1 , 2, 3 ou 4 peptides ou polypeptides selon la revendication 1 à 3 et des virus atténués.
28. Vaccin polyvalent selon la revendication 24 caractérisé en ce qu'il contient outre les polypeptides selon l'une des revendications 1 à 3, un ou plusieurs antigènes viraux, bactériens ou parasitaires.27. Polyvalent vaccine according to claim 24, characterized in that it contains 1, 2, 3 or 4 peptides or polypeptides according to claim 1 to 3 and attenuated viruses. 28. Multipurpose vaccine according to claim 24 characterized in that it contains, in addition to the polypeptides according to one of claims 1 to 3, one or more viral, bacterial or parasitic antigens.
29. Vaccin polyvalent caractérisé en ce qu'il comprend un mélange d'acides nucléiques porteurs de séquences codant pour un polypeptide selon l'une des revendications 1 à 3.29. A versatile vaccine characterized in that it comprises a mixture of nucleic acids carrying sequences coding for a polypeptide according to one of claims 1 to 3.
30. Composition antigénique constituée de polypeptides ou glycopeptides issus de la protéine E, preM, NS1 ou NS3 du virus de la Dengue, caractérisée en ce qu'elle comporte au moins 3 antigènes de 3 sérotypes différents de ce virus, et de préférence un antigène pour chacun des 4 sérotypes.
30. Antigenic composition consisting of polypeptides or glycopeptides derived from protein E, preM, NS1 or NS3 of the Dengue virus, characterized in that it comprises at least 3 antigens of 3 different serotypes of this virus, and preferably an antigen for each of the 4 serotypes.
Applications Claiming Priority (3)
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|---|---|---|---|
| FR9513489A FR2741077B1 (en) | 1995-11-14 | 1995-11-14 | VERSATILE ANTI-DENGUE VACCINE |
| FR9513489 | 1995-11-14 | ||
| PCT/FR1996/001801 WO1997018311A1 (en) | 1995-11-14 | 1996-11-14 | Polyvalent anti-dengue vaccine |
Publications (1)
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|---|---|
| EP0861324A1 true EP0861324A1 (en) | 1998-09-02 |
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| EP96939121A Withdrawn EP0861324A1 (en) | 1995-11-14 | 1996-11-14 | Polyvalent anti-dengue vaccine |
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| JP (1) | JP2000500969A (en) |
| KR (1) | KR19990067581A (en) |
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| BR (1) | BR9611477A (en) |
| CA (1) | CA2235334A1 (en) |
| FR (1) | FR2741077B1 (en) |
| WO (1) | WO1997018311A1 (en) |
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| US6455509B1 (en) * | 1996-06-04 | 2002-09-24 | The United States Of America As Represented By The Secretary Of The Navy | Dengue nucleic acid vaccines that induce neutralizing antibodies |
| BR9815551A (en) | 1997-07-31 | 2000-10-31 | Hawaii Biotech Group | Recombinant dimeric wrap vaccine against flaviviral infection |
| FR2767324B1 (en) * | 1997-08-14 | 2001-04-06 | Pasteur Institut | USE OF RECOMBINANT ENVELOPE PROTEINS FOR THE DIAGNOSIS OF DENGUE VIRUS |
| US6432411B1 (en) | 1999-07-13 | 2002-08-13 | Hawaii Biotechnology Group | Recombinant envelope vaccine against flavivirus infection |
| WO2001059093A2 (en) * | 2000-02-10 | 2001-08-16 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | FULL-LENGTH INFECTIOUS cDNA CLONES OF TICK BORNE FLAVIVIRUS |
| AU2010256845B2 (en) * | 2009-06-01 | 2016-09-22 | Takeda Vaccines, Inc. | Compositions and methods for administration of vaccines against dengue virus |
| WO2016034974A1 (en) * | 2014-09-01 | 2016-03-10 | International Centre For Genetic Engineering And Biotechnology | Vaccine |
| TW201620546A (en) * | 2014-09-02 | 2016-06-16 | 賽諾菲巴斯德公司 | Vaccine compositions |
| CN106290847A (en) * | 2015-05-13 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of dengue virus NS1 antigen emulsion technique detection kit |
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| WO1992003161A1 (en) * | 1990-08-27 | 1992-03-05 | The United States Of America, Represented By The Secretary, United States Department Of Commerce | Flavivirus envelope proteins with increased immunogenicity for use in immunization against virus infection |
| US5843752A (en) * | 1995-05-12 | 1998-12-01 | Schering Corporation | Soluble active hepatitis C virus protease |
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1995
- 1995-11-14 FR FR9513489A patent/FR2741077B1/en not_active Expired - Fee Related
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1996
- 1996-11-14 AU AU76286/96A patent/AU718740B2/en not_active Ceased
- 1996-11-14 KR KR1019980703604A patent/KR19990067581A/en not_active Application Discontinuation
- 1996-11-14 JP JP9518632A patent/JP2000500969A/en active Pending
- 1996-11-14 CA CA002235334A patent/CA2235334A1/en not_active Abandoned
- 1996-11-14 CN CN96198317A patent/CN1202201A/en active Pending
- 1996-11-14 BR BR9611477-0A patent/BR9611477A/en not_active Application Discontinuation
- 1996-11-14 EP EP96939121A patent/EP0861324A1/en not_active Withdrawn
- 1996-11-14 WO PCT/FR1996/001801 patent/WO1997018311A1/en not_active Application Discontinuation
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| WO1997018311A1 (en) | 1997-05-22 |
| BR9611477A (en) | 1999-12-28 |
| FR2741077B1 (en) | 1998-01-23 |
| CN1202201A (en) | 1998-12-16 |
| AU7628696A (en) | 1997-06-05 |
| CA2235334A1 (en) | 1997-05-22 |
| FR2741077A1 (en) | 1997-05-16 |
| KR19990067581A (en) | 1999-08-25 |
| JP2000500969A (en) | 2000-02-02 |
| AU718740B2 (en) | 2000-04-20 |
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